Targeting CCR2/5 in the treatment of nonalcoholic steatohepatitis (NASH) and fibrosis: opportunities and challenges

KEYWORDS:

• Nonalcoholic steatohepatitis
• liver fibrosis
• pharmacotherapy
• hepatocellular carcinoma
• cenicriviroc
• NAFLD
• macrophage
• CCR2 inhibition
• CCR5 inhibition

1. Introduction

In parallel to the obesity pandemic, nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in adults worldwide [1]. Especially its inflammatory form, nonalcoholic steatohepatitis (NASH), but ‘simple steatosis’ as well, can progress to fibrosis, cirrhosis and hepatocellular carcinoma. In these patients, the degree of liver fibrosis is the main histological predictor of liver-related and overall mortality [2].

Although this is an area of intense investigation, there is currently no approved pharmacotherapy for NAFLD. Whereas most compounds intend to correct aberrant metabolism by targeting insulin resistance and hepatic fat accumulation, chemokine (receptor) inhibitors act on inflammatory pathways with the ultimate aim to attenuate, or even reverse, scar tissue formation [3]. In this regard, inhibitors of either C-C chemokine receptors (CCR) 2 and 5 or their respective ligands are of interest for treating NAFLD. CCR2/5 inhibitors have proven to be effective in reducing fibrosis in animal models of NASH and fibrosis through targeting disease-promoting liver macrophages and hepatic stellate cells and have advanced to clinical trials.

2. Rationale for the use of CCR2/5 inhibitors in NASH

While numerous pathways are involved in the pathophysiology of NASH, advances in basic and translational research have implicated macrophage-mediated inflammation and hepatic stellate cell activation as key drivers of progression at multiple stages of the disease. In the liver, tissue-resident and self-renewing Kupffer cells, which have homeostatic, metabolic and tolerogenic functions, can be distinguished from pro-inflammatory monocyte-derived macrophages (MoMF), derived from circulating monocytes that infiltrate the liver upon injury (e.g. after fat accumulation), in a CCR2-dependent manner (reviewed extensively in [4,5]) (Figure 1). Indeed, CCR2⁺ macrophages accumulate in the periportal areas in patients with NASH and advanced fibrosis [6]. These MoMF promote inflammation and angiogenesis, and directly stimulate hepatic stellate cell activation [4]. Hepatic stellate cells are the main source of matrix-producing myofibroblasts [7]. Research in preclinical animal models, including the streptozotocin/high-fat diet, western diet and methionine/choline-deficient (MCD) diet models, supports the concept that blocking CCL2-CCR2-mediated macrophage infiltration improves NASH and fibrosis [6,8,9]. These papers demonstrated efficacy in established steatohepatitis, i.e. if treatment was initiated after induction of liver injury [6,8]. A similar effect has recently been reported in a model for alcoholic liver disease [10]. In line, human liver cirrhosis is characterized by the presence of scar-associated macrophages with a fibrogenic phenotype [11].

Figure 1. CCR2 and CCR5 as targets in NASH and liver fibrosis. The accumulation of hepatic fat causes cellular stress and the release of chemokines, notably CCL2 (= MCP-1), from hepatocytes, Kupffer cells, endothelial and stellate cells. This promotes the infiltration of circulating CCR2⁺ monocytes, which differentiate into monocyte-derived macrophages. These cells contribute to the progression of NASH by maintaining an inflammatory environment and by activating hepatic stellate cells, promoting collagen deposition. These CCR2⁺ Ly6C^high macrophages can mature into Ly6C^low restorative macrophages which boost resolution of inflammation. CCR5 is expressed by stellate cells and is involved in their profibrogenic activation and proliferation. This concept is based on experimental mouse models of steatohepatitis and fibrosis.

Pro-inflammatory macrophages can mature and acquire a restorative phenotype, which is involved in the regression of fibrosis [4]. Therefore, timing the inhibition of macrophage infiltration could be crucial, if CCR2 inhibitors are being used in patients with fibrosis. Notably, CCR2/5 inhibition did not delay fibrosis regression during reversal of MCD diet-induced steatohepatitis in mice and thioacetamide-induced fibrosis in rats [6,8]. As fibrosis regression can lead to a spike in pro-inflammatory monocyte infiltration, CCL2 blockade even enhanced fibrosis resolution in one study [12].

CCR5 is expressed by lymphocyte subsets as well as hepatic stellate cells, in which it regulates migration and proliferation. Indeed, in the bile duct ligation rodent model, both the broad chemokine inhibitor 35 k as well as CCR5 deficiency reduced liver fibrosis [13]. Carbon tetrachloride-induced fibrosis in mice was similarly attenuated by CCL5 inhibition [14]. In the human stellate cell line LX-2, the CCR5 inhibitor maraviroc blocked cell cycle progression and inhibited collagen accumulation [15].

Taken together, these data underpin the potential of CCR2/5 inhibition in the treatment of fibrotic NASH.

3. Clinical efficacy on NASH and fibrosis

The CENTAUR trial (NCT02217475) is the only phase IIb using a CCR2/CCR5 inhibitor, namely cenicriviroc (CVC) [16], in the field of NAFLD/NASH so far. The main eligibility criteria were histological evidence of NASH (joint presence of steatosis, ballooning and lobular inflammation) with a NAFLD activity score (NAS) ≥4 and F1 to F3 stage fibrosis. In order to select a population at higher risk of disease progression, patients either had a NAS ≥5, type 2 diabetes, or overweight/obesity with bridging fibrosis and/or ≥1 component of the metabolic syndrome. The primary outcome was a ≥ 2-point improvement in the NAS without worsening of fibrosis at a liver biopsy after 1 year of treatment. Key secondary outcomes were the complete histological resolution of NASH without worsening of fibrosis, and improvement in fibrosis without worsening of steatohepatitis.

289 patients were included in the study and were randomized 2:1:1 to 150 mg CVC for two years; placebo during year one and 150 mg CVC during year two, and placebo for two years. The primary endpoint was not met, as CVC did not significantly increase the proportion of patients with a ≥ 2-point improvement in NAS. On the other hand, twice as many patients on CVC achieved fibrosis improvement without worsening of NASH (20% vs. 10%, p = 0.0234) after one year [16].

Full results from the second year of follow-up have not yet been published. Tentative data presented at the EASL 2018 meeting (abstract GS-002) indicate that patients treated with CVC for two years compared to two years of placebo had similar rates of ≥1 fibrosis stage resolution, although a numerically higher proportion of patients experienced ≥2 fibrosis stages improvement.

CVC appeared to have a favorable safety profile. No life-threatening adverse events occurred in this trial, and an equal proportion of patients discontinued the drug due to adverse events (CVC 7% vs. placebo 8.3%) [16].

4. Future developments

CVC is now under evaluation in the phase III AURORA trial (NCT03028740), which will include patients with NASH and fibrosis stages F2 and F3. Based on the outcome data in CENTAUR, the primary outcome after 1 year is improvement in histological fibrosis (≥1 stage) without worsening of steatohepatitis. This outcome will be used as a surrogate by the FDA for interim approval of CVC [17]. The long-term efficacy will be assessed after 5 years and should provide data on ‘hard endpoints’, namely the composite of progression to cirrhosis, liver-related clinical outcomes and overall mortality.

Aside from CVC, other CCR2, CCR5 or CCR2/5 inhibitors will likely advance to clinical trials, either in isolation or in combination with other drugs, specifically for the treatment of NASH-related fibrosis.

5. Expert opinion

5.1. Improving fibrosis without treating NASH

CCR2/5 and other chemokine inhibitors are one of the few classes of drugs under development that do not affect the upstream metabolic mechanisms leading to fibrosis progression, such as steatosis or hepatocyte ballooning. Surprisingly, CVC did not improve the severity of steatohepatitis, despite the clear effect on macrophage accumulation in various murine models. One possible explanation is that the histological evaluation of inflammation in patients is not comprehensive enough, as compared to the detailed immunohistochemical and flow cytometric analyses that have been performed on macrophage subsets in mice. In support of an anti-inflammatory effect, the circulating markers high-sensitive CRP, fibrinogen and interleukin-1β and −6 were significantly reduced in CVC-treated patients in the CENTAUR trial [16]. Nevertheless, it remains to be shown that their antifibrotic effect is durable in the long term and is not counteracted by continued metabolic stress and inflammation.

Therefore, it makes sense to combine CCR2/5 inhibitors with one or more drugs acting on upstream metabolic mechanisms, such as insulin resistance or lipotoxicity. As the proportion of patients clearly benefitting from CVC (and other drugs under investigation) is rather low, combination therapy might have synergistic effects. One may speculate that combining a CCR2/5 inhibitor with strong anti-steatotic drugs (such as recombinant fibroblast growth factor (FGF)-21 or FGF-19) can improve NASH and fibrosis more effectively than either drug alone. In the TANDEM phase II trial (NCT03517540), the combination of CVC and the farnesoid X receptor (FXR) agonist tropifexor will be evaluated.

5.2. Cardiovascular effects of CCR2/5 inhibition

The main cause of death in NAFLD patients is cardiovascular disease, the incidence of which is related to the stage of fibrosis [2]. As such, a key issue is whether these compounds will reduce cardiovascular events, either directly or through decreasing liver fibrosis. Unfortunately, there is a paucity of data on cardiovascular parameters, both from preclinical research and human trials. The CCR2/5 inhibitor PF-04634817 has been investigated in patients with vascular complications of diabetes (who are at high risk of NAFLD), yet it only mildly reduced albuminuria in patients with diabetic nephropathy [18], and was inferior to the angiogenesis inhibitor ranibizumab for the treatment of diabetic macular edema [19]. Long-term follow-up data, which will be collected in the AURORA trial for cenicriviroc, will be required to definitively answer this question.

5.3. Translation to cirrhosis and liver cancer

The antifibrotic property of CCR2/5 inhibitors raises the question whether they could be of clinical use in patients with liver cirrhosis. So far, these patients were specifically excluded from major trials. One phase I trial in patients with cirrhosis indicated that 150 mg CVC resulted in higher plasma levels in patients with Child B (n = 8) but not Child A (n = 7) cirrhosis compared to healthy controls [20]. Importantly, the drug was well tolerated and there was no increase in markers of bacterial translocation, in accordance with data showing that CVC does not increase susceptibility to infections [16,20].

It has been shown that CCL2 is overexpressed in both humans and mice with hepatocellular carcinoma (HCC), which is a negative prognostic marker. Moreover, blocking CCL2/CCR2, genetically or pharmacologically, inhibits tumor growth and metastasis, and enhances survival in an HCC mouse model. This effect was attributed to inhibition of monocyte infiltration and reversal of the immunosuppression status of the tumor microenvironment by activation of a CD8 + T cell response [21,22]. Depletion of CCR5 reduced immune cell infiltration, fibrogenesis and tumor formation in mice [23] and resulted in decreased migration and invasiveness of malignant HCC cells in vitro [24,25]. These preclinical data at least favor the exploration of CCR2/5 inhibitors for the treatment (or prevention) of HCC.

Declaration of interest

F Tacke has received funding by Allergan, Galapagos, Inventiva and Bristol Myers Squibb. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

Additional information

Funding

S Lefere is supported by the Research Foundation – Flanders (fellowship 11W4716N) and F Tacke is supported by the German Research Foundation (DFG; Ta434/3-1, Ta434/5-1, CRC1382 and SFB/TRR57).