Hepatitis C is a major healthcare problem, and current therapies have achieved sustained response in more than a half of infected patients. Factors associated with nonresponse are host-viral genotype 1, high viral load, advanced fibrosis, and insulin resistance.1 Sustained virological response (SVR) decreases when insulin sensitivity is impaired.1, 2 Also, impaired fasting glucose has been independently associated with lower SVR rate.3 Insulin resistance is thought to be promoted by hepatitis C virus (HCV) itself and, after clearance of the virus, insulin resistance improves concomitantly with the reduction in the risk of glucose abnormalities and diabetes.1, 3, 4 HCV proteins lead to insulin resistance, promoting the degradation of insulin receptor substrate 1.5 Transgenic mice expressing core HCV protein developed insulin resistance and steatosis.6 Insulin resistance and steatosis promoted by the virus have been found to be associated with improvement in viral fitness,7 and this seems to be a defense mechanism against viral clearance. Lifestyle, exercise, diet, and insulin-sensitizing drugs could improve insulin resistance. Metformin is an oral biguanide that lowers blood glucose and insulin secretion and improves the individual's lipid profile, mainly because of suppression of hepatic glucose output and increased glucose uptake in skeletal muscle.8 Because insulin resistance can be successfully treated with biguanides, we proposed that sustained virological response could be improved by adding metformin to the standard of care for patients with chronic hepatitis C genotype 1 and insulin resistance.
Insulin resistance affects sustained virological response (SVR) in chronic hepatitis C. To know whether adding metformin to standard antiviral treatment improves SVR, we conducted a prospective, multicentered, randomized, double-blinded, placebo-controlled trial in 19 Spanish hospitals, including 123 consecutive patients with genotype 1 chronic hepatitis C and insulin resistance. Patients were randomized to receive either metformin (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (180 μg/week) and ribavirin (1000–1200 mg/day). The primary end point was SVR, and secondary endpoints were viral clearance at weeks 12, 24, and 48, and changes in the homeostasis model assessment (HOMA) index over the first 24 weeks. There were no differences between arms at baseline. In the intent-to-treat analysis, SVR was observed in 53% versus 42% in arm A and arm B, respectively (P = NS). In the subgroup analyses, SVR was higher in females (n = 54) receiving metformin: arm A, 58% (15/26) versus 29% (8/28) arm B (P = 0.03). In the per protocol analysis (PPA; n = 101), SVR was 67% in arm A and 49% in arm B (P = 0.06). Viral decline during the first 12 weeks was greater in females receiving metformin: −4.88 (1.18) versus −4.0 (1.44) (P = 0.021), whereas no differences were seen in males. The triple therapy was well tolerated, but diarrhea was more often seen in arm A (34% versus 11%; P < 0.05). Conclusion: Adding metformin to peginterferon and ribavirin was safe and improved insulin sensitivity. Although the study failed to show a statistically significant difference between arms, it did show an improved SVR in females. (HEPATOLOGY 2009.)
Patients and Methods
Selection of Patients.
Eligible patients were those who were 18 years of age or older, were infected with genotype 1 (determined by INNO-LIPA assay, Innogenetics), and had a quantifiable serum HCV RNA for more than 6 months together with an elevated serum alanine aminotransferase level, nondiabetic, and homeostasis model assessment (HOMA) index greater than 2 (Table 1). Liver biopsy was not mandatory. Liver fibrosis was calculated using the Sydney index (a noninvasive method for the prediction of liver fibrosis based on a formula that includes age, plasma cholesterol concentration, aspartate aminotransferase, alcohol consumption, and HOMA index9 that has been validated in Spanish patients with chronic hepatitis C).10 Patients were ineligible if they had other liver diseases, showed contraindications to peginterferon or ribavirin, or had previously received antiviral drugs.
|Characteristic||Arm A||Arm B||P|
|Age, years||47.1 (8.5)||47.7 (8.9)||0.69|
|HOMA-IR||4.31 (2.24)||4.42 (2.62)||0.78|
|Viral load (lg10)||6.33 (0.73)||6.48 (0.76)||0.28|
|HVL, >400,000 IU/mL||54/59 (92%)||58/64 (91%)||0.95|
|Fibrosis; Sydney Index||0.44 (0.27)||0.46 (0.32)||0.75|
|Weight, kg||78.7 (14.6)||83.0 (18.3)||0.16|
|ALT, U/L||90.1 (69.4)||98.7 (85.3)||0.54|
|AST, U/L||58.4 (41.8)||66.5 (60.5)||0.43|
|GGT, U/L||65.1 (54.2)||84.2 (72)||0.12|
|Platelets, ×103/μL||219 (54)||213 (54)||0.58|
|Cholesterol, mg/dL||171 (27)||178 (39)||0.26|
Patients were randomly assigned into this prospective, multicentered, randomized, double-blinded, placebo-controlled trial (registered in ClinicalTrials.gov; #NCT00546442). The study was conducted in 19 Spanish centers. All sites received approval from their ethics committees. Each patient provided written informed consent for participation. Patients were randomly assigned in a 1:1 ratio to receive either metformin 425 mg three times daily for the first month and 850 mg three times daily from week 4 to 48 (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (Pegasys; Roche Farma, Madrid, Spain) 180 μg weekly and ribavirin 1000 to 1200 mg daily. Patients were followed-up over 24 weeks to confirm SVR.
An overnight (12-hour) fasting blood sample was taken for routine analyses, including aminotransferases (alanine aminotransferase, aspartate aminotransferase), alkaline phosphatase, gamma-glutamyltransferase, platelets, glucose, cholesterol, and triglycerides. All patients had positive anti-HCV as measured using EIA3 (Abbott Laboratories, Chicago, IL) and positive HCV RNA in serum. Hepatitis B surface antigen, anti-hepatitis B core, and anti-human immunodeficiency virus were tested using commercially available kits (Abbott Laboratories, Chicago, IL). All patients were negative for hepatitis B surface antigen and anti-human immunodeficiency virus. Serum insulin levels were measured by electrochemiluminescence immunoassay, using an autoanalyzer Elecsys 1010/2010 (Elecsys Modular Analytics E170; Roche, Basel, Switzerland). Blood lactate levels was measured by a commercially available kit (Immulite Diagnostic Products, Los Angeles, CA). The insulin resistance index was calculated on the basis of fasting values of plasma glucose and insulin according to the HOMA model formula: Insulin resistance (HOMA IR) = fasting insulin (mUI/L) × fasting glucose (mmol/L) ÷ 22.5
The primary end-point was SVR, defined as undetectable serum HCV RNA level (<10 IU/mL) at 24 weeks after the conclusion of treatment.
The secondary end-points were: (1) viral clearance (HCV RNA < 10 IU/mL) at weeks 12, 24, and 48; (2) changes in the HOMA index over the first 24 weeks. Virological breakthrough was defined as a detectable HCV RNA level during treatment in patients who had had undetectable HCV RNA at week 24. Virological relapse was defined as a detectable HCV RNA level during follow-up in patients who had had undetectable HCV RNA at week 48.
Safety was assessed by means of clinical examinations and laboratory tests at weeks 4, 8, 12, 24, 48, and 72. Stepwise reductions of peginterferon alfa-2a, ribavirin, and metformin were permitted in managing clinically significant adverse events, or laboratory abnormalities recorded.
Sample size estimation was based on the assumption that metformin improving insulin resistance could increase SVR rate in 28%.1 The estimated sample size was 124 patients, with a 10% rate of loss-to-follow-up, type I error of 0.05, and type II of 0.2 (Fisher's exact two-sided test).
Intention-to-treat analysis included all 123 randomized cases, and a per-protocol analysis (PPA) included 101 patients. Excluded from the PPA were patients with adherence to less than 80% of the dose and duration of peginterferon (n = 9) and ribavirin (n = 4) and those with daily average intake of metformin less than 50% (1275 mg/day) (n = 9) (Fig. 1). All data were analyzed using SPSS version 15.0 for Windows (Chicago, IL). Comparisons between paired groups were with the Mann-Whitney U test, the student t test, chi-squared, or Fisher's exact test. Backward logistic regression was applied in the multivariate analysis.
Funding and Study Logistics.
The study was funded by a nonrestrictive grant from Roche Farma SA. The funding body had no part in the collection and evaluation of data, or in the writing of the manuscript and the decision to publish. The trial was designed by the principal investigator (M.R.-G.) together with the academic investigators constituting the TRIC-1 group (see Appendix). The logistics of data collection were managed by p-value S.A. (Sevilla, Spain) and the academic investigators, and a copy was logged with the study promoter. The principal investigator (M.R.-G.) together with p-value S.A. performed the statistical analyses and vouch for the integrity and completeness of the data.
Of 125 patients initially recruited, 123 were randomly assigned to a treatment group and received at least one dose of medication; two patients were excluded because of not having met the full criteria for inclusion (one patient was already taking metformin, and the other was found to be infected by a different viral genotype). The two treatment groups were well matched for age, sex, and other variables measured on entry into the trial (Table 1).
In the intention-to-treat analysis (n = 123), viral clearance was seen in 54% (32/59) of patients versus 48% (31/64) at week 12; in 75% (45/59) versus 75% (48/64) at week 24; in 71% (42/59) versus 63% (40/64) at week 48; and in 53% (31/59) versus 42% (27/64) at week 72 (in other words, SVR rate) in arm A and arm B, respectively. Virological breakthrough was slightly higher in patients receiving placebo 17% (8/48) versus 7% (3/45), but this difference was not statistically significant. Relapse rate was similar in both arms: 11 of 59 patients (19%) in arm A and 12 of 64 patients (19%) in arm B; P = 0.98.
In the PPA (n = 101), viral clearance was achieved in 67% (31/46) versus 53 (29/55), P = 0.13 at week 12; in 96% (44/46) versus 78% (43/55), P = 0.01 at week 24; in 91% (42/46) versus 69% (38/55), P < 0.01 at week 48; in 67% (31/46) versus 49% (27/55), P = 0.06 at week 72 in arm A and arm B, respectively.
In females (n = 54), adding metformin to peginterferon alfa-2a plus ribavirin resulted in a doubling of the SVR rate in 15 of 26 patients (58%) in arm A versus 8 of 28 patients (29%) in arm B; P = 0.03) (Fig. 2). Viral decline during the first 12 weeks was greater in females taking metformin than placebo (mean [standard deviation (SD)]) −4.88 (1.18) versus −4.0 (1.44); P = 0.02), whereas no viral decline was noted in males. Furthermore, no cases of breakthrough were seen in females with metformin therapy (females with HCV RNA negative at week 24 (n = 41), with 4 of 20 patients experiencing breakthrough in arm B but none in arm A (0 of 21); P = 0.05.
Changes in Insulin Resistance.
The decline in HOMA index in patients receiving metformin was significantly higher than in patients receiving placebo. In arm A, the HOMA index decreased from 4.3 (2.2) to 2.6 (1.7) and in arm B from 4.6 (2.7) to 3.8 (2.1); P = 0.001. However, no difference was seen in weight loss in either treatment arm. By the end-of-follow-up, women receiving metformin had a mean body weight decrease of 1.3 kg (SD: 5.4) versus 1.1 kg (SD: 5.6) in women receiving standard care; P = NS. In men, body weight decreased by 1.1 kg (SD: 6.1) in patients receiving metformin versus 0.9 kg (SD: 5.5) in patients on placebo; P = NS. Chages in body weight did not influence SVR. However, HOMA less than 2 at week 24 was more often seen in arm A (55%) than in arm B (14%), and this goal was associated with higher SVR (see below).
Predictors of SVR.
In the univariate analysis, higher baseline viral load and calculated fibrosis (the noninvasive Sydney Index) were associated with nonresponse of 6.52 (0.73) versus 6.28 (0.75) log10 HCV RNA (P = 0.08) and 0.52 (0.32) versus 0.38 (0.25) in the Sydney Index (P = 0.01). During treatment, the HCV RNA decline in the first 12 weeks of 3.04 (1.65) versus 1.11 (0.35) log10 HCV RNA, P = 0.0001, and the HOMA-IR decline during the first 24 weeks was associated with sustained response; that is, 19 of 28 patients (68%) with a HOMA-IR less than 2 at week 24 achieved SVR, whereas 25 of 56 patients (45%) with HOMA at 24 weeks greater than 2 achieved SVR (P = 0.05).
Using reverse stepwise logistic multivariate regression analysis, the independent variable related to SVR was HCV RNA at week 12 (hazard ratio, 0.15; 95% confidence interval: 0.07-0.34) (Table 2).
|Parameter||Univariate Analysis||Multivariate Analysis BLR|
|Baseline VL; log10||6.52 (0.73)||6.28 (0.75)||0.08||0.71 (0.39–1.29)||0.22|
|HOMA-IR||4.61 (2.7)||4.1 (2.1)||0.24||1.07 (0.86–1.34)||0.82|
|Weight; kg||80.5 (18)||81.5 (12.2)||0.75||1.05 (065–1.45)||0.56|
|Age; years||48.2 (9.2)||46.5 (8.2)||0.30||1.01 (0.96–1.08)||0.77|
|Sydney Index||0.52 (0.32)||0.38 (0.25)||0.01||0.77 (0.04–15.5)||0.86|
|VL w12; log10||3.04 (1.65)||1.11 (0.35)||0.0001||0.15 (0.07–0.34)||0.0001|
|♂||34/69 (49%)||35/69 (51%)||0.37||0.79 (0.33–1.86)||0.93|
|♀||31/54 (57%)||23/54 (43%)|
|A||28/59 (48%)||31/59 (53%)||0.25||1.51 (0.66–3.43)||0.25|
|B||37/64 (59%)||27/64 (42%)|
|HOMA 24 weeks|
|≤2||9/28 (32%)||19/28 (68%)||0.045||1.12 (0.84–1.49)||0.43|
|>2||31/56 (56%)||25/56 (45%)|
The percentage of patients reporting adverse events or serious adverse events was similar in the two groups. The adverse events reported were those typical of interferon-based treatments, which include fatigue, headache, insomnia, and myalgia. Diarrhea was more often seen in patients assigned to arm A than in patients receiving placebo (36% versus 11%; P < 0.05). However, the diarrhea was well tolerated and did not lead to any withdrawals from the trial. Two patients in each treatment group had to discontinue because of adverse events (Table 2). Doses of metformin or placebo were modified in 37% of patients in arm A and 38% in arm B (P = NS). Serum lactate was monitored during treatment, and no patient developed lactic acidosis or hyperlactatemia.
The combination of metformin, peginterferon alfa-2a, and ribavirin was well tolerated by the patients. It improved insulin resistance in more than 50% of patients and increased SVR rate in 10% of patients with hepatitis C genotype 1 and HOMA greater than 2. This was related to a trend toward a low rate of breakthrough in patients receiving metformin; the response at week 24 and relapse rate were similar in both arms. However, in the PPA, viral clearance was significantly higher in the metformin group at weeks 24 and 48, which supports the proposition of a better antiviral activity of peginterferon alfa-2a and ribavirin in patients receiving metformin. Despite raising SVR by 10% (a clinically relevant increase), this difference did not reach statistical significance, which could be attributable, at least in part, to a type 2 error in sample size calculation. That there is a trend in the intention-to-treat analysis and significance in the PPA and female subanalysis strongly supports this hypothesis. Despite the sample calculation being based on the available data, our subsequent findings of the study suggest that there were not enough patients enrolled in the study to achieve statistically significant differences. Metformin is an oral biguanide, which is one of the most widely prescribed therapeutic agents for the control of type 2 diabetes. Metformin controls glucose levels by suppression of hepatic glucose output, by increasing insulin-mediated glucose disposal, and by decreasing fatty acid oxidation and synthesis of very-low-density lipoprotein.11 During HCV replication, core protein promotes unfolded protein response that causes dysfunction of endoplasmic reticulum and mitochondria.12, 13 Indeed, HCV proteins promote insulin receptor substrate 1 degradation by several mechanisms, including oxidative stress, down-regulation of PPAR, and enhancing TNF production in a genotype-dependent manner. In genotype 1, the degradation of insulin receptor substrate 1 has been shown to be induced by mammalian target of rapamycin and, in genotype 3, by suppressor of cytokine signaling 7 and peroxisome proliferator-activated receptor-gamma (PPAR-γ). Thus, metformin could be useful in the management of this comorbidity because of its ability to increase the binding of insulin to its receptors and to increase phosphorylation as well as the tyrosine kinase activity of insulin receptors14 through, mainly, the action of phosphorylation of hepatic adenosine-monophosphate-activated protein kinase by STK11 (formerly LKB1).15 Pioglitazone was shown not to improve either viral response or insulin sensitivity in five previously nonresponding individuals.16 In treatment-naïve patients, pioglitazone in combination with peginterferon alfa-2b and ribavirin improved insulin sensitivity and hepatic steatosis.17 Elgouhari et al.18 had shown that the combination therapy improved early virological kinetics over the first 4 weeks and the end-of-treatment response, but not SVR.18 The low number of patients (n = 40) precluded subset analysis and comparisons with the current study. However, the mechanism of action of insulin-sensitizer drugs in the setting of patients with hepatitis C appears to be different and could be related to viral genotype. Metformin improved insulin sensitivity in patients with hepatitis C genotype 1. Theoretically, PPAR-γ agonist appears to be indicated in the management of patients with genotype 3 HCV infection (in whom insulin resistance appears as a consequence of decreased PPAR-γ activity). Furthermore, metformin could be preferred because it acts directly in the liver, whereas PPAR-γ agonist shows an effect on peripheral metabolism. Further prospective studies using different insulin sensitizers in a large cohort of patients with hepatitis C with different genotypes are required to demonstrate whether these data could be translated into clinical practice.
In women, metformin increased the SVR rate significantly. This sex-related difference has not been previously reported in the management of hepatitis C. Spontaneous viral clearance has been seen more often in women than in men.19 However, no sex-related impact has been reported on the clinical course of the infection or in SVR rate. Some weak evidence supports the influence of sex on the therapeutic effects of metformin: (1) metabolic syndrome pathophysiology and diabetes-related complications appear to be vary in relation to sex, and vitamin K supplementation appears to improve insulin resistance in men but not in women20; (2) some therapeutic effects of metformin appear to be sex dependent, that is, metformin promotes greater short-term weight loss in women than in men, but these differences were not observed in diabetic control subjects21, 22; (3) STK11 (formerly LKB1) plays a major role in metformin sensitivity,23 and some genetic alterations appear to have a clinical impact that is sex dependent, such as the risk of malignancies in Peutz-Jegher syndrome.24 In women, metformin therapy has been associated with a greater decrease of HCV RNA during the first 12 weeks of treatment and a lower breakthrough rate. These data support the hypothesis that, in women, metformin improves the antiviral activity of peginterferon and ribavirin. It remains to be explored whether genetic alterations in the STK11 gene or nuclear factors such as estrogens are implicated in the ability of metformin to improve antiviral activity.
Two biguanides (phenformin and buformin) have been withdrawn from the pharmacopeias because of evidence of lactic acidosis associated with their use. Despite cases of lactic acidosis having been reported with the use of metformin, a recent systematic review demonstrated no increased risk for lactic acidosis in patients without contraindications to metformin use.25 In the current study, blood lactate level was monitored during treatment, and no patients developed lactic acidosis or hyperlactatemia. Metformin was associated with mild diarrhea, which was well tolerated, and no patient withdrew from the study because of this adverse event. Thus, metformin used together with peginterferon alfa-2a and ribavirin in the treatment of hepatitis C seems to be safe and well tolerated. The perceived contraindication of metformin for patients with alterations in liver function needs to be reevaluated.
In our study, metformin improved insulin sensitivity, with more than half the number of patients achieving HOMA less than 2 by week 24. This end point was also related to the probability of achieving SVR. Nevertheless, metformin-induced improvement in insulin sensitivity is not the sole action of this drug, and the mechanism by which metformin improves the antiviral activity of peginterferon + ribavirin warrants further investigation.
In summary, treating patients with hepatitis C genotype 1 and insulin resistance using metformin improves insulin sensitivity, is safe, and increases SVR rate in patients who reached HOMA lower than 2 at week 24 of therapy and in women, in whom, in the current study, the therapy doubled the SVR rate.
Editorial assistance was provided by Dr. Peter R. Turner.
The Spanish TRIC-1 group members (and their affiliations):
Lourdes Grande, Ana Madrazo, Santiago Durán (Hospital Universitario de Valme, Sevilla, Spain); María Luisa Moreno, Celia Pérez (Hospital General de Valencia,Valencia, Spain); Mercedes Robles (Hospital Virgen de la Victoria, Málaga, Spain); Ignacio de la Torre, Juan Revilla (Hospital Puerta de Hierro, Madrid, Spain); Luis Rodríguez, Ana Gila (Hospital San Cecilio, Granada, Spain); María Luisa Gutiérrez, Sonia Alonso (Hospital Universitario Fundación Alcorcín, Madrid, Spain); Dolors Gimenez (Hospital del Mar, Barcelona, Spain); Juan Manuel Herrerías, María Isabel Carmona (Hospital Universitario Virgen Macarena, Sevilla, Spain); Manuel De la Mata, Francisca Nuñez (Hospital Universitario Reina Sofía, Córdoba, Spain); Antonio Olveira, José Carlos Erdozain (Hospital La Paz, Madrid, Spain); Óscar Nuñez (Hospital Universitario Gregorio Marañon, Madrid, Spain); Javier García-Samaniego, Miriam Romero, Angelica Moreno (Hospital Carlos III, Madrid, Spain); Ricardo Moreno-Otero, María Trapero (Hospital de la Princesa, Madrid, Spain); Ramón Planas, Rosa Morillas, Pilar Giner (Hospital Universitari Germans Trias i Pujol, Badalona, Spain); Francisco Jorquera, Josí Luis Olcoz (Hospital General de León, León, Spain); Blas Dalmau (Hospital Parc Taulí, Barcelona, Spain); Manuel García-Bengoechea, Juan J. Arenas (Hospital de Donostia, San Sebastian, Spain); Eduardo Rodríguez (Hospital General de Alarcos, Ciudad Real, Spain); Rafael Martvín-Vivaldi (Hospital Universitario Virgen de las Nieves, Granada, Spain); Carmen Ramírez, Cristina Carreño, José Moreno-Cruzado (Roche Farma, SA, Madrid, Spain)