pyrimidinones and Disease-Models--Animal

Liver fibrosis is the common pathological basis of all chronic liver diseases, and is the necessary stage for the progression of chronic liver disease to cirrhosis. As one of pathogenic factors, inflammation plays a predominant role in liver fibrosis

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Hepatic Stellate Cells; Hepatocytes; Humans; Imidazoles; Inflammation; Inflammation Mediators; Liver; Liver Cirrhosis; Molecular Targeted Therapy; Protein Kinase Inhibitors; Pyrimidinones; Signal Transduction; Sulfoxides; Ursodeoxycholic Acid

Telbivudine, beta-L-2'-deoxythymidine (LdT), is a new beta-L-nucleoside analogue with potent inhibitory activity against the hepatitis B virus. In in vitro studies and animal models, telbivudine has demonstrated potent and specific antiviral activity against hepatitis B. Additionally, in preclinical animal toxicology studies, telbivudine showed no adverse side effects or adverse effects on mitochondrial function. The promising results of the early in vitro and animal telbivudine studies prompted the development and initiation of Phase I and II human clinical trials. The Phase I clinical study demonstrated that end-of-treatment virological response rates were better for telbivudine recipients at multiple dosing levels as compared with placebo patients. The subsequent Phase IIb human clinical study demonstrated superior antiviral efficacy of telbivudine, significantly better ALT normalisation and better hepatitis B e-antigen loss as compared with lamivudine. Telbivudine was well tolerated with no identified safety issues. Virological breakthrough with telbivudine was significantly lower than with lamivudine.

Topics: Animals; Antiviral Agents; Disease Models, Animal; Hepatitis B, Chronic; Humans; Lamivudine; Nucleosides; Pyrimidinones; Randomized Controlled Trials as Topic; Reverse Transcriptase Inhibitors; Telbivudine; Thymidine

Topics: Acute Disease; Animals; Bone Marrow; Bone Marrow Transplantation; Cyclophosphamide; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Leukemia; Lymphoma; Mechlorethamine; Neoplastic Stem Cells; Photosensitivity Disorders; Podophyllotoxin; Pyrimidinones; Rats; Transplantation, Autologous

This is the first prospective study of a combination therapy involving a cardenolide and a MEK inhibitor for metastatic melanoma. Whereas BRAF mutant melanomas can exhibit profound responses to treatment with BRAF and MEK inhibitors, there are fewer options for BRAF wild-type melanomas. In preclinical studies, we discovered that cardenolides synergize with MEK inhibitor to promote the regression of patient-derived xenografts irrespective of BRAF mutation status. We therefore conducted a phase 1B study of digoxin 0.25 mg and trametinib 2 mg given orally once daily in 20 patients with advanced, refractory, BRAF wild-type melanomas. The most common adverse events were rash, diarrhea, nausea, and fatigue. The response rate was 4/20 or 20% with response durations of 2, 4, 6, and 8 months. The disease control rate (including partial responses and stable disease) was 13/20 or 65% of patients, including 5/6 or 83% of patients with NRAS mutant melanomas and 8/14 or 57% of NRAS wild-type melanomas. Patients with stable disease had disease control for 2, 2, 2, 4, 5, 6, 7, 10, and 10 months. Xenografts from four patients recapitulated the treatment responses observed in patients. Based on these pilot results, an expansion arm of digoxin plus MEK inhibitor is warranted for NRAS mutant metastatic melanoma patients who are refractory or intolerant of immunotherapy.. Digoxin plus trametinib is well tolerated and achieves a high rate of disease control in BRAF wild-type metastatic melanoma patients.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Combined Chemotherapy Protocols; Digoxin; Disease Models, Animal; Female; Humans; Male; Melanoma; Mice; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Retreatment; Treatment Outcome; Xenograft Model Antitumor Assays

Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear.. To strengthen the PI3Kβ target validation using the novel, short-acting inhibitor AZD6482.. AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC(50) 0.01 μm). A maximal anti-platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti-thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3-h infusion. The ex vivo anti-platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin-induced human adipocyte glucose uptake in vitro (IC(50) of 4.4 μm). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm. In man, the homeostasis model analysis (HOMA) index increased by about 10-20% at the highest plasma concentration of 5.3 μm.. This is the first human target validation for PI3Kβ inhibition as anti-platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at 'supratherapeutic' plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.

Topics: Adipocytes; Adolescent; Adult; Animals; Bleeding Time; Blood Platelets; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Double-Blind Method; Fibrinolytic Agents; Glucose; Hemostasis; Hemostatics; Humans; Insulin Resistance; Male; ortho-Aminobenzoates; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidinones; Rats; Signal Transduction; Thrombosis; Time Factors; Young Adult

In a previous in vitro study, dihydropyrimidinone-derived selenoesteres demonstrated antioxidant properties, metal chelators and inhibitory acetylcholinesterase (AChE) activity, making these compounds promising candidates for Alzheimer's Disease (AD) treatment. However, these effects have yet to be demonstrated in an in vivo animal model; therefore, this study aimed to evaluate the safety and efficacy of eight selenoester compounds in a Caenorhabditis elegans model using transgenic strains for amyloid-beta peptide (Aβ) aggregation. The L1 stage worms were acutely exposed (30 min) to the compounds at concentrations ranging from 5 to 200 μM and after 48 h the maintenance temperature was increased to 25 ° C for Aβ expression and aggregation. After 48 h, several parameters related to phenotypic manifestations of Aβ toxicity and mechanistic elucidation were analyzed. At the concentrations tested no significant toxicity of the compounds was found. The selenoester compound FA90 significantly reduced the rate of paralyzed worms and increased the number of swimming movements compared to the untreated worms. In addition, FA90 and FA130 improved egg-laying induced by levamisole and positively modulated HSP-6 and HSP-4 expression, thereby increasing reticular and mitochondrial protein folding response in C. elegans, which could attenuate Aβ aggregation in early exposure. Therefore, our initial screening using an alternative model demonstrated that FA90, among the eight selenoesters evaluated, was the most promising compound for AD evaluation screening in more complex animals.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Animals; Caenorhabditis elegans; Disease Models, Animal; Levamisole; Neuroprotective Agents; Organisms, Genetically Modified; Organoselenium Compounds; Oviposition; Pyrimidinones

Metastatic progression of tumors is driven by genetic alterations and tumor-stroma interaction. To elucidate the mechanism underlying the oncogene-induced gastric tumor progression, we have developed an organoid-based model of gastric cancer from GAstric Neoplasia (GAN) mice, which express Wnt1 and the enzymes COX2 and microsomal prostaglandin E synthase 1 in the stomach. Both p53 knockout (GAN-p53KO) organoids and KRAS

Topics: Animals; Disease Models, Animal; Epithelial-Mesenchymal Transition; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Neoplasm Metastasis; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Signal Transduction; Stomach Neoplasms; Tumor Hypoxia; Tumor Microenvironment; Tumor Suppressor Protein p53

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is an inflammatory immune disease that is characterized by infiltrating inflammatory cells in the prostate and pelvic or by perineal pain. Receptor CXCR3modulates immune and inflammatory responses; however, the effects of CXCR3 antagonist AMG487 in the context of CP/CPPS are unknown. Therefore, we investigated the effect of AMG487 in experimental autoimmune prostatitis (EAP) mice and explored the potential functional mechanisms.. The EAP model was induced by intradermally injecting a mixture of prostate antigens and complete Freund's adjuvant on Days 0 and 28. To evaluate the effect of AMG487 on EAP mice, treatment with AMG487 and vehicle solution was conducted for the indicated period. Then, procedures were performed, including behavioral test, to evaluate the pain response to stimulation before the mice were killed and a histological assessment to evaluate the inflammation after the mice were killed. Immunofluorescence, flow cytometry, and Western blot assay were used to analyze the functional phenotype and regulation mechanism of AMG487 on T helper type 1 (Th1) cells and macrophages.. We found high expression of CXCR3 in human benign prostate tissues with inflammation and EAP mice. The elevated CXCR3 in prostate tissues correlates with the severity of inflammation. CXCR3 antagonist AMG487 treatment ameliorated the inflammatory changes and the pelvic pain of EAP mice. AMG487 inhibits Th1 cell differentiation through the IL-12/STAT4pathway and inhibits pro-inflammatory M1 macrophages through the lipopolysaccharide/NF-κB p65signaling. AMG487 could inhibit the secretion of inflammatory mediators in EAP mice.. CXCR3 antagonist AMG487 could ameliorate the inflammatory changes and the pelvic pain of EAP mice by diminishing Th1 cell differentiation and inhibiting macrophage M1 phenotypic activation. Thus, the results imply that AMG487 has the potential as an effective therapeutic agent in the prevention and treatment of EAP.

Topics: Acetamides; Animals; Autoimmune Diseases; Cell Differentiation; Chronic Pain; Disease Models, Animal; Humans; Inflammation; Macrophages; Male; Mice; Pelvic Pain; Phenotype; Prostatitis; Pyrimidinones; Receptors, CXCR3

We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes.. Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized.. In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance.. In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Glioma; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Sirolimus

Alzheimer's disease (AD) pathology is associated with complex interactions among multiple factors, involving an intertwined network of various signaling pathways. The polypharmacological approach is an emerging therapeutic strategy that has been proposed to overcome the multifactorial nature of AD by targeting multiple pathophysiological factors including amyloid-β (Aβ) and phosphorylated tau. We evaluated a blood-brain barrier penetrating phosphodiesterase 5 (PDE5) inhibitor, mirodenafil (5-ethyl-2-7-n-propyl-3,5-dihydrro-4H-pyrrolo[3,2-d]pyrimidin-4-one), for its therapeutic effects on AD with polypharmacological properties.. To evaluate the potential of mirodenafil as a disease-modifying AD agent, mirodenafil was administered to test its effects on the cognitive behaviors of the APP-C105 AD mouse model using the Morris water maze and passive avoidance tests. To investigate the mechanisms of action that underlie the beneficial disease-modifying effects of mirodenafil, human neuroblastoma SH-SY5Y cells and mouse hippocampal HT-22 cells were used to show mirodenafil-induced alterations associated with the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG)/cAMP-responsive element-binding protein (CREB) pathway, apoptotic cell death, tau phosphorylation, amyloidogenesis, the autophagy-lysosome pathway, glucocorticoid receptor (GR) transcriptional activity, and the Wnt/β-catenin signaling.. Here, mirodenafil is demonstrated to improve cognitive behavior in the APP-C105 mouse model. Mirodenafil not only reduced the Aβ and phosphorylated tau burdens in vivo, but also ameliorated AD pathology induced by Aβ through the modulation of the cGMP/PKG/CREB signaling pathway, glycogen synthase kinase 3β (GSK-3β) activity, GR transcriptional activity, and the Wnt/β-catenin signaling in neuronal cells. Interestingly, homodimerization and nuclear localization of GR were inhibited by mirodenafil, but not by other PDE5 inhibitors. In addition, only mirodenafil reduced the expression levels of the Wnt antagonist Dickkopf-1 (Dkk-1), thus activating the Wnt/β-catenin signaling.. These findings strongly suggest that the PDE5 inhibitor mirodenafil shows promise as a potential polypharmacological drug candidate for AD treatment, acting on multiple key signaling pathways involved in amyloid deposition, phosphorylated tau burden, the cGMP/PKG/CREB pathway, GSK-3β kinase activity, GR signaling, and the Wnt/β-catenin signaling. Mirodenafil administration to the APP-C105 AD mouse model also improved cognitive behavior, demonstrating the potential of mirodenafil as a polypharmacological AD therapeutic agent.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; beta Catenin; Cyclic GMP; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; Humans; Mice; Neuroblastoma; Phosphodiesterase 5 Inhibitors; Phosphorylation; Pyrimidinones; Sulfonamides; tau Proteins

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Enhancer Elements, Genetic; Female; Histone Deacetylase Inhibitors; Humans; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Ovarian Neoplasms; Protein Kinase Inhibitors; Pyridones; Pyrimidinones

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease

Topics: Animals; Antimalarials; Artemisinins; Cell Line, Tumor; Disease Models, Animal; Dogs; Drug Resistance; Female; Hep G2 Cells; Humans; Liver; Macaca fascicularis; Madin Darby Canine Kidney Cells; Malaria, Falciparum; Male; Mice; Mice, Inbred BALB C; Plasmodium cynomolgi; Plasmodium falciparum; Plasmodium yoelii; Pyrimidinones

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of

Topics: Animals; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Female; Imidazoles; Immunotherapy; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Targeted Therapy; Monomeric GTP-Binding Proteins; Mutation; Oximes; Programmed Cell Death 1 Receptor; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidines; Pyrimidinones; Skin Neoplasms; Sulfones; T-Lymphocytes, Regulatory

The uric acid metabolism pathway is more similar in primates and humans than in rodents. However, there are no reports of using primates to establish animal models of hyperuricaemia (HUA).. To establish an animal model highly related to HUA in humans.. Inosine (75, 100 and 200 mg/kg) was intraperitoneally administered to adult male rhesus monkeys (. Inosine (200 mg/kg) effectively increased the SUA level in rhesus monkeys from 51.77 ± 14.48 at 0 h to 178.32 ± 14.47 μmol/L within 30 min and to peak levels (201.41 ± 42.73 μmol/L) at 1 h. PNP mRNA level was increased, whereas XO mRNA and protein levels in the liver were decreased by the inosine group compared with those in the control group. No changes in mRNA and protein levels of the renal uric acid transporter were observed. Ulodesine, allopurinol and febuxostat eliminated the inosine-induced elevation in SUA in tested monkeys.. An acute HUA animal model with high reproducibility was induced; it can be applied to evaluate new anti-HUA drugs

Topics: Acute Disease; Allopurinol; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Febuxostat; Hyperuricemia; Imino Furanoses; Inosine; Macaca mulatta; Male; Pyrimidinones; Reproducibility of Results; Uric Acid

We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson's disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein (αSyn) spread in a PD mouse model.. The acute effects of single-dose treatment with DDL-112 on interleukin-1β-induced extracellular vesicle (EV) release in brain tissue of Thy1-αSyn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant αSyn aggregates in the PD model were determined.. In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in αSyn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Enzyme Inhibitors; Exosomes; Mice, Transgenic; Naphthalenes; Parkinson Disease; Protein Aggregates; Pyrimidinones; Sirtuins; Sphingomyelin Phosphodiesterase

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775)

Topics: Animals; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cells, Cultured; Computational Biology; Disease Models, Animal; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Profiling; Homologous Recombination; Humans; Mice; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Rad51 Recombinase; Radiation-Sensitizing Agents; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

Mutations in SET-binding protein 1 (SETBP1) are associated with poor outcomes in myeloid leukemias. In the Ras-driven leukemia, juvenile myelomonocytic leukemia, SETBP1 mutations are enriched in relapsed disease. While some mechanisms for SETBP1-driven oncogenesis have been established, it remains unclear how SETBP1 specifically modulates the biology of Ras-driven leukemias. In this study, we found that when co-expressed with Ras pathway mutations, SETBP1 promoted oncogenic transformation of murine bone marrow in vitro and aggressive myeloid leukemia in vivo. We demonstrate that SETBP1 enhances the NRAS gene expression signature, driving upregulation of mitogen-activated protein kinase (MAPK) signaling and downregulation of differentiation pathways. SETBP1 also enhances NRAS-driven phosphorylation of MAPK proteins. Cells expressing NRAS and SETBP1 are sensitive to inhibitors of the MAPK pathway, and treatment with the MEK inhibitor trametinib conferred a survival benefit in a mouse model of NRAS/SETBP1-mutant disease. Our data demonstrate that despite driving enhanced MAPK signaling, SETBP1-mutant cells remain susceptible to trametinib in vitro and in vivo, providing encouraging preclinical data for the use of trametinib in SETBP1-mutant disease.

Topics: Animals; Bone Marrow; Carrier Proteins; Cells, Cultured; Disease Models, Animal; GTP Phosphohydrolases; Humans; Leukemia, Myelomonocytic, Juvenile; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutation; Nuclear Proteins; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.

Topics: Acrylonitrile; Aniline Compounds; Animals; Bone and Bones; Disease Models, Animal; Gain of Function Mutation; High-Throughput Nucleotide Sequencing; Humans; Lymphangiogenesis; Lymphatic Vessels; Mice; Osteolysis, Essential; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Signal Transduction; Tertiary Lymphoid Structures

Addressing the global need for the development of safe and potent NSAIDs, new series of oxadiazolo and thiadiazolo fused pyrmidinones were synthesized and initially tested for their analgesic activity. All tested compounds showed promising analgesic activity compared with the reference standard indomethacin. Moreover, anti-inflammatory activity evaluation, ulcerogenic liability, and in vitro COX-1, COX-2 enzyme inhibition assays were also performed for the most active derivatives. The methoxyphenyl piperazinyl derivative 3d showed analgesic activity surpassing indomethacin with protection of 100%, and 83%; respectively. Also 3d showed good anti-inflammatory activity with relatively lower ulcer index compared with other tested compounds, and potent COX-1 and COX-2 inhibitory activity with IC

Topics: Analgesics; Animals; Anti-Inflammatory Agents; Behavior, Animal; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Drug Design; Edema; Humans; Male; Mice; Pyrimidinones; Quantitative Structure-Activity Relationship; Rats; Rats, Wistar

WEE1 plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response (DDR). Inhibition of WEE1 can increase the instability of the genome and have anti-tumor effects in some solid tumors. However, it has certain limitations for multiple cancer cells from different lineages. Therefore, we consider the use of synthetic lethal interactions to enhance the therapeutic effect. Our experiments proved that WEE1 inhibitor (WEE1i) can activate the ataxia telangiectasia and RAD3-related (ATR) pathway and that blockage of ATR dramatically sensitized the WEE1i-induced cell death. The tumor-selective synthetic lethality between bioavailable WEE1 and ATR inhibitors led to tumor remission in vivo. Mechanistically, the combination promoted the accumulation of cytosolic double-strand DNA, which subsequently activated the stimulator of the interferon gene (STING) pathway and induced the production of type I interferon and CD8+ T cells, thereby inducing anti-tumor immunity. Furthermore, our study found that immune checkpoint programmed death-ligand 1 is upregulated by the combination therapy, and blocking PD-L1 further enhances the effect of the combination therapy. In summary, as an immunomodulator, the combination of WEE1i with ATR inhibitor (ATRi) and immune checkpoint blockers provides a potential new approach for cancer treatment.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; DNA; DNA Damage; DNA, Neoplasm; Drug Synergism; Female; G2 Phase Cell Cycle Checkpoints; Genomic Instability; Humans; Immunity; Immunotherapy; Indoles; Interferon Type I; M Phase Cell Cycle Checkpoints; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Morpholines; Ovarian Neoplasms; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Sulfonamides; Tumor Microenvironment; Tumor Stem Cell Assay; Up-Regulation

Monocarboxylate transporter 1 (MCT1) is a regulator of cell metabolism and a therapeutic target for cancer treatment. Understanding the changes in tumour function accompanying MCT1 inhibition will better characterise the anti-tumour effects of MCT1 inhibitors, potentially enabling the identification of pharmacodynamic biomarkers for the clinical development of these agents.. We assessed the impact of the MCT1 inhibitor AZD3965 on tumour metabolism and immune cell infiltration as key determinants of tumour biological function in the MCT1-dependent Raji B cell lymphoma model.. Treatment of Raji xenograft-bearing severe combined immunodeficiency mice with AZD3965 led to inhibition of tumour growth paralleled with a decrease in tumour choline, as detected by non-invasive in vivo proton nuclear magnetic resonance spectroscopy. This effect was attributed to inhibition of phosphocholine de novo synthesis following decreased choline kinase α protein and messenger RNA expression that correlated with the AZD3965-induced build-up in intracellular lactate. These changes were concomitant with increased tumour immune cell infiltration involving dendritic and natural killer cells.. Our data provide new insights into the metabolic and cellular changes that occur in the tumour microenvironment following MCT1 blockade, which may contribute to the anti-tumour activity of AZD3965 and could have potential as pharmacodynamic biomarkers of MCT1 inhibition.

Topics: Animals; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Lipid Metabolism; Mice; Monocarboxylic Acid Transporters; Pyrimidinones; Thiophenes

Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC-8) to examine the expression of C-X-C motif chemokine receptor 4 (CXCR4) and β-catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up-regulated in tubules, with a concomitant activation of β-catenin. CXCR4 expression level was positively correlated with the expression of β-catenin target MMP-7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and β-catenin signalling, protected against tubular injury and renal fibrosis. CXCR4-induced renal fibrosis was inhibited by treatment with ICG-001, an inhibitor of β-catenin signalling. In HKC-8 cells, overexpression of CXCR4 induced activation of β-catenin and deteriorated cell injury. These effects were inhibited by ICG-001. Stromal cell-derived factor (SDF)-1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC-8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3β mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF-1α-induced activation of β-catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and β-catenin activation in renal fibrosis in association with JAK/STAT/GSK3β pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.

Topics: Amino Acid Motifs; Animals; Benzylamines; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Chemokine CXCL12; Cyclams; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Gene Knockdown Techniques; Glycogen Synthase Kinase 3 beta; Humans; Janus Kinase 2; Kidney; Matrix Metalloproteinase 7; Mice; Pyrimidinones; Receptors, CXCR4; Renal Insufficiency, Chronic; STAT3 Transcription Factor; STAT6 Transcription Factor

Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL.. To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents.. Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

Topics: Animals; Computational Biology; Disease Models, Animal; Disease Susceptibility; Down Syndrome; Gene Expression Profiling; Humans; Immunophenotyping; Leukemia, B-Cell; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; Oncogenes; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; ras Proteins; Signal Transduction

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Clonal Evolution; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Synergism; Female; GTP Phosphohydrolases; Humans; Leukemia, Myelomonocytic, Chronic; Membrane Proteins; Mice; Mutation; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Receptor, Notch1; RNA, Small Interfering; Xenograft Model Antitumor Assays

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19

Topics: Acetamides; Animals; Antigens, CD19; Arthritis, Experimental; Arthritis, Rheumatoid; Autoimmunity; B-Lymphocytes; Cells, Cultured; Cytokines; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred DBA; NF-kappa B; Pyrimidinones; Receptors, CXCR3; Signal Transduction

As standard treatments for cancer, DNA-damaging chemotherapeutic agents and irradiation therapy improve survival in patients with various cancers. Wee1, a kinase associated with the cell cycle, causes G2/M cell cycle arrest to allow repair of injured DNA in cancer cells, and a Wee1 inhibitor has been confirmed to lead to apoptosis in cancer cells. Recently, there has been renewed interest in exploring the immune environment which plays a significant role in tumour suppression. A Wee1 inhibitor combined with radiotherapy has been tested in lung, pancreatic, and prostate cancer and melanoma in vivo or in vitro. There is still no research evaluating the immunoregulatory effects of AZD1775 plus high-dose irradiation (IR) in vivo. T cell killing and CD8+ T cell depletion assays demonstrated that the combination of AZD1775 and IR delayed tumour growth in breast cancer mouse models. Additionally, combination treatment also suppressed the expression of PD-L1, a co-inhibitor, through the STAT3-IRF1 axis. The importance and originality of this study are that it explores the internal and external mechanisms of AZD1775 combined with a single high dose of IR and provides a rationale for applying the combination therapy described above in a clinical trial.

Topics: Animals; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Cycle Proteins; Cell Line, Tumor; Combined Modality Therapy; Dendritic Cells; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Mice; Mice, Inbred BALB C; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Radiation, Ionizing

Inhibitors of cAMP-phosphodiesterase 4 (PDE4) exert a number of promising therapeutic benefits, but adverse effects, in particular emesis and nausea, have curbed their clinical utility. Here, we show that PAN-selective inhibition of PDE4, but not inhibition of PDE3, causes a time- and dose-dependent accumulation of chow in the stomachs of mice fed ad libitum without changing the animals' food intake or the weight of their intestines, suggesting that PDE4 inhibition impairs gastric emptying. Indeed, PDE4 inhibition induced gastric retention in an acute model of gastric motility that traces the passage of a food bolus through the stomach over a 30 minutes time period. In humans, abnormal gastric retention of food is known as gastroparesis, a syndrome predominated by nausea (>90% of cases) and vomiting (>80% of cases). We thus explored the abnormal gastric retention induced by PDE4 inhibition in mice under the premise that it may represent a useful correlate of emesis and nausea. Delayed gastric emptying was produced by structurally distinct PAN-PDE4 inhibitors including Rolipram, Piclamilast, Roflumilast, and RS25344, suggesting that it is a class effect. PDE4 inhibitors induced gastric retention at similar or below doses commonly used to induce therapeutic benefits (e.g., 0.04 mg/kg Rolipram), thus mirroring the narrow therapeutic window of PDE4 inhibitors in humans. YM976, a PAN-PDE4 inhibitor that does not efficiently cross the blood-brain barrier, induced gastroparesis only at significantly higher doses (≥1 mg/kg). This suggests that PDE4 inhibition may act in part through effects on the autonomic nervous system regulation of gastric emptying and that PDE4 inhibitors that are not brain-penetrant may have an improved safety profile. The PDE4 family comprises four subtypes, PDE4A, B, C, and D. Selective ablation of any of these subtypes in mice did not induce gastroparesis per se, nor did it protect from PAN-PDE4 inhibitor-induced gastroparesis, indicating that gastric retention may result from the concurrent inhibition of multiple PDE4s. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. Acute gastric retention induced by PDE4 inhibition is alleviated by treatment with the widely used prokinetic Metoclopramide, suggesting a potential of this drug to alleviate the side effects of PDE4 inhibitors. Finally, given that the cause of gastroparesis remains largely idiopathic,

Topics: Aminopyridines; Animals; Benzamides; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclopropanes; Disease Models, Animal; Female; Gastroparesis; Mice; Mice, Nude; Phosphodiesterase 4 Inhibitors; Pyridines; Pyrimidinones; Rolipram

To examine vibegron effects on lower urinary tract dysfunction (LUTD) in mice with spinal cord injury (SCI).. Female mice underwent Th8-9 spinal cord transection and were orally administered vehicle or vibegron after SCI. We evaluated urodynamic parameters at 4 weeks after SCI with or without vibegron. Fibrosis- and ischemia-related messenger RNA (mRNA) and protein levels of collagen and elastin were measured in bladders of vehicle- and vibegron-treated SCI mice, and spinal intact mice.. Non-voiding contractions (NVCs) were significantly fewer (15.3 ± 8.9 vs 29.7 ± 11.4 contractions; P < .05) and the time to the first NVC was significantly longer (1488.0 ± 409.5 vs 782.7 ± 399.7 seconds; P < .01) in vibegron-treated SCI mice vs vehicle-treated SCI mice. mRNAs levels of collagen types 1 and 3, transforming growth factor-β1 (TGF-β1), and hypoxia-inducible factor-1α (HIF-1α) were significantly upregulated in vehicle-treated SCI mice compared with spinal intact and vibegron-treated SCI mice (Col 1: 3.5 vs 1.0 and 2.0-fold; P < .01 and P < .05, Col 3: 2.1 vs 1.0 and 1.2-fold; P < .01 and P < .05, TGF-β1: 1.2 vs 1.0 and 0.9-fold; P < .05 and P < .05, HIF-1α: 1.4 vs 1.0 and 1.0-fold; P < .05 and P < .01). Total collagen and elastin protein levels in vehicle- and vibegron-treated SCI mice did not differ.. Vibegron reduced NVCs, delayed the first NVC, and improved collagen types 1 and 3, TGF-β1, and HIF-1α mRNA expression in SCI mice. Vibegron might be effective for SCI-induced LUTD.

Topics: Adrenergic beta-3 Receptor Agonists; Animals; Disease Models, Animal; Female; Mice; Pyrimidinones; Pyrrolidines; Rats, Sprague-Dawley; Spinal Cord Injuries; Treatment Outcome; Urinary Bladder, Neurogenic; Urination; Urodynamics

Herein, we report spiropyrimidinetriones (SPTs) incorporating N-linked azole substituents on a benzisoxazole scaffold with improved Gram-positive antibacterial activity relative to previously described analogues. SPTs have an unusual spirocyclic architecture and represent a new antibacterial class of bacterial DNA gyrase and topoisomerase IV inhibitors. They are not cross-resistant to fluoroquinolones and other DNA gyrase/topoisomerase IV inhibitors used clinically. The activity of the SPTs was assessed for DNA gyrase inhibition, and the antibacterial activity across Gram-positive and Gram-negative pathogens with N-linked 1,2,4-triazoles substituted on the 5-position provides the most worthwhile profile. Directed nucleophilic and electrophilic chemistry was developed to vary this 5-position with carbon, nitrogen, or oxygen substituents and explore structure-activity relationships including those around a target binding model. Compounds with favorable pharmacokinetic parameters were identified, and two compounds demonstrated cidality in a mouse model of

Topics: Animals; Anti-Bacterial Agents; Azoles; Disease Models, Animal; DNA Gyrase; Dose-Response Relationship, Drug; Isoxazoles; Mice; Microbial Sensitivity Tests; Molecular Structure; Pyrimidinones; Rats; Rats, Wistar; Spiro Compounds; Staphylococcal Infections; Staphylococcus aureus; Structure-Activity Relationship; Topoisomerase II Inhibitors

Pneumonia is one of the commonest causes of death worldwide. High‑temperature requirement A2 (HtrA2) is a proapoptotic mitochondrial serine protease involved in caspase‑dependent or caspase‑independent cell apoptosis. UCF‑101 (5‑[5‑(2‑nitrophenyl) furfuryl iodine]‑1,3‑diphenyl‑2‑thiobarbituric acid), an inhibitor of HtrA2, has a protective effect on organs in various diseases by inhibiting cell apoptosis. The aim of the present study was to explore whether UCF‑101 has a protective effect on lungs in pneumonia. A lipopolysaccharide (LPS)‑induced pneumonia model was established in rats. UCF‑101 (2 µmol/kg) was used for treatment. Lung injury was detected by hematoxylin and eosin staining. Pro‑inflammatory cytokines and oxidative stress‑related factors were detected using corresponding test kits. TUNEL staining was used to measure the amount of cell apoptosis. Apoptosis‑associated proteins were detected by western blot assay. The present study indicated pulmonary injury induced by LPS. Treatment with UCF‑101 clearly alleviated this pulmonary damage and restored the levels of pro‑inflammatory cytokines and oxidative stress‑related factors. In addition, UCF‑101 significantly reduced LPS‑induced cell apoptosis, the release of HtrA2 and cytochrome from mitochondria to the cytoplasm and inhibited the expression of pro‑apoptotic proteins. UCF‑101 also restored the ATP level. The present results demonstrated that UCF‑101 acts as a positive regulator of acute pneumonia by inhibiting inflammatory response, oxidative stress and mitochondrial apoptosis. The present study suggests UCF‑101 as a potential candidate for pneumonia therapy.

Topics: Animals; Apoptosis; Cytokines; Disease Models, Animal; Gene Expression Regulation; Lipopolysaccharides; Male; Nerve Tissue Proteins; Oxidative Stress; Pneumonia; Pyrimidinones; Rats; Serine-Arginine Splicing Factors; Thiones

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Benzodioxoles; Biomarkers, Tumor; Cell Line, Tumor; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mice; Molecular Targeted Therapy; Pancreatic Neoplasms; Protein Kinase Inhibitors; Purines; Pyridones; Pyrimidinones; Signal Transduction; Survival Rate; Treatment Outcome; Xenograft Model Antitumor Assays

There is an urgent need to develop better materials to provide anatomical support to the pelvic floor without compromising its function.. Our aim was to assess outcomes after simulated vaginal prolapse repair in a sheep model using three different materials: (1) ultra-lightweight polypropylene (PP) non-degradable textile (Restorelle) mesh, (2) electrospun biodegradable ureidopyrimidinone-polycarbonate (UPy-PC), and (3) electrospun non-degradable polyurethane (PU) mesh in comparison with simulated native tissue repair (NTR). These implants may reduce implant-related complications and avoid vaginal function loss.. A controlled trial was performed involving 48 ewes that underwent NTR or mesh repair with PP, UPy-PC, or PU meshes (n=12/group). Explants were examined 60 and 180 d (six per group) post-implantation.. Posterior rectovaginal dissection, NTR, or mesh repair.. Implant-related complications, vaginal contractility, compliance, and host response were assessed. Power calculation and analysis of variance testing were used to enable comparison between the four groups.. There were no visible implant-related complications. None of the implants compromised vaginal wall contractility, and passive biomechanical properties were similar to those after NTR. Shrinkage over the surgery area was around 35% for NTR and all mesh-augmented repairs. All materials were integrated well with similar connective tissue composition, vascularization, and innervation. The inflammatory response was mild with electrospun implants, inducing both more macrophages yet with relatively more type 2 macrophages present at an early stage than the PP mesh.. Three very different materials were all well tolerated in the sheep vagina. Biomechanical findings were similar for all mesh-augmented repair and NTR. Constructs induced slightly different mid-term inflammatory profiles.. Product innovation is needed to reduce implant-related complications. We tested two novel implants, electrospun and an ultra-lightweight polypropylene textile mesh, in a physiologically relevant model for vaginal surgery. All gave encouraging outcomes.

Topics: Animals; Biocompatible Materials; Disease Models, Animal; Female; Gynecologic Surgical Procedures; Materials Testing; Models, Animal; Polypropylenes; Prosthesis Design; Pyrimidinones; Sheep; Surgical Mesh; Textiles; Treatment Outcome; Uterine Prolapse

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cells, Cultured; Disease Models, Animal; MicroRNAs; Motor Activity; Neurons; Neuroprotective Agents; Pyrimidinones; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Time Factors; Transcriptome; Wnt Signaling Pathway

The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models.. Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats.. We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.

Topics: Animals; Benzylamines; Cell Movement; Cell Proliferation; Cells, Cultured; Chalcones; Chemokine CXCL2; Cyclams; Disease Models, Animal; Heterocyclic Compounds; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Macrophages; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pericytes; Pulmonary Artery; Pyrimidinones; Rats, Wistar; Receptors, CXCR4; Signal Transduction; Vascular Remodeling; Vascular Resistance

Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/β-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/β-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis in vitro. ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/β-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/β-catenin signal can be a therapeutic target for endometriosis.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Endometriosis; Female; Heterocyclic Compounds, 2-Ring; Humans; Mice; Piperazines; Pyrimidinones; Signal Transduction

Activated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV).. Microscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals.. Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.

Topics: Acetamides; Animals; Cell Line; Chlorocebus aethiops; Dengue; Dengue Virus; Disease Models, Animal; Encephalitis Virus, Japanese; Encephalitis, Japanese; Humans; Interferons; Mice; Monocytes; Platelet Factor 4; Pyrimidinones; THP-1 Cells; Vero Cells; Virus Replication

During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored.. We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects.. Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-. Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

Topics: Animals; Biopsy, Needle; Cells, Cultured; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibrosis; Immunohistochemistry; Mechanistic Target of Rapamycin Complex 1; Mice; Molecular Targeted Therapy; Pyridones; Pyrimidinones; Random Allocation; Reference Values; Renal Insufficiency, Chronic; Signal Transduction

The biological effects of nitric oxide are mediated via protein S-nitrosylation. Levels of S-nitrosylated protein are controlled in part by the denitrosylase, S-nitrosoglutathione reductase (GSNOR). The objective of this study was to examine whether GSNOR inhibition improves outcomes after cardiac arrest and cardiopulmonary resuscitation (CA/CPR).. Adult wild-type C57BL/6 and GSNOR-deleted (GSNOR. GSNOR activity was increased in plasma and multiple organs of mice, including brain in particular. Levels of protein S-nitrosylation were decreased in the brain 6 hours after CA/CPR. Administration of SPL-334.1 attenuated the increase in GSNOR activity in brain, heart, liver, spleen, and plasma, and restored S-nitrosylated protein levels in the brain. Inhibition of GSNOR attenuated ischemic brain injury and improved survival in wild-type mice after CA/CPR (81.8% in SPL-334.1 versus 36.4% in placebo; log rank P=0.031). Similarly, GSNOR deletion prevented the reduction in the number of S-nitrosylated proteins in the brain, mitigated brain injury, and improved neurological recovery and survival after CA/CPR. Both GSNOR inhibition and deletion attenuated CA/CPR-induced disruption of blood brain barrier. Post-CA patients had higher plasma GSNOR activity than did preoperative cardiac surgery patients or healthy volunteers ( P<0.0001). Plasma GSNOR activity was positively correlated with initial lactate levels in postarrest patients (Spearman correlation coefficient=0.48; P=0.045).. CA and CPR activated GSNOR and reduced the number of S-nitrosylated proteins in the brain. Pharmacological inhibition or genetic deletion of GSNOR prevented ischemic brain injury and improved survival rates by restoring S-nitrosylated protein levels in the brain after CA/CPR in mice. Our observations suggest that GSNOR is a novel biomarker of postarrest brain injury as well as a molecular target to improve outcomes after CA.

Topics: Aldehyde Oxidoreductases; Animals; Benzoates; Disease Models, Animal; Heart; Heart Arrest; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Oxidation-Reduction; Pyrimidinones; Resuscitation; Treatment Outcome

Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib, and afatinib do not affect basal secretion but amplify carbachol-stimulated secretion by 2- to 3-fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, was blocked by CF transmembrane conductance regulator (CFTR) and K+ channel inhibitors, and involved EGFR binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution, and channel inhibitors. Rats that were administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from approximately 60% to greater than 80%, which was reduced by up to 75% by the K+ channel inhibitors clotrimazole or senicapoc or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation and support the therapeutic efficacy of channel inhibitors.

Topics: Acetamides; Afatinib; Animals; Cell Membrane Permeability; Clotrimazole; Colon; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Disease Models, Animal; Epithelial Cells; ErbB Receptors; Female; Humans; Intestinal Mucosa; Neoplasms; Oxazines; Potassium Channel Blockers; Potassium Channels; Protein Kinase Inhibitors; Pyrimidinones; Pyrroles; Rats; Trityl Compounds

Topics: Albuminuria; Angiotensin II; Angiotensinogen; Animals; beta Catenin; Blood Pressure; Blood Urea Nitrogen; Bridged Bicyclo Compounds, Heterocyclic; Collagen Type I; Creatinine; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Fibronectins; Gene Expression Regulation; Hypertension; Kidney; Male; Nephrectomy; Peptidyl-Dipeptidase A; Plasminogen Activator Inhibitor 1; Pyrimidinones; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Renin; Renin-Angiotensin System; Wnt Proteins; Wnt Signaling Pathway

To explore the influence of micro ribonucleic acid (miR)-154 on myocardial apoptosis in rats with acute myocardial infarction (AMI), and to analyze whether Wnt/β-catenin signaling pathway was involved in the regulation.. The Sprague-Dawley (SD) rat model of AMI was established via ligation of left anterior descending artery. Rats were randomly divided into model group (M group, n=12) and ICG-001 intervention group (I group, n=12). At the same time, sham operation group (S group, n=12) was established. In I group, ICG-001 (5 mg/kg) was intraperitoneally injected every day after operation. Meanwhile, an equal amount of normal saline was injected in rats of S group and M group. 21 d after operation, the cardiac function of rats in each group was detected via echocardiography. After that, the rats were immediately executed. MI area in each group was detected via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial apoptosis level in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Moreover, the changes of apoptotic proteins in rat myocardial cells were detected via Western blotting. Moreover, the expression level of miR-154 in myocardial cells of rats was detected via quantitative polymerase chain reaction (qPCR). Furthermore, the influence of miR-154 on Wnt/β-catenin signaling pathway was detected via Western blotting.. Compared with S group, left ventricular ejection fraction (LVEF, %) and left ventricular fractional shortening (LVFS, %) were significantly decreased in M group (p<0.01). However, left ventricular internal diameter at end-diastole (LVIDd) and left ventricular internal diameter at end-systole (LVIDs) were significantly increased (p<0.01). In I group, LVEF (%) and LVFS (%) were significantly higher than those of M group (p<0.05), whereas LVIDs and LVIDd were significantly lower (p<0.05). MI area in M group was remarkably larger than that of S group (p<0.01). Meanwhile, MI area in I group was significantly smaller than that of M group (p<0.01). Compared with S group, the number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly increased in M group (p<0.01). However, the expression level of B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax) was significantly decreased (p<0.01). The number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly declined in I group when compared with those of M group (p<0.01). However, the expression level of Bcl-2/Bax was significantly increased in I group (p<0.01). The expression level of miR-154 in myocardial cells of M group and I group was remarkably increased when compared with that of S group (p<0.01). Furthermore, the expression levels of β-catenin and Cyclin D1 in myocardial cells of M group were remarkably higher than those of S group and I group (p<0.01).. AMI significantly increases the expression level of miR-154. Moreover, miR-154 can activate Wnt/β-catenin signaling pathway, eventually promoting myocardial apoptosis.

Topics: Animals; Apoptosis; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; MicroRNAs; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Pyrimidinones; Rats; Rats, Sprague-Dawley; Stroke Volume; Ventricular Function, Left; Wnt Proteins; Wnt Signaling Pathway

KRAS is one of the driver oncogenes in non-small-cell lung cancer (NSCLC) but remains refractory to current modalities of targeted pathway inhibition, which include inhibiting downstream kinase MEK to circumvent KRAS activation. Here, we show that pulsatile, rather than continuous, treatment with MEK inhibitors (MEKis) maintains T cell activation and enables their proliferation. Two MEKis, selumetinib and trametinib, induce T cell activation with increased CTLA-4 expression and, to a lesser extent, PD-1 expression on T cells in vivo after cyclical pulsatile MEKi treatment. In addition, the pulsatile dosing schedule alone shows superior anti-tumor effects and delays the emergence of drug resistance. Furthermore, pulsatile MEKi treatment combined with CTLA-4 blockade prolongs survival in mice bearing tumors with mutant Kras. Our results set the foundation and show the importance of a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy to optimally enhance tumor delay and promote long-term anti-tumor immunity.

Topics: Animals; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; CTLA-4 Antigen; Disease Models, Animal; Female; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Programmed Cell Death 1 Receptor; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Survival Rate; T-Lymphocytes

Hyperactivation of the RAS-RAF-MEK-ERK signaling pathway is exploited by glioma cells to promote their growth and evade apoptosis. MEK activation in tumor cells can increase replication of ICP34.5-deleted herpes simplex virus type 1 (HSV-1), but paradoxically its activation in tumor-associated macrophages promotes a pro-inflammatory signaling that can inhibit virus replication and propagation. Here we investigated the effect of blocking MEK signaling in conjunction with oncolytic HSV-1 (oHSV) for brain tumors.. Infected glioma cells co-cultured with microglia or macrophages treated with or without trametinib were used to test trametinib effect on macrophages/microglia. Enzyme-linked immunosorbent assay, western blotting, and flow cytometry were utilized to evaluate the effect of the combination therapy. Pharmacokinetic (PK) analysis of mouse plasma and brain tissue was used to evaluate trametinib delivery to the CNS. Intracranial human and mouse glioma-bearing immune deficient and immune competent mice were used to evaluate the antitumor efficacy.. Oncolytic HSV treatment rescued trametinib-mediated feedback reactivation of the mitogen-activated protein kinase signaling pathway in glioma. In vivo, PK analysis revealed enhanced blood-brain barrier penetration of trametinib after oHSV treatment. Treatment by trametinib, a MEK kinase inhibitor, led to a significant reduction in microglia- and macrophage-derived tumor necrosis factor alpha (TNFα) secretion in response to oHSV treatment and increased survival of glioma-bearing mice. Despite the reduced TNFα production observed in vivo, the combination treatment activated CD8+ T-cell mediated immunity and increased survival in a glioma-bearing immune-competent mouse model.. This study provides a rationale for combining oHSV with trametinib for the treatment of brain tumors.

Topics: Animals; Blood-Brain Barrier; Brain Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Glioblastoma; Glioma; Herpesvirus 1, Human; Humans; Immunocompetence; Macrophages; Mice; Microglia; Mitogen-Activated Protein Kinase Kinases; Oncolytic Virotherapy; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; RAW 264.7 Cells; Survival Rate; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates. For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens. Cancer, however, is a disease that develops mainly during juvenile and adult stage. Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive. We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects. For the current study, we used a previously established medaka melanoma model. As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression. By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model. Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.

Topics: Animals; Animals, Genetically Modified; Biomarkers, Tumor; Cell Line, Tumor; Cisplatin; Computational Biology; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Oryzias; Pyridones; Pyrimidinones; Transcriptome; Xenograft Model Antitumor Assays

The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance.. Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.

Topics: Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Disease Progression; Drug Resistance, Neoplasm; Drug Synergism; Histone Deacetylase Inhibitors; Humans; MAP Kinase Signaling System; Melanoma; Mice; Panobinostat; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Receptor Tyrosine Kinase-like Orphan Receptors; Receptor, IGF Type 1; Receptors, G-Protein-Coupled; Signal Transduction; Transcription Factors; Uveal Neoplasms; Xenograft Model Antitumor Assays

Transforming growth factor β (TGF-β) is the key cytokine involved in causing fibrosis through cross-talk with major profibrotic pathways. However, inhibition of TGF-β to prevent fibrosis would also abrogate its anti-inflammatory and wound-healing effects. β-catenin is a common co-factor in most TGF-β signaling pathways. β-catenin binds to T-cell factor (TCF) to activate profibrotic genes and binds to Forkhead box O (Foxo) to promote cell survival under oxidative stress. Using a proximity ligation assay in human kidney biopsies, we found that β-catenin/Foxo interactions were higher in kidney with little fibrosis, whereas β-catenin/TCF interactions were upregulated in the kidney of patients with fibrosis. We hypothesised that β-catenin/Foxo is protective against kidney fibrosis. We found that Foxo1 protected against rhTGF-β1-induced profibrotic protein expression using a CRISPR/cas9 knockout of Foxo1 or TCF1 in murine kidney tubular epithelial C1.1 cells. Co-administration of TGF-β with a small molecule inhibitor of β-catenin/TCF (ICG-001), protected against kidney fibrosis in unilateral ureteral obstruction. Collectively, our human, animal and in vitro findings suggest β-catenin/Foxo as a therapeutic target in kidney fibrosis.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Disease Models, Animal; Fibrosis; Forkhead Box Protein O1; Gene Knockout Techniques; Hepatocyte Nuclear Factor 1-alpha; Humans; Kidney; Kidney Diseases; Male; Mice; Pyrimidinones; Signal Transduction; Transforming Growth Factor beta1

Intracerebroventricular (icv) streptozotocin (STZ) injection decreases cerebral insulin signal pathway function and produces multiple effects that resemble the molecular, pathological, and behavioural features of Sporadic Alzheimer's disease (SAD). We previously reported that yonkenafil (yonk), the analogue of sildenafil and a novel PDE5 inhibitor exerts an anti-amyloidogenesis effect by regulating the Aβ level and inhibiting the expression of β-amyloid precursor protein in the APP/PS1 transgenic mice model. In this study, the effects of yonk on cognitive behaviors as well as the pathological features in streptozotocin-induced SAD rat model were investigated. The results demonstrated that administration of yonk at doses of 3 and 10 mg/kg for three weeks significantly improved cognitive deficits, attenuated STZ-induced neuronal death, inhibited the over-activation of microglia and astrocytes and the levels of pro-inflammatory markers, as well as decreased PDE5 protein expression in the hippocampus. Furthermore, yonk (3 mg/kg) notably prevented changes in tau hyperphosphorylation, decreased IRS-1and JNK phosphorylation and increased the GSK3β (ser9) phosphorylation induced by STZ. In summary, these data suggested that yonk significantly reversed STZ-induced memory deficits by inhibiting the over-activation of microglia and astrocytes, as well as ameliorated the levels of pro-inflammatory makers and tau hyperphosphorylation through regulating GSK3β signalling pathway.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; Encephalitis; Insulin; Male; Phosphodiesterase 5 Inhibitors; Phosphorylation; Pyrimidinones; Pyrroles; Rats, Wistar; Streptozocin; tau Proteins

Transforming growth factor-β (TGF-β) plays crucial roles in the development of focal segmental glomerulosclerosis, but key molecular pathways remain unknown. Here, we identified the regulation of mammalian target of rapamycin complex1 (mTORC1) by TGF-β via ERK1/2 in the Adriamycin-induced murine model of focal segmental glomerulosclerosis. Adriamycin administration elicited early activation of TGF-β-ERK1/2-mTORC1 in podocytes, which persisted at later stages of albuminuria and glomerulosclerosis. Phosphorylation of the TGF-β receptor-I (TGF-βRI), Smad3, ERK1/2 and ribosomal protein S6 were evident in the glomeruli of adriamycin-treated mice. Targeting TGFβ-RI and mTORC1 with pharmacological inhibitors suppressed TGF-β signaling in glomeruli and significantly reduced albuminuria, glomerulosclerosis, protein levels of collagen 4α3, plasminogen activator inhibitor-1, and vimentin and restored mRNA levels of podocyte markers. Low dose US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor trametinib/GSK1120212 blunted TGF-β1-induced mTORC1 activation in podocytes, ameliorated up-regulation of TGF-β, plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, fibronectin and α-smooth muscle actin and prevented albuminuria and glomerulosclerosis with improved serum albumin. In cultured podocytes, this pathway was found to be associated with translation of fibrogenic collagen 4α3 and plasminogen activator inhibitor-1, without influencing their transcription. Notably, rapamycin suppressed upstream p-TGF-βRI, p-Smad3 and p-ERK1/2, and trametinib down-regulated upstream p-Smad3 in ex vivo and in vivo studies, indicating that harmful paracrine signaling among glomerular cells amplified the TGF-β-ERK1/2-mTORC1 axis by forming a positive feedback loop. Thus, an accentuated TGF-β-ERK1/2-mTORC1 pathway is suggested as a central upstream mediator to develop proteinuria and glomerulosclerosis. Hence, preventing activation of this vicious loop by trametinib may offer a new therapeutic strategy for glomerular disease treatment.

Topics: Animals; Cell Line; Disease Models, Animal; Doxorubicin; Drug Evaluation, Preclinical; Glomerulosclerosis, Focal Segmental; Humans; Kidney Glomerulus; Male; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Mice; Phosphorylation; Proteinuria; Pyridones; Pyrimidinones; Rats; Transforming Growth Factor beta

As a potent and selective allosteric inhibitor of MEK, TAK-733 has been shown to exert anti-cancer effects for a wide range of cancers both in vitro and in vivo. However, its effects on inhibiting growth have never been investigated in the cardiovascular system, where regulation of abnormal vascular smooth muscle cell growth in neointimal hyperplasia is an important area of focus. Angiotensin II was used to mimic inflammatory neointimal hyperplasia in an in vitro environment, and balloon catheter-induced injury with an infusion of angiotensin II was used to generate an in vivo rat restenosis model under inflammatory conditions. TAK-733 exerted anti-proliferative and anti-migratory effects on human vascular smooth muscle cells. These multiple effects of TAK-733 were evaluated using various assays, such as cell cycle analysis and wound healing. Interestingly, TAK-733 did not induce apoptosis in smooth muscle cells but only reduced the proliferation rate; additionally, it did not affect EC viability. TAK-733 also exhibited anti-inflammatory activity, as observed by attenuated monocyte adhesion to smooth muscle cells via inhibition of ICAM1 and VCAM1 overexpression. The in vivo study demonstrated that neointimal hyperplasia after balloon injury and angiotensin II stimulation was suppressed by TAK-733, and downregulation of the inflammatory signal and enhanced re-endothelialization were observed. TAK-733 may have therapeutic potential for treating neointimal hyperplasia by attenuating smooth muscle cell proliferation, migration, and inflammation. Thus, TAK-733 could be a promising drug candidate for treating patients with restenosis.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Graft Occlusion, Vascular; Humans; Inflammation; Male; Mice; Neointima; Pyridones; Pyrimidinones; Rats; Rats, Sprague-Dawley; RAW 264.7 Cells

Dihydropyrimidinone derivatives containing piperidine moiety were synthesised in a good yield. All the compounds were confirmed by elemental analysis and spectral data. Anti-ulcer activity of novel dihydropyrimidinone-piperidine hybrids (1-18) was evaluated. Among them, four compounds (3, 8, 11 and 15) were found to be most active in 80% ethanol-induced ulcer experimental animal model. All the potent compounds were further evaluated for anti-ulcer activity by different in vivo anti-ulcer models to study the effect of compounds on anti-secretory and cytoprotective activities. All the active compounds inhibited the formation of gastric ulcers and increased the formation of gastric mucin secretion. Compound 15 was found to be the most potent compound of the series as anti-ulcer agent. Additional experimental studies on lead compound 15 will result in a new class of orally active molecule for anti-ulcer activity.

Topics: Animals; Anti-Ulcer Agents; Crystallography, X-Ray; Disease Models, Animal; Dose-Response Relationship, Drug; Ethanol; Models, Molecular; Molecular Structure; Piperidines; Pyrimidinones; Rats; Rats, Wistar; Stomach Ulcer; Structure-Activity Relationship

The role of KRAS, when activated through canonical mutations, has been well established in cancer

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Esophageal Neoplasms; Gene Amplification; Humans; Mice; Mitogen-Activated Protein Kinase Kinases; Piperidines; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidines; Pyrimidinones; Stomach Neoplasms

Pancreatic cancer is resistant to treatment and needs precision individualized therapy to improve the outcome of this disease. Previously, we demonstrated that trametinib (TRA), a MEK inhibitor, could inhibit a pancreatic cancer patient-derived orthotopic xenograft (PDOX). In the present study, we show that gemcitabine (GEM) in combination with TRA was more effective than TRA alone. We implanted a patient pancreatic cancer orthotopically in the pancreatic tail of nude mice to establish the PDOX model. After seven weeks of tumor growth, we divided 32 pancreatic-cancer PDOX nude mice into 4 groups of eight: untreated control; GEM (once a week for 2 weeks); TRA (14 consecutive days); GEM + TRA (GEM: once a week for 2 weeks, TRA:14 consecutive days). We found that treated mice on day 14 had significantly reduced tumor volume in comparison to untreated control. TRA and the combination of GEM + TRA therapy significantly inhibited tumor development in comparison to GEM alone. However, GEM + TRA inhibited the PDOX tumor growth significantly greater than TRA alone. These results suggest the clinical potential of the combination of TRA and GEM for pancreatic cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Deoxycytidine; Disease Models, Animal; Gemcitabine; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Pyridones; Pyrimidinones; Xenograft Model Antitumor Assays

Targeting poly ADP-ribose polymerase (PARP) has been recently identified as a promising option against gastric cancer (GC). However, PARP inhibitors alone achieve limited efficacy. Combination strategies, especially with homologous recombination (HR) impairment, are of great hope to optimize PARP inhibitor's efficacy and expand target populations but remains largely unknown. Herein, we investigated whether a WEE1/ Polo-like kinase 1 (PLK1) dual inhibitor AZD1775 reported to impair HR augmented anticancer activity of a PARP inhibitor olaparib and its underlying mechanisms.. GC cell lines and in vivo xenografts were employed to determine antitumor activity of PARP inhibitor combined with WEE1/PLK1 dual inhibitor AZD1775. Western blot, genetic knockdown by siRNA, flow cytometry, Immunohistochemistry were performed to explore the underlying mechanisms.. AZD1775 dually targeting WEE1/PLK1 enhanced effects of olaparib on growth inhibition and apoptotic induction in GC cells. Mechanistic investigations elucidate that WEE1/PLK1 blockade downregulated several HR-related proteins and caused an accumulation in γH2AX. As confirmed in both GC cell lines and mice bearing GC xenografts, these effects were enhanced by AZD1775-olaparib combination compared to olaparib alone, suggesting that disrupting HR-mediated DNA damage repairs (DDR) by WEE1/PLK1 blockade might be responsible for improved GC cells' response to PARP inhibitors. Given the DNA damage checkpoint as a primary target of WEE1 inhibition, our data also demonstrate that AZD1775 abrogated olaparib-activated DNA damage checkpoint through CDC2 de-phosphorylation, followed by mitotic progression with unrepaired DNA damage (marked by increased pHH3-stained and γH2AX-stained cells, respectively).. PARP inhibitor olaparib combined with WEE1/PLK1 dual inhibitor AZD1775 elicited potentiated anticancer activity through disrupting DDR signaling and the DNA damage checkpoint. It sheds light on the combination strategy of WEE1/PLK1 dual inhibitors with PARP inhibitors in the treatment of GC, even in HR-proficient patients.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; DNA Damage; DNA Repair; Drug Synergism; Female; Humans; Mice; Nuclear Proteins; Phthalazines; Piperazines; Polo-Like Kinase 1; Poly(ADP-ribose) Polymerase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrazoles; Pyrimidines; Pyrimidinones; Stomach Neoplasms; Xenograft Model Antitumor Assays

Mounting evidence suggests that RAF-mediated MEK activation plays a crucial role in paradox MAPK (re)activation, leading to resistance and therapeutic failure with agents hitting a single step along the MAPK cascade.. We examined the molecular and functional effects of single and combined BRAF (dabrafenib), pan-RAF (RAF265), MEK (trametinib) and EGFR/HER2 (lapatinib) inhibition, using Western Blot and conservative isobologram analysis to assess functional synergism, and explored genetic determinants of synergistic interactions. Immunoprecipitation based assays were used to detect the interaction between BRAF and CRAF. The Mann-Whitney U test was used for comparing quantitative variables.. Here we demonstrated that a combination of MEK and BRAF inhibitors overcomes paradoxical MAPK activation (induced by BRAF inhibitors) in BRAF-wt/RAS-mut NSCLC and PDAC in vitro. This results in growth inhibitory synergism, both in vitro and in vivo, in the majority (65%) of the cellular models analyzed, encompassing cell lines and patient-derived cancer stem cells and organoids. However, RAS mutational status is not the sole determinant of functional synergism between RAF and MEK inhibitors, as demonstrated in KRAS isogenic tumor cell line models. Moreover, in EGFR-driven contexts, paradoxical MAPK (re)activation in response to selective BRAF inhibition was dependent on EGFR family signaling and could be offset by simultaneous EGFR/HER-2 blockade.. Overall, our data indicate that RAF inhibition-induced paradoxical MAPK activation could be exploited for therapeutic purposes by simultaneously targeting both RAF and MEK (and potentially EGFR family members) in appropriate molecular contexts. KRAS mutation per se does not effectively predict therapeutic synergism and other biomarkers need to be developed to identify patients potentially deriving benefit from combined BRAF/MEK targeting.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Humans; Imidazoles; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Neoplastic Stem Cells; Oximes; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Xenograft Model Antitumor Assays

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibodies; Disease Models, Animal; Disease Progression; Entorhinal Cortex; Humans; Mice; Models, Biological; Naphthalenes; Pyrimidinones; tau Proteins; Treatment Failure

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents; Autophagy; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Mice; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Radiation Tolerance; Reactive Oxygen Species; Signal Transduction; Xenograft Model Antitumor Assays

Uremic vascular calcification is a regulated cell-mediated process wherein cells in the arterial wall transdifferentiate to actively calcifying cells resulting in a process resembling bone formation. Wnt signalling is established as a major driver for vessel formation and maturation and for embryonic bone formation, and disturbed Wnt signalling might play a role in vascular calcification. ICG-001 is a small molecule Wnt inhibitor that specifically targets the coactivator CREB binding protein (CBP)/β-catenin-mediated signalling. In the present investigation we examined the effect of ICG-001 on vascular calcification in uremic rats. Uremic vascular calcification was induced in adult male rats by 5/6-nephrectomy, high phosphate diet and alfacalcidol. The presence of uremic vascular calcification in the aorta was associated with induction of gene expression of the Wnt target gene and marker of proliferation, cyclinD1; the mediator of canonical Wnt signalling, β-catenin and the matricellular proteins, fibronectin and periostin. Furthermore, genes from fibrosis-related pathways, TGF-β and activin A, as well as factors related to epithelial-mesenchymal transition, snail1 and vimentin were induced. ICG-001 treatment had significant effects on gene expression in kidney and aorta from healthy rats. These effects were however limited in uremic rats, and treatment with ICG-001 did not reduce the Ca-content of the uremic vasculature.

Topics: Animals; beta Catenin; Biomarkers; Bone and Bones; Bridged Bicyclo Compounds, Heterocyclic; CREB-Binding Protein; Disease Models, Animal; Gene Expression Regulation; Kidney Failure, Chronic; Male; Minerals; Organ Specificity; Pyrimidinones; Rats; Uremia; Vascular Calcification; Wnt Signaling Pathway; X-Ray Microtomography

We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Chemokine CXCL12; Disease Models, Animal; Drug Evaluation, Preclinical; Fluorescence Resonance Energy Transfer; Humans; Hypereosinophilic Syndrome; Hypersensitivity; Male; Mice, Inbred BALB C; Models, Molecular; Pyrimidinones; Receptors, CXCR4; Structure-Activity Relationship

Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in patients with TNBC, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell-cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775.. Mono- and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient-derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cell lines.. Cyclin E overexpression (i) is enriched in TNBCs with high recurrence rates, (ii) sensitizes TNBC cell lines and PDX models to AZD1775, (iii) leads to CDK2-dependent activation of DNA replication stress pathways, and (iv) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug-mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy.

Topics: Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle Proteins; Cell Line, Tumor; Cyclic N-Oxides; Cyclin E; Disease Models, Animal; DNA Repair; DNA Replication; Drug Resistance, Neoplasm; Gene Expression; Humans; Indolizines; Mice; Mice, Knockout; Models, Biological; Nuclear Proteins; Prognosis; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyridinium Compounds; Pyrimidinones; Stress, Physiological; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

The gut-vascular barrier (GVB) has recently been depicted to dampen the bacterial invasion of the bloodstream. The intestinal mucosa is a tissue rich in small vessels including capillaries. In this study, the protective effect of berberine on GVB in small bowel mucosa was investigated.. The rat cecal ligation and puncture (CLP) sepsis model was employed to evaluate the effect of berberine on serum endotoxin level and intestinal vascular permeability to Evans blue in vivo. The rat intestinal microvascular endothelial cells (RIMECs) treated by lipopolysaccharide (LPS) were used to assess the effect of berberine on endothelial permeability to FITC-labeled dextran, transendothelial electrical resistance (TEER), and tight junction (TJ) and adherens junction (AJ) expression in vitro.. After 24-hr CLP operation the serum endotoxin concentration and gut vascular permeability were significantly increased, while berberine markedly reduced endotoxin level and vascular leakage. In vitro, LPS not only dramatically increased endothelial permeability of RIMECs to FITC-dextran, but also decreased TEER and inhibited claudin-12, beta-catenin and VE-cadherin expression. These effects of LPS were antagonized by berberine. In addition, our in vivo and vitro studies also confirmed that the effect of berberine on GVB could be partially abolished by ICG001.. Berberine exerted a protective effect on GVB function in sepsis, which was strictly related to the modulation of the Wnt/beta-catenin signaling pathway.

Topics: Animals; Antigens, CD; Berberine; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cadherins; Capillary Permeability; Claudins; Disease Models, Animal; Endothelium, Vascular; Endotoxins; Intestinal Mucosa; Lipopolysaccharides; Male; Protective Agents; Pyrimidinones; Rats; Rats, Long-Evans; Sepsis; Survival Rate; Tight Junctions; Wnt Signaling Pathway

Protein kinase inhibitors (PKIs) are currently tested in clinical studies (phase I-III) as an alternative strategy against (recurrent) ovarian cancer. Besides their anti-tumour efficacy, several PKIs have also shown radiosensitizing effects when combined with external beam radiation. Based on these results we asked if the addition of PKIs offers a therapeutic opportunity to improve radioimmunotherapy (RIT) against ovarian cancer. Five PKIs (alisertib, MK1775, MK2206, saracatinib, temsirolimus) were chosen for cytotoxicity screenings based on their current clinical trials in the treatment of ovarian cancer and their influence on cell cycle regulation and DNA damage repair pathways. We combined selected PKIs with. PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, γH2A.X) was analysed by western blot and fluorescence microscopy. Flow cytometric measurements were performed to evaluate levels of apoptosis based on mono- or combination treatments. The best combination was used for in vivo combination therapy studies in nude mice with SKOV3ip and IGROV1 human ovarian cancer xenografts. Bonferroni correction was used to determine statistical significance for multiple comparisons.. The highest cytotoxicity against both cell lines was observed for MK1775 and alisertib. Combinations including. Our results warrant further evaluation of combination of MK1775 and radioimmunotherapy.

Topics: Animals; Antineoplastic Agents, Immunological; Apoptosis; Cell Line, Tumor; Cell Survival; Disease Models, Animal; DNA Breaks, Double-Stranded; Female; Humans; Immunoconjugates; Lutetium; Mice; Neural Cell Adhesion Molecule L1; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Pyrimidinones; Radioimmunotherapy; Radioisotopes; Xenograft Model Antitumor Assays

the pathogenesis of sepsis-associated encephalopathy (SAE) is multifactorial, involving neurotransmitter alterations, inflammatory cytokines, oxidative damage, mitochondrial dysfunction, apoptosis, and other factors. Mitochondria are major producers of reactive oxygen species, resulting in cellular injury. Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell death; it is translocated from mitochondria to the cytosol after an apoptotic insult. We previously found that UCF-101, a specific inhibitor of Omi/HtrA2, has neuroprotective effects on cerebral oxidative injury and cognitive impairment in septic rats. In this study, the mechanisms and molecular pathways underlying these effects were investigated.. Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham-operated laparotomy and were administered vehicle or UCF-101 (10 µmol/kg). The hippocampus was isolated for subsequent analysis. Omi/HtrA2 expression in the mitochondria or cytosol was evaluated by immunofluorescence or western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was utilized to evaluate levels of apoptosis, and western blotting was used to evaluate apoptosis-related proteins, such as cleaved caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). Tight junction expression was assessed by immunofluorescence and western blotting. Mitochondrial function, inflammatory cytokines, and oxidative stress were also assayed. In addition, a wet/dry method was used to evaluate brain edema and Evans blue extravasation was used to evaluate blood-brain barrier (BBB) integrity.. After CLP treatment, the hippocampus exhibited a mild increase in Omi/HtrA2 expression; cytosolic Omi/HtrA2 expression increased significantly, whereas mitochondrial Omi/HtrA2 expression was reduced, indicating that CLP-induced oxidative stress resulted in the translocation of Omi/HtrA2 from mitochondria to the cytosol. Hippocampal cleaved caspase-3, caspase-9, and PARP levels were significantly higher in animals treated with CLP than in sham-operated animals, while XIAP expression was lower. Treatment with UCF-101 prevented the mobilization of Omi/HtrA2 from mitochondria to the cytosol, attenuated XIAP degradation, and decreased cleaved caspase-3, caspase-9, and PARP expression as well as apoptosis. UCF-101 also reversed the decreased mitochondrial complex I, II, and III respiration and the reduced ATP caused by CLP. In addition, UCF-101 treatment resulted in a significant improvement in BBB integrity, as demonstrated by increased occludin, claudin-5, and zonula occludens 1 levels and reduced Evans blue extravasation. No significant effects of UCF-101 on brain edema were found. Inflammatory cytokines and oxidative stress were significantly higher in the CLP-treated group than in the sham-operated group. However, the inhibition of Omi/HtrA2 by UCF-101 significantly alleviated these responses.. Our data indicated that Omi/ HtrA2 regulates a mitochondria-dependent apoptotic pathway in a murine model of septic encephalopathy. Inhibition of Omi/HtrA2 by UCF-101 leads to neuroprotection by inhibiting the cytosolic translocation of Omi/HtrA2 and antagonizing the caspase-dependent apoptosis pathway. Therapeutic interventions that inhibit Omi/HtrA2 translocation or protease activity may provide a novel method to treat SAE.

Topics: Animals; Apoptosis; Caspase 3; Cytosol; Disease Models, Animal; Dynamins; Electron Transport Chain Complex Proteins; GTP Phosphohydrolases; High-Temperature Requirement A Serine Peptidase 2; Hippocampus; Male; Malondialdehyde; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Occludin; Poly(ADP-ribose) Polymerases; Pyrimidinones; Rats; Rats, Sprague-Dawley; Sepsis; Thiones; X-Linked Inhibitor of Apoptosis Protein

Arterial thrombosis is triggered by tissue factor, which is a transmembrane glycoprotein can be released into the blood circulation after plaque rupture. Animal models with reflecting ruptured plaque lesions will be useful to understand efficacy of anticoagulant. In this study, we sought to improve a common arteriovenous shunt model in rabbits, aiming for a model of thrombosis stimulated with tissue factor, and to investigate the anti-thrombotic effect of a direct factor Xa inhibitor TAK-442 in the model.. In the model where thrombus was stimulated with a thrombogenic silk thread soaked with recombinant human tissue factor, thrombus formation was significantly reduced by TAK-442 at more than 37.5 µg/kg, accompanied with prolonged plasma hemostatic parameters. Although efficacious doses of anti-coagulants in ordinary arteriovenous thrombosis models are widely reported to be higher than those in venous thrombosis models, TAK-442 showed its efficacy in the present arteriovenous shunt thrombosis model, with equivalent sensitivity in a previously reported venous model. TAK-442 might be effective under conditions thrombus formed is more influenced by tissue factor pathway.

Topics: Animals; Arteriovenous Shunt, Surgical; Disease Models, Animal; Factor Xa Inhibitors; Fibrinolytic Agents; Male; Pyrimidinones; Rabbits; Sulfones; Thrombosis

Melanoma is an aggressive cutaneous malignancy, but advances over the past decade have resulted in multiple new therapeutic options, including molecularly targeted therapy, immunotherapy, and oncolytic virus therapy. Talimogene laherparepvec (T-VEC) is a herpes simplex type 1 oncolytic virus, and trametinib is a MEK inhibitor approved for treatment of melanoma. Therapeutic responses with T-VEC are often limited, and BRAF/MEK inhibition is complicated by drug resistance. We observed that the combination of T-VEC and trametinib resulted in enhanced melanoma cell death in vitro. Further, combination treatment resulted in delayed tumor growth and improved survival in mouse models. Tumor regression was dependent on activated CD8

Topics: Animals; B7-H1 Antigen; Basic-Leucine Zipper Transcription Factors; Biological Products; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cytotoxicity, Immunologic; Dendritic Cells; Disease Models, Animal; Herpesvirus 1, Human; Humans; Immunocompetence; Immunotherapy; Lymphocyte Activation; Melanoma; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; Oncolytic Virotherapy; Oncolytic Viruses; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Repressor Proteins; Survival Analysis; T-Lymphocytes; Treatment Outcome; Tumor Microenvironment; Virus Replication; Xenograft Model Antitumor Assays

Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/β-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a β-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of β-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Enzyme Inhibitors; Hepatitis C, Chronic; Histocytochemistry; Injections, Intraperitoneal; Liver Cirrhosis; Mice, Transgenic; Pyrimidinones; Treatment Outcome; Wnt Signaling Pathway

The aim of this study was to profile somatic mutation spectrum in gallbladder cancers (GBCs), and determine the role of MAP kinase pathway in GBC by a series of in vitro and in vivo studies. We performed targeted massively parallel sequencing of DNA isolated from GBCs and matched blood from 14 GBC patients to search for mutations in 504 genes commonly altered in human cancers. We identified recurrent mutations enriched in several major signaling pathways including MAP kinase, Wnt/β-catenin and NF-κB pathways. Immunohistochemistry analysis further validated overactivation of MAP kinase and Wnt pathways in a panel of GBC samples. By treating GBC cells with MEK inhibitor trametinib, we found that trametinib not only dramatically inhibited the activity of MAPK/ERK pathway, but also blocked the Wnt/β-catenin signaling through decreasing β-catenin expression or suppressing nucleus translocation of β-catenin. Moreover, trametinib inhibited the proliferation of GBC cell in a dose- and time-dependent manner, induced GBC cell apoptosis, and inhibited GBC cell migration and invasion. Growth of xenograft tumors derived from GBC cell line NOZ in nude mice was also significantly inhibited by trametinib. Our data highlight the critical role of MAP kinase pathways in GBC pathogenesis, and may represent therapeutic targets for this cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; DNA Mutational Analysis; Gallbladder Neoplasms; High-Throughput Nucleotide Sequencing; Humans; MAP Kinase Signaling System; Mice; Molecular Targeted Therapy; Mutation; NF-kappa B; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction; Tumor Stem Cell Assay; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Pediatric high-grade gliomas (pedHGG) belong to the most aggressive cancers in children with a poor prognosis due to a lack of efficient therapeutic strategies. The β-catenin/Wnt-signaling pathway was shown to hold promising potential as a treatment target in adult high-grade gliomas by abrogating tumor cell invasion and the acquisition of stem cell-like characteristics. Since pedHGG differ from their adult counterparts in genetically and biologically we aimed to investigate the effects of β-catenin/Wnt-signaling pathway-inhibition by the β-catenin/CBP antagonist ICG-001 in pedHGG cell lines. In contrast to adult HGG, pedHGG cells displayed minimal detectable canonical Wnt-signaling activity. Nevertheless, low doses of ICG-001 inhibited cell migration/invasion, tumorsphere- and colony formation, proliferation in vitro as well as tumor growth in vivo/ovo, suggesting that ICG-001 affects pedHGG tumor cell characteristics independent of β-catenin/Wnt-signaling. RNA-sequencing analyses support a Wnt/β-catenin-independent effect of ICG-001 on target gene transcription, revealing strong effects on genes involved in cellular metabolic/biosynthetic processes and cell cycle progression. Among these, high mRNA expression of cell cycle regulator JDP2 was found to confer a better prognosis for pedHGG patients. In conclusion, ICG-001 might offer an effective treatment option for pedHGG patients functioning to regulate cell phenotype and gene expression programs in absence of Wnt/β-catenin signaling-activity.

Topics: Adolescent; Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Movement; Cell Self Renewal; Cell Survival; Cell Transformation, Neoplastic; Chick Embryo; Child; Child, Preschool; CREB-Binding Protein; Databases, Genetic; Disease Models, Animal; Glioma; Humans; Kaplan-Meier Estimate; Neoplastic Stem Cells; Prognosis; Pyrimidinones; Wnt Signaling Pathway; Young Adult

A pancreatic ductal adenocarcinoma (PDAC), obtained from a patient, was grown orthotopically in the pancreatic tail of nude mice to establish a patient-derived orthotopic (PDOX) model. Seven weeks after implantation, PDOX nude mice were divided into the following groups: untreated control (n = 7); gemcitabine (100 mg/kg, i.p., once a week for 2 weeks, n = 7); cobimetinib (5 mg/kg, p.o., 14 consecutive days, n = 7); trametinib (0.3 mg/kg, p.o., 14 consecutive days, n = 7); trabectedin (0.15 mg/kg, i.v., once a week for 2 weeks, n = 7); temozolomide (25 mg/kg, p.o., 14 consecutive days, n = 7); carfilzomib (2 mg/kg, i.v., twice a week for 2 weeks, n = 7); bortezomib (1 mg/kg, i.v., twice a week for 2 weeks, n = 7); MK-1775 (20 mg/kg, p.o., 14 consecutive days, n = 7); BEZ-235 (45 mg/kg, p.o., 14 consecutive days, n = 7); vorinostat (50 mg/kg, i.p., 14 consecutive days, n = 7). Only the MEK inhibitors, cobimetinib and trametinib, regressed tumor growth, and they were more significantly effective than other therapies (p < 0.0001, respectively), thereby demonstrating the precision of the PDOX models of PDAC and its potential for individualizing pancreatic-cancer therapy.

Topics: Animals; Antineoplastic Agents; Azetidines; Cell Line, Tumor; Deoxycytidine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gemcitabine; Humans; Mice; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Piperidines; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Xenograft Model Antitumor Assays

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G

Topics: Animals; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Male; Mice; Nuclear Proteins; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyridines; Pyrimidines; Pyrimidinones; Retinoblastoma Protein; Sarcoma; Xenograft Model Antitumor Assays

Although PARP inhibitors target

Topics: Animals; Antineoplastic Agents; BRCA1 Protein; BRCA2 Protein; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Disease Models, Animal; DNA Mutational Analysis; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Humans; Mice; Mutation; Nuclear Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Selection, Genetic; Xenograft Model Antitumor Assays

In the present study, a series of tetrahydropyridopyrimidinone derivatives, possessing potent dopamine D

Topics: Animals; Antipsychotic Agents; Behavior, Animal; Catalepsy; Disease Models, Animal; Dogs; Half-Life; Humans; Inhibitory Concentration 50; Mice; Microsomes, Liver; Pyrimidinones; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1A; Receptor, Serotonin, 5-HT2A; Receptors, Dopamine D2; Structure-Activity Relationship

The targeting of two independent but synergistic enzymatic activities, histone deacetylases (HDACs, class I and HDAC6) and phosphodiesterase 5 (PDE5), has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). Here we report the discovery of a new first-in-class small-molecule (CM-414) that acts as a dual inhibitor of PDE5 and HDACs. We have used this compound as a chemical probe to validate this systems therapeutics strategy, where an increase in the activation of cAMP/cGMP-responsive element-binding protein (CREB) induced by PDE5 inhibition, combined with moderate HDAC class I inhibition, leads to efficient histone acetylation. This molecule rescued the impaired long-term potentiation evident in hippocampal slices from APP/PS1 mice. Chronic treatment of Tg2576 mice with CM-414 diminished brain Aβ and tau phosphorylation (pTau) levels, increased the inactive form of GSK3β, reverted the decrease in dendritic spine density on hippocampal neurons, and reversed their cognitive deficits, at least in part by inducing the expression of genes related to synaptic transmission. Thus, CM-414 may serve as the starting point to discover balanced dual inhibitors with an optimal efficacy and safety profile for clinical testing on AD patients.

Topics: Alzheimer Disease; Animals; Behavior, Animal; Disease Models, Animal; Female; Hippocampus; Histone Deacetylase Inhibitors; Mice; Mice, Transgenic; Motor Activity; Neuronal Plasticity; Phosphodiesterase 5 Inhibitors; Primary Cell Culture; Pyrazoles; Pyrimidinones

Aberrant activation of β-catenin signaling by both WNT-dependent and WNT-independent pathways has been demonstrated in asthmatic airways, which is thought to contribute critically in remodeling of the airways. Yet, the exact role of β-catenin in asthma is very poorly defined. As we have previously reported abnormal expression of β-catenin in a toluene diisocyanate (TDI)-induced asthma model, in this study, we evaluated the therapeutic efficacy of two small molecules XAV-939 and ICG-001 in TDI-asthmatic male BALB/c mice, which selectively block β-catenin-mediated transcription.. Male BALB/c mice were sensitized and challenged with TDI to generate a chemically induced asthma model. Inhibitors of β-catenin, XAV-939, and ICG-001 were respectively given to the mice through intraperitoneally injection.. TDI exposure led to a significantly increased activity of β-catenin, which was then confirmed by a luciferase assay in 16HBE transfected with the TOPFlash reporter plasmid. Treatment with either XAV-939 or ICG-001 effectively inhibited activation of β-catenin and downregulated mRNA expression of β-catenin-targeted genes in TDI-asthmatic mice, paralleled by dramatically attenuated TDI-induced hyperresponsiveness and inflammation of the airway, alleviated airway goblet cell metaplasia and collagen deposition, decreased Th2 inflammation, as well as lower levels of TGFβ1, VEGF, HMGB1, and IL-1β.. The results showed that β-catenin is a principal mediator of TDI-induced asthma, proposing β-catenin as a promising therapeutic target in asthma.

Topics: Airway Remodeling; Animals; Anti-Asthmatic Agents; Asthma; beta Catenin; Biomarkers; Bridged Bicyclo Compounds, Heterocyclic; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Immunoglobulin E; Immunohistochemistry; Lymphocytes; Male; Mice; Molecular Targeted Therapy; Pyrimidinones; Signal Transduction; Toluene 2,4-Diisocyanate

Previously, a BRAF-V600E-mutant melanoma obtained from the right chest wall of a patient was grown orthotopically in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. Trametinib (TRA), an MEK inhibitor, caused tumor regression. In contrast, another MEK inhibitor, cobimetinib (COB) could slow but not arrest growth or cause regression of the melanoma PDOX. First-line therapy temozolomide (TEM) could slow but not arrest tumor growth or cause regression. In addition, vemurafenib (VEM) was not effective even though VEM is supposed to target the BRAF-V600E mutation. We also previously demonstrated that tumor-targeting with S. typhimurium A1-R combined with TEM was significantly more effective than either S. typhimurium A1-R alone or TEM alone on the melanoma PDOX with the BRAF-V600E mutation. The present study used this PDOX model of melanoma to test its sensitivity to VEM combined with S. typhimurium A1-R compared to VEM alone and VEM combined with COB. VEM combined with S. typhimurium A1-R was significantly more effective than VEM alone or VEM combined with COB (P = 0.0216) which is currently first line therapy for advanced melanoma with a BRAF-V600E mutation. J. Cell. Biochem. 118: 2314-2319, 2017. © 2017 Wiley Periodicals, Inc.

Topics: Aged; Animals; Azetidines; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Indoles; Melanoma; Mice; Mice, Nude; Microscopy, Confocal; Microscopy, Electron, Transmission; Mutation; Piperidines; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Salmonella typhimurium; Sulfonamides; Vemurafenib; Xenograft Model Antitumor Assays

Queuine is a modified pyrrolopyrimidine nucleobase derived exclusively from bacteria. It post-transcriptionally replaces guanine 34 in transfer RNA isoacceptors for Asp, Asn, His and Tyr, in almost all eukaryotic organisms, through the activity of the ancient tRNA guanine transglycosylase (TGT) enzyme. tRNA hypomodification with queuine is a characteristic of rapidly-proliferating, non-differentiated cells. Autoimmune diseases, including multiple sclerosis, are characterised by the rapid expansion of T cells directed to self-antigens. Here, we demonstrate the potential medicinal relevance of targeting the modification of tRNA in the treatment of a chronic multiple sclerosis model—murine experimental autoimmune encephalomyelitis. Administration of a de novo designed eukaryotic TGT substrate (NPPDAG) led to an unprecedented complete reversal of clinical symptoms and a dramatic reduction of markers associated with immune hyperactivation and neuronal damage after five daily doses. TGT is essential for the therapeutic effect, since animals deficient in TGT activity were refractory to therapy. The data suggest that exploitation of the eukaryotic TGT enzyme is a promising approach for the treatment of multiple sclerosis.

Topics: Animals; Brain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Genetic Therapy; Mice, Inbred C57BL; Multiple Sclerosis; Pentosyltransferases; Pyrimidinones; Pyrroles; RNA, Transfer; Thioguanine

Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although β-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of β-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC β-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of β-catenin inhibitors on vascular SMC growth.. We used an inducible, conditional genetic deletion of β-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC β-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC β-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking β-catenin showed decreased mRNA expression of. SMC β-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological β-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting β-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

Topics: Animals; Apoptosis; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Carotid Arteries; Carotid Artery Injuries; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Genotype; Humans; Male; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Phenotype; Pyrimidinones; Signal Transduction; Time Factors; Triazines; Vascular Remodeling

Migraine headache is a neurological disorder affecting millions worldwide. However, little is known about the mechanisms contributing to migraine. Recent genome-wide association studies have found single nucleotide polymorphisms in the gene encoding transient receptor potential channel M8. Transient receptor potential channel M8 is generally known as a cold receptor but it has been implicated in pain signaling and may play a role in migraine pain.. In order to investigate whether transient receptor potential channel M8 may contribute to the pain of migraine, the transient receptor potential channel M8 activator icilin was applied to the dura mater using a rat behavioral model of headache. Cutaneous allodynia was measured for 5 hours using Von Frey filaments.. Dural application of icilin produced cutaneous facial and hind paw allodynia that was attenuated by systemic pretreatment with the transient receptor potential channel M8-selective antagonist AMG1161 (10 mg/kg p.o.). Further, the anti-migraine agent sumatriptan (0.6 mg/kg s.c.) or the non-selective NOS inhibitor L-NAME (20 mg/kg i.p.) also attenuated allodynia when given as a pretreatment.. These data indicate that transient receptor potential channel M8 activation in the meninges produces behaviors in rats that are consistent with migraine and that are sensitive to pharmacological mechanisms known to have efficacy for migraine in humans. The findings suggest that activation of meningeal transient receptor potential channel M8 may contribute to the pain of migraine.

Topics: Animals; Disease Models, Animal; Hyperalgesia; Male; Migraine Disorders; Pyrimidinones; Rats; Rats, Sprague-Dawley; TRPM Cation Channels

This study was aimed to investigate the treatment mechanisms of 5-[5-(2-nitrophenyl) furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (UCF-101) in cerebral ischemia-reperfusion (CIR) model rats. Total of 54 healthy male Wistar rats were randomly assigned into three groups, namely sham group, vehicle group, and UCF-101 group. The CIR-injured model was established by right middle cerebral artery occlusion and reperfusion. Neurological function was assessed by an investigator according to the Longa neurologic deficit scores. Meanwhile, the cerebral tissue morphology and apoptotic neurons were evaluated by H&E and TUNEL staining, respectively. Additionally, the expressions of caspase 3, p-p38, and p-ERK were detected by immunohistochemistry or/and Western blotting assays. As results, neurologic deficit and pathological damage were obviously enhanced and TUNEL positive neurons were significantly increased in CIR-injured rats, as compared with those in sham group. Furthermore, the expressions of caspase 3, p-p38, and p-ERK were also significantly increased in vehicle group than those in sham group (P < 0.05). However, UCF-101 treatment could markedly weaken the neurologic deficit with lower scores and improve pathological condition. After UCF-101 treatment, TUNEL positive neurons as well as the expression of caspase 3 were significantly decreased than those in vehicle group (P < 0.05). Besides, p-p38 was decreased while p-ERK was increased in UCF-101 group than those in vehicle group (P < 0.05). Therefore, we concluded that the protective effects of UCF-101 might be associated with apoptosis process and MAPK signaling pathway in the CIR-injured model.

Topics: Animals; Apoptosis; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Male; MAP Kinase Signaling System; Pyrimidinones; Rats, Wistar; Reperfusion Injury; Thiones

Sunitinib and pazopanib are antiangiogenic tyrosine kinase inhibitors (TKI) used to treat metastatic renal cell carcinoma (RCC). However, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. It is possible that treatment outcomes in RCC patients could be improved by rationally combining TKIs with other agents. Here, we address whether inhibition of the Ras-Raf-MEK-ERK1/2 pathway is a rational means to improve the response to TKIs in RCC. Using a xenograft model of RCC, we found that tumors that are resistant to sunitinib have a significantly increased angiogenic response compared with tumors that are sensitive to sunitinib in vivo. We also observed significantly increased levels of phosphorylated ERK1/2 in the vasculature of resistant tumors, when compared with sensitive tumors. These data suggested that the Ras-Raf-MEK-ERK1/2 pathway, an important driver of angiogenesis in endothelial cells, remains active in the vasculature of TKI-resistant tumors. Using an in vitro angiogenesis assay, we identified that the MEK inhibitor (MEKI) trametinib has potent antiangiogenic activity. We then show that, when trametinib is combined with a TKI in vivo, more effective suppression of tumor growth and tumor angiogenesis is achieved than when either drug is utilized alone. In conclusion, we provide preclinical evidence that combining a TKI, such as sunitinib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for RCC.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Endothelial Cells; Female; Humans; Indoles; Kidney Neoplasms; MAP Kinase Signaling System; Neovascularization, Pathologic; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Pyrroles; Sunitinib; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

The α1-adrenergic receptors (α1-ARs), which belong to a G protein-coupled receptor family, consist of three highly homologous subtypes known as α1A-ARs, α1B-ARs, and α1D-ARs. Our previous findings suggested that α1A-ARs are an important target for imipramine and electroconvulsive therapy. The current study sought to evaluate whether S-(+)-niguldipine and B8805-033, two selective antagonists of α1A-ARs, can evoke antidepressant-like effects in the forced swim test in rats. Both compounds were administered at three time points (24, 5, and 1 h before testing), and the effects of three doses (2, 5, and 10 mg/kg) of each compound were investigated. S-(+)-Niguldipine produced no antidepressant-like effects other than a 14% reduction in immobility time at the highest dose. Although B8805-033 at a dose of 2 mg/kg did not influence the rats' behavior, higher B8805-033 doses (5 and 10 mg/kg) produced significant reductions in immobility time (approximately 42 and 44% vs. controls, respectively; P<0.01). However, this effect was abolished by the concomitant administration of WAY100135, a serotonin receptor antagonist, suggesting that the observed antidepressant-like effects of B8805-033 are unrelated to α1A-ARs. Nevertheless, given the current dearth of selective α1A-AR agonists, the question of whether this particular subtype could be involved in antidepressant therapy mechanisms remains unresolved.

Topics: Adrenergic alpha-1 Receptor Antagonists; Animals; Antidepressive Agents; Dihydropyridines; Dioxins; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Piperazines; Pyrimidinones; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Swimming; Time Factors

To investigate the potential roles that p16 (CDKN2A) and RB activation have in sensitization to MEK inhibitor in resistant KRAS-mutant non-small cell lung cancer cells (NSCLC) in vitro and in vivo.. Cell viability was measured with MTS assays. Effects of administration of radiation and combination drug treatments were evaluated by clonogenic assay, flow cytometry, and Western blots. DNA repair was assessed using immunofluorescent analysis. Finally, lung cancer xenografts were used to examine in vivo effects of drug treatment and radiation therapy.. In this study, we showed that sensitivity to MEK inhibitor correlated to the RB/p16/CDK4 pathway and knockdown of RB induced resistance in cell lines sensitive to MEK inhibitor. Also, overexpression of p16 and inhibition of CDK4 had the ability to sensitize normally resistant cell lines. Our data indicated that the MEK inhibitor (trametinib, GSK112012) cooperated with the CDK4/6 inhibitor (palbociclib, PD0332991) to strongly reduce cell viability of KRAS-mutant NSCLCs that were resistant to the MEK inhibitor in vitro and in vivo. In addition, we report for the first time that resistance of KRAS-mutant NSCLCs to MEK inhibitor is, at least partly, due to p16 mutation status, and we described a drug combination that efficiently reactivates the RB tumor suppressor pathway to trigger radiosensitizing effects, apoptosis, and cell-cycle arrest.. Our findings suggest that MEK inhibitor in combination with CDK4/6 inhibitor has significant anti-KRAS-mutant NSCLC activity and radiosensitizing effect in preclinical models, potentially providing a novel therapeutic strategy for patients with advanced KRAS-mutant NSCLCs.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; DNA Repair; Drug Synergism; Female; Humans; Lung Neoplasms; Mutation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyridines; Pyridones; Pyrimidinones; Radiation Tolerance; Radiation-Sensitizing Agents; ras Proteins; Retinoblastoma Protein; Xenograft Model Antitumor Assays

We have developed a Drosophila lung cancer model by targeting Ras1(G12V)--alone or in combination with PTEN knockdown--to the Drosophila tracheal system. This led to overproliferation of tracheal tissue, formation of tumor-like growths, and animal lethality. Screening a library of FDA-approved drugs identified several that improved overall animal survival. We explored two hits: the MEK inhibitor trametinib and the HMG-CoA reductase inhibitor fluvastatin. Oral administration of these drugs inhibited Ras and PI3K pathway activity, respectively; in addition, fluvastatin inhibited protein prenylation downstream of HMG-CoA reductase to promote survival. Combining drugs led to synergistic suppression of tumor formation and rescue lethality; similar synergy was observed in human A549 lung adenocarcinoma cells. Notably, fluvastatin acted both within transformed cells and also to reduce whole-body trametinib toxicity in flies. Our work supports and provides further context for exploring the potential of combining statins with MAPK inhibitors such as trametinib to improve overall therapeutic index.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Drug Combinations; Drug Screening Assays, Antitumor; Drug Synergism; Fatty Acids, Monounsaturated; Fluvastatin; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; IMP Dehydrogenase; Indoles; Lung Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Pyridones; Pyrimidinones; Signal Transduction; Survival Rate

To evaluate the distribution of a daily phosphodiesterase type 5 inhibitor dose (mirodenafil) in rat plasma and bladder and prostate tissue in a model of atherosclerosis-induced chronic pelvic ischemia.. Thirty-two 18-week-old male Sprague Dawley rats were divided into two groups. Group I (n = 16) comprised a chronic pelvic ischemia model treated with mirodenafil and group II (n = 16) comprised a sham-operated model also treated with mirodenafil. The mirodenafil concentrations in each organ were measured at specific time points after 14 days of daily mirodenafil administration. The drug distribution ratio of group I to group II of each organ was measured, and the bladder tissue-to-plasma and prostate tissue-to-plasma ratios were calculated.. The mean drug concentration in the bladder of the rats in group I did not differ significantly from that of group II after mirodenafil administration. In the prostate, the mean drug concentration of group I was significantly higher than that of group II at 1 and 4 hours after drug administration. The drug concentration was higher in the bladder tissue than in the prostate tissue and the bladder tissue-to-plasma ratio was significantly higher than the prostate tissue-to-plasma ratio.. Our results suggest that mirodenafil levels might be sufficient in the target tissue after daily treatment in an ischemia-induced aging model. Considering the difficulties of tissue distribution study in human subjects, the results of this investigation provided meaningful evidence of the application of daily doses of mirodenafil for treating lower urinary tract symptoms in an aging population.

Topics: Animals; Chronic Disease; Disease Models, Animal; Ischemia; Male; Pelvis; Phosphodiesterase 5 Inhibitors; Prostate; Pyrimidinones; Rats; Rats, Sprague-Dawley; Sulfonamides; Tissue Distribution; Urinary Bladder

Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu

Topics: Adult; Allosteric Regulation; Animals; Autistic Disorder; Autopsy; Disease Models, Animal; Female; Gene Expression Regulation; Hippocampus; Humans; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Knockout; Motor Cortex; Neuronal Plasticity; Pyrazoles; Pyrimidinones; Receptor, Metabotropic Glutamate 5; Rett Syndrome; Seizures; Signal Transduction; Young Adult

Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of premature infants. Connective tissue growth factor (CTGF) plays an important role in tissue development and remodeling. We have previously shown that targeted overexpression of CTGF in alveolar type II epithelial cells results in BPD-like pathology and activates β-catenin in neonatal mice.. Utilizing this transgenic mouse model and ICG001, a specific pharmacological inhibitor of β-catenin, we tested the hypothesis that β-catenin signaling mediates the effects of CTGF in the neonatal lung. Newborn CTGF mice and control littermates received ICG001 (10 mg/kg/dose) or placebo (dimethyl sulfoxide, equal volume) by daily i.p. injection from postnatal day 5 to 15. Alveolarization, vascular development, and pulmonary hypertension (PH) were analyzed.. Administration of ICG001 significantly downregulated expression of cyclin D1, collagen 1a1, and fibronectin, which are the known target genes of β-catenin signaling in CTGF lungs. Inhibition of β-catenin signaling improved alveolar and vascular development and decreased pulmonary vascular remodeling. More importantly, the improved vascular development and vascular remodeling led to a decrease in PH.. β-Catenin signaling mediates the autocrine and paracrine effects of CTGF in the neonatal lung. Inhibition of CTGF-β-catenin signaling may provide a novel therapy for BPD.

Topics: Animals; Animals, Newborn; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Bronchoalveolar Lavage; Bronchopulmonary Dysplasia; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibronectins; Hyperoxia; Hypertension, Pulmonary; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Transgenic; Pulmonary Alveoli; Pyrimidinones; Signal Transduction

The mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway is an essential component of innate immunity necessary for mediating proinflammatory responses in the setting of sepsis. We previously demonstrated that the mitogen-activated protein kinase 1/2 inhibitor trametinib prevents endotoxin-induced renal injury in mice. We therefore assessed efficacy of trametinib in a more clinically relevant experimental model of sepsis.. Controlled in vivo laboratory study.. University animal research laboratory.. Male C57BL/6 mice.. Mice were subjected to cecal ligation and puncture to induce sepsis or underwent sham operation as controls. Six hours after cecal ligation and puncture, mice were randomized to four experimental groups as follows: 1) sham control; 2) sham control + trametinib (1 mg/kg, IP); 3) cecal ligation and puncture; and 4) cecal ligation and puncture + trametinib. All animals received buprenorphine (0.05 mg/kg, SC) and imipenem/cilastatin (14 mg/kg, SC) in 1.5 mL of warm saline (40 mL/kg) at the 6-hour time point. Mice were euthanized at 18 hours after induction of cecal ligation and puncture.. Trametinib inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase signaling 6 hours after cecal ligation and puncture attenuated increases in circulating proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) and hypothermia at 18 hours. Trametinib also attenuated multiple organ injury as determined by serum creatinine, alanine aminotransferase, lactate dehydrogenase, and creatine kinase. At the organ level, trametinib completely restored peritubular capillary perfusion in the kidney. Restoration of microvascular perfusion was associated with reduced messenger RNA expression of well-characterized markers of proximal tubule injury. mitogen-activated protein kinase/extracellular signal-regulated kinase blockade attenuated cecal ligation and puncture-mediated up-regulation of cytokines (tumor necrosis factor-α, interleukin-1β) and restored interleukin-6 to control levels in the renal cortex, indicating the protective effects on the proximal tubule occur primarily through modulation of the proinflammatory response in sepsis.. These data reveal that the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor trametinib attenuates systemic inflammation and multiple organ damage in a clinically relevant model of sepsis. Because trametinib has been safely used in humans, we propose that this drug might represent a translatable approach to limit organ injury in septic patients.

Topics: Acute Kidney Injury; Animals; Cytokines; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Inflammation; Inflammation Mediators; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Multiple Organ Failure; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; RNA, Messenger; Sepsis

Cetuximab is a monoclonal antibody that is effective in the treatment of metastatic colorectal cancer (mCRC). Cetuximab blocks epidermal growth factor receptor (EGFR)-ligand interaction and inhibits downstream RAS-ERK activation. However, only some activating mutations in RAS affect cetuximab efficacy, and it is not clear what else mediates treatment success. Here we hypothesized that cetuximab induces immunogenic cell death (ICD) that activates a potent antitumor response. We found that cetuximab, in combination with chemotherapy, fostered ICD in CRC cells, which we measured via the endoplasmic reticulum (ER) stress response and an increase in phagocytosis by dendritic cells. ICD induction depended on the mutational status of the EGFR signaling pathway and on the inhibition of the splicing of X-box binding protein 1 (XBP1), an unfolded protein response (UPR) mediator. We confirmed the enhanced immunogenicity elicited by cetuximab in a mouse model of human EGFR-expressing CRC. Overall, we demonstrate a new, immune-related mechanism of action of cetuximab that may help to tailor personalized medicine.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Calreticulin; Camptothecin; Cell Death; Cell Line, Tumor; Cetuximab; Colorectal Neoplasms; Dendritic Cells; Disease Models, Animal; Endoplasmic Reticulum Stress; Fluorouracil; HCT116 Cells; HT29 Cells; Humans; Indoles; Irinotecan; Leucovorin; Mice; Panitumumab; Phagocytosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Sulfonamides; Unfolded Protein Response; Vemurafenib; X-Box Binding Protein 1

Class IA PI3K pathway activation resulting from PTEN deficiency has been associated with lack of sensitivity of melanoma to BRAF kinase inhibitors. Although previous studies have shown synergistic activity when pan-PI3K inhibitors were combined with MAPK inhibitors in the treatment of melanoma exhibiting concurrent genetic abnormalities, overlapping adverse events in patients limit optimal dosing and clinical application. With the aim of specifically targeting PTEN-deficient cancers and minimizing the potential for on-target toxicity when inhibiting multiple PI3K isoforms, we developed a program to discover PI3Kβ-selective kinase inhibitors and identified SAR260301 as a potent PI3Kβ-selective, orally available compound, which is now in clinical development. Herein, we provide a detailed biological characterization of SAR260301, and show that this compound has outstanding biochemical and cellular selectivity for the PI3Kβ isoform versus the α, δ, and γ isoforms and a large panel of protein and lipid kinases. We demonstrate that SAR260301 blocks PI3K pathway signaling preferentially in PTEN-deficient human tumor models, and has synergistic antitumor activity when combined with vemurafenib (BRAF inhibitor) or selumetinib (MEK inhibitor) in PTEN-deficient/BRAF-mutated human melanoma tumor models. Combination treatments were very well tolerated, suggesting the potential for a superior safety profile at optimal dosing using selective compounds to inhibit multiple signaling pathways. Together, these experiments provide a preclinical proof-of-concept for safely combining inhibitors of PI3Kβ and BRAF or MEK kinase modulators to improve antitumor activity in PTEN-deficient/BRAF-mutant melanoma, and support the evaluation of SAR260301-based combinations in clinical studies. Mol Cancer Ther; 15(7); 1460-71. ©2016 AACR.

Topics: Administration, Oral; Animals; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Disease Models, Animal; Drug Synergism; Female; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Melanoma; Mice; Models, Molecular; Molecular Conformation; Mutation; Phosphoinositide-3 Kinase Inhibitors; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyrimidinones; Signal Transduction; Xenograft Model Antitumor Assays

Airway smooth muscle cell (ASMC) phenotypic modulation is one of the key factors contributing to asthma. Temperature changes may induce asthma, and these changes are known to be related to the temperature-sensitive transient receptor potential channels (TS-TRPs). The present study was designed to investigate the cellular functions of cold-sensitive channels, TRPM8 and TRPA1, in the phenotypic modulation of ASMCs.. A rat asthma model was constructed and the expression of TS-TRPs in ASM was tested. Using the agonists and antagonists for both TRPM8 and TRPA1, the effects of cold-sensitive channels on the phenotypic modulation of ASMCs were evaluated by measurement of contractile protein expression and cell proliferation and migration. Signaling pathways and matrix metalloproteinase-2 (MMP-2) activity were assayed with Western blotting and gelatin zymography.. TRPM8 and TRPA1 were decreased in the ASM of the rat asthma model. Icilin and menthol, agonists for TRPM8 and TRPA1, inhibited ASMC proliferation and migration induced by fetal bovine serum (FBS) or platelet-derived growth factor (PDGF). Moreover, icilin reversed the FBS-induced inhibition of the expression of contractile phenotype markers, smooth muscle α-actin, and SM22α. Icilin also antagonized the activation of p38 and MMP-2 and the repression of p21 caused by FBS.. Our findings show, for the first time, that the activation of TRPM8 and TRPA1 inhibits ASMC proliferative phenotype. These data suggest that TRPM8 and TRPA1 agonists may be promising new therapies for asthma.

Topics: Actins; Airway Remodeling; Animals; Asthma; Bronchi; Calcium Channel Agonists; Cell Movement; Cell Proliferation; Cold Temperature; Disease Models, Animal; Female; Matrix Metalloproteinase 2; Menthol; Microfilament Proteins; Muscle Contraction; Muscle Proteins; Myocytes, Smooth Muscle; p21-Activated Kinases; p38 Mitogen-Activated Protein Kinases; Phenotype; Platelet-Derived Growth Factor; Pyrimidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Trachea; TRPA1 Cation Channel; TRPM Cation Channels

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Death; Cell Proliferation; Colonic Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Feedback, Physiological; Female; Humans; Imidazoles; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mutant Proteins; Mutation; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins p21(ras); Pyridazines; Pyridones; Pyrimidinones; Receptor, Fibroblast Growth Factor, Type 1; Xenograft Model Antitumor Assays

Asthma is a heterogeneous chronic inflammatory disease, characterized by the development of structural changes (airway remodelling). β-catenin, a transcriptional co-activator, is fundamentally involved in airway smooth muscle growth and may be a potential target in the treatment of airway smooth muscle remodelling.. We assessed the ability of small-molecule compounds that selectively target β-catenin breakdown or its interactions with transcriptional co-activators to inhibit airway smooth muscle remodelling in vitro and in vivo.. ICG-001, a small-molecule compound that inhibits the β-catenin/CREB-binding protein (CBP) interaction, strongly and dose-dependently inhibited serum-induced smooth muscle growth and TGFβ1-induced production of extracellular matrix components in vitro. Inhibition of β-catenin/p300 interactions using IQ-1 or inhibition of tankyrase 1/2 using XAV-939 had considerably less effect. In a mouse model of allergic asthma, β-catenin expression in the smooth muscle layer was found to be unaltered in control versus ovalbumin-treated animals, a pattern that was found to be similar in smooth muscle within biopsies taken from asthmatic and non-asthmatic donors. However, β-catenin target gene expression was highly increased in response to ovalbumin; this effect was prevented by topical treatment with ICG-001. Interestingly, ICG-001 dose-dependently reduced airway smooth thickness after repeated ovalbumin challenge, but had no effect on the deposition of collagen around the airways, mucus secretion or eosinophil infiltration.. Together, our findings highlight the importance of β-catenin/CBP signalling in the airways and suggest ICG-001 may be a new therapeutic approach to treat airway smooth muscle remodelling in asthma.

Topics: Airway Remodeling; Animals; Anti-Asthmatic Agents; Asthma; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; CREB-Binding Protein; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Matrix; Female; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Inbred BALB C; Muscle, Smooth; Ovalbumin; Pyrimidinones

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms' tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.

Topics: Albumins; Animals; beta Catenin; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Computational Biology; Creatinine; Disease Models, Animal; Gene Expression Regulation; Humans; Kidney; Kidney Tubules; Ligands; Mice; Mice, Inbred BALB C; Podocytes; Promoter Regions, Genetic; Proteinuria; Pyrimidinones; ras Proteins; Renin-Angiotensin System; Wnt Signaling Pathway

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase-activating protein neurofibromin. Non-bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFβ. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo-based cell culture approach based on the use of Nf1(flox/flox) bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell-autonomous manner, independent of developmental growth plate-derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1-deficient osteoprogenitors blunts the pro-osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1-deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx (-/-) mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol-based delivery method. Collectively, these results provide novel evidence for a cell-autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Regeneration; Cell Differentiation; Disease Models, Animal; Drug Delivery Systems; Humans; MAP Kinase Kinase Kinases; Mesenchymal Stem Cells; Mice; Mice, Knockout; Nanoparticles; Neurofibromatosis 1; Neurofibromin 1; Protein Kinase Inhibitors; Pseudarthrosis; Pyridones; Pyrimidinones

Several neurophysiological abnormalities have been described in Huntington's disease, including auditory gating deficit, which are considered to reflect impaired brain information-processing. Since transgenic animal models of Huntington's disease capture basic neuropathology of the disorder, auditory gating was studied in BACHD (line5) transgenic rats and Q175 transgenic mice, together with local field gamma power in the hippocampus and primary auditory cortex. Using clinically equivalent acoustic-stimulation paradigms, impaired auditory gating was detected in transgenic BACHD rats under anesthesia and in freely-moving condition. In addition, transgenic BACHD rats showed a lower level of hippocampal and cortical field gamma band power compared to wild-type counterpart, which might be related to their compromised mitochondrial function. Systemic administration of the recently developed phosphodiesterase 9A (PDE9A) inhibitor PF-04447943 dose-dependently improved gating deficit in transgenic BACHD rats in both brain regions. Q175 mice, including wild-type, heterozygote and homozygote mice showed similarly poor gating, and administration of PF-04447943 was without effect. Treatment of transgenic BACHD rats with daily administration of PF-04447943 (1mg/kg) over 7-days resulted in an improvement in their auditory gating both in the hippocampus and primary auditory cortex as evaluated 24h after the last treatment. In fact, differences in auditory gating between wild-type and transgenic BACHD rats were totally abolished after sub-chronic treatment with the PDE9A inhibitor. Our findings indicate that BACHD transgenic rats show abnormal auditory gating with features resembling those of Huntington's disease patients, which could be considered as potential translational biomarker for drug development in treatment of this disease.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Acoustic Stimulation; Animals; Auditory Cortex; Disease Models, Animal; Electrophysiology; Enzyme Inhibitors; Evoked Potentials, Auditory; Hippocampus; Huntington Disease; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pyrazoles; Pyrimidinones; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Sensory Gating

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001-0.03). Interestingly, BRAF(V600E) and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733-mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Humans; Immunoblotting; Kinetics; Melanoma; Mice, Nude; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Xenograft Model Antitumor Assays

Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 μM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.

Topics: Animals; Binding Sites; Cell Line; Cell Proliferation; Disease Models, Animal; Dopaminergic Neurons; Drug Evaluation, Preclinical; Forkhead Box Protein O3; Forkhead Transcription Factors; Histone Deacetylase Inhibitors; Humans; MCF-7 Cells; Molecular Docking Simulation; Neuroprotective Agents; Parkinson Disease; Protein Binding; Protein Structure, Tertiary; Pyrimidinones; Rats; Sirtuin 2; Structure-Activity Relationship; Thiophenes

Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide.. In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging.. GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors.. Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM.

Topics: Animals; Blood-Brain Barrier; Cell Cycle Proteins; Dacarbazine; Disease Models, Animal; DNA Damage; Glioblastoma; Humans; Mice; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Temozolomide; Tumor Burden; Xenograft Model Antitumor Assays

New pyridopyrimidine derivatives were defined using a novel HTS in silico docking method (FRIGATE). The target protein was a dUTPase enzyme (EC 3.6.1.23; Rv2697) which plays a key role in nucleotide biosynthesis of Mycobacterium tuberculosis (Mtb). Top hit molecules were assayed in vitro for their antimycobacterial effect on Mtb H37Rv culture. In order to enhance the cellular uptake rate, the TB820 compound was conjugated to a peptid-based carrier and a nanoparticle type delivery system (polylactide-co-glycolide, PLGA) was applied. The conjugate had relevance to in vitro antitubercular activity with low in vitro and in vivo toxicity. In a Mtb H37Rv infected guinea pig model the in vivo efficacy of orally administrated PLGA encapsulated compound was proven: animals maintained a constant weight gain and no external clinical signs of tuberculosis were observed. All tissue homogenates from lung, liver and kidney were found negative for Mtb, and diagnostic autopsy showed that no significant malformations on the tissues occurred.

Topics: Animals; Antitubercular Agents; Delayed-Action Preparations; Disease Models, Animal; Female; Guinea Pigs; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Nanoconjugates; Pharmaceutical Vehicles; Polyesters; Pyridines; Pyrimidines; Pyrimidinones; Pyrrolidines; Tuberculosis

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Drug Screening Assays, Antitumor; Germinal Center Kinases; GTP Phosphohydrolases; Humans; Leukemia; Membrane Proteins; Mice; Mutation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidinones; Signal Transduction; Small Molecule Libraries; Xenograft Model Antitumor Assays

Extracellular signal-regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using Apc(Δ716) mice, a mouse model of familial adenomatous polyposis and early-stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight-week treatment of Apc(Δ716) mice with trametinib, a small-molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX-2 level in Apc(Δ716) tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX-2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in Apc(Δ716) tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in Apc(Δ716) mice, at least in part, through COX-2 induction in tumor stromal cells.

Topics: Animals; Antineoplastic Agents; Chemokine CCL2; Cyclooxygenase 2; Disease Models, Animal; Female; Genes, APC; Intestinal Polyps; Male; Mice; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Pyridones; Pyrimidinones

A new series of thienopyrimidinones is synthesized and evaluated as selective phosphodiesterase 7 (PDE7) inhibitors for the treatment of inflammatory diseases. The modification of the substituents on thienopyrimidinone revealed that an isopropylamino group at the 2-position was favorable for aqueous solubility. The introduction of 3-pyrrolidines at the 7-position resulted in good solubility, highly potent activity, and good PDE7 selectivity. Among the synthesized compounds, compound 46 exhibited the greatest inhibition of ear edema in a phorbol ester-induced mouse model.

Topics: Animals; Cyclic Nucleotide Phosphodiesterases, Type 7; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Male; Mice; Mice, Inbred ICR; Models, Molecular; Molecular Structure; Phorbol Esters; Pyrimidinones; Solubility; Structure-Activity Relationship; Substrate Specificity

Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Animals; Autoimmune Diseases; Biphenyl Compounds; Cells, Cultured; Disease Models, Animal; Gene Expression; Immunization; Leukocytes; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Peptides; Phospholipases A2; Pyrimidinones; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; Uveitis

Persistent itch is a common symptom of allergic contact dermatitis (ACD) and represents a significant health burden. The chemokine CXCL10 is predominantly produced by epithelial cells during ACD. Although the chemokine CXCL10 and its receptor CXCR3 are implicated in the pathophysiology of ACD, it is largely unexplored for itch and pain accompanying this disorder. Here, we showed that CXCL10 and CXCR3 mRNA, protein, and signaling activity were upregulated in the dorsal root ganglion after contact hypersensitivity (CHS), a murine model of ACD, induced by squaric acid dibutylester. CXCL10 directly activated a subset of cutaneous dorsal root ganglion neurons innervating the area of CHS through neuronal CXCR3. In behavioral tests, a CXCR3 antagonist attenuated spontaneous itch- but not pain-like behaviors directed to the site of CHS. Injection of CXCL10 into the site of CHS elicited site-directed itch- but not pain-like behaviors, but neither type of CXCL10-evoked behaviors was observed in control mice. These results suggest that CXCL10/CXCR3 signaling mediates allergic itch but not inflammatory pain in the context of skin inflammation. Thus, upregulation of CXCL10/CXCR3 signaling in sensory neurons may contribute to itch associated with ACD. Targeting the CXCL10/CXCR3 signaling might be beneficial for the treatment of allergic itch.

Topics: Acetamides; Animals; Anti-Allergic Agents; Cells, Cultured; Chemokine CXCL10; Dermatitis, Allergic Contact; Disease Models, Animal; Ganglia, Spinal; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Neurons; Pruritus; Pyrimidinones; Receptors, CXCR3; Receptors, G-Protein-Coupled; Signal Transduction; Skin; Up-Regulation

A novel series of arylpiperazinylalkyl purine-2,4-diones (4-27) and purine-2,4,8-triones (31-38) was synthesized and tested to evaluated their affinity for the serotoninergic (5-HT1A, 5-HT6, 5-HT7) and dopaminergic (D2) receptors. Compounds with purine-2,4-dione nucleus generally had affinity values higher than the corresponding purine-2,4,8-trione compounds. A spectrum of receptor activities was observed for compounds with a substituent at the 7-position of the imidazo[2,1-f]purine-2,4-dione system and some potent 5-HT1A (18, 25), 5-HT7 (14) and mixed 5-HT1A/5-HT7 (8, 9) receptor ligands with additional affinity for dopamine D2 receptors (15) has been identified. Moreover, docking studies proved that a substituent at the 7-position of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione could be essential for receptor affinity and selectivity, especially towards 5-HT1A and 5-HT7. The results of the preliminary pharmacological in vivo studies of selected derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione, including 9 as a potential anxiolytic, 8 and 15 as potential antidepressants, and 18 and 25 as potential antidepressant and anxiolytic agents.

Topics: Animals; Anti-Anxiety Agents; Antidepressive Agents; Binding Sites; Disease Models, Animal; Models, Molecular; Molecular Structure; Piperazine; Piperazines; Purines; Pyrimidinones; Structure-Activity Relationship

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.

Topics: Administration, Topical; Animals; Casein Kinase II; Cell Line; Choroidal Neovascularization; Crystallography, X-Ray; Disease Models, Animal; Enzyme Inhibitors; Humans; Macular Degeneration; Mice; Models, Molecular; Molecular Docking Simulation; Niacinamide; Piperidines; Protein Serine-Threonine Kinases; Pyrimidinones; Small Molecule Libraries; Structure-Activity Relationship; Thiadiazoles

Secondary death of neural cells plays a key role in the physiopathology and the functional consequences of traumatic spinal cord injury (SCI). Pharmacological manipulation of cell death pathways leading to the preservation of neural cells is acknowledged as a main therapeutic goal in SCI. In the present work, we hypothesize that administration of the neuroprotective cell-permeable compound ucf-101 will reduce neural cell death during the secondary damage of SCI, increasing tissue preservation and reducing the functional deficits. To test this hypothesis, we treated mice with ucf-101 during the first week after a moderate contusive SCI. Our results reveal that ucf-101 administration protects neural cells from the deleterious secondary mechanisms triggered by the trauma, reducing the extension of tissue damage and improving motor function recovery. Our studies also suggest that the effects of ucf-101 may be mediated through the inhibition of HtrA2/OMI and the concomitant increase of inhibitor of apoptosis protein XIAP, as well as the induction of ERK1/2 activation and/or expression. In vitro assays confirm the effects of ucf-101 on both pathways as well as on the reduction of caspase cascade activation and apoptotic cell death in a neuroblastoma cell line. These results suggest that ucf-101 can be a promising therapeutic tool for SCI that deserves more detailed analyses.

Topics: Animals; Apoptosis; Caspases; Disease Models, Animal; Inhibitor of Apoptosis Proteins; Locomotion; MAP Kinase Signaling System; Mice, Inbred C57BL; Movement Disorders; Neurons; Neuroprotective Agents; Pyrimidinones; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Thiones

The aim of this study was to evaluate the potential of (18)F-fluorodeoxyglucose-positron emission tomography ((18)F-FDG-PET) for monitoring the therapeutic efficacy of TAK-733, an inhibitor of mitogen-activated protein kinase kinase, in nude rats bearing A549 (human lung carcinoma) xenografts.. TAK-733 was administered orally by gavage to nude xenograft rats for 2 weeks, at dosage levels of 0 (0.5% w/v methylcellulose solution), 1, 3, and 10 mg/kg/day (n = 8/dose). Tumor size was measured before treatment (day 0), and on days 1, 3, 7, 9, 11, and 14. PET scans were performed pretreatment (day 0), and on days 2, 4, 7, 10, and 14. Tracer accumulations in tumor tissue were quantified as the mean standard uptake value (SUVmean).. No deaths or treatment-related body weight losses occurred during the study period. TAK-733 showed dose-dependent inhibition of tumor growth and (18)F-FDG uptake in tumor tissue. At a dosage of 10 mg/kg, TAK-733 treatment produced a statistically significant reduction in tumor weight from day 11 compared with the vehicle group (P < 0.05). Tumor growth was inhibited in the 10 mg/kg group with a treated/control value of 31% on day 14. The SUVmean on day 2 in this dosage group was statistically lower than that observed on day 0, and that seen in the vehicle group on day 2 (P < 0.05 for both comparisons). Furthermore, this reduction in SUVmean at 10 mg/kg was maintained over time. In the two lower dosage groups (1 and 3 mg/kg), SUVmean gradually increased over time.. (18)F-FDG-PET enabled early determination of late anti-tumor activity in response to TAK-733 treatment.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Fluorodeoxyglucose F18; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Positron-Emission Tomography; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Rats; Treatment Outcome

Although the majority of patients with HPV(+) oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV(+) head and neck squamous cell carcinoma (HNSCC).. Clonogenic survival assays and an orthotopic mouse model of HPV(+) oral cancer were used to examine the in vitro and in vivo sensitivity of HPV(+) HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms.. We found that AZD-1775 displays single-agent activity and enhances the response of HPV(+) HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV(+) HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV(+) HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV(-) HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment.. AZD-1775 selectively sensitizes HPV(+) HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV(+) HNSCC tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, p53; Head and Neck Neoplasms; Humans; Inhibitory Concentration 50; Male; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nuclear Proteins; Papillomavirus Infections; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Squamous Cell Carcinoma of Head and Neck; Tumor Burden; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

The purpose of the experiment was to compare the effects of nifekalant and amiodarone on the return of spontaneous circulation (ROSC), survival, as well as on the hemodynamic parameters in a swine model of prolonged ventricular fibrillation (VF).. After 8 min of untreated VF, bolus doses of epinephrine (adrenaline) and either nifekalant, or amiodarone, or saline (n = 10 per group), were administered after randomization. Cardiopulmonary resuscitation (CPR) was commenced immediately after drug administration and defibrillation was attempted 2 min later. CPR was resumed for another 2 min after each defibrillation attempt and the same dose of adrenaline was given every 4th minute during CPR.. Forty-eight hour survival was significantly higher with nifekalant compared to amiodarone (p < 0.001) and saline (p = 0.02), (9/10 vs. 0/10 vs. 3/10, respectively). Systolic aortic pressure, diastolic aortic pressure and coronary perfusion pressure were significantly higher with nifekalant during CPR and immediate post-resuscitation period (p < 0.05). The animals in the amiodarone group had a slower heart rate at the 1st and 45th min post-ROSC (p < 0.001 and p = 0.006, respectively). The number of electric shocks required for terminating VF, time to ROSC and adrenaline dose were significantly higher with amiodarone compared to nifekalant (p < 0.001).. Nifekalant showed a more favorable hemodynamic profile and improved survival compared to amiodarone and saline in this swine model.

Topics: Amiodarone; Animals; Blood Pressure; Cardiopulmonary Resuscitation; Disease Models, Animal; Electric Countershock; Epinephrine; Female; Heart Arrest; Heart Rate; Pyrimidinones; Survival Analysis; Swine; Ventricular Fibrillation

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease. Post-mortem hallmarks of MSA neuropathology include oligodendroglial α-synuclein (αSYN) inclusions, striatonigral degeneration, olivopontocerebellar atrophy, and increased microglial activation that accompanies the wide spread neurodegeneration. Recently, we demonstrated upregulation of myeloperoxidase (MPO) in activated microglia and provided evidence for the role of microglial MPO in the mediation of MSA-like neurodegeneration (Stefanova et al. Neurotox Res 21:393-404, 2015). The aim of the current study was to assess the therapeutic potency of MPO inhibition (MPOi) in a model of advanced MSA. We replicated the advanced pathology of MSA by intoxicating transgenic PLP-α-synuclein transgenic mice with 3-nitropropionic acid (3NP). After onset of the full-blown pathology, MSA mice received either MPOi or vehicle over 3 weeks. Motor phenotype and neuropathology were analyzed to assess the therapeutic efficacy of MPOi compared to vehicle treatment in MSA mice. MPOi therapy initiated after the onset of severe MSA-like neuropathology in mice failed to attenuate motor impairments and neuronal loss within the striatum, substantia nigra pars compacta, inferior olives, pontine nuclei, and cerebellar cortex. However, we observed a significant reduction of microglial activation in degenerating brain areas. Further, nitrated αSYN accumulation was reduced in the striatonigral region. In summary, delayed-start MPOi treatment reduced microglial activation and levels of nitrated αSYN in a mouse model of advanced MSA. These effects failed to impact on motor impairments and neuronal loss in contrast to previously reported disease modifying efficacy of early-start therapy with MPOi in MSA.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Enzyme Inhibitors; Humans; Male; Mice, Transgenic; Microglia; Motor Activity; Multiple System Atrophy; Myelin Proteolipid Protein; Neurons; Neuroprotective Agents; Nitro Compounds; Peroxidase; Propionates; Pyrimidinones; Pyrroles; Severity of Illness Index; Treatment Outcome

Immucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response.. BALB/c mice were infected with 107 amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85-89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime.. Immucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.

Topics: Adenine; Adenosine; Animals; Antibodies, Protozoan; Antiprotozoal Agents; Blood Chemical Analysis; Disease Models, Animal; Drug-Related Side Effects and Adverse Reactions; Female; Gene Expression; Immunophenotyping; Interferon-gamma; Interleukin-10; Leishmania; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Mesocricetus; Mice, Inbred BALB C; Parasite Load; Purine Nucleosides; Pyrimidinones; Pyrrolidines; Spleen; T-Lymphocyte Subsets; Treatment Outcome; Tumor Necrosis Factor-alpha

The 3D QSAR studies based on generation of common pharmacophore hypotheses (CPHs) were performed separately for the series of 1,2,3,4-tetrahydropyrimidin-5-yl-acetic acid and 2-(4-sulfonylphenyl)pyrimidine analogs for their in-vivo anti-inflammatory activity and in-vitro COX-2 inhibitory activity respectively. The main idea of selecting two different series was to develop two 3D QSAR models for same scaffold (1,2,3,4-tetrahydropyrimidine/pyrimidine) for same target, but with different aspects of activity. The aim of study was to screen designed compounds and select new compounds with increased COX-2 selectivity. The best 3D QSAR model from both group was employed as 3D search query to screen the designed 4,5,6-triphenyl-1,2,3,4-tetrahydropyrimidine derivatives. The new compounds showing good predicted activity were selected for experimental studies. Among the synthesized compounds, 5c and 5f showed highest anti-inflammatory activity.

Topics: Animals; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Drug Design; Edema; Female; Male; Models, Chemical; Models, Molecular; Molecular Structure; Pyrimidines; Pyrimidinones; Quantitative Structure-Activity Relationship; Rats; Sulfones; Thiones

The morphogen pathways Hedgehog, Wnt and Notch are attractive targets for antifibrotic therapies in systemic sclerosis. Interference with stem cell regeneration, however, may complicate the use of morphogen pathway inhibitors. We therefore tested the hypothesis that combination therapies with low doses of Hedgehog, Wnt and Notch inhibitors maybe safe and effective for the treatment of fibrosis.. Skin fibrosis was induced by bleomycin and by overexpression of a constitutively active TGF-β receptor type I. Adverse events were assessed by clinical monitoring, pathological evaluation and quantification of Lgr5-positive intestinal stem cells.. Inhibition of Hedgehog, Wnt and Notch signalling dose-dependently ameliorated bleomycin-induced and active TGF-β receptor type I-induced fibrosis. Combination therapies with low doses of Hedgehog/Wnt inhibitors or Hedgehog/Notch inhibitors demonstrated additive antifibrotic effects in preventive as well as in therapeutic regimes. Combination therapies were well tolerated. In contrast with high dose monotherapies, combination therapies did not reduce the number of Lgr5 positive intestinal stem cells.. Combined inhibition of morphogen pathways exerts additive antifibrotic effects. Combination therapies are well tolerated and, in contrast to high dose monotherapies, may not impair stem cell renewal. Combined targeting of morphogen pathways may thus help to overcome dose-limiting toxicity of Hedgehog, Wnt and Notch signalling.

Topics: Amyloid Precursor Protein Secretases; Animals; Bleomycin; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Drug Therapy, Combination; Fibrosis; Hedgehog Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Protein Serine-Threonine Kinases; Pyrimidinones; Receptor, Transforming Growth Factor-beta Type I; Receptors, Notch; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Veratrum Alkaloids; Wnt Proteins; Wnt Signaling Pathway

Since information regarding the effects of pH on the extent of nifekalant-induced repolarization delay and torsades de pointes remains limited, we assessed it with a Langendorff heart model of guinea pigs. First, we investigated the effects of pH change from 7.4 to 6.4 on the bipolar electrogram simulating surface lead II ECG, monophasic action potential (MAP), effective refractory period (ERP), and terminal repolarization period (TRP) and found that acidic condition transiently enhanced the ventricular repolarization. Next, we investigated the effects of pH change from 6.4 to 7.4 in the presence of nifekalant (10 μM) on the ECG, MAP, ERP, TRP, and short-term variability (STV) of MAP90 and found that the normalization of pH prolonged the MAP90 and ERP while the TRP remained unchanged, suggesting the increase in electrical vulnerability of the ventricle. Meanwhile, the STV of MAP90 was the largest at pH 6.4 in the presence of nifekalant, indicating the increase in temporal dispersion of repolarization, which gradually decreased with the return of pH to 7.4.Thus, a recovery period from acidosis might be more dangerous than during the acidosis, because electrical vulnerability may significantly increase for this period while temporal dispersion of repolarization remained increased.

Topics: Acidosis; Action Potentials; Animals; Anti-Arrhythmia Agents; Disease Models, Animal; Electrocardiography; Guinea Pigs; Humans; Hydrogen-Ion Concentration; Male; Perfusion; Pyrimidinones; Time Factors; Torsades de Pointes

Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of preterm infants. The development of pulmonary hypertension (PH) significantly increases the mortality and morbidity of this disease. β-Catenin signaling plays an important role in tissue development and remodeling. Aberrant β-catenin signaling is associated with clinical and experiment models of BPD. To test the hypothesis that inhibition of β-catenin signaling is beneficial in promoting alveolar and vascular development and preventing PH in experimental BPD, we examined the effects of ICG001, a newly developed pharmacological inhibitor of β-catenin, in preventing hyperoxia-induced BPD in neonatal rats. Newborn rat pups were randomized at postnatal day (P)2 to room air (RA) + DMSO (placebo), RA + ICG001, 90% FiO2 (O2) + DMSO, or O2 + ICG001. ICG001 (10 mg/kg) or DMSO was given by daily intraperitoneal injection for 14 days during continuous exposure to RA or hyperoxia. Primary human pulmonary arterial smooth muscle cells (PASMCs) were cultured in RA or hyperoxia (95% O2) in the presence of DMSO or ICG001 for 24 to 72 hours. Treatment with ICG001 significantly increased alveolarization and reduced pulmonary vascular remodeling and PH during hyperoxia. Furthermore, administering ICG001 decreased PASMC proliferation and expression of extracellular matrix remodeling molecules in vitro under hyperoxia. Finally, these structural, cellular, and molecular effects of ICG001 were associated with down-regulation of multiple β-catenin target genes. These data indicate that β-catenin signaling mediates hyperoxia-induced alveolar impairment and PH in neonatal animals. Targeting β-catenin may provide a novel strategy to alleviate BPD in preterm infants.

Topics: Animals; Animals, Newborn; Apoptosis; beta Catenin; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Bronchopulmonary Dysplasia; Cell Proliferation; Disease Models, Animal; Extracellular Matrix; Fluorescent Antibody Technique; Humans; Hyperoxia; Hypertension, Pulmonary; Immunoenzyme Techniques; Myocytes, Smooth Muscle; Pulmonary Alveoli; Pyrimidinones; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Although extracellular adenosine 5'-triphosphate (eATP) has a crucial role in the sensitization phase of contact hypersensitivity (CHS), the mechanism by which hapten causes keratinocyte cell death and ATP release is unknown. We examined the time course of cell death, reactive oxygen species (ROS) production, and ATP release in HaCaT cells and in normal human keratinocytes after exposure to nonmetal haptens, NiCl2, or irritants. Both haptens and irritants caused cell death of keratinocytes but with different time courses. N-acetylcysteine (NAC) significantly reduced only nonmetal hapten-induced cell death as assessed by propidium iodide exclusion. We examined the effects of antioxidants and pannexin (Panx) inhibitors on cell death, ROS production, and ATP release by chemical-treated HaCaT cells. Nonmetal hapten-induced cell death, but not NiCl2- or irritant-related cell death, was dependent on reactivity to thiol residues in the cells. NAC reduced cell death and ATP release, whereas antioxidants and Panx inhibitors did not inhibit cell death but significantly attenuated ATP release. Panx1 small interfering RNA (siRNA) also suppressed ATP release from hapten-exposed HaCaT cells. Intraperitoneal injection of a Panx1 inhibitor attenuated murine CHS. These findings suggest that nonmetal hapten reactivity to thiol residues causes membrane disruption of keratinocytes and ROS production that leads to ATP release through opening of Panx hemichannels.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Benzopyrans; Cell Death; Cells, Cultured; Connexins; Cystine; Dermatitis, Contact; Dinitrochlorobenzene; Disease Models, Animal; Female; Haptens; Humans; Irritants; Keratinocytes; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Nickel; Protein Structure, Quaternary; Protein Structure, Tertiary; Pyrimidinones; Reactive Oxygen Species; RNA, Small Interfering

Recently, the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified as molecular sensors for cold, and it has been suggested that they play a crucial role in allodynia by modulating voltage-gated calcium channel currents (ICa(V)). The aim of this study was to analyze the modulation of ICa(V) by the TRPM8-agonist icilin in vitro and to investigate the analgesic effect of icilin in a neuropathic pain model in vivo. Whole cell patch-clamp recordings were performed on isolated naïve and injured rat dorsal root ganglia (DRG) neurons, and the analgesic efficacy of icilin applied topically to the paws or intrathecally was tested in rats after spinal nerve ligation (SNL). ICa(V) (depolarization from -80 to 0mV) in naïve DRG neurons was reduced dose dependently (0.002-200µM) by icilin (18-80%). Subtype isolation of calcium channels show a marked reduction of L-type channel currents compared to N-type channel currents. The effects of icilin on ICa(V) were not significantly different in non-injured and SNL-injured DRG neurons. In vivo, neither topical (10-200µM) nor intrathecal application of icilin (0.1nM to 1µM) affected tactile allodynia or thermal hyperalgesia after SNL, but it increases cold allodynia 6h after application. We conclude that the icilin-induced modulation of ICa(V) in DRG neurons is unlikely to mediate analgesic effects or contribute directly to the pathogenesis of cold allodynia in the rat SNL model, but it is a potential mechanism for the analgesic effects of icilin in other pain models.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, N-Type; Cells, Cultured; Cold Temperature; Disease Models, Animal; Dose-Response Relationship, Drug; Ganglia, Spinal; Hyperalgesia; Ligation; Male; Membrane Potentials; Neuralgia; Neurons; Patch-Clamp Techniques; Peripheral Nerve Injuries; Pyrimidinones; Rats; Rats, Wistar; Spinal Nerves; TRPM Cation Channels

Vasopressin V1B receptor antagonists may be effective for the treatment of depression and anxiety and the objective of this study was to characterize the pharmacological profiles of two newly synthesized arginine vasopressin receptor 1B (V1B receptor) antagonists, TASP0233278 and TASP0390325.. We investigated the in vitro profiles of TASP0233278 and TASP0390325. In addition, the effect of TASP0390325 on the increase in plasma adrenocorticotropic hormone (ACTH) levels induced by corticotropin-releasing factor (CRF)/desmopressin (dDAVP) was investigated. We also investigated the antidepressant and anxiolytic profiles of TASP0233278 and TASP0390325 in animal models.. Both TASP0233278 and TASP0390325 showed a high affinity and potent antagonist activity for V1B receptors. Oral administration of TASP0390325 antagonized the increase in plasma ACTH levels induced by CRF/dDAVP in rats, indicating that TASP0390325 blocks the anterior pituitary V1B receptor in vivo. Oral administration of TASP0233278 or TASP0390325 also exerted antidepressant effects in two models of depression (a forced swimming test and an olfactory bulbectomy model). Moreover, TASP0233278 improved depressive-like behaviour induced by repeated treatment with corticosterone, a model that has been shown to be resistant to treatment with currently prescribed antidepressants. In addition to depression models, TASP0233278 or TASP0390325 exerted anxiolytic effects in several anxiety models (social interaction, elevated plus-maze, stress-induced hyperthermia, separation-induced ultrasonic vocalization and sodium lactate-induced panic-like responses in panic-prone rats).. TASP0233278 and TASP0390325 are potent and orally active V1B receptor antagonists with antidepressant and anxiolytic activities in rodents.

Topics: Administration, Oral; Animals; Anti-Anxiety Agents; Antidepressive Agents; Antidiuretic Hormone Receptor Antagonists; CHO Cells; Corticosterone; Cricetulus; Depression; Disease Models, Animal; Humans; Indoles; Male; Mice; Proline; Pyridines; Pyrimidinones; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Vasopressin

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

Topics: Administration, Oral; Alternative Splicing; Animals; Cells, Cultured; Coumarins; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Isocoumarins; Longevity; Mice; Muscular Atrophy, Spinal; Pyrimidinones; RNA, Messenger; Sequence Deletion; Small Molecule Libraries; Survival of Motor Neuron 2 Protein

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS-rapidly accelerated fibrosarcoma kinase (RAF)-MEK-ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Disease Models, Animal; Drug Therapy, Combination; ErbB Receptors; Humans; Male; Mitogen-Activated Protein Kinases; Mutation; Pancreatic Neoplasms; Panitumumab; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Receptor, ErbB-2; Signal Transduction; Trastuzumab; Tumor Burden; Xenograft Model Antitumor Assays

Treatment of myocardial infarction within the first 1 to 2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbβ3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbβ3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly by autoinjector to facilitate its use in the prehospital setting. Here, we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models.. RUC-4 was ≈ 20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60-80 mg/mL). It shared RUC-2's specificity for αIIbβ3 versus αVβ3, did not prime the receptor to bind fibrinogen, or induce changes in β3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human but not mouse platelets. Intramuscular injection of RUC-4 in nonhuman primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5 to 24 hours.. RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a prehospital therapy of myocardial infarction, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.

Topics: Animals; Binding Sites; Blood Platelets; Carotid Stenosis; Chlorides; Disease Models, Animal; Emergency Medical Services; Ferric Compounds; Fibrinolytic Agents; Humans; Macaca fascicularis; Male; Mice; Mice, Transgenic; Molecular Dynamics Simulation; Myocardial Infarction; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Protein Conformation; Pyrimidinones; Solubility; Thiadiazoles; Thrombosis; von Willebrand Factor

Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.

Topics: Animals; Biological Transport; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Female; Glycolysis; Humans; Lactates; Metabolomics; Monocarboxylic Acid Transporters; Neoplasms; Oxidative Stress; Pyrimidinones; Radiation Tolerance; Symporters; Thiophenes; Tumor Burden; Xenograft Model Antitumor Assays

Fibrosis is a major socioeconomic burden, but effective antifibrotic therapies are not available in the clinical routine. There is growing evidence for a central role of Wnt signalling in fibrotic diseases such as systemic sclerosis, and we therefore evaluated the translational potential of pharmacological Wnt inhibition in experimental dermal fibrosis.. We examined the antifibrotic effects of PKF118-310 and ICG-001, two novel inhibitors of downstream canonical Wnt signalling, in the models of prevention and treatment of bleomycin-induced dermal fibrosis as well as in experimental dermal fibrosis induced by adenoviral overexpression of a constitutively active transforming growth factor (TGF)-β receptor I.. PKF118-310 and ICG-001 were well tolerated throughout all experiments. Both therapeutic approaches showed antifibrotic effects in preventing and reversing bleomycin-induced dermal fibrosis as measured by skin thickness, hydroxyproline content and myofibroblast counts. PKF118-310 and ICG-001 were effective in inhibiting TGF-β receptor I-driven fibrosis as assessed by the same outcome measures.. Blockade of canonical Wnt signalling by PKF118-310 and ICG-001 showed antifibrotic effects in different models of skin fibrosis. Both therapies were well tolerated. Although further experimental evidence for efficacy and tolerability is necessary, inhibition of canonical Wnt signalling is a promising treatment approach for fibrosis.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Fibrosis; Mice; Mice, Inbred DBA; Pyrimidinones; Scleroderma, Systemic; Signal Transduction; Skin; Skin Diseases; Treatment Outcome; Triazines; Wnt Signaling Pathway

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

Topics: Animals; Calcitonin Gene-Related Peptide; Calcium Signaling; Chemokines; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Humans; Inflammation; Inflammation Mediators; Ion Channel Gating; Mice; Mice, Knockout; Pyrimidinones; Trinitrobenzenesulfonic Acid; TRPM Cation Channels; TRPV Cation Channels

The novel antibiotic MBX-500, dosed at 100, 200, or 400 mg/kg twice daily for 7 days, was evaluated for the treatment of Clostridium difficile infection (CDI) in the gnotobiotic pig model. MBX-500 increased survival at all doses and at high doses improved clinical signs and reduced lesion severity, similar to vancomycin. Our results show that MBX-500 is an effective antibiotic for the treatment of diarrhea associated with CDI and prevents severe systemic disease.

Topics: Animals; Anti-Bacterial Agents; Clostridioides difficile; Clostridium Infections; Colon; Diarrhea; Disease Models, Animal; Drug Evaluation, Preclinical; Fluoroquinolones; Germ-Free Life; Kaplan-Meier Estimate; Pyrimidinones; Severity of Illness Index; Swine; Treatment Outcome; Vancomycin

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; DNA Damage; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Humans; Membrane Proteins; Mice; Neoplasms; Nuclear Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Tumor Burden; Xenograft Model Antitumor Assays

Novel thienopyrimidine derivatives of azetidinone possessing the combined features of the cholesterol absorption inhibitor drug ezetimibe and potential antihyperlipidemic 2-substitutedthienopyrimidin-4-ones were synthesized and characterized by spectroscopic data and elemental analysis. These compounds were evaluated for their lipid-lowering activity in Wistar albino rats. Some of them showed significant lipid-lowering effects comparable to those of the standard drug, gemfibrozil, at the same dose levels.

Topics: Animals; Azetidines; Biomarkers; Cholesterol; Disease Models, Animal; Drug Design; Gemfibrozil; Hyperlipidemias; Hypolipidemic Agents; Intestinal Absorption; Intestinal Mucosa; Intestines; Molecular Structure; Polyethylene Glycols; Pyrimidinones; Rats; Rats, Wistar; Structure-Activity Relationship; Triglycerides

The adult mammalian heart has limited capability for self-repair after myocardial infarction. Therefore, therapeutic strategies that improve post-infarct cardiac function are critically needed. The small molecule ICG-001 modulates Wnt signaling and increased the expression of genes beneficial for cardiac regeneration in epicardial cells. Lineage tracing experiments, demonstrated the importance of β-catenin/p300 mediated transcription for epicardial progenitor contribution to the myocardium. Female rats given ICG-001 for 10 days post-occlusion significantly improved ejection fraction by 8.4%, compared to controls (P<0.05). Taken together, Wnt modulation via β-catenin/CBP inhibition offers a promising therapeutic strategy towards restoration of myocardial tissues and an enhancement of cardiac functions following infarction.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Growth Differentiation Factor 15; Muscle Contraction; Myoblasts, Cardiac; Myocardial Infarction; Myocardium; Paracrine Communication; Pericardium; Proto-Oncogene Proteins c-kit; Pyrimidinones; Rats; Regeneration; Wnt Signaling Pathway

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; Naphthalenes; Pyrimidinones; Sirtuin 1; Transplantation, Heterologous

The term "stress-related mucosal disease" (SRMD) represents conditions ranging from superficial mucosal damage to focal deep mucosal damage in the stomach, of which pathogenesis is deduced to be violent mucosal ischemia or excess oxidative stress, but not fully clarified yet. Under the hypothesis that mucosal cell apoptosis subsequent to endoplasmic reticulum (ER) stress might play a crucial role, we evaluated the efficacy and mechanism that novel acid pump antagonist (APA), revaprazan, alleviated water immersion restraint stress (WIRS) induced SRMD in rats.. In order to define whether WIRS-induced SRMD is associated with ER stress, we checked the alteration in the expression of ER stress markers including GRP78, CHOP, XBP-1, BiP as well as apoptosis in WIRS-induced SRMD. The efficacy of revaprazan on either alleviating ER stress or attenuating SRMD was compared with proton pump inhibitor (PPI) and gastroprotectant.. Ten hours of WIRS induced a severe degree of SRMD, in which ER stress markers including CHOP, XBP1, and BiP were significantly overexpressed in the gastric tissues. However, these markers of ER stress were significantly decreased in the group pretreated with revaprazan compared to PPI or gastroprotectant, accompanied with a significant reduction in apoptotic index. In addition to ER stress, revaprazan imposed anti-inflammatory benefit to limit SRMD based on significant levels of inflammatory cell apoptosis.. Endoplasmic reticulum stress accompanied with drastic apoptosis was implicated in the development of SRMD, but revaprazan could rescue the stomach from SRMD through alleviating ER stress in epithelial cells much better than either PPI or gastroprotectant.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cell Line; Cytoprotection; Disease Models, Animal; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gastric Mucosa; Heat-Shock Proteins; Immersion; Inflammation Mediators; Metabolic Detoxication, Phase II; Mice; Proton Pump Inhibitors; Pyrimidinones; Rats; Rats, Wistar; Regulatory Factor X Transcription Factors; Severity of Illness Index; Stomach Diseases; Stress, Physiological; Stress, Psychological; Tetrahydroisoquinolines; Time Factors; Transcription Factor CHOP; Transcription Factors; X-Box Binding Protein 1

Multiple system atrophy (MSA) is a rare and fatal α-synucleinopathy characterized by a distinctive oligodendrogliopathy with glial cytoplasmic inclusions and associated neuronal multisystem degeneration. The majority of patients presents with a rapidly progressive parkinsonian disorder and atypical features such as early autonomic failure and cerebellar ataxia. We have previously reported that complete MSA pathology can be modeled in transgenic mice overexpressing oligodendroglial α-synuclein under conditions of oxidative stress induced by 3-nitropropionic acid (3-NP) including striatonigral degeneration, olivopontocerebellar atrophy, astrogliosis, and microglial activation. Here, we show that myeloperoxidase (MPO), a key enzyme involved in the production of reactive oxygen species by phagocytic cells, is expressed in both human and mouse MSA brains. We also demonstrate that in the MSA mouse model, MPO inhibition reduces motor impairment and rescues vulnerable neurons in striatum, substantia nigra pars compacta, cerebellar cortex, pontine nuclei, and inferior olives. MPO inhibition is associated with suppression of microglial activation but does not affect 3-NP induced astrogliosis in the same regions. Finally, MPO inhibition results in reduced intracellular aggregates of α-synuclein. This study suggests that MPO inhibition may represent a novel candidate treatment strategy against MSA-like neurodegeneration acting through its anti-inflammatory and anti-oxidative properties.

Topics: Aged; alpha-Synuclein; Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gliosis; Humans; Male; Mice; Mice, Transgenic; Microglia; Middle Aged; Motor Activity; Multiple System Atrophy; Nerve Degeneration; Peroxidase; Pyrimidinones; Pyrroles

The development of early diagnostic and prognostic tools for the visualization of amyloid-β (Aβ) deposits is one important focus of current imaging research. In patients with Alzheimer's disease (AD), non-invasive and efficient detection of soluble and aggregated Aβ is important to determine the immediate success of intervention trails. The novel near infrared-fluorescence (NIRF) probe THK-265 efficiently penetrates the blood-brain barrier and has a strong and efficient binding to cerebral Aβ. Ex vivo microscopy of i) THK-265-labeling of plaques in paraffin-embedded tissue and ii) cerebral cryo-sections after intravenous injection of THK-265 confirmed a systematic increase of the NIRF signal corresponding to Aβ plaque number and size during disease progression. Furthermore, we investigated different stages of plaque formation in amyloid-β protein precursor transgenic mice in vivo after intravenous application of THK-265 to evaluate different aggregation levels with NIRF signals. The intensity of the NIRF signal correlated well with the plaque burden, indicating its utility for direct monitoring of Aβ aggregation progression. In summary, our results support the use of the NIRF probe THK-265 for the diagnosis and direct visualization of amyloid deposits and open the possibility for efficient, pre-symptomatic monitoring of Aβ deposition in the aging brain.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; Disease Models, Animal; Disease Progression; Fluorescent Dyes; Mice; Mice, Transgenic; Plaque, Amyloid; Pyrimidinones; Thiones

Our aim was to differentiate human (h) embryonic stem (ES) cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI) and type II (AEII) cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF). A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB) differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.. Omission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP) transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC(-/-) mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.. This study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.

Topics: Animals; Bleomycin; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Lineage; Collagen; Disease Models, Animal; Embryonic Stem Cells; Gene Expression Regulation; Humans; Lung; Mice; Phenotype; Pulmonary Fibrosis; Pyrimidinones; Stem Cell Transplantation; Transplantation, Heterologous

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.. To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118-310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118-310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118-310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118-310 treated mice were unable to seed the growth of secondary tumors after transplant.. These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.

Topics: Animals; beta Catenin; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Female; Gene Expression Profiling; Mammary Glands, Animal; Mice; Neoplastic Stem Cells; Pyrimidinones; Receptor, ErbB-2; Triazines; Wnt Proteins; Wnt Signaling Pathway

Agents with α-2 adrenoreceptor (AR) agonistic action have reportedly suppressed tachyarrhythmias..  We hypothesized that α-2 AR agonists would have an inhibitory effect on abnormal repolarization-related ventricular tachyarrhythmias (VTs). To test this hypothesis, the effects of 2 clinically available α-2 AR agonists (dexmedetomidine and clonidine) on the occurrence of VTs were assessed in a methoxamine-sensitized rabbit model of acquired long QT syndrome (Study 1: n=45). In control rabbits, administration of methoxamine and nifekalant almost invariably caused VTs (14/15). In contrast, incidence of VT significantly decreased during the treatment with dexmedetomidine (1μg·kg(-1)·min(-1): 5/12 [P<0.01 vs. control]) or with clonidine (33.3μg·kg(-1)·min(-1): 10/18 [P<0.01]). To verify that VTs in this animal model are triggered by early afterdepolarization (EAD), the monophasic action potential on the left ventricular surface was recorded in 28 open-chest rabbits (Study 2). EAD-like hump was less frequently detected during treatment with clonidine or dexmedetomidine (2/14) than in saline-treated rabbits (9/10, P<0.005). Presence of a hump was significantly related to the advent of VTs (P<0.05)..  Agents with α-2 AR agonistic action have an inhibitory effect on VTs in a rabbit model of long QT syndrome. Alpha-2 AR agonists, especially dexmedetomidine, may be a therapeutic choice for abnormal repolarization-related VTs that are resistant to conventional treatment. 

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-2 Receptor Agonists; Animals; Anti-Arrhythmia Agents; Clonidine; Dexmedetomidine; Disease Models, Animal; Heart Conduction System; Humans; Long QT Syndrome; Methoxamine; Pyrimidinones; Rabbits; Tachycardia

High throughput screening identified the pyridothienopyrimidinone 1 as a ligand for the metabotropic glutamate receptor 1 (mGluR1=10 nM). Compound 1 has an excellent in vivo profile; however, it displays unfavorable pharmacokinetic issues and metabolic stability. Therefore, using 1 as a template, novel analogues (10i) were prepared. These analogues displayed improved oral exposure and activity in the Spinal Nerve Ligation (SNL) pain model.

Topics: Administration, Oral; Animals; Chronic Pain; Disease Models, Animal; Heterocyclic Compounds, 3-Ring; Humans; Pyrimidinones; Rats; Receptors, Metabotropic Glutamate; Structure-Activity Relationship; Thiophenes

Anesthesia sometimes suppresses ventricular tachyarrhythmias (VT) resistant to conventional pharmacological treatment.. To know (1) whether deep anesthesia inhibits abnormal repolarization-related VT and (2) if α2-adrenoreceptor (AR) agonistic action is associated with the antiarrhythmic effect of anesthetics, the incidence of VT in a rabbit model of acquired long QT syndrome using different anesthetic regimen was assessed. In Study 1 (n = 30), 15 rabbits were lightly anesthetized with ketamine (123 ± 46 mg/kg) and an α2-AR agonist, xylazine (9.4±3.0mg/kg), while combination of these anesthetics at high doses were used in the other 15 rabbits (343 ± 78 mg/kg and 38.9 ± 3.0 mg/kg). Administration of α1-AR stimulant, methoxamine and nifekalant (Ikr blocker) caused VT in all lightly anesthetized rabbits. In contrast, VT was observed only in 1 of the 15 deeply anesthetized rabbits (P < 0.01). In Study 2 (n = 15), 10 rabbits were anesthetized with high-dose ketamine and low-dose xylazine. In the other 5 rabbits, low-dose ketamine and high-dose xylazine were used. QTc interval in the latter was longer than that of the former (399 ± 56 ms vs. 494 ± 57 ms, P < 0.01). Although no VT appeared in high/low-rabbits, VT occurred in 3 out of 5 low/high-rabbits (P < 0.05).. These results suggest that (1) deep anesthesia suppresses abnormal repolarization-related VT and (2) antiarrhythmic effect of anesthesia on this type of VT is not dependent on α2-AR agonistic action.

Topics: Adrenergic alpha-2 Receptor Agonists; Anesthesia, General; Anesthetics, Combined; Animals; Anti-Arrhythmia Agents; Blood Pressure; Disease Models, Animal; Dose-Response Relationship, Drug; Electrocardiography; Ketamine; Long QT Syndrome; Male; Methoxamine; Pyrimidinones; Rabbits; Tachycardia, Ventricular; Time Factors; Xylazine

Hypertension is the most common comorbidity and major risk factor in patients with erectile dysfunction. The pharmacokinetics of mirodenafil, used for the treatment of erectile dysfunction, after the intravenous and oral administration (20 mg/kg) to 6-week-old rats (with blood pressure within the normotensive range) and 16-week-old spontaneously hypertensive rats (SHRs) and their age-matched control normotensive Kyoto-Wistar (KW) rats, and 16-week-old deoxycorticosterone acetate-salt-induced hypertensive rats (DOCA-salt rats) and their age-matched control Sprague-Dawley (SD) rats were compared. It was found that time-averaged renal clearance (Cl(r)) was of minor importance and that time-averaged non-renal clearance (Cl(nr)) was dominant. In both 6- and 16-week-old SHRs, the Cl(nr)s and areas under the curve (AUCs) of intravenous mirodenafil were significantly smaller and greater than those of the controls, but in 16-week-old DOCA-salt rats, they were comparable to the controls. Although the AUC of oral mirodenafil in 16-week-old SHRs was comparable to the controls, the Cl(nr)s (or total body clearances, Cls) of intravenous mirodenafil and intestinal intrinsic clearances were significantly smaller than the controls and comparable to the controls for both 6- and 16-week-old SHRs, unlike in the 16-week-old DOCA-salt rats. The above data suggest that the significantly smaller Cl(nr) and greater AUC of intravenous mirodenafil and comparable AUC of oral mirodenafil in 16-week-old SHR could be due to the hereditary characteristics of SHRs, and not due to the hypertensive state itself.

Topics: Animals; Desoxycorticosterone; Disease Models, Animal; Hypertension; Male; Pyrimidinones; Random Allocation; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Sulfonamides

Icilin is a transient receptor potential cation channel subfamily M (TRPM8) agonist that produces behavioral activation in rats and mice. Its hallmark overt pharmacological effect is wet-dog shakes (WDS) in rats. The vigorous shaking associated with icilin is dependent on NMDA receptor activation and nitric oxide production, but little else is known about the biological systems that modulate the behavioral phenomenon. The present study investigated the hypothesis that alpha(2)-adrenoceptor activation inhibits icilin-induced WDS. Rats injected with icilin (0.5, 1, 2.5, 5mg/kg, i.p.) displayed dose-related WDS that were inhibited by pretreatment with a fixed dose of clonidine (0.15 mg/kg, s.c.). Shaking behavior caused by a fixed dose (2.5mg/kg) of icilin was also inhibited in a dose-related manner by clonidine pretreatment (0.03-0.15 mg/kg, s.c.) and reduced by clonidine posttreatment (0.15 mg/kg, s.c.). Pretreatment with a peripherally restricted alpha(2)-adrenoceptor agonist, ST91 (0.075, 0.15 mg/kg), also decreased the incidence of shaking elicited by 2.5mg/kg of icilin. Pretreatment with yohimbine (2mg/kg, i.p.) enhanced the shaking induced by a low dose of icilin (0.5mg/kg). The imidazoline site agonists, agmatine (150mg/kg, i.p.) and 2-BFI (7 mg/kg, i.p.), did not affect icilin-evoked shaking. These results suggest that alpha(2)-adrenoceptor activation inhibits shaking induced by icilin and that increases in peripheral, as well as central, alpha(2)-adrenoceptor signaling oppose the behavioral stimulant effect of icilin.

Topics: Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Agmatine; Analysis of Variance; Animals; Behavior, Animal; Clonidine; Disease Models, Animal; Dose-Response Relationship, Drug; Head Movements; Male; Movement Disorders; Pyrimidinones; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Time Factors; Yohimbine

Published evidence suggests that phosphoinositide 3 kinase-β (PI3K-β) plays an important role in platelet aggregation and shear activation. TGX-221 is a selective PI3K-β inhibitor with a good separation of anti-thrombotic efficacy and bleeding (therapeutic index) in rats. Our goal was to further evaluate potential of a PI3K-β inhibitor as an anti-thrombotic agent by determining the therapeutic index in another species and efficacy model. Reported effects of TGX-221 in rats were also confirmed.. TGX-221 (0.3 + 0.3, 1 + 1, 3 + 3 mg/kg + mg/kg/hr, i.v.) or vehicle was given to mice starting 15 min prior to FeCl(3) arterial thrombosis (AT), tail or kidney bleeding time (BT) procedures.. Integrated blood flow over 30 min (%baseline mean ± SEM) improved (p < 0.05) with TGX-221 doses 1 + 1 (49 ± 13.9%) and 3+3 (88 ± 10.6%) versus 0.3 + 0.3 (10 ± 0.8%) and vehicle (10 ± 0.6%). Vascular patency (non-occluded/total arteries) improved (p < 0.01) with TGX-221 doses of 3 + 3 (7/8), but not 0.3 + 0.3 (0/8) or 1 + 1 (4/8) versus vehicle (0/8). Tail BT (sec) increased (p < 0.05) with TGX-221 doses of 3 + 3 (median 1560) and 1 + 1 (1305) versus vehicle (225). Mean renal BT (sec) increased (p < 0.05) in all TGX-221 groups (3 + 3: 510 + 26; 1 + 1: 478 + 41; 0.3 + 0.3: 246 + 37) versus vehicle (123 + 9). For comparison, a reference agent, aspirin (30 mpk, i.p.) increased tail BT 1.9X and renal BT 2.6X.. The novel finding of a clear impact on hemostasis by TGX-221 was demonstrated by increased bleeding in two models in mice at anti-thrombotic doses. The results suggest a narrower therapeutic index for this PI3K-β inhibitor than previously recognized, at least for this species.

Topics: Animals; Bleeding Time; Blood Platelets; Carotid Artery Thrombosis; Disease Models, Animal; Fibrinolytic Agents; Hemorrhage; Humans; Kidney; Male; Mice; Mice, Inbred C57BL; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyrimidinones; Rats; Rats, Sprague-Dawley; Tail

Numerous changes occur during aging and Alzheimer's disease (AD) progression, including a decline in cholinergic functioning and cognition, as well as alterations in gene expression and activity in the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Donepezil, the current standard of care for Alzheimer's disease, improves cholinergic functioning and has demonstrated effects on multiple domains of cognition, including memory and attention in both preclinical species and patients. We previously found that increasing activation of the NO/cGMP pathway via phosphodiesterase 9 (PDE9) inhibition also improves memory in rodents and suggested that PDE9 might be a promising target for novel treatments for AD. Here we investigated whether PDE9 inhibition also enhances attention using a novel attention task in rats. We validated this task using several pharmacological manipulations and showed that the selective PDE9 inhibitor PF-04447943 produced effects similar to those of donepezil. These data confirm and extend the hypothesis that PDE9 inhibition might serve as a novel treatment for AD and age-related cognitive decline.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cognition Disorders; Disease Models, Animal; Male; Nootropic Agents; Phosphodiesterase Inhibitors; Pyrazoles; Pyrimidinones; Rats; Rats, Wistar; Scopolamine

Kidney fibrosis, a typical characteristic of chronic renal disease, is associated with tubular epithelial cell apoptosis. The results of our recent studies have shown that Omi/HtrA2 (Omi), a proapoptotic mitochondrial serine protease, performs a crucial function in renal tubular epithelial apoptotic cell death in animal models of acute kidney injury, including cisplatin toxicity and ischemia-reperfusion insult. However, the role of Omi in tubulointerstitial disease-associated fibrosis in the kidney remains to be clearly defined. We evaluated the potential function and molecular mechanism of Omi in ureteral obstruction-induced kidney epithelial cell apoptosis and fibrosis. The mice were subjected to unilateral ureteral obstruction (UUO) via the ligation of the left ureter near the renal pelvis. UUO increased the protein level of Omi in the cytosolic fraction of the kidney, with a concomitant reduction in the mitochondrial fraction. UUO reduced the X-linked inhibitor of apoptosis protein (XIAP), a substrate of Omi, and pro-caspase-3, whereas it increased cleaved poly(ADP-ribose) polymerase (cleaved PARP) and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells. When mice were treated with ucf-101, an inhibitor of the proteolytic activity of Omi (6.19 microg/day ip), on a daily basis beginning 2 days before UUO and continuing until the end of the experiment, the Omi inhibitor protected XIAP cleavage after UUO and reduced the increment of PARP cleavage and the numbers of TUNEL-positive cells. Furthermore, the Omi inhibitor significantly attenuated UUO-induced increases in fibrotic characteristics in the kidney, including the atrophy and dilation of tubules, expansion of the interstitium, and increases in the expression of collagens, alpha-smooth muscle actin, and fibronectin. In conclusion, Omi/HtrA2 is associated with apoptotic signaling pathways in tubular epithelial cells activated by unilateral ureteral obstruction, thereby resulting in kidney fibrosis.

Topics: Actins; Animals; Apoptosis; Caspase 3; Disease Models, Animal; Epithelial Cells; Fibrosis; High-Temperature Requirement A Serine Peptidase 2; In Situ Nick-End Labeling; Kidney Diseases; Kidney Tubules; Male; Mice; Mice, Inbred BALB C; Mitochondria; Mitochondrial Proteins; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Pyrimidinones; Serine Endopeptidases; Signal Transduction; Thiones; Time Factors; Ureteral Obstruction; X-Linked Inhibitor of Apoptosis Protein

To evaluate the value of the hepatitis B virus (HBV) replication mouse model with regard to several aspects of the study of HBV biology.. To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents, the interferon inducer polyinosinic-polytidylin acid (polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1, and the inhibiting effect of these agents on HBV DNA replication was evaluated. To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant, telbivudine resistance mutants (rtM204I, ayw subtype), adefovir resistance mutants (rtA181V + rtN236T, ayw subtype) and HBx-minus mutants were injected respectively, and their corresponding HBV DNA replication intermediates in mouse liver were assessed.. Compared with the wild type HBV replication mouse model without antiviral agent treatment, the HBV DNA replication intermediates of the polyIC-treated group were decreased 1-fold; while in the entecavir- and adefovir-treated groups, the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold, respectively. For the mouse models injected with telbivudine resistance mutant, adefovir resistance mutant and HBx-minus mutant, HBV DNA replication intermediates could still be detected, but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold, 5.6-fold and 2.9-fold respectively, compared with the mouse model with wild type HBV plasmid.. The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.

Topics: Animals; Antiviral Agents; Disease Models, Animal; Drug Resistance, Viral; Enzyme-Linked Immunosorbent Assay; Hepatitis B; Hepatitis B virus; Humans; Liver; Male; Mice; Mice, Inbred BALB C; Mutation; Nucleosides; Pyrimidinones; Telbivudine; Thymidine; Virus Replication

To compare the efficacy of nifekalant and amiodarone in the treatment of cardiac arrest in a porcine model.. After 4min of untreated ventricular fibrillation, animals were randomly treated with nifekalant (2mgkg(-1)), amiodarone (5mgkg(-1)) or saline placebo (n=12 pigs per group). Precordial compression and ventilation were initiated after drug administration and defibrillation was attempted 2min later. Hemodynamics were continuously measured for 6h after successful resuscitation.. Compared with saline, nifekalant and amiodarone equally decreased the number of electric shocks, defibrillation energy, epinephrine dose, and duration of cardiopulmonary resuscitation required for successful resuscitation (P<0.01). The incidence of restoration of spontaneous circulation (ROSC) and the 24-h survival rate were higher in both antiarrhythmic drug groups (P<0.05) vs. the saline group. Furthermore, post-resuscitation myocardial dysfunction at 4-6h after successful resuscitation was improved in animals given antiarrhythmic drugs as compared with the saline group (P<0.05). There were no differences between nifekalant and amiodarone for any of these parameters.. The effect of nifekalant was similar to that of amiodarone for improving defibrillation efficacy and for the treatment of cardiac arrest. Administration of either nifekalant or amiodarone before defibrillation increased the ROSC and 24-h survival rates and improved post-resuscitation cardiac function in this porcine model.

Topics: Amiodarone; Animals; Anti-Arrhythmia Agents; Cardiopulmonary Resuscitation; Disease Models, Animal; Dose-Response Relationship, Drug; Heart Arrest; Heart Rate; Male; Pyrimidinones; Swine; Treatment Outcome; Ventricular Fibrillation; Ventricular Function

Thrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Topics: Administration, Oral; Animals; Anticoagulants; Antithrombins; Azetidines; Benzylamines; Blood Coagulation; Disease Models, Animal; Dose-Response Relationship, Drug; Factor V; Factor Xa; Factor Xa Inhibitors; Feedback, Physiological; Hemorrhage; Humans; Male; Phospholipids; Prothrombin Time; Pyrimidinones; Rats; Rats, Sprague-Dawley; Risk Assessment; Sulfones; Thrombin; Thromboplastin; Venous Thrombosis

Many observations report the cardioprotective effects of inhibitors of aldose reductase in different models of ischaemia-reperfusion injury in diabetic myocardium. In this paper, the inhibitory effects of the new pyrido[1,2-a]-pyrimidin-4-one derivative PPO, whose aldose reductase-inhibitory and antioxidant effects were shown in a previous study, were evaluated.. The effect of PPO was evaluated on aldose reductase from hearts of diabetic and non-diabetic rats, and compared with that of the reference drug epalrestat. Moreover, the two drugs were tested on isolated and Langendorff-perfused diabetic and non-diabetic hearts submitted to ischaemia-reperfusion cycle.. Epalrestat showed equivalent levels of potency in inhibiting the activity of the enzyme in the diabetic and in the non-diabetic hearts. On the contrary, the inhibitory potency of PPO was decreased in the diabetic organs. In the diabetic hearts submitted to ischaemia-reperfusion, an increased level of heart aldose reductase activity was recorded, and both PPO and epalrestat produced cardioprotective effects, suggesting that aldose reductase is deeply involved in the process of ischaemia-reperfusion injury in diabetic myocardium. In non-diabetic hearts, where aldose reductase has a lower activity, epalrestat failed to produce significant protection, while PPO still maintained cardioprotective effects, which may be reasonably attributed to useful 'ancillary' effects - such as antioxidant activity - independent from the aldose reductase inhibition.. Therefore PPO, a new molecule endowed with both aldose reductase-inhibitory effects and antioxidant activity, may represent the prototype of a new class of multitarget drugs, focused on two different steps deeply involved in the pathogenesis of ischaemic injury of diabetic hearts.

Topics: Aldehyde Reductase; Animals; Antioxidants; Cardiotonic Agents; Diabetes Mellitus, Experimental; Disease Models, Animal; Enzyme Inhibitors; Male; Myocardial Reperfusion Injury; Myocardium; Pyridines; Pyrimidinones; Rats; Rats, Wistar; Rhodanine; Thiazolidines

The mammalian neuropeptide, melanin-concentrating hormone, interacts with two G protein-coupled receptors, melanin-concentrating hormone receptor (MCHR) 1 and MCHR2; however, only MCHR1 is expressed in rats and mice. In the present study, we evaluated MCHR1 antagonism in preclinical models believed to be predictive of antiobesity and antidepressant activity. Central activity of the selective MCHR1 antagonist, GW803430 [6-(4-chloro-phenyl)-3-[3-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-3H-thieno[3,2-d]pyrimidin-4-one], was evaluated using ex vivo binding with autoradiography. Effective doses of GW803430 (1 and 3 mg/kg p.o.) were correlated with antiobesity activity in a 14-day study of diet-induced obese rats. GW803430 was evaluated subsequently for antidepressant-like effects in mice and rats. Acute and subchronic administration reduced immobility time in the mouse forced-swim test at doses of 3 (acute) and 3 and 10 (chronic) mg/kg p.o., an effect that was absent in MCHR1(-/-) mice. Combined subeffective doses of GW803430 (0.3 and 1 mg/kg p.o.) and imipramine (5 mg/kg) produced a robust antidepressant-like response. The compound was also active in the tail suspension test at a dose of 10 mg/kg p.o. GW803430 (30 mg/kg p.o.) significantly reduced submissive behaviors at weeks 2 and 3, a model of submissive behavior that may predict antidepressant onset. GW803430 decreased marble burying in mice at doses of 3, 10, and 30 mg/kg p.o., an assay that detects anxiolytic-like effects. Thus, GW803430 produces robust antiobesity and antidepressant-like effects in rats and mice at doses that compete for central MCHR1 in vivo. As such, MCHR1 should be considered as a promising target for future drug discovery efforts.

Topics: Animals; Anti-Obesity Agents; Antidepressive Agents; Autoradiography; Behavior, Animal; Body Weight; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Male; Mice; Mice, Knockout; Motor Activity; Obesity; Protein Binding; Pyrimidinones; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Receptors, Somatostatin; Social Dominance; Swimming; Thiophenes

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Bone Resorption; Disease Models, Animal; Inflammation; Osteoclasts; Pharmacokinetics; Protein Kinase Inhibitors; Protein Structure, Tertiary; Pyrimidinones; Rats; Receptor, Macrophage Colony-Stimulating Factor; Structure-Activity Relationship

To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H(2)O(2)-induced oxidative stress in human RPE, in the Ccl2(-)(/)(-)Cx3cr1(-)(/)(-) double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice.. Oxidative stress was induced in ARPE-19 cells by 1 mM H(2)O(2) for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice.. H(2)O(2)-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H(2)O(2)-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi(-)(/)(-) photoreceptors and RPE.. These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Cell Survival; Chemokine CCL2; CX3C Chemokine Receptor 1; Cytosol; Disease Models, Animal; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; High-Temperature Requirement A Serine Peptidase 2; Humans; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Protein Transport; Pyrimidinones; Receptors, Chemokine; Retinal Degeneration; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Serine Endopeptidases; Thiones; X-Linked Inhibitor of Apoptosis Protein

It has been reported that mirodenafil is primarily metabolized via hepatic cytochrome P450 (CYP) 1A1/2, 2B1/2, 2D1 and 3A1/2 in rats. It has also been reported that the protein expression of hepatic CYP3A1 and intestinal CYP1A1 and 3A1/2 increases and that of hepatic CYP2D1 decreases in rats with acute renal failure induced by uranyl nitrate (U-ARF rats). Thus, the pharmacokinetics of mirodenafil were studied in control and U-ARF rats.. The pharmacokinetic parameters of mirodenafil and SK3541 (a metabolite of mirodenafil) were compared after the intravenous and oral administration of mirodenafil at a dose of 20 mg/kg to U-ARF and control rats.. After interavenous administration of mirodenafil to U-ARF rats, the total area under the concentration-time curve (AUC) of mirodenafil was significantly smaller (36.5% decrease) than controls, possibly due to the significantly faster non-renal clearance (66.1% increase; because of increase in the protein expression of hepatic CYP3A1) than controls. After the oral administration of mirodenafil to U-ARF rats, the AUC of mirodenafil was also significantly smaller (47.8% decrease) due to the increase in the protein expression of hepatic CYP3A1 and intestinal CYP1A1 and 3A1/2 compared with controls.. After both intravenous and oral administration of mirodenafil to U-ARF rats, the AUC(SK3541)/AUC(mirodenafil) ratios were comparable with that in controls and this could be due to further metabolism of SK3541 in rats.

Topics: Acute Kidney Injury; Administration, Oral; Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP3A; Disease Models, Animal; Injections, Intravenous; Intestinal Mucosa; Liver; Male; Microsomes; Pyrimidinones; Rats; Rats, Sprague-Dawley; Sulfonamides; Uranyl Nitrate

To examine whether chronic treatment with a type 5 phosphodiesterase inhibitor (PDE5I) could suppress corporal apoptosis via potentiation of Akt signalling in diabetic erectile dysfunction.. Sprague-Dawley rats (12 wk old) were divided into three groups (n=12 in each): normal control, diabetes (DM), and diabetes treated with PDE5I (DM+PDE5I). The rats in the diabetic groups received a single injection of streptozotocin (50mg/kg), and from 8 wk after establishment of diabetes, DM and DM+PDE5I were treated with vehicle and PDE5I (SK-3530, 10mg/kg), respectively, for 4 wk. After 12 wk of streptozotocin injections, six rats in each group underwent cavernosometry with cavernous nerve electrostimulation (2V, 0.2 ms, 50s, 2.5-20 Hz). The penile tissues from the remaining six rats were used for immunohistochemical evaluation of apoptosis, immunoblotting for the phosphorylation of Akt and its downstream molecule Bad, and a colorimetric assay of caspase activity.. Rats in the DM group showed markedly lower erectile parameters than those in the control group, whereas rats in the DM+PDE5I group showed normalized results. Despite persistent hyperglycaemia, PDE5I treatment significantly reduced the mean apoptotic index (39.6+/-4.6 vs. 21.3+/-1.7, p<0.05). Densitometry revealed significantly higher levels of Akt and Bad phosphorylation, implying inhibition of pro-apoptotic stimuli. PDE5I treatment also significantly inhibited the activities of cavernosal caspase 3 and caspase 9, the main effectors of apoptosis.. Chronic treatment with PDE5I activated Akt signalling, which suppressed pro-apoptotic stimuli and maintained erectile function in rat model of diabetic erectile dysfunction.

Topics: Animals; Apoptosis; Diabetes Complications; Disease Models, Animal; Erectile Dysfunction; Male; Muscle, Smooth; Penis; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfones

Topics: Animals; Apoptosis; Diabetes Complications; Disease Models, Animal; Erectile Dysfunction; Male; Muscle, Smooth; Penis; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfones

The clinical use of HIV protease inhibitors is associated with insulin resistance and other metabolic changes that increase long-term cardiovascular risk. Since the failing heart has increased reliance on glucose, the influence of drug exposure on glucose homeostasis, myocardial glucose uptake, cardiac function, and survival was determined in TG9 mice, an established transgenic model of dilated cardiomyopathy generated by cardiac-specific overexpression of Cre-recombinase, as these animals progressed to overt heart failure. Beginning on day of life 75, TG9 mice and nontransgenic littermate controls were given a daily 10 mg/kg intraperitoneal injection of HIV protease inhibitors (ritonavir, lopinavir/ritonavir 4:1, atazanavir, atazanavir/ritonavir 4:1) or vehicle. Glucose tolerance testing, measurement of in vivo myocardial 2-deoxyglucose uptake, and echocardiography were performed before and 30 min following drug administration. The progression of dilated cardiomyopathy in TG9 animals was accompanied by impaired glucose tolerance, which was acutely exacerbated by exposure to ritonavir. Ritonavir and lopinavir precipitated acute, decompensated heart failure and death from pulmonary edema in TG9 mice. However, atazanavir, which does not inhibit glucose transport, had no effect. These studies demonstrate that, in the presence of dilated cardiomyopathy, HIV protease inhibitors that impair glucose transport induce acute, decompensated heart failure. The potential for HIV protease inhibitors to contribute to or exacerbate cardiomyopathy in human patients warrants further investigation.

Topics: Animals; Atazanavir Sulfate; Blood Glucose; Cardiomyopathy, Dilated; Deoxyglucose; Disease Models, Animal; Echocardiography; Glucose Tolerance Test; Glucose Transporter Type 4; Heart Failure; HIV Protease Inhibitors; Humans; Insulin Resistance; Lopinavir; Mice; Myocardium; Oligopeptides; Pulmonary Edema; Pyridines; Pyrimidinones

Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5',4':5,6]pyrimido[1,2- a]benzimidazol-5(6 H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5',4':5,6]pyrimido-[1,2- a]benzimidazol-5(6 H)-one ( 25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis; Benzimidazoles; Crystallography, X-Ray; Disease Models, Animal; Drug Design; Drug Evaluation, Preclinical; Enzyme Activation; Female; Injections, Intradermal; Interleukin-2; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Male; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Structure; Protein Kinase Inhibitors; Pyrimidinones; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Reproducibility of Results; Stereoisomerism; Structure-Activity Relationship; T-Lymphocytes

It is still difficult to manage chronic glomerulonephritis with corticosteroids because of safety concerns, especially for patients with mild symptoms and infants. Therefore, an alternative approach is greatly required. Pemirolast potassium (CAS 100299-08-9) is an antiallergic drug with high safety.. Two glomerulonephritis rat models were prepared to examine the pharmacological actions of pemirolast potassium: one reversible model prepared with the anti-Thy-1 antibody, and another irreversible model by unilateral nephrectomy and with the anti-Thy-1 antibody. Pemirolast potassium was administered to 10 Japanese chronic glomerulonephritis patients concurrently affected by allergic rhinitis in order to examine its efficacy for mild proteinuria.. Pemirolast potassium 1 and 10 mg/kg markedly inhibited proteinuria in the reversible model. In the irreversible model, pemirolast potassium 3 mg/kg showed a significant decrease in the incidence of glomerulosclerosis. In chronic glomerulonephritis patients, pemirolast potassium, 10 mg twice daily, for 6 months, significantly reduced the severity of proteinuria.. Our research suggested the efficacy of pemirolast potassium in glomerulonephritis. A well-controlled study is considered necessary to validate pemirolast potassium as a therapeutic drug for glomerulonephritis.

Topics: Adolescent; Adult; Animals; Anti-Allergic Agents; Autoantibodies; Disease Models, Animal; Female; Glomerulonephritis; Histamine Antagonists; Humans; Kidney; Male; Middle Aged; Nephrectomy; Pilot Projects; Proteinuria; Pyridines; Pyrimidinones; Rats; Rats, Sprague-Dawley; Rats, Wistar; Rhinitis, Allergic, Seasonal; Thyroid Gland

The increase in inward current, primarily L-type Ca2+ current, facilitates torsades de pointes (TdP). Because human atrial natriuretic peptide (ANP) moderates the L-type Ca2+ current, in our study it was hypothesized that ANP counteracts TdP.. We tested the effect of ANP, guanosine 3', 5'-cyclic monophosphate analogue (8-bromo cGMP) and hydralazine on the occurrence of TdP in a rabbit model. In control rabbits, administration of methoxamine and nifekalant almost invariably caused TdP (14/15). In contrast, ANP (10 microg . kg(-1) . min(-1)) markedly abolished TdP (2/15), whereas hydralazine failed to show a comparable anti-arrhythmic action (10/15). TdP occurred only in 1 of 15 rabbits treated with 8-bromo cGMP. Presence of early afterdepolarization-like hump in the ventricular monophasic action potential was associated with the occurrence of TdP.. Results suggest that ANP affects TdP in the rabbit model, and that this anti-arrhythmic effect of ANP is not necessarily shared by other vasodilating agents.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Atrial Natriuretic Factor; Calcium Channels, L-Type; Cyclic GMP; Disease Models, Animal; Electrocardiography; Humans; Hydralazine; Male; Methoxamine; Pyrimidinones; Rabbits; Sympathomimetics; Torsades de Pointes; Vasodilator Agents

Cold allodynia is a poorly understood symptom of neuropathic pain. Two members of the transient receptor potential (TRP) family of proteins, TRPM8 and TRPA1, may contribute to cold somatosensation. The aim of the present study was to investigate the usefulness of icilin as a pharmacological tool to study primary afferent fibre responses to cold stimuli and to determine whether there are differences in the responses of spinal neurones to cooling of peripheral receptive fields in control versus neuropathic rats. The effects of icilin, a TRPM8 and TRPA1 agonist, on intracellular Ca(2+) ([Ca(2+)](i)) responses of small diameter adult dorsal root ganglia (DRG) neurones were determined. Icilin (10 nM-10 microM) produced a concentration-related increase in [Ca(2+)](i) in DRG neurones, which was attenuated by the non-selective TRP channel antagonist ruthenium red (10 microM). In vivo electrophysiology in naïve, sham-operated and SNL rats demonstrated that application of ice to receptive fields evoked firing of wide dynamic range (WDR) neurones, which was significantly greater in SNL rats than naïve and sham-operated rats. Intraplantar injection of icilin did not evoke firing of WDR neurones in naïve, sham-operated or SNL rats but inhibited mechanically-evoked responses of WDR neurones in naïve and sham-operated rats, whilst facilitating mechanically-evoked responses in SNL rats. Icilin increased both innocuous (sham-operated and SNL rats) and noxious (SNL rats) receptive field sizes of WDR neurones. Our data suggests that icilin modulates the mechanosensitivity of dorsal horn neurones. The differing effects of ice and icilin on dorsal horn neurones indicate different mechanisms of action.

Topics: Analysis of Variance; Animals; Ankyrins; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Cold Temperature; Disease Models, Animal; Dose-Response Relationship, Drug; Evoked Potentials; Ganglia, Spinal; Hyperesthesia; Ligation; Male; Mechanoreceptors; Neuralgia; Neurons; Pain Threshold; Pyrimidinones; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Nerves; TRPA1 Cation Channel; TRPC Cation Channels; TRPM Cation Channels

Vascular inflammation and enhanced production of angiotensin II (ANG II) are involved in the pathogenesis of hypertension and diabetes, disease states that predispose the afflicted individuals to ischemic disorders. In light of these observations, we postulated that ANG II may play a role in promoting leukocyte rolling (LR) and adhesion (LA) in postcapillary venules after exposure of the small intestine to ischemia-reperfusion (I/R). Using an intravital microscopic approach in C57BL/6J mice, we showed that ANG II type I (AT(1)) or type II (AT(2)) receptor antagonism (with valsartan or PD-123319, respectively), inhibition of angiotensin-converting enzyme (ACE) with captopril, or calcitonin gene-related peptide (CGRP) receptor blockade (CGRP8-37) prevented postischemic LR but did not influence I/R-induced LA. However, both postischemic LR and LA were largely abolished by concomitant AT(1) and AT(2) receptor blockade or chymase inhibition (with Y-40079). Additionally, exogenously administered ANG II increased LR and LA, effects that were attenuated by pretreatment with a CGRP receptor antagonist or an NADPH oxidase inhibitor (apocynin). Our work suggests that ANG II, formed by the enzymatic activity of ACE and chymase, plays an important role in inducing postischemic LR and LA, effects that involve the engagement of both AT(1) and AT(2) receptors and may be mediated by CGRP and NADPH oxidase.

Topics: Acetophenones; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin II Type 2 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Calcitonin Gene-Related Peptide; Captopril; Cell Adhesion; Chymases; Disease Models, Animal; Endothelial Cells; Imidazoles; Intestines; Ischemia; Leukocyte Rolling; Leukocytes; Male; Mice; Mice, Inbred C57BL; Microscopy, Video; NADPH Oxidases; Pyridines; Pyrimidinones; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Calcitonin Gene-Related Peptide; Reperfusion Injury; Tetrazoles; Valine; Valsartan; Venules

Cold hypersensitivity is a common sensory abnormality accompanying peripheral neuropathies and is difficult to treat. Progress has been made in understanding peripheral mechanisms underlying neuropathic pain but little is known concerning peripheral mechanisms of cold hypersensitivity. The aim of this study was to analyze the contribution of uninjured primary afferents to the cold hypersensitivity that develops in neuropathic rats. Rats with a lumbar 5 (L5) and L6 spinal nerve ligation (SNL, Chung model) but not sham, developed mechanical allodynia, evidenced by decreased paw withdrawal thresholds and increased magnitude of response to von Frey stimulation. Cold hypersensitivity also developed in SNL but not sham rats, evidenced by enhanced nociceptive behaviors induced by placement on a cold plate (6 degrees C) or application of icilin (a transient receptor potential M8 (TRPM8)/transient receptor potential A1 (TRPA1) receptor agonist) to nerve-injured hind paws. Single fiber recordings demonstrated that the mean conduction velocities of intact L4 cutaneous A delta- and C-fibers were not different between naive and SNL rats; however, mechanical thresholds of the A delta- but not the C-fibers were significantly decreased in SNL compared with naive. There was a higher prevalence of C-mechanoheat-cold (CMHC) fibers in SNL compared with naive, but the overall percentage of cold-sensitive C-fibers was not significantly increased compared with naive. This was in contrast to the numerous changes in A delta-fibers: the percentage of L4 cold sensitive A delta-, but not C-fibers, was significantly increased, the percentage of L4 icilin-sensitive A delta-, but not C-fibers, was significantly increased, the icilin-induced activity of L4 A delta-, but not C-fibers, was significantly increased. Icilin-induced activity was blocked by the TRPA1 antagonist Ruthenium Red. The results indicate plasticity in both A delta- and C-uninjured fibers, but A delta fibers appear to provide a major contribution to cold hypersensitivity in neuropathic rats.

Topics: Action Potentials; Analysis of Variance; Animals; Calcium; Cold Temperature; Disease Models, Animal; Dose-Response Relationship, Drug; Ganglia, Spinal; Hyperalgesia; Male; Nerve Fibers; Neural Conduction; Neuronal Plasticity; Neurons, Afferent; Pain Threshold; Peripheral Nervous System Diseases; Physical Stimulation; Pyrimidinones; Rats; Rats, Sprague-Dawley; Reaction Time; Statistics, Nonparametric

Control of the glucose level in the blood plasma has been achieved in vitro and in vivo by administration of vanadium and zinc in form of inorganic salts. It has been shown that elements are poorly absorbed in their inorganic forms and required high doses which have been associated with undesirable side effects. Many researchers, therefore, have focused on metal complexes that were prepared from VOSO(4) or ZnSO(4) and low-molecular-weight bidentate ligands. Seven kinds of 1-hydroxy-4,6-disubstituted and 1-hydroxy-4,5,6-trisubstituted-2(1H)-pyrimidinones were synthesized by reaction of N-benzyloxyurea and beta-diketones and subsequent removal of the protecting group. Six kinds of 1-hydroxy-4-(substituted)amino-2(1H)-pyrimidinones were synthesized by the substitution reaction of 1-benzyloxy-4-(1',2',4'-triazol-1'-yl)-2(1H)-pyrimidinone with various alkyl amines or amino acids. Treatment with VOSO(4) and ZnSO(4) or Zn(OAc)(2) afforded vanadyl(IV) and zinc(II) complexes which were characterized by means of (1)H NMR, IR, EPR, and UV-vis spectroscopies, and combustion analysis. The in vitro insulin-mimetic activity of these complexes was evaluated from 50% inhibitory concentrations (IC(50)) on free fatty acid (FFA) release from isolated rat adipocytes treated with epinephrine. Vanadyl complexes of 4,6-disubstituted-2(1H)-pyrimidinones showed higher insulin-mimetic activities than those of 4,5,6-trisubstituted ones. On the other hand, Zn(II) complexes showed lower insulin-mimetic activities than VOSO(4) and ZnSO(4) as positive controls. It was found that the balance of the hydrophilicity and/or hydrophobicity is important for higher insulin-mimetic activity. The in vivo insulin-mimetic activity was evaluated with streptozotocin (STZ)-induced diabetic rats. Blood glucose levels were lowered from hyperglycemic to normal levels after the treatment with bis(1,2-dihydro-4,6-dimethyl-2-oxo-1-pyrimidinolato)oxovanadium(IV) by daily intraperitoneal injections. The improvement in glucose tolerance was also confirmed by an oral glucose tolerance test.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Disease Models, Animal; Drug Evaluation, Preclinical; Electron Spin Resonance Spectroscopy; Hypoglycemic Agents; Inhibitory Concentration 50; Insulin; Ligands; Male; Molecular Mimicry; Molecular Structure; Organometallic Compounds; Pyrimidinones; Rats; Rats, Wistar; Vanadates; Zinc

Class III antiarrhythmic agents have been widely used to suppress ventricular tachyarrhythmias in patients with heart failure because they have been shown to have positive inotropic effects as well. However, it remains to be examined whether those agents also exert positive inotropic effects in failing hearts. We addressed this important issue in a rat model of heart failure. We used Nifekalant as a representative class III antiarrhythmic agent. Four weeks after a s.c. injection of 60 mg/kg monocrotaline (MCT) or vehicle (Ctr) into rats, we obtained trabeculae from right ventricles and measured the developed force and intracellular Ca(2+) ([Ca(2+)](i)) by the fura-2 microinjection method. The sarcoplasmic reticulum (SR) Ca(2+) content was assessed by the rapid-cooling contracture (RCC) technique. MCT rats exhibited right ventricular hypertrophy induced by pressure overload. The protein expression of SR Ca(2+) ATPase type 2 (SERCA2) and the SERCA2/phospholamban ratio in MCT rats was lower with a slower decline of Ca(2+) transients and a reduced amplitude of RCCs. Nifekalant concentration-dependently increased the force, peak [Ca(2+)](i), and the amplitude of RCCs in Ctr rats but not in MCT rats with identical prolongation of the action potential. Under the SR inhibited with cyclopiazonic acid and ryanodine, Nifekalant increased the force in Ctr rats but not in MCT rats. These results indicate that the positive inotropic effects of Nifekalant is reduced in failing hearts, probably due to the depressed SR Ca(2+) uptake and reduced reserve of the trans-sarcolemmal Ca(2+) transport, warranting a caution in the antiarrhythmic therapy with a class III antiarrhythmic agent in heart failure.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Calcium; Cardiotonic Agents; Disease Models, Animal; Heart Failure; Monocrotaline; Myocardial Contraction; Pyrimidinones; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum

We established a new and facile model to investigate allergic mechanism and assess the effect of antiallergic compounds. Male Wistar rats were actively or passively sensitized. Active sensitization was performed by injection of both dinitrophenylated-ovalbumin (DNP-OA) and Bordetella pertussis. Nine days later, DNP-OA was injected into the right hind footpad. This antigen challenge induced a biphasic footpad swelling that consisted of an early-phase (EPR) and a late-phase response (LPR). In rats passively sensitized with rat anti-DNP-OA serum, DNP-OA induced only EPR. The EPR was suppressed by disodium cromoglycate, a mast cell stabilizer, but not by cyclosporin A, an immunosuppressant, while the LPR was suppressed by cyclosporin A. Furthermore, to investigate these two allergic responses determined by the interactions between the hapten and the carrier proteins, two distinct haptenated antigens were created. DNP-Ascaris (DNP-As) induced a marked EPR and LPR in DNP-As-sensitized rats. However, DNP-As induced only EPR in DNP-OA-sensitized rats, indicating that the usage of the same carrier protein in both sensitization and challenge was necessary for induction of LPR. These data suggest that this actively sensitization model in which EPR and LPR are functionally distinguishable should be useful for evaluating the efficacy of antiallergic compounds.

Topics: Aminopyridines; Animals; Anti-Allergic Agents; Antigens; Cromolyn Sodium; Cyclosporine; Dinitrobenzenes; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Evaluation, Preclinical; Edema; Haptens; Hindlimb; Hypersensitivity, Delayed; Male; ortho-Aminobenzoates; Ovalbumin; Passive Cutaneous Anaphylaxis; Promethazine; Pyridines; Pyrimidinones; Quinolones; Rats; Rats, Wistar

Because nifekalant hydrochloride (NIF) displayed a superior defibrillating effect on ventricular tachycardia/fibrillation (VT/VF) in cardiopulmonary arrest (CPA) patients, despite some QT prolongation, its effect on transmural dispersion of repolarization (TDR) in the left ventricle (LV) in an animal model of CPA was investigated.. Eight beagle dogs were created with a myocardial infarction under anesthesia, and then VT/VF induction by continuous stimulation and cardiopulmonary resuscitation (CPR) were repeated. NIF (0.3 mg/kg) was administered under acidotic conditions (pH 7.26). The QTc interval measured by Y-lead ECG showed no significant prolongation before and after NIF. The activation recovery interval (ARI) measured by 64-lead LV surface mapping showed minimum ARI prolongation (40%) by NIF without maximum ARI prolongation, and as a result the ARI dispersion decreased by 67%. The repolarization time (RPT) with the plunge electrode showed 13-19% prolongation in the subendocardium and subepicardium with CPR, but NIF prolonged the RPT in the middle layer alone (17%), and as a result Plunge-TDR decreased by 82% (n=8, p<0.05).. Administration of NIF during CPR decreased the TDR by RPT prolongation selectively in the middle layer. Because the subendocardial and subepicardial RPTs after CPR were already prolonged before NIF administration, it may have been the reason why the QT-prolonging effect of NIF was not reflected in the body surface ECG.

Topics: Animals; Anti-Arrhythmia Agents; Cardiopulmonary Resuscitation; Disease Models, Animal; Dogs; Heart Arrest; Heart Conduction System; Humans; Pyrimidinones; Tachycardia, Ventricular; Ventricular Fibrillation

We developed a colitis model in Syrian hamsters (Mesocricetus auratus) to investigate the relationship between colitis and neutrophil elastase (NE). Colitis was induced by a single intracolonic dose of trinitrobenzene sulfonic acid (TNBS; 90 mg/ml) dissolved in 15% (vol/vol) ethanol. The ulcer area, tissue myeloperoxidase (MPO) activity, and luminal NE activity all were increased on Days 1 and 5, corresponding with the acute inflammatory histopathological changes. These acute inflammatory parameters subsequently decreased by Day 14, and chronic inflammatory histopathological changes became evident. Recurrence of inflammation was not observed during the period up to Day 28. To evaluate our colitis model, the effects of prednisolone were examined. Prednisolone was administered orally once on the day before induction of colitis, and animals were treated twice daily thereafter. Although prednisolone had little effect on the tissue MPO activity, prednisolone inhibited the ulcer area and NE activity. In addition, the effects of an NE-specific inhibitor (ONO-6818) on our TNBS-induced colitis model were examined. In the subcutaneous treatment study, ONO-6818 was administered once before the induction of colitis. Although ONO-6818 had little effect on the tissue MPO activity, the ulcer area and NE activity were decreased in the ONO-6818-treated group. The inhibitory effects on the ulcer area and NE activity were confirmed after oral treatment with ONO-6818 after induction of colitis. We conclude that our colitis model is useful for investigating the relationship between colitis and NE, and inhibition of NE activity can prevent the progression of ulceration.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Colon; Cricetinae; Disease Models, Animal; Leukocyte Elastase; Male; Mesocricetus; Oxadiazoles; Prednisolone; Pyrimidinones; Random Allocation; Trinitrobenzenesulfonic Acid

1. Regional haemodynamic responses to a continuous, 4-day infusion of the selective phosphodiesterase type 5 inhibitor, UK-357,903 (0.133 or 1.33 mg x kg(-1) h(-1)) were measured in conscious spontaneously hypertensive rats, and compared with those of enalapril (1 mg x kg(-1) h(-1)). 2. Both doses of UK-357,903 caused modest reductions in mean blood pressure that were not dose-dependent and only significantly different from the vehicle effects on Day 1 of the study (mean -11.8 and -15.3 mmHg for low and high doses, respectively). UK-357,903 had mesenteric and hindquarters vasodilator effects, which were, again, similar for both dose levels and only significantly different from vehicle on Day 1. Neither dose of UK-357,903 affected renal vascular conductance or heart rate. 3. Although the haemodynamic effects of UK-357,903 were not clearly dose-related and some appeared to wane with time, geometric mean plasma levels of UK-357,903 increased in proportion to dose, and were sustained throughout the infusion period. Furthermore, plasma cyclic guanosine monophosphate, a biomarker of phosphodiesterase 5 inhibition, was persistently elevated, and increased with increasing dose. 4. Enalapril caused a fall in mean blood pressure on day 1 (-14.1 mmHg) that was associated with dilatation in renal, mesenteric and hindquarters vascular beds. The haemodynamic effects of enalapril were sustained or increased over the 4-day infusion, although plasma free drug levels were stable. 5. In conclusion, we have shown regional and temporal changes in the haemodynamic effects of UK-357,903, which may be due to activation of compensatory mechanisms, but there were no signs of functional compensation to the cardiovascular effects of enalapril.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin I; Animals; Cardiovascular Physiological Phenomena; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Dose-Response Relationship, Drug; Enalapril; Hemodynamics; Hypotension; Infusions, Intravenous; Male; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Pyrimidinones; Radioimmunoassay; Rats; Rats, Inbred SHR; Renin; Sulfones; Time Factors

Sildenafil (Pfizer Pharmaceuticals, Sandwich, Kent, UK) has been associated with pulmonary vasorelaxation. A more potent Sildenafil analogue (UK 343-664 [Pfizer Pharmaceuticals]) has been developed, but its effects in vivo have not been studied. This study evaluated the effects of UK 343-664 (Pfizer) during acute pulmonary hypertension.. Fourteen adult swine were anesthetized with 1 minimum alveolar concentration isoflurane and were mechanically ventilated with an FIO(2) of 50%. End tidal CO(2) was maintained between 32 and 36 mm Hg. Micromanometer tipped catheters were placed in the ascending aorta, pulmonary artery, and right ventricle. Pulmonary flow was measured with a perivascular probe using transit time ultrasound.. Pulmonary hypertension was induced with a continuous infusion of the thromboxane analogue U46619. Animals were randomized into two groups. Group 1 (n = 9) received 500 microg of UK 343-664 (Pfizer) intravenously for more than 2 minutes. Group 2 (n = 5) served as the control group. Data were recorded continuously for 60 minutes. Statistical analyses were performed with the analysis of variance and t tests. A p less than 0.05 was considered significant.Pulmonary hypertension was achieved in all animals. The administration of UK 343-664 (Pfizer) was associated with a significant decrease in pulmonary artery pressure (30.3%; p < 0.05) and pulmonary vascular resistance (42%; p < 0.05) with mild systemic vasodilatation. These effects were partially maintained at 30 minutes (a 17.3% and 39% decrease, respectively; p < 0.05).. The administration of UK 343-664 (Pfizer) was associated with predominant pulmonary vasodilatation without systemic hypotension. This may represent a significant advance in the treatment of acute pulmonary hypertension. Potential clinical implications for this new phosphodiesterase enzyme type V (PDEV) inhibitor merit further study.

Topics: Acute Disease; Animals; Disease Models, Animal; Hypertension, Pulmonary; Piperazines; Purines; Pyrimidinones; Random Allocation; Sildenafil Citrate; Sulfones; Swine; Vasodilator Agents

The pharmacological profile of the new CCK1 receptor antagonist IQM-97,423, (4aS,5R)-2-benzyl-5-(tert-butylaminocarbonyl-tryptophyl)amino-1,3-dioxoperhydropyrido-[1,2-c]pyrimidine, was examined in in vitro and in vivo studies and compared with typical CCK1 antagonists such as devazepide and lorglumide. IQM-97,423 showed a high affinity at [3H]-pCCK8-labeled rat pancreatic CCK1 receptors, and was virtually devoid of affinity at brain CCK2 receptors. IQM-97,423 antagonized CCK8S-stimulated alpha-amylase release from rat pancreatic acini with a potency similar to devazepide and much higher than lorglumide. In the guinea pig isolated longitudinal muscle-myenteric plexus preparation, IQM-97,423 produced a full antagonism of the contractile response elicited by CCK8S and a weaker effect on the contraction elicited by CCK4, suggesting a selective antagonism at CCK1 receptors. The protective effect of IQM-97,423 and devazepide was tested in two models of acute pancreatitis in rats, induced by injection of cerulein or by combined bile and pancreatic duct obstruction. The new compound fully prevented the cerulein-induced increase in plasma pancreatic enzymes and in pancreas weight with a potency similar to devazepide. In common bile-pancreatic duct ligature-induced acute pancreatitis, IQM-97,423 partially prevented, like devazepide, the increase in plasma pancreatic enzyme activity and in pancreas weight. Consequently, the pyridopyrimidine derivative IQM-97,423 is a potent and highly selective CCK1 receptor antagonist with preventive effects in two experimental models of acute pancreatitis and a potential therapeutic interest.

Topics: Acute Disease; alpha-Amylases; Animals; Binding, Competitive; Cerebral Cortex; Cholecystokinin; Devazepide; Disease Models, Animal; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mice; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Neuromuscular Junction; Pancreatitis; Peptide Fragments; Proglumide; Pyrimidinones; Rats; Rats, Wistar; Receptor, Cholecystokinin A

We investigated the effects of a novel oral neutrophil elastase inhibitor (ONO-6818) on acute lung injury and pulmonary emphysema induced by human neutrophil elastase (HNE). Young male Wistar rats were divided into four treatment groups: (1) control group (saline); (2) HNE group (HNE 200 U + 0.5% carboxymethyl-cellulose [solution for ONO-6818]); (3) low-dose ONO-6818 group (HNE 200 U + ONO-6818 10 mg/kg); and (4) high-dose ONO-6818 group (HNE 200 U + ONO-6818 100 mg/kg). Saline and HNE were applied via the trachea using a microsprayer. ONO-6818 was administered orally 1 hour before HNE application. Six hours after HNE application, neutrophil counts and hemoglobin concentration in bronchoalveolar lavage fluid and lung tissue myeloperoxidase activity were determined. Eight weeks after the application, FRC, TLC, lung compliance, and mean linear intercept were estimated. ONO-6818 attenuated dose-dependently HNE-induced increases in lung myeloperoxidase activity, hemoglobin, and neutrophil count in bronchoalveolar lavage fluid. Furthermore, it significantly attenuated HNE-induced increases in FRC, TLC, lung compliance, and mean linear intercept. ONO-6818 inhibited acute lung injury induced by HNE by minimizing lung hemorrhage and accumulation of neutrophils in the lung. ONO-6818 also inhibited the development of HNE-induced emphysematous changes including lung hyperinflation, degradation of elastic recoil, and airspace enlargement.

Topics: Administration, Oral; Animals; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Drug Evaluation, Preclinical; Emphysema; Functional Residual Capacity; Humans; Leukocyte Count; Leukocyte Elastase; Lung Compliance; Lung Volume Measurements; Male; Neutrophils; Oxadiazoles; Pyrimidinones; Rats; Rats, Wistar; Respiratory Mechanics; Sputum

To evaluate the antidiabetic and hypolipidaemic potential of a novel thiazolidinedione, PMT13, in different animal models of insulin resistance.. PPAR transactivation study was performed in HEK293T cells using ligand binding domains of PPARalpha, gamma and delta. Insulin-resistant db/db and ob/ob mice were treated orally with different doses of PMT13 at 0.3-10 mg/kg/day for 15 and 14 days respectively. Zucker fa/fa rats were treated with 3 mg/kg (p.o.) dose of the compound. Plasma glucose, triglyceride, free fatty acid and insulin levels were measured. Liver glucose 6-phosphatase (G6-Ptase) and adipose lipoprotein lipase activity was measured in treated mice. Isolated rat aortic preparations preconstricted with phenylephrine were used to study the vascular relaxation potential of PMT13 in presence of insulin. A 28-day oral toxicity study was performed in Wistar rats.. PMT13 showed similar PPARgamma activation as rosiglitazone, but failed to show any activity against PPARalpha or PPARdelta. In obese and diabetic db/db and ob/ob mice, PMT13 showed better reduction in plasma glucose, triglyceride and insulin levels than rosiglitazone and an improvement in glucose tolerance. In insulin-resistant Zucker fa/fa rat model, PMT13 treatment showed better reduction in plasma triglyceride, free fatty acid and insulin levels than that of rosiglitazone. Treated mice showed decreased G6-Ptase activity in liver. The LPL activity was increased in post-heparin plasma and epididymal fat of treated db/db mice. In an isolated, precontracted rat aortic preparation, PMT13 treatment significantly increased insulin-induced relaxation. A 28-day oral toxicity study in rats showed no treatment-related adverse effects.. Our studies indicate that PMT13 is a potent activator of PPARgamma with antidiabetic, hypolipidaemic and insulin-sensitizing properties. Additionally, PMT13 inhibited liver G6-Ptase activity and increased lipoprotein lipase activity. It showed improvement in insulin-induced vasorelaxation. The compound also showed a good safety margin. Therefore, PMT13 can be a potential drug candidate for future development.

Topics: Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Hypoglycemic Agents; Insulin Resistance; Liver; Mice; Mice, Obese; Pyrimidines; Pyrimidinones; Rats; Rats, Zucker; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors

Arterially perfused, decerebrate preparations of the insectivore, Suncus murinus were made to determine whether the emetic reflex could be activated in such a preparation using a range of stimuli shown to be emetic in conscious or anaesthetised Suncus. Efferent phrenic and vagus nerve activities and electromyograms (EMGs) from the temporalis, abdominal oesophagus and trapezius muscles were recorded, as well as longitudinal shortening of the oesophagus and dorso-ventral movements of the thorax. The preparations swallowed spontaneously every 0.6 to 6.5 min. The duration of a swallow was 3.1 +/- 0.3 s (recorded as the time taken for the oesophagus to shorten and recover to its resting position) and the oesophagus shortened by 3.5 +/- 0.4 mm during a swallow. The emetic reflex was activated by electrical stimulation (30 Hz, 10-20 V, 0.2 ms pulse width, for 30 s) of abdominal vagal afferents (latency < 30 s) or by arterial perfusion with either 40 nM of the capsaicin analogue resiniferatoxin (latency 1.7 +/- 0.6 min), 6 microM nicotine (latency 1.6 +/- 0.1 min) or 1 microM of the phosphodiesterase IV inhibitor CP-80,633 (latency 8.9 +/- 3.9 min). These emetic stimuli produced somatic and visceral movements in Suncus preparations indicative of activation of the emetic reflex. There were pronounced contractions of the thorax that occurred simultaneously with oesophageal shortening and mouth opening, separated by thorax expansion and a burst of phrenic nerve activity. During emetic-like episodes, oesophageal shortenings were only 0.84 +/- 0.1 s in duration, faster than the duration of shortening observed during swallowing (cf. swallowing, 3.1 +/- 0.3 s; P < 0.0001). The shortening of the oesophagus during emetic-like episodes was 6.2 +/- 0.4 mm, which was greater than the shortening seen during swallowing (cf. swallowing, 3.5 +/- 0.4 mm; P < 0.0001). We conclude that the emetic reflex can be activated in our Suncus preparations and that this non-sentient small adult animal model can now be used to study the neurophysiology and pharmacology of swallowing and emesis.

Topics: Animals; Aorta, Thoracic; Decerebrate State; Deglutition; Disease Models, Animal; Diterpenes; Electric Stimulation; Esophagus; Female; Heart Rate; Male; Naloxone; Narcotic Antagonists; Nicotine; Nicotinic Agonists; Perfusion; Phrenic Nerve; Physical Stimulation; Pyrimidinones; Reflex; Respiratory Mechanics; Shrews; Vagus Nerve; Video Recording; Vomiting

Because interferon-gamma, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells.

Topics: Animals; Cells, Cultured; Chromones; Dermatitis, Atopic; Disease Models, Animal; Ear; Edema; Enzyme Inhibitors; Eosinophils; Hypersensitivity; Immunosuppressive Agents; Leukotriene Antagonists; Male; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Oxadiazoles; Pyrimidinones; Skin; Tacrolimus; Th1 Cells; Th2 Cells

To investigate the development of airway hyperresponsiveness in infantile guinea pigs, animals (10 days old) were immunized twice and challenged by inhalation of 1% ovalbumin for 10 min with 7 days intervals. Similar to adult guinea pigs, infantile ones developed an increased airway responsiveness to acetylcholine 24 hr after antigen challenge. There was a marked increase in the number of total leukocytes, eosinophils and lymphocytes in bronchoalveolar lavage fluid (BALF). Suplatast tosilate (suplatast) and pemirolast potassium (pemirolast) given orally throughout the experiments suppressed the development of airway hyperresponsiveness in infantile animals. They showed similar potency in the suppression of eosinophil accumulation in BALF and lung tissue, while suplatast inhibited lymphocyte accumulation stronger than pemirolast. Collectively, the present model of airway hyperresponsiveness in infantile guinea pigs may be useful in predicting the efficacy of antiallergic agents in the treatment of asthmatic children.

Topics: Animals; Anti-Allergic Agents; Antigens; Arylsulfonates; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Guinea Pigs; Histamine Antagonists; Leukocyte Count; Male; Ovalbumin; Pyridines; Pyrimidinones; Respiratory Hypersensitivity; Sulfonium Compounds

1. The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[N alpha-tert-butoxicarbonyl)L-tryptophyl] amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158. 2. IQM-95,333 displaced [3H]-CCK-8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50 > 10 microM). IQM-95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. 3. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCKA receptors. 4. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCKA receptor-mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. 5. Low doses (50-100 micrograms kg-1, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCKA receptors. 6. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10-5,000 micrograms kg-1). Typical CCKA and CCKB antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range. 7. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. 8. In conclusion, IQM-95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic-like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

Topics: Amylases; Animals; Anorexia; Anti-Anxiety Agents; Benzodiazepinones; Carbamates; Cholecystokinin; Devazepide; Diazepam; Disease Models, Animal; Fenfluramine; Guinea Pigs; Hormone Antagonists; Locomotion; Male; Mice; Phenylurea Compounds; Pyrimidinones; Rats; Rats, Wistar; Receptors, Cholecystokinin; Selective Serotonin Reuptake Inhibitors

We studied the electrophysiologic and antifibrillatory properties of MS-551 (1,3-dimethyl-6-((2-[N-hydroxy-ethyl)-3-(4-nitrophenyl) propylamino] ethylamino) 2,4(1H,3H) pyrimidinedione hydrochloride) in a conscious canine model of sudden cardiac death. Three to 5 days after surgically induced myocardial infarction (MI: 2-h occlusion of the left anterior descending coronary artery, LAD), animals were subjected to programmed electrical stimulation (PES) to identify those at risk for sudden cardiac death. MS-551 was administered (2.0, 3.0, or 4 x 2.0 mg/kg intravenously, i.v.). Vehicle-treated animals received 0.9% sodium chloride solution for injection. MS-551 (multiple-dose regimen) increased ventricular effective refractory period (VERP) from 112 +/- 4 to 137 +/- 4 ms (p < 0.05) as compared with vehicle treatment, which did not alter VERP (125 +/- 6 to 121 +/- 5 ms). MS-551 prolonged QTc interval from a predrug value of 293 +/- 8 to 333 +/- 18 ms postdrug. The size of surgically induced MI did not differ among groups: 2.0 mg/kg, 23 +/- 4%; 3.0 mg/kg, 28 +/- 2%; 4 x 2.0 mg/kg, 25 +/- 3%; and vehicle, 28 +/- 3% of the left ventricle. Single bolus administration of MS-551 (2.0 or 3.0 mg/kg i.v.) did not confer significant protection against sudden cardiac death. However, repeated administration of MS-551 protected against sudden cardiac death in 8 of 10 dogs as compared with 2 of 12 in the vehicle-treated group (p < 0.05). The data indicate that a multiple-dose regimen of MS-551 provides protection against ischemia-induced ventricular fibrillation (VF) in the postinfarcted heart. The mechanism by which MS-551 achieves its antifibrillatory effect most likely depends on its ability to prolong VERP of myocardium without altering ventricular conduction velocity.

Topics: Animals; Anti-Arrhythmia Agents; Chromatography, High Pressure Liquid; Death, Sudden, Cardiac; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Electric Stimulation; Electrocardiography; Electrophysiology; Male; Myocardial Infarction; Pyrimidinones; Random Allocation; Ventricular Fibrillation

We studied the electrophysiologic and antifibrillatory effects of the class III agent MS-551 in a rabbit isolated heart model in which ventricular fibrillation (VF) occurs reproducibly under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener, pinacidil. Ten minutes after MS-551 or vehicle administration, addition of pinacidil (1.25 microM) to the buffer was followed by a 12-min hypoxic period and 40-min reoxygenation. At a low concentration of MS-551 (1.0 microM), VF occurred in 5 of 6 hearts, the same incidence as in the control group (5 of 6). In contrast 0 of 6 hearts treated with 15 microM MS-551 developed VF (p < 0.05 vs. vehicle). Ventricular effective refractory period (VERP) was determined in a separate group of isolated hearts (n = 13). Pinacidil alone, during normoxic perfusion, decreased VERP 48 +/- 11% (p < 0.05) 15 min after exposure. Five minutes of hypoxia alone also decreased VERP (57 +/- 8%, p < 0.05). Under normoxic conditions, MS-551 increased ERP 31 +/- 10% (p < 0.05 vs. baseline). VERP prolongation by MS-551 was reduced in the presence of pinacidil but remained 22 +/- 6% (p < 0.05) above baseline. The results suggest that VERP shortening owing to pinacidil-mediated ATP-dependent K+ channel opening is associated with development of VF in isolated heart. MS-551 attenuates the pinacidil-mediated decrease in VERP and prevents pinacidil+hypoxia-reoxygenation-induced VF. Because pinacidil and hypoxia open myocardial KATP channels, putatively decreasing VERP, MS-551 may exert its antifibrillatory effect through partial blockade of KATP channels.

Topics: Animals; Anti-Arrhythmia Agents; Disease Models, Animal; Dogs; Guanidines; Heart; Heart Atria; Hypoxia; In Vitro Techniques; Pinacidil; Pyrimidinones; Rabbits; Vasodilator Agents; Ventricular Fibrillation

We examined the effects of MS-551 (1,3-dimethyl-6-[(2-[N-(2-hydroxyethyl)-3-(4- nitrophenyl)propylamino]ethylamino] 2,4(1H,3H)-pyrimidinedione hydrochloride), a new class III antiarrhythmic drug, on programmed electrical stimulation (PES)-induced ventricular arrhythmias, the effective refractory period (ERP), intraventricular conduction, and hemodynamics in a canine myocardial infarction (MI) model. MS-551 was administered intravenously (i.v.) in two consecutive doses; the first dose (low dose) was 0.5 mg/kg/min after a bolus injection of 0.3 mg/kg, and a second dose (high dose) was 0.1 mg/kg/min after a bolus injection of 0.3 mg/kg. PES induced ventricular tachycardia (VT) or ventricular fibrillation (VF) in 10 of 12 animals. MS-551 abolished or lessened the ventricular arrhythmias in 7 of 10 animals at both doses. ERP was significantly prolonged by MS-551 in both the normal and infarcted zones in a dose-dependent fashion. Ventricular conduction of a premature excitation induced by a premature stimulation with various coupling intervals was decreased only at a coupling interval approximating that of ERP. MS-551 at either low or high dose did not significantly change the heart rate (HR), mean blood pressure (MBP), cardiac output (CO), or maximum rate of increase in left ventricular pressure (LVP) significantly. MS-551 produced a suppression of the PES-induced ventricular arrhythmias through prolongation of ERP without having any significant effect on hemodynamics in a canine MI model.

Topics: Animals; Anti-Arrhythmia Agents; Disease Models, Animal; Dogs; Electrocardiography; Hemodynamics; Myocardial Infarction; Pyrimidinones; Refractory Period, Electrophysiological; Tachycardia, Ventricular

The effects of the new inotropic agent, SDZ 218-135 [(+)-(S)-4-[3-(4-diphenyl-methyl-1-piperazinyl)-2-hydroxy-propyl]- 6-(2-hydroxyethyl)-5-methyl-1,2,4-triazolo-[1,5-a]pyrimidin-7(4H)-one], were investigated using in vitro and in vivo techniques. In isolated rat atria, SDZ 218-135 elicited a dose-dependent increase in contractile force (+50% at 10 microM), which was paralleled by an increase in functional refractory period. In anesthetized rats SDZ 218-135 enhanced left ventricular (+)dP/dtmax by 100% at 10 mg/kg without influencing heart rate, arterial blood pressure, and cardiac output. In contrast to its predecessor, DPI 201-106, cardiac relaxation remained essentially unimpaired. The positive inotropic action was also maintained in a rabbit model of depressed heart function after myocardial infarction, where SDZ 218-135 increased peak acceleration of blood in the aorta. The prolongation of the effective refractory period in rat atria suggested possible antiarrhythmic effects. Indeed, SDZ 218-135 showed a dose-dependent marked reduction in reperfusion arrhythmias after coronary artery occlusion in rats. This effect was most likely due to a Class III action, since SDZ 218-135 significantly increased action potential duration (+10% at 10 microM/l) of the isolated guinea pig papillary muscle. In conclusion, SDZ 218-135 is a novel positive inotropic agent with an interesting profile of action. It does not impair cardiac relaxation and shows antiarrhythmic effects in a model of reperfusion-induced arrhythmias. The in vivo and in vitro data are consistent with a mechanism of action via sodium channel agonism.

Topics: Adenosine Triphosphate; Anesthesia; Animals; Arrhythmias, Cardiac; Cardiotonic Agents; Disease Models, Animal; Electrophysiology; Female; Guinea Pigs; Heart Atria; Hemodynamics; Isometric Contraction; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Ovum; Papillary Muscles; Phosphodiesterase Inhibitors; Piperazines; Potassium; Propranolol; Pyrimidinones; Rabbits; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Triazoles

The effects of FK664, a novel positive inotropic agent, and enoximone on pentobarbital-induced heart failure were compared in dog heart-lung preparations. Both FK664 and enoximone improved the cardiac function curve in a dose-dependent manner and restored it to the control level at drug concentrations of 1 microgram/ml and 10 micrograms/ml, respectively. Therefore, the cardiotonic potency of FK664 appears to be 10 times that of enoximone. These agents were almost equal in force-rate separation of cardiac effect. Neither of the agents produced arrhythmia at any dose tested. These results suggest that FK664 may be a potent cardiotonic agent for the treatment of heart failure.

Topics: Animals; Cardiotonic Agents; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Enoximone; Extracorporeal Circulation; Heart; Heart Failure; Imidazoles; Lung; Pyrimidinones

The antiarrhythmic effects of MS-551, which prolongs cardiac action potential duration without affecting the maximum upstroke velocity of the action potential, were assessed in three different canine ventricular arrhythmia models: 1) ventricular tachycardia (VT) induced by electrical stimuli 3-5 days after myocardial infarction, 2) spontaneous ventricular tachyarrhythmias 24-48 hr after two-stage coronary ligation and 3) ventricular tachyarrhythmias induced by digitalis. Intravenous administration of MS-551 (0.1-1 mg/kg) decreased the susceptibility in 10 dogs out of 13 to VT or ventricular fibrillation evoked by programmed electrical stimulation (PES) delivered to the ventricular septum 3-5 days after myocardial infarction. Oral administration of MS-551 (3 mg/kg) also decreased the susceptibility to VT evoked by PES in 7 out of 10 conscious postinfarction dogs. Concurrently, intravenous (0.1-1 mg/kg) or oral (3 mg/kg) administration of MS-551 produced increases in the ventricular effective refractory periods (ERP) by 7 +/- 1% - 17 +/- 3% or 13 +/- 2%, respectively. Similarly, d-sotalol (0.3-3 mg/kg, i.v. and 10 mg/kg, p.o.) decreased the susceptibility to VT with increased ERP. However, MS-551 (1 and 10 mg/kg, i.v.) failed to inhibit both canine two-stage coronary ligation arrhythmia and digitalis arrhythmia. These results suggest that MS-551 is a pure class III antiarrhythmic drug which may be effective in the treatment of life-threatening reentrant tachyarrhythmias, but not in automaticity arrhythmias.

Topics: Administration, Oral; Animals; Anti-Arrhythmia Agents; Digitalis Glycosides; Disease Models, Animal; Dogs; Electric Stimulation; Electrocardiography; Female; Heart Rate; Injections, Intravenous; Male; Pyrimidinones; Tachycardia

The photosensitizing dye merocyanine 540 (MC 540) was evaluated as a means for purging malarially infected red cells from murine blood using the rodent malarial pathogens, Plasmodium yoelii and Plasmodium berghei, as models of human malaria. Malarially infected red cells bound more MC 540 and were more sensitive to MC 540-sensitized photoirradiation than were noninfected erythroid cells. Extracorporeal exposure of infected red cells to the dye and white light prevented the transmission of the disease in a transfusion model. P. berghei-infected red cells were more resistant to the antimalarial activity of MC 540 than were P. yoelii-infected cells, presumably because P. berghei preferentially infects reticulocytes whereas P. yoelii infects mature red cells. The possibility of using photoirradiation sensitized by MC 540 or related dyes to purge malarially infected donor blood is discussed.

Topics: Animals; Blood Transfusion; Disease Models, Animal; Erythrocytes; Female; Flow Cytometry; Malaria; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Microscopy, Fluorescence; Photochemotherapy; Plasmodium; Plasmodium berghei; Plasmodium yoelii; Pyrimidinones; Radiation-Sensitizing Agents

Four experiments were conducted to evaluate the effect of diet and the administration of H2-antagonists in feed on gastric ulcer formation and performance of growing-finishing swine. Pigs receiving a finely ground diet (less than lmm) grew faster (.73 vs .68 kg/d, P less than .01) and had better feed utilization (3.47 vs 3.76, P less than .01) than pigs receiving a cracked corn-based diet. Incidence of ulcers in the esophageal region of the stomach of pigs fed the finely ground diet was greater (P less than .01) than in pigs fed cracked corn. The average daily gain of pigs receiving the finely ground diet was inversely related to ulcer incidence (r = .403, P less than .01, df = 59). The addition of 5, 10, 20 or 100 ppm of the H2-antagonist, metiamide, or 6, 18 or 54 ppm of SK&F 93479 to the finely ground diet did not improve pig performance or affect the incidence of gastric ulceration. The addition of 2, 6 and 18 ppm of SK&F 93479 to a corn-soy diet containing 4.5% alfalfa meal caused a reduction in gastric ulceration (P less than .05) and improved feed utilization by 3.2% (P less than .05). These data suggest that finely ground diets improve the performance of growing-finishing swine, but increase the incidence of ulcers in the esophageal region of the stomach. Severe gastric ulceration adversely affects swine performance. Feeding H2-antagonists does not reduce the ulcerogenic properties of finely ground diets, suggesting factors other than gastric acid secretion are involved in ulcerogenesis. The use of H2-antagonists in corn-soy diets improves feed utilization and reduces ulceration.

Topics: Animal Feed; Animals; Disease Models, Animal; Female; Food Additives; Histamine H2 Antagonists; Male; Metiamide; Particle Size; Pyrimidinones; Stomach Ulcer; Swine; Swine Diseases; Thiourea

A series of 4,4-disubstituted tetrahydro- and 4,4-disubstituted hexahydro-3H-pyrido[1,2-c]pyrimidin-3-ones (4 and 5, respectively) were prepared from 2-aryl-2-(2-piperidinyl)-4-[N,N-bis (1-methylethyl)amino] butanamides (2). Individual racemates of the piperidinyl amides 2 were converted to pure racemic diaza bicyclic compounds that were evaluated for antiarrhythmic activity in the Harris dog model and anticholinergic activity in a muscarinic receptor binding assay. Selected compounds were subsequently evaluated for hemodynamic effects in anesthetized dogs where blood pressure depression and negative inotropic activity were assessed. Of this group, 4a (R = CH3) and 5a (R = CH3) showed the most favorable pharmacological profiles; the former compound was chosen for toxicity testing over the latter due to its lack of noncompetitive inhibition of acetylcholine-induced contractions of guinea pig ileum segments. Clinical evaluation is now under way.

Topics: Animals; Arrhythmias, Cardiac; Blood Pressure; Brain; Chemical Phenomena; Chemistry; Depression, Chemical; Disease Models, Animal; Dogs; Female; Guinea Pigs; Heart; Magnetic Resonance Spectroscopy; Male; Myocardial Contraction; Pyridines; Pyrimidinones; Quinuclidinyl Benzilate; Rats; Receptors, Muscarinic; X-Ray Diffraction