Pirfenidone and Advanced Liver Fibrosis.

The study will be conducted in compliance with International Standard good clinical practices (GCPs) and the Declaration of Helsinki. The protocol was approved by local Institutional Review Board and registered in clinical trials.gov.

Clinical and Laboratory Evaluation Blood counts and liver function tests (bilirubin, albumin, prothrombin time expressed as international normalized ratio (INR), serum transaminases, glucose, and creatinine) are measured at 12-week intervals. Patient's somatometric measurements (height and body weight), vital signs, and frequency of adverse events (AE) are recorded. Liver enzymes will be scored as stable, improving, or worsening.

Study End Points The primary efficacy endpoint is a reduction of fibrosis score by at least 30% either in Fibro Test units or kilo Pascals (kPa) according to hepatic elastography or a reduction of 1 point on the METAVIR scale. Secondary efficacy endpoints include improvement in alanine aminotranferase (ALT) and/or aspartate aminotransferase (AST), albumin, serum concentrations of TGFbeta, IL-1 and IL-6 and endothelin, and Child-Pugh and MELD scores. Worsening MELD is defined as switching from a lower-score to a higher-score and improving as switching from a higher-score to a lower-score, where Group 1 was MELD ≤ 9, Group 2 10-19, and Group 3 > 20.

Primary safety endpoints include clinical side effects, blood profile abnormalities, overall survival, and pharmacokinetic (PK) findings. Secondary safety endpoints included quality of life scores.

Evaluation and Classification of Fibrosis Outcomes Fibrosis-regression profile (FRP): decreases >30% in FT score or 30% in kPa in liver stiffness measurement (LSM) or decreasing 1 point on the METAVIR score comparing baseline and M12 measurements.

Fibrosis-stabilization profile (FSP): stable FT results or kPa measurements (variations lower than 30%) or METAVIR score.

Fibrosis-progression profile (FPP): increases greater than 30% in FT score or kPa or increasing 1 point on METAVIR.

Specific Evaluation and Classification of Biochemical Outcomes Biochemical markers: Blood parameters determined after overnight fasting include: albumin, prothrombin time, total bilirubin, ALT, AST, Alkaline phosphatase (AP), and gamma-glutamyltransferase (GGT), measured in fresh serum within 8h of collection on an automated biochemistry analyzer (Hitachi 917; Roche Diagnostics).

alpha-2-macroglobulin (A2M), apolipoprotein-A1, and haptoglobin levels will be assayed by nephelometry (Image; Beckman Coulter).

Fibro Test®: blinded Fibrotest measurements will be performed on fresh serum and according to the recommended pre-analytic and analytic methods.

Hepatic elastography: Transient Elastography (TE) is performed according to published recommendations using the Fibro-Scan® M probe. LSM is expressed in kPa. Only procedures with at least 10 validated measurements, >60% success rate, and an interquartile range <30% of the median will be considered reliable. Patients with ascites will be offered real time shear wave elastography (Aixplorer, Supersonic Imagine).

Cytokines and pirfenidone: Serum concentrations of Interleukin 6 (IL-6), transforming growth factor beta (TGF-β1), endothelin 1 (ET-1), and tumor necrosis factor alpha (TNF-α) will be quantified by ELISA in an automated enzyme-linked immunosorbent assay (EIA) analyzer Coda Microplate System (Bio-Rad Laboratories, Inc., Hercules, California, USA) and values normalized against serum of healthy volunteers with normal liver function and LSM <5 kPa (F0).

Evaluation of Safety Profile Monitoring for safety and toxicity will be performed throughout the study. When necessary, appropriate medical intervention will be provided.

Quality of life assessment: All patients will fill out the Euro-Qol Index survey, including the visual analog scale evaluation at baseline and at 12 months. Investigators incorporate the nonutility-based Short Form-36v2 survey, which provides a detailed profile of health-related quality of life.

Ethical considerations. The study will be conducted in accordance with the Declaration of Helsinki and the E6 Good Clinical Practice Standards International Conference on Harmonization (ICH).

Statistical Data Analysis: Investigators will be using the Statistical Package for the Social Sciences (SPSS) Statistical software 6.12 for Windows. Descriptive statistics include mean, standard deviation standard error. A paired or unpaired t Test will be used to compare means before and after study medication administration. Values <5% will be considered significant. Kaplan Meier survival analyses will include all patients on active study-medication treatment despite duration of exposure. Sample size calculation was based on the following assumptions: (1) mean expected baseline elastography score of 27.4 kPa, standard deviation of 15.7; expected estimated-fibrosis reduction rate higher than 30% (delta); (4) alpha error of 1%; (5) an accepted beta error of 10% (power = 90%); (6) two tails. The final number of patients required to find a significant difference using the G power statistical program was 61 patients.