Chronic HCV patients with normal ALT values respond well to rebetron therapy

HEPATOLOGYVol. 34, No. 4, Pt. 2, 2 0 0 1

AASLD ABSTRACTS

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HIGH-DAILY INDUCTION DOSE OF INTERFERON IN COMBINATION W I T H RIBAVIRIN IN CHRONIC HEPATITIS C N O N RESPONDER PATIENTS : A RANDOMISED CONTROLLED TRIAL. Vincent Leroy Mr, Nadine Dutertre, CHU Grenoble, Grenoble France; Albert Tran, CHU Nice, Nice France; Sylvie Naveau, CHU Clamart, Clamart France; A r m a n d Abergel, CHU Clermond-Ferrand, C l e r m o n d - F e r r a n d France; Marc Bourliere, Hopital SaintJoseph, Marseflle France; Agnes Plages, Jean-Pierre Zarski, CHU Grenoble, Grenoble France

CHRONIC HCV PATIENTS W I T H NORMAL ALl" VALUES RESPOND W E L L T O REBETRON THERAPY. A n n L Silverman, David Ternes, William Beaumont Hosp, Royal Oak, MI; Lorraine Comanor, Indep Clin Researcher, Palo Alto, CA; Stuart C Gordon, William Beaumont Hosp, Royal Oak, MI

The aim of this prospective randomised study was to evaluate the efficacy and tolerability of two schedules of high daily dose of IFN alpha 2a in combination with ribavirin in non responder patients. Methods : 38 patients were recruited from 5 centers. They had histologically proven chronic hepatitis, ALT > N, and were previously non responders to standard regimen of IFN (3 MUI tiw at least 3 mouths) or IFN + ribavirin (at least 6 months). IFN schedule was 18 MUI/D for 2 days followed by 9 MUUD for 12 days (Group i : n = 2 0 ) or 6 MUUD for 14 days (Group 2 : n = 18) ; followed in both groups by IFN 6 MUUD for 14 days, 3 MUI/D for 5 months and 3 MUI tiw for 6 months. HCV RNA was quantified by PCR (Amplicor Monitor 2) at D1, D2, D3, D4, W2 and W4. End-points were serum HCV RNA negativation by PCR at the end of daily period (W24) and at the end of treatment (W48). Results : Both groups were similar concerning clinical, biochemical and virological characteristics (genotype i : 80%). Median viralload was 6.1 Log cop/ml in group 1 and 6.0 in group 2. Previous treatment was IFN monotherapy in 30 patients (79%) and combination IFN + ribavirin in 8 (21%).In intention-to-treat analysis, the rate of virological response was 30% in group 1 (6/20) and 28% in group 2 (6/18). In both groups, a biphasic decline in viral load was observed. Delta Log D1-D3 (first slope) was 1.3 in group 1 and 0.9 in group 2 (NS). The second decline slope was lower than first slope, without difference between both groups (Delta Log D3-W2 = 0.7 and 0.4, respectively). Decline first slope was similar between responders and non responders at 524 (Delta Log D1-D3 = 1.4 vs 1.1). By contrast, Delta Log D3-W2 was higher in responders than non responders (2.3 vs 1.3, p<0.01). A decline of less than 2 log at W2 and W4 was associated with non response in 88% and 100% of cases, respectively. Among responder patients at W24, 6 are currently under treatment and 6 were evaluated at W48. Among them, 2/'3 (group 1) and 3/3 (group 2) achieved virological relapse at W48. Thus, the rate o f end-of-treatment response was 10% (1/10) in group 1 and 0% (0/11) in group 2 (per protocole analysis).Mains side effects were asthenia (72%), myalgies (59%), nausea (19%), neutropenia (63%), thrompbopenia (53%) and anemia (12%). Treatment was stopped for side effects in 7 patients, 5 from group i and 2 from group 2. Causes were angor (n=3), epilepsia (n--- 1) and severe asthenia (n=3). Conclusions : Despite the use of induction high daily dose of IFN in combination with ribavirin, a very tow response rate was oberved in patients non responders to standard treatment. HCV RNA decline at W2 and W4 seems to be predictive of virological response at month 6. In the future, assessment of viral load decline could be of interest in theses patients during treatment with PEG-IFN.

Background: Normalization of ALT was the treatment endpoint of the original hepatitis C antiviral trials. Current studies continue to exclude patients with normal baseline ALT values. Aim: To determine whether chronic HCV patients with normal ALT levels respond to combination interferon/ribavirin. Methods: 16 HCV RNA positive patients with normal ALT a n d one with ALI" < 1.2 X ULN for at least 3 consecutive m o n t h s received daily interferon alpha 5 MU for one month, plus ribavirin (1000-1200 mg/d), followed by standard Rebetrou. Patients were monitored at baseline~ weeks 1,2,4,8~12,16,20,24,28 by the quantitative VERSANT HCV 3.0 (bDNA) assay (cut-off 615 IU/mL) u p until the time they went below the cut-off at w h i c h point they were reflexed to the Bayer HCV Qualitative Test based on transcription mediated amplification (TMA, limit of detection, 5 IU/ml). Patients whose viral loads were below the bDNA assay cut-off at week 24 were treated for a n additional 24 weeks. Those w h o h a d quantifiable virus at week 24 terminated therapy, All patients were followed for 72 weeks. End of treatment (EOT) a n d sustained response (SR) were defined as undetectable I-ICV RNA by TMA at treatment week (TW) 48 a n d follow-up (FU) week 24, respectively. Results: 7 men and 10 w o m e n (mean age, 45.3 years) were treated; 80% were genotype 1. Two patients dropped out at treatment weeks 2 and 14. Using intent to treat analysis, 8/17 (47%) were b o t h EOT a n d sustained responders; 6 of these 8 were genotype 1. Seven of 8 SR patients h a d HCV RNA levels below the bDNA cut-off at T W 4, but only 4 h a d undetectable HCV RNA by TMA at this time point. At week 12, all SR were below the bDNA cut-off, while 6 h a d undetectable HHCVRNA. One SR did not have undetectable HCV RNA b y TMA until after 16 weeks of treatment. All four patients with low baseline ALT levels ( < 20 U/L) achieved SR. No ALT flares were observed in a n y of the patients. Conclusion: A cohort of chronic HCV patients with normal ALT values responded well to combination interferon/ribavirin therapy, with a sustained response rate of 47%.

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INTRAHEPATIC INNATE AND ADAPTIVE IMMUNE RESPONSES IN HCV INFECTED CHIMPANZEES. Miehelina Nascimbeni, Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, MD; Marian Major, Kathleen Mihalik, CBER, FDA, Bethesda, MD; Charles M Rice, The Rockefeller University, New York, NY; Stephen M Feinstone, CBER, FDA, Bethesda, MD; Barbara Rehermann, Liver Diseases Section, NIDDK, National Institutes of Heahh, Bethesda, MD

INDUCIBLE NITRIC OXIDE SYNTHASE IS NECESSARY FOR ALCOHOLINDUCED LIVER INJURY: STUDIES W I T H KNOCK-OUT MICE. Gavin E Arteel Dr, Erwin G a e b d e Dr, Michael D Wheeler Dr, Takehiko Uesugi Dr, UNC-Chapel Hill, Chapel Hill, NC; Maria B Kadiiska Dr, Henry D C o n n o r Dr, Ronald P Mason Dr, NIEHS, RTP, NC; Ronald G T h u r m a n Dr, UNC-Chapel Hill, Chapel Hill, NC

The uninfected liver is an immunologieally distinct organ containing ceils of the innate a n d adaptive i m m u n e response. The differential contribution of these cell populations to resolution a n d pathogenesis of hepatitis C is unknown. Liver infiltrating cells were isolated from biopsies of 8 naive, 4 recovered, 4 acutely a n d 5 persistently infected chimpanzees a n d from 3 chimpanzees with very low viral load (intermittently positive s e r u m RT-PCR). The infecting virus was m o n o c l o n a l HCV genotype l a in all cases. Direct ex vivo FACS analysis for CD3, CD56, CD4, CD8 a n d DR allowed calculation of the absolute n u m b e r of cells with these surface markers per em biopsy. In addition, a recovered animal challenged with h o m o l o g o u s virus was studied prospectively a n d in vitro e x p a n d e d CD4/CD8 single a n d double positive T cell lines were evaluated for antigen specificity, proliferation, cytokine secretion a n d cytotoxicity. The livers of naive animals contained p r e d o m i n a n t l y C D 3 + C D 5 6 - T ceils (7xl0*/cm biopsy), fewer C D 3 + C D 5 6 + NKT cells (0.7x10 ~') a n d C D 3 - C D 5 6 + NK cells (2.3x104). During acute hepatitis C, the n u m b e r of T cells increased b y a factor of 4, NKT cells by a factor of 5 a n d NK cells b y a factor of 3. After recovery, a slight, 2.4-fold increase of the NKT cell n u m b e r was maintained, while the n u m b e r of T a n d NK ceils decreased. In persistently infected animals, not only the n u m b e r of NKT, b u t also of T cells was elevated. The intrahepatic T cell population of persistently infected antreals (10 4-10 5 viral Geq/ml) contained 21 o~ C D 4 + cells a n d 61 O~ C D 8 + cells while those animals with very low viral load (intermittently positive serum RT-PCR) displayed relatively more intrahepatic C D 4 + cells (32%) a n d fewer C D 8 + cells (43%). 40-60% of intrahepatic C D 4 + T cells expressed the activation m a r k e r DR (compared to only 8-19% C D 8 + T cells) a n d 43% coexpressed CD8a/]3. U p o n challenge with h o m o l o g o u s virus, increased n u m b e r s of intrahepatic T cells (12x) a n d NK cells (3x) coincided with transient viremia. At this time, 90% of these CD4CD8 double positive, 51% of CD4 single positive a n d 45% of CD8 single positive T cells were activated a n d DR positive. Also, more CD4CD8 double positive (87%) than CD4 single positive (47%) than CD8 single positive cell lines (0%) were HCV specific. All CD4CD8 double positive cell lines expressed CD8a/]3, TCRct/~8 a n d p r o d u c e d I F N y in response to HCV nonstructural proteins. In conclusion, b o t h innate a n d adaptive i m m u n e responses appear to play a role in HCV infection. Especially CD4 single a n d CD4CD8 double positive cells are highly activated a n d HCV specific.

Alcoholconsumption causes oxidativestress in liver and increases the formationof the a.hydroxyethyl free radical. Recently,it was shown that injury and a-hydroxyethyl free radical production is completely blocked in mice deficient in the superoxide-producing enzyme NADPH oxidase (p47Ph°x knockout mice), indicating a key role of superoxide (02• ) production from NADPH oxidase in the progression of alcoholic liver injury. One mechanism to explain the role of OzO-in the production of the c¢-hydroxyethylradical adduct is the formation of hydroxyl radical (tHe) via superoxide dismutase (SOD) and transition metals. Alternatively, Kupffer cells also produce the free radical nitric oxide (NOe) from the inducible form of nitric oxide synthase (iNOS) when stimulated. Nitric oxide and O2e-combine ~a a diffusion-limited reaction to form peroxynitrite (ONTO-), another potent oxidant capable of forming a-hydroxyethyl free radicals during its decay. The purpose of the current study was to therefore test the hypothesis that iNOS is involved in oxidative stress and liver damage due to alcohol consumption, iNOS knockout (B6.129P2Nos2~mlL°*9or wild-type (C57BId6) mice were fed high-fat control or ethanol-containing diets for 4 weeks enterally via a gastric tube surgicallyimplanted in the stomach. The amount of ethanol in the diet was increased over the course of the study from 5% to 8% to obtain optimal delivery of calories without compromising growth or survival. The initial rate of ethanol delivery (16 g/kg/ day) was increased in a step-wise manner 1 g/kg every 2 days until the end of the first week and then I g/kg/4 days until the end of the experiment. At sacrifice, blood and liver tissue were collected for further analysis. There were no significant differencesin body weight gains between the groups studied, indicating that animals were adequately nourished. As reported previously by severalgroups, daily urine alcohol concentrations fluctuated between 0 and 500 mg/dL.Wild-type and iNOS knockout mice had similar patterns of urine alcohol cycling and averageand peak urine alcohol levels were not significantlydifferent between the groups with values ~200 mg/dL and -450 mg/dL, respectively.Four weeks of enteml alcohol feeding significantly increased liver to body weight ratios from -5.5% to -9.0% in wild-type mice; the effect of alcohol diet on liver to body weight ratios was significantlyblunted in iNOS knockout mice by about 50%. Serum ALT levels were increased significantly about 4-fold over control values (29 _~ 4 IU/L) by entcral ethanol (113 ± 20) in wild-type mice; this effectwas significantlyblunted in iNOSknockout mice by -60%. Enteral ethanol also caused severe fatty accumulation, mild inflammation,and necrosis in the liver in wild-type mice (total pathology score = 5.2 -T.0.6), but did not cause pathology in iNOS knockout mice (total pathology score = 0.7 - 0.3). Further, the accumulation of 4-hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (reactive nitrogen species formation) protein addacts caused by alcohol was blocked completelyin iNOSknockout mice. These data clearly show that knocking out iNOS in mice confers almost complete protection against the damaging effects of ethanol to liver, supporting the hypothesis that iNOS is required for the pathogertesisof early alcohol-induced hepatitis. The finding that both lipid peroxidation and tyrosine nitration is blocked in iNOS knockout mice indicates that NOe production is involved in the formation of damaging reactive species. The fact that nearly identical protection can be observed with mice deficient in NADPH oxidase indicates that the damaging oxidant in alcoholic liver iujury is dependent on the production of both O2e and NOe, suggestirtga role for ONtO in alcoholic liver injury (supported, in part, by NIAAA).