Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

Effect of a High-Fat Meal on Single-Dose Rencofilstat (CRV431) Oral Bioavailability in Healthy Human Subjects.

Rencofilstat (RCF) is a novel cyclophilin inhibitor under development for the treatment of nonalcoholic steatohepatitis and hepatocellular carcinoma. This phase 1, randomized, open-label study in healthy participants assessed the relative bioavailability of a single dose of RCF 225-mg soft gelatin capsules in both fasted and high-fat conditions. Forty-four participants were enrolled to either the fasted (n = 24) or the high-fat fed (n = 20) arm. Noncompartmental pharmacokinetics were evaluated following a single 225-mg oral dose. Administration of RCF with a high-fat meal led to increases in maximum concentration, area under the concentration-time curve (AUC) from time 0 to 24 hours, and AUC from time 0 to infinity fed-to-fasted geometric mean ratios of 102.2%, 114.5%, and 132.9%, respectively. All AUC geometric mean ratios were outside of the 80% to 125% range, suggesting that a high-fat meal can increase the extent of RCF exposure. Time to maximum concentration increased from 1.5 to 1.8 hours in the fasted and high-fat groups, respectively, suggesting slightly delayed absorption. High fat intake may delay gastric emptying while increasing the absorption and bioavailability of RCF. No treatment-emergent adverse events were observed in the fasted group, and 1 treatment-emergent adverse event occurred in the high-fat meal group. The differences in observed whole-blood concentrations are unlikely to have clinically relevant effects given the wide therapeutic index of RCF demonstrated in previous phase 1 studies.

Rencofilstat, a cyclophilin inhibitor: A phase 2a, multicenter, single-blind, placebo-controlled study in F2/F3 NASH.

Rencofilstat (RCF) demonstrated antifibrotic effects in preclinical models and was safe and well tolerated in Phase 1 studies. The aim of this Phase 2a study was safety, tolerability, pharmacokinetics, and exploration of efficacy biomarkers in subjects with nonalcoholic steatohepatitis (NASH). This Phase 2a, multicenter, single-blind, placebo-controlled study randomized 49 presumed F2/F3 subjects to RCF 75 mg once daily (QD), RCF 225 mg QD, or placebo for 28 days. Primary safety and tolerability endpoints were explored using descriptive statistics with post hoc analyses comparing active to placebo groups. Pharmacokinetics were evaluated using population pharmacokinetics methods. Efficacy was explored using biomarkers, transcriptomics, and lipidomics. RCF was safe and well tolerated, with no safety signals identified. The most frequently reported treatment-emergent adverse events were constipation, diarrhea, back pain, dizziness, and headache. No clinically significant changes in laboratory parameters were observed, and RCF pharmacokinetics were unchanged in subjects with NASH. Alanine transaminase (ALT) reduction was greater in active subjects than in placebo groups. Nonparametric analysis suggested that ALT reductions were statistically different in the 225-mg cohort compared with matching placebo: -16.3 ± 25.5% versus -0.7 ± 13.4%, respectively. ProC3 and C6M reduction was statistically significant in groups having baseline ProC3 > 15.0 ng/ml. RCF was safe and well tolerated after 28 days in subjects with presumed F2/F3 NASH. Presence of NASH did not alter its pharmacokinetics. Reductions in ALT, ProC3, and C6M suggest direct antifibrotic effects with longer treatment duration. Reductions in key collagen genes support a mechanism of action via suppression and/or regression of collagen deposition. Conclusion: These results support advancement of rencofilstat into a larger and longer Phase 2b study.

Cyclophilin inhibition as a potential treatment for nonalcoholic steatohepatitis (NASH).

Introduction: Cyclophilins are a family of diverse regulatory enzymes that have been studied for over 30 years; they participate in many pathophysiological processes. Genetic deletion or pharmacologic inhibition of cyclophilins has shown therapeutic effects in a wide spectrum of disease models, including liver disorders, and hence may be beneficial in treating nonalcoholic steatohepatitis (NASH).Areas Covered: This articles briefly describes cyclophilin isomerases and the main classes of cyclophilin antagonists; it then summarizes data showing cyclophilin participation in the major pathophysiological activities that occur in NASH.Expert Opinion: Optimization of therapeutic outcomes in the treatment of NASH may be best realized by targeting multiple pathologic pathways, especially when treating advanced stages of the disease. A preferred approach for achieving this goal is to use compounds such as cyclophilin inhibitors that simultaneously target multiple disease processes. The pleiotropic benefits of this drug class derive from the extraordinary functionality of prolyl isomerization as a regulatory mechanism and its evolutionary diversification into many biochemical pathways. Nonimmunosuppressive analogs of cyclosporine A are the most thoroughly characterized cyclophilin inhibitors and show significant potential to attenuate several of the major pathophysiological events in NASH - mitochondrial dysfunction, cellular injury and death, inflammation, and in particular, fibrosis.

A Pan-Cyclophilin Inhibitor, CRV431, Decreases Fibrosis and Tumor Development in Chronic Liver Disease Models.

Previous studies show that cyclophilins contribute to many pathologic processes, and cyclophilin inhibitors demonstrate therapeutic activities in many experimental models. However, no drug with cyclophilin inhibition as the primary mode of action has advanced completely through clinical development to market. In this study, we present findings on the cyclophilin inhibitor, CRV431, that highlight its potential as a drug candidate for chronic liver diseases. CRV431 was found to potently inhibit all cyclophilin isoforms tested-A, B, D, and G. Inhibitory constant or IC50 values ranged from 1 to 7 nM, which was up to 13 times more potent than the parent compound, cyclosporine A (CsA), from which CRV431 was derived. Other CRV431 advantages over CsA as a nontransplant drug candidate were significantly diminished immunosuppressive activity, less drug transporter inhibition, and reduced cytotoxicity potential. Oral dosing to mice and rats led to good blood exposures and a 5- to 15-fold accumulation of CRV431 in liver compared with blood concentrations across a wide range of CRV431 dosing levels. Most importantly, CRV431 decreased liver fibrosis in a 6-week carbon tetrachloride model and in a mouse model of nonalcoholic steatohepatitis (NASH). Additionally, CRV431 administration during a late, oncogenic stage of the NASH disease model resulted in a 50% reduction in the number and size of liver tumors. These findings are consistent with CRV431 targeting fibrosis and cancer through multiple, cyclophilin-mediated mechanisms and support the development of CRV431 as a safe and effective drug candidate for liver diseases. SIGNIFICANCE STATEMENT: Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. In this study, CRV431 is shown to potently inhibit multiple cyclophilin isoforms, possess several optimized pharmacological properties, and decrease liver fibrosis and tumors in mouse models of chronic liver disease, which highlights its potential to be the first approved drug primarily targeting cyclophilin isomerases.

In Vitro Phase I Metabolism of CRV431, a Novel Oral Drug Candidate for Chronic Hepatitis B.

The cytochrome P450-mediated Phase I in vitro metabolism of CRV431 was studied using selective chemical inhibition and recombinant human enzymes. Additionally, the metabolic profile of CRV431 in human, rat, and monkey liver microsomes was investigated. Liver microsomes were incubated for 0-80 min with CRV431, and the metabolite profile was assessed by electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS). CRV431 was extensively metabolized through oxidation to produce various hydroxylated and demethylated species. Species identified included monohydroxylated CRV431 (two distinct products), dihydroxylated CRV431, demethylated CRV431 (two distinct products), demethylated and hydroxylated CRV431 (two distinct products), didemethylated and hydroxylated CRV431, and didemethylated and dihydroxylated CRV431. The magnitude of metabolism was greatest in monkey, followed by human, followed by rat. Importantly, all of the species identified in human microsomes were correspondingly identified in monkey and/or rat microsomes. Human liver microsome studies using selective chemical inhibition, as well as studies using recombinant human cytochrome P450 enzymes, revealed that the major enzymes involved are cytochromes P450 3A4 and 3A5. Enzymes 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 are not involved in the in vitro metabolism of CRV431. This information will be useful for the further development of CRV431 both preclinically and clinically.

Cytochrome P450 3A and P-glycoprotein drug-drug interactions with voclosporin.

AIMS: Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. Pharmacokinetic drug interactions between voclosporin and a CYP3A inhibitor, inducer and substrate and a P-glycoprotein inhibitor and substrate were evaluated. METHODS: Voclosporin 0.4 mg kg(-1) was administered to 24 subjects in each of five studies, as follows: every 12 h (Q12H) alone and concomitantly with ketoconazole 400 mg once daily (QD); single dose before and single dose after rifampin 600 mg QD; Q12H where midazolam 7.5 mg was administered as a single dose alone before voclosporin and with last the dose of voclosporin; Q12H alone and concomitantly with verapamil 80 mg every 8 h; and Q12H with digoxin 0.25 mg QD. The noncompartmental pharmacokinetic parameters maximal concentration (Cmax ) and area under the concentration-time curve (AUC) were obtained, and geometric least squares mean ratios and 90% confidence intervals were evaluated. RESULTS: Ketoconazole increased voclosporin Cmax (6.4-fold) and AUC (18-fold); rifampin reduced voclosporin AUC (0.9-fold); voclosporin did not change exposure of midazolam or α-hydroxy-midazolam; verapamil increased voclosporin Cmax (2.1-fold) and AUC (2.7-fold); and voclosporin increased digoxin Cmax (0.5-fold), AUC (0.25-fold) and urinary excretion (0.2-fold). CONCLUSIONS: Administration of voclosporin concomitantly with strong inhibitors and inducers of CYP3A resulted in increased and decreased exposures, respectively, and should be considered contraindicated. Drug-drug interactions involving voclosporin and CYP3A substrates are not expected. Administration of voclosporin concomitantly with inhibitors and substrates of P-glycoprotein resulted in increased voclosporin and substrate exposures, respectively. Appropriate concentration and safety monitoring is recommended with co-administration of voclosporin and P-glycoprotein substrates and inhibitors.

Voclosporin food effect and single oral ascending dose pharmacokinetic and pharmacodynamic studies in healthy human subjects.

Voclosporin (VCS) is a novel calcineurin (CN) inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the single ascending dose pharmacokinetics (PK) and pharmacodynamics (PD, CN activity) of VCS and the effect of food. VCS was administered orally in single doses of 0.25 through 4.5 mg/kg in 62 subjects in the single ascending dose study and as a single oral 1.5 mg/kg dose to 18 subjects after fasting, consumption of a low-fat and high-fat meal. Non-compartmental PK, PD, and PKPD correlation were evaluated. Following single oral doses, systemic exposure increased in a linear manner and demonstrated 1:1 dose-proportional, first-order linear PK above 1.5 mg/kg. VCS inhibited CN activity in a dose-related fashion with maximal inhibition peaking at 3.0 mg/kg. PKPD correlation indicated an EC50 of 78.3 ± 6.8 ng/mL. Administration of VCS with a low-fat and high-fat meal decreased C(max) by 29% and 53%, respectively, and AUC(inf) by 15% and 25%, respectively. Following ascending single doses of VCS, exposure increased in a linear fashion. A food effect on exposure was demonstrated, with a more pronounced effect following a high-fat meal. VCS concentrations were also found to correlate with CN activity.

ISA247: quality of life results from a phase II, randomized, placebo-controlled study.

BACKGROUND: Psoriasis is a chronic skin condition that can negatively affect a patient's quality of life (QoL), often hindering social functioning. ISA247, a novel psoriatic agent, has shown clinical efficacy in moderate to severe psoriasis sufferers, but its effect on QoL is currently not reported. OBJECTIVE: The objective of this study was to assess the effect of ISA247 on the QoL in patients with stable, plaque-type psoriasis. METHODS: A phase II, randomized, double-blind, placebo-controlled, parallel-group, multicenter study assessed the effects of ISA247 doses of 0.5 mg/kg/d (n = 77) or 1.5 mg/kg/d (n = 83) compared with placebo (n = 41) for 12 weeks. QoL was assessed using the Dermatology Life Quality Index (DLQI) and Psoriasis Disability Index (PDI) scales. RESULTS: ISA247 treatment (pooled groups) significantly improved QoL scores as assessed by both the DLQI and the PDI compared with those receiving placebo (p < .05). Treatment with the higher dose of 1.5 mg/kg/d demonstrated a significantly greater response to many of the QoL scales compared with the 0.5 mg/kg/d group (p < .05). CONCLUSIONS: ISA247 appears to improve the QoL while also providing effective treatment for chronic, moderate to severe, plaque-type psoriasis.

A randomized, multicenter, double-blind, placebo-controlled phase 2 trial of ISA247 in patients with chronic plaque psoriasis.

BACKGROUND: Use of current oral calcineurin inhibitors for the treatment of psoriasis is limited by toxicity. OBJECTIVE: Evaluate the safety and efficacy of ISA247, a new oral calcineurin inhibitor, in plaque psoriasis patients. METHODS: This 12-week, randomized, double-blind, placebo-controlled, parallel-group study included 201 plaque psoriasis patients with > or = 10% body surface area involvement. Patients were randomized to placebo, ISA247 0.5 mg/kg/d, and ISA247 1.5 mg/kg/d groups. End points included a 2-point reduction in the Static Global Assessment score and a 75% reduction in the Psoriasis Area and Severity Index. RESULTS: A 2-point SGA reduction was achieved in 0% (placebo), 15.6% (0.5 mg/kg/d), and 45.1% (1.5 mg/kg/d) (P < .0001). A 75% reduction in the Psoriasis Area and Severity Index was achieved in 0% (placebo), 18.2% (0.5 mg/kg/d), and 66.7% (1.5 mg/kg/day) (P < .0001). While serum creatinine increased in patients treated with ISA247 1.5 mg/kg/d, it remained within the normal range. LIMITATIONS: Longer-term studies are needed to evaluate the effect of ISA247 on renal function. CONCLUSION: ISA247 appears safe and effective for treating moderate to severe psoriasis.

Influence of cimetidine co-administration on the pharmacokinetics of acebutolol enantiomers and its metabolite diacetolol in a rat model: the effect of gastric pH on double-peak phenomena.

Acebutolol (AC) is a chiral beta-adrenergic receptor-blocking agent, which has been shown to be clinically effective in hypertension. The plasma concentration-time profiles of AC exhibit two peaks following oral administration of racemate for both R- and S-enantiomers. In the present study, the absorption of AC after a single dose was studied as a function of gastric pH in male Sprague-Dawley rats. Furthermore, the effect of cimetidine (CIM) on pharmacokinetic parameters of AC and its metabolite diacetolol (DC) was evaluated. CIM (50 mg kg(-1)) was administered via jugular vein 30 min prior to AC administration to elevate the intragastric pH. AC (50 mg kg(-1)) was administered orally by gavage and serial blood samples were collected before and for 8h after AC administration. Plasma samples were assayed for AC and DC, pharmacokinetic parameters were estimated and compared with those of control. The concentration-time profiles and the pharmacokinetics of AC were unchanged after co-administration of CIM. The oral absorption of AC, as assessed by the area under the plasma concentration-time curve (AUC) and the amount of unchanged drug recovered in the urine were not affected by CIM. The amount of metabolite recovered in the urine and the rate of absorption, however, were significantly altered. These are unlikely to be of clinically importance as we have found that the extent of absorption was not changed. We, therefore, concluded that intragastric elevation of pH has no effect either on generation of multiple peaking or on pharmacokinetic parameters of AC.

Amelioration of accelerated collagen induced arthritis by a novel calcineurin inhibitor, ISA(TX)247.

OBJECTIVE: To examine the efficacy and toxicity of ISA(TX)247, a novel calcineurin inhibitor, in comparison to cyclosporine (cyclosporin A, CSA) and placebo in established collagen induced arthritis. ISA(TX)247 has up to 3-fold greater potency than CSA in an in vitro whole blood calcineurin inhibition assay and in in vivo solid organ and cell transplantation models. Phase I clinical trials show no discernible nephrotoxicity. METHODS: Type II collagen immunized DBA/Lac J mice with established arthritis were randomized to treatment with ISA(TX)247 (125/250/500 microg/mouse), CSA (250/500 microg/mouse), or drug vehicle, by daily intraperitoneal injection for 10 days from the onset of clinical arthritis. RESULTS: A significant dose dependent reduction in clinical severity was observed in ISA(TX)247 treated but not in CSA treated animals 10 days after the onset of established arthritis, and when examined by area under the curve analysis during the treatment period. Significant improvement in paw swelling (p < 0.001), synovial histology (p < 0.001), and articular cartilage damage scores (p = 0.002) was also noted in ISA(TX)247 treated animals, even in the 125 pg dose group (p = 0.03 for paw swelling and synovial histology). By comparison, CSA had no significant effect on either synovial inflammation or articular cartilage damage. ISA(TX)247 (500 microg dose group) was the only treatment to significantly decrease the development of proximal interphalangeal joint erosions (p < 0.05). A significant reduction in Type II collagen antibody titer was noted in ISA(TX)247 animals in both 250 microg (p = 0.02) and 500 microg (p = 0.004) dosage groups, but only in the 500 microg group for CSA (p = 0.004). Treatment was well tolerated, with no significant toxicity in ISA(TX)247 groups. CONCLUSION: ISA(TX)247 demonstrates efficacy and safety in the treatment of established collagen induced arthritis. Together with its improved potency and nephrotoxicity profile in comparison to CSA, this agent warrants further clinical investigation in autoimmune disease. Phase II studies in rheumatoid arthritis have been initiated.