Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and enhances CB2 gene expression in patients with chronic hepatitis C

Chronic hepatitis C (CHC) is one of the most common etiologies for liver fibrosis and will eventually progress to cirrhosis or even to hepatocellular carcinoma [1, 2]. Nowadays, it is estimated that up to 3% of the world population is affected by CHC, thus, a great deal of drugs designed to clear the liver from the infectious viral component in this disease, have been developed [2‐6]. Nevertheless, fibrotic sequels eventually leading to dysfunctional liver activity in these patients are far from being resolved. In this frame of mind, pirfenidone (PFD) (5 methyl-1-phenil-2 (1H)-pyridone) has proved anti-fibrotic and anti-inflammatory properties in a wide number of animal models of fibrosis. PFD effects are mediated in part through inhibition of NF-κ-B activation, these mechanisms included inhibition of PDGF, hepatic stellate cells (HSC) proliferation, reduction of TNF-α and IFN-α levels and decrease in iNOS/NO induction [7, 8]_. Also, PFD down-regulates TGF-β1, TIMP-1, MMP-2 mRNA and collagen deposition [9, 10]_. Previously, our group demonstrated that one-year treatment with 1200 mg/day of oral PFD to patients with established liver fibrosis, decreased liver necroinflammation, steatosis and at less extent, fibrosis. Collagen I, TGF-β1 and TIMP-1 mRNAs were also down-regulated [11]_. Among the many factors known to influence hepatic fibrosis progression (gender, age at HCV-infection, alcohol consumption), cannabinoids consumption has also been studied [12‐14]. Recently, Zampino and cols. have highlighted that HCV-related clinical conditions like fibrosis, cirrhosis and hepatocellular carcinoma are the result of liver and systemic chronic inflammation [15]. On the other hand, cannabinoids signal through a G protein-coupled receptors called CB1 and CB2 which are absent or expressed in low levels in non injured livers, but strongly up-regulated in fibrotic-liver, especially in hepatic myofibroblasts and vascular endothelial cells [16‐18]. Several studies have shown that endogenous and exogenous cannabinoid ligands and their receptors play a key role in the pathogenesis of chronic liver injury [19‐21]. Based in our previous report [11], we aimed in this work to implement a 24 months clinical protocol with 1200 mg/day of oral PFD to analyze its effect in CB1 and CB2 cannabinoid receptors expression, serum levels of IL-6, TNF-α, TGF-β1 and necroinflammation and fibrosis scores. We reasoned that fibrosis stage in liver tissue from patients with CHC reported in this communication deserved further consideration, since an extended period with PFD treatment could result in an enhanced resolution of fibrosis as determined by liver biopsy. Furthermore, a couple of interesting articles recently published on the use of PFD in the treatment of two fibrotic diseases as diabetic nephropathy and pulmonary fibrosis suggest that PFD improves organ functionality demonstrating a benefit when it is used to treat fibrotic human pathologies [22, 23]. Specifically, this very interesting study entitled “A Phase 3 Trial of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis” deserves a further and careful consideration [23].

Our group has demonstrated that PFD is an anti-inflammatory and anti-fibrogenic agent in experimental models of cirrhosis [9] and in humans affected with hepatic fibrosis caused by different etiologies [11, 30‐32]_. Moreover, PFD has shown to improve necroinflammation, steatosis and liver regeneration in a sub-set of patients with established advanced liver fibrosis caused by HCV infection [11]. It had also been demonstrated that PFD has an anti-fibrogenic action inhibiting capsular contracture in mammary implants and an antioxidant role in different experimental models of cirrhosis [31‐33]_. Moreover, two randomized, double-blind, multicentre studies recently published; used PFD in the treatment of pulmonary fibrosis and diabetic nephropathy, pointing out the relevance of PFD in the clinical scenario when it comes to treat patients with these fibrotic illnesses [22, 23]. Moreover, in the present study we evaluated the effect of 24 months treatment with 1200 mg/kg of PFD in HCV chronically-infected patients to elucidate whether PFD therapy has an effect on serum expression of fibrogenic and pro-inflammatory molecules as well as other markers. At the molecular level, PFD possess a potent anti-TNF-α and anti-TGF-β1 action and on other pro-inflammatory cytokines [34‐36]. It has been confirmed in this two-years follow-up trial that IL-6 experienced a significant decrease in serum levels in most of patients as well. This fact indicates that the inflammatory pathway IL-6/TNF-α is being affected by PFD treatment. According to this, necroinflammation score also reduces in 82% of patients after treatment; a number that is significant greater than the 53.3% achieved with one-year treatment. Interestingly, we showed that 2-year PFD treatment compared to 1-year treatment augments the number of patients that achieved fibrosis reduction (30% in the one-year follow up vs. 67% in the two-year study). This improvement in liver fibrosis was histologically monitored using Ishak score, and a considerably 2-point decrease (mean value) in liver fibrosis score was achieved at the end of treatment compared with patients-initial data. This information correlates with observed reduction on serum levels of hepatic enzymes and bilirubin that reflects a functional restoration in the liver due to PFD treatment.

As known, steatosis is the result of the accumulation of fat in hepatocytes and it has been associated with rapid progression of liver fibrosis [37, 38]. The percentage of patients that reduced fat-occupied liver area with 2-year treatment was roughly the same as those in the one-year period (60% vs 61.5%). It is important to keep in mind, that studies have reported that agonist of CB1 receptor promotes steatosis and strong evidence argues for a steatogenic role of the cannabinoid system [14, 39‐41]. Therefore, histological findings in steatosis reduction can be related with diminution in CB1-mRNA levels observed in this study. In this framework, the endocannabinoid system plays an important role in liver fibrosis. In murine models of chronic liver-injury, CB1 receptor antagonism by pharmacological or genetic mechanisms reduced fibrotic area, TGF-β1 expression and accumulation of fibrogenic cells [29]. In opposition to CB1 receptor antagonism effects, CB2 receptor agonist counteracts liver fibrosis and induces inhibition and apoptosis of hepatic myofibroblasts and stellate cells [19]. Thus, we looked at the effects of PFD therapy in the expression of these molecules. To our knowledge, this report is the first attempt to search for a possible relation between PFD and CB1 and CB2 mRNA expression. After treatment, patients showed CB1 mRNA reduced to almost half of the initial level (even when no statistical difference was obtained) demonstrating benefits of treatment associated with steatosis decline; while CB2 mRNA levels were over-expressed approximately by 50% which correlates with the improvement in fibrotic score and necroinflammation. In this context, results from Coppola et al. related to rs35761398 variant of CB2 receptor gene (CNR2) in Italian HCV-chronic infected patients, demonstrated that QQ allele is associated with more severe inflammation and hepatocellular necrosis. The influence of this polymorphism in the response to pirfenidone in Hispanic patients could be evaluated in future studies [42].

As reported, CBs liver expression can be detected mainly in hepatocytes and stellate cells [19, 29, 43]. In this protocol, CBs mRNAs were detected in liver homogenates. Thus, we believe this reflects the organ microenvironment that induces liver improvement. As observed, PFD was satisfactorily tolerated for the two-years period at 1200 mg/day, given that 7/28 patients did not develop any secondary effect and 21 patients developed just negligible side effects like gastritis, nausea and rash. Several limitations of the study must be recognized, though our findings strongly support that PFD reduces liver fibrosis, necroinflammation and steatosis. Also, treatment for a period of two years is well-tolerated and increasing time of treatment renders enhanced benefits as observed in this HCV-liver fibrosis patient cohort, i.e. recovering in hepatic markers, TGF-β1 and pro-inflammatory cytokines serum levels reduction as well as, mRNA levels of CB2 mRNA increase.

In conclusion, there is an evident advantage of two-years treatment over the one-year period and administration of PFD induced only minor side effects, which were resolved after 2/3 months of PFD intake. In addition, histopathologic results showed improvement in terms of the progression of fibrosis and stage of inflammation, as well as decrease in the percentage of steatosis after two years of treatment with PFD. Also, this study is the first to show that PFD decreases serum levels of TGF-β1 and IL-6 and gene expression increases anti-fibrogenic CB2 receptor. However, it is important to remember that viral clearance is indispensable to cure the disease and to resolve liver damage. In this context, direct antiviral agents (DAA) and immune system boosters (some of them with proven efficacy) are the standard of care for chronic HCV infected patients. However, these treatments are not available for general population who is not covered under the social services (i.e. regular health care in North-America) in developing countries like Mexico. This is due to the elevated cost of such a treatments, which make them inaccessible to non-affiliated patients. Then, in this majority of untreated patients with DAA, an alternative anti-fibrogenic therapy could impact their health and quality of life. Besides, the combination of pirfenidone with DAAs can be useful in patients infected with genotypes that can be hardly eliminated with standard therapies and can also be evaluated in other liver diseases.