piperidines and Scleroderma--Systemic

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

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

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

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

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

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

BACKGROUNDSystemic sclerosis (SSc) is an autoimmune, connective tissue disease characterized by vasculopathy and fibrosis of the skin and internal organs.METHODSWe randomized 15 participants with early diffuse cutaneous SSc to tofacitinib 5 mg twice a day or matching placebo in a phase I/II double-blind, placebo-controlled trial. The primary outcome measure was safety and tolerability at or before week 24. To understand the changes in gene expression associated with tofacitinib treatment in each skin cell population, we compared single-cell gene expression in punch skin biopsies obtained at baseline and 6 weeks following the initiation of treatment.RESULTSTofacitinib was well tolerated; no participants experienced grade 3 or higher adverse events before or at week 24. Trends in efficacy outcome measures favored tofacitnib. Baseline gene expression in fibroblast and keratinocyte subpopulations indicated IFN-activated gene expression. Tofacitinib inhibited IFN-regulated gene expression in SFRP2/DPP4 fibroblasts (progenitors of myofibroblasts) and in MYOC/CCL19, representing adventitial fibroblasts (P < 0.05), as well as in the basal and keratinized layers of the epidermis. Gene expression in macrophages and DCs indicated inhibition of STAT3 by tofacitinib (P < 0.05). No clinically meaningful inhibition of T cells and endothelial cells in the skin tissue was observed.CONCLUSIONThese results indicate that mesenchymal and epithelial cells of a target organ in SSc, not the infiltrating lymphocytes, may be the primary focus for therapeutic effects of a Janus kinase inhibitor.TRIAL REGISTRATIONClinicalTrials.gov NCT03274076.FUNDINGPfizer, NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01 AR070470, NIH/NIAMS K24 AR063120, Taubman Medical Research Institute and NIH P30 AR075043, and NIH/NIAMS K01 AR072129.

Topics: Biomarkers; Endothelial Cells; Fibroblasts; Humans; Keratinocytes; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Pyrroles; Scleroderma, Systemic; Treatment Outcome

Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by fibrosis of the skin and internal organs, autoimmunity-driven damage and vasculopathy. The current approved disease-modifying treatments have limited efficacy, and treatment is guided toward alleviating organ complications. Thus, there is an unmet need for discovering new effective treatment options. There is recent evidence that the JAK/STAT signaling pathway is markedly activated in SSc patients. To assess the efficacy and safety of tofacitinib (TOF) on skin and musculoskeletal involvement as compared to methotrexate (MTX) in systemic sclerosis (SSc). In this 52-week pilot study, 66 patients with SSc were enrolled: 33 patients received 5 mg of oral TOF twice a day; 33 received 10 mg of MTX weekly. The proportion of dcSSc and lcSSc patients was similar (dcSSc: 42% TOF group and 36% MTX group; lcSSc: 58% TOF group and 64% MTX group). The primary outcome was the change in the modified Rodnan skin score (mRSS). Secondary outcomes included ultrasound (US) skin thickness and musculoskeletal involvement (US10SSc score). Digital ulcers (DUs) and adverse events (AEs) were documented through the treatment. Both groups had similar characteristics and medians on the outcome measures at baseline. At week 52, the TOF median mRSS was significantly lower than the MTX (p < 0.001) with a mean reduction of 13 points versus MTX 2.57. The mean percent improvement in the TOF group was 44% higher than in the MTX group. TOF median US skin thickness was significantly lower than MTX (p < 0.001), with a mean reduction of 0.31 mm versus 0.075 mm in the MTX group. The US10SSc median score was significantly lower in the TOF group (p = 0.002); mean reduction of 10.21 versus 5.27 in the MTX group. Healing of DUs with no new occurrences was observed in the TOF group. There was no significant difference between the groups in the number of AEs from baseline to week 52. TOF showed greater efficacy than MTX in reducing mRSS, skin thickness and musculoskeletal involvement in SSc and a satisfactory safety profile.

Topics: Adult; Female; Humans; Immunosuppressive Agents; Male; Methotrexate; Middle Aged; Pilot Projects; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Scleroderma, Systemic; Severity of Illness Index; Skin; Skin Ulcer; Ultrasonography

OPC-28326 is a selective alpha-adrenergic antagonist with preferential binding to the alpha(2C)-adrenergic receptor (alpha(2C)-AR) subtype. This study observed the effect of OPC-28326 on skin temperature and digital blood flow following an acute cold challenge in patients with Raynaud's phenomenon secondary to scleroderma.. The study was designed as a single-center, double-blind, placebo-controlled, randomized, 3-period crossover study of OPC-28326 (oral doses of 10 mg or 40 mg) or placebo. The primary outcome measures were the time to recover 50% and 70% of the fall (induced by cold challenge) in baseline digital skin temperature.. Twelve of 13 enrolled patients completed the study. The mean time to achieve 50% and 70% recovery of the change in prechallenge digital skin temperature was shorter after the OPC-28326 40-mg dose than after placebo (50% recovery at 5.8 minutes versus 10.0 minutes [P = 0.02]; 70% recovery at 13.8 minutes versus 19.5 minutes [P = 0.01]). These recovery times tended to be shorter in the 10 mg OPC-28326 group as well, but the difference versus placebo was not significant (50% recovery at 9.0 minutes versus 10.0 minutes [P = 0.65]; 70% recovery at 15.3 minutes versus 19.5 minutes [P = 0.07]). Total digital blood flow tended to be lower prior to the cold challenge and after administration of 40 mg OPC-28326, as compared with that after placebo, but the difference was not significant. Symptoms that were potentially drug-related were reported more frequently with 40 mg OPC-28326 than with 10 mg OPC-28326 or with placebo, but none were serious or sustained.. OPC-28326 at doses of 10 mg and 40 mg was well tolerated during this study. The shorter time to skin temperature recovery after 40 mg OPC-28326 suggests that selective alpha(2C)-AR blockade improves digital skin perfusion during recovery from cooling in patients with Raynaud's phenomenon secondary to scleroderma.

Topics: Administration, Oral; Adolescent; Adrenergic alpha-Antagonists; Adult; Aged; Aged, 80 and over; Aniline Compounds; Cold Temperature; Cross-Over Studies; Dose-Response Relationship, Drug; Double-Blind Method; Female; Fingers; Humans; Male; Middle Aged; Piperidines; Raynaud Disease; Regional Blood Flow; Scleroderma, Systemic; Skin Temperature; Vasodilation

In a double-blind crossover study lower esophageal sphincter pressure and distal esophageal motility were studied in 10 patients with progressive systemic sclerosis or mixed connective tissue disease, following a single intravenous dose of cisapride or placebo. The measurements were carried out under basal conditions and 30 min after intravenous administration of 10 mg cisapride or placebo. No effects on lower esophageal sphincter pressure or distal esophageal motility were observed.

Topics: Adult; Aged; Cisapride; Double-Blind Method; Esophagogastric Junction; Esophagus; Female; Humans; Male; Middle Aged; Mixed Connective Tissue Disease; Peristalsis; Piperidines; Pressure; Scleroderma, Systemic; Serotonin Antagonists

1. Cisapride is a novel prokinetic drug which facilitates or restores motility throughout the gastrointestinal tract. Its mechanism of action is thought to involve enhancement of acetylcholine release in the myenteric plexus of the gut. 2. The effect of intravenous cisapride 10 mg on gastro-oesophageal dysfunction was investigated in 20 patients with systemic sclerosis, using a double-blind, randomised, cross-over, placebo-controlled manometric study design. 3. The increase in lower oesophageal sphincter pressure was significantly higher after cisapride (mean +/- s.e. mean, 8.3 +/- 2.1 cm H2O) than after placebo (mean +/- s.e. mean. 0.1 +/- 0.3 cm H2O) (P less than 0.001). The increase in the number of fundic gastric contractions during the 30 min study period was significantly higher after cisapride (mean +/- s.e. mean, 7.7 +/- 2.3) than after placebo (mean +/- s.e. mean, 0.9 +/- 0.6) (P less than 0.01). 4. No serious clinical adverse effects were observed. 5. The study demonstrates that intravenous cisapride induces a significant increase in lower oesophageal sphincter pressure and in the number of fundic gastric contractions, which may be beneficial in the treatment of scleroderma gastro-oesophageal dysfunction. Further long-term studies of the effect of oral cisapride in patients with systemic sclerosis are warranted.

Topics: Cisapride; Double-Blind Method; Esophagogastric Junction; Female; Gastric Fundus; Gastrointestinal Motility; Humans; Male; Manometry; Middle Aged; Piperidines; Scleroderma, Systemic

Prokinetic agents might be useful in patients with progressive systemic sclerosis (PSS) who have disturbed function of the lower esophageal sphincter and impaired acid-clearance of the tubular esophagus. We therefore compared, by means of esophageal manometry, the effect of 20 mg cisapride orally vs. placebo in 12 patients with progressive systemic sclerosis and proven esophageal dysfunction as well as in 10 healthy volunteers in a double-blind, prospective trial. An increase of the lower esophageal resting pressure from 18.1 +/- 2.4 mm Hg to 23.9 +/- 8.1 mm Hg* after cisapride administration was observed in healthy volunteers, and from 10.9 +/- 3.2 mm Hg to 13.6 +/- 4.0 mm Hg* in the PSS patients. The amplitudes of peristaltic waves in the distal part of the esophagus were increased by cisapride from 83.8 +/- 10.6 mm Hg to 95.6 +/- 15.5 mm Hg* in volunteers and from 28.9 +/- 12.8 mm Hg to 36.8 +/- 16.2 mm Hg in patients (*:P less than 0.05). These results indicate that cisapride has a therapeutic rationale in the treatment of esophageal dysfunction in PSS; further clinical investigations are justified.

Topics: Adult; Cisapride; Double-Blind Method; Esophageal Motility Disorders; Esophagus; Female; Humans; Male; Manometry; Middle Aged; Piperidines; Prospective Studies; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Serotonin Antagonists

The effects of cisapride on gastric emptying, esophageal emptying, and gastrointestinal symptoms were evaluated in 8 patients with progressive systemic sclerosis who had delayed gastric emptying of the solid or liquid component of a meal, or both. A double-isotope technique was used to measure gastric emptying, and esophageal emptying was measured as the time for a bolus of the solid meal to enter the stomach. Gastrointestinal symptoms were assessed by a questionnaire. On 2 days each patient received cisapride (10 mg) or placebo intravenously, 5 min before an esophageal and gastric emptying test. After these 2 days each subject took cisapride (10 mg q.i.d., p.o.) for 1 mo. Cisapride improved solid and liquid gastric emptying (p less than 0.001), but had no significant effect on esophageal emptying (p less than 0.1). Upper gastrointestinal symptoms were reduced after cisapride (p less than 0.001), and no side effects were reported. These results indicate that gastroparesis is a treatable cause of morbidity in progressive systemic sclerosis.

Topics: Adult; Cisapride; Esophagus; Female; Gastric Emptying; Gastrointestinal Diseases; Humans; Male; Middle Aged; Peristalsis; Piperidines; Scleroderma, Systemic

Ketanserin, a selective antagonist of the 5-HT2 receptor, was evaluated in a 4-week open pilot trial of 30 patients with Raynaud's phenomenon. Moderate or marked relief was reported in 15 of 18 (83%) patients with systemic sclerosis, whereas only 4 of 12 (33%) patients with Raynaud's phenomenon of other etiology received such benefit (P less than 0.01). These subjective ratings were supported by the results of serial digital strain gauge plethysmography during controlled cold challenge. Additional clinical findings suggested that ketanserin therapy facilitated the healing of ischemic digital ulcerations and reduced hand edema in patients with systemic sclerosis. These findings lend support to the hypothesis that serotonin is an important element in the pathogenesis of systemic sclerosis.

Topics: Adult; Aged; Clinical Trials as Topic; Female; Fingers; Humans; Ketanserin; Male; Middle Aged; Pilot Projects; Piperidines; Plethysmography; Raynaud Disease; Receptors, Serotonin; Regional Blood Flow; Scleroderma, Systemic; Serotonin Antagonists

The serotonin receptor blocker ketanserin was given orally in a double blind crossover study to 10 patients with connective tissue disorders and Raynaud's phenomenon. Eight of the 10 patients improved clinically on ketanserin and none on placebo. Digital blood flow was assessed with laser Doppler flowmetry (LDF), photoplethysmography, and skin temperature measurements. Laser Doppler flowmetry was the most useful method, showing a significant reduction in recovery time after a standard cold provocation. Although the resting flow was not significantly improved, digital ulcers healed in four out of five patients, providing evidence of increased nutritive flow. The results of this study suggest that orally administered ketanserin may be an effective and well tolerated treatment for Raynaud's phenomenon associated with connective tissue disorders, especially scleroderma.

Topics: Adult; Clinical Trials as Topic; Connective Tissue Diseases; Double-Blind Method; Female; Fingers; Humans; Ketanserin; Microcirculation; Middle Aged; Piperidines; Raynaud Disease; Scleroderma, Systemic; Serotonin Antagonists; Ultrasonography

Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is important in the process of inflammation and fibrosis. The adenosine 5'-monophosphate-activated protein kinase (AMPK) enzyme can affect JAK/STAT pathway. Tofacitinib is a pan-JAK inhibitör. Metformin activates AMPK enzyme. We aimed to investigate the therapeutic efficacy of tofacitinib and metformin on IL-17 and TGF-β cytokines, skin fibrosis and inflammation in mouse model of systemic sclerosis (SSc). 40 Balb/c female mice were divided into 4 groups: (control, sham (BLM), tofacitinib and metformin). The mice in the tofacitinib group received oral tofacitinib (20 mg/kg/daily) and mice in the metformin group received oral metformin (50 mg/kg/day) for 28 days. At the end of 4th week, all groups of mice were decapitated and tissue samples were taken for analysis. Histopathological analysis of skin tissue was performed, and mRNA expressions of collagen 3A, IL-17 and TGF-β were assessed by real-time PCR and ELISA. Repeated BLM injections had induced dermal fibrosis. Moreover, the tissue levels of collagen 3A, IL-17 and TGF-β were elevated in the BLM group. Tofacitinib and metformin mitigated dermal fibrosis. They reduced dermal thickness and tissue collagen 3A, IL-17 and TGF-β levels. Tofacitinib and metformin demonstrated anti-inflammatory and anti-fibrotic effects in the mouse model of SSc.

Topics: Animals; Drug Therapy, Combination; Female; Fibrosis; Metformin; Mice; Mice, Inbred BALB C; Piperidines; Pyrimidines; Scleroderma, Systemic; Skin

Topics: Humans; Piperidines; Pyrimidines; Scleroderma, Diffuse; Scleroderma, Systemic; Skin

Systemic sclerosis (SSc) is a connective tissue disease with a significant morbidity and reduced survival of patients. Effective treatment and clinical control of the disease remain challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies only alleviate symptoms and slow disease progression. Here, we investigated the therapeutic potential of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in an in vitro model of autoimmunity.. PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release, and activation of the transcription factor NFκB were evaluated.. Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α mainly from the effector B cell population in patients with SSc. Importantly, small doses of ibrutinib (0.1 μM) preserved the production of immunoregulatory IL-10 while effectively inhibiting hyperactivated, profibrotic effector B cells. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment.. Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.

Topics: Adenine; Adult; Agammaglobulinaemia Tyrosine Kinase; Aged; Aged, 80 and over; B-Lymphocytes; Cells, Cultured; Cytokines; Female; Humans; Male; Middle Aged; NF-kappa B; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Scleroderma, Systemic

To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients.. In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNɑ or TGFβ and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays.. SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNɑ or TGFβ did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFɑ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib.. Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.

Topics: Adult; Aged; Becaplermin; Benzimidazoles; Cells, Cultured; Cytokines; Female; Fibroblasts; Humans; Inflammation; Macrophages; Male; Middle Aged; Monocytes; Piperidines; Platelet Factor 4; Receptors, Platelet-Derived Growth Factor; Scleroderma, Systemic

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-B

Topics: B-Lymphocyte Subsets; B-Lymphocytes; Cell Differentiation; Cell Proliferation; Cells, Cultured; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Memory; Interleukin-4; Janus Kinase Inhibitors; Ki-1 Antigen; Lymphocyte Activation; Piperidines; Pyrimidines; Scleroderma, Systemic; Th2 Cells

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 μM or 0.1 μM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of

Topics: Animals; Anti-Inflammatory Agents; Cell Differentiation; Chemokine CXCL13; Female; Gene Expression Regulation; Hypochlorous Acid; Janus Kinase 1; Janus Kinase 2; Janus Kinase 3; Lung; Lung Diseases, Interstitial; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Nitriles; Piperidines; Primary Cell Culture; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Pyrroles; Receptors, Immunologic; Scleroderma, Systemic; Suppressor of Cytokine Signaling 3 Protein

Topics: Animals; B-Lymphocytes; Cytokines; Humans; Piperidines; Pyrimidines; Rituximab; Scleroderma, Systemic; Th1 Cells; Th2 Cells

Topics: Arthritis; Female; Humans; Middle Aged; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Pyrroles; Scleroderma, Systemic

Topics: Adult; Humans; Janus Kinase Inhibitors; Male; Piperidines; Pyrimidines; Pyrroles; Scleroderma, Systemic

The hallmarks of systemic sclerosis (SSc) are autoimmunity, microangiopathy and fibrosis. Skin fibrosis is accompanied by attrition of the dermal white adipose tissue layer, and alterations in the levels and function of adiponectin. Since these findings potentially implicate adiponectin in the pathogenesis of SSc, we employed a novel pharmacological approach to augment adiponectin signaling using AdipoRon, an orally active adiponectin receptor agonist. Chronic treatment with AdipoRon significantly ameliorated bleomycin-induced dermal fibrosis in mice. AdipoRon attenuated fibroblast activation, adipocyte-to-myofibroblast transdifferentiation, Th2/Th17-skewed polarization of the immune response, vascular injury and endothelial-to-mesenchymal transition within the lesional skin. In vitro, AdipoRon abrogated profibrotic responses elicited by TGF-β in normal fibroblasts, and reversed the inherently-activated profibrotic phenotype of SSc fibroblasts. In view of these broadly beneficial effects on all three cardinal pathomechanisms underlying the clinical manifestations of SSc, pharmacological augmentation of adiponectin signaling might represent a novel strategy for the treatment of SSc.

Topics: Administration, Oral; Animals; Bleomycin; Disease Models, Animal; Female; Fibrosis; Humans; Inflammation; Mice, Inbred C57BL; Piperidines; Receptors, Adiponectin; Scleroderma, Systemic; Signal Transduction; Skin; Vascular System Injuries

Systemic sclerosis (SSc) is a multi-organ fibrotic disease with few treatment options. Activated fibroblasts are the key effector cells in SSc responsible for the excessive production of collagen and the development of fibrosis. Platelet-derived growth factor (PDGF), a potent mitogen for cells of mesenchymal origin, has been implicated in the activation of SSc fibroblasts. Our aim was to examine the therapeutic potential of crenolanib, an inhibitor of PDGF receptor signaling, in cultured fibroblasts and in angiotensin II-induced skin and heart fibrosis. Crenolanib effectively inhibited proliferation and migration of SSc and healthy control fibroblasts and attenuated basal and transforming growth factor-β-induced expression of CCN2/CTGF and periostin. In contrast to healthy control fibroblasts, SSc fibroblasts proliferated in response to PDGFAA, whereas a combination of PDGFAA and CCN2 was required to elicit a similar response in healthy control fibroblasts. PDGF receptor α mRNA correlated with CCN2 and other fibrotic markers in the skin of SSc patients. In mice challenged with angiotensin II, PDGF receptor α-positive cells were increased in the skin and heart. These PDGF receptor α-positive cells co-localized with PDGF receptor β, procollagen, and periostin. Treatment with crenolanib attenuated the skin and heart fibrosis. Our data indicate that inhibition of PDGF signaling presents an attractive therapeutic approach for SSc.

Topics: Animals; Benzimidazoles; Cell Adhesion Molecules; Cells, Cultured; Connective Tissue Growth Factor; Disease Models, Animal; Fibroblasts; Gene Expression Regulation; Humans; Immunoblotting; Mice; Mice, Inbred C57BL; Piperidines; Real-Time Polymerase Chain Reaction; Receptors, Platelet-Derived Growth Factor; RNA; RNA, Messenger; Scleroderma, Systemic; Signal Transduction

Systemic sclerosis (SSc) IgGs affecting the M(3)-muscarinic receptor (M(3)-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of M(3)-R and IgG-M(3)-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 +/- 1.2% caused by 10(-4) M BeCh was significantly attenuated to 8.3 +/- 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on K(+) depolarization and alpha(1)-adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-M(3)-R complex. SSc IgGs attenuated M(3)-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies.

Topics: Adrenergic alpha-Agonists; Adult; Aged; Aged, 80 and over; Anal Canal; Animals; Autoantibodies; Benzofurans; Bethanechol; Case-Control Studies; Dose-Response Relationship, Drug; Female; Humans; Immunoglobulins; In Vitro Techniques; Male; Middle Aged; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Myocytes, Smooth Muscle; Phenylephrine; Piperidines; Potassium Chloride; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Scleroderma, Systemic

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

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

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

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

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

The intestine is involved in about half of the cases with progressive-systemic sclerosis. Intestinal transit disturbances which are caused by neuropathy of the enteric nerve system occur frequently. However, upto-date only few studies which determined the effect of prokinetic drugs exist. Patients with intestinal involvement caused by progressive-systemic sclerosis were treated with the prokinetic drugs cisapride (20 mg, TID; n = 9), erythromycin (250 mg, TID; n = 7) and octreotide (50 micrograms s. c., at night time; n = 5) over a period of four weeks. At study entry and after each treatment period the transit times through the stomach, small and large intestine were evaluated by use of the metal-detector test. Gastric emptying was only accelerated by erythromycin (42 +/- 3 min vs. 54 +/- 6 min; p = 0.0422), whereas treatment with cisapride and octreotide did not result in significant changes (48 +/- 4 min; p = 0.3743 and 44 +/- 4 min; p = 0.1975; resp.). Small intestinal transit times were not altered significantly by cisapride (108 +/- 15 min vs. 108 +/- 9 min; p = 0.2733), crythromycin (92 +/- 8 min; p = 0.0707) or octreotide (106 +/- 12 min; p = 0.8927). Furthermore colonic transit was not fastened by none of the prokinetic agents (study entry: 68 +/- 12 h; cisapride: 88 +/- 12 h; p = 0.0569; erythromycin 77 +/- 14 h; p = 0.7349; octreotide 107 +/- 14 h; p = 0.8927). Four patients were withdrawn from the study because of diarrhea. Prokinetic drugs do not seem to have a major impact on intestinal transit times in patients with progressive-systemic sclerosis. The use of these drugs is limited because of frequent side effects.

Topics: Aged; Cisapride; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythromycin; Female; Gastric Emptying; Gastrointestinal Agents; Gastrointestinal Motility; Gastrointestinal Transit; Humans; Male; Middle Aged; Octreotide; Piperidines; Scleroderma, Systemic; Sympathomimetics; Treatment Outcome

Manometric study was performed to investigate the effects of cisapride, a new non-antidopaminergic gastrointestinal prokinetic compound, on interdigestive lower esophageal sphincter pressure (LESP) and gastroduodenal motility using infused catheter technique. The subjects consisted of 9 healthy volunteers and 29 patients with progressive systemic sclerosis (19), reflux esophagitis (8) and others (2). 4 mg of cisapride was given by bolus injection, continuous infusion or oral administration. The following results were obtained: Intravenous and oral cisapride increased LESP compared with basal pressure. Especially, bolus injection of cisapride caused a significant elevation of LESP during 30 minutes after administration. After administration of cisapride, gastroduodenal motility was accelerated gradually, then inducing IMC-like contractions. By bolus injection, IMC-like contractions were induced in healthy subjects more frequently than in patients group. On the other hand, motility index of stomach and duodenum showed persistent increase in patients group compared with healthy subjects. Cisapride-induced IMC-like contractions initiated from LES and upper part of stomach and mediated to duodenum, though aborad migration was not confirmed in the present study.

Topics: Adolescent; Adult; Aged; Cisapride; Duodenum; Electromyography; Esophagitis, Peptic; Esophagogastric Junction; Gastrointestinal Motility; Humans; Manometry; Middle Aged; Piperidines; Scleroderma, Systemic

Topics: Adolescent; Humans; Ketanserin; Leg Ulcer; Male; Piperidines; Scleroderma, Systemic