Eric Lefebvre

Efficacy and safety study of cenicriviroc for the treatment of non-alcoholic steatohepatitis in adult subjects with liver fibrosis: CENTAUR Phase 2b study design

BACKGROUND Non-alcoholic steatohepatitis (NASH) is often accompanied by liver fibrosis, which can progress to cirrhosis; C-C chemokine receptors type 2 and 5 (CCR2/CCR5), which mediate interactions driving inflammation and fibrosis, are promising treatment targets. Cenicriviroc (CVC), a dual-CCR2/CCR5 antagonist, has potent anti-inflammatory and antifibrotic activity in animal models; in HIV-positive subjects it reduced soluble CD14 levels, aspartate aminotransferase-to-platelet count ratio index, and non-invasive hepatic fibrosis risk scores; favorable tolerability was demonstrated in ~600 subjects. Efficacy and safety of CVC 150 mg for treating NASH with liver fibrosis are being evaluated over 2 years (primary endpoint at Year 1 [Y1]). DESIGN Phase 2b, randomized, double-blind, placebo-controlled, multinational study (CENTAUR; NCT02217475). Adults with histological evidence of NASH, non-alcoholic fatty liver disease activity score (NAS) ≥ 4, and liver fibrosis (stages 1-3 NASH clinical research network system) enrolled. Subjects have increased risk of progression to cirrhosis due to ≥1 characteristic: type 2 diabetes; body mass index > 25 kg/m(2) with ≥1 feature of metabolic syndrome; bridging fibrosis and/or NAS ≥ 5. Liver biopsy evaluation at Screening, Y1, and Year 2 (Y2). OBJECTIVES Assess histologic improvement (≥2-point in NAS with ≥1-point improvement in >1 category) without worsening of fibrosis at Y1 (primary); evaluate complete NASH resolution without worsening of fibrosis at Y2 (key secondary). DISCUSSION CENTAUR is the first prospective study evaluating an oral agent exclusively enrolling subjects with NASH and liver fibrosis, with increased risk of developing cirrhosis. It will compare shorter versus longer CVC treatment and assess correlations between decreased inflammation and fibrosis.

A randomized, placebo‐controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis

The aim of this study was to evaluate cenicriviroc (CVC), a dual antagonist of CC chemokine receptor types 2 and 5, for treatment of nonalcoholic steatohepatitis (NASH) with liver fibrosis (LF). A randomized, double‐blind, multinational phase 2b study enrolled subjects with NASH, a nonalcoholic fatty liver disease activity score (NAS) ≥4, and LF (stages 1‐3, NASH Clinical Research Network) at 81 clinical sites. Subjects (N = 289) were randomly assigned CVC 150 mg or placebo. Primary outcome was ≥2‐point improvement in NAS and no worsening of fibrosis at year 1. Key secondary outcomes were: resolution of steatohepatitis (SH) and no worsening of fibrosis; improvement in fibrosis by ≥1 stage and no worsening of SH. Biomarkers of inflammation and adverse events were assessed. Full study recruitment was achieved. The primary endpoint of NAS improvement in the intent‐to‐treat population and resolution of SH was achieved in a similar proportion of subjects on CVC (N = 145) and placebo (N = 144; 16% vs. 19%, P = 0.52 and 8% vs. 6%, P = 0.49, respectively). However, the fibrosis endpoint was met in significantly more subjects on CVC than placebo (20% vs. 10%; P = 0.02). Treatment benefits were greater in those with higher disease activity and fibrosis stage at baseline. Biomarkers of systemic inflammation were reduced with CVC. Safety and tolerability of CVC were comparable to placebo. Conclusion: After 1 year of CVC treatment, twice as many subjects achieved improvement in fibrosis and no worsening of SH compared with placebo. Given the urgent need to develop antifibrotic therapies in NASH, these findings warrant phase 3 evaluation. (Hepatology 2018;67:1754‐1767).

Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte‐derived macrophages (MoMFs) massively accumulated in injured liver within 12‐24 hours, whereas the number of tissue‐resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C‐C motif) receptor 2 (CCR2), given that Ccr2−/− mice display reduced infiltration of monocytes and attenuated liver injury post‐APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2+ monocytes infiltrate liver as early as 8‐12 hours post‐APAP overdose and form dense cellular clusters around necrotic areas. CCR2+ MoMFs express a distinct pattern of inflammatory, but also repair‐associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post‐APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C‐C motif) ligand (CCL2; by the inhibitor, mNOX‐E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP‐induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a proinflammatory polarization. Conclusion: Infiltrating monocyte‐derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667‐1682)

Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis

Macrophages are key regulators of liver fibrosis progression and regression in nonalcoholic steatohepatitis (NASH). Liver macrophages comprise resident phagocytes, Kupffer cells, and monocyte‐derived cells, which are recruited through the chemokine receptor C‐C motif chemokine receptor 2 (CCR2). We aimed at elucidating the therapeutic effects of inhibiting monocyte infiltration in NASH models by using cenicriviroc (CVC), an oral dual chemokine receptor CCR2/CCR5 antagonist that is under clinical evaluation. Human liver tissues from NASH patients were analyzed for CCR2+ macrophages, and administration of CVC was tested in mouse models of steatohepatitis, liver fibrosis progression, and fibrosis regression. In human livers from 17 patients and 4 controls, CCR2+ macrophages increased parallel to NASH severity and fibrosis stage, with a concomitant inflammatory polarization of these cluster of differentiation 68+, portal monocyte‐derived macrophages (MoMF). Similar to human disease, we observed a massive increase of hepatic MoMF in experimental models of steatohepatitis and liver fibrosis. Therapeutic treatment with CVC significantly reduced the recruitment of hepatic Ly‐6C+ MoMF in all models. In experimental steatohepatitis with obesity, therapeutic CVC application significantly improved insulin resistance and hepatic triglyceride levels. In fibrotic steatohepatitis, CVC treatment ameliorated histological NASH activity and hepatic fibrosis. CVC inhibited the infiltration of Ly‐6C+ monocytes, without direct effects on macrophage polarization, hepatocyte fatty acid metabolism, or stellate cell activation. Importantly, CVC did not delay fibrosis resolution after injury cessation. RNA sequencing analysis revealed that MoMF, but not Kupffer cells, specifically up‐regulate multiple growth factors and cytokines associated with fibrosis progression, while Kupffer cells activated pathways related to inflammation initiation and lipid metabolism. Conclusion: Pharmacological inhibition of CCR2+ monocyte recruitment efficiently ameliorates insulin resistance, hepatic inflammation, and fibrosis, corroborating the therapeutic potential of CVC in patients with NASH. (Hepatology 2018;67:1270‐1283)

Prolonged cenicriviroc therapy reduces hepatic fibrosis despite steatohepatitis in a diet-induced mouse model of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease projected to become the leading cause of cirrhosis and liver transplantation in the next decade. Cenicriviroc (CVC), a dual chemokine receptor 2 and 5 antagonist, prevents macrophage trafficking and is under clinical investigation for the treatment of human NASH fibrosis. We assessed the efficacy and durability of short and prolonged CVC therapy in a diet‐induced mouse model of NASH, the choline deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) model. C57BL/6 mice received 4 or 14 weeks of standard chow or the CDAHFD. CVC (10 mg/kg/day and 30 mg/kg/day for 4 weeks and 20 mg/kg/day and 30 mg/kg/day for 14 weeks) was initiated simultaneously with the CDAHFD. At 4 and 14 weeks, livers were harvested for histology and flow cytometric analyses of intrahepatic immune cells. High‐dose CVC (30 mg/kg/day) therapy in CDAHFD mice for 4 or 14 weeks inhibited intrahepatic accumulation of Ly6Chigh bone marrow‐derived macrophages. Prolonged CVC therapy (14 weeks) yielded no significant differences in the total intrahepatic macrophage populations among treatment groups but increased the frequency of intrahepatic anti‐inflammatory macrophages in the high‐dose CVC group. Despite ongoing steatohepatitis, there was significantly less fibrosis in CDAHFD mice receiving high‐dose CVC for 14 weeks based on histologic and molecular markers, mirroring observations in human NASH CVC trials. CVC also directly inhibited the profibrotic gene signature of transforming growth factor‐β‐stimulated primary mouse hepatic stellate cells in vitro. Conclusion: CVC is a novel therapeutic agent that is associated with reduced fibrosis despite ongoing steatohepatitis. Its ability to alter intrahepatic macrophage populations and inhibit profibrogenic genes in hepatic stellate cells in NASH livers may contribute to its observed antifibrotic effect. (Hepatology Communications 2018;2:529‐545)

Pharmacological inhibition of CCR2/5 signaling prevents and reverses alcohol‐induced liver damage, steatosis and inflammation in mice

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C‐C chemokine receptor type 2 (Ccr2) and C‐C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C‐C chemokine ligand types 2 (CCL2), and C‐C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as “prevention” throughout the alcohol feeding or as “treatment” started after the development of ALD. Alcohol‐induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen‐1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol‐related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi) and reduced proinflammatory Ly6Chi MØ in livers of alcohol‐fed mice. CVC increased liver T‐cell numbers and attenuated Il‐2 expression without an effect on CD69+ or CD25+ T‐cell expression. In vitro, CVC inhibited CCL2‐induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation‐related protein (Adrp), whereas it augmented acyl‐coenzyme A oxidase 1 (Acox‐1), proliferator‐activated receptor gamma co‐activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide‐induced liver injury (TNF‐α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP‐ribose polymerase [PARP] and caspase‐3 [CASP‐3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol‐induced steatohepatitis and liver damage.

The Critical Role of Chemokine (C–C Motif) Receptor 2-Positive Monocytes in Autoimmune Cholangitis

The therapy of primary biliary cholangitis (PBC) has lagged behind other autoimmune diseases despite significant improvements in our understanding of both immunological and molecular events that lead to loss of tolerance to the E2 component of pyruvate dehydrogenase, the immunodominant autoepitope of PBC. It is well known that Ly6Chi monocytes are innate immune cells infiltrating inflammatory sites that are dependent on the expression of C–C motif chemokine receptor 2 (CCR2) for emigration from bone marrow. Importantly, humans with PBC have a circulating monocyte pro-inflammatory phenotype with macrophage accumulation in portal tracts. We have taken advantage of an inducible chemical xenobiotic model of PBC and recapitulated the massive infiltration of monocytes to portal areas. To determine the clinical significance, we immunized both CCR2-deficient mice and controls with 2OA-BSA and noted that CCR2 deficiency is protective for the development of autoimmune cholangitis. Importantly, because of the therapeutic potential, we focused on inhibiting monocyte infiltration through the use of cenicriviroc (CVC), a dual chemokine receptor CCR2/CCR5 antagonist shown to be safe in human trials. Importantly, treatment with CVC resulted in amelioration of all aspects of disease severity including serum total bile acids, histological severity score, and fibrosis stage. In conclusion, our results indicate a major role for Ly6Chi monocytes and for CCR2 in PBC pathogenesis and suggest that inhibition of this axis by CVC should be explored in humans through the use of clinical trials.

APOBEC3G/3A Expression in Human Immunodeficiency Virus Type 1-Infected Individuals Following Initiation of Antiretroviral Therapy Containing Cenicriviroc or Efavirenz

Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro. However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4+ cell count and CD4+/CD8+ cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.

Rates of and Factors Associated With Placebo Response in Trials of Pharmacotherapies for Nonalcoholic Steatohepatitis: Systematic Review and Meta-analysis

Background & Aims: It is important to know the extent of the placebo effect in designing randomized controlled trials for patients with nonalcoholic steatohepatitis (NASH), to accurately calculate sample size and define treatment endpoints. Methods: We performed a systematic review and meta‐analysis of the placebo groups from randomized controlled trials of adults with NASH that provided histologic and/or magnetic resonance image‐based assessments. We identified trials through a comprehensive search of MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and Scopus, from each databases inception through January 2, 2018. Results: We identified 39 randomized controlled trials, comprising 1463 patients who received placebo. Histologic assessment data (the nonalcoholic fatty liver disease activity scores, NAS) were available from 956 patients; magnetic resonance spectroscopy data were available from 295 patients and magnetic resonance proton density fat fraction measurements from 61 patients. Overall, 25% of patients given placebo had an improvement in NAS by 2 or more points (95% CI, 21%–29%) with a small amount of heterogeneity (I2 = 27%). There were improvements by at least 1 point in steatosis scores of 33% ± 3% of patients, in hepatocyte ballooning scores of 30% ± 3% of patients, in lobular inflammation scores of 32% ± 3% of patients, and in fibrosis scores of 21% ± 3% of patients, with a moderate amount of heterogeneity among trials (I2 range, 51%–63%). Patients given placebo had a statistically significant improvement in NAS (by 0.72 ± 0.19), with a large amount of heterogeneity (I2 = 96%). Univariate and multivariate meta‐regression showed that trials with a higher baseline NAS, those conducted in South America, and those in which patients had a decrease in body mass index, were associated with greater improvements in NAS among patients given placebo. Patients given placebo had significant reductions in intrahepatic triglyceride, measured by magnetic resonance spectroscopy (by 1.45% ± 0.54%) with moderate heterogeneity (I2 = 40%), and in magnetic resonance proton density fat fraction (by 2.43 ± 0.89), without heterogeneity (I2 = 0). Mean serum levels of alanine and aspartate aminotransferases decreased significantly (by 11.7 ± 3.8 U/L and 5.9 ± 2.1 U/L, respectively; P < .01 for both). Conclusions: In a meta‐analysis of randomized controlled trials of NASH, patients given placebo have significant histologic, radiologic, and biochemical responses. The placebo response should be considered in designing trials of agents for treatment of NASH.