Clinical trial: low plasma cholesterol and oxidative stress predict rapid virological response to standard therapy with peginterferon and ribavirin in HCV patients

Authors


Prof. F. Angelico, Department of Experimental Medicine, Sapienza University, Rome, Policlinico Umberto 1, 155 Viale del Policlinico, 00161 Rome, Italy.
E-mail: Francesco.angelico@uniroma1.it

Summary

Background  Rapid virological response (RVR) is the best predictor of sustained response to standard HCV treatment.

Aim  To evaluate predictive factors of RVR.

Methods  Sixty-five patients (mean age 52.6 ± 13.8; 37 genotype-1, and 28 genotypes-2/3) were consecutively treated with pegIFN-alpha2a or 2b once weekly plus daily ribavirin based on body weight for 24 or 48 weeks, according to genotype. RVR was defined as undetectable HCV-RNA at week 4.

Results  Twenty-seven percent of patients achieved RVR in genotypes 1 and 60.7% in genotypes 2/3 (< 0.01). Rapid responders had higher mean serum baseline total and LDL-cholesterol levels (< 0.01). RVR was 20.0% in the bottom tertile of total cholesterol and 63.6% in the top tertile (< 0.01). HCV-RNA levels at week 4 were positively correlated with baseline serum insulin (< 0.01), HOMA-IR (< 0.01), body mass index (< 0.05) and number of components of metabolic syndrome (< 0.01) and negatively correlated with cholesterol levels (< 0.05). At multivariate analysis, age, LDL-cholesterol, HCV genotype and serum 8-iso-PGF2alpha, a marker of oxidative stress, were independent predictors of RVR.

Conclusions  Our prospective study supports a role of low serum total and LDL-cholesterol and of oxidative stress as positive independent predictive factors of poor RVR in HCV patients.

Introduction

Chronic hepatitis C is a leading cause of chronic liver disease in Western countries. HCV-related liver disease is a major public health issue because of its propensity to progress to chronic hepatitis, cirrhosis and hepatocellular carcinoma. The current standard of care for patients with chronic hepatitis C is the combination of pegylated interferon-α andribavirin. Unfortunately, approximately forty-five percent of genotype 1 patients do not exhibit a sustained response (SVR), while standard treatment appears highly effective for patients with HCV genotypes 2 or 3.1, 2

Viral kinetics are considered the basis for the study of response to therapy. The decrease in viral load within 24 h after administration of a single test dose of pegINF-alfa predicts the outcome of treatment with a specificity of 100% and sensitivity of 83%.3, 4

Recently, rapid virological response (RVR) to standard treatment, defined as an undetectable serum HCV RNA level at week 4 of treatment, has become a new tool for predicting treatment outcomes in HCV patients and the best predictor of SVR.5–7 Moreover, RVR may identify patients for whom a shorter course of therapy may be appropriate. 8

Many retrospective studies have addressed the issue of identifying factors predicting the response to standard anti-viral treatment in HCV patients.9, 10 Viral factors such as genotype and quasispecies and viral load have a high predictive value for outcome of therapy. 11 Conversely, host predictive factors have a low negative predictive value.

Some retrospective studies identified high levels of LDL and total cholesterol as predictors of SVR.12–14 Insulin resistance, liver steatosis and the metabolic syndrome have also been found to be associated with a greater risk of treatment failure in patients with hepatitis C treated with combination therapy.15–18 Other baseline host characteristics including younger age, high platelet count and low body mass index have been suggested to influence treatment response favourably.9, 10, 15–18 However, most of the above findings have been obtained by retrospective observational cohort studies in which insulin resistance and/or serum lipids have not been evaluated and the metabolic syndrome has not been diagnosed according to current working definitions.19

Our aim was to investigate the predictive factors of RVR in a prospective study of HCV patients consecutively treated with the standard Peg-IFN-alpha2/ribavirin combination therapy.

Methods

Patients

Sixty-five HCV Italian patients (42 men and 23 women), aged 18–75 years (mean age 52.6 ± 13.8; 37 genotype 1 and 28 genotype 2/3), were included in the study. Patients were consecutively recruited from two University hospitals of Rome between December 2006 and November 2007. Patients were considered eligible if they met the following inclusion criteria: age >18 years; positive serum HCV-RNA by standard polymerase chain reaction. Patients were excluded if they had: presence of hepatitis B surface antigen (HBsAg) in serum or markers of HIV coinfection; recent or active alcohol and/or drug abuse; platelet count below 70 000/mL or leucocyte count below 3000/mL; evidence of clinical cirrhosis and evidence of autoimmune or genetic liver diseases, malignancies or renal, haematological, cardiopulmonary, neurological and other gastrointestinal diseases. None of the patients was taking statins or other medications active on lipoprotein metabolism.

Study protocol

This was a prospective study of patients with chronic HCV infection. All patients fulfilling the inclusion/exclusion criteria started treatment with Peg-IFN-alpha2a (180 mcg/week) or Peg-IFN-alpha2b (1.5 mcg/kg/week) subcutaneously once weekly plus daily ribavirin based on body weight for 24 or 48 weeks, according to genotype. Clinical, biochemical and virological features were evaluated at baseline and after 4 weeks. RVR was defined as undetectable HCV-RNA at week 4 of treatment. Patients were also classified as: complete-rapid-responders (patients with undetectable serum HCV-RNA at week 4); partial-rapid-responders (patients with a serum HCV-RNA reduction > 2 log at week 4); nonrapid-responders (patients with a serum HCV-RNA decrease <2 log at week 4 from pre-treatment viral load). The end-point of the study was to define the predictive value of metabolic factors on the rate of RVR in HCV difficult-to-treat (genotypes 1) and easy-to-treat (genotypes 2/3) patients.

Subjects underwent a complete clinical and biochemical workup including full medical history and physical examination. Arterial blood pressure was measured on the right arm with the subjects in a sitting position and after a 5-min rest, using a mercury sphygmomanometer: the average of two measurements, 1 min apart, was considered. Height and weight were recorded with subjects wearing light clothing without shoes using a professional balance beam (Seca, Hamburg, Germany) and body mass index (BMI) was calculated as weight (kg) divided by height (m2). Waist circumference was measured at the level midway between the lowest rib margin and the iliac crest.

Following the new National Cholesterol Education Program Adult Treatment Panel III criteria,19 metabolic syndrome was diagnosed by the concomitant presence of at least three of the following five clinical features: central obesity (defined as waist circumference ≥102 cm for men and ≥88 cm for women), fasting blood glucose ≥100 mg/dL or drug treatment for increased blood glucose levels, triglycerides ≥150 mg/dL or drug treatment for increased triglyceride levels, HDL-cholesterol <40 mg in men and <50 mg/dL in women/dL, arterial systolic/diastolic blood pressure ≥130/≥85 mm/Hg or drug treatment for increased blood pressure.

Blood samples were collected at baseline and at week 4 of treatment. After an overnight fast and supine rest for at least 10 min, blood samples were taken in tubes containing 3.8% sodium citrate and centrifuged at 5000 × g for 10 min to obtain plasma, which was stored at −80 °C until use. Serum total cholesterol, HDL-cholesterol and triglycerides were measured using an Olympus AN 560 apparatus following enzymatic colorimetric method. LDL-cholesterol levels were calculated according to the Friedwald formula.20 Plasma insulin levels were assayed by commercially available radioimmunoassay. The homeostasis model assessment (HOMA-IR) was used to estimate insulin resistance using the formula: glucose (mmol/L) × [insulin (mU/L)/22.5].21

Serum HCV-RNA concentrations were measured using a quantitative multicycle reversed transcribed polymerase chain reaction method, with a lower limit of sensitivity of <15 UI/mL (PCR real time). HCV genotypes were determined after hybridisation of biotin-labelled polymerase chain reaction products to oligonucleotide probes bound in strips on nitrocellulose membranes (Inno-LiPA, Innogenetics, Brussels, Belgium). The HCV genotypes were designated according to the nomenclature proposed by Lau JY et al.22

Plasma 8-iso-Prostaglandin F was measured by a8-iso-Prostaglandin F specific EIA kit (Cayman chemical). 8-iso-Prostaglandin F concentration was expressed as pmol/mL. Intra- and inter-assay coefficients of variation were 2.1% and 4.5% respectively.

Serum high-sensitivity C-Reactive Protein (hs-CRP) was assayed by an immunonephelometric method (N-High Sensitivity CRP, Dade Behring Marburg Gmbh). Intra-assay and inter-assay coefficients of variation were 3% and 2.5% respectively.

Each subject signed a study consent before enrolment in the trial. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Statistical analysis

Statistical analysis was performed using the spss statistical software V15.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± s.d. for continuous variables. Student’s t-test for unpaired data was used for comparison of mean values. Proportions and categorical variables were tested by the chi-square test and the two-tailed Fisher exact method, when appropriate. All P values were two-tailed; a P value of less than 0.05 was considered as indicating statistical significance. Multivariate analyses were carried out using stepwise logistic regression models, which considered as dependent variables the dichotomous response variable presence or absence of rapid virological response. The following were entered as independent variables in the different models: age, gender, BMI, waist circumference, diabetes mellitus, metabolic syndrome, blood pressure, basal HOMA-IR, serum HCV-RNA, ALT values, total and LDL cholesterol levels, HCV genotype (1 vs. 2/3), serum 8-iso-Prostaglandin F, hs-CPR and type of peg-INF- alpha administered.

Results

The study was performed in 65 HCV patients. Mean age was 52.3 ± 13.6; most patients were men (64.6%) and had a predominant genotype 1 infection (56.9%). Twenty percent had type 2 diabetes mellitus, 30.8% were overweight and 21.5% were obese; prevalence of MS was 24.6%. The baseline characteristics of HCV patients according to viral genotypes 1 or 2/3 are reported in Table 1.

Table 1.   Some clinical and metabolic characteristics of HCV patients with genotypes 1 or 2/3
 Viral genotype
12/3
n = 37n = 28
  1. * Defined according to ATPIII criteria.19

  2.  P < 0.02.

Males (%)70.357.1
Age (years)51.0 ± 12.354.0 ± 15.2
Tx Peg-IFN-alpha2a (%)57.154.1
Body weight (kg)76.5 ± 15.871.1 ± 14.8
Body mass index (kg/m2)26.1 ± 5.125.5 ± 5.0
Waist circumference (cm)92.8 ± 13.188.5 ± 12.5
Diabetes mellitus (%)21.617.9
Obesity (BMI ≥30) (%)24.317.9
Platelets × 103167 ± 53 156 ± 82
Fasting glucose (mg/dL)98.3 ± 27.0105.8 ± 28.5
Fasting insulin (umol/L)13.4 ± 7.312.0 ± 8.5
HOMA-IR3.18 ± 1.903.47 ± 3.53
No. risk factors*1.46 ± 1.231.32 ± 1.36
Hyperglycaemia (%)*37.835.7
High blood pressure (%)*40.528.6
High waist circumference (%)*29.728.6
Low HDL cholesterol (%)*32.428.6
High triglycerides (%)*13.510.7
Metabolic syndrome (%)24.325.0
Total cholesterol (mg/dL)172.3 ± 35.0178.1 ± 42.5
HDL cholesterol (mg/dL)49.8 ± 14.648.3 ± 18.5
LDL cholesterol (mg/dl)100.4 ± 31.6119.4 ± 42.0
Triglycerides (mg/dL)108.7 ± 50.192.9 ± 37.4
HCV-RNA × 103 (UI/mL)5.366 ± 9.9503.972 ± 4.801
AST (UI/mL)68.5 ± 71.677.7 ± 69.5
ALT (UI/mL)102.0 ± 81.2108.0 ± 116.7
hs-CRP (mg/dL)1.53 ± 1.943.50 ± 3.46†
8-iso-Prostaglandin F (pmol/mL)53.1 ± 59.243.1 ± 58.3

After the first 4 weeks of treatment, 27 patients (41.5%) achieved RVR (27.0% in genotype 1 and 60.7% in genotypes 2/3, = 0.01). Sustained virological response was 45.2% in genotype 1 and 62.5% in genotypes 2/3. SVR was 79.2% in rapid responders and 37.1% in non-rapid responders (= 0.004). In patients with genotype 1, SVR was 77.8% in rapid responders and 34.8% in non-rapid responders (< 0.05); the corresponding percentages of SVR in patients with genotypes 2/3 were 80.0% and 41.7% (P = N.S.) respectively.

There were no dropouts and none of the patients had to reduce the drug dosage. The demographic and baseline clinical data of patients according to rapid virological response are reported in Table 2.

Table 2.   Some clinical and metabolic characteristics of rapid responders and non-rapid responders HCV patients to standard pegylated interferon-α/ribavirin therapy
 Rapid virological response†
NonrespondersRespondersP
n = 38n = 27
  1. * Defined according to ATPIII criteria.19

  2.  HCV RNA negative at week 4.

Males (%)65.759.30.308
Genotype 1 (%)71.137.00.007
Tx Peg-IFN-alpha2a (%)55.355.60.591
Age (years)53.4 ± 14.251.6 ± 13.50.716
Body weight (kg)72.2 ± 12.377.0 ± 19.00.218
Body mass index (kg/m2)25.2 ± 4.526.7 ± 5.60.256
Waist circumference (cm)90.5 ± 12.091.5 ± 14.90.768
Diabetes mellitus (%)26.311.10.115
Platelets × 103153 ± 59175 ± 750,198
Fasting glucose (mg/dL)101.3 ± 30.599.5 ± 19.6 0.649
Fasting insulin (umol/L)13.5 ± 7.411.7 ± 8.30.424
HOMA-IR3.3 ± 2.23.1 ± 3.30.775
No. risk factors*1.6 ± 1.31.2 ± 1.10.351
Hyperglycaemia (%)*45.425.90.098
High blood pressure (%)*37.133.30.490
High waist circumference (%)*25.737.00.187
Low HDL cholesterol (%)*40.022.20.162
High triglycerides (%)*14.311.10.560
Metabolic syndrome (%)25.725.90.530
Total cholesterol (mg/dL)163.3 ± 28.6190.9 ± 44.40.003
HDL cholesterol (mg/dL)46.9 ± 15.253.5 ± 18.10.155
LDL cholesterol (mg/dL)96.7 ± 26.4126.7 ± 46.60.005
Triglycerides (mg/dL)95.6 ± 39.3111.3 ± 52.80.177
HCV-RNA × 103 (UI/mL)5939 ± 101993561 ± 41710.345
AST (UI/mL)77.0 ± 49.955.0 ± 58.00.092
ALT (UI/mL)109.2 ± 75.988.1 ± 105.90.253
hs-CRP (mg/dL)2.01 ± 2.902.88 ± 2.770.279
8-iso-Prostaglandin F (pmol/mL)51.3 ± 61.444.6 ± 55.60.335
SVR (%)  37.1%  79.2%0.004

Rapid viral responders had statistically significant higher mean serum baseline total cholesterol (163.3 ± 28.6 mg/dL vs. 190.9 ± 44.4 mg/dL; = 0.003) and LDL cholesterol (96.7 ± 26.4 mg/dL vs. 126.7 ± 46.6 mg/dL; = 0.005) levels and a lower prevalence of HCV genotype 1 as compared with rapid nonresponders.

Before treatment, mean serum total cholesterol was 157.1 ± 27.6 mg/dL in 14 patients with no RVR, 167.0 ± 29.1 mg/dL in 24 with partial RVR and 190.9 ± 44.4 in 27 who had complete RVR (< 0.01). The corresponding figures for basal serum LDL cholesterol were 94.3 ± 17.9 mg/dL, 98.5 ± 31.9 mg/dL and 126.7 ± 44.4 mg/dL respectively (< 0.02). The prevalence of RVR was 20.0% in the bottom tertile of basal total cholesterol (15.4% in genotype 1 and 28.6% in genotypes 2/3) and 63.6% in the top tertile (33.3% in genotype 1 and 100.0% in genotypes 2/3) (= 0.01).

Rapid virological response was similar in 35 patients treated with Peg-IFN-alpha2a and in 30 treated with Peg-IFN-alpha2b (41.7% and 41.4% respectively, =0.591); the percentage of patients treated with Peg-IFN-alpha2a was not statistically different between responders and nonresponders in all patients (55.6 vs. 55.3 respectively, = 0.561) (Table 2) and in those with genotypes 1 and 2/3.

At bivariate analysis, serum HCV-RNA levels at week 4 were positively correlated with baseline serum insulin (< 0.01), HOMA-IR (< 0.01), body mass index (< 0.05) and no. of components of the metabolic syndrome (< 0.01) and negatively correlated with plasma total cholesterol levels (< 0.05) (Table 3).

Table 3.   Linear correlation coefficients between HCV-RNA at week 4 of treatment and at six months after dropping treatment and some clinical and biochemical variables at baseline examination
 HCV-RNA at week 4HCV-RNA at week 6 off therapy
rPrP
  1. * Defined according to ATPIII criteria.19

Age0.034N.S.0.048N.S.
HCV-RNA0.253<0.050.238N.S.
ALT−0.069N.S.−0.088N.S.
Fasting glucose0.073N.S.0.213N.S.
Total cholesterol−0.271<0.05−0.062N.S.
HDL cholesterol−0.123N.S.0.045N.S.
LDL cholesterol−0.181N.S.0.079N.S.
Triglycerides−0.014N.S.0.011N.S.
Insulinemia0.418<0.010.277<0.05
HOMA-IR0.315<0.010.280<0.05
8-iso-Prostaglandin F−0.100N.S.−0.159N.S.
hs-RCP−0.050N.S.−0.049N.S.
Waist circumference0.203N.S.0.231N.S.
BMI0.307<0.010.323<0.01
No risk factors*0.382<0.010.345<0.01

Compared with nonresponders, rapid responders tended to have lower mean serum 8-iso-Prostaglandin F levels (44.6 ± 55.6 pmol/lL vs. 51.3 ± 61.4 pmol/mL) and a higher percentage decrease in 8-iso-Prostane levels at week 4 of anti-viral treatment (46% vs. 19%), although the above comparisons did not reach statistical significance.

Several multiple logistic regression models were constructed to evaluate the independent predictors of RVR. We initially tested covariates that may have had clinical relevance and those that showed univariate associations with RVR. In the final regression model, younger age, non-1 HCV genotypes, high total cholesterol levels and low serum 8-iso-Prostaglandin F levels were independent predictors of RVR, after controlling for gender, the metabolic syndrome and serum HCV-RNA and ALT basal levels (predictive value of the model: R2 = 0.41) (Table 4).

Table 4.   Independent factors predictive of rapid virological response at week 4
FactorCategoryOR (95% CI)P
  1. Only variables that achieved statistical significance (< 0.05) on multivariate logistic regression are shown.

Age (years)≥5010.031
<5014.3 (1.2–162.6)
HCV GenotypeSubtype 110.027
Subtype non-16.6 (1.2–35.6)
Total cholesterol (mg/dL)<16510.003
≥16562.3 (4.0–958.6)
8-iso-Prostaglandin F (pmol/mL)≥5010.049
<5010.0 (1.0–99.7)

Discussion

Response rate to standard of care for HCV treatment with a combination of peg-IFN and ribavirin is about 50% and remains a major concern in patient care. Recently, RVR has become a valuable new tool for better treatment tailoring and optimization and patient motivation. Our findings confirm RVR as a good predictor of SVR both in genotypes 1 and non-1. In fact, both in patients with genotypes 1 and in those with genotypes 2/3, almost 80% of patients who had an RVR achieved an SVR.

However, so far, only few data derived from retrospective studies7–13 are available on the clinical and metabolic features of HCV patients without RVR to peg-INF and ribavirin standard therapy. Moreover, conflicting results have been reported and the clinical relevance of some host’s conditions such as the metabolic syndrome, diabetes mellitus, obesity and dyslipidemia remains under debate.

The present study is the first prospective trial evaluating the predictive value of viral, clinical and metabolic characteristics in relationship with RVR in patients with chronic hepatitis C.

As expected, in our study population, the RVR rate was much lower in patients with genotype 1 than in those with genotypes 2 or 3, (27.0% vs. 60.7%, respectively).1, 2, 23 No association was found in the present study between RVR and baseline HCV-RNA levels, although the latter was the only independent positive predictor of RVR in a post hoc analysis of data collected during a phase III study performed in HCV genotype 1 patients.7

At multivariate analysis, we identified several independent host factors capable of predicting RVR after peg-INF/ribavirin treatment. The most statistically significant finding was the association between serum cholesterol and treatment outcome in patients with chronic HCV. We identified low baseline cholesterol as an independent predictor of lack of RVR. The association between low serum cholesterol and poor RVR to HCV standard treatment was demonstrated by univariate and multivariate analyses. In fact, even after controlling for age and viral genotype, higher levels of pre-treatment cholesterol levels significantly increased the probability of RVR. This suggests that having higher LDL or total cholesterol levels prior to treatment may favour a good outcome of therapy, given that achievement of RVR is considered the strongest predictor of sustained response. The relationship between lipid profile prior to treatment and response to HCV treatment was already described by several retrospective studies.12, 13, 24 Long-term reversal of hypocholesterolemia is related to sustained viral response in genotype 3, HCV patients.25 Moreover, the LDL receptor has been proposed as a candidate receptor for HCV and a competitive inhibition of C virus binding to the LDL receptor by LDL has been shown in vitro,26 suggesting a role for serum lipids in regulating HCV clearance. Recently, an anti-viral in vivo effect of statins has been demonstrated and their use in combination with standard anti-viral therapy has been also hypothesized,27, 28 although atorvastatin treatment does not appear to inhibit HCV-RNA replication in vivo.29 Low serum cholesterol may also indicate more severe liver disease, progression of fibrosis and subsequent reduced treatment response; in our study, a routine liver biopsy to assess the degree of fibrosis was not performed; however, HCV patients with clinically defined cirrhosis and those with early biochemical and/or echographic signs of disease progression were excluded.

In our study, plasma 8-iso-prostaglandin F2α, an endogenously formed end product of lipid peroxidation and a reliable marker of in vivo systemic free radical generation and oxidative stress,30 was an independent predictor of rapid nonresponse at multivariate logistic analysis. In addition, mean plasma 8-isoprostane levels after 4 weeks on anti-viral treatment were decreased by 46% in patients with RVR and only by 19% in those without RVR, suggesting that enhanced oxidative stress may also be implicated in the pathophysiological mechanisms of nonresponse to anti-viral therapy. We have previously demonstrated that lipid peroxidation is increased in liver cirrhosis.31 Further studies have suggested that oxidative stress may play an important role in the pathogenesis of chronic liver diseases and it has been shown that noncirrhotic HCV patients have an increased oxidative stress and lower levels of antioxidant markers.32 In fact, in a study performed in patients with chronic liver diseases, lipid peroxidation, as assessed by plasma values of 8-isoprostane, was increased in those with non-alcoholic fatty liver disease and chronic hepatitis C, as compared to healthy controls;33 moreover, in the same study, consistent with our results, peg-interferon treatment resulted in a dramatic decrease in 8-isoprostane levels, concomitant with disappearance of HCV-RNA. Finally, a combination of anti-viral and antioxidant therapy such as vitamin E has been suggested to enhance the overall response rate of HCV patients to peg-interferon and ribavirin therapy.34–36

Previous retrospective studies have suggested that insulin-resistance syndrome could contribute to treatment failure in HCV patients.17 Insulin resistance, measured by the homeostasis model (HOMA-IR), has been found to be independently associated with reduced sustained response rate to peginterferon plus ribavirin.18, 37–39 Recently, it has been suggested that measures to modify insulin resistance may be effective in enhancing sustained virological response before or during anti-viral treatment.18, 40 Of interest, in our study, is although at univariate analysis body mass index, basal serum insulin, HOMA-IR levels and the clustering of metabolic risk, factors were all significantly correlated with HCV-RNA serum levels at week four of treatment, they were not independently associated with RVR and the prevalence of the metabolic syndrome did not differ between rapid responders and nonresponders. These negative findings add to the controversy in current medical literature regarding the role of abdominal obesity and the metabolic syndrome in treatment outcome of HCV patients.15–17, 39–42

In conclusion, the results of our prospective study support a role of low serum total and LDL cholesterol and of oxidative stress as independent predictive factors of poor RVR in HCV treated patients. Less conclusive findings were observed for insulin resistance and the metabolic syndrome, which, in our experience, were not independently associated with a lack of RVR.

Acknowledgement

Declaration of personal and funding interests: None.

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