piperidines and halofuginone

Halofuginone is a clinically active derivative of febrifugine that was first isolated from the Chinese herb Dichroa febrifuga. The beneficial biological effects of halofuginone on various diseases including parasitic diseases, cancer, fibrosis, and autoimmune disorders have been investigated. Halofuginone has reduced toxic side effects when compared to febrifugine, an advantage that has led to the commercial availability of halofuginone-based antiparasitic drugs for animal use, and to human clinical trials for the treatment of tumors and fibrosis. This review summarizes advances in determining the mechanism of action of halofuginone, focusing on its antiparasitic role in malaria, cryptosporidiosis, coccidiosis, toxoplasmosis, and leishmaniasis. We discuss mechanistic insights into halofuginone's primary mode of action which involves inhibition of the prolyl-tRNA synthetase enzyme, which is crucial in protein synthesis. Halofuginone exemplifies the untapped wealth of plant-derived compounds in disease therapeutics.

Topics: Animals; Antiprotozoal Agents; Fibrosis; Humans; Malaria; Piperidines; Quinazolinones

A prior systematic review on the efficacy of halofuginone (HFG) treatment to prevent or treat cryptosporidiosis in bovine calves was inconclusive. We undertook an updated synthesis and meta-analyses on key outcomes for the treatment of calves with HFG. Evaluated outcomes were oocyst shedding, diarrhoea, mortality and weight gain. Experiments had to describe results for same age animals in contemporary arms. Most doses were 100-150 mcg kg-1 day-1. Results were subgrouped by study design, experiments with the lowest risk of bias and lack of industry funding. Eighteen articles were found that described 25 experiments. Most evidence came from randomized controlled trials in Europe. Significantly lower incidence of oocyst shedding, diarrhoea burden and mortality was reported when treatment started before calves were 5 days old. Most studies reported on outcomes for animals up to at least 28 days old. Publication bias was possible in all outcomes and seemed especially likely for diarrhoea outcomes. Beneficial results when HFG treatment was initiated in calves older than 5 days were also found. Prophylactic treatment to prevent cryptosporidiosis is effective in preventing multiple negative outcomes and is beneficial to calf health and will result in a reduction of environmental contamination by Cryptosporidium oocysts.

Topics: Animals; Cattle; Cattle Diseases; Coccidiostats; Cryptosporidiosis; Cryptosporidium parvum; Diarrhea; Feces; Oocysts; Piperidines; Quinazolinones; Weight Gain

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

Topics: Anti-Bacterial Agents; Escherichia coli; Fatty Alcohols; Humans; Isoleucine-tRNA Ligase; Mupirocin; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; RNA, Transfer, Leu; Species Specificity; Structure-Activity Relationship; Thermus thermophilus; Transfer RNA Aminoacylation

The quinazolinone-containing 2,3-disubstituted piperidines febrifugine and isofebrifugine have been the subject of significant research efforts since their occurrence in Dichroa febrifuga and their anti-malarial actions were first described in the late 1940s. Subsequently they have also been shown to be present in other plants belonging to the hydrangea family and various analogues of febrifugine have been prepared in attempts to tune biological properties. The most notable analogue is termed halofuginone and a substantial body of work now demonstrates that this compound possesses potent human disease relevant activities. This review focuses on the literature associated with efforts dedicated towards uncovering the structures of febrifugine and isofebrifugine, the development of practical methods for their synthesis and the syntheses of structural analogues.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antimalarials; Cyclization; Cycloaddition Reaction; Humans; Piperidines; Plasmodium falciparum; Quinazolines; Quinazolinones; Stereoisomerism

Halofuginone (HF) is a derivative of dichroine which is the extract of traditional Chinese medicine. It is widely used as an efficient anticoccidial drug. Recent studies have found that HF has unique biological activities, showing great potential capacities in the treatment of autoimmune diseases. In the article, we summarized the therapeutic effects of HF in a variety of autoimmune diseases and its mechanism, providing references for further clinical studies of HF.

Topics: Autoimmune Diseases; Humans; Medicine, Chinese Traditional; Piperidines; Quinazolinones

Fibrosis, which can be defined as an abnormal or excessive accumulation of extracellular matrix (ECM), particularly fibrillar collagens, is a key driver of progressive organ dysfunction in many inflammatory and metabolic diseases, including idiopathic pulmonary fibrosis (IPF), cirrhosis, nephropathy, and oral submucous fibrosis (OSF). It has been estimated to contribute to ∼45% of deaths in the developed world. Therefore, agents that target specific fibrotic pathways, with the consequence of slowing, arresting, or even reversing the progression of tissue fibrogenesis, are urgently needed. 7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4(3H)-quinazolinone (halofuginone), an analog of febrifugine, which specifically targets the pathogenesis of ECM proteins, inhibits tissue fibrosis and regeneration and even affects the development of tumors in various tissues. Four modes of actions of halofuginone against fibrosis have been presented: 1) Inhibition of mothers against decapentaplegic homolog 3 (Smad3) phosphorylation downstream of the TGF-β signaling pathway, 2) reduction of collagen amounts, 3) decreases in ECM protein, and 4) selective prevention of Th17 cell differentiation. In this review, we will mainly focus on the rationale for halofuginone against fibrosis.

Topics: Animals; Collagen; Extracellular Matrix Proteins; Fibrosis; Humans; Models, Biological; Piperidines; Quinazolinones; Th17 Cells

Halofuginone is an analog of febrifugine-an alkaloid originally isolated from the plant Dichroa febrifuga. During recent years, halofuginone has attracted much attention because of its wide range of beneficial biological activities, which encompass malaria, cancer, and fibrosis-related and autoimmune diseases. At present two modes of halofuginone actions have been described: (1) Inhibition of Smad3 phosphorylation downstream of the TGFβ signaling pathway results in inhibition of fibroblasts-to-myofibroblasts transition and fibrosis. (2) Inhibition of prolyl-tRNA synthetase (ProRS) activity in the blood stage of malaria and inhibition of Th17 cell differentiation thereby inhibiting inflammation and the autoimmune reaction by activation of the amino acid starvation and integrated stress responses. This review deals with the history and origin of this natural product, its synthesis, its known modes of action, and it's various biological activities in pre-clinical animal models and in humans.

Topics: Animals; Antimalarials; Antineoplastic Agents; Antiprotozoal Agents; Apoptosis; Clinical Trials as Topic; Humans; Piperidines; Quinazolinones

Strictures of the urethra are the most common cause of obstructed micturition in younger men and there is frequent recurrence after initial treatment. This review was performed to determine the best strategy for stricture recurrence prevention following urethral endoscopic management.. We reviewed the published literature in PubMed, the Cochrane Library, and Google Scholar focusing on this intractable problem regardless of language restrictions. Outcomes of interest included the study methods and the applied strategy's efficacy. The level of evidence and grade of recommendations of included studies were appraised with an Oxford Centre for Evidence-Based Medicine Scale.. Currently, numerous techniques, including catheterization, repeated dilation, brachytherapy, and intraurethral use of various antifibrosis agents, have been employed to oppose the process of wound contraction or regulate the extracellular matrix. But unfortunately, none of these techniques or agents have demonstrated efficacy with enough evidence.. Although lots of strategies are available, still, we do not have a suitable, single optimum solution for all the conditions. The clinical decision of stricture-recurrence-prevention techniques should be carefully tailored to every individual patient. As the studies are not sufficient, more efforts are warranted to address this interesting but challenging issue.

Topics: Animals; Anti-Bacterial Agents; Brachytherapy; Catheterization; Dilatation; Endoscopy; Fibrosis; Humans; Piperidines; Quinazolinones; Rats; Secondary Prevention; Steroids; Urethra; Urethral Stricture; Urinary Catheterization

The trans-2,3-disubstituted piperidine, quinazolinone-containing natural product febrifugine (also known as dichroine B) and its synthetic analogue, halofuginone, possess antimalarial activity. More recently studies have also shown that halofuginone acts as an agent capable of reducing fibrosis, an indication with clinical relevance for several disease states. This review summarizes historical isolation studies and the chemistry performed which culminated in the correct structural elucidation of naturally occurring febrifugine and its isomer isofebrifugine. It also includes the range of febrifugine analogues prepared for antimalarial evaluation, including halofuginone. Finally, a section detailing current opinion in the field of halofuginone's human biology is included.

Topics: Antimalarials; Humans; Molecular Structure; Parasitic Sensitivity Tests; Piperidines; Plasmodium; Quinazolines; Quinazolinones; Structure-Activity Relationship

Organ fibrosis and architectural remodeling can severely disrupt tissue function, often with fatal consequences. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli, and the key cellular mediator of fibrosis comprises the myofibroblasts which, when activated, serve as the primary collagen-producing cells. Complex links exist between fibrosis, regeneration and carcinogenesis, and the concept that all organs contain common tissue fibrosis pathways that could be potential therapeutic targets is an attractive one. Because of the major impact of fibrosis on human health there is an unmet need for safe and effective therapies that directly target fibrosis. Halofuginone inhibits tissue fibrosis and regeneration, and thereby affects the development of tumors in various tissues along the gastrointestinal tract. The high efficacy of halofuginone in reducing the fibrosis that affects tumor growth and tissue regeneration is probably due to its dual role in inhibiting the signaling pathway of transforming growth factor β, on the one hand, and inhibiting the development of Th17 cells, on the other hand. At present halofuginone is being evaluated in a clinical trial for other fibrotic indication, and any clinical success in that trial would allow the use of halofuginone, also for all other fibrotic indications, including those of the gastrointestinal tract.

Topics: Animals; Antineoplastic Agents; Digestive System Diseases; Digestive System Neoplasms; Fibrosis; Gastrointestinal Agents; Gastrointestinal Tract; Humans; Piperidines; Quinazolinones; Regeneration; Signal Transduction

Muscular dystrophies (MDs) include different inherited diseases that all result in progressive muscle degeneration, impaired locomotion and often premature death. The major focus of MD research has been on alleviating the primary genetic deficit - using gene therapy and myoblast-transfer approaches to promote expression of the deficient or mutated genes in the muscle fibers. Although promising, these approaches have not yet entered into clinical practice and unfortunately for MD patients, there is currently no cure. Thus, the development of complementary and supportive therapies that slow disease progression and improve patients' quality of life is critically important. The main features of MDs are sarcolemmal instability and increased myofiber vulnerability to mechanical stress, resulting in myofiber degeneration. Fibrosis, with progressive replacement of muscle tissue, is a prominent feature in some MDs, preventing complete regeneration and hampering muscle functions. TGFβ is the leading candidate for activating fibroblasts and eliciting overproduction of extracellular matrix (ECM) proteins. Halofuginone, an inhibitor of Smad3 phosphorylation downstream of TGFβ signaling, inhibits the activation of fibroblasts and their ability to synthesize ECM, regardless of their origin or location. In animal models of MDs with prominent muscle fibrosis, halofuginone treatment has resulted in both prevention of collagen production in young animals and resolution of established fibrosis in older ones: the reduction in muscle collagen content was associated with improved muscle histopathology and major improvements in muscle function. Recently, these halofuginone-dependent improvements were also observed in MD with minor fibrosis involvement, probably due to a direct effect of halofuginone on muscle cells, resulting in myotube fusion that is dependent on Akt and MAPK pathway activation. In summary, halofuginone improves muscle histopathology and muscle functions in various MDs, via inhibition of muscle fibrosis on the one hand, and increased myotube fusion on the other.

Topics: Animals; Collagen; Fibrosis; Gene Expression Profiling; Humans; Mice; Mice, Inbred mdx; Models, Biological; Muscle, Skeletal; Muscular Dystrophies; Muscular Dystrophy, Animal; Phosphorylation; Piperidines; Quinazolinones; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Wound healing plays a major role in the development of acquired subglottic stenosis. Pharmacologic treatment of subglottic stenosis must address both physiologic and pathologic healing processes. The relevant Pubmed and Ovid databases from 1960 to 2007 were systematically searched. Several modulating agents have been tested. Most of them were poorly investigated. Three modalities were thoroughly studied-steroids and antibiotics, mitomycin, and antireflux medications. However, there are conflicting data regarding their role in preventing and treating subglottic stenosis. Current data support to some extent the textbook suggestions of antibiotics, steroids, and antireflux treatment. As no other treatment options exist, we recommend using these modalities for pharmacologic modulation of subglottic stenosis. Mitomycin should still be considered as an unproven treatment; its use may be considered as an adjunct.

Topics: Acetylcysteine; Aminopropionitrile; Animals; Antimetabolites; Colchicine; Fluorouracil; Humans; Hyperbaric Oxygenation; Laryngostenosis; Mitomycin; Nucleic Acid Synthesis Inhibitors; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Secondary Prevention; Wound Healing

Halofuginone seems to reduce diarrhoea and oocyst shedding in calves with cryptosporidiosis, but provides no complete cure. To develop more precise estimates of the effects of halofuginone on calf cryptosporidiosis, meta-analyses were performed, including studies on prophylactic and therapeutic treatment. Meta-analysis increases statistical power because several trials are evaluated together, increasing the effective sample size and possibility of detecting true effects. In total, 20 cohort or clinical studies (in 16 publications) investigating halofuginone treatment in calves were identified. One study was excluded because treated calves and control calves were not investigated in parallel. Four studies (three publications) were excluded because only abstracts were available. Thus, 15 studies from 12 publications, with 10-311 calves were included for data extraction. Of these, five studies from three publications could not be used for meta-analysis because they did not report the data needed. Effects on infection prevalence, diarrhoeal prevalence and mortality were investigated. For prophylactic treatment, halofuginone had an effect on infection and diarrhoeal prevalence on study days 4 and 7, but the control group had significantly lower infection prevalence than the halofuginone treated group on study day 21. Heterogeneity was detected on study days 14 and 21 and publication bias was detected on study days 7 and 14. Mortality was not affected. For therapeutic treatment, a shortage of studies in combination with heterogeneity made interpretations uncertain, and we could not determine if halofuginone treatment benefits calves.

Topics: Animals; Cattle; Cattle Diseases; Clinical Trials as Topic; Coccidiostats; Cryptosporidiosis; Mass Screening; Meta-Analysis as Topic; Piperidines; Prevalence; Quinazolinones; Sweden

Uterine leiomyomas are a common disorder resulting in significant morbidity for women and substantial economic impact on the health care system. Current therapies include conservative surgery, hysterectomy, and hormonal therapy. Conservative surgical therapy often fails because of recurrence, and hysterectomy dramatically limits reproductive options. Radiologic therapies are associated with considerable risk of morbidity and mortality and are not likely to be compatible with reproduction. Hormonal therapies such as gonadotropin-releasing hormone (GnRH) analogues or progestins with or without estrogen are utilized by many patients, but long-term use of either is often responsible for unacceptable morbidity and hormonal therapies are not compatible with reproduction. Newer hormonal alternatives such as progesterone antagonists and selective agonists as well as "add-back" estrogen therapy in addition to GnRH analogues have been developed and show promise. However, no hormonal therapy that significantly alters estrogen and progesterone production or function is likely to be compatible with reproduction. Thus, it is important to develop novel nonhormonal therapies for medical treatment of leiomyomas. Other laboratories have evaluated pirfenidone, halofuginone, heparin, and interferon-alpha (IFN-alpha). Recent work in our laboratory suggests potential use of two additional classes of compounds, thiazolidinediones and tocopherol analogs. The rationale, evidence, and potential for the use of each of these compounds in the treatment of leiomyomas are discussed.

Topics: Bexarotene; Female; Heparin; Humans; Interferon-alpha; Leiomyoma; ortho-Aminobenzoates; Piperidines; Pyridones; Quinazolines; Quinazolinones; Reproduction; Tetrahydronaphthalenes; Thiazolidinediones; Tocopherols; Uterine Neoplasms

Chronic graft-versus-host disease (cGvHD) and systemic sclerosis (scleroderma [SSc]) share clinical characteristics, including skin and internal organ fibrosis. Fibrosis, regardless of the cause, is characterized by extracellular matrix deposition, of which collagen type I is the major constituent. The progressive accumulation of connective tissue results in destruction of normal tissue architecture and internal organ failure. In both SSc and cGvHD, the severity of skin and internal organ fibrosis correlates with the clinical course of the disease. Thus, there is an unmet need for well-tolerated antifibrotic therapy. Halofuginone is an inhibitor of collagen type I synthesis in cells derived from various tissues and species and in animal models of fibrosis in which excess collagen is the hallmark of the disease. Halofuginone decreased collagen synthesis in the tight skin mouse (Tsk) and murine cGvHD, the 2 experimental systems that show many features resembling those of human GvHD. Inhibition of collagen synthesis by halofuginone is achieved by inhibiting transforming growth factor beta-dependent Smad3 phosphorylation. Dermal application of halofuginone caused a decrease in collagen content at the treated site of a cGvHD patient, and reduction in skin scores was observed in a pilot study with SSc patients. The results of the human studies provide basis for using halofuginone treatment for dermal fibrosis. As a first step toward future treatment of internal organ involvement, an oral administration study was performed in which halofuginone was well tolerated and plasma levels surpassed the predicted therapeutic exposure.

Topics: Administration, Cutaneous; Animals; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Collagen Type I; Fibrosis; Graft vs Host Disease; Humans; Mice; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Scleroderma, Systemic

Angiogenesis is the process by which tumours induce a blood supply, crucial for growth and metastasis. Evidence for its role in bladder carcinogenesis, its usefulness as a marker of patient prognosis, and potential anti-angiogenic therapies for future development are discussed in this chapter.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Cyclohexanes; DNA-Binding Proteins; Drug Combinations; Endothelial Growth Factors; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Mice; Mice, Nude; Neovascularization, Pathologic; Nuclear Proteins; O-(Chloroacetylcarbamoyl)fumagillol; Piperidines; Quinazolines; Quinazolinones; Sesquiterpenes; Thymidine Phosphorylase; Transcription Factors; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

While the biology of the pathogenesis of scleroderma is continually being better understood, there still is no single agent or therapeutic combination that has a clear impact on the disease process. Traditional medications (colchicine, potassium aminobenzoate (potaba), D-penicillamine) are disappointing in clinical practice despite anecdotal evidence of benefit. Furthermore, the most popular traditional drug, D-penicillamine, failed to clearly show benefit when tested in a well-designed clinical trial comparing conventional high dose with a very low dose (125 mg po. every other day [corrected]) [1]. Currently, most success in managing scleroderma and improving quality of life is secondary to organ-specific therapy, such as management of a renal crisis with an ACE inhibitor, treatment of Raynaud's phenomenon with calcium channel blockers, or control of serious gastrointestinal reflux disease with a proton pump inhibitor. In this review we will focus on novel therapies that are currently being tested in the treatment of scleroderma and have the potential of modifying the disease process and overall clinical outcome. We have attempted to review the rationale for each agent, recognising that its true biological effect will only be determined in clinical trials.

Topics: Autoimmune Diseases; Bone Marrow Transplantation; Cysteine; Humans; Interferon-gamma; Nitric Oxide; Piperidines; Prostaglandins; Quinazolines; Quinazolinones; Relaxin; Scleroderma, Systemic; Tetracycline; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Optimal management for scleroderma (systemic sclerosis) is likely to require treatment of the underlying disease process, which remains incompletely understood, and also of the organ-based complications of this heterogeneous condition. Clinical trials evaluating several potential agents have been completed recently, including D-penicillamine and interferon alpha. Unfortunately none of these studies has suggested significant efficacy. This article focuses on new treatment approaches using existing therapeutic agents, such as prostacyclin, and considers the potential usefulness of new agents (eg, relaxin, halofuginone) or strategies such as intensive immunosuppression with peripheral stem cell rescue. Ultimately, a better understanding of disease pathogenesis may facilitate the development of targeted therapy against key events or mediators, but for the present better evaluation of existing agents and a focus on optimizing protocols for organ-based complications, such as pulmonary vascular disease or hypertensive renal crisis, are important goals.

Topics: Clinical Trials as Topic; Combined Modality Therapy; Female; Hematopoietic Stem Cell Transplantation; Humans; Immunosuppressive Agents; Male; Minocycline; Nitric Oxide; Piperidines; Prognosis; Quinazolines; Quinazolinones; Relaxin; Scleroderma, Systemic; Treatment Outcome

1. Fibrosis is characterized by extracellular matrix deposition, of which collagen type I is the major constituent. The progressive accumulation of connective tissue resulted in destruction of normal tissue architecture and function. 2. Fibrosis is a common response to various insults or injuries and can be the outcome of any perturbation in the cellular function of any tissue. 3. Halofuginone was found to inhibit collagen alpha 1(I) gene expression and collagen synthesis in a variety of cell cultures including human fibroblasts derived from patients with excessive skin collagen type I synthesis. 4. Halofuginone was found to inhibit collagen alpha 1(I) gene expression and collagen synthesis in animal models characterized by excessive deposition of collagen. In these models, fibrosis was induced in various tissues such as skin, liver, lung, etc. Halofuginone was injected intraperitoneally, added to the foodstuff or applied locally. 5. Halofuginone decreased skin collagen in a chronic graft-versus-host disease patient. 6. The ability of extremely low concentrations of halofuginone to inhibit collagen alpha 1(I) synthesis specifically and transiently at the transcriptional level suggests that this material fulfills the criteria for a successful and effective anti-fibrotic therapy.

Topics: Animals; Collagen; Fibrosis; Humans; Liver Cirrhosis; Piperidines; Postoperative Complications; Protein Synthesis Inhibitors; Pulmonary Fibrosis; Quinazolines; Quinazolinones; Skin; Tissue Adhesions

Halofuginone lactate (HL) is registered in several countries for the prevention of calf cryptosporidiosis, but the compound's utility in the presence of co-infection with other enteropathogens is not well understood. We performed a randomized controlled field trial of the efficacy of HL for the prevention of natural calf cryptosporidiosis, in the presence of co-infection with rotavirus and Salmonella Typhimurium. Newborn calves on one farm were sequentially enrolled and allocated to a full dose (n=15), half dose (n=15), or a placebo control group (n=15), using a randomized block design. The Cryptosporidium oocysts in fecal specimens collected on Days 6, 8, 10, 14 and 20 were counted and the severity of the diarrhea was assessed using fecal consistency scores (solid, semisolid, or liquid). The oocyst numbers and fecal consistency scores were statistically compared between the groups. Ninety one percent of the calves shed Cryptosporidium parvum oocysts during the trial. The full dose group had a longer prepatent period than the control group, but no statistical difference in the number of oocysts was identified between the groups after controlling for the effects of sex and breed. The fecal consistency scores and mortality rates did not differ between the groups. These results indicated that the anti-Cryptosporidium activity and clinical benefit of HL were limited. It is concluded that in order to maximize the clinical efficacy of HL in the field, diagnostic efforts should aim to rule out the presence of other enteropathogens.

Topics: Animals; Cattle; Cattle Diseases; Cryptosporidiosis; Feces; Female; Male; Oocysts; Piperidines; Quinazolinones; Rotavirus; Rotavirus Infections; Salmonella Infections, Animal; Salmonella typhimurium

Halofuginone (stenorol) has been used as an effective anticoccidial reagent for decades but very little is known about its mode of action. In this study, chickens were inoculated with Eimeria tenella oocysts on 14-day-old and medicated with halofuginone at days 0, 1, 2, 3, 4, 5 and 6 post inoculations (groups 0, 1, 2, 3, 4, 5 and 6, respectively). Chickens in group 7 were taken as challenge-unmedicated control and in group 8 unchallenged-unmedicated control. The survival rate, body weight gains (BWG), oocysts production, cecal scores, bloody diarrhea and histological examinations were analyzed to evaluate the anticoccidial efficacy of halofuginone and to initially elucidate its mechanisms. Results showed that halofuginone which acted as a coccidiostatic can significantly enhance the BWG, and decrease both the oocyst shedding and cecal destruction caused by E. tenella infection. The histological slide examination noted that halofuginone was effective when provided 0-2 days post inoculation but only partially effective when applied 3-7 days post infection. The second-generation schizonts treated with halofuginone appeared vacuolated and degenerated. It is concluded that halofuginone can inhibit the parasite's invasion of host cecal hypothetical cell at the early stages of life cycle and later disturb the parasite's development by vacuolation of the schizonts. The resulting abnormal schizonts could not divide into schizoites and were eventually eliminated by the host's immune response.

Topics: Animals; Chickens; Coccidiosis; Coccidiostats; Eimeria tenella; Piperidines; Poultry Diseases; Quinazolinones

Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1 or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G(0) /G(1) cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1 and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Female; G1 Phase; Humans; JNK Mitogen-Activated Protein Kinases; Male; Membrane Potential, Mitochondrial; Mice; Mice, SCID; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Piperidines; Proto-Oncogene Proteins c-bcl-2; Quinazolinones; Resting Phase, Cell Cycle; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

Using a novel blinded intrapatient vehicle control design, we conducted a phase II study of topically administered halofuginone, an angiogenesis inhibitor that inhibits collagen type-I and matrix metalloproteinases (MMPs), in patients with AIDS-related Kaposi sarcoma. Serial Kaposi sarcoma biopsies assessed treatment effects on angiogenic factors and Kaposi sarcoma herpesvirus-latency associated nuclear antigen-1 (KSHV-LANA). We observed marked heterogeneity of KSHV-LANA expression. Although the small number of subjects whose response could be evaluated precluded definitive assessment of halofuginone's efficacy, we observed a significant decrease in type-I collagen only in halofuginone-treated lesions, but no effect on MMP-2. The trial design is applicable to future studies of topical agents.

Topics: Administration, Topical; Adult; AIDS-Related Opportunistic Infections; Antineoplastic Agents; Female; Humans; Male; Middle Aged; Piperidines; Quinazolinones; Sarcoma, Kaposi; Single-Blind Method; Young Adult

Halofuginone (tempostatin) is a synthetic derivative of a quinazolinone alkaloid showing anti-angiogenic, anti-metastatic and anti-proliferative effects in preclinical studies. The objectives of this phase I study were to assess the dose-limiting toxicities (DLTs), to determine the maximum tolerated dose (MTD) and to study the pharmacokinetics (PKs) of halofuginone when administered once or twice daily orally to patients with advanced solid tumours.. Patients were treated with escalating doses of halofuginone at doses ranging from 0.5 to 3.5 mg/day. For pharmacokinetic analysis plasma sampling was performed during the first and second course and assayed using a validated high-performance liquid chromatographic assay with mass spectrometric detection.. Twenty-four patients received a total of 106 courses. The 'acute' MTD was reached at 3.5 mg/day, with nausea, vomiting, and fatigue as DLT. The recommended dose for chronic administration was defined as 0.5mg/day with the requirement of 5HT3 antagonists to control nausea and vomiting considered as DLT. Several patients experienced bleeding complications on treatment with halofuginone in which a causal relationship could not be excluded. The PKs of halofuginone were linear over the dose range studied with a large interpatient variability.. In this study the DLT of halofuginone was nausea, vomiting, and fatigue. The recommended dose for phase II studies of halofuginone is 0.5mg administered orally, once daily.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Fatigue; Humans; Maximum Tolerated Dose; Middle Aged; Nausea; Neoplasms; Piperidines; Quinazolines; Quinazolinones; Vomiting

In a 5-year survey regarding its prevalence and importance in five German state veterinary laboratories Cryptosporidium was diagnosed annually in 19-36% of faecal samples either submitted to the laboratories or taken post mortem. In approximately half of the cases no other enteropathogens were detected. However, only 73% of 30 laboratories participating in a questionnaire survey routinely tested for this parasite, and the majority of researchers considered cryptosporidiosis to be of minor importance. In a placebo-controlled field study 152 suckling calves were treated daily against cryptosporidiosis either with sulfadimidine or with halofuginone (Halocur, Intervet) over 1 week. Treatment by oral drench started at the onset of diarrhoea in the herd. Oocyst excretion, faecal consistency and health status were recorded five times for a 3-week period. Oocyst excretion peaked 7-14 days in the placebo group after the onset of diarrhoea, and during that period prevalence and intensity of excretion were significantly lower in the halofuginone-treated group compared to the sulfadimidine and the placebo control groups. The health status (diarrhoea, dehydration) declined in all groups but was significantly (P<0.05-0.001) better in the halofuginone group in the first 2 weeks. Halofuginone effectively (P<0.05-0.001) reduced oocyst excretion and improved the health status of the treated animals, while sulfadimidine had no effect against Cryptosporidium.

Topics: Animals; Anti-Infective Agents; Cattle; Cattle Diseases; Coccidiostats; Cryptosporidiosis; Cryptosporidium; Dairying; Diarrhea; Feces; Female; Germany; Oocysts; Piperidines; Prevalence; Quinazolines; Quinazolinones; Sulfamethazine; Surveys and Questionnaires

The effect of continuous administration, weekly 24 h administration, phased withdrawal and phased inclusion of dietary halofuginone at 3 mg kg-1 on skin tensile strength was tested in Ross I broiler pullets. Controls received monensin continuously at 100 mg kg-1. A total of 2,592 birds were used in 2 lots of 1,296 in 2 separate programmes. Continuous dietary administration of halofuginone affected skin tensile strength negatively (P less than 0.05), with an equally significant recovery following a 7 d withdrawal period. A polynomial regression analysis indicates that skin strength may undergo a slight compensatory overstrengthening reaction within 7 d of withdrawal of halofuginone. Weekly 24 h administration of halofuginone reduced differences in skin strength between treatment and controls to non-significant levels. Differences in skin strengths between controls and the halofuginone phased inclusion programme decreased as the birds aged, being significant (P less than 0.05) on Days 14 and 28 and non-significant on Days 21, 35, 42 and 49. A weakening of skin tensile strength of broiler carcasses, as far as it may be influenced by halofuginone, can be alleviated through different shuttle programmes involving halofuginone. Such programmes would include intermittent dosing, a withdrawal period, administration to older birds or a combination of these programmes.

Topics: Animals; Chickens; Coccidiostats; Piperidines; Quinazolines; Quinazolinones; Skin; Skin Physiological Phenomena; Tensile Strength

Halofuginone was administered to both sexes of 2 broiler strains, Ross and Hubbard, in 4 dietary treatments, 0, 1.5, 3 and 6mg kg-1. Controls received dietary monensin at 100 mg kg-1. Group feed intake and body mass were recorded weekly. Skin tensile strength was monitored on Days 28, 35, 42 and 49 using a standardised cut of breast skin from carcasses randomly drawn from each treatment, strain and sex combination. Halofuginone inclusion level was inversely correlated (r = -0.36) with skin tensile strength and affected both sexes of the 2 strains (P less than 0.01). Between sexes, the skins of cockerels were consistently stronger than those of hens (P less than 0.01). The effect of age (P less than 0.07) and that of strain (P less than 0.06) tended towards significance at the 5% level. The singular effect of halofuginone on skin tensile strength was masked by the interactions treatment and sex (P less than 0.01); treatment and age (P less than 0.02); treatment and strain (P less than 0.06); age and strain (P less than 0.01). An inclusion level of 6mg kg-1, double the recommended level, could reduce growth and skin tensile strength significantly.

Topics: Animals; Chickens; Coccidiostats; Female; Food Additives; Male; Piperidines; Quinazolines; Quinazolinones; Skin; Skin Physiological Phenomena; Tensile Strength

Systemic lupus erythematosus (SLE) is a multisystemic, inflammatory autoimmune disease. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells participated in the pathogenesis of SLE. MDSCs has been considered a potential therapeutic target for lupus. As traditional Chinese medicine, Halofuginone (HF) has the extensive immunomodulatory effects on some autoimmune disorders. Our research was dedicated to discovering therapeutic efficacy of HF for lupus to explore novel mechanisms on MDSCs. We found that HF prominently alleviated the systemic symptoms especially nephritis in Imiquimod-induced lupus mice, and simultaneously repaired the immune system, reflected in the alteration of autoantibodies. HF diminished the quantity of MDSCs in lupus mice, and induced apoptosis of MDSCs. Through RNA sequencing performed on the sorted MDSC from lupus mice and HF-treated lupus mice, B lymphoid tyrosine kinase (Blk, a non-receptor cytoplasmic tyrosine kinase) was screened as the target molecule of HF. It's proven that HF had two independent effects on Blk. On the one hand, HF increased the mRNA expression of Blk in MDSCs by inhibiting the nuclear translocation of p65/p50 heterodimer. On the other hand, HF enhanced the kinase activity of Blk in MDSCs through direct molecular binding. We further investigated that Blk suppressed the phosphorylation of downstream ERK signaling pathway to increase the apoptosis of MDSCs. In conclusion, our study illustrated that HF alleviated the disease progression of lupus mice by targeting Blk to promote the apoptosis of MDSCs, which indicated the immunotherapeutic potential of HF to treat lupus.

Topics: Animals; Lupus Erythematosus, Systemic; Mice; Myeloid-Derived Suppressor Cells; Piperidines; Quinazolinones

5-aminolevulinic acid-mediated PDT (ALA-PDT) has been used in a variety of skin diseases including cSCC (cutaneous squamous cell carcinoma). Halofuginone (HL) is a less-toxic febrifugine derivative and has inhibitory effects on a variety of cancer cells. For now, there are no published study focusing on the combination use of ALA-PDT with HL to improve clinical efficacy of cSCC.. In this study, we will examine the effectiveness of combined treatment of ALA-PDT and HL in cSCC as well as its underlying mechanism.. The human epidermoid carcinoma cell line SCL-1 was treated with ALA-PDT or/ and HL, and cell viability, cell migration, ROS production, apoptosis were evaluated by CCK-8, colony formation, scratch assay, DCFH-DA probe, flow cytometry, respectively. The protein expression of NRF2 signaling was examined by western blot.. HL strengthened ALA-PDT's inhibition of SCL-1 cell viability, migration, as well as NRF2 related β-catenin, p-Erk1/2, p-Akt and p-S6K1 expression. Overexpression of NRF2 conferred resistance to co-treatment's effects on c-Myc, Cyclin D1, Bcl-2, as well as cell proliferation. HL also strengthened ALA-PDT's inhibition of tumor volume in cSCC mouse model and elevated ROS generation of ALA-PDT.. HL enhances the anti-tumor effect of ALA-PDT in vitro and in vivo. HL has the potential to enhance the anti-tumor effect of ALA-PDT in cSCC via inhibiting NRF2 signaling.

Topics: Aminolevulinic Acid; Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Mice; NF-E2-Related Factor 2; Photochemotherapy; Photosensitizing Agents; Piperidines; Quinazolinones; Skin Neoplasms

Dense tumor stroma is the physiological barrier in drug delivery that prevents anticancer drugs from entering the tumor, thereby seriously limiting the drugs' therapeutic effect. In this study, a Janus nanoplatform consisting of periodic mesoporous organosilica-coated platinum nanoplatforms (JPMO-Pt) and anti-stroma drug halofuginone (HF) (denoted as JPMO-Pt-HF), was developed to deplete the tumor stroma and synergistically treat breast cancer in BALB/c mice. The prepared JPMO-Pt had a uniform size of 245 nm, a good dispersion, an excellent in vitro and in vivo biocompatibility, and a high loading capacity for HF (up to 50 μg/mg). The antitumor experiments showed that the survival rate of 4 T1 cells exhibited an obvious downward trend when the cells were incubated with the JPMO-Pt-HF and irradiated with 808 nm laser. Moreover, the cell survival rate was only about 10% at 48 h when the HF concentration was 2.0 μg/mL. Notably, JPMO-Pt-HF under irradiation had an excellent synergistic therapeutic effect on tumor cells. In vivo antitumor experiment further showed that the JPMO-Pt-HF, in combination with laser irradiation, could minimize tumor growth, showing significantly better effects than those observed for the case of monotherapy involving photothermal therapy (PTT) (152 vs. 670 mm

Topics: Animals; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Humans; Hyperthermia, Induced; Mice; Mice, Inbred BALB C; Nanoparticles; Phototherapy; Photothermal Therapy; Piperidines; Quinazolinones

Our previous study found that the combination of halofuginone (HF) and artemisinin (ATS) synergistically arrest colorectal cancer (CRC) cells at the G1/G0 phase of the cell cycle; however, it remains unclear whether HF-ATS induces cell death. Here we report that HF-ATS synergistically induced caspase-dependent apoptosis in CRC cells. Specifically, both

Topics: Antineoplastic Agents; Apoptosis; Artemisinins; Autophagy; Caspase 8; Caspase 9; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Enzyme Activation; Humans; Piperidines; Quinazolinones; Receptor Cross-Talk

Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.

Topics: Eukaryotic Initiation Factor-2; Phosphorylation; Piperidines; Proline; Protein Biosynthesis; Quinazolinones

The Keap1-Nrf2 system is the master regulator of the cellular response against oxidative and xenobiotic stresses. Constitutive activation of Nrf2 is frequently observed in various types of cancers. Nrf2 hyperactivation induces metabolic reprogramming in cancer cells, which supports the increased energy demand required for rapid proliferation and confers high-level resistance against anticancer radio/chemotherapy. Hence, Nrf2 inhibition has emerged as an attractive therapeutic strategy to counter such acquired resistance in Nrf2-activated tumors. We previously identified Halofuginone (HF) as a promising Nrf2 inhibitor. In this study, we pursued preclinical characterization of HF and found that while HF markedly reduced the viability of cancer cells, it also caused severe hematopoietic and immune cell suppression in a dose-dependent manner. Hence, to overcome this toxicity, we decided to employ a nanomedicine approach to HF. We found that encapsulation of HF into a polymeric micelle (HF micelle; HFm) largely relieved the systemic toxicity exhibited by free HF while maintaining the tumor-suppressive properties of HF. LC-MS/MS analysis revealed that the reduction in the magnitude of adverse effects was the result of the ability to release HF from the HFm core in a slow and sustained manner. These results thus support the contention that HFm will potentially counteract Nrf2-activated cancers in the clinical settings.

Topics: Adenocarcinoma of Lung; Chromatography, Liquid; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; Micelles; Nanoparticles; NF-E2-Related Factor 2; Oxidative Stress; Piperidines; Quinazolinones; Tandem Mass Spectrometry

Halofuginone (HF)-loaded TPGS polymeric micelles (HTPM) were successfully fabricated using the thin-film hydration technique. HTPM via intravenous injection have been demonstrated to exert an excellent anticancer effect against triple-negative breast cancer (TNBC) cells and subcutaneous xenografts. In the present study, we further explored the potential treatment effect and mechanism of orally administered HTPM alone and in combination with surgical therapy on TNBC in subcutaneous and orthotopic mouse models.. Herein, the stability and in vitro release behavior of HTPM were first evaluated in the simulated gastrointestinal fluids. Caco-2 cell monolayers were then used to investigate the absorption and transport patterns of HF with/without encapsulation in TPGS polymeric micelles. Subsequently, the therapeutic effect of orally administered HTPM was checked on subcutaneous xenografts of TNBC in nude mice. Ultimately, orally administered HTPM, combined with surgical therapy, were utilized to treat orthotopic TNBC in nude mice.. Our data confirmed that HTPM exhibited good stability and sustained release in the simulated gastrointestinal fluids. HF was authenticated to be a substrate of P-glycoprotein (P-gp), and its permeability across Caco-2 cell monolayers was markedly enhanced via heightening intracellular absorption and inhibiting P-gp efflux due to encapsulation in TPGS polymeric micelles. Compared with HF alone, HTPM showed stronger tumor-suppressing effects in subcutaneous xenografts of MDA-MB-231 cells when orally administered. Moreover, compared with HTPM or surgical therapy alone, peroral HTPM combined with partial surgical excision synergistically retarded the growth of orthotopic TNBC. Fundamentally, HTPM orally administered at the therapeutic dose did not cause any pathological injury, while HF alone led to weight loss and jejunal bleeding in the investigated mice.. Taken together, HTPM could be applied as a potential anticancer agent for TNBC by oral administration.

Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Humans; Mice; Mice, Nude; Micelles; Piperidines; Polymers; Quinazolinones; Triple Negative Breast Neoplasms; Vitamin E

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world's most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies.

Topics: Amino Acyl-tRNA Synthetases; Antimalarials; Humans; Piperidines; Plasmodium; Plasmodium falciparum; Quinazolinones; RNA, Transfer

We previously found that pharmacological inhibition of prolyl-tRNA synthetase by halofuginone has potent activity against Nosema ceranae, an important pathogen of honey bees. However, we also observed that prolyl-tRNA synthetase inhibition is toxic to bees, suggesting further work is necessary to make this a feasible therapeutic strategy. As expected, we found that pharmacological inhibition of prolyl-tRNA synthetase activity resulted in robust induction of select canonical ATF4 target genes in honey bees. However, our understanding of this and other cellular stress responses in general in honey bees is incomplete. Thus, we used RNAseq to identify novel changes in gene expression after halofuginone treatment and observed induction of genes involved in ribosome biogenesis, translation, tRNA synthesis, and ribosome-associated quality control (RQC). These results suggest that halofuginone, potentially acting through the Integrated Stress Response (ISR), promotes a transcriptional response to ribosome functional impairment in honey bees rather than the response designed to oppose amino acid limitation, which has been observed in other organisms after ISR induction. In support of this idea, we found that cycloheximide (CHX) administration also induced all tested target genes, indicating that this gene expression program could be induced by ribosome stalling in addition to tRNA synthetase inhibition. Only a subset of halofuginone-induced genes was upregulated by Unfolded Protein Response (UPR) induction, suggesting that mode of activation and cross-talk with other cellular signaling pathways significantly influence ISR function and cellular response to its activation. Future work will focus on understanding how the apparently divergent transcriptional output of the ISR in honey bees impacts the health and disease of this important pollinator species.

Topics: Animals; Antifungal Agents; Bees; Eating; Nosema; Organelle Biogenesis; Piperidines; Quinazolinones; Ribosomes; Transcription, Genetic; Transcriptional Activation

Currently available drugs being used to treat leishmaniasis have several shortcomings, including high toxicity, drug administration that requires hospitalization, and the emergence of parasite resistance against clinically used drugs. As a result, there is a dire need for the development of new antileishmanial drugs that are safe, affordable, and efficient. In this study, two new series of synthesized quinazolinone derivatives were investigated as potential future antileishmanial agents, by assessing their activities against the Leishmania (L.) donovani and L. major species. The cytotoxicity profiles of these derivatives were assessed in vitro on Vero cells. The compounds were found to be safer and without any toxic activities against mammalian cells, compared to the reference drug, halofuginone, a clinical derivative of febrifugine. However, they had demonstrated poor antileishmanial growth inhibition efficacies. The two compounds that had been found the most active were the mono quinazolinone 2d and the bisquinazolinone 5b with growth inhibitory efficacies of 35% and 29% for the L. major and L. donovani 9515 promastigotes, respectively. These outcomes had suggested structural redesign, inter alia the inclusion of polar groups on the quinazolinone ring, to potentially generate novel quinazolinone derivatives, endowed with effective antileishmanial potential.

Topics: Animals; Antiprotozoal Agents; Cell Survival; Chlorocebus aethiops; Leishmania; Leishmania donovani; Piperidines; Quinazolinones; Structure-Activity Relationship; Vero Cells

Keloids are characterized by increased deposition of fibrous tissue in the skin and subcutaneous tissue following an abnormal wound healing process. Although keloid etiology is yet to be fully understood, fibroblasts are known to be key players in its development. Here we analyze the antifibrotic mechanisms of Halofuginone (HF), a drug reportedly able to inhibit the TGF-β1-Smad3 pathway and to attenuate collagen synthesis, in an in-vitro keloid model using patient-derived Keloid Fibroblasts (KFs) isolated from fibrotic tissue collected during the "Scar Wars" clinical study (NCT NCT03312166). TGF-β1 was used as a pro-fibrotic agent to stimulate fibroblasts response under HF treatment. The fibrotic related properties of KFs, including survival, migration, proliferation, myofibroblasts conversion, ECM synthesis and remodeling, were investigated in 2D and 3D cultures. HF at 50 nM concentration impaired KFs proliferation, and decreased TGF-β1-induced expression of α-SMA and type I procollagen production. HF treatment also reduced KFs migration, prevented matrix contraction and increased the metallo-proteases/inhibitors (MMP/TIMP) ratio. Overall, HF elicits an anti-fibrotic contrasting the TGF-β1 stimulation of KFs, thus supporting its therapeutic use for keloid prevention and management.

Topics: Actins; Adult; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Humans; Keloid; Male; Middle Aged; Myofibroblasts; Piperidines; Procollagen; Quinazolinones; Skin; Transforming Growth Factor beta1; Young Adult

Small molecules are immunochemically classified as hapten that lacking of at least two epitopes, usually using competitive format for establishing immunoassays. However, theoretically, noncompetitive immunoassay format is more sensitive and has a wider analytical range. In the present study, a novel hapten of halofuginone was synthesized and used to produce a monoclonal antibody (mAb). By analyzing the binding kinetics, we found that the affinity of analyte-enzyme to mAb was much greater than that of analyte, which could result in a low sensitivity of competitive assay format. Based on this, we established a novel noncompetitive immunoassay by using a replacement approach. The noncompetitive format has obvious advantages in sensitivity and analytical range, which promoted approximately 3.5- and 5-fold, respectively, compared to the competitive immunoassay. Ultimately, the newly designed noncompetitive immunoassay in this work will provide insights as well as alternative method to traditional small molecule competitive assays.

Topics: Antibodies, Monoclonal; Epitopes; Haptens; Immunoassay; Limit of Detection; Piperidines; Quinazolinones

Halofuginone hydrobromide (HF) is a synthetic analogue of the naturally occurring quinazolinone alkaloid febrifugine, which has potential therapeutic effects against breast cancer, however, its poor water solubility greatly limits its pharmaceutical application. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of vitamin E, which can self-assemble to form polymeric micelles (PMs) for encapsulating insoluble anti-tumor drugs, thereby effectively enhancing their anti-cancer effects.. HF-loaded TPGS PMs (HTPMs) were manufactured using a thin-film hydration technique, followed by a series of characterizations, including the hydrodynamic diameter (HD), zeta potential (ZP), stability, drug loading (DL), encapsulation efficiency (EE), and in vitro drug release. The anti-cancer effects and potential mechanism of HTPMs were investigated in the breast cell lines MDA-MB-231 and MCF-7, and normal breast epithelial cell line Eph-ev. The breast cancer-bearing BALB/c nude mouse model was successfully established by subcutaneous injection of MDA-MB-231 cells and used to evaluate the in vivo therapeutic effect and safety of the HTPMs.. The optimized HTPMs had an HD of 17.8±0.5 nm and ZP of 14.40±0.1 mV. These PMs exhibited DL of 12.94 ± 0.46% and EE of 90.6 ± 0.85%, along with excellent storage stability, dilution tolerance and sustained drug release in pH-dependent manner within 24 h compared to free HF. Additionally, the HTPMs had stronger inhibitory effects than free HF and paclitaxel against MDA-MB-231 triple-negative breast cancer cells, and little toxicity in normal breast epithelial Eph-ev cells. The HTPMs induced cell cycle arrest and apoptosis of MDA-MB-231 by disrupting the mitochondrial membrane potential and enhancing reactive oxygen species formation. Evaluation of in vivo anti-tumor efficacy demonstrated that HTPMs exerted a stronger tumor inhibition rate (68.17%) than free HF, and exhibited excellent biocompatibility.. The findings from this study indicate that HTPMs holds great clinical potential for treating triple-negative breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Compounding; Female; Humans; Membrane Potential, Mitochondrial; Mice, Inbred BALB C; Mice, Nude; Micelles; Paclitaxel; Piperidines; Polymers; Quinazolinones; Reactive Oxygen Species; Treatment Outcome; Triple Negative Breast Neoplasms; Vitamin E

Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH).. Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca. Halofuginone increased voltage-gated K

Topics: Animals; Calcium; Hypertension, Pulmonary; Hypoxia; Mice; Myocytes, Smooth Muscle; Pharmaceutical Preparations; Phosphatidylinositol 3-Kinases; Piperidines; Pulmonary Artery; Quinazolinones

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

Topics: Animals; Cell Survival; Cells, Cultured; Codon; Gene Ontology; Liver; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Piperidines; Polyribosomes; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Quinazolinones; RNA, Transfer; Signal Transduction; Stress, Physiological

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

Topics: Angiotensin-Converting Enzyme 2; Animals; Cells, Cultured; Chlorocebus aethiops; COVID-19; COVID-19 Drug Treatment; High-Throughput Screening Assays; Homoharringtonine; Humans; Lung; Mice; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; SARS-CoV-2; Serine Endopeptidases; Spike Glycoprotein, Coronavirus; Virus Internalization

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(-)-halofuginone enantiomers on motor coordination and diaphragm histopathology in

Topics: Animals; Disease Models, Animal; Fibrosis; Male; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Piperidines; Quinazolinones

As the central regulator of the oxidative stress response, nuclear factor erythroid 2-related factor 2 (Nrf2) is attracting great interest as a therapeutic target for various cancers, and the possible clinical applications of novel Nrf2 inhibitors have been explored in Nrf2-activated cancers. In the present study, we specifically investigated halofuginone, which is derived from a natural plant alkaloid. We found that halofuginone administration decreased the number of pancreatic intraepithelial neoplasias in pancreas-specific

Topics: Aldehyde Dehydrogenase; Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Deoxycytidine; Dose-Response Relationship, Drug; Gemcitabine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NF-E2-Related Factor 2; Pancreatic Neoplasms; Piperidines; Quinazolinones

Topics: Angiogenesis Inhibitors; Animals; Disease Models, Animal; Epithelium; Female; Nasal Mucosa; Piperidines; Quinazolinones; Rats; Treatment Outcome; Wound Healing; Wounds and Injuries

Acute lung injury (ALI) is characterized by neutrophilic infiltration, uncontrolled oxidative stress and inflammatory processes. Despite various therapeutic regimes having been performed, there remains no effective pharmacotherapy available to treat ALI. Halofuginone (HF), a ketone isolated from Dichroa febrifuga, exhibits significant anti‑inflammatory and antifibrotic effects. Dexamethasone (DEX), a synthetic glucocorticoid, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. The present study aimed to investigate the effects of the combination of HF and DEX in the treatment of ALI. The present results suggested that the simultaneous administration of HF and DEX markedly decreased the level of pro‑inflammatory cytokines and increased the level of anti‑inflammatory cytokines, as assessed by western blot analysis. In addition, HF and DEX effectively decreased nuclear factor‑κB activity via suppressing the phosphorylation of P65 in lipopolysaccharide (LPS)‑induced human pulmonary alveolar epithelial cells (HPAEpiC) and lung tissues extracted from ALI rats, as determined by immunofluorescence. Furthermore, in vivo experiments demonstrated that the combination of HF and DEX in LPS‑induced ALI rats defended against lung fibrosis, perivascular inflammation, congestion and edema of pulmonary alveoli, as assessed by histopathological analysis, TUNEL staining and immunohistochemistry assay. Taken together, the present study indicated the synergistic effect of HF and DEX on LPS‑induced ALI in HPAEpiC cells and a rat model. These results offer a novel therapeutic approach for the treatment of ALI.

Topics: Acute Lung Injury; Alveolar Epithelial Cells; Animals; Cell Survival; Dexamethasone; Disease Models, Animal; Drug Synergism; Humans; Inflammation; Lipopolysaccharides; NF-kappa B; Phosphorylation; Piperidines; Quinazolinones; Rats, Sprague-Dawley; Signal Transduction

Topics: Animals; Chickens; Chromatography, Liquid; Coccidiosis; Eggs; Food Analysis; Humans; Lactones; Lasalocid; Liquid-Liquid Extraction; Monensin; Nigericin; Piperidines; Poultry; Pyrans; Quinazolinones; Robenidine; Tandem Mass Spectrometry; United States; United States Food and Drug Administration

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Topics: Amino Acids; Amino Acyl-tRNA Synthetases; Animals; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Cell Line; Fibroblasts; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Lung; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Piperidines; Primary Cell Culture; Protein Serine-Threonine Kinases; Quinazolinones; RNA-Binding Proteins; RNA-Seq; Signal Transduction; Synovial Membrane; Synoviocytes; Trans-Activators

Malignant mesothelioma is a rare but aggressive form of malignancy, which is difficult to diagnose and is resistant to current chemotherapeutic treatment options. Molecular techniques have been used to investigate the mechanisms of action and the beneficial therapeutic effects of halofuginone (HF) in several cancers but not malignant mesotheliomas. In this study, the antiproliferative and apoptotic effects of HF were investigated through its ability to deregulate EGFR downstream signalling cascade proteins in the pathologically aggressive malignant mesothelioma and non-small-cell lung cancer cells. We showed that administration of HF at nanomolar concentrations induced a dose-dependent reduction in the viability of cancer cells, made cell cycle arrest, inhibited proliferation of cancer cells via STAT3 and ERK1/2 pathways and triggered the apoptotic cascade via p38MAPK. We demonstrated that the apoptotic cell death mechanism was mediated by enhanced activation of caspase-3 and concomitant PARP cleavage, downregulation of Bcl-2 and upregulation of Bax in both malignant mesothelioma and lung cancer cells. In particular, we demonstrated that cancer cells were more sensitive to HF treatment than normal mesothelial cells. Taken together, this study suggests that HF exerts its anticancer effects in lung-derived cancers by targeting signal transduction pathways mainly through deregulation of ERK1/2, STAT3 and p38MAPK to reduce cancer cell viability, induce cell cycle arrest and apoptotic cell death. Thus, HF might be considered as a potential agent against malignant mesothelioma and/or lung cancer cells.

Topics: Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Humans; Lung; Lung Neoplasms; MAP Kinase Signaling System; Mesothelioma, Malignant; p38 Mitogen-Activated Protein Kinases; Piperidines; Proto-Oncogene Proteins c-bcl-2; Quinazolinones; Signal Transduction; STAT3 Transcription Factor

Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf

Topics: Animals; Disease Models, Animal; Dysferlin; Fibrosis; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Piperidines; Protein Synthesis Inhibitors; Quinazolinones

Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifuga that facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in the epithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored the anti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factor β1 (TGF-β1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherin and increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim), and α-smooth muscle actin (α-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedly prevented the EMT induced by TGF-β1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cell EMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complex nuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which modulates the SMAD signaling pathway. These results suggested that HF inhibits TGF-β1-induced EMT in IPEC-J2 cells through the eIF2α/SMAD signaling pathway. Our findings suggest that HF can serve as a potential anti-EMT agent in intestinal fibrosis therapy.

Topics: Actins; Animals; Antineoplastic Agents; Cadherins; Cell Line; Enterocytes; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-2; Fibronectins; Gene Expression Regulation; Phosphorylation; Piperidines; Protein Isoforms; Quinazolinones; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Swine; Transforming Growth Factor beta1; Vimentin

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).\ \ This article has been retracted at the request of the Editor-in-Chief and the corresponding author. The corresponding author confessed that the paper was written and submitted by a third-party organization without his/her or any authors’ supervision. Therefore, the editor decided to retract the paper as concern has been raised regarding the integrity of the data. The author apologized for this misconduct.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; DNA Damage; Dose-Response Relationship, Drug; Esophageal Neoplasms; Forkhead Box Protein O3; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Piperidines; Quinazolinones; Signal Transduction

Atherosclerosis has become a major cause of mortality for several years, however the underlying mechanism of this disorder is still complicated. Endothelial dysfunction is a hallmark in the beginning of atherosclerosis. Lipopolysaccharides (LPS) is an important risk factor contributing to endothelial dysfunction. This study demonstrates that Halofuginone, an anti-malarial drug, possesses protective effects on human umbilical vein endothelial cells (HUVECs) against LPS-induced endothelial dysfunction. Through this study, we demonstrate that Halofuginone ameliorates LPS-induced attachment of THP-1 cells to HUVECs by inhibiting the expressions of adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Halofuginone also suppresses the production of pro-inflammatory cytokines, including tumor necrosis factor α. (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6). Furthermore, Halofuginone reduces the overproduction of reactive oxygen species (ROS) by regulating the expression of NADPH oxidase 2 (NOX-2). Mechanistically, we find the protective effects of Halofuginone depend on the expression of Kruppel-like factor 2 (KLF2), which is mediated by extracellular regulated protein kinases 5 (ERK5). Totally, our findings demonstrate that Halofuginone possesses a protective function in endothelial cells, indicating a therapeutic potential to modulate endothelial dysfunction in atherosclerosis.

Topics: Cell Adhesion; Cells, Cultured; Cytokines; Human Umbilical Vein Endothelial Cells; Humans; Kruppel-Like Transcription Factors; Lipopolysaccharides; Mitogen-Activated Protein Kinase 7; Monocytes; Piperidines; Quinazolinones; RNA, Small Interfering; THP-1 Cells

Advanced glycation end products (AGEs) have been reported to serve an important role in the stiffening of cardiac tissues and myocardial cell injury. Serious myocardial cell injury can result in various heart diseases with high mortality. Halofuginone (HF), which possesses marked anti‑inflammatory and antifibrotic effects, has recently been applied to inhibit the effects of cardiac stress. The present study aimed to investigate the potential effects of HF and its underlying mechanism in the treatment of AGEs‑induced H9C2 cardiomyocyte damage. The western blot results of the present study demonstrated that HF may reduce the expression levels of myocardial injury markers, including myoglobin, creatine kinase MB and cardiac troponin I. In addition, flow cytometric analysis indicated that the production of reactive oxygen species (ROS) was significantly decreased by HF. Additionally, endoplasmic reticulum (ER) stress was suppressed in response to treatment with HF, as observed by low expression levels of ER stress‑associated proapoptotic proteins (CCAAT/enhancer‑binding protein homologous protein and cleaved caspase‑12); overexpression of prosurvival proteins (growth arrest and DNA damage‑inducible protein GADD34 and binding immunoglobulin protein) was also reported. Furthermore, the expression levels of microtubule‑associated proteins 1A/1B light chain 3B (LC3)II/LC3I and Beclin 1 were elevated, whereas P62 expression levels were reduced following treatment with HF. These findings, together with immunofluorescence staining of LC3, indicated that HF may induce autophagy. Finally, the protective effects of HF on AGEs‑treated H9C2 cells were reversed following treatment with the inhibitor 3‑methyladenine, as indicated by inhibition of autophagy, and increases in apoptosis, ROS production and the ER stress response. Collectively, the findings of the present study suggested that the protective effects of HF against AGEs‑induced myocardial cell injury may be associated with the induction of autophagy and amelioration of ROS‑mediated ER stress and apoptosis. These findings may contribute to the development of a novel therapeutic method to inhibit the progression of myocardial cell injury.

Topics: Animals; Apoptosis; Autophagic Cell Death; Cell Line; Endoplasmic Reticulum Stress; Glycation End Products, Advanced; Myocytes, Cardiac; Piperidines; Quinazolinones; Rats

Velez J, Lange MK, Zieger P et al. Long-term use of yeast fermentation products in comparison to halofuginone for the control of cryptosporidiosis in neonatal calves. Vet Parasitol 2019; 269: 57–64 KRYPTOSPORIDIEN SIND WELTWEIT VORKOMMENDE PATHOGENE PROTOZOEN VON WIRBELTIEREN. CRYPTOSPORIDIUM PARVUM ZäHLT ZU DEN WICHTIGSTEN DURCHFALLERREGERN BEIM NEONATALEN KALB. NACH ORALER AUFNAHME KOMMT ES HäUFIG ZU EINER STARKEN VERMEHRUNG IM MIKROVILLISAUM DES DARMEPITHELS GEFOLGT VON MALABSORPTION MIT AKUTER WäSSRIGER DIARRHö, SCHWäCHE UND ABMAGERUNG BEI Z. T. STARK GESTöRTEM ALLGEMEINBEFINDEN. DER HOCHGRADIGEN FäKALEN AUSSCHEIDUNG VON OOZYSTEN FOLGT EINE MONATELANGE PERSISTENZ DIESER INFEKTIöSEN STADIEN. DIE INFEKTION IST NACH MEHREREN TAGEN SELBSTLIMITIEREND.: ZUR BEHANDLUNG STEHEN 2 WIRKSTOFFE MIT ANTIKRYPTOSPORIDIALER WIRKUNG ZUR VERFüGUNG, HALOFUGINON UND PAROMOMYCIN, WOBEI NUR HALOFUGINON EINE ZULASSUNG ZUR KRYPTOSPORIDIOSETHERAPIE HAT. NEBEN EINER ENGEN THERAPEUTISCHEN BREITE BEWIRKT HALOFUGINON BEI AKUTER ERKRANKUNG KEINE VERBESSERUNG DES KLINISCHEN ZUSTANDS. AKTUELLEN STUDIEN ZUFOLGE REDUZIERTE DIE 4-WöCHIGE VERFüTTERUNG VON FERMENTATIONSPRODUKTEN DER HEFE SACCHAROMYCES CEREVISIAE AN NEUGEBORENE, KRYPTOSPORIDIUMINFIZIERTE KäLBER DIE SCHäDIGUNG DER DüNNDARMZOTTEN IM VERGLEICH ZU KONTROLLTIEREN. DIES DEUTET AUF EINE PROPHYLAKTISCHE WIRKSAMKEIT GEGENüBER DER INFEKTION HIN. ZIEL DER STUDIE WAR, DIE WIRKUNG VON GVO-FREIEN SACCHAROMYCES CEREVISIAE-FERMENTATIONSPRODUKTEN MIT DER EINER HALOFUGINON-BEHANDLUNG GEGEN EINE CRYPTOSPORIDIUM PARVUM-INFEKTION BEI KäLBERN IN EINEM MILCHVIEHBETRIEB ZU VERGLEICHEN.

Topics: Animals; Cattle; Cryptosporidiosis; Fermentation; Piperidines; Quinazolinones

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, including increased life span, protection against age-associated disorders, and improved metabolic fitness and immunity. Cells respond to conditions of AA starvation by activating the amino acid starvation response (AAR). Here, we showed that mimicking AAR with halofuginone (HF) enhanced the magnitude and affinity of neutralizing, antigen-specific antibody responses in mice immunized with dengue virus envelope domain III protein (DENVrEDIII), a potent vaccine candidate against DENV. HF enhanced the formation of germinal centers (GCs) and increased the production of the cytokine IL-10 in the secondary lymphoid organs of vaccinated mice. Furthermore, HF promoted the transcription of genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymph nodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhanced GC responses and may promote the establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augment the efficacy of vaccines against dengue and other infectious diseases.

Topics: Amino Acids; Animals; Antibodies, Neutralizing; Antibodies, Viral; Antibody Formation; Dengue Vaccines; Interleukin-10; Mice; Mice, Inbred BALB C; Piperidines; Quinazolinones

Along with infections, ultrafiltration failure due to the toxicity of glucose-containing peritoneal dialysis (PD) solutions is the Achilles' heel of PD method. Triggered by the protective effect of general control nonderepressible-2 (GCN-2) kinase activation against high-glucose conditions in other cell types, we evaluated whether the same occurs in human peritoneal mesothelial cells. We activated GCN-2 kinase with halofuginone or tryptophanol, and assessed the impact of this intervention on glucose transporter-1, glucose transporter-3, and sodium-glucose cotransporter-1, glucose influx, reactive oxygen species (ROS), and the events that result in glucotoxicity. These involve the inhibition of glyceraldehyde 3-phosphate dehydrogenase and the diversion of upstream glycolytic products to the aldose pathway (assessed by D-sorbitol), the lipid synthesis pathway (assessed by protein kinase C activity), the hexosamine pathway (determined by O-linked β-N-acetyl glucosamine-modified proteins), and the advanced glycation end products generation pathway (assessed by methylglyoxal). Then, we examined the production of the profibrotic transforming growth factor-β1 (TGF-β1), the pro-inflammatory interleukin-8 (IL-8). Cell apoptosis was assessed by cleaved caspase-3, and mesothelial to mesenchymal transition (MMT) was evaluated by α-smooth muscle actin protein. High-glucose conditions increased glucose transporters, glucose influx, ROS, all the high-glucose-induced harmful pathways, TGF-β1 and IL-8, cell apoptosis, and MMT. Halofuginone and tryptophanol inhibited all of the above high glucose-induced alterations, indicating that activation of GCN-2 kinase ameliorates glucotoxicity in human peritoneal mesothelial cells, preserves their integrity, and prevents MMT. Whether such a strategy could be applied in the clinic to avoid ultrafiltration failure in PD patients remains to be investigated.

Topics: Cells, Cultured; Dialysis Solutions; Epithelial Cells; Epithelial-Mesenchymal Transition; Glucose; Humans; Peritoneal Dialysis; Peritoneum; Piperidines; Primary Cell Culture; Protein Serine-Threonine Kinases; Quinazolinones; Reactive Oxygen Species; Tryptophan

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest forms of cancer, in part, because it is largely refractory to current therapies. The failure of most standard therapies in PDA, as well as promising immune therapies, may be largely ascribed to highly unique and protective stromal microenvironments that present significant biophysical barriers to effective drug delivery, that are immunosuppressive, and that can limit the distribution and function of antitumor immune cells. Here, we utilized stromal reengineering to disrupt these barriers and move the stroma toward normalization using a potent antifibrotic agent, halofuginone. In an autochthonous genetically engineered mouse model of PDA, halofuginone disrupted physical barriers to effective drug distribution by decreasing fibroblast activation and reducing key extracellular matrix elements that drive stromal resistance. Concomitantly, halofuginone treatment altered the immune landscape in PDA, with greater immune infiltrate into regions of low hylauronan, which resulted in increased number and distribution of both classically activated inflammatory macrophages and cytotoxic T cells. In concert with a direct effect on carcinoma cells, this led to widespread intratumoral necrosis and reduced tumor volume. These data point to the multifunctional and critical role of the stroma in tumor protection and survival and demonstrate how compromising tumor integrity to move toward a more normal physiologic state through stroma-targeting therapy will likely be an instrumental component in treating PDA. SIGNIFICANCE: This work demonstrates how focused stromal re-engineering approaches to move toward normalization of the stroma disrupt physical barriers to effective drug delivery and promote antitumor immunity.

Topics: Animals; Biophysical Phenomena; Carcinoma, Pancreatic Ductal; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Humans; Macrophages; Mice; Pancreatic Neoplasms; Piperidines; Quinazolinones; Random Allocation; Stromal Cells; Tumor Microenvironment

Arboviruses represent a group of pathogens that can spread efficiently throughout human populations by hematophagous arthropod vectors. The mosquito-borne (re)emerging Chikungunya and Dengue viruses belong to the alphavirus and flavivirus genus, respectively, with no approved therapeutics or safe vaccines for humans. Transmitted by the same vector Aedes spp., these viruses cause significant morbidity and mortality in endemic areas. Due to the increasing likelihood of co-circulation and co-infection with viruses, we aimed to identify a pharmacologically targetable host factor that can inhibit multiple viruses and show that a potent antagonist of prolyl tRNA synthetase (halofuginone) suppresses both Chikungunya and Dengue viruses. Host tRNA synthetase inhibition may signify an additional approach to combat present and future epidemic pathogens.

Topics: Aedes; Amino Acyl-tRNA Synthetases; Animals; Antiviral Agents; Cells, Cultured; Chikungunya Fever; Chikungunya virus; Dengue; Dengue Virus; Fibroblasts; Foreskin; Host Microbial Interactions; Humans; Insect Proteins; Male; Mosquito Vectors; Piperidines; Quinazolinones; Receptor, Interferon alpha-beta

In Duchenne muscular dystrophy (DMD), the progressive loss of muscle and its ability to function is associated with significant fibrosis, representing the major disease complication in patients. Halofuginone, a halogenated analog of the naturally occurring febrifugine, has been shown to prevent fibrosis in various animal models, including those of muscular dystrophies. Here, two optically active enantiomers of deoxyhalofuginone - a halofuginone analogue in which the hydroxy group in position 3 was removed from the piperidinyl entity - were evaluated with respect to their effect on muscle histopathology in mdx mice. Male mdx mice were treated with either deoxyhalofuginone (as single enantiomers or in racemic form), or halofuginone, for 10 weeks, starting at the age of 4 weeks. Halofuginone caused a significant reduction in total collagen content, degenerative areas, as well as in utrophin and phosphorylated-Smad3 levels in the mdx diaphragms. However, neither the deoxyhalofuginone enantiomers, nor its racemic form had any effect on these parameters. A positive effect of the deoxyhalofuginone (+)-enantiomer was observed on myofiber diameters; however, it was lesser than that of halofuginone. It is concluded that the hydroxy group plays a key role in halofuginone's effects related to fibrosis in DMD, and points towards the transforming growth factor β/Smad3 signaling pathway being involved in this inhibition. Elucidation of the structure-function relationship of halofuginone, in relation to inhibiting fibrosis in muscular dystrophies, is of the utmost importance for creating the next generation of anti-fibrotic therapies that will be more efficacious and less toxic, hence improving life quality of patients.

Topics: Animals; Disease Models, Animal; Fibrillar Collagens; Fibrosis; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Phosphorylation; Piperidines; Quinazolinones; Signal Transduction; Smad3 Protein; Utrophin

We have previously identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum as the functional target of the natural product febrifugine and its synthetic analogue halofuginone (HFG), one of the most potent antimalarials discovered to date. However, our studies also discovered that short-term treatment of asexual blood stage P. falciparum with HFG analogues causes a 20-fold increase in intracellular proline, termed the adaptive proline response (APR), which renders parasites tolerant to HFG. This novel resistance phenotype lacks an apparent genetic basis but remains stable after drug withdrawal. On the basis of our findings that HFG treatment induces eIF2α phosphorylation, a sensitive marker and mediator of cellular stress, we here investigate if eIF2α-signaling is functionally linked to the APR. In our comparative studies using a parasite line lacking PfeIK1, the Plasmodium orthologue of the eIF2α-kinase GCN2 that mediates amino acid deprivation sensing, we show that HFG activity and the APR are independent from PfeIK1 and eIF2α signaling.

Topics: Amino Acyl-tRNA Synthetases; Antimalarials; Drug Resistance; Eukaryotic Initiation Factor-2; Humans; Malaria, Falciparum; Phosphorylation; Piperidines; Plasmodium falciparum; Proline; Protozoan Proteins; Quinazolinones; Signal Transduction

Halofuginone, which is the main active ingredient of Dichroa fabrifuga, was used to inhibit the synthesis of type I collagen and played increasingly important roles in tumor therapy. This study aims to investigate the protective effects of halofuginone on human umbilical vein endothelial cells (HUVECs) from H2O2-induced apoptosis and oxidative stress.. Propidium iodide and Annexin-V double staining assay was used to measure the apoptosis. Cell viability assay, the measurements of reactive oxygen species (ROS) parameters malondialdehyde and superoxide dismutase, western-blot assays, and quantitative PCR were used to elucidate the effects and mechanisms of halofuginone in protecting H2O2-induced injury.. The results showed that halofuginone counteracted H2O2-induced cell viability decline and PCNA downregulation. Furthermore, halofuginone decreased ROS levels and protected HUVECs from H2O2-induced apoptosis. In detail, it showed that H2O2 induced a transient activation of Mitogen-activated protein kinases members ERK1/2 and p38, whereas induced a sustained activation of c-Jun N-terminal kinase (JNK), which play dominant roles in triggering apoptosis. Inhibition of JNK activation also inhibited H2O2-mediated apoptosis. Finally, it was shown that halofuginone upregulated VEGF expressions, which functioned by inhibiting sustained JNK activation, thus protecting HUVECs.. Halofuginone has powerful effects in protecting HUVECs from H2O2-induced apoptosis, via upregulating VEGF and inhibiting overactivated JNK phosphorylation. Halofuginone might be a promising preventive drug for cardiovascular diseases.

Topics: Apoptosis; Cells, Cultured; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Piperidines; Proto-Oncogene Proteins c-bcl-2; Quinazolinones; Vascular Endothelial Growth Factor A

Traditional Chinese medicine (TCM) are both historically important therapeutic agents and important source of new drugs. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been shown to exert strong antiproliferative effects that differ markedly among various cell lines. However, whether HF inhibits MCF-7 cell growth in vitro and underlying mechanisms of this process are not yet clear. Here, we offer the strong evidence of the connection between HF treatment, exosome production and proliferation of MCF-7 cells. Our results showed that HF inhibits MCF-7 cell growth in both time- and dose-dependent manner. Further microRNA (miRNA) profiles analysis in HF treated and nontreated MCF-7 cell and exosomes observed that six miRNAs are particularly abundant and sorted in exosomes. miRNAs knockdown experiment in exosomes and the MCF-7 growth inhibition assay showed that exosomal microRNA-31 (miR-31) modulates MCF-7 cells growth by specially targeting the histone deacetylase 2 (HDAC2), which increases the levels of cyclin-dependent kinases 2 (CDK2) and cyclin D1 and suppresses the expression of p21. In conclusion, these data indicate that inhibition of exosome production reduces exosomal miR-31, which targets the HDAC2 and further regulates the level of cell cycle regulatory proteins, contributing to the anticancer functions of HF. Our data suggest a new role for HF and the exosome production in tumorigenesis and may provide novel insights into prevention and treatment of breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Exosomes; Female; Histone Deacetylase 2; Humans; MCF-7 Cells; Medicine, Chinese Traditional; MicroRNAs; Piperidines; Quinazolinones

In the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway.. As an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting.. In three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects.. We demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses.

Topics: Amino Acids; Autophagy; Eukaryotic Initiation Factor-2; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Microtubule-Associated Proteins; Piperidines; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Quinazolinones; Sequestosome-1 Protein

Neurodegeneration caused with retinal ischemia or high intraocular pressure is irreversible in general. We have focused on the role of hypoxia-inducible factor (HIF) in retinal homeostasis and revealed that HIF inhibition may be effective against retinal neovascular and neurodegeneration. In this study, we performed in vitro screening of natural products and found halofuginone, which is a derivative of febrifugine extracted from hydrangea, as a novel HIF inhibitor. Administration of halofuginone showed a significant neuroprotective effect by inhibiting HIF-1α expression in a murine retinal ischemia-reperfusion model histologically and functionally. These results indicate that halofuginone can be a neuroprotective agent in ischemic retinal degenerative diseases.

Topics: 3T3 Cells; Animals; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Piperidines; Quinazolinones; Reperfusion Injury; Retinal Degeneration; Retinal Vessels

Traditional Chinese medicines have been recognized as especially promising anticancer agents in modern anticancer research. Halofuginone (HF), an analog of quinazolinone alkaloid extracted from Dichroa febrifuga, is widely used in traditional medicine. However, whether HF inhibits the growth of breast cancer cells and/or reduces the migration and invasion of MCF-7 human breast cancer cells, as well as the underlying mechanisms in vitro, remains unclear. In this study, we report that an HF extract inhibits the growth of MCF-7 cells and reduces their migration and invasion, an important feature of potential anticancer agents. In addition, HF significantly increases the activation of autophagy, which is closely associated with tumor metastasis. As STMN1 and p53 have been closely implicated in breast cancer progression, we analyzed their expression in the context of HF extract treatment. Western blot analysis showed that HF suppresses STMN1 and p53 expression and activity in an autophagy-dependent manner. Collectively, these data indicate that activation of autophagy reduces expression of STMN1 and p53, and the migration and invasion of cancer cells contributes to the anti-cancer effects of the HF. These findings may provide new insight into breast cancer prevention and therapy.

Topics: Autophagy; Breast Neoplasms; Cell Movement; Female; Humans; MCF-7 Cells; Neoplasm Invasiveness; Piperidines; Quinazolinones; Stathmin; Tumor Suppressor Protein p53

Most low back pain is caused by intervertebral discs (IVD) degeneration, a disease that prevalence is increasing with age. Halofuginone, an analog of ferbrifugine isolated from plant Dichroa febrifuga, has drawn much attention in recent years for the wide range of bioactivities in malaria, cancer, fibrotic and autoimmune diseases. In this study, we evaluated the benefit effects of halofuginone in IVD degeneration treatment in a validated rabbit puncture model. Halofuginone treatment could attenuate disc degeneration by suppressing the decrease of discs height and nucleus pulposus signal strength. Besides, halofuginone treatment could suppress mRNA and protein expression of collagen I in nucleus pulposus. This might possibly due to the inactivation of transform growth factor-β (TGFβ) signal pathway by down-regulating p-Samd3 and up-regulating inhibitory Smad7. Then, we evaluated the effects of halofuginone treatment on nuclear factor of kappa B (NF-κB) signal pathway and its downstream pro-inflammatory cytokines. The level of p-p65 and p-IκBα was down-regulated in halofuginone treated group, indicating the inactivation of NF-κB signal pathway. The mRNA expression of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8) was decreased in nucleus pulposus too, indicating the down-regulation of pro-inflammatory cytokines. In conclusion, halofuginone treatment could attenuate IVD degeneration and this was possibly due to suppressing of collagen I production and inactivation of TGFβ and NF-κB signal pathway in nucleus pulposus of degenerated discs. These results suggest that halofuginone has the potential for IVD degeneration treatment, but more research is needed to validate this.

Topics: Animals; Collagen Type I; Female; Intervertebral Disc Degeneration; NF-kappa B; Piperidines; Quinazolinones; Rabbits; Signal Transduction; Transforming Growth Factor beta

There are no standard guidelines for the treatment of cryptosporidiosis in reptiles. The aim of this study was to evaluate the efficacy of two cryptosporidiosis therapies in captive green iguanas. Eight green iguanas aged 2-6 years, including 6 (1 ♂ and 5 ♀) animals with chronic diarrhea, received treatment for cryptosporidiosis. The presence of Cryptosporidium sp. oocysts was determined in 8 iguanas (100%), Isospora sp. oocysts were detected in 3 animals (37.5%), and Oxyuridae eggs were observed in 5 iguanas (62.5%). The animals were divided into two therapeutic groups (A and B). Group A iguanas were administered halofuginone (Halocur, 0,50 mg/ml Intervet Productions S.A., France) at a dose of 110 mg/kg body weight (BW) every 7 days for 5 weeks. Group B animals were administered sulfadiazine and trimethoprim (Norodine Vet Oral Paste sulfadiazine 288,3 mg/g, trimethoprim 58 mg/g, ScanVet Animal Health A/S, Denmark) at 75 mg/kg BW per os every 5 days for 5 weeks and spiramycin and metronidazole (Stomorgyl, spiramycin 1500000 IU, metronidazole 250 mg, Merial, France) at 200 mg/kg BW every 5 days for 5 weeks. Both groups received hyperimmune bovine colostrum and subcutaneous fluids. Before treatment, the average number of Cryptosporidium sp. oocysts in 1 g of feces was determined at 1.71 * 10

Topics: Animals; Coccidiostats; Cryptosporidiosis; Cryptosporidium; Feces; Iguanas; Intestinal Mucosa; Oocysts; Piperidines; Poland; Quinazolinones; Sulfadiazine; Treatment Outcome; Trimethoprim

Topics: Animals; Animals, Newborn; Cattle; Cattle Diseases; Coccidiostats; Cryptosporidiosis; Germany; Piperidines; Quinazolinones

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1β (IL-1β) and provides protection from intestinal inflammation in mice. HF inhibits IL-1β through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1β mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1β production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1β is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

Topics: Adaptation, Physiological; Amino Acids; Animals; Autophagy; Cells, Cultured; Colitis; Gene Expression Regulation; Inflammasomes; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Piperidines; Protein Biosynthesis; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Quinazolinones; Reactive Oxygen Species; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Sodium Dodecyl Sulfate; Starvation; Stress, Physiological; T-Cell Intracellular Antigen-1

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Topics: Animals; Disease Models, Animal; Dysferlin; Humans; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Myoblasts; Phosphatidylinositol 3-Kinases; Piperidines; Quinazolinones; Synaptotagmins

The aim of this study is to evaluate the anti-inflammatory and anti-fibrotic effects of halofuginone in caustic esophageal burn injury in rats.. Corrosive esophageal injury (CEI) was produced in male Wistar albino rats by instilling NaOH solution (1 ml, 37.5%) into the distal esophagus. Rats were decapitated on the 3rd day (early group) or 28th day (late group), and treated daily with either saline or halofuginone (100 µg/kg/day; i.p.), continued on alternate days after the third day. Histopathological evaluation and measurement of nitric oxide (NO), peroxynitrite (ONOO-) and oxygen-derived radicals by chemiluminescence (CL) were made in the distal 2 cm of the esophagus. Non-irrigated proximal esophageal samples were assessed for the levels of nuclear factor (NF)-κB, caspase-3, glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) activity.. GSH, MDA, NF-κB and caspase-3 levels, and MPO activity in the proximal esophagus were not different among groups. Increased number of TUNEL (+) cells in the irrigated esophagus of the early and late caustic injury groups was reduced by halofuginone treatment. High microscopic damage scores in both early and late CEI groups were decreased with halofuginone treatment. NO, ONOO- and CL levels, which were elevated in the saline-treated early CEI group, were reduced by halofuginone treatment, but reduced NO and ONOO- levels in the late period of saline-treated group were increased by halofuginone.. In addition to its anti-fibrotic effects, current findings demonstrate that halofuginone exerts antioxidant and anti-apoptotic actions and supports therapeutic potential for halofuginone in CEI-induced oxidative stress.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Burns, Chemical; Caspase 3; Esophagus; Glutathione; Male; Malondialdehyde; NF-kappa B; Nitric Oxide; Oxidative Stress; Peroxidase; Piperidines; Quinazolinones; Rats; Rats, Wistar; Reactive Oxygen Species

Our objectives were to investigate the therapeutic effect of halofuginone (HF) in the treatment of autoimmune thyroid diseases (AITDs) and explore its underlying mechanism of action.. The Graves' disease (GD) model was generated by immunizing female BALB/c mice with adenovirus expressing the TSH receptor A subunit (Ad-TSHR289). The Ad-TSHRA+HF and Ad- TSHRA+DMSO groups were injected intraperitoneally with HF or the vehicle control (DMSO), respectively. The autoimmune thyroiditis (AIT) group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with the vehicle control (DMSO) during the study period. The AIT/HF group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with HF. The frequencies of splenic Th17 cells, Tregs and Bregs were determined by flow cytometry. The mRNA levels of IL-17, forkhead box P3 (Foxp3), RORγt and IL-10 were determined by real-time PCR.. In both Ad-TSHRA+DMSO and Ad-TSHRA+HF groups, 10 out of 15 mice displayed serum T4 and TSAb levels above 3 SD beyond the mean control levels. The number of CD4+CD25+Foxp3+ T lymphocytes in the GD model was significantly increased in the HF group compared with the DMSO group (P < 0.05). The mRNA level of Foxp3 was significantly increased in the Ad-TSHRA+HF group compared with the Ad-TSHRA+DMSO group (P < 0.05). However, neither the abundance of CD4+IL-17+ T cell subpopulation nor the mRNA expression level of RORγt differed significantly between the Ad-TSHRA+HF and Ad-TSHRA+DMSO groups (P > 0.05). The serum TgAb titer was significantly reduced in the AIT/HF group compared with the AIT group (P < 0.01). The differences in the number of CD4+CD25+Foxp3+ T lymphocytes and the mRNA levels of Foxp3 between the AIT/HF and AITgroups were not significant (P > 0.05). However, the number of CD4+IL-17+ T cells and the mRNA levels of IL-17 and RORγt were significantly increased in HF-treated mice compared with the non-treated AIT-induced mice (P < 0.05).. Treatment with HF significantly decreased the incidence of AIT by decreasing the number of CD4+IL-17+ T cells.

Topics: Animals; Disease Models, Animal; Female; Graves Disease; Mice; Mice, Inbred BALB C; Mice, Transgenic; Piperidines; Quinazolinones; Random Allocation; Th17 Cells; Thyroiditis, Autoimmune; Treatment Outcome

The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure.. Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner.. Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

Topics: Amino Acids; Amino Acyl-tRNA Synthetases; Animals; Autophagy; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Fibrosis; Heart Failure; Humans; Hypertrophy, Left Ventricular; Induced Pluripotent Stem Cells; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Piperidines; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Quinazolinones; Stress, Physiological; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Autophagy has a key role in metabolism and impacts on tumorigenesis. Our previous study found that halofuginone (HF) exerts anticancer activity in colorectal cancer (CRC) by downregulating Akt/mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. But whether and how HF regulates autophagy and metabolism to inhibit cancer growth remains an open question. Here, we unveil that HF activates ULK1 by downregulation of its phosphorylation site at Ser757 through Akt/mTORC1 signaling pathway, resulting in induction of autophagic flux under nutrient-rich condition. On the other hand, HF inactivates ULK1 by downregulation of its phosphorylation sites at Ser317 and Ser777 through LKB1/AMPK signaling pathway, resulting in autophagic inhibition under nutrient-poor condition. Furthermore, Atg7-dependent autophagosome formation is also induced under nutrient-rich condition or blocked in nutrient-poor environment, respectively, upon HF treatment. More interestingly, we also found that HF inhibits glycolysis under nutrient-rich condition, whereas inhibits gluconeogenesis under nutrient-poor condition in an Atg7-dependent manner, suggesting that autophagy has a pivotal role of glucose metabolism upon HF treatment. Subsequent studies showed that HF treatment retarded tumor growth in xenograft mice fed with either standard chow diet or caloric restriction through dual regulation of autophagy in vivo. Together, HF has a dual role in autophagic modulation depending on nutritional conditions for anti-CRC.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Autophagy; Cell Line, Tumor; Colorectal Neoplasms; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Piperidines; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Quinazolinones; Signal Transduction; Xenograft Model Antitumor Assays

Topics: Animals; Antiprotozoal Agents; Chickens; Coccidia; Molecular Structure; Piperidines; Poultry Diseases; Quinazolinones

Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

Topics: Animals; Chondrocytes; Collagen Type I; Collagen Type II; Extracellular Matrix; Female; Male; Piperidines; Quinazolinones; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Osteoarthritis (OA) is the most common degenerative condition of the weight‑bearing joints worldwide without effective medical therapy. In order to investigate whether administration of halofuginone (HF) may attenuate OA, the present study allocated 3‑month‑old male mice into Sham group, vehicle‑treated anterior cruciate ligament transection (ACLT) group and HF‑treated ACLT group. The present study determined that HF treatment reduced the expression of matrix metallopeptidase‑13 and collagen X in articular cartilage. Additionally, it lowered the Osteoarthritis Research Society International‑Modified Mankin score and prevented the loss of articular cartilage from Safranin O and Fast Green staining. HF reduced the progression of osteoarthritis by downregulating abnormally elevated TGF‑β1 activity in articular cartilage. Administration of HF may be a potential preventive therapy for OA.

Topics: Animals; Anterior Cruciate Ligament; Anti-Inflammatory Agents; Cartilage, Articular; Collagen Type X; Disease Models, Animal; Gene Expression Regulation; Male; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Osteoarthritis; Piperidines; Quinazolinones; Signal Transduction; Transforming Growth Factor beta1

The hepatitis B virus (HBV) has caused acute and chronic liver diseases in ~350 million infected people worldwide. Halofuginone (HF) is a plant alkaloid which has been demonstrated to play a crucial role in immune regulation. Our present study explored the function of HF in the immune response of HBV-infected Sprague Dawley (SD) rats. Plasmid containing pCDNA3.1-HBV1.3 was injected in SD rats for the construction of an acute HBV-infected animal model. Our data showed that HF reduced the high concentrations of serum hepatitis B e-antigen, hepatitis B surface antigen, and HBV DNA induced by HBV infection. HF also reduced the number of T helper (Th)17 cells and the expression of interleukin (IL)-17 compared with the pCDNA3.1-HBV1.3 group. Moreover, pro-inflammatory cytokine levels (IL-17, IL-23, interferon-γ, and IL-2) were downregulated and anti-inflammatory cytokine levels (IL-4 and IL-13) were upregulated by HF. Through further research we found that the expression of AMP-activated protein kinase (AMPK) and IKBA which suppressed NF-κB activation was increased while the expression of p-NF-κB P65 was decreased in pCDNA3.1-HBV1.3+HF group compared with pCDNA3.1-HBV1.3 group, indicating that HF may work through the activation of AMPK. Finally, our conjecture was further verified by using the AMPK inhibitor compound C, which counteracted the anti-inflammation effect of HF, resulting in the decreased expression of AMPK, IKBA and increased expression of p-NF-κB P65 and reduced number of Th17 cells. In our present study, HF was considered as an anti-inflammatory factor in acute HBV-infected SD rats and worked through AMPK-mediated NF-κB p65 inactivation. This study implicated HF as a potential therapeutic strategy for hepatitis B.

Topics: Acute Disease; AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Cytokines; Disease Models, Animal; DNA, Viral; Female; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Inflammation; Piperidines; Quinazolinones; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Transcription Factor RelA

To evaluate effects of halofuginone (H) on radiation-induced lung injury (RILI), 60 rats were divided into six groups: Group (G) 1 control, G2 radiotherapy (RT) only, G3 and G4 2. 5 and 5 μg H and G5 and G6 RT + 2.5 and 5 μg H groups, respectively. A single dose of 12 Gy RT was given to both lungs. H was applied intraperitoneally with daily doses, until animals were killed at 6 and 16 weeks after RT. At 6th and 16th weeks of RT, five rats from each group were killed. Lung tissues were dissected for light and electron microscopy. Chronic inflammation, fibrosis and transforming growth factor-beta (TGF)-β scores of all study groups were significantly different at 6th and 16th week ( p < 0.001). Chronic inflammation, fibrosis and TGF-β scores of G2 were higher than G5 and G6 at 6th and 16th weeks of RT. At 16th week, fibrosis and TGF-β scores of G5 were higher than G6 ( p = 0.040 and 0.028, respectively). Electron microscopical findings also supported these results. Therefore, H may ameliorate RILI. The effect of the H was more prominent at higher dose and after long-term follow-up. These findings should be clarified with further studies.

Topics: Animals; Female; Lung; Lung Injury; Piperidines; Pulmonary Fibrosis; Quinazolinones; Radiation Injuries, Experimental; Radiation-Protective Agents; Radiation, Ionizing; Rats, Wistar; Transforming Growth Factor beta

Vocal fold injury results in severe voice alteration that limits occupational function and social interaction. An ovine model of laryngeal injury has been developed, validated and utilized to examine laryngeal wound healing and the effect of a novel collagen inhibitor (halofuginone) on surgical wound healing. The study design includes basic research and animal model.. An ovine laryngeal model was utilized to study controlled vocal fold injury and healing. Twenty-five sheep were divided into five groups. Sheep underwent right vocal fold injury preceded or followed by administration of halofuginone orally, topically or intralesionally. Biopsies were taken at commencement, 1 month and larynges explanted at 3 months. Specimens were examined for elastin and collagen density and epithelial changes. Pearson correlation statistics and Student's t-tests were used to assess inter-relationships.. All sheep tolerated halofuginone. One sheep death occurred in an untreated sheep. Vocal fold tissue demonstrated a predictable histological response to injury. Elastin was significantly reduced post-injury in the glottis. Halofuginone administered orally for 10 weeks prevented elastin loss and demonstrated a trend of reducing collagen density post-injury.. In an ovine laryngeal injury model, administration of a fibrosis inhibitor resulted in altered elastin and collagen deposition after injury in the glottis. Further investigation is warranted to examine whether these tissue changes affect vocal fold dynamics.

Topics: Administration, Oral; Administration, Topical; Animals; Collagen; Disease Models, Animal; Elastin; Fibrosis; Injections, Intralesional; Piperidines; Quinazolinones; Sheep; Treatment Outcome; Vocal Cords; Wound Healing

Fibroblast-like synoviocytes (FLSs) display an aggressive phenotype that is a critical factor in cartilage destruction in rheumatoid arthritis (RA). Increased FLS migration and proliferation are essential to the pathology of RA. Halofuginone has been found to inhibit cell migration and proliferation in cancer cells. However, whether halofuginone has a role in the treatment of RA FLSs is unclear. In this study, we found that halofuginone reduced migration, invasion, cell proliferation and MMPs expression in RA FLSs. In addition, we demonstrated that halofuginone inhibited reorganization of the actin cytoskeleton during cell migration. To gain insight into the molecular mechanisms, we evaluated the effect of halofuginone on the MAPK and AKT pathways. Our results indicated that halofuginone inhibited the activity of MAPK and AKT. Taken together, these results suggest that halofuginone may protect against joint destruction in RA by regulating synoviocyte migration, invasion and cell proliferation by inhibiting MAPK and AKT activation.

Topics: Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Cell Movement; Cell Proliferation; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Humans; Male; Middle Aged; Oncogene Protein v-akt; Piperidines; Quinazolinones; Signal Transduction; Synoviocytes; Tumor Necrosis Factor-alpha

The KEAP1-NRF2 system regulates the cellular defence against oxidative and xenobiotic stresses. NRF2 is a transcription factor that activates the expression of cytoprotective genes encoding antioxidative, detoxifying and metabolic enzymes as well as transporters. Under normal conditions, KEAP1 represses NRF2 activity by degrading the NRF2 protein. When cells are exposed to stresses, KEAP1 stops promoting NRF2 degradation, and NRF2 rapidly accumulates and activates the transcription of target genes. Constitutive accumulation of NRF2 via a variety of mechanisms that disrupt KEAP1-mediated NRF2 degradation has been observed in various cancer types. Constitutive NRF2 accumulation confers cancer cells with a proliferative advantage as well as resistance to anti-cancer drugs and radiotherapies. To suppress the chemo- and radio-resistance of cancer cells caused by NRF2 accumulation, we conducted high-throughput chemical library screening for NRF2 inhibitors and identified febrifugine derivatives. We found that application of the less-toxic derivative halofuginone in a low dose range rapidly reduced NRF2 protein levels. Halofuginone induced a cellular amino acid starvation response that repressed global protein synthesis and rapidly depleted NRF2. Halofuginone treatment ameliorated the resistance of NRF2-addicted cancer cells to anti-cancer drugs both in vitro and in vivo. These results provide preclinical proof-of-concept evidence for halofuginone as an NRF2 inhibitor applicable to treatment of chemo- and radio-resistant forms of cancer.

Topics: A549 Cells; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression; Humans; Inhibitory Concentration 50; Male; Mice, Inbred BALB C; Mice, Nude; NF-E2-Related Factor 2; Oxidative Stress; Piperidines; Quinazolinones; Xenograft Model Antitumor Assays

Fibrosis is the main complication of muscular dystrophies. We identified moesin, a member of the ezrin-radixin-moesin family, in dystrophic muscles of mice representing Duchenne and congenital muscular dystrophies (DMD and CMD, respectively) and dysferlinopathy, but not in the wild type. High levels of moesin were also observed in muscle biopsy specimens from DMD, Ullrich CMD, and merosin-deficient CMD patients, all of which present high levels of fibrosis. The myofibroblasts, responsible for extracellular matrix protein synthesis, and the macrophages infiltrating the dystrophic muscles were the source of moesin. Moesin-positive cells were embedded within the fibrotic areas between the myofibers adjacent to the collagen type I fibers. Radixin was also synthesized by the myofibroblasts, whereas ezrin colocalized with the myofiber membranes. In animal models and patients' muscles, part of the moesin was in its active phosphorylated form. Inhibition of fibrosis by halofuginone, an antifibrotic agent, resulted in a major decrease in moesin levels in the muscles of DMD and CMD mice. In summary, the results of this study may pave the way for exploiting moesin as a novel target for intervention in MDs, and as part of a battery of biomarkers to evaluate treatment success in preclinical studies and clinical trials.

Topics: Adult; Animals; Child; Child, Preschool; Collagen Type I; Cytoskeletal Proteins; Diaphragm; Homozygote; Humans; Immunohistochemistry; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred mdx; Microfilament Proteins; Muscle, Skeletal; Muscular Dystrophies; Myofibroblasts; Phosphorylation; Piperidines; Quinazolinones

Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf(-/-) mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf(-/-) mice, halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the halofuginone-induced cell survival, suggesting this pathway's involvement in mediating halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.

Topics: Animals; Apoptosis; Cell Cycle; Cell Survival; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Muscle Fibers, Skeletal; Muscular Dystrophies, Limb-Girdle; Muscular Dystrophy, Duchenne; Phosphatidylinositol 3-Kinases; Piperidines; Proto-Oncogene Proteins c-akt; Quinazolinones; Satellite Cells, Skeletal Muscle; Signal Transduction

Examine whether osteoarthritis (OA) progression can be delayed by halofuginone in anterior cruciate ligament transection (ACLT) rodent models.. 3-month-old male C57BL/6J (wild type; WT) mice and Lewis rats were randomised to sham-operated, ACLT-operated, treated with vehicle, or ACLT-operated, treated with halofuginone. Articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. Immunostaining, flow cytometry, RT-PCR and western blot analyses were conducted to detect relative protein and RNA expression. Bone micro CT (μCT) and CT-based microangiography were quantitated to detect alterations of microarchitecture and vasculature in tibial subchondral bone.. Halofuginone attenuated articular cartilage degeneration and subchondral bone deterioration, resulting in substantially lower OARSI scores. Specifically, we found that proteoglycan loss and calcification of articular cartilage were significantly decreased in halofuginone-treated ACLT rodents compared with vehicle-treated ACLT controls. Halofuginone reduced collagen X (Col X), matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5) and increased lubricin, collagen II and aggrecan. In parallel, halofuginone-attenuated uncoupled subchondral bone remodelling as defined by reduced subchondral bone tissue volume, lower trabecular pattern factor (Tb.pf) and increased thickness of subchondral bone plate compared with vehicle-treated ACLT controls. We found that halofuginone exerted protective effects in part by suppressing Th17-induced osteoclastic bone resorption, inhibiting Smad2/3-dependent TGF-β signalling to restore coupled bone remodelling and attenuating excessive angiogenesis in subchondral bone.. Halofuginone attenuates OA progression by inhibition of subchondral bone TGF-β activity and aberrant angiogenesis as a potential preventive therapy for OA.

Topics: Animals; Anterior Cruciate Ligament; Bone and Bones; Bone Remodeling; Bone Resorption; Cartilage, Articular; Disease Models, Animal; Disease Progression; Male; Mice; Mice, Inbred C57BL; Osteoarthritis; Osteoclasts; Piperidines; Quinazolinones; Random Allocation; Rats; Rats, Inbred Lew; Transforming Growth Factor beta

Increasing evidence suggests that interleukin (IL)-17A plays an important role in chronic lung allograft dysfunction (CLAD), characterized by airway and lung parenchymal fibrosis, after lung transplantation. Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation. The purpose of this study was to examine the effect of halofuginone on CLAD development using a minor alloantigen‒mismatched mouse orthotopic lung transplant model.. C57BL/6 recipient mice received an orthotopic left lung transplant from C57BL/10 donors, mismatched for minor antigens. Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant.. Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 ± 4.7% vs 52.5 ± 13.8%, p < 0.01), as well as significantly reduced parenchymal fibrosis (5.5 ± 2.3% vs 35.9 ± 10.9%, p < 0.05). Immunofluorescent staining for IL-17A demonstrated a decreased number and frequency of IL-17A‒positive cells in halofuginone-treated lung grafts on Day 28, as compared with controls. Halofuginone treatment also decreased IL-17A and IL-22 transcripts at Day 14, transforming growth factor-β1 and matrix metalloproteinase-2 transcripts at Days 14 and 28.. The beneficial effect of halofuginone on development of airway and lung parenchymal fibrosis in the mouse lung transplant model highlights the important role of IL-17A in CLAD and merits further pre-clinical and clinical studies.

Topics: Animals; Chronic Disease; Disease Models, Animal; Graft Rejection; Interleukin-17; Lung Transplantation; Male; Mice; Mice, Inbred C57BL; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Th17 Cells; Transplantation, Homologous

Viral myocarditis (VMC) is an inflammation of heart muscle in infants and young adolescents. This study explored the function of halofuginone (HF) in Coxsackievirus B3 (CVB3) -treated suckling mice. HF-treated animal exhibited higher survival rate, lower heart/body weight, and more decreased blood sugar concentration than CVB3 group. HF also reduced the expressions of interleukin(IL)-17 and IL-23 and the numbers of Th17 cells. Moreover, HF downregulated pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels. The expressions of transforming growth factor(TGF-β1) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) p65/ tumor necrosis factor-α (TNF-α) proteins were decreased by HF as well. Finally, the overexpression of TGF-β1 counteracted the protection effect of HF in CVB3-treated suckling mice. In summary, our study suggests HF increases the survival of CVB3 suckling mice, reduces the Th17 cells and pro-inflammatory cytokine levels, and may through downregulation of the TGF-β1-mediated expression of NF-κB p65/TNF-α pathway proteins. These results offer a potential therapeutic strategy for the treatment of VMC.

Topics: Animals; Anti-Inflammatory Agents; Coxsackievirus Infections; Enterovirus B, Human; Mice; Mice, Inbred BALB C; Myocarditis; NF-kappa B; Piperidines; Quinazolinones; Signal Transduction; Th17 Cells; Transforming Growth Factor beta1

Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model?. HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis.. Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas.. Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group.. Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF.. Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P < 0.05) reduction in tumor volume. The HF-induced volume reduction was accompanied by increased apoptosis and decreased cell proliferation. In contrast, there was no significant change in the collagen content either at the transcript or protein level between UL xenografts in control and HF groups. HF treatment did not change the expression level of ovarian steroid hormone receptors. No adverse pathological effects were observed in other tissues from mice undergoing treatment at these doses.. While this study did test the effects of HF on human leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear.. The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery.. This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests.

Topics: Animals; Antineoplastic Agents; Apoptosis; Body Weight; Cell Proliferation; Female; Humans; Immunohistochemistry; Leiomyoma; Mice, Inbred NOD; Mice, SCID; Piperidines; Quinazolinones; Uterine Neoplasms; Xenograft Model Antitumor Assays

Hypoxia and inflammation have been identified as the hallmarks of colitis, intertwined with metabolism. Here, we report that halofuginone (HF), an antiparasitic drug, attenuates dextran sulfate sodium (DSS)-induced colitis in mice, as represented by attenuating the disease activity index, inhibiting colonic shortening, ameliorating colonic lesions and histological signs of damage, reducing colonic myeloperoxidase activity, and suppressing the production of pro-inflammatory cytokines in colon tissue. Intriguingly, the hypoxia-inducible factor 1alpha (HIF-1α) and tumor necrosis factor alpha were also suppressed by HF treatment in colon tissues, exhibiting a tissue-specific effect. To further reveal the metabolic signatures upon HF treatment, mass spectrometry-based metabolomic analysis of the small molecular metabolites in liver, spleen and colon tissues was performed. As a result, we found that HF treatment counteracted the levels of acylcarnitines, including palmitoyl-l-carnitine, isobutyrylcarnitine, vaccenylcarnitine, and myristoylcarnitine, in colon tissues with DSS induction, but no significant change in the levels of acylcarnitines was observed in liver or spleen tissues. The metabolic signatures may indicate that incomplete fatty acid oxidation (FAO) in the colon could be restored upon HF treatment as the tissue-specific metabolic characterization. Taken together, our findings uncovered that the HF potentiated anti-inflammatory effect in DSS-induced colitis in mice and its underlying mechanisms could be associated with the inhibition of HIF-1α and reduced levels of acylcarnitines, suggesting that both the inhibition of HIF-1α and the counteraction of incomplete FAO might be useful in the prevention and treatment of inflammatory bowel disease.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Body Weight; Colitis; Cytokines; Dextran Sulfate; Disease Models, Animal; Energy Metabolism; Enzyme Activation; Fatty Acids; Gene Expression; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Liver; Male; Metabolic Networks and Pathways; Mice; Models, Biological; Oxidation-Reduction; Peroxidase; Phenotype; Piperidines; Quinazolinones; RNA, Messenger; Spleen

Phototherapy with UV light is a standard treatment for psoriasis, yet the mechanisms underlying the therapeutic effects are not well understood. Studies in human and mouse keratinocytes and in the skin tissues from human patients and mice showed that UV treatment triggers ubiquitination and downregulation of the type I IFN receptor chain IFNAR1, leading to suppression of IFN signaling and an ensuing decrease in the expression of inflammatory cytokines and chemokines. The severity of imiquimod-induced psoriasiform inflammation was greatly exacerbated in skin of mice deficient in IFNAR1 ubiquitination (Ifnar1(SA)). Furthermore, these mice did not benefit from UV phototherapy. Pharmacologic induction of IFNAR1 ubiquitination and degradation by an antiprotozoal agent halofuginone also relieved psoriasiform inflammation in wild-type but not in Ifnar1(SA) mice. These data identify downregulation of IFNAR1 by UV as a major mechanism of the UV therapeutic effects against the psoriatic inflammation and provide a proof of principle for future development of agents capable of inducing IFNAR1 ubiquitination and downregulation for the treatment of psoriasis.

Topics: Animals; Cell Line; Chemokines; Cytokines; Disease Models, Animal; Down-Regulation; Humans; Inflammation; Keratinocytes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Psoriasis; Quinazolinones; Receptor, Interferon alpha-beta; Signal Transduction; Skin; Ubiquitination; Ultraviolet Therapy

Combinational drug therapy is one of the most promising strategies in modern anticancer research. Traditional Chinese medicine (TCM) formulas represent a wealth of complex combinations proven successful over centuries of clinical application. One such formula used to treat a variety of diseases, including cancer, contains two herbs, whose main active components are Halofuginone (HF) and Artemisinin (ATS). Here we studied the anticancer synergism of HF and ATS in various cancer cell lines and in a xenograft nude mice model. We found that the HF-ATS combination arrested more cells at the G1/G0 phase than either one alone, with the concomitant increased levels of CDK2 inhibitors, p21Cip1 and p27Kip1. By knocking down p21Cip1 and p27Kip1 separately or simultaneously in HCT116 cells and MCF-7 cells, we found that p21Cip1 was required for HF induced G1/G0 arrest, whereas p21Cip1 and p27Kip1 were both required for ATS or HF-ATS combination-mediated cell cycle arrest. Moreover, HF-ATS combination synergistically inhibited tumor growth in xenograft nude mice, and this was associated with the increased levels of p21Cip1 and p27Kip1. Collectively, these data indicate that the upregulation of p21Cip1 and p27Kip1 contributes to the synergistic anticancer effect of the HF-ATS combination.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Artemisinins; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Synergism; Female; G1 Phase; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Piperidines; Quinazolinones; Resting Phase, Cell Cycle; Up-Regulation

The aim of this study was to determine whether there is an association between Cryptosporidium infections in calves and immunological factors, as well as farm-related factors or the application of the anti-cryptosporidiosis drug Halofuginone. From January to June 2010, 63 cow-calf-pairs from 20 different farms near Zürich, Switzerland have been investigated. Each cowcalf- pair was visited three times within the first 6 weeks of life to collect data of the farm and animals, as well as blood, faecal, colostral and milk samples. An ELISA using sporozoite antigen was developed for the specific detection of anti-Cryptosporidium-IgG in blood- and colostral serum. The IgG concentration in the bloodand colostral serum was determined using radial immuno diffusion test (RID). White blood cell isolation and differential blood cell counts and California Mastitis Test were performed. Bacteriological studies on quarter-milk-samples were carried out. Cryptosporidium oocysts were diagnosed with the modified Ziehl-Neelsen staining, other protozoa with the SAFC method and Eimeria oocysts and helminth eggs were diagnosed with the combined sedimentation/floatation test. ELISAs were performed for the detection of rota- and coronavirus, E. coli F5 and Cryptosporidium spp. in bovine feces (bio-X Diagnostics®, Belgium). The highest prevalence of Cryptosporidium oocysts was 54.0% and found 7 to 20 days post natum, whereas 47.1% were suffering from diarrhea. The transfer of total IgG with the colostrum and the humoral immunity of the calf could not prevent any infection with Cryptosporidium, but the severity of the diarrhea symptoms decreased with increasing total IgG concentrations. Calves housed in open sheds showed significantly more often diarrhea, i. e. they shed more Cryptosporidium oocysts during the first 4 days and 7 to 20 days post natum, respectively. Halofuginone (Halocur®) is approved for prophylaxis against cryptosporidiosis, but it showed no effect on the excretion of Cryptosporidium oocysts in the present study.. Ziel dieser Studie war es, einen Zusammenhang zwischen dem Vorkommen von Cryptosporidien und immunologischen Faktoren, sowie Betriebsfaktoren und die Verwendung von Halofuginon, zu untersuchen. Von Januar bis Juni 2010 wurden im Kanton Zürich 63 Mutter- Kalb-Paare aus 20 verschiedenen Betrieben untersucht. Innerhalb von 6 Wochen erfolgten zu ausgewählten Zeitpunkten drei Besuche. Dabei wurden Bestandsdaten erhoben, wie auch Blut-, Kot-, Kolostrum-, respektive Milchproben untersucht. Zum Nachweis von anti-Cryptosporidien-IgG im Blut- und Kolostrum- Serum wurde ein ELISA mit Sporozoiten-Antigen entwickelt. Die IgG-Konzentration im Kolostrum- und Blutserum wurde mittels RID (Radialer Immundiffusionstest) bestimmt. Zusätzlich wurde bei der Kuh und beim Kalb ein Differenzialblutbild erstellt und beim Muttertier ein Schalmtest durchgeführt. Bakteriologische Untersuchungen erfolgten an Vierviertelgemelksproben. Cryptosporidien-Oozysten wurden durch eine modifizierte Ziehl-Neelsen-Färbung, andere Protozoen durch die SAFC-Methode nachgewiesen und mittels kombiniertem Sedimentations-/Flotationsverfahren wurden Helminthen-Eier und Eimeria-Oozysten erfasst. Ein ELISA diente dem Nachweis von Rota- und Coronaviren, E. coli F5 und Cryptosporidium spp. in bovinem Faeces (Bio-X Diagnostics®, Belgien). Die Prävalenz von Cryptosporidien-Infektionen war zwischen dem 7. und 20. Lebenstag der Kälber am höchsten (54.0%), wobei 47.1% dieser Kälber an Durchfall litten. Offenställe steigerten signifikant das Risiko für Durchfall, bzw. für Cryptosporidien-Ausscheidung zwischen dem 1. und 4., bzw. zwischen dem 7. und 20. Lebenstag. Die Übertragung von Total-IgG mit dem Kolostrum und die humorale Immunität des Kalbes konnten keine Infektion mit Cryptosporidien verhindern, der Schweregrad der Durchfallsymptomatik nahm aber mit zunehmender total IgG-Konzentration ab. Der für die Prophylaxe gegen Cryptosporidiose zugelassene Wirkstoff Halofuginon zeigte in dieser Studie keine Wirkung auf die Ausscheidung von Cryptosporidien-Oocysten.. Le but de la présente étude était d’étudier s’il existe un rapport entre l’apparition de cryptosporidies et des facteurs immunologiques, des facteurs liés à l’exploitation ainsi qu’à l’usage d’halofuginone. De janvier à juin 2010, on a examiné 63 paires mère-veau provenant de 20 exploitations du canton de Zürich. Au cours de 6 semaines on a effectué, à des moments choisis, trois visites. A ces occasions, des données relatives à l’exploitation ainsi que des échantillons de sang, de selles, de colostrum respectivement de lait ont été collectés. On a développé un test ELISA avec des antigènes de sporozoïtes pour mettre en évidence la présence IgG anti-cryptosporidies dans le sang et dans le colostrum. La concentration en IgG dans le colostrum et dans le sérum a été mesurée avec un test d’immunodiffusion radiale (RID). En outre on a réalisé une image sanguine différentielle des vaches et des veaux et effectué un test de Schalm chez les vaches. Un examen bactériologique a été réalisé sur un échantillon provenant des quatre quartiers. Les oocystes de cryptosporidies ont été mis en évidence au moyen d’une coloration de Ziehlk-Neelsen modifiée, les autres protozoaires ont été mis en évidence par la méthode SAFC et les oeufs d’helminthes ainsi que les oocystes d’Eimeria par un processus de sédimentation-flottation combiné. Un test ELISA a été utilisé pour les rota- et les coronavirus, les E. coli F5 et Cryptosporidium spp. dans les selles des bovins (Bio-X Diagnostics®, Belgique). La prévalence d’infections par des cryptosporidies était maximale entre le 7ème et le 20ème jour de vie des veaux (50.4%), 47.1% de ces veaux souffrant de diarrhée. Les stabulations libres augmentaient de façon significative le risque de diarrhée et d’excrétion de cryptosporidies entre le 1er et le 4ème jour respectivement entre le 7ème et le 20ème jour. La transmission d’IgG et l’immunité humorale des veaux n’empêchaient pas l’infection par des cryptosporidies mais la gravité de la diarrhée diminuait avec l’augmentation de la concentration des IgG totales. L’halofuginone, substance enregistrée pour la prophylaxie de la cryptosporidiose, n’a pas montré, dans cette étude, d’efficacité pour empêcher l’excrétion d’oocystes de cryptosporidies.. Lo scopo di questo studio è di indagare sulla correlazione tra la presenza di Cryptosporidium e fattori immunologici e aziendali nonché l’uso di alofuginone. Da gennaio a giugno 2010, nel Canton Zurigo sono state esaminate 63 coppie madre-vitello provenienti da 20 diverse aziende. Nello spazio di 6 settimane sono state eseguite, in momenti determinati, tre visite che hanno raccolto dati sulla mandria e esaminati campioni di sangue, escrementi, colostro e latte. Per rilevare la presenza di anti-IgG Cryptosporidium nel sangue e nel siero di colostro è stato sviluppato un test ELISA con un antigene sporozoite. La concentrazione di IgG nel siero di colostro e nel siero sanguigno è stata determinata mediante RID (test di immunodiffusione radiale). Inoltre, è stato eseguito sulla bovina e sul vitello un emocromo differenziale e sulla vacca madre un CMT. Esami batteriologici sono stati eseguiti su campioni di latte proveniente dai quattro quarti. Oocisti di Cryptosporidium sono stati rilevati da una colorazione di Ziehl-Neelsen, gli altri protozoi con il metodo SAFC mentre con una combinazione di processi di sedimentazione/flottazione sono state rilevate uova di elminti e oocisti di Eimeria. Un test ELISA è stato utilizzato per provare la presenza di Rotavirus e Coronavirus, E. Coli F5 e Cryptosporidium spp nelle feci bovine (Bio-X Diagnostics®, Belgio). La prevalenza di infezioni da Cryptosporidium si situava tra il 7° e il 20° giorno di vita del vitello ad alto livello (54.0%); il 47.1% di questi vitelli soffriva di diarrea. Il rischio di diarrea aumentava significativamente in caso di stalle aperte rispettivamente le secrezioni di Cryptosporidium tra il 1° e il 4° risp. il 7° e il 20° giorno di vita. La trasmissione con il colostro di IgG totali e l’immunità umorale dei vitelli non hanno potuto prevenire l’infezione da Cryptosporidium ma la gravità dei sintomi della diarrea diminuiva con l’aumento della concentrazione di IgG totali. In questo studio, l’approvato principio attivo alofuginone per la profilassi contro la cryptosporidiosi non ha mostrato nessun effetto sulla secrezione di oocisti di Cryptosporidium.

Topics: Animals; Cattle; Cattle Diseases; Coccidiostats; Cryptosporidiosis; Enzyme-Linked Immunosorbent Assay; Housing, Animal; Immunoglobulin G; Piperidines; Prevalence; Quinazolinones; Risk Factors; Switzerland; Time Factors

Radiotherapy is used to treat many different human tumors. Paradoxically, radiation can activate TGF-β1 signaling and induce the epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor progression. This study investigated the inhibitory effects of halofuginone, a plant-derived alkaloid that has been shown to inhibit TGF-β1 signaling, on radiation-induced EMT and explored the underlying mechanisms using a Lewis lung carcinoma (LLC) xenograft model. The cells and animals were divided into five treatment groups: Normal Control (NC), Halofuginone alone (HF), Radiotherapy alone (RT), Radiotherapy combined with Halofuginone (RT+HF), and Radiotherapy combined with the TGF-β1 inhibitor SB431542 (RT+SB). Radiation induced EMT in lung cancer cells and xenografts, as evidenced by increased expression of the mesenchymal markers N-cadherin and Vimentin, and reduced expression of the epithelial markers E-cadherin and Cytokeratin. Further, radiotherapy treatment increased the migration and invasion of LLC cells. Halofuginone reversed the EMT induced by radiotherapy in vitro and in vivo, and inhibited the migration and invasion of LLC cells. In addition, TGF-β1/Smad signaling was activated by radiotherapy and the mRNA expression of Twist and Snail was elevated; this effect was reversed by halofuginone or the TGF-β1 inhibitor SB431542. Our results demonstrate that halofuginone inhibits radiation-induced EMT, and suggest that suppression of TGF-β1 signaling may be responsible for this effect.

Topics: Animals; Antineoplastic Agents; Benzamides; Carcinoma, Lewis Lung; Cell Movement; Dioxoles; Epithelial-Mesenchymal Transition; Female; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Piperidines; Quinazolinones; Smad Proteins; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

Topics: Amino Acyl-tRNA Synthetases; Antimalarials; Catalytic Domain; Crystallography; Humans; Models, Molecular; Multiprotein Complexes; Piperidines; Plasmodium falciparum; Protozoan Proteins; Quinazolinones; Structure-Activity Relationship; Toxoplasma

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials.

Topics: Amino Acyl-tRNA Synthetases; Animals; Antimalarials; Computer-Aided Design; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Design; Drug Resistance; Enzyme Inhibitors; Erythrocytes; Liver; Malaria, Falciparum; Mice; Models, Molecular; Molecular Structure; Molecular Targeted Therapy; Piperidines; Plasmodium falciparum; Protozoan Proteins; Quinazolines; Quinazolinones; Structure-Activity Relationship; Time Factors

Duchenne Muscular Dystrophy is characterized by: near absence of dystrophin in skeletal muscles; low percentage of revertant myofibers; up-regulation of utrophin synthesis; and a high degree of muscle fibrosis. In patient quadriceps femoris biopsies (n = 6, ages between 3-9 years) an inverse correlation was observed between the levels of collagen type I - representing fibrosis - and the levels of utrophin. This correlation was independent of the patient's age and was observed in the entire muscle biopsy sections. In the mdx mice diaphragm (n = 6/group), inhibition of fibrosis by halofuginone resulted in increases in the levels of utrophin. The utrophin/fibrosis relationships were not limited to collagen type I, but also applied to other constituents of the fibrosis machinery. The inverse correlation was found also in old mdx mice with established fibrosis. In addition, inhibition of collagen type I levels was associated with increases in the numbers of revertant myofibers, both as single myofibers and in clusters in the diaphragm and the gastrocnemius. In summary, our results demonstrate an inverse correlation between the level of muscle fibrosis and the level of utrophin and that of the number of revertant myofibers. These findings may reveal common links between the fibrotic and utrophin-synthesis pathways and offer new insights into the regulation of utrophin synthesis.

Topics: Animals; Biopsy; Child; Child, Preschool; Collagen; Collagen Type I; Gene Expression Regulation; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Piperidines; Quadriceps Muscle; Quinazolinones; Utrophin

Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because we recently demonstrated that TGF-β/Smad cascade plays a crucial role in osteosarcoma metastatic progression, we investigated the effect of halofuginone, identified as an inhibitor of the TGF-β/Smad3 cascade, on osteosarcoma progression. A preclinical model of osteosarcoma was used to evaluate the impact of halofuginone on tumor growth, tumor microenvironment and metastasis development. In vivo experiments showed that halofuginone reduces primary tumor growth and lung metastases development. In vitro experiments demonstrated that halofuginone decreases cell viability mainly by its ability to induce caspase-3 dependent cell apoptosis. Moreover, halofuginone inhibits the TGF-β/Smad3 cascade and the response of TGF-β key targets involved in the metastases dissemination process such as MMP-2. In addition, halofuginone treatment affects the "vicious cycle" established between tumor and bone cells, and therefore the tumor-associated bone osteolysis. Together, these results demonstrate that halofuginone decreased primary osteosarcoma development and associated lung metastases by targeting both the tumor cells and the tumor microenvironment. Using halofuginone may be a promising therapeutic strategy against tumor progression of osteosarcoma specifically against lung metastases dissemination.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Bone Remodeling; Cell Line, Tumor; Cell Proliferation; Disease Progression; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Osteosarcoma; Piperidines; Quinazolinones; Signal Transduction; Transfection

Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-β (TGF-β) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model.. NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 μg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells.. HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-β-signaling.. Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.

Topics: Animals; Disease Models, Animal; Humans; Immunophenotyping; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Neovascularization, Pathologic; Phosphorylation; Piperidines; Quinazolinones; Smad2 Protein; Tumor Cells, Cultured

The Akt/mTORC1 pathway plays a central role in the activation of Warburg effect in cancer. Here, we present for the first time that halofuginone (HF) treatment inhibits colorectal cancer (CRC) growth both in vitro and in vivo through regulation of Akt/mTORC1 signaling pathway. Halofuginone treatment of human CRC cells inhibited cell proliferation, induced the generation of reactive oxygen species and apoptosis. As expected, reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase in pentose phosphate pathway upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated Akt/mTORC1 signaling pathway. Moreover, metabolomics delineated the slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells upon HF treatment, to support our notion that HF regulates Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells, showing the anticancer activity of HF through metabolic regulation of Akt/mTORC1 in CRC.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Female; Glucose; Glucose Transporter Type 1; Glycolysis; HCT116 Cells; Hexokinase; Humans; In Situ Nick-End Labeling; Lipids; Mechanistic Target of Rapamycin Complex 1; Medicine, Chinese Traditional; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Pentose Phosphate Pathway; Piperidines; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolinones; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases

Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-β production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity.

Topics: Amino Acids; Animals; Apoptosis; Autoimmunity; Cells, Cultured; Cytokines; Disease Models, Animal; Gene Expression Regulation; Immune Tolerance; Indoleamine-Pyrrole 2,3,-Dioxygenase; Inflammation; Lupus Erythematosus, Systemic; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Piperidines; Protein Serine-Threonine Kinases; Quinazolinones; Signal Transduction

To investigate the effects of Th1/Th17 cell imbalance on the pathogenesis of acute graft-versus-host disease (GVHD) in mice.. In a murine GVHD model of C57BL/6 (H-2(b)), a low dose of halofuginone (HF) was applied for treating the recipients in order to result in Th1/Th17 imbalance. Rechipient mice were divided into GVHD group (without HF intervention) and GVHD plus HF group (treated by HF). The recipients were monitored for survival rate, clinical scores of acute GVHD, contents of circulatory Th1 and Th17 cells, Th1/Th17 ratio and serum level of IFN-γ and IL-17A. Expression levels of IFN-γ and IL-17A in target organs were analyzed by using real-time PCR, and the target organs were delivered for histological examinations.. Recipients treated with HF showed that all the mortality, circulatory Th1/Th17 ratio and clinical score were higher than those in the mice without HF intervention (P < 0.05). Circulatory Th1/Th17 ratio positively correlates with clinical score (P < 0.001). HF administration reduces the expression level of intestinal IL-17A and increases intrahepatic and intestinal IFN-γ level (P < 0.05), HF treatment aggravates GVHD in liver and small intestine with augmented hepatic and intestinal inflammation.. Th1/Th17 imbalance contributes to the pathogenesis of acute GVHD.

Topics: Animals; Disease Models, Animal; Graft vs Host Disease; Interferon-gamma; Interleukin-17; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Piperidines; Quinazolinones; Th1 Cells; Th17 Cells

Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe's equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity.

Topics: Animals; Blotting, Western; Cell Death; Cell Proliferation; Cells, Cultured; Cyclosporine; Drug Synergism; Epithelial Cells; Flow Cytometry; Humans; Kidney Tubules; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Piperidines; Proline; Quinazolinones; Receptors, Antigen, T-Cell; Sirolimus; T-Lymphocytes

Primary liver cancer is a common cancer and the mortality of liver cancer ranks the second of all malignancy-related deaths in China. The most common primary liver cancer is hepatocellular carcinoma, accounting for approximately 90% of the total. Because liver is the largest parenchymatous organ in the body undertaking all kinds of important metabolic functions, liver cancer inevitably causes greater harms and its treatment is extremely difficult. Currently, there are still no effective drugs for the treatment of patients with advanced inoperable liver cancer. We observed the strong inhibitory activity of halofuginone on HepG2 cell growth and the cell cycle and apoptosis assays showed that halofuginone arrested the cell cycle and inhibited the induction. And we found that halofuginone inhibits tumor cell cycle possibly by up-regulating p15 and p21 of expression. Then, we found that the proportion of cleaved PARP, caspase-3, 8 and 9 in HepG2 cells increased after halofuginone treatment. And the results showed that halofuginone down-regulated Mcl-1 and c-IAP1 expression. Finally, our results showed halofuginone regulated the activities of JNK and MEK/ERK signaling pathways in hepatocellular carcinoma cells. In summary, this study shows that halofuginone can inhibit the in vitro growth, arrest the cell cycle and induce the apoptosis of HepG2 cells. Its mechanisms of action may be related to the regulation of associated protein expression, up-regulation of JNK, and inhibition of MEK/ERK signaling pathway.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Invasiveness; Piperidines; Quinazolinones; Signal Transduction

Esophageal endoscopic submucosal dissection (ESD) is an effective minimally invasive therapy for early esophageal cancer and high-grade Barrett dysplasia. However, esophageal stricture formation after circumferential or large ESD has limited its wide adoption. Mitomycin C (MMC), halofuginone (Hal), and transforming growth factor β3 (TGF-β3) exhibits antiscarring effects that may prevent post-ESD stricture formation.. Using endoscopic mucosectomy (EEM) technique, an 8- to 10-cm-long circumferential esophageal mucosal segment was excised in a porcine model. The site was either untreated (control, n = 6) or received 40 evenly distributed injections of antiscarring agent immediately and at weeks 1 and 2. High and low doses were used: MMC 5 mg (n = 2), 0.5 mg (n = 2); Hal 5 mg (n = 2), 1.5 mg (n = 2), 0.5 mg (n = 2); TGF-β3 2 μg (n = 2), 0.5 μg (n = 2). The degree of stricture formation was determined by the percentage reduction of the esophageal lumen on weekly fluoroscopic examination. Animals were euthanized when strictures exceeded 80 % or the animals were unable to maintain weight.. The control group had a luminal diameter reduction of 78.2 ± 10.9 % by 2 weeks and were euthanized by week 3. Compared at 2 weeks, the Hal group showed a decrease in mean stricture formation (68.4 % low dose, 57.7 % high dose), while both TGF-β3 dosage groups showed no significant change (65.3 % low dose, 76.2 % high dose). MMC was most effective in stricture prevention (53.6 % low dose, 35 % high dose). Of concern, the esophageal wall treated with high-dose MMC appeared to be necrotic and eventually led to perforation. In contrast, low dose MMC, TGF-β3 and Hal treated areas appeared re-epithelialized and healthy.. Preliminary data on MMC and Hal demonstrated promise in reducing esophageal stricture formation after EEM. More animal data are needed to perform adequate statistical analysis in order to determine overall efficacy of antiscarring therapy.

Topics: Angiogenesis Inhibitors; Animals; Cicatrix; Disease Models, Animal; Dissection; Drug Therapy, Combination; Esophageal Diseases; Esophageal Stenosis; Esophagoscopy; Follow-Up Studies; Injections; Intestinal Mucosa; Mitomycin; Nucleic Acid Synthesis Inhibitors; Piperidines; Quinazolinones; Swine; Transforming Growth Factor beta3; Wound Healing

Halofuginone (HF) is extracted from Dichroa febrifuga, a plant used in traditional medicine. We report that the HF extract inhibits the growth of breast cancer cells and induces the generation of reactive oxygen species (ROS) and apoptosis, an important feature of potential anticancer agents. In addition, HF significantly reduces the migration and invasion of MCF-7 and MDA-MB-231 human breast cancer cells after 12-O-tetraecanoylphorbol-13-acetate (TPA) stimulation. As matrix metalloproteinase-9 plays a critical role in tumor metastasis, we analyzed its expression with the HF extract treatment. Western blot analysis and gelatin zymography showed that HF suppresses MMP-9 expression and activity concentration-dependently. HF also decreases the nuclear protein levels of nuclear factor kappa B (NF-κB) and c-fos (AP-1), critical transcription factors regulating MMP-9 expression through binding the MMP-9 promoter region. Luciferase assays showed that HF decreases TPA-induced MMP-9 promoter binding activities of NF-κB and AP-1. Taken together, these are the first results indicating that halofuginone may represent a promising new agent for breast cancer chemotherapy.

Topics: Apoptosis; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; MCF-7 Cells; NF-kappa B; Piperidines; Proto-Oncogene Proteins c-akt; Quinazolinones; Transcription Factor AP-1

The aim of the present study was to investigate the therapeutic effect of halofuginone (HF) in the treatment of idiopathic thrombocytopenic purpura (ITP) and explore the underlying mechanism. Sixty ITP mice were divided into four groups including control group, low dose group (25 mg/kg HF), medium dose group (50 mg/kg HF), and high dose group (100 mg/kg HF). Corresponding dose of HF was administrated by gavage daily in HF groups for 7 days, and the same volume of saline was given in control group. Platelet counts were 28.87 ± 3.91 × 10(9)/L, 57.13 ± 2.75 × 10(9)/L, 86.73 ± 3.06 × 10(9)/L and 89.73 ± 2.84 × 10(9)/L in control group, low dose group, medium dose group, and high dose group respectively, on day 7 after intragastrically administration of HF or saline. Compared with control group, three HF groups showed significantly increased levels of INF-γ and IL-2 (all P < 0.05), and significantly decreased concentrations of IL-4 and IL-10(all P < 0.05). The expression of T-bet mRNA increased and the expression of GATA-3 mRNA decreased (all P < 0.05) in ITP mice after intragastric administration with different dose of HF. HF significantly recovered peripheral platelet counts in ITP mice through promoting Th1 cell differentiation and attenuating Th2 differentiation in ITP mice.

Topics: Animals; CD3 Complex; Cell Differentiation; Cytokines; Female; GATA3 Transcription Factor; Male; Mice; Mice, Inbred BALB C; Piperidines; Platelet Count; Purpura, Thrombocytopenic, Idiopathic; Quinazolinones; RNA, Messenger; T-Box Domain Proteins; Th1 Cells; Th2 Cells

Anti-inflammation strategy is one of the proposed therapeutic approaches to hepatic fibrosis. T helper (Th) 17 cells, which play a detrimental role in experimental murine models of inflammatory diseases, have been demonstrated to participate in the pathogenesis of liver damage. The inhibitory effect of halofuginone (HF), an active component of extracts derived from the plant alkaloid febrifugine, on collagen synthesis has been shown in animal models of the fibrotic disease. The aim of this study was to clarify the in vivo effect of HF on Th17 cells in concanavalin A-induced fibrosis rats. Haematoxylin-eosin (HE) staining and Masson staining were performed to observe collagen deposition. The presence of INF-gamma, TNF-alpha, IL-6, IL-17, IL-1beta, IL-33 and IL-10 in serum and the presence of ROR-γt, IL-17, TGF-β1 and α-SMA in liver tissue were detected. Flow cytometry was performed to analyse the percentage of Th17 cells. We observed significantly lower levels of INF-gamma, TNF-alpha, IL-6, IL-17, IL-1beta, TGF-β1 and α-SMA in HF-treated group of rats, and the percentage of Th17 cells in splenic lymphocyte was decreased well. Histological examination demonstrated that HF significantly reduced the severity of liver fibrosis in HF-treated rats. We concluded that HF (10 mg/kg) exerts an antifibrotic impact on Th17 cells and its relative cytokines in rats with ConA-induced fibrosis.

Topics: Actins; Alanine Transaminase; Albumins; Animals; Aspartate Aminotransferases; Cell Differentiation; Concanavalin A; Disease Models, Animal; Interferon-gamma; Interleukin-10; Interleukin-17; Interleukin-1beta; Interleukin-33; Interleukin-6; Interleukins; Liver; Liver Cirrhosis; Male; Nuclear Receptor Subfamily 1, Group F, Member 3; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Wistar; Th17 Cells; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The IL-23 pathway is genetically linked to autoimmune disease in humans and is required for pathogenic Th17 cell function in mice. However, because IL-23R-expressing mature Th17 cells are rare and poorly defined in mice at steady-state, little is known about IL-23 signaling. In this study, we show that the endogenous CCR6(+) memory T cell compartment present in peripheral lymphoid organs of unmanipulated mice expresses Il23r ex vivo, displays marked proinflammatory responses to IL-23 stimulation in vitro, and is capable of transferring experimental autoimmune encephalomyelitis. The prolyl-tRNA synthetase inhibitor halofuginone blocks IL-23-induced Stat3 phosphorylation and IL-23-dependent proinflammatory cytokine expression in endogenous CCR6(+) Th17 cells via activation of the amino acid starvation response (AAR) pathway. In vivo, halofuginone shows therapeutic efficacy in experimental autoimmune encephalomyelitis, reducing both established disease progression and local Th17 cell effector function within the CNS. Mechanistically, AAR activation impairs Stat3 responses downstream of multiple cytokine receptors via selective, posttranscriptional suppression of Stat3 protein levels. Thus, our study reveals latent pathogenic functions of endogenous Th17 cells that are regulated by both IL-23 and AAR pathways and identifies a novel regulatory pathway targeting Stat3 that may underlie selective immune regulation by the AAR.

Topics: Amino Acids; Animals; Encephalomyelitis, Autoimmune, Experimental; Inflammation; Interleukin-23; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Knockout; Phosphorylation; Piperidines; Quinazolinones; Receptors, CCR6; Receptors, Interleukin; STAT3 Transcription Factor; Th17 Cells

Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that halofuginone would reduce apoptosis--the presumed cause of satellite-cell depletion during muscle degradation-in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when halofuginone was added to cultured myofibers. No apparent effect of halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which halofuginone improves muscle pathology and function in muscular dystrophies.

Topics: Actins; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Survival; Diaphragm; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Humans; Macrophages; Mice; Mice, Inbred mdx; Mixed Function Oxygenases; Muscular Dystrophy, Duchenne; Myoblasts; Myofibrils; PAX7 Transcription Factor; Phosphatidylinositol 3-Kinases; Piperidines; Primary Cell Culture; Quinazolinones; Signal Transduction

The small molecule halofuginone has been shown to inhibit fibrosis, angiogenesis, and tumor progression. This study was undertaken to evaluate the effects of halofuginone in preventing autoimmune arthritis in mice.. The effects of halofuginone on joint diseases were assessed by clinical scoring and histologic analysis. Protein expression levels were confirmed by immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry, and/or Western blotting. The expression levels of messenger RNA (mRNA) for various molecules were determined by real-time polymerase chain reaction (PCR). Proliferation of osteoclast precursors was assessed by bromodeoxyuridine uptake. Osteoclast differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and area of resorbed bone.. Treatment with halofuginone suppressed the development of autoimmune arthritis and reciprocally regulated Th17 cells and FoxP3+ Treg cells. These effects of halofuginone on Th17 differentiation involved increased signaling of ERK and reduction of STAT-3 and NF-ATc1 expression. Furthermore, halofuginone induced the expression of indoleamine 2,3-dioxygenase (IDO) in dendritic cells, leading to reduced production of Th17 cells. In addition, halofuginone prevented the formation and activity of osteoclasts through suppression of transcription factors, such as activator protein 1 and NF-ATc1, and inhibited cell cycle arrest by the committed osteoclast precursors via expression of Ccnd1 encoding cyclin D1.. Taken together, our results suggest that halofuginone is a promising therapeutic agent for the treatment of Th17 cell-mediated inflammatory diseases and bone diseases.

Topics: Animals; Arthritis; Autoimmune Diseases; Cell Differentiation; Disease Models, Animal; Disease Progression; Forkhead Transcription Factors; Male; Mice; Mice, Inbred DBA; Mice, Knockout; NFATC Transcription Factors; Osteoclasts; Osteoprotegerin; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Th17 Cells

Th1/Th17 imbalance had been indicated to mediate several kinds of inflammatory diseases. We deduce that Th1/Th17 imbalance might also contribute to the pathogenesis of acute graft-versus-host disease (GVHD). This study is to investigate the relation between Th1/Th17 imbalance and acute GVHD.. We applied a murine GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient by treating the recipients with low dose of halofuginone (HF), which is competent in selectively inhibiting Th17 differentiation and facilitating Th1 differentiation. Recipient mice were monitored for survival rate, body weight change, clinical symptoms and pathological evidence of acute GVHD. We also measured the proportions of Th1 and Th17 cells in circulation and expression levels of IFN-γ and IL-17A in tissues involved in GVHD.. Firstly, we confirm the existence of Th1/Th17 imbalance in acute GVHD and Th1/Th17 imbalance positively correlates with severity of acute GVHD. Secondly, low dose of HF augments Th1/Th17 imbalance by driving the Th1/Th17 balance to a Th1-dominant reaction. Finally, augmented Th1/Th17 imbalance leads to aggravated systemic GVHD. An increased Th1-type reaction results in aggravated hepatic and intestinal GVHD, and inhibiting Th17 differentiation is sufficient to alleviate pulmonic impairment.. Our study is indicative for a critical role of Th1/Th17 imbalance in the pathogenesis of murine GVHD.

Topics: Acute Disease; Animals; Disease Progression; Graft vs Host Disease; Interferon-gamma; Interleukin-17; Lymphocyte Count; Mice, Inbred BALB C; Mice, Inbred C57BL; Organ Specificity; Piperidines; Quinazolinones; Th1 Cells; Th17 Cells

Three major processes, constrictive vessel remodeling, intimal hyperplasia (IH), and retarded re-endothelialization, contribute to restenosis after vascular reconstructions. Clinically used drugs inhibit IH but delay re-endothelialization and also cause constrictive remodeling. Here we have examined halofuginone, an herbal derivative, for its beneficial effects on vessel remodeling and differential inhibition of IH versus re-endothelialization.. Two weeks after perivascular application to balloon-injured rat common carotid arteries, halofuginone versus vehicle (n=6 animals) enlarged luminal area 2.14-fold by increasing vessel size (adaptive remodeling; 123%), reducing IH (74.3%) without inhibiting re-endothelialization. Consistent with its positive effect on vessel expansion, halofuginone reduced collagen type 1 (but not type 3) production in injured arteries as well as that from adventitial fibroblasts in vitro. In support of its differential effects on IH versus re-endothelialization, halofuginone produced greater inhibition of vascular smooth muscle cell versus endothelial cell proliferation at concentrations ≈50 nmol/L. Furthermore, halofuginone at 50 nmol/L effectively blocked Smad3 phosphorylation in smooth muscle cells, which is known to promote smooth muscle cell proliferation, migration, and IH, but halofuginone had no effect on phospho-Smad3 in endothelial cells.. Periadventitial delivery of halofuginone dramatically increased lumen patency via adaptive remodeling and selective inhibition of IH without affecting endothelium recovery. Halofuginone is the first reported small molecule that has favorable effects on all 3 major processes involved in restenosis.

Topics: Adaptation, Biological; Angiogenesis Inhibitors; Angioplasty, Balloon; Animals; Carotid Arteries; Carotid Artery Injuries; Cell Proliferation; Cells, Cultured; Collagen Type I; Endothelium, Vascular; Fibroblasts; Humans; Hyperplasia; Male; Models, Animal; Myocytes, Smooth Muscle; Organ Specificity; Piperidines; Postoperative Complications; Quinazolinones; Rats; Rats, Sprague-Dawley; Smad3 Protein; Vascular Remodeling

Drug resistance remains a major public health challenge for malaria treatment and eradication. Individual loci associated with drug resistance to many antimalarials have been identified, but their epistasis with other resistance mechanisms has not yet been elucidated.. We previously described two mutations in the cytoplasmic prolyl-tRNA synthetase (cPRS) gene that confer resistance to halofuginone. We describe here the evolutionary trajectory of halofuginone resistance of two independent drug resistance selections in Plasmodium falciparum. Using this novel methodology, we discover an unexpected non-genetic drug resistance mechanism that P. falciparum utilizes before genetic modification of the cPRS. P. falciparum first upregulates its proline amino acid homeostasis in response to halofuginone pressure. We show that this non-genetic adaptation to halofuginone is not likely mediated by differential RNA expression and precedes mutation or amplification of the cPRS gene. By tracking the evolution of the two drug resistance selections with whole genome sequencing, we further demonstrate that the cPRS locus accounts for the majority of genetic adaptation to halofuginone in P. falciparum. We further validate that copy-number variations at the cPRS locus also contribute to halofuginone resistance.. We provide a three-step model for multi-locus evolution of halofuginone drug resistance in P. falciparum. Informed by genomic approaches, our results provide the first comprehensive view of the evolutionary trajectory malaria parasites take to achieve drug resistance. Our understanding of the multiple genetic and non-genetic mechanisms of drug resistance informs how we will design and pair future anti-malarials for clinical use.

Topics: Biological Evolution; Drug Resistance; Genomics; Humans; Malaria, Falciparum; Mutation; Piperidines; Plasmodium falciparum; Protozoan Proteins; Quinazolinones; Sequence Analysis, DNA

Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiation and cytokine production in activated T cells. The present study was designed to examine the biological effects of HF against T cell receptor and interleukin (IL)-2 stimulated T cell proliferation. T cell proliferation in cultured murine splenocytes was determined by methylthiazol tetrazolium assay. Cell apoptosis was mainly determined by fluorescence-activated cell sorting with Annexin-V and 7-aminoactinomycin D staining. Here, we showed that HF significantly suppressed T cell proliferation in naïve splenocyte cultures in response to alloantigen or anti-CD3 antibody (IC₅₀, 2-2.5 nM; P<0.0001), or in activated T cell cultures in response to IL-2 (IC₅₀, 16 nM; P<0.0001) in a dose-dependent manner. HF did neither attenuate IL-2 production in anti-CD3 antibody activated T cells nor disrupt STAT5 signaling in IL-2-stimulated T cells, but its anti-T cell proliferation was correlated with an increase in cell apoptosis and a decrease in proline uptake in culture medium. Further experiments showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation and cell apoptosis. In conclusion, these data suggest that HF interferes with proline incorporation or uptake, resulting in apoptosis via amino acid starvation response in T cells in the response to antigen/mitogen or IL-2 stimulation.

Topics: Animals; Antiprotozoal Agents; Apoptosis; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Culture Media; Dose-Response Relationship, Drug; Flow Cytometry; In Situ Nick-End Labeling; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Piperidines; Proline; Quinazolinones; Receptors, Antigen, T-Cell; Spleen; T-Lymphocytes

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.

Topics: Acetonitriles; Angiogenesis Inhibitors; Binding Sites; Cell Differentiation; Computer Simulation; Curcumin; Cyclohexenes; Endothelial Cells; Endothelium, Vascular; ErbB Receptors; Fibroblast Growth Factor 2; Neovascularization, Physiologic; Piperidines; Quinazolinones; Vascular Endothelial Growth Factor Receptor-1

Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its effects by acting as a high-affinity inhibitor of the enzyme glutamyl-prolyl-tRNA synthetase (EPRS). In order to elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyl-tRNA synthetase domain of human EPRS (hPRS) at 2.4 Å resolution (hPRS-apo), of hPRS complexed with ATP and the substrate proline at 2.3 Å resolution (hPRS-sub) and of hPRS complexed with HF at 2.62 Å resolution (hPRS-HF) are presented. These structures show plainly that motif 1 functions as a cap in hPRS, which is loosely opened in hPRS-apo, tightly closed in hPRS-sub and incorrectly closed in hPRS-HF. In addition, the structural analyses are consistent with more effective binding of hPRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development of more potent and selective hPRS inhibitors for promoting inflammatory resolution.

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Catalytic Domain; Crystallography, X-Ray; Glutamate-tRNA Ligase; Humans; Mutagenesis; Piperidines; Proline; Protein Binding; Protein Conformation; Protein Synthesis Inhibitors; Quinazolinones; Substrate Specificity

Halofuginone (HF) is an active component of extracts derived from the plant alkaloid febrifugine and has shown therapeutic promise in animal models of fibrotic disease. Our main objectives were to clarify the suppressive effect of HF on concanavalin A (ConA)-induced liver fibrosis. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while orally administration of HF significantly decreased the levels of the transaminases. In addition, the levels of hyaluronic acid (HA), procollagen III (PCIII) and TGF-β1 in the serum and collagen I, α-SMA, tissue inhibitors of metalloproteinase 2 (TIMP2) and Smad3 in the liver tissue were significantly lowered with the treatment of HF. Histological examination also demonstrated that HF significantly reduced the severity of liver fibrosis. Since ConA-induced liver fibrosis is caused by the repeated activation of T cells, immunomodulatory substances might be responsible for the suppressive effect of HF. We found that the production of nuclear factor (NF)-kB in the serum was increased in ConA-treated group, while decreased significantly with the treatment of HF. The changes of inflammatory cytokines tumor necrosis factor (TNF-α), IL-6 and IL-1β in the serum followed the same rhythm. All together, our findings indicate that orally administration HF (10ppm) would attenuate the liver fibrosis by suppressing the synthesis of collagen I and inflammation-mediated liver injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Concanavalin A; Drugs, Chinese Herbal; Interleukin-1beta; Interleukin-6; Liver Cirrhosis; Male; Piperidines; Quinazolinones; Rats, Wistar; Transforming Growth Factor beta1

The objective of the present study was to evaluate the protective effects of halofuginone against renal ischemia/reperfusion (I/R) injury.. Male Wistar albino rats were unilaterally nephrectomized and the left renal pedicles were occluded for 45 min to induce ischemia and then reperfused for 6 h (early) or for 72 h (late). The rats were treated intraperitoneally with either halofuginone (100 μg/kg/day) or saline 30 min prior to ischemia and the dose was repeated in the late reperfusion groups. In the sham groups, rats underwent unilateral nephrectomy and were treated at similar time points. The animals were decapitated at either 6 h or 72 h of reperfusion and trunk blood and kidney samples were obtained.. I/R injury increased renal malondialdehyde levels, myeloperoxidase activity and reactive oxygen radical levels, and decreased the renal glutathione content. Halofuginone treatment was found to reduce oxidative I/R injury and improve renal function in the rat kidney, as evidenced by reduced generation of reactive oxygen species, depressed lipid peroxidation and myeloperoxidase activity, and increased glutathione levels.. The present findings demonstrate the anti-inflammatory and antioxidant effects of halofuginone in renal I/R injury, supporting its potential use where renal I/R injury is inevitable.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Fibrosis; Glutathione; Kidney Diseases; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Nephrectomy; Oxidative Stress; Peroxidase; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury

Febrifugine is the active component of the Chinese herb Chang Shan (Dichroa febrifuga Lour.), which has been used for treating malaria-induced fever for about 2,000 years. Halofuginone (HF), the halogenated derivative of febrifugine, has been tested in clinical trials for potential therapeutic applications in cancer and fibrotic disease. Recently, HF was reported to inhibit T(H)17 cell differentiation by activating the amino acid response pathway, through inhibiting human prolyl-transfer RNA synthetase (ProRS) to cause intracellular accumulation of uncharged tRNA. Curiously, inhibition requires the presence of unhydrolysed ATP. Here we report an unusual 2.0 Å structure showing that ATP directly locks onto and orients two parts of HF onto human ProRS, so that one part of HF mimics bound proline and the other mimics the 3' end of bound tRNA. Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS. Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment. Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Antimalarials; Binding Sites; Crystallography, X-Ray; Herbal Medicine; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Ligands; Medicine, Chinese Traditional; Models, Molecular; Piperidines; Proline; Quinazolines; Quinazolinones; RNA, Transfer

Absence of, or loss-of-function mutations in the dysferlin gene (dysf) result in dysferlinopathy, characterized by increased muscle inflammation, collagen deposition and deterioration in muscle function. We evaluated halofuginone efficacy in improving muscle histopathology in mice with deleted dysf transmembrane domain. Quadriceps sublumbar and longissimus muscles of 9-month-old dysf-/- mice treated with halofuginone for 4 months exhibited a reduction in centrally-nucleated myofibers, inflammatory infiltrates and collagen content. Late onset of dysferlinopathy makes it ideal for evaluating the efficacy of early treatments on late outcome. The dysf-/- mice were treated with halofuginone for 3 to 4 months starting at 1, 5 or 9 months of age, and quadricep muscle histopathology was evaluated at 12 months. Collagen content and number of centrally nucleated myofibers decreased after early halofuginone treatment, administered when myofibers with central nuclei and inflammatory infiltrates are evident, but there was almost no fibrosis. When administered at the beginning of fibrosis it resulted in a further decrease in the number of centrally-nucleated myofibers with no additional decrease in collagen levels. Cardiac fibrosis was almost completely abolished following early halofuginone treatment. Halofuginone inhibited Smad3 phosphorylation and its translocation to the nucleus and increased the activity of matrix metalloproteinases 9 and 2 responsible for resolution of pre-existing collagen. Macrophage and myofibroblast invasion into the dystrophic muscle at the site of myofibers with central nuclei was inhibited by halofuginone. These results suggest that early halofuginone treatment can prevent the late outcome of dysferlinopathy and can cause resolution of the established fibrosis when administered at later stages.

Topics: Animals; Collagen; Disease Models, Animal; Dysferlin; Fibrosis; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Phosphorylation; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Smad3 Protein; Treatment Outcome

The lung is one of the most sensitive organs to ionizing radiation, and damage to normal lung tissue remains a major dose limiting factor for patients receiving radiation to the thorax. Radiation induced lung injury (RILI) which is also named as "radiation pneumonpathy" is a continuous process and regarded as the result of an abnormal healing response. It has been shown that transforming growth factor β-1 (TGF-β1) plays an integral role in the radiation induced lung fibrosis formation by promoting the chemoattraction of fibroblasts and their conversion to myofibroblasts. Halofuginone is a, low molecular weight plant derived alkaloid, isolated from the Dichroa febrifuga plant that exhibits antifibrotic activity and inhibition of type I collagen synthesis. Halofuginone has been shown to protect against radiation induced soft tissue fibrosis by virtue of inhibiting various members of TFG-β signaling pathway. By the light of these findings, we hypothesize that Halofuginone may be able to ameliorate the radiation induced lung fibrosis.

Topics: Animals; Fibrosis; Humans; Lung Injury; Piperidines; Quinazolinones; Radiation Pneumonitis; Radiation-Protective Agents; Transforming Growth Factor beta

Topics: Amino Acids; Anti-Inflammatory Agents; Cell Survival; Humans; Molecular Targeted Therapy; Piperidines; Quinazolinones; Second Messenger Systems; Signal Transduction

Dietary restriction, or reduced food intake without malnutrition, increases life span, health span, and acute stress resistance in model organisms from yeast to nonhuman primates. Although dietary restriction is beneficial for human health, this treatment is not widely used in the clinic. Here, we show that short-term, ad libitum feeding of diets lacking essential nutrients increased resistance to surgical stress in a mouse model of ischemia reperfusion injury. Dietary preconditioning by 6 to 14 days of total protein deprivation, or removal of the single essential amino acid tryptophan, protected against renal and hepatic ischemic injury, resulting in reduced inflammation and preserved organ function. Pharmacological treatment with halofuginone, which activated the amino acid starvation response within 3 days by mimicking proline deprivation, was also beneficial. Both dietary and pharmacological interventions required the amino acid sensor and eIF2α (eukaryotic translation initiation factor 2α) kinase Gcn2 (general control nonderepressible 2), implicating the amino acid starvation response and translational control in stress protection. Thus, short-term dietary or pharmacological interventions that modulate amino acid sensing can confer stress resistance in models of surgical ischemia reperfusion injury.

Topics: Animals; Dietary Proteins; Humans; Kidney; Liver; Mice; Mice, Inbred C57BL; Organ Specificity; Piperidines; Proline; Protein Deficiency; Protein Serine-Threonine Kinases; Quinazolinones; Reperfusion Injury; Stress, Physiological; Tryptophan

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

Topics: Amino Acyl-tRNA Synthetases; Animals; Cell Differentiation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Mice; Mice, Inbred C57BL; Piperidines; Quinazolines; Quinazolinones; Structure-Activity Relationship; Th17 Cells

Burn scar formations can cause disfiguration and loss of dermal function. The purpose of this study was to examine whether application of modified silicone gel sheets with an antifibrotic drug halofuginone-eluting hybrid surface produce an effect on scar development. There were a total of 2 animal groups. The athymic nude mice (nu/nu) of both groups underwent transplantation of full-thickness human skin grafts onto their backs and setting of partial thickness burn injury. The status of local scar development was observed over a period of 3 months after the application of silicone gel sheets and also after application of surface-modified halofuginone-eluting silicone gel sheets. Subsequently, via real-time polymerase chain reaction, the cDNA levels from key mediators of scar formation (transforming growth factor beta, COL1A1, connective tissue growth factor, fibroblast growth factor 2, matrix metalloproteinase 2, matrix metalloproteinase 9) were established and statistically evaluated. In comparison with uncoated silicone gel sheets, the application of halofuginone-eluting silicone gel sheets lead to a significant difference in gene expression activity in scar tissue. Halofuginone-eluting hybrid surface silicone gel sheets significantly increase the antiscarring effect of adhesive silicone gel sheets by deceleration and downregulation of scar development by normalization of the expression activity.

Topics: Animals; Burns; Cicatrix, Hypertrophic; Coated Materials, Biocompatible; Humans; Mice; Mice, Nude; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Silicone Gels; Skin Transplantation

Due to its ability to disrupt transforming growth factor beta (TGF-β) signaling, halofuginone has been successfully used to treat various fibrotic disorders. Here we investigated the antifibrotic potential of halofuginone in human corneal fibroblasts.. Human corneal fibroblasts were isolated from human donor corneas for in vitro experiments. TGF-β was used to stimulate pro-fibrotic responses from corneal fibroblasts under halofuginone treatment. The expression of alpha smooth muscle actin (α-SMA) and fibronectin was analyzed by western blots. Phalloidin toxin was used to stain cultures for stress fiber assemblies. Quantitative reverse transcription PCR (qRT-PCR) and immunostaining were used to analyze the expression of type I collagen mRNA and protein, respectively. The expression of Smad2, Smad3, phospho-Smad2, and phospho-Smad3 was determined by western blots.. Halofuginone was well tolerated by human corneal fibroblasts up to 10 ng/ml as demonstrated by a cell viability assay. At this concentration, TGF-β-induced expression of the fibrotic markers α-SMA, fibronectin, and type I collagen was significantly reduced. Interestingly, under our experimental conditions, halofuginone treatment led to reduced protein expression of Smad3, which was both dose- and time-dependent.. Our results suggest that halofuginone may exert its antifibrotic effects in the cornea via a novel molecular mechanism and may be used as an antifibrotic agent for corneal fibrosis treatment.

Topics: Actins; Biomarkers; Cell Survival; Cells, Cultured; Collagen Type I; Cornea; Dose-Response Relationship, Drug; Fibroblasts; Fibronectins; Fibrosis; Gene Expression Regulation; Humans; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1

Anti-angiogenic therapy is a promising approach for the treatment of increased angiogenesis in certain diseases. We aimed to investigate the local anti-angiogenic effect of silicone implants coated with Halofuginone, an angiogenesis inhibitor that inhibits synthesis of collagen-type-I and matrix metalloproteinases. The degree of angiogenesis was observed after implantation of surface modified Halofuginone eluting silicone implants into a submuscular pocket in rats over a period of 3 months. Subsequently, key mediators of angiogenesis (TGF-beta-1, bFGF, COL1A1, MMP-2, MMP-9, VEGF and PDGF) were established by immunohistological staining and RT-PCR and statistically evaluated. In comparison to uncoated silicone implants, Halofuginone eluting silicone implants lead to a significant local decrease of angiogenesis. Halofuginone eluting hybrid surface silicone implants have a significant local anti-angiogenic effect by down-regulating the expression activity of key mediators of angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Coated Materials, Biocompatible; Down-Regulation; Drug Delivery Systems; Gene Expression Regulation; Materials Testing; Models, Biological; Neovascularization, Physiologic; Piperidines; Prostheses and Implants; Quinazolinones; Rats; Rats, Sprague-Dawley; Silicones

Malaria is a devastating parasitic disease that afflicts one-third of the world's population. Antimalarial drugs in common use address few targets, and their efficacy is being undermined by parasite resistance. Most therapeutics target blood-stage malaria, whereas only few compounds are active against malaria's liver stage, the first stage of the Plasmodium parasite's life cycle within the human host. The identification of inhibitors active against liver-stage malaria would benefit the development of chemical probes to elucidate the poorly understood biology of this phase of the parasite life cycle and could provide agents to prevent and eliminate the disease. Herein we report the development of a live-cell parasite traversal assay in 384-well format amenable to high-throughput screening that exploits the wounding of liver cells by the parasite. This method identifies small molecules that may inhibit the parasite's actin-myosin motor system. The traversal assay, in addition to established methods, was used to evaluate the activity of halofuginone, a synthetic halogenated derivative of the natural alkaloid febrifugine, against liver-stage Plasmodium berghei parasites. Halofuginone was found to inhibit P. berghei sporozoite load in HepG2 cells with an IC(50) value of 17 nM. While the compound does not affect parasite traversal through human liver cells, an inhibition time course assay indicates that it affects essential processes in both early- and late-stage parasite development.

Topics: Animals; Antimalarials; Cell Line, Tumor; Cytokinesis; High-Throughput Screening Assays; Humans; Inhibitory Concentration 50; Liver; Malaria; Mice; Models, Biological; Piperidines; Plasmodium berghei; Plasmodium falciparum; Quinazolinones; Sporozoites; Structure-Activity Relationship

Osteoporosis is characterized by enhanced activity of osteoclasts relative to that of osteoblasts. Thus, the principal means of treating the most common form of osteoporosis, namely that attending menopause, is inhibition of osteoclast formation or function. We have demonstrated that the inflammatory cytokine, IL-17, mediates estrogen-deficient osteoporosis, in mice, and that genetic blockade of its function prevents ovariectomy-induced bone loss. We herein report that the febrifugine derivative, halofuginone, a small molecule drug, reduces abundance of Th-17 cells in mice and prevents estrogen-deficient osteoporosis by diminishing bone resorption without impacting osteogenesis. In keeping with IL-17 mediating its osteoclastogenic effects by promoting RANK ligand expression by osteoblasts, halofuginone does not directly inhibit the bone resorptive cell. Thus, halofuginone, which is presently FDA-approved for treatment of scleroderma, is a candidate therapeutic for post-menopausal osteoporosis.

Topics: Animals; Body Weight; Bone Density Conservation Agents; Bone Resorption; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Drug Evaluation, Preclinical; Estrogens; Interleukin-17; Mice; Osteoclasts; Osteogenesis; Osteoporosis; Ovariectomy; Piperidines; Quinazolinones; RANK Ligand

Interleukin-17 (IL-17), which is important for host defens, has been implicated in autoimmune and chronic inflammatory diseases. As knockout mice lack IL-17 expression in δγT, NKT-like cells, studies investigating the association between TH17 cells and cutaneous graft-versus-host disease (GVHD) in animal models have reported conflicting results. To determine the role of TH17 cells in cutaneous GVHD, we developed an acute GVHD model using C57BL/6(H-2(b)) donors to BABL/c (H-2(d)) recipients. Blood samples and skin were examined for inflammation and infiltrating cells using histology and fluorescence-activated cell sorter (FACS) on days 6 and 15 after bone marrow transplantation. We found donor T cells to mediate severe cutaneous inflammation, which was ameliorater by administration of halofuginone (HF) to the recipients. Mechanistically, we demonstrate the severe tissue damage during this disorder to be associated with the production of IL-17 and the expansion of IL-17-producing CD4(+) cells. Specific inhibition of TH17 differentiation and function by HF reduced disease severity. Thus, TH17 cells are sufficient to induce acute cutaneous GVHD.

Topics: Acute Disease; Animals; Bone Marrow Transplantation; Cell Differentiation; Cell Separation; Cells, Cultured; Disease Models, Animal; Female; Flow Cytometry; Graft vs Host Disease; Inflammation Mediators; Interleukin-17; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Piperidines; Quinazolinones; Skin; Th1 Cells; Th17 Cells; Time Factors

Uterine leiomyomas, or fibroids, are the most frequent gynecological tumors in premenopausal women with as many as 65% of women becoming clinically symptomatic. Uterine fibroids are benign myometrial tumors that produce large quantities of extracellular matrix proteins. Despite its high morbidity, the molecular basis underlying the development of uterine leiomyomas is not well understood. Domestic hens of Gallus gallus domesticus develop oviductal leiomyomas similar to those found in humans. We investigated the natural history of chicken leiomyomas, in vivo expression of protein biomarkers, and in vitro expression of ovarian steroid receptors. Based on the analysis of 263 hens, tumor prevalence, tumor number per hen, and tumor size increased as the hens aged. Immunohistochemistry for alpha-smooth muscle actin (SMA) and desmin confirmed the smooth muscle phenotype of the chicken leiomyomas. Intense collagen expression was detected in these oviductal leiomyomas by Mason's trichrome, and the tumors also showed increased expression of TGFB3 and collagen type I mRNAs. Consistent with human leiomyomas, chicken fibroids displayed increased BCL2 and estrogen (E) and progesterone (P) receptor expression. Chicken leiomyomas were dissociated for in vitro culture. Cells from explants were positive for SMA, desmin, and E and P receptors until the fourth passage. These cells also displayed a response similar to human cells when challenged with halofuginone, an antifibrotic agent. Our findings indicate that the chicken is an excellent complementary model for studies involving the pathophysiology of human uterine leiomyomas.

Topics: Aging; Animals; Antineoplastic Agents; Chickens; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Leiomyoma; Oviducts; Piperidines; Poultry Diseases; Prevalence; Primary Cell Culture; Quinazolinones; Tumor Cells, Cultured; Uterine Neoplasms

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.. Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.. Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.. The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Ceruletide; Collagen; Extracellular Matrix; Fibrosis; Humans; Male; Mice; Mice, Transgenic; Myofibroblasts; Neoplasm Invasiveness; Pancreatic Neoplasms; Piperidines; Quinazolinones; Stromal Cells

Halofuginone, a low-molecular-weight quinazolinone alkaloid that inhibits collagen α1(I), has been shown to suppress cancer growth, metastasis, and angiogenesis. These activities were attributed in part to the inhibition of matrix metalloproteinase-2 (MMP-2). The present study was carried out to explore the molecular mechanism underlying this effect. We found a marked (50%) inhibition in MMP-2 gelatinolytic activity in human breast cancer MDA-MB-435 cells pretreated with as little as 50 ng/ml of halofuginone, a concentration that markedly inhibited their invasive and proliferative capacities. We further show that both early growth response 1 (Egr-1) and Nab-2 (corepressor of Egr1 activation) are upregulated by halofuginone in a dose-dependent and time-dependent (up to 5 h) manner. Using MMP-2 reporter gene and chromatin immunoprecipitation analyses, we found that Egr-1 binds to the MMP-2 promoter and inhibits its activity. Altogether, our results identify the downstream elements (Egr-1, Nab-2, and MMP-2) by which halofuginone exerts its antitumoral effect, thereby advancing its potential therapeutic application as an anticancer drug.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Early Growth Response Protein 1; Female; Gene Expression; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Piperidines; Promoter Regions, Genetic; Quinazolinones; Repressor Proteins; Transcription Factors; Up-Regulation

Postoperative adhesion formation continues to be a significant surgical complication, and methods for preventing abdominopelvic adhesions remain limited. Halofuginone (HF) is a type-1 collagen synthesis inhibitor and may enhance the effects of a physical barrier in preventing adhesion formation. We evaluated the effectiveness of a HF infused keratin hydrogel on preventing adhesions in a rat cecal abrasion model.. Laparotomy and standardized cecal abrasion was performed on 58 retired-breeder Sprague Dawley female rats to induce intra-abdominal adhesions. Rats were randomized to: no treatment; Interceed absorbable adhesion barrier; keratin hydrogel alone; or keratin hydrogel infused with 22 μg/mL of HF. Necropsies were performed at postop d-14 to assess the extent and tenacity of adhesions and grade histologic inflammation and fibrosis using a standard scoring system. Serum, liver, kidneys, and lungs were harvested to evaluate tissue HF concentrations. Protein and drug elution curves were generated to assess the release of HF from the hydrogel.. Treatment with Keratin-HF hydrogel resulted in significantly fewer abdominal adhesions than any other treatment, and significantly less dense adhesions compared with Interceed or keratin hydrogel alone. Subset histologic analysis did not reveal qualitative differences. HF was undetectable in serum and kidneys, and detected at negligible concentrations in liver and lungs. Keratin-HF hydrogel drug release in phosphate-buffered solution (PBS) was sustained over 7 d and correlated with keratin protein degradation.. Keratin-HF hydrogel is a novel therapeutic agent that may provide a better method for preventing the development of postoperative adhesions using a combined physical barrier and pharmacologic approached.

Topics: Animals; Cecum; Female; Hydrogel, Polyethylene Glycol Dimethacrylate; Keratins; Piperidines; Quinazolinones; Rats; Rats, Sprague-Dawley; Tissue Adhesions

Halofuginone, isolated from Dichroa febreifuga, is a potent inhibitor of skin collagen in chronic graft-versus-host disease (GVHD). To evaluate the effect of halofuginone on the development of cutaneous GVHD, we developed a murine model based on BALB/c (H-2d) as recipients with transplantation of C57BL/6(H-2b) bone marrow plus splenocytes. Halofuginone or its vehicle dimethyl sulfoxide (DMSO) was given introperitoneally at a dose of 5 ug/mouse daily from one day before transplantation until 20 days post-transplantation. Halofuginone-treated recipients showed only very mild appearance of cutaneous GVHD, whereas DMSO-treated recipients rapidly showed manifestation of severe cutaneous GVHD, indicating a protective effect of halofuginone in cutaneous GVHD. After injected with halofuginone, we observed a decrease in the number of CD4(+) interleukin (IL)-17(+) cells and a parallel increase in that of CD4(+) interferon (IFN)-gamma(+) cells in peripheral blood. This shift between CD4(+) IL-17(+) cells and CD4(+) IFN-gamma(+) cells developed through modulation of cytokine profile indicated by a marked increase in the levels of IFN-gamma, tumor necrosis factor (TNF)-alpha, and IL-6. The level of IL-10 was not changed obviously. Mechanistically, we demonstrate that severe tissue damage was associated with the production of IL-17 and expansion of CD4(+)IL-17(+) cells during this disorder. Specific inhibition of Th17 differentiation by halofuginone reduced disease severity. Our results indicate a significant role of halofuginone in suppressing cutaneous GVHD, apparently through effect on inhibition of Th17 cells differentiation.

Topics: Angiogenesis Inhibitors; Animals; Bone Marrow Transplantation; Cell Differentiation; Chronic Disease; Cytokines; Female; Graft vs Host Disease; Male; Mice; Mice, Inbred BALB C; Piperidines; Quinazolinones; Skin Diseases; Th17 Cells; Time Factors; Transplantation, Homologous

TGF-β derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-β signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-β signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-β, and TGF-β-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-β target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205 Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti-TGF-β strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-β receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-β signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Growth Processes; Cell Line, Tumor; Disease Progression; Female; Gene Expression; Humans; Melanoma; Mice; Mice, Nude; Piperidines; Quinazolinones; Signal Transduction; Xenograft Model Antitumor Assays

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody's assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.

Topics: Antibody Specificity; Antigens; Calibration; Enzyme-Linked Immunosorbent Assay; Humans; Limit of Detection; Peptide Library; Piperidines; Quinazolinones; Single-Chain Antibodies

Urethral strictures are from periurethral spongiofibrosis that develops as a result of urethral trauma, disease, or iatrogenic injury. The spongy tissue that surrounds the strictured urethra has an altered ratio of collagen, with increased collagen type I relative to type III. We evaluated the ability of a urethral catheter that was coated with halofuginone (HF), a potent type I collagen inhibitor, to prevent spongiofibrosis formation in a rat model.. HF was coated on silicone catheters and release kinetics were measured. Success of impregnation was evaluated with scanning electron microscopy, serial weights, and drug elution data. Urethral strictures were induced in rats using electrocautery. Half the animals had placement of an HF-coated catheter while the others had uncoated silicone controls. Animals were sacrificed at predetermined time points, and urethral tissue was either processed for staining with Masson trichrome and anti-alpha-1 collagen or digested to determine HF concentration. Serum drug levels were also determined in treated animals. Slides were graded by a pathologist who was blinded to treatment to determine collagen deposition.. HF was coated successfully on silicone catheters. Local urethral concentration of HF was tenfold higher than serum concentration in treated rats. Animals with HF-coated catheters had no new type I collagen deposition after urethral injury. Control animals had increased periurethral collagen type I deposition, typical of urethral stricture formation.. HF can be coated successfully on silicone catheters. HF successfully inhibits periurethral type I collagen deposition after urethral injury. This may become an important therapy to prevent urethral stricture formation or recurrence after endoscopic therapy.

Topics: Animals; Catheters; Cicatrix; Collagen Type I; Disease Models, Animal; Male; Piperidines; Quinazolinones; Rats; Rats, Sprague-Dawley; Staining and Labeling; Stents; Urethra; Urethral Diseases

Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC).. To monitor the course of infection we exploited a new in vivo imaging technique.. i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of βdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment.. These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis.

Topics: Animals; beta-Defensins; Candida albicans; Candidiasis, Vulvovaginal; Disease Models, Animal; Female; Immunity; Interleukin-17; Interleukin-23; Lymph Nodes; Mice; Neutrophil Infiltration; Piperidines; Quinazolinones; Th17 Cells; Vagina

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

Topics: Animals; Blood Cell Count; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, SCID; Oncogene Proteins, Fusion; Piperidines; Protein Serine-Threonine Kinases; Quinazolinones; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

To investigate the effects of the antifibrotic drug halofuginone on extracellular matrix production, cell proliferation, and apoptosis of cultured myometrial and leiomyoma smooth muscle cells.. Comparative and controlled experimental research study.. University research laboratory.. Leiomyoma and myometrial tissues were obtained from eight different patients at the time of elective hysterectomy.. The effects of halofuginone on cell proliferation were assessed by tritiated thymidine uptake assays and cell count assays. Effects on TGFbeta1, collagen type I, and collagen type III mRNA levels were assessed by quantitative real-time polymerase chain reaction. Effects on apoptosis were assayed using a chemiluminescent assay to measure changes in caspase 3 and 7.. Halofuginone inhibited cell proliferation of both leiomyoma and autologous myometrial cells in a dose-dependent manner by inhibiting DNA synthesis within 24 hours and later inducing apoptosis (as measured by increased caspase 3/7) by 48-72 hours. Halofuginone also significantly reduced collagen type I (alpha1) and collagen type III (alpha1) mRNA levels, as well as the profibrotic factor TGFbeta1 mRNA levels in both cell types.. These results provide evidence to support the use of the antifibrotic drug halofuginone as a novel drug treatment for uterine leiomyomas.

Topics: Apoptosis; Cell Proliferation; Collagen Type I; Collagen Type III; Female; Fibrosis; Growth Inhibitors; Humans; Leiomyoma; Myocytes, Smooth Muscle; Myometrium; Piperidines; Quinazolinones; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

To evaluate the effect of halofuginone on trinitrobenzene sulfonic acid (TNBS)-induced colonic injury, rats were given halofuginone (40 microg/kg, intraperitoneally) or saline 1 h before the induction of colitis, and the injections were continued twice daily for 3 days until they were decapitated. High macroscopic and microscopic damage scores, elevated colonic wet weights, colonic myeloperoxidase activity, malondialdehyde and tissue collagen level, and luminol chemiluminescence values, and marked reduction in glutathione level of the saline-treated colitis group were all reversed by treatment with halofuginone. In conclusion, halofuginone exerts beneficial effects in TNBS-induced colonic inflammation in rats. The anti-inflammatory effects of halofuginone appear to involve suppression of neutrophil accumulation, preservation of endogenous glutathione, and inhibition of reactive oxidant generation. Halofuginone also shows antifibrotic effect via inhibition of tissue collagen production. The present data encourage possible use of the antifibrotic halofuginone as an anti-inflammatory agent in improving oxidative injury in colitis.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Collagen; Collagen Type I; Colon; Female; Glutathione; Luminescence; Male; Malondialdehyde; Organ Size; Peroxidase; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Sprague-Dawley; Trinitrobenzenesulfonic Acid

Transforming growth factor beta (TGF-beta) is implicated in radiation-induced fibrosis of normal tissues in patients receiving radiotherapy. Inhibiting the TGF-beta signaling pathway by various means has been shown to reduce radiation-induced fibrosis in pre-clinical studies. The present study evaluated the effects of interfering with the TGF-beta signaling pathway on the radiosensitivity of selected human tumor cell lines using the plant-derived alkaloid, halofuginone. Halofuginone treatment inhibited cell growth, halted cell cycle progression, decreased radiation-induced DNA damage repair, and decreased TGF-beta receptor II protein levels, leading to increased cellular radiosensitization. These data further support the goal of manipulating the TGF-beta pathway to achieve a positive increase in the therapeutic gain in clinical radiotherapy.

Topics: Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Survival; Flow Cytometry; Humans; Piperidines; Quinazolinones; Radiation Tolerance; Signal Transduction

The asymmetric dihydroxylation of amino-functionalized vinyl sulfone 19 has been used for the 3-step preparation of 3-hydroxylpiperidine 24 in 86% enantiomeric excess. This enantiomerically enriched building block was used then to synthesize the naturally occurring antimalarial alkaloid febrifugine 1 and its antiangiogenic analogue, halofuginone 3.

Topics: Catalysis; Hydroxylation; Molecular Structure; Piperidines; Quinazolines; Quinazolinones; Stereoisomerism; Sulfones

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.

Topics: Animals; Cell Fusion; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Muscle Fibers, Skeletal; Myoblasts; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperidines; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolinones; Signal Transduction; Smad3 Protein

In this study, we assessed the effects of halofuginone and methylprednisolone on hypopharyngeal and esophageal stricture that can develop following radiation to the head and neck of rats. Rats were divided into four groups randomly and 18 Gy radiation was given to the head and neck regions of all rats except the control group. Group 1 (Control Group): No radiation or drugs were administered. Group 2 (Radiation Group): only radiation was applied without any drugs. Group 3 (Halofuginone Group): halofuginone 100 microg/kg per day was given intraperitoneally. Group 4 (Methylprednisolone Group): methylprednisolone 1 mg/kg per day was administered intramuscularly. In all groups, 90 days after application of radiation, sections of the proximal esophagus and hypopharynx were examined for fibrosis, fibroblast proliferation, vascularization, epithelial atypia, necrosis, polymorphonuclear leukocytes, mononuclear cells, and stenosis index by light microscope and the hydroxyproline levels were assessed biochemically. Fibrosis, epithelial atypia and hydroxyproline levels were found to be significantly higher in the radiation group compared to the control group (P < 0.05). We did not observe fibrosis in either the halofuginone or the control groups. Fibrosis was also significantly lower in the methylprednisolone group than the radiation group (P < 0.05). The differences of the stenosis index scores between the groups were not statistically significant (P < 0.05). Vascularization was similar in all groups. We think that especially halofuginone is a drug that can be used safely to prevent fibrosis due to radiotherapy, but further studies are needed.

Topics: Animals; Anti-Inflammatory Agents; Esophageal Stenosis; Esophagus; Female; Hydroxyproline; Hypopharynx; Injections, Intramuscular; Injections, Intraperitoneal; Methylprednisolone; Piperidines; Premedication; Protein Synthesis Inhibitors; Quinazolinones; Radiation Pneumonitis; Rats; Rats, Wistar

Most solid tumors consist of neoplastic and nonneoplastic cells and extracellular matrix components. In the pancreas, activated stellate cells (PSCs) are the source of the extracellular matrix proteins. We evaluated the significance of PSC activation in tumor establishment and development in mouse xenografts.. Xenografts were established by implanting human pancreatic cancer cells (MiaPaca-2) subcutaneously or orthotopically by injecting them into the spleen. Fibrosis was induced by cerulein. Collagen level was evaluated by Sirius red staining. Prolyl 4-hydroxylase β and stellate cell activation-associated protein (Cygb/STAP) were determined by immunohistochemistry.. Halofuginone inhibited subcutaneous tumor development implanted with Matrigel and reduced collagen and prolyl 4-hydroxylase β levels. Few tumors, which developed slowly, were observed after MiaPaca-2 implantation without Matrigel. Increase in tumor number and rate of development were observed with addition of PSCs from control pancreas, and further increase was observed when the PSCs were from cerulein-treated mice. Preincubation of the PSCs with halofuginone elicited Cygb/STAP level reduction and tumor growth inhibition. More tumors developed orthotopically in cerulein-treated mice than in controls; this was prevented by halofuginone.. Extracellular matrix production by activated PSCs is essential for tumor establishment and growth. Thus, inhibition of PSC activation is a viable means of reducing pancreatic tumor development.

Topics: Animals; Cell Line, Tumor; Extracellular Matrix; Fibrosis; Humans; Male; Mice; Neoplasm Transplantation; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Piperidines; Quinazolinones; Transplantation, Heterologous

The objective of the study is to determine whether topical halofuginone (HF) application has an impact on prolonging the time for healing of myringotomy incision, and to investigate histopathologic effects of HF on tympanic membrane (TM) in rat model. Forty rats with normal eardrums were involved in this study. The study group and control group consisted of 30 and 10 rats, respectively. A posterior incision 1 mm in diameter was made on healthy eardrums of the rats. Following incision, gelfoam soaked with HF hydrobromide of 30 mg/dl was applied on the perforation in study group, whereas gelfoam soaked with isotonic saline was applied on the perforation in control group. On days 1, 3, 7, 10, 14, 18, 21, 24, 27, and 30, otoendoscopic evaluation of eardrums under general anesthesia was conducted and perforations were screened. A rat of each group was killed in control days and TMs were dissected to evaluate histopathological changes. The average times for patency of perforation in study and control groups were 21.43 and 7.50 days, respectively. The difference was found to be statistically significant (p < 0.05). Histopathological evaluation revealed that HF reduces hyalinisation and fibrosis in eardrum, when compared with the control group. In conclusion, HF significantly delays closure time of myringotomies in rat model. However, this delay may not be enough for recovery of otitis media with effusion.

Topics: Administration, Topical; Animals; Disease Models, Animal; Gelatin Sponge, Absorbable; Middle Ear Ventilation; Otitis Media with Effusion; Piperidines; Quinazolinones; Random Allocation; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Time Factors; Tympanic Membrane; Wound Healing

In muscular dystrophies (MD) the loss of muscle and its ability to function are associated with fibrosis. We evaluated the efficacy of halofuginone in reducing fibrosis in the dy(2J)/dy(2J) mouse model of congenital MD. Mice were injected intraperitoneally with 5 microg of halofuginone 3 times a week for 5 or 15 weeks, starting at the age of 3 weeks. Halofuginone caused a reduction in collagen synthesis in hindlimb muscles. This was associated with reductions in the degenerated area, in cell proliferation, in the number of myofibers with central nuclei, with increased myofiber diameter, and with enhanced motor coordination and balance. Halofuginone caused a reduction in infiltrating fibroblasts that were located close to centrally nucleated myofibers. Our results suggest that halofuginone reduced the deleterious effects of fibrosis, thus improving muscle integrity. Halofuginone meets the criteria for a novel antifibrotic therapy for MD patients.

Topics: Animals; Cell Count; Cell Proliferation; Collagen; Fibrosis; Laminin; Mice; Mice, Knockout; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Animal; Piperidines; Quinazolinones; Rotarod Performance Test

Capsular fibrosis is one of the most severe complications that can occur in connection with silicone breast implants. Should this case arise, a periprosthetic deposition of fibroid tissue may evolve. Transforming growth factor (TGF)-beta is one of the most important mediators in relation to such processes.. The chinazolinone derivative halofuginone is a type I collagen synthesis inhibitor that interferes with the TGF-beta signaling pathway. The work at hand examines the local antifibrotic effectiveness of halofuginone lactate, which has been biotechnologically bound to the silicone implant's surface. The experiments in relation to this were conducted in vivo on two groups of seven Sprague-Dawley rats. Group I received untreated silicone implants, and group II received halofuginone-coated silicone implants.. Submusculary embedded halofuginone-coated silicone implants have shown no systemic side effects. The histologic and immunohistologic examinations of the periprostatic capsules revealed a significant decrease of CD68 histiocytes, TGF-beta, fibroblasts, collagen type I and type III, and capsular thickness after a 3-month period.. The results confirmed a decrease in foreign body responses to halofuginone surface-modified silicone implants and mark their potential for obtaining a lessened capsular fibrosis by way of a local antifibrotic effect.

Topics: Animals; Breast Implants; Coated Materials, Biocompatible; Collagen Type I; Disease Models, Animal; Female; Fibrosis; Foreign-Body Reaction; Mammary Glands, Animal; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Sprague-Dawley; Silicon; Treatment Outcome

Following IV injection of doxycycline in a dose of 20 mg kg(-1) b.wt., its serum concentration was best fitted in two-compartment open model in chickens fed either on control or on anticoccidials-containing rations. Diclazuril and halofuginone resulted in a significant short distribution half-life (t(½α)) (7.17±0.39 and 11.88±1.05 min, respectively) and increased total body clearance (Cl(tot)) 0.37±0.024 and 0.295±0.034 L/kg/h, respectively. Following oral dosing the tested drug absorbed with t(½ab) of 41.38±1.6, 17.48±0.86 and 41.83±1.8 min, respectively and their C(max) values (3.18±0.18, 5.425±0.48 and 0.986±0.037 μg/ml) were attained at 2.07±0.097, 1.403±0.074 and 2.55±0.106 h. For doxycycline alone and in presence of diclazuril and halofuginone, respectively. Systemic bioavailability was 22.64±3.46, 86.74±9.23 and 22.38±3.09%, respectively. Following IM injection t(½ab) were 9.096±1.34 for doxycycline alone, 16.24±2.21 and 15.6±1.7 min in the presence of diclazuril and halofuginone, respectively. C(max) was 3.10±0.28, 4.63±0.57 and 0.55±0.07 μg/ml reached at 0.8±0.083, 1.13±0.126 and 1.21±0.105 h. For the antibiotic alone, and in presence of either diclazuril and halofuginone, respectively. Systemic bioavailability was 22.41±3.86, 88.97±12.9 and 12.31±0.99% in chickens fed on anticoccidial-free, diclazuril- and halofuginone-containing rations, respectively. Both the tested anticoccidials induced higher doxycycline tissue residues in all tested tissue samples.

Topics: Animal Feed; Animals; Anti-Bacterial Agents; Chickens; Coccidiosis; Coccidiostats; Doxycycline; Drug Interactions; Drug Therapy, Combination; Female; Half-Life; Injections, Intramuscular; Injections, Intravenous; Male; Nitriles; Piperidines; Quinazolinones; Triazines

Our objective was to determine whether coating the amniotic membrane with halofuginone, a type 1 collagen synthase inhibitor, with or without the hemostasis-inducing substance chitosan, reduced the number and severity of adhesions in the rat uterine horn injury model. Sixty retired breeder Sprague-Dawley rats underwent midline laparotomy and a zone of ischemia was created in the left uterine horn of each animal. Rats were randomized to one of six treatment groups: (1) untreated control, (2) oxidized regenerated cellulose (Interceed®) (ORC), (3) plain amnion, (4) amnion coated on both sides with 0.5% solution of halofuginone (HAH), (5) amnion coated on one side with 0.5% halofuginone and on the other side with chitosan (CAH), or (6) amnion coated on both sides with chitosan (CAC). The zone of ischemia in each left uterine horn was wrapped in each treatment. Rats were sacrificed 2 weeks after laparotomy, and adhesions were counted and scored for severity. Data were analyzed using Chi square and a p < 0.05 was considered significant. Our results showed that there were no differences in the percentage of animals with adhesions in the untreated, ORC, plain amnion, or CAC groups. No adhesions formed in any animal in the HAH group and only 14% of the animals developed adhesions to the uterine horn in the CAH group (p < 0.05). The percentage of animals with moderate and severe adhesions did not differ between untreated controls and the ORC groups, but were significantly reduced in all four of the amnion groups: plain amnion, HAH, CAH, and CAC (p < 0.05). Amnion coated with halofuginone alone or in combination with chitosan reduced the percentage of animals with adhesions, as well as the percentage of animals with moderate and severe adhesions compared to untreated controls and the ORC group in the rat uterine horn injury model. Amnion alone or coated with chitosan reduced the percentage of rats with moderate and severe adhesions, but not the percentage of rats with adhesions of any type compared to both untreated controls and the ORC group in the rat uterine horn injury model.

Topics: Abdomen; Amnion; Angiogenesis Inhibitors; Animals; Cellulose, Oxidized; Chitosan; Female; Ischemia; Laparotomy; Piperidines; Postoperative Complications; Quinazolinones; Random Allocation; Rats; Rats, Sprague-Dawley; Tissue Adhesions; Treatment Outcome; Uterine Diseases; Uterus

Transforming growth factor-beta (TGF-beta) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-beta(1) signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-beta(1) in cultured mesangial cells. In addition, the expression of TGF-beta type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-beta(1). An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.

Topics: Animals; Collagen Type I; Diabetic Nephropathies; Extracellular Matrix; Fibronectins; Mesangial Cells; Mice; Mice, Inbred Strains; Oxidative Stress; Piperidines; Quinazolinones; Rats; Smad2 Protein; Transforming Growth Factor beta

Chronic pancreatitis is characterized by inflammation and fibrosis. We evaluated the efficacy of halofuginone, an inhibitor of collagen synthesis and myofibroblast activation, in preventing cerulein-induced pancreas fibrosis.. Collagen synthesis was evaluated by in situ hybridization and staining. Levels of prolyl 4-hydroxylase beta (P4Hbeta), cytoglobin/stellate cell activation-associated protein (Cygb/STAP), transgelin, tissue inhibitors of metalloproteinases, serum response factor, transforming growth factor beta (TGFbeta), Smad3, and pancreatitis-associated protein 1 (PAP-1) were determined by immunohistochemistry. Metalloproteinase activity was evaluated by zymography.. Halofuginone prevented cerulein-dependent increase in collagen synthesis, collagen cross-linking enzyme P4Hbeta, Cygb/STAP, and tissue inhibitors of metalloproteinase 2. Halofuginone did not affect TGFbeta levels in cerulein-treated mice but inhibited serum response factor synthesis and Smad3 phosphorylation. In culture, halofuginone inhibited pancreatic stellate cell (PSC) proliferation and TGFbeta-dependent increase in Cygb/STAP and transgelin synthesis and metalloproteinase 2 activity. Halofuginone increased c-Jun N-terminal kinase phosphorylation in PSCs derived from cerulein-treated mice. Halofuginone prevented the increase in acinar cell proliferation and further increased the cerulein-dependent PAP-1 synthesis.. Halofuginone inhibits Smad3 phosphorylation and increases c-Jun N-terminal kinase phosphorylation, leading to the inhibition of PSC activation and consequent prevention of fibrosis. Halofuginone increased the synthesis of PAP-1, which further reduces pancreas fibrosis. Thus, halofuginone might serve as a novel therapy for pancreas fibrosis.

Topics: Animals; Blotting, Western; Cells, Cultured; Ceruletide; Collagen; Cytoglobin; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Globins; Immunohistochemistry; In Situ Hybridization; Injections, Intraperitoneal; Male; Mice; Mice, Inbred ICR; Pancreas; Pancreatitis-Associated Proteins; Piperidines; Procollagen-Proline Dioxygenase; Protein Disulfide-Isomerases; Protein Synthesis Inhibitors; Proteins; Quinazolinones; Serum Response Factor; Signal Transduction; Smad3 Protein; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

The aim of the study is to ascertain the antifibrotic effect of topically applied halofuginone after acute subglottic injury.. After standardized trauma to subglottic area, rats were divided into two groups: the study group that received treatment and the control group that did not. The subjects were treated with topical application of cottonoid soaked in 30 mg/dL halofuginone solution for 5 minutes after subglottic trauma. The larynx specimens were examined histopathologically by light microscopy to assess fibrosis, epithelialization, inflammation, and necrosis.. The fibrosis indexes of the treated group were significantly less than those of the control group (P < 0.05).. Topically applied halofuginone hydrobromide decreases fibrosis/scar tissue formation secondary to experimentally induced acute subglottic trauma.

Topics: Administration, Topical; Animals; Fibrosis; Laryngostenosis; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric

Febrifugine and its derivatives including halofuginone which possess very high activity against malaria were prepared synthetically from easily available starting material, 3-hydroxy picoline, and using simple reaction conditions. Synthesis of 2-amino-5, 6-methylenedioxy benzoic acid, (which is an intermediate for the process) is described. The selectivity enhancement in nitration of 3, 4-methylenedioxybenzaldehyde towards 6-nitro isomer was done with the help of surfactant. The antimalarial activity of synthesized compounds was determined by using in vitro assays against chloroquine sensitive (D6), chloroquine resistant (W2) Plasmodium falciparum strains for susceptibility and two mammalian cell lines (neuronal cell line NG108 and macrophage cell line J774) for cytotoxicity. The IC(50)s of halofuginone was observed to be the best among the synthesized derivatives of febrifugine.

Topics: Animals; Antimalarials; Benzoates; Cell Line; Piperidines; Plasmodium falciparum; Quinazolines; Quinazolinones

Topics: Amino Acids; Animals; Autoimmunity; Cell Differentiation; Eukaryotic Initiation Factor-2; Evolution, Molecular; Gene Expression Regulation; Humans; Interleukin-17; Lymphopoiesis; Mice; Multiple Sclerosis; Phosphorylation; Piperidines; Protein Biosynthesis; Protein Serine-Threonine Kinases; Quinazolinones; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer

A central challenge for improving autoimmune therapy is preventing inflammatory pathology without inducing generalized immunosuppression. T helper 17 (TH17) cells, characterized by their production of interleukin-17, have emerged as important and broad mediators of autoimmunity. Here we show that the small molecule halofuginone (HF) selectively inhibits mouse and human TH17 differentiation by activating a cytoprotective signaling pathway, the amino acid starvation response (AAR). Inhibition of TH17 differentiation by HF is rescued by the addition of excess amino acids and is mimicked by AAR activation after selective amino acid depletion. HF also induces the AAR in vivo and protects mice from TH17-associated experimental autoimmune encephalomyelitis. These results indicate that the AAR pathway is a potent and selective regulator of inflammatory T cell differentiation in vivo.

Topics: Activating Transcription Factor 4; Amino Acids; Animals; Autoimmunity; Cell Differentiation; Cytokines; Encephalomyelitis, Autoimmune, Experimental; Eukaryotic Initiation Factor-2; Gene Expression; Humans; Interleukin-17; Lymphopoiesis; Mice; Mice, Inbred C57BL; Phosphorylation; Piperidines; Protein Serine-Threonine Kinases; Quinazolinones; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer

Fibrosis of normal tissues often accompanies radiation treatment of cancer. Activation of the transforming growth factor-beta (TGF-beta) signaling pathway is thought to play a major role in radiation-induced fibrosis and has prompted the development and assessment of low molecular weight inhibitors of the pathway. Previous studies with halofuginone have shown it to inhibit TGF-beta signaling in vitro and protect mice from radiation-induced leg contraction (a model for soft tissue fibrosis). The current study confirms these findings for HaCaT cells stimulated with exogenous TGF-beta treatment. Reducing the halifuginone treatment from 7 days/week (used previously) to 5 days/week post-radiation exposure provided significant protection against radiation-induced leg contraction in mice 3 and 4 months post-radiation treatment. Halofuginone treatment was shown to attenuate TGF-beta signaling molecules taken from irradiated skin including TGF-betaRII, pSmad3, Smad7, and TSP1. The latter, TSP1, a co-activator of TGF-beta may serve as a suitable biomarker for monitoring the efficacy of halofuginone should it be evaluated in a clinical setting for protection against radiation-induced fibrosis.

Topics: Animals; Cell Line; Contracture; Female; Fibrosis; Humans; Leg; Mice; Mice, Inbred C3H; Piperidines; Protein Serine-Threonine Kinases; Quinazolinones; Radiotherapy; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The aim of this study was to investigate the putative beneficial effect of halofuginone on gentamicin-induced nephrotoxicity in rats. Sprague-Dawley rats were treated with gentamicin sulphate (GEN; 80 mg/kg) or saline intraperitoneally (i.p.) for 7 days. Halofuginone was administered (0.1 mg/kg/day; i.p.) following GEN or saline injections. Blood and urine samples were collected to measure the renal function tests. Kidneys were excised for histological evaluation and for the measurement of malondialdehyde (MDA), glutathione (GSH), myeloperoxidase (MPO) activity, and chemiluminescence (CL). Halofuginone treatment to animals with GEN-induced renal injury caused a significant decrease in serum blood urea nitrogen level and reduced the elevated MDA, GSH content, and MPO activity. It was also effective in reversing the elevated CL values of rats with GEN-induced nephrotoxicity and preserving renal morphology, as examined microscopically. In conclusion, halofuginone was beneficial in GEN-induced acute nephrotoxicity. The mechanism could be attributed, at least in part, to decreased tissue leukocyte infiltration and reactive metabolite production.

Topics: Animals; Antioxidants; Chlorides; Drug Interactions; Female; Gentamicins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kidney; Kidney Function Tests; Kidney Tubules; Male; Malondialdehyde; Oxidative Stress; Piperidines; Plant Extracts; Quinazolinones; Rats; Rats, Sprague-Dawley; Renal Insufficiency

The aim of this paper is to assess the effects of halofuginone, a specific inhibitor of synthesis of collagen Type 1, on fibrogenetic process in an experimental model of early pancreatic fibrosis.. Thirty rats were divided into three equal groups: group 1, sham laparotomy; group 2, severe hyperstimulation and obstruction pancreatitis (SHOP) with no treatment; group 3, SHOP with halofuginone treatment group. SHOP model was induced by complete pancreatic duct obstruction and daily cerulein hyperstimulation (50 microg/kg, intraperitoneally). Halofuginone was administered daily from the operative day (5 mg/kg, intraperitoneally). All of the animals were sacrificed, and blood and pancreatic tissue samples were obtained for biochemical and histopathological examination on the 5th postoperative day.. No mortality was observed in any group. Serum amylase, lipase, hyaluronic acid, and nitric oxide levels were significantly higher in groups 2 and 3 compared with group 1 (P < 0.05), but were significantly lower in group 3 compared with group 2 (P < 0.05). No significant differences were observed regarding serum malondialdehyde and glutathione levels between groups 1 and 3. Tissue hydroxyproline levels were found to be significantly higher in groups 2 and 3 compared with group 1 (P < 0.001), but were significantly lower in group 3 compared with group 2 (P < 0.001). Although tissue hydroxyproline levels were significantly higher in the halofuginone treatment group compared with the control group, histopathological evaluation did not reveal a significant difference between these groups regarding collagen deposition. When group 3 was compared with group 2, halofuginone significantly reduced inflammation and acinar atrophy in the pancreas as well (P < 0.05).. Halofuginone was found to be effective in reducing SHOP-related inflammation, acinar atrophy, and fibrosis in the pancreas.

Topics: Amylases; Animals; Collagen Type I; Disease Models, Animal; Female; Fibrosis; Hyaluronic Acid; Lipase; Nitric Oxide; Pancreas; Pancreatic Diseases; Pancreatitis; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Wistar

The aim of this study was to investigate the preventive effect of halofuginone on posterior glottic stenosis (PGS) in an animal model.. A randomized, controlled animal study.. Sixteen male New Zealand White rabbits were used for this study. After the mucosa of posterior glottis was removed for producing PGS, the study group (eight rabbits) was given intraperitoneal halofuginone at 0.1 mg/kg/day for 4 weeks and saline was injected into peritoneum in the control group. At 4 weeks after injury, postsurgical changes of posterior glottis were evaluated by gross and histologic examination.. PGS was induced by the mucosal stripping of the posterior glottis. The halofuginone-treated group showed less scarring and granulation tissue formation. Also, the degree of synechia was significantly less than that of control group. Histologic analysis showed the decreased fibrosis in the halofuginone-treated group.. This study suggests that halofuginone can be helpful in preventing PGS after laryngeal injury.

Topics: Animals; Disease Models, Animal; Fibrosis; Glottis; Injections, Intraperitoneal; Laryngostenosis; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rabbits; Random Allocation

Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFbeta in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.

Topics: Age Factors; Animals; Blotting, Western; Cell Line; Cells, Cultured; Collagen; Diaphragm; Fibrosis; Immunohistochemistry; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Motor Activity; Muscles; Muscular Dystrophy, Animal; Myoblasts; Myocardium; Piperidines; Postural Balance; Protein Synthesis Inhibitors; Quinazolinones; Rotarod Performance Test; Smad3 Protein

We describe an outbreak of cryptosporidiosis in Stone curlews kept in a mixed-species rearing unit in Dubai. Cryptosporidium was the predominant intestinal pathogen detected, although microbiological investigations revealed a concurrent Salmonella infantis infection in two of the 29 Stone curlew chicks that died. Nineteen of 29 birds had catarrhal enteritis associated with histopathological findings of numerous Cryptosporidium developmental stages at the mucosal surface. Catarrhal enteritis was present without associated Cryptosporidium oocysts in five cases. Histology of the intestine, faecal examination by direct microscopy and antigenic detection by immunochromatography revealed the presence of Cryptosporidium spp. associated with catarrhal enteritis in intestinal sections and faeces. Clinical and histopathological outcomes of infection were severe, including disruption of intestinal epithelial integrity, the presence of numerous endogenous Cryptosporidium stages in intestinal epithelia and the excretion of large numbers of sporulated oocysts. The application of polymerase chain reaction and restriction fragment length polymorphism techniques at two 18S rRNA and one Cryptosporidium oocyst wall protein gene locus confirmed the presence of Cryptosporidium parvum DNA in faecal samples.

Topics: Animals; Antiprotozoal Agents; Bird Diseases; Birds; Cryptosporidiosis; Disease Outbreaks; Feces; Piperidines; Quinazolinones; Spiramycin; Triazines; United Arab Emirates

Tumors are complex tissues composed of neoplastic cells, soluble and insoluble matrix components and stromal cells. Here we report that in melanoma, turn-over of type I collagen (Col(I)), the predominant matrix protein in dermal stroma affects melanoma progression. Fibroblasts juxtaposed to melanoma cell nests within the papillary dermis display high levels of Col(I) mRNA expression. These nests are enveloped by collagen fibers. In contrast, melanoma-associated fibroblasts within the reticular dermis express Col(I) mRNA at a level that is comparable to its expression in uninvolved dermis and reduced amount of collagen protein can be observed. To determine the significance of Col(I) expression in melanoma, we pharmacologically inhibited its transcription in a porcine cutaneous melanoma model by oral administration of halofuginone. When administered before melanoma development, it reduced melanoma incidence and diminished the transition from microinvasive toward deeply invasive growth by limiting the development of a tumor vasculature. Whereas invasive melanoma growth has been correlated with increased blood vessel density previously, our data for the first time demonstrate that the proangiogenic effect of Col(I) expression by fibroblasts and vascular cells precedes the development of invasive melanomas in a de novo tumor model.

Topics: Angiogenesis Inhibitors; Animals; Collagen Type I; Humans; Immunohistochemistry; In Situ Hybridization; Melanoma; Neoplasm Invasiveness; Neovascularization, Pathologic; Piperidines; Quinazolinones; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Swine; Swine, Miniature

The effect of halofuginone (Halo) on established fibrosis in older mdx dystrophic muscle was investigated. Mice (8 to 9 mo) treated with Halo (or saline in controls) for 5, 10, or 12 wk were assessed weekly for grip strength and voluntary running. Echocardiography was performed at 0, 5, and 10 wk. Respiratory function and exercise-induced muscle damage were tested. Heart, quadriceps, diaphragm, and tibialis anterior muscles were collected to study fibrosis, collagen I and III expression, collagen content using a novel collagenase-digestion method, and cell proliferation. Hepatocyte growth factor and alpha-smooth muscle actin proteins were assayed in quadriceps. Halo decreased fibrosis (diaphragm and quadriceps), collagen I and III expression, collagen protein, and smooth muscle actin content after 10 wk treatment. Muscle-cell proliferation increased at 5 wk, and hepatocyte growth factor increased by 10 wk treatment. Halo markedly improved both cardiac and respiratory function and reduced damage and improved recovery from exercise. The overall impact of established dystrophy and dysfunction in cardiac and skeletal muscles was reduced by Halo treatment. Marked improvements in vital-organ functions implicate Halo as a strong candidate drug to reduce morbidity and mortality in Duchenne muscular dystrophy.

Topics: Actins; Age Factors; Animals; Cell Proliferation; Collagen Type I; Collagen Type III; Diaphragm; Disease Models, Animal; Fibrosis; Heart; Hepatocyte Growth Factor; Mice; Mice, Inbred mdx; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Myocardium; Physical Endurance; Piperidines; Protein Synthesis Inhibitors; Quadriceps Muscle; Quinazolinones; Respiratory Mechanics; Running; Time Factors; Ventricular Function, Left

Topics: Age Factors; Animals; Collagen Type I; Collagen Type III; Disease Models, Animal; Fibrosis; Heart; Mice; Mice, Inbred mdx; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Myocardium; Physical Endurance; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Respiratory Mechanics; Time Factors; Ventricular Function, Left

Advanced hepatic fibrosis is characterized by excessive extracellular matrix deposition, where collagen and proteoglycans are the main constituents of scar tissue. In previous studies, we showed that heparanase, a heparan sulfate-degrading enzyme, and vascular endothelial growth factor (VEGF) play an important role during liver development and remodeling. In this communication, we investigated the relationship between heparanase and VEGF in thioacetamide-induced liver fibrosis in rats. Our study shows that heparanase mRNA expression levels correlate with those of VEGF during the induction and recovery stages of liver fibrosis. We further demonstrated that treating fibrotic rat livers with halofuginone (HF), a multipotent antifibrogenic drug, and subsequently subjecting them to hydrodynamics-based transfection with human VEGF-165 resulted in elevated expression of heparanase mRNA. Moreover, these rats demonstrated an improved capacity to regenerate following 70% partial hepatectomy. In vitro, HF stimulated heparanase and VEGF mRNA expression in hepatic stellate cells. Taken together, our results suggest that in addition to the known multiple functions of HF, it also enhances heparanase and VEGF expression and promotes liver regeneration. Accordingly, HF seems to possess ideal properties required to become an excellent antifibrogenic agent in humans.

Topics: Animals; Glucuronidase; Hydroxyproline; Liver Cirrhosis, Experimental; Liver Regeneration; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thioacetamide; Transfection; Up-Regulation; Vascular Endothelial Growth Factor A

Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-beta (TGF-beta), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-beta-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-beta. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-beta-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by halofuginone. This study thus highlights the role of TGF-beta signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.

Topics: Acid Phosphatase; Animals; Antineoplastic Agents; Cluster Analysis; Cytoglobin; Disease Progression; Fibrillin-1; Fibrillins; Gene Expression Profiling; Gene Expression Regulation; Globins; Hepatocytes; Humans; Isoenzymes; Liver Cirrhosis; Male; Microfilament Proteins; Nuclear Proteins; Osteopontin; Phosphorylation; Piperidines; Quinazolinones; Rats; Rats, Wistar; Signal Transduction; Smad2 Protein; Smad3 Protein; Substrate Specificity; Tartrate-Resistant Acid Phosphatase; Thioacetamide; Transforming Growth Factor beta

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R (2), 0.98-0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 microM) and monensin (0.00225 to 0.144 microM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10(5) oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 microM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 microM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 microM or more, inhibition was at least 90%; 0.05 microM still yielded 80% inhibition, whereas at concentrations below 0.00625 microM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.

Topics: Animals; Cell Line; Coccidiostats; Cryptosporidium; Dose-Response Relationship, Drug; Humans; Monensin; Piperidines; Quinazolinones

Stromal myofibroblasts play an important role in tumor progression. The transition of fibroblasts to myofibroblasts is characterized by expression of smooth muscle genes and profuse synthesis of extracellular matrix proteins. We evaluated the efficacy of targeting fibroblast-to-myofibroblast transition with halofuginone on tumor progression in prostate cancer and Wilms' tumor xenografts. In both xenografts, low doses of halofuginone treatment, independent of the route of administration, resulted in a trend toward inhibition in tumor development. Moreover, halofuginone synergizes with low dose of docetaxel in prostate cancer and vincristine and dactinomycin in Wilms' tumor xenografts, resulting in significant reduction in tumor volume and weight comparable to the effect observed by high doses of the respective chemotherapies. In prostate cancer and Wilms' tumor xenografts, halofuginone, but not the respective chemotherapies, inhibited the synthesis of collagen type I, alpha-smooth muscle actin, transgelin, and cytoglobin, all of which are characteristics of activated myofibroblasts. Halofuginone, as the respective chemotherapies, increased the synthesis of Wilms' tumor suppressor gene product (WT-1) and prostate apoptosis response gene-4 (Par-4), resulting in apoptosis/necrosis. These results suggest that targeting the fibroblast-to-myofibroblast transition with halofuginone may synergize with low doses of chemotherapy in achieving a significant antitumoral effect, avoiding the need of high-dose chemotherapy and its toxicity without impairing treatment efficacy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Docetaxel; Drug Synergism; Fibroblasts; Humans; Male; Mice; Mice, Nude; Muscles; Myoblasts; Piperidines; Prostatic Neoplasms; Quinazolinones; Taxoids; Tumor Cells, Cultured; Wilms Tumor

Halofuginone is a novel antifibrotic agent that can reverse the fibrotic process by specific inhibition of collagen type I synthesis.. To evaluate the effect of Halo on the development of glomerulosclerosis and interstitial fibrosis in the 5/6 nephrectomy rat model. Male Wistar rats were assigned to undergo 5/6 NX or sham operation, and then divided into three groups: 5/6 NX rats (NX-Halo and NX-Control) and sham. Systolic blood pressure, proteinuria and body weight were determined every 2 weeks. At sacrifice (10 weeks) creatinine clearance was evaluated and remnant kidneys removed for histologic examination, sirius red staining and in situ hybridization. Systolic blood pressure increased progressively in both 5/6 NX groups. Halo slowed the increase in proteinuria in 5/6 NX rats. As expected, creatinine clearance was lower in 5/6 NX groups when compared to sham rats. Creatinine clearance was significantly higher in the NX-Halo group at the end of the study period. Histologic examination by light microscopy showed significantly less severe interstitial fibrosis and glomerulosclerosis in Halo-treated rats. The increase in collagen alpha1 (I) gene expression and collagen staining after nephrectomy was almost completely abolished by Halo.. Halofuginone reduced proteinuria as well as the severity of interstitial fibrosis and glomerulosclerosis in 5/6 NX rats. The renal beneficial effect of Halo was also demonstrated by the blunted decrease in creatinine clearance observed in the treated animals.

Topics: Animals; Blood Pressure; Case-Control Studies; Disease Models, Animal; Fibrosis; Kidney; Male; Nephrectomy; Piperidines; Protein Synthesis Inhibitors; Proteinuria; Quinazolinones; Rats; Rats, Wistar

The aim of the study was to assess the antifibrotic effect of systemically applied halofuginone after subglottic injury.. After standardized trauma to subglottic area, rats were divided into two groups: a study group that received treatment and a control group that did not. The rats were treated with 0.1 mg/kg/day intraperitoneal halofuginone injection for 30 days. The larynx specimens were examined histopathologically under light microscope for epithelization, inflammation, necrosis, and fibrosis.. The fibrosis indexes of the treated group were significantly less than those of the control group (P < .01).. Systemically applied halofuginone hydrobromide decreases fibrosis/scar tissue formation secondary to experimentally induced acute subglottic trauma.

Topics: Animals; Cicatrix; Epithelium; Female; Fibrosis; Glottis; Injections, Intraperitoneal; Laryngitis; Laryngostenosis; Larynx; Male; Necrosis; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Random Allocation; Rats; Rats, Sprague-Dawley

This study looked at measurement of endotoxaemia as a tool in determining prognosis and probable response to treatment in scouring lambs. One hundred eighty-three lambs in the first 15-20 days of life, from eight Merino sheep farms located in the region of La Serena, south-west Spain, were used in this experiment. Scouring and normal/control lambs were selected following a clinical examination, the scouring group was further divided into subgroups, specifically those that did or did not survive 72 h following treatment. At the time of the clinical examination, faecal and blood samples were taken. Faecal culture and commercial faecal antigen tests for detection of enteropathogens in faeces and serum endotoxin measurement using chromogenic lymulus amoebocyte lysate (LAL) were carried out. Scouring lambs received 0.07 mg/kg liveweight halofuginone once a day for 3 days, a single oral dose of 0.20 mg/kg liveweight of spectinomycin and oral rehydration fluid. The pathogens isolated were Cryptosporidium spp. and Escherichia coli. The case fatality rate was 51% in the scouring lambs. Postmortem findings were consistent with enterotoxigenic E. coli infection. The concentration of endotoxin was 0.18 +/- 0.12 ng/ml in the control group, 0.35 +/- 0.17 ng/ml in the surviving lambs and 0.46 +/- 0.14 ng/ml in the non-surviving lambs. Significant differences between groups were found. Case fatality rate of the scouring lambs with endotoxaemia below 0.30 ng/ml was 0%, while it was 100% above 0.50 ng/ml. These results may be utilized as a prognostic indicator in lambs affected by E. coli and Cryptosporidium that will help aid in decision-making as to whether to treat a lamb or not based on its chances of survival.

Topics: Age Factors; Animals; Animals, Newborn; Cryptosporidium; Decision Making; Diarrhea; Endotoxemia; Escherichia coli; Feces; Fluid Therapy; Piperidines; Prognosis; Protein Synthesis Inhibitors; Quinazolinones; Sheep; Sheep Diseases; Survival Analysis

Topics: Administration, Topical; Cell Proliferation; Gene Expression Regulation, Enzymologic; Humans; Keloid; Matrix Metalloproteinase 2; Models, Biological; Models, Theoretical; Piperidines; Protein Synthesis Inhibitors; Quinazolinones

It is a major interest in the field of hematopoietic stem cell transplantation to reduce scarring of healing wounds with overdeposition of collagen due to radiation injury or graft-versus-host disease. Halofuginone (HF) inhibits collagen alpha1(I) gene expression and overdeposition of collagen. We examined the effect of HF on the healing of full-depth incision wounds inflicted in normal skin or skin areas compromised by local preirradiation with 18 Gy. Preirradiation significantly decreased the tensile strength of the healing wounds at day 14 (by approximately 60%, p < 0.0001). In contrast, HF treatment did not significantly decrease the strength of wounds inflicted in both normal and preirradiated skin. Histological evaluation revealed that HF induced moderate thinning of the dermis accompanied by elevated thickness of the epidermis and enhanced rejoining of subdermal muscles in the wound area. HF only minimally reduced total collagen deposition in both groups, with minor changes in the level of more matured fibrillar collagen network. Our study demonstrates that HF does not significantly affect wound strength. This encourages the possible use of HF as an antifibrotic agent with minimal complications for post-hematopoietic stem cell transplantation complications including radiation toxicity and graft-versus-host disease.

Topics: Animals; Cicatrix; Collagen Type I; Drug Evaluation, Preclinical; Hematopoietic Stem Cell Transplantation; Mice; Mice, Inbred C3H; Piperidines; Quinazolinones; Skin; Specific Pathogen-Free Organisms; Tensile Strength; Transplantation Conditioning; Wound Healing; Wounds, Penetrating

Halofuginone, an alkaloid isolated from the plant Dichroa febrifuga, has been shown to be a potent inhibitor of tissue fibrosis. We herein demonstrate that, at concentrations below 10(-7) M, halofuginone does not affect the cell cycle but efficiently induces extracellular signal-regulated kinases(1,2) (ERK(1,2)), p38 and Jun NH2-terminal kinases(1,2) (JNK(1,2)) phosphorylation. In addition, at these non cytotoxic concentrations, halofuginone diminishes the capacity of fibroblasts to contract mechanically unloaded collagen lattices, an effect that is specifically blocked by the ERK inhibitors PD98059 and U0126, not by inhibitors of the JNK or p38 pathways. These data thus indicate that the inhibitory effect of halofuginone on fibroblast contractile activity, a key function for wound healing implicated in the development of tissue fibrosis, is an ERK-mediated mechanism.

Topics: Blotting, Western; Butadienes; Cell Line; Cell Proliferation; Cell Survival; Collagen Type I; Dose-Response Relationship, Drug; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flavonoids; Humans; Imidazoles; MAP Kinase Signaling System; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Piperidines; Protein Synthesis Inhibitors; Pyridines; Quinazolinones; Time Factors

Halofuginone inhibits fibrosis by decreasing type I collagen synthesis and tumor growth through an anti-angiogenic mechanism. In vitro data suggested that halofuginone inhibits angiogenesis through upregulating thrombospondin-1 (TSP-1) expression and by inhibiting cell proliferation. To determine whether thrombospondin-1 (TSP-1) is necessary for inhibition of tumor growth and angiogenesis by halofuginone, we tested the effect of halofuginone on mammary tumor growth in polyoma middle T antigen, TSP-1 null (TSP-1-/-PyT) transgenic mice. After 30 days of treatment, we found a significant decrease in tumor weight in these mice and the extent of tumor growth inhibition was comparable to that found in TSP-1 expressing PyT mice (TSP-1+/+PyT). However, no significant difference in tumor weight was observed after 60 days of halofuginone treatment between control and treated mice in both genotypes. Interestingly, type I collagen level was lower in the halofuginone treated TSP-1+/+PyT tumors at 30 days, but this was not observed in the TSP-1-/-PyT mice. Levels of type I collagen did not correlate with blood vessel number as a decrease in the number of vessels was observed in the halofuginone treated tumors from both the TSP-1+/+PyT and TSP-1-/-PyT mice as compared to control tumors. Because halofuginone has been shown to inhibit type I collagen synthesis by inhibiting the TGF-beta signaling pathway, we measured Smad 2/3 phosphorylation levels and found that halofuginone inhibited Smad 2/3 phosphorylation in cells derived from TSP-1+/+PyT tumors. We also found that it inhibited Smad 2/3 phosphorylation in cells treated with the TGF-beta activating sequence of TSP-1, TSR2+RFK. Our data demonstrate that halofuginone inhibits mammary tumor growth in a transgenic mouse model via a TSP-1 independent pathway, by decreasing tumor angiogenesis and by inhibiting TGF-beta signaling.

Topics: Animals; Antigens, Viral, Tumor; Antineoplastic Agents; Breast Neoplasms; Collagen Type I; Mice; Mice, Transgenic; Neovascularization, Pathologic; Piperidines; Polyomavirus; Quinazolines; Quinazolinones; Smad2 Protein; Smad3 Protein; Thrombospondin 1; Transforming Growth Factor beta; Tumor Burden

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

Topics: Animals; Cell Line; Cell Movement; Cell Proliferation; Cells, Cultured; Collagenases; Enzyme Induction; Hepatocytes; Liver Cirrhosis; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Piperidines; Quinazolines; Quinazolinones; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

Tyrosine phosphatase PRL-1 is one of the immediate-early genes up-regulated during liver regeneration and is apparently involved in cell proliferation. Previously, we have demonstrated that halofuginone, an inhibitor of collagen type I synthesis, prevents liver fibrosis and improves cirrhotic liver regeneration. In this study, we evaluated the effect of halofuginone on PRL-1 expression, its cellular localization in vitro and during liver regeneration, and fibrosis progression in vivo. In culture, halofuginone increased PRL-1 expression in primary rat hepatocytes and in hepatocellular carcinoma (HCC) cell lines, the former being more sensitive to halofuginone. The halofuginone-dependent increase in PRL-1 gene expression was correlated with an increase in the transcription factor early growth response-1 (Egr-1) and inversely correlated with the inhibition of cell proliferation. Halofuginone arrested HepG2 and Huh7 cell lines at the G1 phase, whereas Hep3B cells were arrested at G2/M, probably because of a reduction in the synthesis of cyclins D1 and B1 in all HCC cells and increased cyclin A in Hep3B cells. Halofuginone also affected the PRL-1 sub-cellular localization that was cell-cycle-dependent. In addition, halofuginone augmented PRL-1 expression in the remnant liver after partial hepatectomy and in chemically induced fibrosis in rats; this was accompanied by increased expression of insulin-like growth factor binding protein 1 (IGFBP-1), another immediate-early gene of regeneration. The regulation of the expression of the early genes of regeneration such as PRL-1 and IGFBP-1 is thus part of the mode of action of halofuginone and results in the prevention of liver fibrosis and improved cirrhotic liver regeneration.

Topics: Animals; Cell Cycle; Cell Proliferation; Cells, Cultured; Early Growth Response Protein 1; Hepatocytes; Immediate-Early Proteins; Insulin-Like Growth Factor Binding Protein 1; Liver Cirrhosis; Liver Regeneration; Male; Piperidines; Protein Tyrosine Phosphatases; Quinazolinones; Rats; Rats, Wistar

Interstitial fibrosis is the final common pathway of renal damage and represents an important therapeutic target. Halofuginone is a nontoxic alkaloid, used as a coccidiostat, and is a potent inhibitor of collagen alpha(1)(I) and matrix metalloproteinase-2 (MMP-2) expression. We thus studied the effects of halofuginone on proliferation, collagen I synthesis, and MMP-2 activity of rat renal papillary fibroblasts in culture.. Fibroblasts were isolated from rat renal papillae and studied during passages 3-4. The cell proliferation was studied in the presence of varying concentrations of halofuginone. The collagen synthesis was studied by [3H]proline uptake, before and after collagenase digestion, at varying concentrations of halofuginone. The MMP-2 activity was determined by zymography. The gelatinolytic activity was determined on gelatin-impregnated polyacrylamide gels containing samples of cell medium after incubation for 24 h with different halofuginone doses.. We studied a phenotype of papillary fibroblasts which stained positive for alpha smooth muscle actin. These cells are phenotypically myofibroblasts. Halufuginone inhibited the proliferation of these cells in a dose-related and reversible manner. Platelet-derived growth factor is known to stimulate fibroblast proliferation. Halofuginone at a concentration of 250 ng/ml almost completely abolished the effect of platelet-derived growth factor. It also almost completely inhibited the MMP-2 activity at doses of 250-350 ng/ml, as shown by zymography.. Halofuginone exhibits antifibrotic effects in rat renal papillary fibroblasts in culture, in terms of inhibition of proliferation and inhibition of MMP-2. These findings could have therapeutic potential.

Topics: Animals; Cell Proliferation; Cells, Cultured; Collagen Type I; Fibroblasts; Kidney Medulla; Male; Matrix Metalloproteinase Inhibitors; Piperidines; Platelet-Derived Growth Factor; Quinazolines; Quinazolinones; Rats; Rats, Wistar

Halofuginone is a low-molecular weight quinazolinone alkaloid coccidiostat that inhibits collagen type I synthesis, extracellular matrix deposition, and angiogenesis. This study was conducted to assess its potential in preventing subglottic stenosis (SGS).. We induced SGS in 10 dogs randomly divided into 2 groups. Each group received treatment between 3 days before and 21 days after the induction of SGS. One group received oral halofuginone 40 microg/kg, and the other was given placebo. The area of the subglottic lumen was measured at baseline and 3 months later. In addition, human tracheal fibroblasts were cultured. The inhibitory effect of halofuginone was compared to the effect of mitomycin.. All dogs survived throughout the study with no side effects. Three months after the operation, no halofuginone-treated dog had SGS, in contrast to a 66% to 80% stenosis rate (mean, 72%) in controls (p < .008). Thick fibrotic tissue was found in the placebo-treated larynges, whereas an almost normal architecture was observed in halofuginone-treated larynges. Halofuginone inhibited the growth of human tracheal fibroblasts by 75%, in comparison with 60% inhibition by mitomycin (no statistically significant difference).. This preliminary study shows that halofuginone is effective in preventing SGS caused by an acute injury. Halofuginone has a potential therapeutic role in preventing SGS in humans.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Dogs; Fibroblasts; Follow-Up Studies; Humans; In Vitro Techniques; Laryngostenosis; Mitomycin; Nucleic Acid Synthesis Inhibitors; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Treatment Outcome

Halofuginone, an inhibitor of collagen synthesis, appears to be a promising antitumoral drug in preclinical studies. We used a relevant rat model of autochthonous, chemically induced, spontaneously metastasizing hepatocellular carcinoma (HCC) to test the efficacy of halofuginone on tumor progression and matrix metalloproteinase (MMP) expression. Following sequential administration of diethylnitrosamine and N-nitrosomorpholine for 14 weeks, all animals developed HCC and then received halofuginone or its solvent for 10 weeks. The final number of liver tumors was lower in the halofuginone group than in the solvent group (57.2 +/- 4.6 vs 68 +/- 5.0; P < .01). The percentage of the lung surface infiltrated by metastasis was much smaller in the halofuginone group (0.3 +/- 0.2%) than in the solvent group (13.5 +/- 10.1%; P < .02). MMP-9 activity was decreased in the halofuginone group by 89% and 63% in non-neoplastic parts of the liver and tumor, respectively. The percentage of active MMP-2 was reduced by 90% in non-neoplastic parts of the liver and by 61% in tumors. This was likely subsequent to a decreased expression of both MMP-14 and tissue inhibitor of matrix metalloproteinase-2, which are required for pro-MMP-2 activation. These results, obtained from a clinically relevant model, further suggest the potential benefit of halofuginone in HCC.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Liver Neoplasms; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Neoplasm Metastasis; Piperidines; Quinazolines; Quinazolinones; Rats; Rats, Inbred F344

Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent.

Topics: Apoptosis; Chemotaxis; Cytokines; Dose-Response Relationship, Drug; Humans; Lymphocyte Activation; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Piperidines; Quinazolines; Quinazolinones; Signal Transduction; T-Lymphocytes

Ninety, seven- to 10-day-old calves were allocated to three groups of 30 and treated daily for seven days with either 100 microg/kg halofuginone hydrobromide or 2.5 mg/kg decoquinate orally or left untreated as controls. The levels of diarrhoea and dehydration were monitored daily for 28 days from the first day of treatment (day 0) and samples of faeces were collected on days 0, 7, 14, 21 and 28, to quantify the excretion of Cryptosporidium parvum oocysts. The calves were weighed on days 3 and 28. The treatments had no effect on the levels of diarrhoea or dehydration, the proportions of diarrhoeic calves or the proportions of calves shedding oocysts. However, unlike decoquinate, halofuginone significantly reduced the excretion of oocysts on day 7 (P<0.0001), and decoquinate increased the average daily weight gain of the calves (P=0.049).

Topics: Animals; Cattle; Cattle Diseases; Coccidiostats; Cryptosporidiosis; Cryptosporidium parvum; Decoquinate; Dehydration; Diarrhea; Feces; Female; Male; Parasite Egg Count; Piperidines; Quinazolinones; Random Allocation; Treatment Outcome; Weight Gain

Fibrous capsule formation around implants remains a difficult problem that has been studied for decades. The etiology is elusive, but the end result is the deposition of a dense collagenous capsule around implanted materials. The purpose of this study was to determine the effects of a type I collagen synthesis inhibitor, halofuginone, on fibrous capsule formation around implanted materials. Silastic disks were implanted subcutaneously into 4 groups of adult male rats for up to 8 weeks. Group 1 received drug throughout the study, group 2 received drug during the first half only, group 3 received drug during the second half only, and the control group received no drug. Implants were removed and histology of the capsules was examined. A collagen index score was calculated from digital images of trichrome-stained histologic sections, which permitted semiquantitative comparison of collagen content among the 4 groups. The collagen index values clearly indicate that halofuginone effectively inhibited collagen deposition within the capsule around the implanted disks. Halofuginone treatment also resulted in a decrease in the collagen index score in rat skin, indicating that halofuginone may affect preexisting collagenous structures. The ability of halofuginone to inhibit collagen deposition in new and preexisting fibrous capsules suggests that it may be a useful adjunct to minimize the formation of capsules around implantable prostheses.

Topics: Animals; Collagen Type I; Colorimetry; Fibrosis; Male; Muscles; Piperidines; Prostheses and Implants; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Silicone Elastomers; Subcutaneous Tissue; Transforming Growth Factor beta

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was < 0.5 microg kg(-1) in egg and < 1 microg kg(-1) in muscle. For dinitrocarbanilide, the CCbeta was estimated at < 3 microg kg(-1) in egg and < 10 microg kg(-1) in chicken muscle.

Topics: Animals; Antibodies; Carbanilides; Chickens; Coccidiostats; Cross Reactions; Drug Residues; Eggs; Enzyme-Linked Immunosorbent Assay; Food Contamination; Muscles; Nicarbazin; Piperidines; Quinazolines; Quinazolinones; Reproducibility of Results

Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of halofuginone, an inhibitor of collagen type I synthesis and angiogenesis, to inhibit WT development in xenografts models.. WTs derived from 2 patients with favorable histology at different disease stages were implanted subcutaneously or orthotopically in the kidneys of nude mice. Halofuginone was administered intraperitoneally (2 mug per mouse every other day) or given in the diet (1 part per million).. Independent of disease stage, tumor location or administration route, halofuginone caused a decrease in angiogenesis that resulted in marked inhibition of tumor development. This result was accompanied by a reduction in collagen synthesis, reduced levels of hepatocyte growth factor receptor MET and increased levels of the tumor suppressor protein WT1. In culture halofuginone increased the synthesis of WT1 in the human WT cell-line SK-NEP-1 and in other cancer cell lines such as hepatocellular carcinoma and prostate cancer. In SK-NEP-1 halofuginone also lowered erb B2 levels and reduced cell proliferation.. These results suggest that halofuginone is a potent inhibitor of WT progression. Because of its unique mode of action, halofuginone may decrease the treatment burden when combined with chemotherapy.

Topics: Angiogenesis Inhibitors; Animals; Cell Line; Child, Preschool; Collagen; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Injections, Intraperitoneal; Kidney Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Piperidines; Proto-Oncogene Proteins c-met; Quinazolines; Quinazolinones; Receptor, ErbB-2; Transplantation, Heterologous; Wilms Tumor; WT1 Proteins

Esophageal strictures caused by caustic injury continue to be a plaguing problem. Halofuginone (HF) has been proven to inhibit the formation of fibrosis in various animal models and human diseases. Its mechanism appears to be through the suppression of the production of collagen alpha1(I) and transforming growth factor-beta signaling pathway. We tried to assess whether HF would have an effect on the formation of strictures after inducing caustic esophageal.. Esophageal injury was caused by injecting 25% NaOH to an isolated esophageal segment. Study group rats were treated with HF orally for 3 consecutive days before the injury and afterward. Control group rats received regular chow. The results were evaluated by upper gastrointestinal series (UGI) and through pathologic studies.. HF treatment resulted in marked improvement in the esophageal strictures. The UGI series showed esophageal patency of 73% (45%-100%) in the treated animals (n = 7) as compared with almost no patency, 11% (5-16%), in the controls (n = 4) (P = .018). The histologic examination showed significantly less stricture and scarring in the treated group. Whereas the ratio between the esophageal wall thickness to mucosal thickness was 2.34 +/- 0.23 in the study group, the control group had a ratio of 9.56 +/- 0.69 (P = .0044). Finally, whereas 86% of the study group survived, all the rats in the control group died by day 20.. HF modulated the wound healing reaction caused by caustic injury of the esophagus in a rat model, resulting in increased esophageal patency, reduction in esophageal wall thickness, and increased survival.

Topics: Animals; Burns, Chemical; Caustics; Collagen Type I; Esophageal Stenosis; In Vitro Techniques; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Radiography; Rats; Sodium Hydroxide; Transforming Growth Factor beta; Wound Healing

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.

Topics: Animals; Blotting, Northern; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cells, Cultured; COS Cells; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Fibrosis; Gene Deletion; Genes, Reporter; Humans; Immunoblotting; MAP Kinase Signaling System; Mice; Mice, Inbred C3H; Microscopy, Confocal; Microscopy, Fluorescence; Piperidines; Plasmids; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Radiation Pneumonitis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad3 Protein; Time Factors; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Halofuginone, an inhibitor of collagen synthesis, prevented and caused resolution of established hepatic fibrosis. A genomic approach in vivo was used to search for additional genes responsible for halofuginone mode of action.. Fibrosis was induced in rats by thioacetamide (TAA) and evaluated by collagen type I gene expression and the levels of collagen, tissue inhibitors of metalloproteinases-2 and smooth-muscle actin. Halofuginone was given in the diet. cDNA from liver biopsies was hybridized on Atlas arrays comprising of 588 genes. The results were confirmed by Northern blots and in situ hybridization.. Insulin-like growth factor binding protein-1 (IGFBP-1) was one of the 13 genes differentially expressed in the fibrotic liver after halofuginone treatment. After 2 and 4 weeks, halofuginone prevented the TAA-induced down-regulation of IGFBP-1 gene expression. Halofuginone also prevented the TAA-dependent changes in IGFBP-3 gene expression. Halofuginone affected IGFBP-1 synthesis in rat hepatocytes and cells of hepatocyte origin and caused time- and dose-dependent increases in the IGFBP-1 gene expression and synthesis by HepG2 cells. The IGFBP-1 secreted by HepG2-inhibited stellate cell motility.. Halofuginone is an anti-fibrotic drug that inhibits collagen synthesis by stellate cells and preventing alteration in the synthesis of IGFBPs by hepatic cells.

Topics: Animals; Cell Movement; Collagen Type I; Hepatocytes; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Male; Oligonucleotide Array Sequence Analysis; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Thioacetamide; Tissue Inhibitor of Metalloproteinase-2

To assess the effects of halofuginone, a specific inhibitor of synthesis of collagen type 1, which is the major constituent of fibrosis, on esophageal stricture formation due to caustic ingestion.. Sixty rats were divided into four equal groups: control group; sham laparotomy group; caustic injury without treatment group; caustic injury with halofuginone treatment group. Caustic injuries were done by 50% sodium hydroxide. Halofuginone was administered by the first postoperative day. All animals were sacrificed on day 21; and the results were evaluated by hydroxyproline levels, stenosis index, lumen diameter, histopathological evaluation, wall thickness, and animal weights.. Mortality differences were significant comparing group 3 with group 1 and 2 (P = 0.006) and group 4 (P = 0.03). According to hydroxyproline levels, the differences are significantly higher (P <0.001) comparing group 3 with group 1, 2, and 4. The P value was considered significant in all other parameters (P <0.001) for all the groups but group 1 versus group 2 (P >0.05).. Halofuginone, a specific inhibitor of collagen type 1 synthesis, significantly reduced esophageal stricture occurrence.

Topics: Animals; Burns, Chemical; Caustics; Collagen Type I; Esophageal Stenosis; Esophagus; Fibrosis; Injections, Intraperitoneal; Male; Models, Animal; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Sodium Hydroxide

Halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha 1 (I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and fibroblast proliferation. It was recently shown to be effective in suppression of bladder carcinoma and glioma. This study sought to evaluate the effect of treatment with halofuginone on growth of hepatocellular carcinoma (HCC) in mice. Athymic Balb/c mice were injected subcutaneously with 10(7) human hepatoma cells (Hep3B), followed by treatment with halofuginone administered in the diet (750 microg/kg) starting on day 3, before tumour innoculation. The control group was received a normal diet. Mice were followed for survival, tumour volume and serum alpha-fetoprotein (alpha FP). The mechanism of the anti-tumour effect of halofuginone was determined in vitro by assessing tumour cell growth, and by measuring the serum concentrations of interferon-gamma (IFN gamma) and interleukin 2 (IL2). Halofuginone treatment induced almost complete tumour suppression in treated mice. Mortality rates were 10% and 50%, in halofuginone-treated and control mice, respectively (P<0.001). No visible tumour was observed in treated mice, as compared with a 364 mm3 tumour in control mice. Serum alpha FP were 0.1 and 212 ng/ml in treated and control mice, respectively (P<0.005). Halofuginone significantly inhibited HCC proliferation in vitro. Maximal inhibition of 64% of tumour cell growth was observed at a concentration of 10(-8) M. The anti-tumour effect was mediated via a significant increase in IFN gamma and IL2 (90 vs. 35, and 210 vs. 34 pg/ml in treated and control groups, respectively, P<0.005). Treatment with halofuginone effectively suppressed the progression of HCC in mice. This effect may be associated with a direct anti-tumour effect, and/or enhancement of a systemic immune response.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Coccidiostats; Drug Screening Assays, Antitumor; Humans; Interferon-gamma; Interleukin-2; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Piperidines; Quinazolines; Quinazolinones

Small-intestinal submucosa (SIS) has been successful as an onlay graft in ureteral repair, but tubularized segment interposition of SIS has been unsuccessful. Our objective was to evaluate whether a type I collagen inhibitor, halofuginone, would prevent stricture formation in tubularized SIS interposition.. We performed either laparoscopic partial ureteral excision followed by an SIS onlay graft (N = 5) or complete laparoscopic ureteral excision followed by an SIS interposition graft (N = 7) in domestic pigs. Animals received either no (N = 3), low-dose (N = 5), or high-dose (N = 4) halofuginone. Animals had ureteral stenting for 2 weeks after surgery and were permitted to survive for 6 or 9 weeks. An intravenous urogram (IVU) was performed prior to sacrifice. Kidneys were examined grossly and histologically.. One animal that received an onlay graft died of an unrelated illness. The remaining four ureteral onlay animals, including one control and two low-dose and one high-dose pig, had grossly normal kidneys at harvest. The IVU was normal in the control and high-dose animal but showed delayed excretion with mild hydroureteronephrosis in the low-dose animals. Pathologic examination of the SIS site revealed circumferential reepithelialization with inflammation and mild fibrosis. All seven tubularized interposition graft kidneys demonstrated either severe hydroureteronephrosis (N = 5) or renal atrophy (N = 2), and all had complete obstruction on IVU. Pathologic examination revealed a stenotic ureteral lumen with extensive surrounding inflammation and fibrosis.. An SIS onlay graft was successful in the porcine model of ureteral injury. Halofuginone, a type I collagen inhibitor, did not demonstrate a significant beneficial effect in this technique. Ureteral tubularized interpositions with SIS are unsuccessful and not improved by halofuginone.

Topics: Animals; Collagen Type I; Female; Intestinal Mucosa; Intestine, Small; Piperidines; Postoperative Complications; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Swine; Ureter; Ureteral Obstruction

A sensitive and very selective high-performance liquid chromatography/tandem mass spectrometric (LC/MS/MS) method for the detection of halofuginone in whole egg has been developed. After deproteinisation with acetonitrile and evaporation of the organic solvent, halofuginone was further isolated by applying immunoaffinity chromatography. The concentrated eluent was injected into the LC/MS/MS system on a C18 column. The precursor ion ([M+H]+) produced by positive electrospray ionisation was selected for fragmentation with argon. Validation parameters such as recovery, linearity and repeatability, decision limit (CCalpha) and detection capability (CCbeta) were determined.

Topics: Animals; Chickens; Chromatography, Affinity; Chromatography, High Pressure Liquid; Coccidiostats; Eggs; Piperidines; Quinazolines; Quinazolinones; Spectrometry, Mass, Electrospray Ionization

An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 micro Polar RP 80A (4 microm, 100 mm x 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.

Topics: Animals; Chromatography, Liquid; Coccidiostats; Dogs; Indicators and Reagents; Mass Spectrometry; Molecular Weight; Piperidines; Quinazolines; Quinazolinones; Reference Standards; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization

Halofuginone (HF) is an antifibrotic agent in rat models of liver fibrosis caused by repetitive intoxications. A beneficial effect of HF on a biliary type of liver fibrosis has not been proven yet.. Bile duct-obstructed rats were given HF from the moment of obstruction onwards and compared with no treatment. After 3 weeks, respectively, 6 weeks, aminopyrine breath test (ABT) and haemodynamic measurements including of portal pressure were carried out. Liver pieces were taken for Sirius red quantitative scoring, as well as for semiquantitative determinations of collagen type I and III RNA levels.. ABT was significantly worse in HF-treated rats as compared with no treatment (P=0.02). Haemodynamic data and collagen type I and III determinations were not significantly different between groups. Biliary fibrosis scores were significantly higher in HF-treated rats as compared with no treatment (P=0.03). More Sirius red staining was associated with more proliferation of bile ductules.. HF may worsen biliary fibrosis. This contrasts sharply with antifibrotic effects in other models of liver fibrosis. Distinctive cellular mechanisms in biliary fibrosis may explain this discrepancy. One should be cautious for chronic application of HF in man with cholestasis.

Topics: Aminopyrine; Animals; Bile Ducts; Breath Tests; Cholestasis, Extrahepatic; Collagen Type I; Collagen Type III; Disease Models, Animal; Ligation; Liver; Liver Cirrhosis, Biliary; Liver Cirrhosis, Experimental; Male; Piperidines; Portal Pressure; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Topics: Animals; Antineoplastic Agents; Coccidiosis; Coccidiostats; Drugs, Chinese Herbal; Humans; Phytotherapy; Piperidines; Poultry; Poultry Diseases; Quinazolines; Quinazolinones; Veterinary Drugs

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.

Topics: Calibration; Coccidiostats; Eggs; Liver; Piperidines; Quinazolines; Quinazolinones; Spectrometry, Mass, Electrospray Ionization

Halofuginone has recently been shown to inhibit tumor progression of various types of cancers. The antitumoral effect was associated with decreased tumor angiogenesis rather than a direct cytostatic effect on the tumor cells. The antiangiogenic action of the drug could be related to its inhibition of collagen type I synthesis, inhibition of matrix metalloproteinases (MMPs), or via both mechanisms because both collagen synthesis and MMP activity have been shown to be involved in angiogenesis. Vascular endothelial growth factor (VEGF), in addition to its effect on endothelial cell proliferation, has been shown to be a potent inducer of MMP expression. Because von Hippel-Lindau (VHL)-associated tumors express high levels of VEGF, it was of interest to ascertain the potential usefulness of halofuginone for treatment of these tumors. Pheochromocytoma tissue fragments obtained at surgery from a VHL type 2a patient were propagated s.c. in male BALB/c nu/nu (nude) mice. For experiments, 2-3-mm tumor fragments were transplanted secondarily s.c. to nude mice. Two treatment groups received halofuginone in standard lab chow at 3 and 5 ppm; control animals received regular chow. All groups were followed for 6 weeks after transplantation. A marked and significant diminution of tumor size and weight was observed in the drug-treated animals (>90% reduction of mean tumor volume for both the 3 and 5 ppm groups). In vivo magnetic resonance imaging analysis of tumors in halofuginone-treated animals showed a significant reduction of vascular functionality. Immunohistochemical studies revealed decreased collagen type I levels and vascular density in treated tumors and gelatinase assays of tumor extracts revealed a reduction of MMP-2 and MMP-9 activity in halofuginone-treated cells. Taken together, our data indicate that therapy directed at blocking MMP activity (presumably related to excessive VEGF expression in VHL) and reduction of type I collagen deposition curtails angiogenesis and thereby presumably tumor growth in this model system.

Topics: Animals; Antineoplastic Agents; Cell Division; Collagen; Collagen Type I; Disease Progression; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Pheochromocytoma; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Temperature; Time Factors; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor A; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein

We developed a reproducible animal model for the induction of urethral stricture in the rabbit and evaluated the role of halofuginone in limiting stricture formation.. A total of 20 New Zealand male rabbits were used in the first phase of the experiment. Bulbar urethral stricture was induced by electrocoagulation. The animals were then randomly assigned to 2 groups of 10 each, which received a diet containing halofuginone or a normal diet. In the second phase electrocoagulation induced stricture was treated with visual internal urethrotomy in 45 rabbits. These rabbits were randomly assigned to 2 groups, namely a halofuginone and a control group.. In the first phase stricture developed in 2 study rabbits (20%) vs 10 controls (100%). In the second phase 37 rabbits were evaluable (8 died). Recurrent stricture was observed in 5 of the 18 study rabbits (27%) vs 14 of the 19 controls (73%).. Halofuginone is effective in limiting the occurrence of de novo urethral stricture and recurrent stricture after visual internal urethrotomy. This antifibrotic molecule may become an important therapy to treat urethral stricture and/or recurrence following endoscopic manipulation of stricture in humans.

Topics: Administration, Oral; Animals; Electrocoagulation; Fibrosis; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rabbits; Secondary Prevention; Urethra; Urethral Stricture

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.

Topics: Angiogenesis Inhibitors; Animals; Antimetabolites, Antineoplastic; Bleomycin; Blotting, Northern; Bronchoalveolar Lavage Fluid; Cell Count; Collagen; Fibroblasts; Gene Expression Regulation; Hydroxyproline; Intubation, Intratracheal; Lung; Male; Piperidines; Pulmonary Fibrosis; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA; Transforming Growth Factor beta

Hepatic fibrosis involves excess deposition of extracellular connective tissue of which collagen type I fibers form the predominant component. Left untreated it develops into cirrhosis, often linked with hepatocellular carcinoma. Owing to the fact that cirrhotic liver regeneration is impaired, resection of hepatocellular carcinoma associated with cirrhosis is questionable. The aim of the present study was to determine the potential of halofuginone, a collagen type I inhibitor, in improving liver regeneration in cirrhotic rats.. Partial hepatectomy (70%) was performed in thioacetamide-induced cirrhotic rats fed a halofuginone-containing diet. Liver regeneration was monitored by mass and proliferating cell nuclear antigen. The Ishak staging system and hydroxyproline content were used to evaluate the level of fibrosis.. Halofuginone administered prior to and following partial hepatectomy did not inhibit normal liver regeneration despite the reduced levels of collagen type I mRNA. When given to rats with established fibrosis, it caused a significant reduction in alpha smooth muscle actin, TIMP-2, collagen type I gene expression and collagen deposition. Such animals demonstrated improved capacity for regeneration.. Halofuginone may prove useful in improving survival of patients with hepatocellular carcinoma and cirrhosis undergoing surgical resection.

Topics: Animals; Collagen Type I; Extracellular Matrix; Liver Cirrhosis; Liver Regeneration; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Thioacetamide

The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor beta signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription.. The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying alpha2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on alpha2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry.. Treatment of fibroblasts with 10(-8)M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the -3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen alpha2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression.. Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun-dependent manner.

Topics: Administration, Topical; Animals; Cells, Cultured; Collagen; Collagen Type I; Drug Synergism; Fibroblasts; Gene Expression; Male; Mice; Mice, Mutant Strains; Mitogens; Phosphorylation; Piperidines; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-jun; Quinazolines; Quinazolinones; RNA, Messenger; Scleroderma, Systemic; Transcription Factor AP-1; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1

The end point of pathogenic events in scleroderma is fibrosis of the skin and internal organs. Fibrosis in scleroderma results from the over synthesis and deposition of collagen in the connective tissue. The morbidity and mortality of the scleroderm is very high and presently there is no specific treatment. Halofuginone is a drug with great potential for the treatment of scleroderma since it inhibits the synthesis of collagen type I by fibroblasts. We have studied the in vivo effect of halofuginone in tight skin (TSK) mice that spontaneously develop a scleroderma-like disease due to a genetic defect. Our results demonstrate that halofuginone prevented the occurrence of skin sclerosis when administered to newborn mice and reduced cutaneous hyperplasia when administered in adult TSK mice. These effects correlated with a decreased number of cells synthesizing collagen gene transcripts and a reduction in the level of autoantibodies specific for human target antigens. These results indicate that halofuginone may have use as a therapeutic in the treatment of fibrotic disease.

Topics: Animals; Animals, Newborn; Collagen; Disease Models, Animal; Fibrosis; Humans; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Scleroderma, Systemic; Skin Diseases

Halofuginone is a drug that has been shown to have an antifibrotic property in vitro and in vivo. Whereas halofuginone shows promise as a therapeutic agent for a variety of diseases including scleroderma, liver cirrhosis, cystic fibrosis, and certain types of cancer, the mechanism of action remains unknown. Using the tight skin mouse (TSK) model for scleroderma, we evaluated the ability of halofuginone to inhibit spontaneous development of dermal fibrosis. We found that administration of a low dose of halofuginone both in adult and newborn animals for 60 d prevented the development of cutaneous hyperplasia (dermal fibrosis). In vitro halofuginone was found to reduce the amount of collagen synthesized by fibroblasts. This effect was due to a reduction in the promoter activity of the type-I collagen genes as treatment of fibroblast cultures with 10(-8) M halofuginone reduced the level of alpha2(I) collagen message detectible by northern blot and greatly reduced the activity of a reporter construct under control of the -3200 to +54 bp alpha2(I) collagen promoter. In addition, analysis of transforming growth factor beta signaling pathways in fibroblasts revealed that halofuginone inhibited transforming-growth-factor-beta-induced upregulation of collagen protein and activity of the alpha2(I) collagen promoter. Further we found that halofuginone blocked the phosphorylation and subsequent activation of Smad3 after transforming growth factor beta stimulation. Apparently the inhibitory property was specific to Smad3 as there was no inhibitory effect on the activation of Smad2 after stimulation with transforming growth factor beta. Our results demonstrate that halofuginone is a specific inhibitor of type-I collagen synthesis and may elicit its effect via interference with the transforming growth factor beta signaling pathway.

Topics: Animals; Cells, Cultured; Collagen; Collagen Type I; DNA-Binding Proteins; Fibroblasts; Gene Expression; Hyperplasia; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Piperidines; Quinazolines; Quinazolinones; Receptors, Transforming Growth Factor beta; Sclerosis; Signal Transduction; Skin; Skin Diseases; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Halofuginone, an inhibitor of collagen type I synthesis, is an anti-angiogenic agent. Here we evaluated the efficacy of halofuginone to inhibit prostate cancer (PC) xenografts representing various phenotypes of the disease.. An androgen-dependent (CWR22), an androgen-independent (PC3), and a neuroendocrine (WISH-PC2) PC xenograft were used. Halofuginone was given orally or injected intraperitoneally. Tumor size, collagen alpha1(I) gene expression (in situ hybridization), collagen content (sirius red staining), angiogenesis (immunohistochemistry with factor VIII antibodies), and apoptosis/necrosis (DNA fragmentation) were evaluated.. Halofuginone inhibited the growth of all subcutaneously implanted xenografts and of WISH-PC2 when transplanted orthotopically. The effect was dose-dependent (WISH-PC2) and accompanied by decrease in plasma PSA levels (CWR22). In all xenografts, halofuginone inhibited collagen alpha1(I) gene expression, reduced collagen content, and endothelial cell number resulting in an increase in apoptosis/necrosis.. Oral administration of halofuginone slowed the progression of PC xenografts representing a broad range of phenotypes. Halofuginone may become a new modality for PC prevention.

Topics: Adenocarcinoma; Administration, Oral; Androgens; Animals; Antineoplastic Agents; Apoptosis; Collagen Type I; Disease Progression; DNA, Neoplasm; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Injections, Intraperitoneal; Male; Mice; Mice, SCID; Necrosis; Neovascularization, Pathologic; Phenotype; Piperidines; Prostatic Neoplasms; Quinazolines; Quinazolinones

Topics: Animals; Bleomycin; Collagen; Disease Models, Animal; Female; Injections, Intraperitoneal; Injections, Subcutaneous; Mice; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Scleroderma, Systemic; Specific Pathogen-Free Organisms; Treatment Outcome

Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone-an inhibitor of collagen synthesis-on thioacetamide (TAA)-induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin-positive cells expressing high levels of the collagen alpha1(I) gene and containing high levels of TIMP-2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen alpha1(I) gene, resulting in low levels of collagen. The level of TIMP-2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen alpha1(I) gene expression, TIMP-2 content, and smooth muscle actin-positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen alpha1(I) gene expression in the SV40-immortalized rat HSC-T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.

Topics: Animals; Cell Division; Liver; Liver Cirrhosis; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Thioacetamide

The effect of dermal application of halofuginone-an inhibitor of collagen type I synthesis-on skin collagen and collagen alpha1(I) gene expression in an animal model of scleroderma and chronic graft versus host disease (cGvHD) was evaluated. Halofuginone-containing cream was applied on the tight-skin mouse (Tsk) and skin biopsies were taken for collagen staining by sirius red and for collagen alpha1(I) gene expression by in situ hybridization. In addition, cell proliferation was evaluated by immunostaining for proliferation cell nuclear antigen (PCNA) alone or in combination with collagen alpha1(I) probe. The number of mast cells was assessed by toluidine blue. Dermal application of halofuginone (0.01%) for 60 days was as good as systemic administration (1 microg/mouse/day) in reducing collagen alpha1(I) gene expression in skin biopsy and almost as good in reducing skin width. Halofuginone was stable and effective only at acidic pH. The effect of halofuginone (0.03%) was time-dependent. After 40 days of daily treatment, a significant reduction in the collagen alpha1(I) gene expression was observed and further decrease was observed after 60 days. The reduction in collagen alpha1(I) gene expression and the reduction in the proliferation of dermal fibroblasts probably occur in the same subset of cells. No effect of halofuginone on the proliferation of keratinocytes or on mast cell number was observed. These results suggest that target-oriented application of halofuginone may become a novel therapy for fibrotic disorders in general and for scleroderma in particular.

Topics: Administration, Topical; Animals; Cell Division; Collagen; Disease Models, Animal; Fibrosis; Male; Mast Cells; Mice; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Skin

Halofuginone (HF) inhibits synthesis of collagen type I and matrix metalloproteinase-2 and is being considered for clinical evaluation as an antineoplastic agent. Pharmacokinetic studies were performed in rodents to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of HF after i.v. delivery and the bioavailability of HF after i.p. and oral delivery.. Studies were performed in CD2F1 mice and Fischer 344 rats. In preliminary toxicity studies in mice single HF i.v. bolus doses between 1.0 and 5.0 mg/kg were used. Pharmacokinetic studies were conducted in mice after administration of 1.5 mg/kg HF. In preliminary toxicity studies in male rats HF i.v. bolus doses between 0.75 and 4.5 mg/kg were used. In pharmacokinetic studies in rats an HF dose of 3.0 mg/kg was used. Compartmental and non-compartmental analyses were applied to the plasma concentration versus time data. Plasma, red blood cells, various organs, and urine were collected for analysis.. HF doses > or = 1.5 mg/kg proved excessively toxic to mice. In mice, i.v. bolus delivery of 1.5 mg/kg HF produced "peak" plasma HF concentrations between 313 and 386 ng/ml, and an AUC of 19,874 ng/ml min, which corresponded to a total body clearance (CLtb) of 75 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. The bioavailability of HF after i.p. and oral delivery to mice was 100% and 0%, respectively. After i.v. bolus delivery to mice, HF distributed rapidly to all tissues, except brain. HF persisted in lung, liver, kidney, spleen, and skeletal muscle longer than in plasma. In the oral study, HF was undetectable in plasma and red blood cells, but was easily detectable in kidney, liver, and lung, and persisted in those tissues for 48 h. Urinary excretion of HF accounted for 7-11% of the administered dose within the first 72 h after i.v. dosing and 15-16% and 16% of the administered dose within 24 and 48 h, respectively, after oral dosing. There were no observed metabolites of HF in mouse plasma or tissues. In rats, i.v. bolus delivery of 3.0 mg/kg produced a "peak" plasma HF concentration of 348 ng/ml, and an AUC of 43,946 ng/ml min, which corresponded to a CLtb of 68 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. After i.v. bolus delivery to rats, HF distributed rapidly to all tissues, with low concentrations detectable in brain and testes. HF was detectable in some tissues for up to 48 h. HF could be detected in rat plasma after a 3 mg/kg oral dose. Peak HF concentration (34 ng/ml) occurred at 90 min, but HF concentrations were less than the lower limit of quantitation (LLQ) by 420 min. Urinary excretion of HF accounted for 8-11% of the administered dose within the first 48 h after i.v. dosing. No HF metabolites were detected in plasma, tissue, or urine.. HF was rapidly and widely distributed to rodent tissues and was not converted to detectable metabolites. In mice, HF was 100% bioavailable when given i.p. but could not be detected in plasma after oral administration, suggesting limited oral bioavailability. However, substantial concentrations were present in liver, kidney, and lungs. HF was present in rat plasma after an oral dose, but the time course and low concentrations achieved precluded reliable estimation of bioavailability. These data may assist in designing and interpreting additional preclinical and clinical studies of HF.

Topics: Animals; Antineoplastic Agents; Blood Proteins; Chromatography, High Pressure Liquid; Male; Mice; Piperidines; Protein Binding; Quinazolines; Quinazolinones; Rats; Rats, Inbred F344; Tissue Distribution

When treating a ureteral narrowing by endopyelotomy or endoureterotomy, the urologist hopes that after the narrowed area is cut, it will heal with a patent lumen. A ureteral stent is usually left in place to provide a framework for healing. In cases of failure, histologic examination shows a predominance of collagen. We evaluated the effects of epidermal growth factor (EGF) and a collagen synthesis inhibitor, halofuginone, as adjuncts to ureteral healing.. Ten female pigs underwent ureteroureteral anastomosis bilaterally, with the left side being stented. They were then randomized to receive EGF (N = 4), halofunginone (N = 3), or saline (control; N = 3). On postoperative day 30, the ureters were examined grossly and histologically.. The lumens of the ureters were significantly larger in the animals that received either EGF or funginone than in the control animals. The epithelium was significantly thicker in the animals that received halofunginone than in the controls or animals receiving EGF. The thickness of the smooth muscle and adventitia was similar in the three groups. Stenting improved the results.. Both EGF and halofuginone show promise as adjuncts to endopyelotomy and endoureterotomy.

Topics: Adjuvants, Pharmaceutic; Anastomosis, Surgical; Animals; Collagen; Drug Evaluation; Epidermal Growth Factor; Female; Piperidines; Quinazolines; Quinazolinones; Random Allocation; Swine; Ureter; Wound Healing

Urethral strictures are narrowing of the urethra caused by fibrosis due to excessive collagen production in response to an insult. We evaluated the effects of halofuginone, a potent inhibitor of collagen alpha1(I) gene expression, on experimentally induced urethral strictures in vivo and on rat urethral fibroblasts in vitro.. Applying coagulation current to the male rat urethra produced urethral strictures. Halofuginone was given to the animals for 7 days, starting on the day of stricture formation, either orally at 1 and 5 ppm in the diet or by injection of 0.03% halofuginone solution into the urethra. All rats were sacrificed on day 21. Collagen alpha1(I) gene expression was evaluated by in situ hybridization, collagen content by sirius red staining and urethral morphology by urethrogram.. Coagulation current produced reproducible strictures with a typical urethrogram appearance, which were associated with increases in collagen alpha1(I) gene expression and collagen content at the stricture site. Halofuginone injected into the urethra or orally at 5 ppm normalized the urethrogram and prevented increases in collagen alpha1(I) gene expression and collagen content. Halofuginone at a concentration of 10-8 M. inhibited the collagen secreted by fibroblasts derived from the rat male urethra, which was due to inhibition of the collagen alpha1(I) gene expression.. Halofuginone prevented stricture formation and may become an important mode of therapy in the prevention of restenosis during urethral stricture formation.

Topics: Animals; Collagen; Disease Models, Animal; Immunohistochemistry; In Situ Hybridization; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Urethral Stricture

Leiomyomas (fibroids) are benign smooth-muscle cell (SMC) tumors of the uterus and are the most common pelvic tumors in women. These tumors occur primarily during the reproductive years and are the most common indication for hysterectomy in women. Unfortunately the only effective treatments for leiomyomas and the associated abnormal uterine bleeding are surgical, involving either hysterectomy, myomectomy, or hysteroscopic removal of the tumors. The goal of this paper is to discuss recent research findings that support the idea of using therapeutic compounds that block the actions of specific growth factors as therapeutic agents for treatment of leiomyomas and abnormal uterine bleeding. Most of the studies were carried out using cell cultures of leiomyoma or myometrial SMCs. Primary cultures of SMCs provide a system for investigation of the roles of growth factors and their receptors in proliferation of normal myometrial and leiomyoma SMCs. Several growth factors have been shown to be present and to have regulatory roles in the proliferation of uterine SMCs. Bioassay and Western blotting of fast protein liquid chromatography fractions of tissue extracts identified platelet-derived growth factor, heparin-binding epidermal growth factor, hepatoma-derived growth factor, and basic fibroblast growth factor in normal myometrium and fibroid tumors. The presence of heparin-binding growth factors suggests a possible focus for therapeutic agents. RG13577 (a heparinlike compound) and halofuginone (an alkyloid) reversibly inhibited DNA synthesis of normal myometrial and leiomyoma cells without toxic effects. Pirfenidone, a known antifibrotic drug, inhibited DNA synthesis and synthesis of collagen type I mRNA in normal and fibroid cells, and decreased collagen type III mRNA only in normal myometrial cells. Another hopeful therapeutic candidate, interferon-Alpha, significantly inhibited growth factor-stimulated proliferation in both normal and leiomyoma cells. These results suggest that future nonsurgical treatments for leiomyomas may include compounds that block the actions of specific growth factors that regulate proliferation and collagen production by uterine SMCs.

Topics: Antineoplastic Agents; Blotting, Western; Chromatography, Liquid; Female; Growth Inhibitors; Growth Substances; Humans; Hysterectomy; Hysteroscopy; Immunohistochemistry; Interferon-alpha; Leiomyoma; Phenoxyacetates; Piperidines; Polymers; Protein Synthesis Inhibitors; Pyridones; Quinazolines; Quinazolinones; Receptors, Growth Factor; Uterine Neoplasms

We have previously demonstrated that halofuginone, a low molecular weight quinazolinone alkaloid, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also effectively suppresses tumor progression and metastasis in mice. These results together with the well-documented role of extracellular matrix (ECM) components and matrix degrading enzymes in formation of new blood vessels led us to investigate the effect of halofuginone on the angiogenic process. In a variety of experimental system, representing sequential events in the angiogenic cascade, halofuginone treatment resulted in profound inhibitory effect. Among these are the abrogation of endothelial cell MMP-2 expression and basement membrane invasion, capillary tube formation, and vascular sprouting, as well as deposition of subendothelial ECM. The most conclusive anti-angiogenic activity of halofuginone was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor (bFGF) -induced neovascularization in response to systemic administration of halofuginone, either i.p. or in the diet. The ability of halofuginone to interfere with key events in neovascularization, together with its oral bioavailability and safe use as an anti-parasitic agent, make it a promising drug for further evaluation in the treatment of a wide range of diseases associated with pathological angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Aorta; Capillaries; Cattle; Dose-Response Relationship, Drug; Endothelium, Vascular; Fibroblast Growth Factor 2; Matrix Metalloproteinase 2; Neovascularization, Physiologic; Piperidines; Quinazolines; Quinazolinones; Rats

The objective of this study was to evaluate the effects of halofuginone-a specific inhibitor of collagen type I synthesis-in preventing uterine horn adhesion formation in rats.. Adhesions were induced by scraping the rat uterine horns until capillary bleeding occurred. Halofuginone was either injected intraperitoneally or administered orally. The number and severity of the adhesions were scored. Collagen alpha1(I) gene expression was evaluated by in situ hybridization; total collagen was estimated by sirius red staining. Collagen synthesis in response to halofuginone was evaluated in cells cultured from the adhesions.. Regardless of the administration procedure, halofuginone reduced significantly the number and severity of the adhesions in a dose-dependent manner. Halofuginone prevented the increase in collagen alpha1(I) gene expression observed in the rats that underwent this procedure, thus affecting only the newly synthesized collagen but not the resident collagen. In cells derived from rat uterine horn adhesions, halofuginone induced dose-dependent inhibition of collagen synthesis.. Upregulation of collagen synthesis appears to play a critical role in the pathophysiologic mechanism of adhesion formation. Halofuginone could be used as an important means of understanding the role of collagen in adhesion formation and might become a novel and promising antifibrotic agent for preventing adhesion formation after pelvic surgery.

Topics: Administration, Oral; Animals; Collagen; Female; Gene Expression; In Situ Hybridization; Injections, Intraperitoneal; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Tissue Adhesions; Uterine Diseases

We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.

Topics: Animals; Antineoplastic Agents; Cell Division; Humans; Male; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Stromal Cells; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.

Topics: Animals; Antineoplastic Agents; Carcinoma; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Gelatin; Humans; Laminin; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Neoplasm Transplantation; Piperidines; Promoter Regions, Genetic; Proteoglycans; Quinazolines; Quinazolinones; Tumor Cells, Cultured; Urinary Bladder Neoplasms

In chronic graft-versus-host disease (cGvHD), skin fibrosis, contractures, and an increase in collagen content form the hallmark. We report a successful treatment of a cGvHD patient by topical application of halofuginone, an inhibitor of collagen alpha1(I) gene expression.. Halofuginone-containing ointment was applied daily on the left side of the neck and shoulder of a cGvHD patient. Collagen alpha1(I) gene expression and collagen content in skin biopsy specimens were evaluated by in situ hybridization and sirius red staining, respectively.. After 3 and 6 months, a marked reduction in skin collagen synthesis was observed, accompanied with increase neck rotation on the treated side. After cessation of treatment, the sclerosis, skin tightness, and collagen alpha1(I) gene expression returned to baseline level. No adverse effects were observed, and no plasma levels of halofuginone could be detected.. Halofuginone may provide a promising novel and safe therapy for cGvHD patients.

Topics: Administration, Topical; Adult; Animals; Collagen; Gene Expression; Graft vs Host Disease; Humans; Male; Neck; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rabbits; Range of Motion, Articular; Sclerosis; Skin; Skin Transplantation

Halofuginone, an inhibitor of collagen alpha1(I) gene expression was used for the treatment of subcutaneously implanted C6 glioma tumors. Halofuginone had no effect on the growth of C6 glioma spheroids in vitro, and these spheroids showed no collagen alpha1(I) expression and no collagen synthesis. However, a significant attenuation of tumor growth was observed in vivo, for spheroids implanted in CD-1 nude mice which were treated by oral or intraperitoneal (4 microg every 48 hours) administration of halofuginone. In these mice, treatment was associated with a dose-dependent reduction in collagen alpha1(I) expression and dose- and time-dependent inhibition of angiogenesis, as measured by MRI. Moreover, halofuginone treatment was associated with improved re-epithelialization of the chronic wounds that are associated with this experimental model. Oral administration of halofuginone was effective also in intervention in tumor growth, and here, too, the treatment was associated with reduced angiogenic activity and vessel regression. These results demonstrate the important role of collagen type I in tumor angiogenesis and tumor growth and implicate its role in chronic wounds. Inhibition of the expression of collagen type I provides an attractive new target for cancer therapy.

Topics: Animals; Antineoplastic Agents; Collagen; Dose-Response Relationship, Drug; In Situ Hybridization; Kinetics; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Time Factors; Tumor Cells, Cultured; Wound Healing

Seven anticoccidial drugs commonly used in poultry (diclazuri), monensin, salinomycin, halofuginone, nicarbazin, robenidine, amprolium, and lasalocid) were tested for residual activity after withdrawal. In each test, the products were given at the recommended level to cages of 10 broiler chickens. Oral inoculation with coccidia was given after withdrawal of medication. Birds pretreated with 1 ppm of diclazuril and inoculated with Eimeria tenella after drug withdrawal had normal weight gain and very low lesion scores. Residual activity depleted gradually over several days, as shown by higher lesion scores when medication was withdrawn for up to 3 days before inoculation. Similar results were observed when young birds were inoculated with a mixture of E. tenella, E. maxima and E. acervulina, and also when birds were given diclazuril to market weight (6 weeks of age) and inoculated with a mixture of six species of Eiméria (The above species plus E. brunetti, E. mitis, and E. necatrix) after withdrawal of medication for 2 days. In contrast, there was no evidence of residual anticoccidial activity with nicarbazin, halofuginone, lasalocid, amprolium, salinomycin or monensin. Overall, the residual activity was unique to diclazuril.

Topics: Amprolium; Animal Feed; Animals; Chickens; Coccidiosis; Coccidiostats; Eimeria tenella; Feces; Female; Lasalocid; Male; Monensin; Nicarbazin; Nitriles; Piperidines; Poultry Diseases; Pyrans; Quinazolines; Quinazolinones; Random Allocation; Triazines

To evaluate the effects of halofuginone, a specific inhibitor of collagen type I synthesis, on the postoperative formation of abdominal adhesions in rats.. Postoperative adhesions remain the leading cause of small bowel obstruction in the Western world. Surgical trauma causes the release of a serosanguineous exudate that forms a fibrinous bridge between two organs. This becomes ingrown with fibroblasts, and subsequent collagen deposition leads to the formation of a permanent adhesion. Most of the drugs used have been clinically ineffective, and none has been specific to a particular extracellular matrix molecule. Therefore, there are serious concerns about the toxic consequences of interfering with the biosynthesis of other collagens, other matrix proteins, or vital collagen-like molecules.. Adhesions were induced by scraping the cecum until capillary bleeding occurred. The adhesions were scored 21 days later. Halofuginone was either injected intraperitoneally (1 microg/25 g body weight) every day, starting on the day of operation, or added orally at concentrations of 5 or 10 mg/kg, starting 4 days before the operation. Collagen alpha1(I) gene expression was evaluated by in situ hybridization, total collagen was estimated by Sirius red staining, and collagen type III was detected by immunohistochemistry.. The adhesions formed between the intestinal walls were composed of collagen and were populated with cells expressing the collagen alpha1(I) gene. Regardless of the administration procedure, halofuginone significantly reduced the number and severity of the adhesions. Halofuginone prevented the increase in collagen alpha1(I) gene expression observed in the operated rats, thus reducing collagen content to the control level. In fibroblasts derived from abdominal adhesions, halofuginone induced dose-dependent inhibition of collagen alpha1(I) gene expression and collagen synthesis. Collagen type III levels were not altered by adhesion induction or by halofuginone treatment.. Upregulation of collagen synthesis appears to have a critical role in the pathophysiology of postoperative adhesions. Halofuginone, an inhibitor of collagen type I synthesis, could be used as an important tool in understanding the role of collagen in adhesion formation, and it may become a novel and promising antifibrotic agent for preventing postoperative adhesion formation.

Topics: Abdomen; Animals; Collagen; Histocytochemistry; In Situ Hybridization; Piperidines; Postoperative Complications; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Tissue Adhesions

Coccidia were isolated from a commercial broiler farm with a history of suspected drug resistance. The sensitivity profiles of the Eimeria spp. isolates against the anticoccidial drugs nicarbazin (NIC), narasin (NAR), halofuginone (HAL), salinomycin (SAL), meticlorpindol plus methylbenzoquate (MET), and monensin (MON) at the recommended dose levels were followed in three battery trials (B1, B2, B3) corresponding to a field study over three periods of commercial broiler keeping (F1, F2, F3). Shuttle programs were performed in F1 (NIC/MON) and in F2 (MET/MON) while only SAL was used in F3. Eimeria acervulina and E. tenella were isolated from indicator birds in F1 while only E. acervulina could be found during F2 and F3. In trial B1 the isolate from F1 was identified as resistant against HAL and partly resistant against NIC and MON, the two drugs that were used in F1. Following the replacement of NIC in the starter feed by MET the respective isolate from F2 showed no resistance against ionophores (trial B2) while partial resistance against HAL was still present. Since SAL was the most efficient drug in B1 and B2 only this drug was applied in F3. Apart from a resistance against HAL no resistance against any of the other tested anticoccidials was found in the isolate from F3. SAL controlled coccidiosis efficiently in the field and best productivity was recorded in F3. This study shows that battery trials have a good predictive value in respect to the efficacy of anticoccidials under the conditions of commercial broiler production.

Topics: Animal Husbandry; Animals; Chickens; Clopidol; Coccidiosis; Coccidiostats; Drug Resistance; Eimeria; Feces; Food Industry; Ionophores; Monensin; Nicarbazin; Piperidines; Poultry Diseases; Predictive Value of Tests; Pyrans; Quinazolines; Quinazolinones; Quinolones

The horse-parasitizing species Babesia equi Laveran, 1901 was redescribed as Theileria equi Mehlhorn, Schein 1998 and, thus, transferred from one valid genus to another. This transfer was needed since it turned out that this horse parasite showed the relevant characteristics of theilerians with regard to biological data, morphological features, biochemical properties, and molecular biological relationships.

Topics: Animals; Antiprotozoal Agents; Arachnid Vectors; Babesia; Erythrocytes; Horse Diseases; Horses; Life Cycle Stages; Lymphocytes; Naphthoquinones; Piperidines; Quinazolines; Quinazolinones; Theileria; Theileriasis; Ticks

Halofuginone, an anticoccidial quinoazolinone, can specifically inhibit collagen type alpha1 (I) synthesis and gene expression, and also inhibits cultured smooth muscle cell proliferation. The aim of this study was to investigate the effect of halofuginone on neointimal formation of rat aorta after culture in a concentration-dependent manner in vitro. Thoracic aorta of Wistar rats was removed and manipulated to damage the endothelium under sterile conditions, and culture for 15 days in halofuginone-free or halofuginone-added culture medium (n = 20). Segments of cultured aorta were studied by histologic and immunohistochemical methods. Proliferation of neointimal layers consisting of loose multilayer cellular structure was observed in the halofuginone-free control group after 15 days of rat aorta culture, and neointimal formation was significantly decreased as an increasing concentration of halofuginone was added. As with precultured fresh aorta, no intimal proliferation was observed in the cultured segments of aorta with 500 ng/ml halofuginone added to culture medium. The proliferation of cell nuclear antigen index was significantly higher in the halofuginone-free control group than that in the halofuginone-added groups. The present results suggest that halofuginone can inhibit neointimal formation of rat aorta after culture in a concentration-dependent fashion in vitro.

Topics: Animals; Aorta; Coccidiostats; Culture Techniques; Dose-Response Relationship, Drug; Male; Muscle, Smooth, Vascular; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Tunica Intima

Proliferation of vascular smooth muscle cells (SMCs) and accumulation of extracellular matrix (ECM) components within the arterial wall in response to local injury are important etiologic factors in vascular proliferative disorders such as arteriosclerosis and restenosis after angioplasty. Fibrillar and nonfibrillar collagens are major constituents of the ECM that modulate cell shape and proliferative responses and thereby contribute to the pathogenesis of intimal hyperplasia. Halofuginone, an anticoccidial quinoazolinone derivative, inhibits collagen type I gene expression. We investigated the effect of halofuginone on (1) proliferation of bovine aortic endothelial cells and SMCs derived from the same specimen and maintained in vitro, (2) ECM deposition and collagen type I synthesis and gene expression, and (3) injury-induced intimal hyperplasia in vivo. DNA synthesis and proliferation of vascular SMCs in response to serum or basic fibroblast growth factor were abrogated in the presence of as little as 0.1 microgram/mL halofuginone; this inhibition was reversible upon removal of the compound. Under the same conditions, halofuginone exerted a relatively small antiproliferative effect on the respective vascular endothelial cells. Halofuginone also inhibited the synthesis and deposition of ECM components by vascular SMCs as indicated both by a substantial reduction in the amount of sulfated proteoglycans and collagen type I synthesis and gene expression. Local administration of halofuginone in the rabbit ear model of crush injury-induced arterial intimal hyperplasia resulted in a 50% reduction in intimal thickening as measured by a morphometric analysis of the neointima/media ratio. The differential inhibitory effect of halofuginone on vascular SMCs versus endothelial cells, its inhibition of ECM deposition and collagen type I synthesis, and its ability to attenuate injury-induced intimal hyperplasia may place halofuginone alone or in combination with other antiproliferative compounds as a potential candidate for prevention of arterial stenosis and accelerated atherosclerosis.

Topics: Animals; Arteries; Cattle; Cell Division; Cells, Cultured; Collagen; Humans; Hyperplasia; Muscle, Smooth, Vascular; Piperidines; Quinazolines; Quinazolinones; Rabbits; Tunica Intima

Ten Eimeria field isolates from North Germany were studied in battery tests for sensitivity to selected anticoccidials. A high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly multiple resistance. Partial or complete resistance to maduramicin was found in 7 field isolates, to monensin in 6, to salinomycin in 5, to nicarbazin in 8, to halofuginone in 7, to robenidine and toltrazuril in 1, and to diclazuril in 2 field isolates. Multiple resistance had developed in 7 of the 10 isolates. Cross-resistance between maduramicin, monensin, and salinomycin occurred in 5 Eimeria isolates. One isolate showed cross-resistance between diclazuril and toltrazuril. From the resistant isolates 15 pure E. acerculina and 5 pure E. brunetti strains were obtained by single oocyst infections. Seven of the E. acerculina and 4 of the E. brunetti strains showed resistance or partial resistance that was also present in the original isolate. Ten of 11 resistant strains were multiply resistant.

Topics: Animals; Chickens; Coccidiosis; Coccidiostats; Drug Resistance; Drug Resistance, Multiple; Eimeria; Germany; Lactones; Male; Monensin; Nicarbazin; Nitriles; Piperidines; Poultry Diseases; Pyrans; Quinazolines; Quinazolinones; Robenidine; Triazines

Hepatic cirrhosis is characterized by excessive deposition of collagen, resulting from an increase in type I collagen gene transcription. We evaluated the effect of halofuginone-a specific inhibitor of collagen type alpha 1(I) gene expression-on dimethylnitrosamine (DMN)-induced liver fibrosis/cirrhosis in rats.. Fibrosis was induced by intraperitoneal injection of DMN. Halofuginone (5 mg/kg) was added to the diet. Collagen was stained with Sirius red and collagen alpha 1(I) gene expression was evaluated by in situ hybridization.. In control rats, a low level of collagen alpha 1(I) gene expression was observed. A high dose of DMN (1%) caused severe fibrosis, as indicated by induction of collagen alpha 1(I) gene expression and increased liver collagen content. Addition of halofuginone before the onset of fibrosis, almost completely prevented the increase in collagen type I gene expression and resulted in lower liver collagen content. Moreover, halofuginone partially prevented the marked decrease in liver weight and reduced the mortality rate. At a lower dose of DMN (0.25%), which causes mild fibrosis, halofuginone prevented the increase in collagen alpha 1(I) gene expression, prevented the increase in liver collagen deposition and reduced plasma alkaline phosphatase activity, all of which are characteristic of liver fibrosis/ cirrhosis.. These results suggest that halofuginone can be used as an important tool to understand the regulation of the collagen alpha 1(I) gene and may become a novel and promising antifibrotic agent for liver fibrosis/ cirrhosis.

Topics: Alkaline Phosphatase; Animals; Collagen; Dimethylnitrosamine; Gene Expression; In Situ Hybridization; Injections, Intraperitoneal; Liver; Liver Cirrhosis, Experimental; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.

Topics: Animals; Carbon Radioisotopes; Cell Division; Cells, Cultured; Collagen; Extracellular Matrix; Gene Expression; Glomerular Mesangium; Piperidines; Proline; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Sulfates; Tritium

The effect of halofuginone, a plant alkaloid known to inhibit collagen type I synthesis, on skin collagen content and skin morphology was evaluated in two in vivo models of scleroderma: the murine chronic graft-versus-host disease (cGvHD) and the tight skin mouse. Skin collagen was assessed by hydroxyproline levels in skin biopsies and by immunohistochemistry using anti-collagen type I antibodies. Daily intraperitoneal injections of halofuginone (1 microgram/mouse) for 52 d starting 3 d before spleen cell transplantation, abrogated the increase in skin collagen and prevented the thickening of the dermis and the loss of the subdermal fat, all of which are characteristic of the cGvHD mice. Halofuginone had a minimal effect on collagen content of the control mice. The halofuginone-dependent decrease in skin collagen content was concentration-dependent and was not accompanied by changes in body weight in either the cGvHD or the control mice. Injections of halofuginone (1 microgram/mouse) for 45 d caused a decrease in the collagen content and dermis width in tight skin mice, but did not affect the dermis width of control mice. Collagen content determination from skin biopsies confirmed the immunohistochemical results in the same mice. The low concentration of halofuginone needed to prevent collagen deposition in fibrotic skin without affecting body weight suggests that halofuginone may serve as a novel and promising anti-fibrotic therapy.

Topics: Animals; Collagen; Disease Models, Animal; Fibrosis; Graft vs Host Disease; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Osmolar Concentration; Piperidines; Quinazolines; Quinazolinones; Scleroderma, Localized; Skin

The therapeutic effect of halofuginone hydrobromide (Steronol) and spiramycin solubilized (Spirasol) applied to clinical Cryptosporidium serpentis infections in captive snakes was investigated. Pathological changes induced by C. serpentis were typical for snake cryptosporidiosis. Spiramycin induced no significant change in the pattern of shedding of fecal Cryptosporidium oocysts; biopsies and necropsies revealed cryptosporidiosis in gastric mucosa of all spiramycin-treated animals. In all, 8 of 21 (38%) halofuginone-treated snakes stopped shedding C. serpentis oocysts; examination of gastric tissue of 6 of these 8 animals revealed cryptosporidiosis in 2 snakes. Hepatotoxic and nephrotoxic pathological changes induced by halofuginone included focal or multifocal, severe, acute liver necrosis; severe liver hemosiderosis; and bilateral, severe, acute diffuse cortical and tubular necrosis and iron deposition. Postprandial regurgitation associated with midbody swelling, observed in 4 halofuginone-treated and 2 spiramycin-treated snakes at 15 and 21 weeks after drug withdrawal, did not coincide with oocyst-positive feces. Neither halofuginone nor spiramycin treatment produced a satisfactory therapeutic outcome when applied against clinical C. serpentis infections in snakes.

Topics: Animals; Coccidiostats; Cryptosporidium; Feces; Intestine, Large; Intestine, Small; Piperidines; Quinazolines; Quinazolinones; Snails; Spiramycin; Stomach

The effect of halofuginone (a plant alkaloid) on collagen alpha 1(I) gene expression and collagen synthesis was evaluated in human skin fibroblasts from patients with chronic graft-versus-host disease (cGvHD) or scleroderma and from a normal individual. Halofuginone caused a dose-dependent inhibition in collagen alpha 1(I) gene expression and collagen synthesis in all cultures tested, the cGvHD fibroblasts being the least sensitive. In normal and scleroderma fibroblasts, concentrations of halofuginone as low as 10(-10) M and 10(-9) M were sufficient to cause a significant reduction in collagen alpha 1(I) gene expression and collagen synthesis, respectively. In addition, halofuginone also inhibited the transforming growth factor beta-induced collagen synthesis. Three days after halofuginone removal, collagen gene expression returned to control levels. The reduction of collagen mRNA transcript levels by halofuginone appeared to be dependent on new protein synthesis because simultaneous treatment of fibroblasts with protein synthesis inhibitors prevents the suppressive effect of halofuginone on collagen alpha 1(I) mRNA gene expression. The ability of extremely low concentrations of halofuginone to inhibit collagen alpha 1(I) synthesis specifically and transiently at the transcriptional level suggests that this material may be an important tool for studying collagen alpha 1(I) gene regulation and may be used as a novel and promising antifibrotic therapy.

Topics: Adult; Coccidiostats; Collagen; Dose-Response Relationship, Drug; Female; Fibroblasts; Graft vs Host Disease; Humans; Middle Aged; Piperidines; Quinazolines; Quinazolinones; Time Factors

Pulmonary fibrosis is a disorder causing a high mortality rate for which therapeutic options are limited. Therefore, the effect of halofuginone, a novel inhibitor of collagen type I synthesis, on bleomycin-induced pulmonary fibrosis was studied in rats. Pulmonary fibrosis was induced by intraperitoneal injections of bleomycin for seven consecutive days, and halofuginone was administered intraperitoneally every second day during the entire experimental period of 42 d. Collagen determination in the lungs and the examination of histologic sections showed that halofuginone significantly reduced fibrosis relative to the untreated control rats. We conclude that halofuginone is a potent in vivo inhibitor of bleomycin-induced pulmonary fibrosis, and that it may potentially be used as a novel therapeutic agent for the treatment of this dysfunction.

Topics: Animals; Bleomycin; Collagen; Lung; Male; Piperidines; Pulmonary Fibrosis; Quinazolines; Quinazolinones; Rats; Rats, Inbred Strains

To test in vivo the effect of previously observed inhibition of collagen type I transcription by the plant alkaloid Halofuginone, deep flexor tendons of 12 chickens were severed and sutured, and Halofuginone was applied topically at the site of surgery. Intact tendons, and severed but untreated tendons served as controls. The effect of the treatment was assessed by histological, biochemical, and biomechanical examinations of the operated and intact tendons three weeks after surgery. The results indicated an almost complete absence of fibrous peritendinous adhesions in the histologic sections of the Halofuginone treated tendons. There was a statistically significant decrease in collagen contents of and in both force and energy required to pull out the Halofuginone treated tendons from their sheath, relative to the untreated controls. Halofuginone had no effect on the cellularity of the healing tissue. We conclude that Halofuginone is a potent inhibitor of fibrous peritendinous adhesions with potentially important clinical applications.

Topics: Animals; Chickens; Collagen; Gene Expression Regulation; Hydroxyproline; Inflammation; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Statistics, Nonparametric; Tendon Injuries; Tissue Adhesions

This study was conducted to assess the effect of the coccidiostat halofuginone (Stenorol) on growth, feed consumption, and survival of Chukar partridge. Halofuginone was fed to three replicates (14 chicks per replicate) of chukar chicks from 2 to 7 d of age at levels of 0, 1.5, 3.0, 6.0 and 12 ppm. Mortality from 2 to 7 d was 0, 0, 0, 11, and 21 birds, respectively, by treatment. Seven-day body weight showed a significant linear decrease with increasing halofuginone level (P < 0.01). On the 7th d, replicates receiving 6.0 and 12.0 ppm halofuginone were transferred to unmedicated feed for the remainder of the test due to excessive mortality. The other groups were continued until 6 wk of age. At 6 wk, chicks fed 6 or 12 ppm halofuginone from 2 to 7 d and then unmedicated feed did not differ in body weight from those fed the unmedicated control diet. A significant difference in mortality was not observed among the other three treatment groups to 6 wk of age. A linear depression in 3-, 4-, 5-, and 6-wk body weight with increasing halofuginone level was observed within the first three treatment levels (P < 0.05). It was concluded that 1.5 ppm halofuginone depressed growth of young chukars and that 6 ppm resulted in increased mortality.

Topics: Animals; Coccidiostats; Dose-Response Relationship, Drug; Feeding Behavior; Growth; Piperidines; Poultry; Quinazolines; Quinazolinones; Weight Gain

Live performance and carcass quality of female broilers were evaluated under four coccidiostat programs (CP) and two feed treatments. The CP consisted of halofuginone (H) and salinomycin (S), fed either continuously (HH and SS) or in rotational programs (HS and SH), during the starter (1 to 21 d) and grower (22 to 35 d) periods, respectively. All groups received an unmedicated withdrawal feed from 36 to 42 d. Feed treatments consisted of a control and a fortified diet high in proline and supplemented with additional ascorbic acid and zinc (50 birds per pen; 4 pens per feed; 8 pens per CP). In addition to live performance and skin puncture strength, carcass quality attributes following processing (at 43 d of age) were assessed. No CP by feed interactions were detected for any of the variables measured. The CP treatments did not differ for live performance. Birds on fortified feed were heavier at 21 d (P < .001) and had an improved feed conversion at 42 d (P < .05). Skin puncture strength was significantly reduced for the birds fed H, either in continuous (HH) or rotational programs (HS and SH). Skin sores-scratches and tears were lowest for the SS and SH groups. The HH treatment resulted in fewer grade A carcasses (P < .001). Halofuginone, when fed continuously or in the starter feed, affected carcass quality of broilers. Higher dietary proline or supplementation with ascorbic acid and zinc did not appear to alleviate the effects of halofuginone on skin quality.

Topics: Animals; Ascorbic Acid; Body Weight; Chickens; Coccidiostats; Female; Food, Fortified; Piperidines; Proline; Pyrans; Quinazolines; Quinazolinones; Skin; Skin Physiological Phenomena; Tensile Strength; Zinc

Live performance and skin characteristics of male and female broilers were evaluated under four coccidiostat feed additive programs. Treatments consisted of halofuginone (H) and salinomycin (S), fed either continuously (HH and SS) or in rotational programs (HS and SH) during the starter (1 to 21 d) and grower (22 to 35 d) periods, respectively. An unmedicated withdrawal feed was provided from 36 to 42 d of age. Body weights, feed efficiency, and mortality (by pen) were determined, in addition to skin puncture strength measurements taken at Days 21, 35, and 42 on five birds per pen. At 43 d of age, all birds were processed and individually graded for skin defects. There were no treatment by sex interactions for any variable measured. Male body weights, feed efficiency, and total mortality exceeded those of females (P < .05). Skin puncture strength was reduced at 21 d in the HH and HS groups, at 35 d in the HH and SH groups, and at 42 d in the HH, HS, and SH groups. Thigh sores and scratches were higher for the HH group (P < .05), and thigh skin tears were higher for the HH and HS groups (P < .01). Males had more swollen hock joints and breast blisters than females (P < .001). Females had more thigh skin tears (P < .01) and broken wings (P < .001) than males. Results of the present study demonstrated that halofuginone affected skin strength of broilers, especially when used continuously or only in the starter feed (1 to 21 d).

Topics: Animals; Body Weight; Chickens; Coccidiostats; Female; Food, Fortified; Male; Piperidines; Pyrans; Quinazolines; Quinazolinones; Sex Factors; Skin; Skin Physiological Phenomena; Tensile Strength

Halofuginone was evaluated for its activity against experimentally induced infection due to Cryptosporidium parvum in rats rendered immunosuppressed with dexamethasone. The drug's activity was dose-related and both prophylaxis and therapy reduced the rate and severity of infection in the small intestine and caecum. Prophylactic treatment reduced infection of the common bile duct, but therapeutic administration did not and neither form of treatment reduced the infection rate in the colon. Intestinal infection recurred at a level comparable to that of untreated controls when treatment was discontinued. Treatment with halofuginone may reduce the severity of acute cryptosporidiosis, but is less efficacious for chronic cryptosporidiosis involving the colon and extraintestinal tissues, a manifestation increasingly seen in the immunocompromised host.

Topics: Animals; Colonic Diseases; Common Bile Duct; Cryptosporidiosis; Cryptosporidium parvum; Dexamethasone; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Feces; Female; Immunosuppression Therapy; Intestine, Large; Intestine, Small; Parasite Egg Count; Piperidines; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Treatment Outcome

To determine if halofuginone hydrobromide, a specific type I collagen inhibitor, could prevent intimal hyperplasia at a vascular anastomosis.. Intimal hyperplasia is characterized by smooth muscle cell proliferation and extracellular matrix accumulation. Halofuginone was used to block collagen production and smooth muscle cell proliferation in cell cultures and in a rabbit model of an end-to-end anastomosis of the right common carotid artery. Animals were fed a nontoxic dose of halofuginone. Eighteen rabbits were fed the inhibitor in a randomized blinded fashion and were examined after 4 weeks by harvesting the arteries after perfusion fixation at physiologic pressures.. Halofuginone inhibited smooth muscle cell proliferation in vitro and had no effect on cell viability. Morphometric quantification verified that halofuginone treatment significantly attenuated anastomotic intimal thickness.. Oral administration of halofuginone inhibits intimal hyperplasia at vascular anastomoses. Intimal hyperplasia inhibition by halofuginone may be a therapeutic option for preventing arterial stenosis in vascular surgery.

Topics: Anastomosis, Surgical; Animals; Blotting, Northern; Carotid Artery, Common; Cell Division; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Hyperplasia; Muscle, Smooth, Vascular; Piperidines; Procollagen; Quinazolines; Quinazolinones; Rabbits; RNA, Messenger; Tropoelastin; Tunica Intima

Two experiments were conducted to determine whether supplemental levels of L-proline in the diets of broiler chickens would mitigate the skin weakening effect caused by continuous feeding of the anticoccidial halofuginone. In Experiment 1, tensile strength and collagen levels in thigh apteria skin were determined at 21 and 42 d of age in male broilers fed 0, .5, and 1% L-proline with either halofuginone (3 mg/kg) or salinomycin (61 mg/kg). In Experiment 2, the same measurements were made on female broilers receiving diets containing halofuginone and supplemented with 0, .5, or 1% L-proline, 1% L-proline through 21 d of age, or 1% L-glutamic acid through 21 d of age, or a diet containing high L-proline feedstuffs (corn gluten meal and ring dried blood meal). In Experiment 1, dermis thickness of thigh apteria was measured in the males at Day 21. Skin strength was increased in male and female broilers fed halofuginone with addition of .5 and 1% L-proline, respectively, at 21 and 42 d of age. Continuous incorporation of synthetic L-proline into diets was shown to improve skin strength in females, whereas diets formulated to contain high levels of L-proline from feedstuffs, 21-d feeding of L-proline, or L-glutamic acid did not increase skin strength.

Topics: Animals; Body Weight; Chickens; Coccidiostats; Collagen; Female; Food, Fortified; Male; Piperidines; Proline; Quinazolines; Quinazolinones; Skin; Skin Physiological Phenomena; Tensile Strength

Continuous feeding of the anticoccidial halofuginone to broilers is associated with reduced skin tensile strength and increased skin tearing during processing. The possible mitigating effect of shuttle administration of halofuginone and salinomycin to female broilers was evaluated. Halofuginone or salinomycin were included in the starter and grower diets in all four possible combinations, with anticoccidial omitted from the finisher diets. Starter, grower, and finisher diets were fed to broilers through 3, 6, and 7 wk of age, respectively. Skin strength of pullets fed a diet based on milo and corn (NW) vs a diet based on corn was also compared in a factorial arrangement. Two further treatments were also included: 1) halofuginone-only NW diet supplemented with 2,500 ppm ascorbic acid from 0 to 7 wk; and 2) NW diet reared on wire floor without anticoccidial treatment. Skin tensile strength was determined at 3, 6, and 7 wk of age. Dietary composition had no effect upon skin strength or BW of broilers. Addition of ascorbic acid to the diet containing halofuginone anticoccidial did not improve skin strength. Continuous feeding of halofuginone reduced skin strength whereas withholding anticoccidial and continuous feeding of salinomycin resulted in high skin strength. When halofuginone was used in shuttle feeding programs with salinomycin, there were no differences in skin strength at 7 wk of age compared to birds that were continuously treated with salinomycin. These results suggest halofuginone may be used in a shuttle program either during the starter or grower phase without adverse affect on skin tensile strength at slaughter.

Topics: Animal Feed; Animals; Chickens; Coccidiostats; Diet; Drug Administration Schedule; Female; Piperidines; Pyrans; Quinazolines; Quinazolinones; Skin; Skin Physiological Phenomena; Tensile Strength

The effect of halofuginone--a plant alkaloid used as a coccidiostat in birds--on collagen metabolism was studied in various avian and mammalian cell cultures. In avian skin fibroblasts halofuginone attenuated the incorporation of [3H]proline into collagenase-digestible proteins (CDP) at concentrations as low as 10(-11) M, without affecting production of [3H]collagenase-nondigestible proteins (NCDP), cell proliferation or collagen degradation. Halofuginone depressed specifically the expression of alpha 1 gene of collagen type I but not that of collagen type II. This was demonstrated in skin fibroblasts and growth-plate chondrocytes using probes containing inserts sequences corresponding to the alpha 1(I) and alpha 1(II) mRNAs. A slight inhibition of the expression of alpha 2(I) was observed in avian skin fibroblasts but not in growth-plate chondrocytes. The inhibition of gene expression of both polypeptides of collagen type I in skin fibroblasts resulted in a decrease in synthesis, as demonstrated by immunoprecipitation with specific type I collagen antiserum. In primary cultures of mouse skin fibroblasts, avian epiphyseal growth plate chondrocytes and a rat embryo cell line--all of which produce and secrete collagen type I--halofuginone inhibited the incorporation of [3H]proline into CDP, the Rat-1 line being the most sensitive to the drug. These results suggest that halofuginone affects specifically type I collagen synthesis by repressing gene-expression. The need for extremely low concentrations of halofuginone to inhibit collagen type I synthesis, regardless of the tissue or animal species, contributes to the potential usefulness of the substance in studying collagen metabolism.

Topics: Animals; Birds; Cell Division; Cells, Cultured; Coccidiostats; Collagen; Embryo, Mammalian; Embryo, Nonmammalian; Fibroblasts; Gene Expression; Growth Plate; Matrix Metalloproteinase Inhibitors; Mice; Piperidines; Proline; Quinazolines; Quinazolinones; Rats; Species Specificity

A floor-pen trial was conducted to investigate the effects of different shuttle programs upon the growth of broilers to 8 wk of age. Nicarbazin, halofuginone, and robenidine, when included in the starter feed for 3 wk, were effective in preventing lesions due to Eimeria. The effects of medication upon performance were apparent, medicated groups gaining more weight by 6 wk and having a lower feed conversion at 6, 7, and 8 wk than the unmedicated controls. There were no significant differences in body weight at 6, 7, or 8 wk or feed conversion at 6 or 7 wk among the medicated groups, whether medication was withdrawn for 7 or 14 days. A decrease in the number of small and medium oocysts in the litter was observed as the trial progressed. Few large oocysts (Eimeria maxima) were seen in the medicated groups. Numbers of oocysts did not increase following withdrawal of medication. Birds from all medicated groups were challenged at 6 wk with oocysts of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. Weight gains were similar to that of the unchallenged controls, indicating that they had acquired immunity to these species of Eimeria.

Topics: Animals; Body Weight; Chickens; Coccidiosis; Coccidiostats; Diet; Eimeria; Feces; Immunity; Male; Nicarbazin; Parasite Egg Count; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Robenidine

A method has been developed for the determination of the coccidiocidic drug halofuginone in feedstuff concentrates which is based on the combination of capillary isotachophoresis and capillary zone electrophoresis in the column-switching mode. The high load capacity of the isotachophoretic step and high sensitivity of the zone electrophoretic step enabled analysis of up to 25 microliters of sample solution containing as little as 10(-8) M halofuginone with excellent reproducibility (R.S.D. about 1%). Attention was paid to the possibility of the existence of transient local isotachophoresis in the zone electrophoretic step, and experimental and theoretical methods of revealing zones migrating isotachophoretically in the background electrolyte were shown.

Topics: Animal Feed; Animals; Coccidiostats; Electrophoresis; Piperidines; Poultry; Quinazolines; Quinazolinones

Broilers were grown to 42 days of age on diets supplemented with salinomycin (60 mg/kg), monensin (99 mg/kg), or halofuginone (3 mg/kg) and continued on unmedicated diets to 49 days of age. There were no significant (P greater than .05) differences among anticoccidials in final body weight, feed conversion, or mortality rates. Samples of birds were processed for dressing percentage and parts yield. Both males and females fed salinomycin had significantly higher breast meat yield as a percentage of postchill weight than those fed halofuginone but not those fed monensin; differences were not significant for breast meat yield of males or females fed monensin or halofuginone. Males fed halofuginone had significantly heavier leg quarters than those fed salinomycin but not those fed monensin. Females fed salinomycin had significantly higher water uptake during chill than those fed monensin or halofuginone. Results of the present study indicate that the anticoccidial used in growing broilers may influence some carcass yield parameters.

Topics: Animals; Body Weight; Chickens; Coccidiostats; Eating; Female; Male; Monensin; Muscle Development; Muscles; Piperidines; Pyrans; Quinazolines; Quinazolinones; Random Allocation

In vivo and in vitro experiments were conducted in an effort to elucidate the mechanism of suppression by halofuginone of skin strength in broilers. In the in vivo study, halofuginone was included at concentrations of 0, 1.5, 3, and 6 mg/kg of diet, corresponding to 0, 50, 100, and 200%, respectively, of the amount recommended for use as a coccidiostat. Each dietary treatment was given to 260 female broiler day-old chickens. Skin tearing was evaluated at the processing plant. Skin collagen and Kjeldahl-nitrogen were determined chemically. At the age of 7 wk, BW and feed efficiency were affected only in birds consuming the diet containing the highest concentration of the drug. Skin tearing increased but skin collagen concentration decreased in a dose-dependent manner. Fibroblasts were obtained by collagenase digestion from chicken skin and cultured. The cultured cells were incubated with various concentrations of halofuginone, monensin, and nicarbazin, and [3H]proline incorporation was evaluated in collagenase-digestible (representing mostly collagen) and nondigestible proteins exported by the cells into the medium. Halofuginone, at a concentration as low as 10(-11) M, inhibited incorporation of [3H]proline into collagenase-digestible proteins, but did not affect incorporation of [3H]proline into collagenase-nondigestible proteins. Even at concentrations as high as 10(-9) M, neither monensin nor nicarbazin affected collagenase-digestible proteins. The in vitro results suggest that halofuginone specifically inhibits collagen synthesis by skin fibroblasts. Results of both in vivo and in vitro trials suggest that the increase of skin tearing during processing, induced by halofuginone, is caused by direct suppression of skin collagen synthesis.

Topics: Animals; Cells, Cultured; Chickens; Coccidiostats; Collagen; Dose-Response Relationship, Drug; Female; Fibroblasts; Monensin; Nicarbazin; Piperidines; Quinazolines; Quinazolinones; Random Allocation; Skin; Weight Gain

Efficacy of virginiamycin (22 mg/kg) in combination with no drug, amprolium, carbarsone, halofuginone, or monensin, was studied. Male and female turkeys were raised to market age in five experiments conducted from 1983 to 1987. Body weights and feed:gain responses to virginiamycin for males and females were positive and significant (P less than .05). Virginiamycin resulted in mean 5.2 and 6.3% body weight responses and 3.3 and 2.2% feed:gain responses for males at 19 or 20 wk of age and for females at 16 or 17 wk of age, respectively. Mortality rates were low in all studies, and were not influenced by virginiamycin. In a processing study, virginiamycin in combination with halofuginone did not affect shrinkage, yield, or market grade. Feed was utilized by males and females 3.9 and 3.0%, respectively, more efficiently than expected with dietary virginiamycin, compared with results predicted by a simulation modeling technique. Profitability was considerably greater with dietary virginiamycin using actual data than with simulated feed consumption data.

Topics: Amebicides; Amprolium; Animal Feed; Animals; Arsanilic Acid; Drug Interactions; Eating; Energy Metabolism; Female; Male; Monensin; Mortality; Piperidines; Quinazolines; Quinazolinones; Sex Characteristics; Turkeys; Virginiamycin; Weight Gain

Two trials were conducted to evaluate the effects of feeding various anticoccidial products to turkeys to 8 wk and then growing to market age (16 wk for hens and 20 wk for toms). Anticoccidials evaluated in the first trial included amprolium at 187.5 mg/kg for 0 to 4 wk and 125 mg/kg for 4 to 8 wk; butynorate at 375 mg/kg for 0 to 8 wk; monensin at both 60 (MON-60) and 100 mg/kg for 0 to 8 wk; zoalene at 187.5 mg/kg for 0 to 4 wk and 125 mg/kg for 4 to 8 wk; and halofuginone at 3 mg/kg for 0 to 8 wk. In the second trial, MON-60 was replaced by a combination of sulfadimethoxine (62.5 mg/kg) plus ormetoprim (37.5 mg/kg) for 0 to 8 wk. In each trial each treatment was fed to four pens of 16 hens and four pens of 12 toms. Several of the anticoccidials significantly influenced the weight of both hens and toms by producing lower weights at the end of the 8-wk feeding period than birds in other treatments. However, after removal of the anticoccidials, compensatory gains were observed in almost every instance. Significant effects of previous anticoccidial feeding were noted on body weight of hens at 16 wk but not on weights of toms at 20 wk.

Topics: Amprolium; Animal Feed; Animals; Coccidiostats; Dietary Fats; Dinitolmide; Eating; Female; Male; Monensin; Organotin Compounds; Piperidines; Pyrimidines; Quinazolines; Quinazolinones; Random Allocation; Sulfadimethoxine; Turkeys; Weight Gain

Two trials were conducted to compare the efficacy of currently approved anticoccidials for turkeys against challenge using a field isolate of mixed Eimeria species; E. adenoides, E. gallopavonis, and E. meleagrimitis. Poults in wire-floored cages were fed unmedicated diets from day-old to 3 wk of age. Diets were supplemented with either amprolium (AMP, 125 mg/kg), butynorate (BUT, 375 mg/kg), monensin (MON-60, 60 mg/kg; MON-100, 100 mg/kg), halofuginone (HAL; 3 mg/kg), zoalene (ZOA; 125 mg/kg), or sulfadimethoxine plus ormetoprim (SUL + ORM, 62.5 mg/kg and 37.5 mg/kg, respectively). After 2 days on the test diets, poults were individually weighed and inoculated with sporulated coccidial oocysts from the field isolate. Total fecal collections were obtained for Days 0 to 5 and 6 to 10 to estimate oocyst output. At 10 days postinoculation, the birds were individually weighed and killed to determine severity of intestinal lesions. The HAL and MON were most effective and AMP, ZOA, and SUL + ORM were least effective in maintaining weight and in reducing the severity of intestinal lesions. All the coccidiostats tested reduced oocyst passage, but poults fed HAL produced fewer oocysts. The results demonstrated differences in efficacy among anticoccidials with the more recently approved drugs providing the best protection against coccidiosis.

Topics: Amprolium; Animals; Coccidiosis; Coccidiostats; Dinitolmide; Feces; Intestines; Male; Monensin; Organotin Compounds; Piperidines; Poultry Diseases; Pyrimidines; Quinazolines; Quinazolinones; Sulfadimethoxine; Turkeys; Weight Gain

Fifty splenectomised calves naturally infected with Theileria buffeli were treated with primaquine phosphate (ICI, UK) and halofuginone lactate (Hoechst, Australia) either separately or in combination. Infections in treated calves were monitored for up to 26 weeks by examining Giemsa stained peripheral blood films for piroplasms and by an immunofluorescent antibody test. When used alone neither of the drugs eliminated infection. The most successful results were obtained when two treatments of halofuginone lactate, at a rate of 1 mg kg-1 body weight and six treatments of primaquine phosphate, at a rate of 2 mg kg-1 body weight, were administered concurrently. No theilerial relapses were observed in 14 of 16 calves so treated, and no antibody to T. buffeli was detected in these calves beyond the twelfth week after treatment. The results have application in the preparation of Theileria-free calves for use in the production of living vaccines against babesiosis and anaplasmosis.

Topics: Animals; Antibodies, Protozoan; Antiprotozoal Agents; Apicomplexa; Cattle; Drug Therapy, Combination; Fluorescent Antibody Technique; Piperidines; Primaquine; Quinazolines; Quinazolinones; Recurrence; Splenectomy; Theileriasis

A high performance liquid chromatography (HPLC) method for the determination of the anticoccidial and antitheilerial drug halofuginone in bovine plasma was developed. Samples were diluted with acetic acid (10%, v/v) and cleaned up on a Bond Elut C8 column. The analyte was eluted from the extraction column and chromatographed by reversed-phase HPLC using decylamine as a competing-ion reagent. Detection was by UV at 243 nm. Recovery from plasma was 75%, and within-day and between-day coefficients of variation were 5.23 and 6.35% respectively. The specificity and sensitivity of this method (limit of detection in plasma, 1 ng/mL) were sufficiently high to enable us to characterize the time course of the drug in plasma after oral administration of therapeutic doses to cattle.

Topics: Administration, Oral; Animals; Cattle; Chromatography, High Pressure Liquid; Coccidiostats; Indicators and Reagents; Microchemistry; Piperidines; Quinazolines; Quinazolinones

The effect of feeding halofuginone at 3 and 6 ppm on the excretion of Salmonella typhimurium by experimentally infected chickens was studied. A standardized procedure was used involving the oral inoculation of 72-h-old specific pathogen-free chickens with 10(8) organisms of a nalidixic acid-resistant mutant of a strain of S. typhimurium. At weekly intervals, cloacal swabs were taken and a semi-quantitative assessment was made of the numbers of Salmonella organisms excreted. When compared with the control group of infected chickens fed unmedicated food, the group fed halofuginone at 3 ppm showed no significant increase in excretion rate. The group fed 6 ppm showed a slight increase in excretion which was statistically significant.

Topics: Animal Feed; Animals; Chickens; Coccidiostats; Colony Count, Microbial; Feces; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Salmonella Infections, Animal; Salmonella typhimurium; Specific Pathogen-Free Organisms

The activities of five anticoccidials were compared against Eimeria species in/of chickens, in controlled in vivo and in vitro laboratory studies. Two more recent and potent market entries (maduramicin and halofuginone) were compared with three older polyether antibiotic anticoccidials (monensin, lasalocid and salinomycin). Halofuginone, lasalocid, maduramicin, monensin and salinomycin were evaluated at 3, 125, 5, 120 and 66 ppm, respectively, of active drug in the diets. At these levels, all five drugs demonstrated significant activity against Eimeria tenella, E. maxima, E. necatrix, E. brunetti and E. acervulina (in vivo). Monensin was least effective against E. tenella, and one of the lesser efficacious drugs against E. necatrix, maduramicin, was least effective against E. maxima. In studies of single Eimeria species infections, comparable weight gains were noted for the drugs. In the mixed Eimeria species infections, however, birds treated with maduramicin had significantly higher weight gains than did birds medicated with monensin. Unlike in vivo potencies, titration in vitro indicated that monensin was most potent (active at 10(-6) mcg ml-1), and maduramicin and lasalocid least potent (inactive at less than or equal to 10(-3) mcg ml-1).

Topics: Animals; Anti-Bacterial Agents; Body Weight; Chickens; Coccidiosis; Coccidiostats; Eimeria; Female; Ionophores; Lactones; Lasalocid; Male; Monensin; Piperidines; Poultry Diseases; Pyrans; Quinazolines; Quinazolinones

Topics: Animal Feed; Chromatography, High Pressure Liquid; Coccidiostats; Piperidines; Quinazolines; Quinazolinones; Solvents; Spectrophotometry, Ultraviolet

Large White male turkeys were raised to 20 wk of age on diets with varying kinds and levels of coccidiosis drug protection. Dietary treatments included unmedicated control (UMC), amprolium (AMP, 125 mg/kg), and 3 levels of halofuginone hydrobromide (HAL, during 0 to 4 and 4 to 8 wk of age, in 3.3; 3.1.5; and 1.5,1.5 mg/kg, respectively). Turkeys on these five treatments were exposed to coccidial oocysts at 14 days of age and again at 61 days of age; turkeys in an identical five treatments were not exposed until 61 days of age. At 28 days, nonexposed turkeys gained well and similarly, whereas exposed UMC showed poor growth and high mortality. At 56 days, regardless of exposure, UMC were lightest, AMP birds were intermediate, and birds in all HAL treatments were heaviest. Similarities in results of exposed and nonexposed birds suggests that nonexposed birds became exposed through tracking of oocysts in the room. Lesion scores of those challenged with oocysts at 61 days showed that all treatments had adequate resistance to coccidiosis. At 20 weeks of age, combining both exposure methods, birds fed HAL (dosage 3.1.5 or 1.5, 1.5 mg/kg) were significantly heavier than UMC and AMP treatments.

Topics: Animal Nutritional Physiological Phenomena; Animals; Body Weight; Coccidiosis; Coccidiostats; Male; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Turkeys

Studies were conducted at six locations over a 7-yr period to evaluate the response of broiler chickens to bacitracin methylene disalicylate (BMD) and roxarsone in the presence of diets containing 3 ppm halofuginone/kg feed. Treatments consisted of a 2 x 2 factorial arrangement with 0 or 55 mg BMD and 0 or 50 mg roxarsone/kg feed. These additives were fed beginning with day-old chicks and were removed 6 days before termination of the study, which varied in length from 48 to 56 days among locations. Body weights were significantly improved (P less than .05) by the addition of either BMD or roxarsone with a significant interaction (P less than .05) between BMD and roxarsone. Roxarsone improved body weights only in the presence of BMD. Feed utilization was significantly (P less than .05) improved by addition of either BMD or roxarsone, with no interaction between the two products.

Topics: Animal Feed; Animals; Arsenicals; Bacitracin; Body Weight; Chickens; Coccidiosis; Coccidiostats; Female; Food Additives; Male; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Roxarsone

Chemoprophylaxis of Cryptosporidium baileyi infections was attempted by feeding 4 groups of chicks diets containing 3 mg of halofuginone/kg of feed, 60 mg of salinomycin/kg, 75 mg of lasalocid/kg, or 110 mg of monensin/kg. Rations were fed 5 days before oral or intratracheal inoculation with oocysts and were continued for 20 days. None of the drugs prevented C baileyi infections. Clinical signs of respiratory tract disease and gross lesions of airsacculitis were observed in intratracheally inoculated birds in all treatment groups and nonmedicated controls. Orally inoculated birds did not develop clinical signs of infection. Pathogenic bacteria were not isolated from the respiratory tract systems of any chicks. Halofuginone delayed the establishment of infections of the bursa of Fabricius and cloaca, but not of the trachea.

Topics: Animals; Chickens; Coccidiostats; Cryptosporidiosis; Lasalocid; Monensin; Piperidines; Poultry Diseases; Pyrans; Quinazolines; Quinazolinones

Cattle were infected with three isolates of Theileria parva and treated with halofuginone lactate during acute clinical disease. The health, weight gain and carrier state of the cattle were monitored for 15 months. Limited treatment rapidly reduced fever and parasitosis but parasite recrudescences occurred and 12 out of 21 treated cattle died. Persistent carrier states were identified with two T. p. parva isolate infections and a transient carrier state with T. p. lawrencei. Three cattle which died from a chronic wasting syndrome during the follow-up period showed exhaustion of lymph nodes but no Theileria macroschizonts were detected in any tissue.

Topics: Animals; Antiprotozoal Agents; Body Weight; Carrier State; Cattle; Leukocyte Count; Lymph Nodes; Male; Piperidines; Quinazolines; Quinazolinones; Theileriasis; Ticks

Topics: Animals; Body Weight; Coccidiostats; Ducks; Eating; Geese; Piperidines; Quinazolines; Quinazolinones

Halofuginone was tested at 0, 1.5, 3.0, or 6.0 ppm in the feed against single and mixed infections of recent field isolates of Eimeria adenoeides, E. meleagrimitis, and E. gallopavonis. Severe infections were produced in unmedicated, infected controls. Weight gains were significantly improved by 1.5 or 3 ppm of halofuginone, and intestinal lesion scores and fecal droppings scores were greatly reduced. Oocyst shedding was reduced 91 to 100% by halofuginone treatments. Treatment with 6 ppm of halofuginone gave good protection but appeared to depress weight gains slightly. Treatment with 3 ppm was optimal for the control of all species of coccidia and for optimum performance.

Topics: Animals; Body Weight; Coccidiosis; Coccidiostats; Feeding Behavior; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Turkeys

The stability of experimentally induced resistance to halofuginone, decoquinate or arprinocid in lines of Eimeria tenella, was examined after 10 passages in unmedicated chickens. There was no loss of resistance to halofuginone or decoquinate. Resistance to arprinocid was unaffected in a line developed in the presence of 150 ppm of the drug but was unstable in a line developed with 60 ppm.

Topics: Adenine; Animals; Chickens; Coccidiosis; Coccidiostats; Decoquinate; Drug Resistance; Eimeria; Hydroxyquinolines; Piperidines; Quinazolines; Quinazolinones

Resistance to halofuginone has been developed by serially passaging the Houghton strain of Eimeria tenella in chickens medicated with progressively greater concentrations of drug. Attempts to develop resistance to 3 ppm (as recommended for use by the manufacturer) or higher concentrations, by the method of Weppelman et al., were unsuccessful.

Topics: Animals; Chickens; Coccidiosis; Coccidiostats; Drug Resistance, Microbial; Eimeria; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones

In a field trial 100 cattle suffering from naturally acquired East Coast fever caused by Theileria parva were treated with halofuginone lactate at a dose of 0.6 to 2.0 mg/kg body weight. Records were kept of exact diagnosis, treatment and follow up observations. The total recovery rate was 87%. The rate was highest in local breeds (up to 100%) and lowest in Friesian cattle (61.5%). Two treatments were administered to nine animals, two of which had relapsed four to six days after the first treatment. Halofuginone was well tolerated when the therapeutic dose was diluted in at least 500 ml of water. It proved to be a reliable and effective drug in the treatment of theileriosis under field conditions.

Topics: Animals; Burundi; Cattle; Piperidines; Quinazolines; Quinazolinones; Theileriasis

Floor-pen studies were conducted in three geographic locations to study the efficacy of halofuginone (3.0 ppm in feed) against important species of coccidia in turkeys. Severe coccidiosis exposure was obtained in studies in Georgia and Colorado and mild coccidiosis in Wisconsin. Coccidiosis caused by contamination of the feed with oocysts of Eimeria adenoeides, E. meleagrimitis, and E. gallopavonis was almost completely controlled by halofuginone at 3 ppm, even though intestinal lesion scores and weight loss were high in two studies. Halofuginone was also highly effective in preventing buildup of infection in pens contaminated by indirect means (movement by caretakers through the pens, etc.). Weight gains and feed conversion were best in poults receiving halofuginone, whether directly or indirectly exposed. There was no evidence that halofuginone caused any untoward reactions in the poults, even though the drug was fed until turkeys reached market weight.

Topics: Animals; Coccidiosis; Coccidiostats; Female; Male; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Turkeys

Experiments were conducted at three geographic locations in the United States (Arkansas, Colorado, and Minnesota) to examine the response of Large White turkeys to bambermycins in the presence of halofuginone. Each location utilized diets commonly used at that station for growing turkeys. All diets contained 3 ppm halofuginone, and were supplemented with 0, 1, 2, or 4 g bambermycins/ton (908 kg). Bambermycins was fed from day-old to marketing; halofuginone was removed from feed for 3 days prior to marketing. Although there were some significant differences in final body weights or feed utilization among locations, there were no significant interactions of location X bambermycins. Body weight of males was significantly (P less than .05) improved by the addition of 2 g bambermycins/ton. Body weight of females was significantly (P less than .05) improved by all levels of bambermycins; 4 g/ton stimulated significantly (P less than .05) superior gains than 1 or 2 g/ton. When body weight for both sexes was combined, bambermycins at 2 and 4 g/ton resulted in significant (P less than .05) improvements in body weight. Feed utilization, expressed as units of feed per unit of gain, was not significantly influenced by dietary treatment. There were no significant differences in mortality related to location or dietary treatment.

Topics: Animals; Anti-Bacterial Agents; Arkansas; Bambermycins; Body Weight; Coccidiostats; Colorado; Female; Food Additives; Male; Minnesota; Piperidines; Quinazolines; Quinazolinones; Sex Factors; Turkeys

Sulphadimidine, amprolium, halofuginone and chloroquine phosphate were administered to buffalo calves 10 days after experimental infection with Eimeria bareillyi. Animals given sulphadimidine or amprolium remained clinically normal and shed only a few oocysts in their faeces. Halofuginone was found partially effective and chloroquine phosphate completely ineffective in preventing faecal oocyst discharge and intestinal lesions. Sulphadimidine and amprolium treated calves gained weight, but chloroquine treated calves suffered progressive weight loss similar to that of infected untreated controls. No significant alterations of haematological values were observed either in the treated calves or in the untreated controls.

Topics: Amprolium; Animals; Buffaloes; Chloroquine; Coccidiosis; Coccidiostats; Eimeria; Intestine, Small; Piperidines; Quinazolines; Quinazolinones; Sulfamethazine

In a series of field trials 48 (55%) of 88 field cases of East Coast fever treated with halofuginone lactate recovered and survived and 40 (45%) died while 31 (86%) of 36 untreated control animals died of East Coast fever and five (14%) recovered. For cases diagnosed and treated early a 100% recovery rate was achieved. A single dose at 1.2 mg/kg body weight per os was adequate. Recovery rate was only 36% for cases diagnosed and treated at an advanced stage. Such cases required two doses at 1.2 mg/kg or a combination of 1.2 and 2.4 mg/kg body weight given at about two days interval. The temperature dropped to normal levels within 48 h of treatment and schizonts started degenerating as early as 24 h after treatment and had disappeared by 48 to 72 h. Piroplasms were not affected by the drug. Recovered animals remained free of East Coast fever for periods up to 12 months of observation despite continuous tick challenge. It was concluded that, with early detection and treatment augmented with proper tick control, outbreaks of East Coast fever can be effectively controlled with halofuginone lactate.

Topics: Animals; Cattle; Piperidines; Quinazolines; Quinazolinones; Theileriasis; Tick Infestations

A line of Eimeria tenella (H) resistant to robenidine, decoquinate and amprolium has been produced by genetic recombination. It was not possible to obtain a cross between this line and lines resistant to clopidol or arprinocid and halofuginone. Recombination between lines resistant to arprinocid and halofuginone resulted in a loss of pathogenicity.

Topics: Adenine; Amprolium; Animals; Chickens; Clopidol; Coccidiostats; Decoquinate; Drug Resistance, Microbial; Eimeria; Guanidines; Hydroxyquinolines; Picolines; Piperidines; Quinazolines; Quinazolinones; Recombination, Genetic; Robenidine

The efficacy of halofuginone (DL-trans-7-bromo-6-chloro-3,3-(3-hydroxy-2-piperidyl) acetonyl-4-(3H) quinazolinone), Wellcome 993 C (2-hydroxy-3-cyclohexyl-1,4-naphthoquinone), and oxytetracycline, all of which have been shown to have a schizonticidal effect in the treatment of bovine theileriosis, and the babesicidal drug diminazene diaceturate, were tested against the schizont stages of Babesia equi in cell culture. The in vitro test system measured DNA synthesis in treated and untreated cell lines. Halofuginone (0.02 microgram/ml) and Wellcome 993 C (5 micrograms/ml) suppressed the incorporation of tritiated thymidine by more than 80%. Oxytetracycline was less effective, while diminazene diaceturate showed no notable effect. The insufficient schizonticidal activity may explain the failure of diminazene diaceturate to cure B. equi infections. Schizonticidal drugs either alone, or in combination with known babesicidal drugs, should be tested in the chemotherapy of B. equi infections.

Topics: Amidines; Animals; Antiprotozoal Agents; Babesia; Cell Line; Diminazene; DNA; Lymphocytes; Naphthoquinones; Oxytetracycline; Piperidines; Quinazolines; Quinazolinones; Thymidine

Seven groups of calves were treated with parvaquone (993C: Clexon, Wellcome) (20 mg kg-1 intramuscularly) or halofuginone lactate (1.2 mg kg-1 orally) on the first, third or sixth day of significant pyrexia following artificial infection with the Hissar strain of Theileria annulata. Eight untreated control animals developed severe theileriosis and five died. All animals treated with parvaquone or halofuginone lactate on the first or third day of fever underwent relatively mild theileriosis and all of them recovered. One of five animals treated with parvaquone and three of six treated with halofuginone on the sixth day of fever died of theileriosis.

Topics: Administration, Oral; Animals; Antiprotozoal Agents; Cattle; Drug Administration Schedule; Injections, Intramuscular; Piperidines; Quinazolines; Quinazolinones; Theileriasis

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Coccidiostats; Meat; Piperidines; Quinazolines; Quinazolinones

Current models for antimalarial drug screening generally measure the survival of drug-treated rodents infected with Plasmodium berghei. Modifications of existing continuous culture methods for P. falciparum allow the rapid, accurate and economical determination of drug effects directly against the human pathogen. Parasite cultures can be maintained in RPMI 1640 medium supplemented with human or rabbit serum or with hypoxanthine-supplemented bovine serum. The antiparasite effects of four drugs, chloroquine, chloramphenicol, clindamycin, and halofuginone, are identical in these sera; drugs can be screened routinely against P. falciparum grown in bovine serum supplemented with hypoxanthine. Drug effects may be rapidly and accurately determined by monitoring the incorporation of 3H-hypoxanthine into parasite nucleic acids. Results obtained with this technique are highly correlated with those derived from visual counting of parasites in thin blood films. Compounds with antimalarial activity in culture may be further screened by measuring the effects of serum obtained from drug-treated rabbits on parasites in culture. The advantages of this system over models currently used for antimalarial screening are discussed.

Topics: Animals; Antimalarials; Cattle; Chloramphenicol; Chloroquine; Clindamycin; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Erythrocytes; Humans; Hypoxanthine; Hypoxanthines; Piperidines; Plasmodium falciparum; Quinazolines; Quinazolinones; Rabbits

Topics: Animal Feed; Chromatography, High Pressure Liquid; Coccidiostats; Piperidines; Quinazolines; Quinazolinones

Halofuginone lactate, given once orally at a dosage rate of 1,2 mg/kg body mass on the 1st, 3rd or 5th days of fever, resulted in the recovery of only 1 out of 5 splenectomized cattle. Three splenectomized animals, treated on the 1st as well as the 4th day of fever, recovered and were then carriers. Six untreated controls all died. The potential value of a chemotherapeutic agent for Theileria parva lawrencei infections in South Africa is discussed.

Topics: Animals; Cattle; Coccidiostats; Piperidines; Quinazolines; Quinazolinones; Theileriasis

The efficacy of Wellcome compound 993C and halofuginone was compared using cattle artificially infected with Theileria parva and using in vitro cultures of T parva. At a total dose of 20 mg/kg administered by the intramuscular route, 993C cured all nine cattle of advanced theileriosis and no major recrudescences of the infection occurred. Halofuginone was given by mouth at 1.2 mg/kg and cured five cattle of early clinical theileriosis, but significant recrudescences of infection occurred. Halofuginone at 1.2 mg/kg cured a further five out of six cattle with advanced theileriosis, but four of the five survivors developed moderate to severe resurgences of infection. The dose of 20 mg/kg of 993C was known to be close to its effective level, but 1.2 mg/kg halofuginone, the dose used by earlier workers, may not have been adequate. All 10 untreated control cattle died of acute theileriosis. The in vitro EC50 of both compounds was 0.003 mg per litre but, whereas the dose response curve of 993C was sigmoid, that of halofuginone was bellshaped. This may reflect toxicity to the host lymphoblastoid cells with higher concentrations of halofuginone.

Topics: Animals; Antiprotozoal Agents; Apicomplexa; Cattle; Coccidiostats; Lymph Nodes; Male; Naphthoquinones; Piperidines; Quinazolines; Quinazolinones; Theileriasis

Coccidia were isolated from litter samples collected in poultry houses in Georgia and other southeastern states and from intestinal scrapings from chickens submitted to diagnostic laboratories. The most common species of coccidia encountered were E. tenella, E. acervulina, and E. maxima. Each of the 41 isolates was tested for sensitivity to eight commercial anticoccidial drugs. Most of the isolates were resistant to some of the drugs judged by the parameters of percent weight gain, percent lesion score reduction, and a sensitivity index score. There was a high frequency of resistance to clopidol, amprolium/ethopabate, nequinate, zoalene, and sulfaquinoxaline. A very small percentage of the isolates tested were resistant to nicarbazin, robenidine, or halofuginone.

Topics: Amprolium; Animals; Chickens; Coccidiostats; Drug Resistance; Eimeria; Intestines; Manure; Nicarbazin; Piperidines; Quinazolines; Quinazolinones; Robenidine

Monensin, lasalocid, salinomycin, nicarbazin, halofuginone, or arprinocid were fed to 1-week-old male broiler chicks at recommended levels and 1.5, 2, 2.5, and 3 times the recommended level, for 3 weeks. Pair-feeding experiments also were conducted to investigate the extent that growth depression with medicated diets could be attributed to the drop in feed consumption. At the recommended level of drugs, growth and feed conversion were not significantly affected. At elevated drug levels, performance was impaired; the adverse effects of drugs became more pronounced with increasing the concentrations in the diets. Weight gain was significantly depressed at 1.5X with arprinocid, halofuginone, and salinomycin, at 1 to 2X with monensin, at 2X with lasalocid, and at 2.5X with nicarbazin. Feed conversion, however, was adversely affected by 2X with halofuginone or 2.5X with salinomycin, nicarbazin, arprinocid, monensin, or lasalocid. The results of the pair-feeding experiments with 2 to 3 times drug levels indicated that most of the growth depression with medicated diets could be attributed to reduced feed consumption, but all drugs except arprinocid caused some additional growth depression.

Topics: Adenine; Animal Feed; Animals; Body Weight; Chickens; Coccidiostats; Growth; Lasalocid; Male; Monensin; Nicarbazin; Piperidines; Quinazolines; Quinazolinones

In goats infected with Sarcocystis capracanis and sheep infected with S. Ovicanis prophylactical application of halofuginone prevented clinical symptoms but also prevented the development of immunity to homologous challenge. Halofuginone was most effective in the treatment of acute sarcosporidiosis at a dose of 0.67 mg/kg b. w. on two successive days in both goats and sheep. In acute bovine theileriosis (Theileria parva and T. annulata) halofuginone was effective at a single dose about of 1.2 to 2 mg/kg b.w. Cattle recovered from theileriosis through treatment were immune to homologous challenge.

Topics: Animals; Cattle; Coccidiostats; Drug Combinations; Goats; Oxytetracycline; Piperidines; Quinazolines; Quinazolinones; Sarcocystosis; Sheep; Sheep Diseases; Sulfadoxine; Theileriasis; Trimethoprim

The effect of halofuginone (Stenorol) on schizonts of Theileria parva was studied by means of the electron microscope. After administration of 1,2 mg halofuginone per kg, it was observed that infected lymphocytes burst apart, whereas the intracellular parasites were at first little affected, showing some expansion of the perinuclear spaces. Once they were released from the cells, the schizonts degenerated completely. Apparently they are not capable of surviving outside the host cells. Probably this is due to the lack of a pellicle, which is characteristic for motile (i.e. normally extracellular) stages. The destruction of parasitized host cells occurred very rapidly, so that no infected cells could be observed 126 h after treatment. Uninfected lymphocytes were not affected by the drug.

Topics: Animals; Apicomplexa; Cattle; Cattle Diseases; Lymphocytes; Piperidines; Quinazolines; Quinazolinones; Theileriasis

Halofuginone and arprinocid, new anticoccidial drugs, were tested to determine the time of peak activity in the life cycle of Eimeria and whether they were coccidiocidal or coccidiostatic. Halofuginone was completely effective if medication was initiated by Day 3 postinoculation, but only partly effective if given on Days 4-7. Arprinocid was effective if given on Days 2-7 postinoculation, but only partly effective if given on Days 3-7. When medication was started on Day 0 and withdrawn on various subsequent days, good activity was recorded with halofuginone if the drug was given up to Day 1 postinoculation, but arprinocid required feeding up to Day 4 postinoculation. In other studies, medication was given until Days 6 or 7 postinoculation, then discontinued, to allow further development of coccidia that were arrested by the drug. With halofuginone, the oocyst passage was low with E. tenella and E. maxima but moderate with E. mivati. With arprinocid, oocyst passage was low with E. maxima and E. mivati but moderate with E tenella. These results suggest that halofuginone is active over a broader part of the life cycle than arprinocid, but both drugs had a predominantly coccidiocidal action.

Topics: Adenine; Animals; Chickens; Coccidiosis; Coccidiostats; Eimeria; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Species Specificity

Piglets which were early-weaned at the age of 21.7 days and experimentally monoinfected with oocysts of Isospora suis showed distinct reductions in zootechnical criteria during an experimental period of 4 weeks. The daily liveweight gains in the infected piglets (group B) was 19.7% lower than in the control group A, which was free of Coccidia. Comparative photographs with the REM showed serious lesions in the small intestine of infected piglets, which are thought to be mainly responsible for the reduced productivity. The application of 150 mg Lasalocid per kg of total feed to infected piglets caused the rate of weight gain to attain the same values as the noninfected controls (group A). Piglets receiving Lasalocid treatment passed oocysts with the faeces which were infectious. On the other hand, infected piglets which were treated with 6 mg Halofuginone per kg of total feed did not contain any oocysts in the faeces. Despite having a higher liveweight at the beginning of the experiment, this group only gained as much liveweight as the infected piglets (group B). This depression in liveweight gains could be explained by the significantly reduced uptake of feed, which was 21.1% lower than in the controls (group A). 6 weeks after the first infection, a re-infection resulted in the appearance of oocysts in the faeces of the piglets which had been treated with Halofuginone. On the other hand, the animals treated with Lasalocid had developed an efficient immunity to Isospora suis.

Topics: Animals; Body Weight; Coccidiosis; Feces; Isospora; Lasalocid; Piperidines; Quinazolines; Quinazolinones; Swine; Weaning

Methods are described for the analysis of the anti-coccidial drug, halofuginone, in chicken tissue at concentrations as low as 1 ppb (0.001 ppm) and in chicken feed at a concentration of 3 ppm, using high-performance liquid chromatography. The tissue analysis involves: enzymatic release of the halofuginone followed by ethyl acetate extraction under basic conditions, partition into ammonium acetate buffer, concentration using Sep-pakTM C18 cartridge. The feed analysis involves: ethyl acetate extraction under basic conditions, partition into hydrochloric acid, concentration using XAD-2 column chromatography. Both methods use high-performance liquid chromatography with ultraviolet detection for the final analysis. Precision and accuracy data for both methods are given.

Topics: Animal Feed; Animals; Chickens; Chromatography, High Pressure Liquid; Coccidiostats; Piperidines; Quinazolines; Quinazolinones; Tissue Distribution

A range of anticoccidial drugs was tested in young pheasants inoculated with three species of eimeria. Eimeria colchici, the most pathogenic species, was completely controlled by clopidol/methyl benzoquate and robenidine hydrochloride with the elimination of oocyst production. Clopidol and arprinocid gave good protection with reduced oocyst output and clopidol also adversely affected oocyst sporulation. Halofuginone and monensin were less effective. Amprolium hydrochloride/sulphaquinoxaline/ethopabate (ASE) and sulphaquinoxaline were ineffective. Both halofuginone and ASE controlled infections caused by E phasiani and E duodenalis. Halofuginone at 3, 6 or 9 ppm in the food produced no toxic effects in pheasants but growth rates of red-legged partridge chicks were retarded by concentrations of 3 and 6 ppm. Halofuginone protected young pheasants in a field trial in which sulphaquinoxaline was less effective.

Topics: Animals; Bird Diseases; Birds; Clopidol; Coccidiosis; Coccidiostats; Piperidines; Quinazolines; Quinazolinones

Topics: Animals; Apicomplexa; Body Temperature; Cattle; Piperidines; Quinazolines; Quinazolinones; Species Specificity; Theileriasis

Three experiments were designed to test the efficacy of salinomycin and stenorol against infection by various Eimeria species on cage reared broiler type chicks. Efficacy was based on a coccidial index. Sixty parts per million salinomycin alone or in combination with 50 ppm 3 nitro significantly improved the index over basal treatments or when 3 nitro was used alone. The differences in index values recorded for coban and salinomycin were not significant. Stenorol significantly improved the index and appeared to be a most effective anticoccidial product. Broiler chickens reared in floor pens to 8 weeks showed a significant reduction in weight gain when the diet contained salinomycin +3 nitro or coban. Stenorol at 3, 6, or 9 ppm reduced body weight, with linear regression for this effect being highly significant (P less than .01). No coccidiosis was observed.

Topics: Animals; Chickens; Coccidiosis; Coccidiostats; Ionophores; Male; Monensin; Piperidines; Poultry Diseases; Pyrans; Quinazolines; Quinazolinones; Roxarsone

The efficacy of Stenorol (halofunginone) was tested against six species of chicken Eimeria in a series of four battery experiments utilizing 3- to 4 1/2-week-old Cobb color-sexed broiler chickens. There were five replicates of eight chickens per replicate for each treatment of an experiment or a total of 1080 birds used in the study. The isolates were predominantly E. tenella, E. maxima, E. acevulina, E. necatrix, E. brunetti, or E. mivati and had previously been proven partially to totally resistant to several commercially available anticoccidial drugs. Halofunginone, at 3 ppm in the ration, was highly effective (P less than .01) against all six isolates as measured by weight gain at D+6 or +7and D+12 or +14 postinoculation; feed efficiency at D-2 to D+12 or +14; morbidity; mortality; dropping score; lesion score (D+6 or +7); and oocyst production during 4 or 5 days postinoculation (D = day of inoculation). The drug was not as effective against E. acervulina as against the other species, and increasing halofuginone to 4 ppm failed to improve activity of the drug signif;cantly against this isolate. However, 3 ppm of drug was effective against two other isolates of E. acervulina (from Alabama and Mississippi); 4 ppm was quite effective (P less than .01) in reducing dropping and lesion scores, but not significantly better than 3 ppm as measureed by other parameters. No relapse occurred after drug withdrawal and halofuginone was found to be cidal rather than static.

Topics: Animals; Body Weight; Chickens; Coccidiosis; Coccidiostats; Drug Resistance; Eimeria; Feeding Behavior; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones

A total of 879 broiler strain chickens ranging from 2 1/2- to 7 1/2 weeks of age was utilized in four battery experiments to determine whether Roxarsone and/or bacitracin MD added to halofuginone were compatible and beneficial in reducing the effects of coccidial infections. The additives were generally beneficial as measured by weight gain and feed efficiency but not as measured by other parameters such as dropping score, lesion score, or oocyst production. The addition of 200 g of bacitracin/ton of feed did not give an additional response above that from 50 g/ton. Roxarsone in the ration was more effective in younger chickens (2 1/2 week old) than older ones (6 weeks, 2 days and 7 weeks, 3 days).

Topics: Animals; Arsenicals; Bacitracin; Body Weight; Chickens; Coccidiosis; Coccidiostats; Drug Combinations; Eimeria; Feeding Behavior; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones; Roxarsone; Salicylates

Halofuginone at 3 ppm in the ration was tested against turkey coccidial infections caused by four species, in a series of eight battery trials of 16 days duration. The drug was evaluated against infections caused by Eimeria meleagrimitis, E. adenoeides, E. gallopavonis, and E. dispersa. As measured by livability, weight gain, feed efficiency, morbidity, dropping score, lesion score, and oocyst production the drug was highly effective in Beltsville Small White turkeys. The drug at 3 ppm appeared to be about equally effective against all four species and almost completely prevented infection or the effects of infection in some experiments, except when the challenge was too severe.

Topics: Animals; Body Weight; Coccidiosis; Coccidiostats; Drug Resistance; Eimeria; Feeding Behavior; Piperidines; Poultry Diseases; Quinazolinones; Turkeys

Methods are described for the analysis of the anti-coccidial drug, halofuginone, at concentrations of 3 ppm in chicken feed, using gas-liquid chromatography and high-performance liquid chromatography. Both methods are based on ethyl acetate extraction, partition into hydrochloric acid and purification and concentration using XAD-2 column chromatography. The precision and accuracy of both methods is given.

Topics: Animal Feed; Animals; Chickens; Chromatography, Gas; Chromatography, High Pressure Liquid; Coccidiostats; Food, Fortified; Methods; Minerals; Piperidines; Quinazolines; Quinazolinones

The activities of monensin, lasalocid and halofuginone against Eimeria tenella, E. brunetti and E. necatrix have been studied under laboratory conditions. Complete control of experimental infections in the chick, separable from toxicity, was not obtained with monensin, but was achieved with the other two compounds at levels of 150 and 6 ppm in the food respectively. All three compounds appear to inhibit coccidial development very early in the life-cycle, and to have a fairly rapid lethal effect, monensin and lasalocid more so than the febrifugine derivative. In vivo observations have been supplemented with in vitro studies. Some discussion of the difficulties of relating laboratory experiments to field performance is given.

Topics: Administration, Oral; Animal Feed; Animals; Chickens; Coccidiosis; Coccidiostats; Eimeria; Feces; Lasalocid; Monensin; Piperidines; Poultry Diseases; Quinazolines; Quinazolinones