Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis

Abstract

Background & aims: Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis.

Methods: Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses.

Results: CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight.

Conclusions: CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).

Conflict of interest statement

Competing Interests: EL and HJ are employees of Tobira Therapeutics, Inc. GM is on the Scientific Advisory Board of Tobira Therapeutics, Inc. and has served on the Board of Directors of Tobira Therapeutics, Inc. and holds stock options in Tobira Therapeutics, Inc. RR, FH, HC, TH and HY have received research funding from Tobira Therapeutics, Inc. LPR, CP, DP and TR have no conflicts of interest to declare. MT is a member of an institution that received clinical trial funding or research grants from Tobira Therapeutics, Inc. GW is a consultant for Tobira Therapeutics, Inc. and holds stock options in Tobira Therapeutics, Inc. SLF is on the Scientific Advisory Board of Tobira Therapeutics, Inc., has performed research studies supported by Tobira Therapeutics, Inc. and holds stock options in Tobira Therapeutics, Inc. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. CVC Effect on Monocyte/Macrophage Migration.

(A) Monocyte/macrophage recruitment reduced in TG-induced mouse model of peritonitis after pre-treatment with CVC (PO BID or QD dosing); (B) cenicriviroc (1 μM) inhibits CCL2-mediated chemotaxis of activated murine macrophages (F4/80+/CD11b+) ex vivo. *p < 0.05 vs. TG + vehicle control; **p < 0.01 vs. TG + vehicle control; ***p < 0.001 vs. TG + vehicle control; † p < 0.001 vs. TG + DEX; ‡ p = 0.018 vs. vehicle control. aVehicle control: 0.5% [w/v] methylcellulose + 1% Tween®-80 (pH ~1.3). BID, twice daily; CCL2, C-C chemokine ligand 2; CVC, cenicriviroc; CVC5, CVC 5 mg/kg/day; CVC20, CVC 20 mg/kg/day; CVC100, CVC 100 mg/kg/day; DEX, dexamethasone; PO, oral gavage; QD, once daily; SEM, standard error of the mean; TG, thioglycollate.

Fig 2. Reduction of Plasma ALT Levels in NASH Model.

*p < 0.05 vs. vehicle control. aVehicle control: 0.5% [w/v] methylcellulose + 1% Tween®-80 (pH ~1.3). ALT, alanine aminotransferase; BID, twice daily; CVC, cenicriviroc; CVC20, CVC 20 mg/kg/day; CVC100, CVC 100 mg/kg/day; NASH, non-alcoholic steatohepatitis; SD, standard deviation.

Fig 3. Reduction in Fibrosis.

(A) Representative micrographs of Sirius red-stained liver sections in the rat TAA model (early intervention and established fibrosis; 100x), mouse NASH model (200x) and mouse UUO model (200x). (B) Reduction in collagen deposition in the rat TAA model (early intervention and established fibrosis), mouse NASH modela and mouse UUO modelb. *p < 0.05 vs. sham control; **p < 0.01 vs. vehicle control; ***p < 0.001 vs. vehicle control; †p < 0.05 vs. UUO + vehicle control. aPerivascular area subtracted; bData presented exclude a single outlier from an animal in the CVC20 group, which had a CVF value >2 SDs higher than any other animal in the group. CVF, Collagen Volume Fraction; CVC, cenicriviroc; NASH, non-alcoholic steatohepatitis; SD, standard deviation; SEM, standard error of the mean; TAA, thioacetamide; UUO, unilateral ureteral obstruction.

Fig 4. CVC Effects on Extracellular Matrix Protein Content.

Mean expression of (A) collagen type I and (B) alpha-SMA in the TAA model compared to vehicle control; mean expression of hydroxyproline content in (C) NASH model and (D) UUO model. alpha-SMA, alpha-smooth muscle actin; BID, twice daily; CVC, cenicriviroc; NASH, non-alcoholic steatohepatitis; QD, once daily; SD, standard deviation; TAA, thioacetamide; UUO, unilateral ureter obstruction.