Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and enhances CB2 gene expression in patients with chronic hepatitis C
© Flores-Contreras et al.; licensee BioMed Central Ltd. 2014
Received: 17 August 2013
Accepted: 14 July 2014
Published: 27 July 2014
The aim of this study was to assess whether two-years treatment with Pirfenidone influences necroinflammation, fibrosis and steatosis, serum levels of TGF-β1, IL-6, TNF-α and CB1 and CB2 gene expression, in patients with chronic hepatitis C (CHC).
Twenty-eight patients out of 34 with CHC virus infection were enrolled in the study and received Pirfenidone (1200 mg/day) for 24 months. Six patients dropped out after 12 months of PFD. Liver biopsies and serum samples were obtained at the beginning and end of treatment. Modified HAI was calculated. CB1 and CB2 gene expression was correlated with fibrosis progression alongside with necroinflammation and steatosis. TGF-β1, IL-6, TNF-α and liver transaminases were measured in serum at two-months intervals. HCV genotype and viral load were also assessed. Quality of life was evaluated by SF36 questionnaires and the prognosis of disease was assessed with Child-Pugh score. The Wilcoxon test matched-pair signed ranks were used to analyze the outcomes.
Intention to treat analyses were performed for biochemistry and clinical parameters. At the end of treatment, necroinflammation grading was reduced in an average of 3.2 points in 82% of patients (p < 0.05) and Ishak’s fibrosis stage decreased 2-points average in 67% of patients (p < 0.05). Steatosis decreased in 61% of patients. IL-6 and TGF-β1 serum levels decreased significantly in 93% and 67% of patients (p < 0.05), respectively, while TNF-α diminished in 47% of patients. ALT and AST tended to normalize in 81% of patients; CB2 mRNA levels increased in 86% and CB1 expression diminished in 29% of patients. Both, quality of life and Child-Pugh score improvements were reported in all patients.
Pirfenidone for two years benefits CHC patients and improves inflammation, fibrosis and steatosis in higher number of patients as previously shown for 12-months treatment with PFD. Additionally, PFD improved TGFβ1 and IL-6 levels and diminished liver expression of anti-fibrogenic receptor CB2.
ClinicalTrials.gov identifier: NCT02161952. Protocol Registration Date: 06/11/2014.
KeywordsPirfenidone Liver fibrosis Necroinflammation Cytokines Cannabinoid receptor 1 Cannabinoid receptor 2 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 . 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 . 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 , 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 .
Consecutive patients seen in our department were enrolled if they met the following criteria: (1) patients with established advanced liver disease caused by hepatitis C virus chronic infection defined by a positive test for anti-HCV antibodies and detectable serum HCV RNA (Amplicor HCV 2.0 PCR test system; Quest Diagnostic, San Juan Capistrano, CA, USA) (2) signing of an informed consent form to allow collection of liver biopsies before and after (3) no anti-fibrotic, antiviral or immunosuppressive drugs for at least 6 months before starting pirfenidone therapy; and (4) no alcohol intake and non-smokers of cannabis for at least 6 months before nor during PFD treatment. The baseline period was defined as an observational period before pirfenidone therapy and each patient was used as its own control.
The no-inclusion criteria were the following: (1) evidences for other forms of liver diseases (2) co-infection with hepatitis B virus or HIV; (3) post-transplant patients; (4) known intolerance to pirfenidone; (5) pregnancy or breast-feeding; (6) gastrointestinal bleeding; (7) malignancy; and (8) patients with concomitant disease such as heart failure, coronary artery disease, diabetes or cancer.
This is an open-label, non-controlled and non-randomized clinical trial, designed to be carried out for 24 months in patients with chronic hepatitis C (CHC). The trial site was Institute of Molecular Biology in Medicine and Gene Therapy, University of Guadalajara, Mexico.
In this study 34 patients older than 18 years of age were enrolled, which had not previously participated in any other clinical protocol. Enrolled patients provided a medical history; complete physical examination was performed and intravenous blood was collected to assess liver function, liver fibrosis markers, complete blood count, blood coagulation profile, urinalysis, electrolytes status and serology (HCV, HBV, and HIV). HCV genotype was determined by LIPA (Quest Diagnostic, San Juan Capistrano, CA, USA) and HCV viral load was measured by PCR Quant (Amplicor HCV 2.0 PCR test system of Quest Diagnostic, San Juan Capistrano, CA, USA). Results were expressed as log10 IU/mL. Abdominal ultrasonography with Doppler assessment and computed tomography scan were performed to assess ascites and liver disease and portal system. Child-Pugh score was used to evaluate the severity of liver disease in all patients. Age at HCV infection, source of contamination and body mass index (BMI) were determined. Duration of HCV infection was estimated as the difference between the date of infection and the date of baseline liver biopsy. Two liver biopsies were obtained; one at baseline and a second one after two years of treatment.
Pirfenidone was administered three times a day in the form of 400 mg capsules manufactured according to standard good manufacturing practices (GMPs); good laboratory practices (GLPs) and sanitary regulations enforced by the Federal Commission for Protection against Sanitary Risks (COFEPRIS).
During the study, patients had twelve medical visits during a 24 months period, in which patients were physically examined, adverse event story was collected and laboratory testing was realized. Patients also completed the 36 item Short-Form Health Survey (SF-36). Health survey was applied to evaluate quality of life in patients before and after PFD treatment with their own initial data as control.
This safety/efficacy, nonrandomized, self-monitored, open phase study was approved by Ethical Committee from Hospital Civil de Guadalajara with registration number 505/05 and the COFEPRIS (Mexican Ministry of Health) with registry number 05330020020126. Furthermore, the protocol was registered in ClinicalTrials.gov identifier: NCT02161952.
Also, this study was undertaken in accordance with the Declaration of Helsinki and with local laws and regulation applicable to the use of drugs in Mexico, and all patients gave informed consent.
Pirfenidone was supplied orally in 400 mg gel capsules three times daily (every 8 hours) for a full dosage of 1200 mg daily. All patients were instructed to take pirfenidone 20 minutes after meals to minimize gastrointestinal symptoms during 24 months. Patient’s compliance of drug intake was assessed using specifically designed drug registration sheets.
Biomarkers were analyzed in plasma from samples collected at baseline visit and end of study visits. Blood biomarkers measured were IL-6, TNF-α and TGF-β1. Cytokine analysis was measured using a conventional automated analyzer (Sincron-Cx7 analyzer) and pre-coated ELISA assay (R & D Systems, Minneapolis, MN, USA). All ELISA kits were utilized as described by the manufacturers.
Liver biopsy was obtained before starting treatment with pirfenidone and second liver biopsy after two years of treatment using Color Doppler Sonography Needle-Guided. Liver biopsy specimens were fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin (H&E) and trichromic Masson. Fibrosis and necroinflammation were analyzed according to the Modified histological activity index (HAI) of Ishak scoring system . Hepatic steatosis was measured according to the percentage of fat vacuoles in 20 random fields using a computer-assisted automated image analyzer (Image Pro-Plus 5.0, Media Cybernetics, Inc, Bethesda, MD, USA). Necroinflammation was scored by the activity index on a scale of 0 to 18. Fibrosis was staged on a scale of 0 to 6 (F0 or F6, F6 defining cirrhosis) with fibrosis stage of 4 or 5 defined as severe or advanced fibrosis. The 50% of patients enrolled in this study displayed advanced liver fibrosis (stage 4-5); 30% of patients had cirrhosis (stage 6) at the beginning of the trial and the 20% of them displayed mild or moderate liver fibrosis (stage 1-3).
A portion of liver biopsy was frozen for RT-PCR analysis. Total RNA from liver biopsy was extracted using Trizol reagent according to the manufacturer instructions (Invitrogen, Carlsbad, CA, USA). RNA was quantified using spectophotometry. Reverse transcription was performed with 2 μg of total RNA for all genes with the cDNA synthesis kit (Cat. 4368814). Using 240 ng of random primer, 2U RNAse inhibitor, 5 mM of DTT, 1 mM of dNTPs and 200U of transcriptase, PCR was performed using the following protocol: 65°C/5 min, 4°C/5 min, 25°C/5 min, 50°C/60 min, 70°C/15 min and 4°C/5 min.
Quantitative real time PCR was performed using a Rotor Gene 3000 Termocycler (Corbett Research, Cambridge shire, UK) under the following conditions: 1 hold for 2 min at 50°C,1 hold for 5 min at 94°C, and 45 cycles of 30 sec at 94°C and 40 sec at 60°C. Specific primers for CB1 and CB2 were acquired from Applied Biosystems, NJ, USA. GAPDH was used as housekeeping gene. For the reaction, 2 μl of cDNA was used in 5 μl of Mix, 0.5 μl of TaqMan probe and primers for CB1 (Cat#:Hs00275634_m1), CB2 (Cat#:Hs00361490_m1) and GAPDH (Cat#:Hs99999905_m1). Gene expression was calculated with the 2-ΔCT method according to Livak et al.
Because values in baseline and treatment periods did not follow a parametric distribution, the Wilcoxon matched-pair signed ranks test was used to analyze the outcomes. Intention to treat analysis (ITT) was also performed. Data are presented as mean ± SD for parametric data. Statistical analysis was performed using Prism software (GraphPad Prism, CA, USA) Significance was defined as a P value <0.05.
Study group characteristics
Causes of Death in patients enrolled in the study
Number of patients
Duration of treatment
< 1 year
Esophagic varices bleeding
Percent of enrolled patients death
Secondary effects associated with pirfenidone treatment for two years
*n = 28
**n = 34
General characteristics of patients
Male, n (%)
Female, n (%)
Age at exposure (yr)
Mean ± SD
27 ± 9
Age at liver biopsy before treatment (yr)
Mean ± SD
56 ± 10
Age at liver biopsy after treatment (yr)
Mean ± SD
58 ± 10
Route of transmission
Blood transfusion, n (%)
Nosocomial, n (%)
Duration of HCV exposure (yr)
Mean ± SD
32 ± 10
Genotype 1, n (%)
Genotype 2, n (%)
Genotype 3, n (%)
ND, n (%)
Change in HCV viral load after treatment
Increase 1 log, n (%)
Unchanged*, n (%)
Body mass index (Kg/m2)
28 ± 7**
Characteristics of dropping-out patients
Severity of disease according to child-pugh score
< 2 years
Intention to treat
Effect of PFD on liver biochemistry and clinical data
Biochemical measurements of patients
< 2 years
Intention to treat
ALT (mean ± SD)
67 ± 25
59 ± 20
85 ± 71
65 ± 34
82 ± 68
64 ± 32
AST (mean ± SD)
95 ± 38
87 ± 38
94 ± 55
75 ± 38
94 ± 54
78 ± 38
Total Bilirubin (mean ± SD)
2 ± 1.7
3.1 ± 1.5
1 ± 0.8
1 ± 0.5
1.4 ± 0.9
1.4 ± 1.1
Direct Bilirubin (mean ± SD)
0.8 ± 0.5
1.5 ± 0.9
0.4 ± 0.3
0.4 ± 0.2
0.5 ± 0.3
0.6 ± 0.6
Indirect Bilirubin (mean ± SD)
1.3 ± 0.9
1.6 ± 0.9
0.8 ± 0.7
0.6 ± 0.4
0.9 ± 0.7
0.8 ± 0.6
Child-Pugh score in enrolled patients
Child-Pugh score was measured in order to indirectly assess the prognosis of chronic liver disease and to correlate with patient survival at two years . The 28 patients that concluded the two-year treatment showed an improved score at the end of the study (Table 4). Hepatitis C patients with established liver fibrosis after PFD treatment had improved Child-Pugh score compared to initial values as seen in Table 4. Treatment allowed patients to remain in a compensated status, since 20 out of 28 patients experienced no change in Child-Pugh A or B score, 5/28 improved their score and only 3 patients decreased Child-Pugh score indicating most severe liver damage. However, when drop-out patients were included, no statistical significance was achieved in this score.
Intention to treat analysis for quality of life
Intention-to-treat analysis for quality of life
Domains (mean ± SD)
69 ± 30
82 ± 23
61 ± 47
86 ± 32
70 ± 26
81 ± 19
55 ± 21
73 ± 18
62 ± 24
73 ± 22
73 ± 29
81 ± 23
68 ± 45
83 ± 38
75 ± 20
83 ± 17
Pirfenidone was well tolerated. All patients tolerated the dosage of 1200 mg/day from the beginning of the treatment; seven patients did not develop any secondary effect. Table 2 summarizes secondary effects known to be associated with pirfenidone for 27/34 patients that showed any or several of them. Patients developed negligible secondary effects, like gastritis (23/34 patients; 64%%), nausea (12/3 patients; 35%) among others. These secondary effects disappeared 3 months after initiating PFD intake. None of patients dropped out of the study due to side effects of treatment. All patients that conclude the study adhered to treatment (they received ≥80% of scheduled doses).
PFD effect in liver histopathology
Effects of PFD on biomarkers
PFD effect on CB1 and CB2 receptors gene expression
Our group has demonstrated that PFD is an anti-inflammatory and anti-fibrogenic agent in experimental models of cirrhosis  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 . 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 . In opposition to CB1 receptor antagonism effects, CB2 receptor agonist counteracts liver fibrosis and induces inhibition and apoptosis of hepatic myofibroblasts and stellate cells . 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 .
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.
Body mass index
Cannabinoid receptor 1
Cannabinoid receptor 2
Chronic Hepatitis C
Histological activity index
Hepatitis C virus
Human immunodeficiency virus
induced Oxide Nitric Synthetase
Nuclear factor kappa-light-chain-enhancer of activated B cells
Platelet-derived growth factor
Real time- Polymerase chain reaction
Tissue inhibitor of metalloproteinases-1
Tumor growth factor- beta1
Tumor necrosis factor-alpha.
Authors acknowledge CONACYT for the grant provided.
Source of funding
Juan Armendariz-Borunda received a grant (#25474) from CONACYT (Science and Technology National Council).
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