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Conference Reports
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The Liver Meeting— 54th Annual Meeting of The American Association for the Study of Liver Diseases, October 24 - 28, Boston, MA—Day 4

Alan Franciscus
Editor-in-Chief, HCV Advocate


Poster 248: RETREATMENT OF STANDARD INTERFERON/RIBAVIRIN NONRESPONDER PATIENTS WITH CHRONIC HEPATITIS C WITH DAILY CONCENSUS INTERFERON AND RIBAVIRIN YIELDS HIGH SUSTAINED RESPONSE RATES

Stephan Kaiser, Holger G Hass, Michael Gregor, University of Tuebingen, Tuebingen, Germany

Objective
Numerous studies have shown that retreatment of chronic hepatitis C non-responders with conventional interferon alfa and ribavirin leads to very low sustained response rates of only about 7%. Furthermore, studies with pegylated interferon and ribavirin in nonresponders to standard interferon and ribavirin have shown only 8 -11 % sustained response rates. However, recently improved response rates have been observed in pilot trials using consensus interferon (interferon alfacon-1, CIFN) even as monotherapy in previous combination therapy non-responders.

Methods
In the present study, the efficacy of CIFN daily dosing and induction therapy followed by CIFN / ribavirin combination treatment in combination therapy non-responders was evaluated. 120 patients (mean age: 46.1 yrs., 79% male) have been included. All patients had elevated ALT levels and were viremic, with 91% having genotype 1. The average weight of patients was 79 kg. Histologic confirmation of chronic inflammation by liver biopsy was obtained in all patients with grading and staging according to the Ishak Score, where 28% of patients showed bridging fibrosis or cirrhosis. Patients were either treated with CIFN at a dosage of 18 ug QD for 4 weeks, followed by 9 ug QD for 8 weeks or with CIFN 27 ug QD for 4 weeks, followed by 8 weeks of CIFN 18 ug QD. Thereafter, treatment was continued in all treatment groups with CIFN at a dose of 9 ug QD with ribavirin (at 10 - 15 mg/kg/d) for another 36 weeks.

Results
Data show that after the initial 12 weeks of CIFN monotherapy, a primary response with undetectable serum HCV-RNA was observed in 43 % of patients in the CIFN 18/9 ug QD group and in 58 % in the CIFN 27/18 ug group. After 24 weeks of CIFN / ribavirin combination therapy, a negative PCR was observed in 62 % in the CIFN 18/9 ug + ribavirin group, and in 71% of the CIFN 27/18 ug + ribavirin group. The subset of patients having reached end-of-treatment and 24 week follow-up show response rates of 59 - 66 % (ETR) and 37 - 43 % (SR), respectively.

Due to side effects the CIFN dose had to be dose reduced in 16 % of patients and discontinued in 7 % of patients. The most common cause for dose reductions were significant reductions in WBC and platelet counts, especially in the 27 ug CIFN group, while no growth factors were used in this study. Three patients experienced garde III thrombocytopenias, while no grade IV neutropenias or thrombocytopenias were observed. The overall tolerability of the CIFN 18/9 ug regimen was of comparable tolerability to a standard therapy with pegylated interferon and ribavirin, while the CIFN 27/18/9 ug regimen was less tolerable during the high dose induction period. However, drop out rates were not different between the two dosing regimen.

Conclusions
CIFN daily dosing / induction therapy together with subsequent ribavirin combination therapy thus shows promising response rates in previous combination therapy non-responders. The data are especially interesting since almost all patients are genotype 1 non-responders. Although a significant proportion of patients experienced a relapse after cessation of therapy, the overall sustained response rates are nevertheless promising showing a response rate about 3-fold higher when compared to rates observed with pegyated interferons. It is concluded that CIFN maybe an effective treatment modality for this difficult-to-treat patient group.

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Poster 252: RANDOMIZED CONTROLLED TRIAL OF PEGINTERFERON ALFA-2A PLUS RIBAVIRIN FOR CHRONIC HEPATITIS C VIRUS-GENOTYPE 4 AMONG EGYPTIAN PATIENTS

Fouad I A Thakeb, Cairo University, Cairo, Egypt; Mahmoud M Omar, Theodor Bilharz Reaserch Instiute, Cairo, Egypt; Mostafa M El Awady, Academy of research and science, Cairo, Egypt; Sherif Y Isshak, Cairo University, Cairo, Egypt.

Background
HCV prevalence in Egypt is approximately 12% (7.2 millions people). Genotype 4 is the most prevalent, appears the least responsive to treatment. We compared the efficacy and safety of Peginterferon alfa-2a (PEG-IFN plus ribavirin (RBV) and interferon alfa-2a (standard IFN) plus RBV, in the treatment of chronic hepatitis C.

Methods
A total of 100 patients with chronic hepatitis C ,genotype 4 were randomly assigned to 48 weeks treatment, 51 patient received 180 µg of PEG-IFN once weekly plus daily RBV (1000 or 1200 mg. depending on body weight) and 49 received 3 million units of IFN thrice weekly plus daily RBV for 48 weeks. Final evaluation depending on intention to treat was done 24 weeks after cessation of therapy.

Results
Patients in both groups were well matched demographically as well as the liver biopsy histology and viral load. A significantly higher proportion of patients who received PEG-IFN plus RBV had a sustained virologic response (defined as the absence of detectable HCV RNA 24 weeks after cessation of therapy) than of patients who received standard IFN plus RBV (35/51 % vs. 8/49 respectively, P<0.001). The overall safety profiles of the two treatment regimens were similar; the incidence of influenza-like symptoms and depression was lower in the group receiving PEG-IFN than in the group receiving standard IFN plus RBV.

Conclusions
In patients with chronic hepatitis C, once-weekly PEG-IFN plus RBV was tolerated as well as standard IFN plus RBV and gave significant improvements in the rate of sustained virologic response (68.6% vs. 16.4 % ).

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Poster 1119: BONE LOSS IS RELATED TO SERUM TRANSAMINASES LEVELS IN CHRONIC HEPATITIS C

Eduardo Redondo-Cerezo, Hospital Universitario Virgen de las Nieves, Granada, Spain; Jorge Luis González-Calvín, Hospital Universitario San Cecilio, Granada, Spain; Rafael Martín-Vivaldi, Flor Nogueras-López, Hospital Universitario Virgen de las Nieves, Granada, Spain; Rosaura Fernández-Pascual, Universidad de Jaén, Granada, Spain; Fernando Escobar-Jiménez, Hospital Universitario San Cecilio, Granada, Spain

Introduction
Low bone mass has been described in association with liver cirrhosis. Nevertheless, little is known about the effect of chronic hepatitis C on bone mass and bone turnover. The aim of our study was to assess bone mineral density (BMD) and bone metabolism markers in these patients.

Patients and Methods
34 chronic hepatitis C patients (serologically and histologically demonstrated) were enrolled (mean age 41 years; range 25-55 years; 24 male, mean age 42 years, range 27-55 years and 10 female, mean age 40, range 25-51 years). All the patients had elevated serum ALT levels at least two fold the upper normal range. Patients with cirrhosis, HBV, HDV or HIV coinfections, alcohol consumption, pregnancy, BMI<20 kg/m2, autoimmune disease, a previous therapy with immunosuppressive drugs, steroids, vitamin D, or any medication related to mineral metabolism, renal impairment, malignancies, cardiac or lung or other liver superimposed diseases, were excluded. 80 healthy volunteers served as control subjects (mean age 40 years; range 23-50). Analysis were made for biochemical liver function tests, serum bone Gla-protein (BGP), as a marker of bone formation, insulin-like growth factor I (IGF-I), insulin-like growth factor binding globulin type 3 (IGFBP-3) 25-hidroxivitamin D (25OHD), intact parathyroid hormone (PTH), free testosterone index (FTI), and urinary deoxypyridinoline (D-Pyr) excretion, as a marker of bone resorption. BMD was studied by dual x-ray absorptiometry at the lumbar spine (LS) and femoral neck (FN) and the results were expressed as Z-score values. Osteopenia was defined as Z-score values between –1.5 and –2.5.

RESULTS
BMD in LS were significantly lower in patients (-1.02±1.24) than in controls (0.05±1.49) (p<0.0001). Osteopenia was found in 60% of the patients. Patients had lower IGF-I levels (103.14±45.19 ng/mL) than controls (158.39±68.12 ng/mL) (p<0.0001). Urine D-Pyr levels were significantly higher (6.18±2.26 nM/mM) in patients than in controls (3.88±1.47 nM/mM) (p<0.0001).). No differences were found for serum alkaline phosphatase, cholinesterase, albumin, BGP, 25-OHD, PTH, and FTI. BMD was inversely correlated with serum levels of ALT (r: -0.36; p<0.04) and urine D-Pyr values (r:-0.55; p<0.01). Also, serum ALT levels were inversely correlated with serum levels of IGF-I (r:-0.37; p<0.05) and 25OHD (r:-0.76; p<0.02).

CONCLUSIONS
Our study shows that patients with chronic hepatitis C have low BMD compared to controls and a high prevalence of osteopenia (60%). The inverse correlation found between bone mass and serum ALT levels suggests a relation with liver damage. Low serum IGF-I levels could also be involved in bone loss. The high urinary D-Pyr excretion rate suggests increased bone resorption in these patients.

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Poster 1120: HEPATITIS C TREATMENT PRACTICE PATTERNS IN THE UNITED STATES: A NATIONAL SURVEY

Sean Hurley, Eric J Lawitz, Brooke Army Medical Center, San Antonio, TX

Background
Hepatitis C affects 1.8% of the population of the United States. The introduction of pegylated interferon has improved the efficacy of antiviral therapy when combined with Ribavirin. Patients adhering to therapy have been shown to achieve the highest sustained virologic responses. Strategies to maintain adherence are not well described or studied. In order to assess clinical practice patterns of clinicians, a nationwide survey was conducted of gastroenterologists and hepatologists.

Methods
The two-page survey assessed demographics of the respondents and then focused on several areas of the management of patients with hepatitis C: the use of physician extenders, the role of liver biopsy, the medical treatment, and the management of side effects in patients on treatment. To date, the survey has been distributed to 3,000 of 5,0000 gastroenterologists and hepatologists and 421 have been returned (14%). Follow-up phone interviews with selected respondents will be performed.

Results
Although most respondents use physician extenders, 28% use credentialed providers (PA-C, APN) to aid in hepatitis C management. Most (73.1%) respondents will order a liver biopsy on all of their hepatitis C patients. Treatment success rates in genotype 2 and 3 patients have not resulted in a decreased rate of pretreatment liver biopsies. Liver biopsies are referred to a radiologist to be preformed in 57.6% of respondents. The test and treat strategy is used by 70% of respondents to determine the need for vaccinations against Hepatitis A/B, however 7% do not routinely recommend vaccination. Uniformly (80.3%) the therapy of choice is pegylated interferon and weight-based ribavirin. Eighty-six percent of respondents treat genotype 2 and 3 patients for 24 weeks. Half of surveyed physicians would recommend treatment for patients with acute Hepatitis C. The emerging data on early virologic response has resulted in 73% of respondents discontinuing therapy if a 2 log or greater reduction in viral load is not achieved at week 12 of therapy. Despite limited data on the efficacy of retreatment of RebetronR nonresponders, 62% of respondents retreat previous nonresponders and 74% retreat previous relapsers. Thirty percent of respondents have used maintenance therapy outside of a clinical trial in nonresponders with advanced liver disease. Dose reduction is the primary strategy to manage side effects and cytopenias. Forty-one percent of respondents will treat cytopenias with growth factors as the initial treatment strategy however insurance reimbursement limits their use more than 50% of the time in 26% of respondents. Ten percent use gabapentin for myalgias/arthralgias and 18% will use pharmaceutical agents to combat fatigue (methylphenidate, modafenil, testosterone). Management of depression includes an 11% rate of prophylactic antidepressants use. Most (74%) of respondents will manage therapy induced depression themselves. SSRI's are the agents of choice with fluoxetine and sertraline the most frequently prescribed.

Conclusion
Physicians' practice patterns are consistent with the recommendations of the 2002 NIH Consensus Conference based on the portion of the statement reviewed in this survey. The primary strategy to manage adverse events is dose reduction. Alternative strategies to manage adverse events are being used across the United States. Prospective randomized placebo controlled trials are needed to critically examine the effectiveness of these alternative strategies.

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Poster 1127: DO OVER-THE-COUNTER ANALGESICS CONTRIBUTE TO DECOMPENSATION OF CIRRHOSIS? - A PROSPECTIVE STUDY

Sakib K Khalid, Yale University, Hepatitis C Resource Center CT-VA Healthcare System, New Haven-West Haven, CT; Jill Addesa, Victor J Navarro, Yale University, New Haven, CT; Guadalupe Garcia-Tsao, Yale University, CT-VA Healthcare System, New Haven-West Haven, CT

Over-the-counter analgesics (OTCA) can theoretically lead to decompensation of cirrhosis. Acetaminophen, an intrinsic hepatotoxin, would do so by producing an acute on chronic liver injury, particularly in the setting of chronic alcohol consumption. Non-steroidal anti-inflammatory drugs (NSAIDs) would do so by blunting the response to diuretics and/or by promoting renal vasoconstriction. The effect of OTCA in precipitating the decompensation of cirrhosis has not been investigated and was the objective of this prospective study.

Methods
In the period comprised between August 2000 and May 2002, 91 cirrhotic patients were admitted consecutively for decompensation of cirrhosis (variceal hemorrhage, ascites, encephalopathy, jaundice, spontaneous bacterial peritonitis or other infections, and/or renal dysfunction). These cases were compared to 153 consecutive cirrhotic patients seen in the outpatient liver clinic in the same period, who had not been hospitalized either in the previous 3 months nor 30 days after inclusion into the study. A second control group of 89 randomly selected non-cirrhotic patients concurrently hospitalized with the cases was added to account for recall bias. All patients underwent a structured questionnaire eliciting drug and alcohol use, as well as detailed information regarding all medications, including OTCA (quantity, frequency, and type of preparation), and alcohol ingestion (30-day timeline follow-back method). Non-parametric statistics (Kruskal-Wallis, Mann-Whitney) and the Fisher’s exact test were used. Results are expressed as medians and ranges.

Results
The decompensating events leading to admission in the 91 cases were encephalopathy (41%), ascites (31%), variceal hemorrhage (23%), SBP or other infection (10%), hepatic hydrothorax (7%), jaundice (5%) and/or renal dysfunction (4%). Within the subgroup of 90 patients in whom alcohol played a role in the etiology of cirrhosis, cases ingested significantly more alcohol in the last 30 days (median 812 vs. 112 grams, p=0.03) and for a longer period (20 days vs. 2 days, p=0.03) compared to controls. In patients with non-alcoholic etiologies, no differences were found between cases and controls regarding alcohol or OTCA ingestion. There was no relationship between the type of decompensating event and the type or amount of OTCA.

Conclusions
Cirrhotic patients ingest significantly less OTCA than non-cirrhotic controls. The ingestion of low quantities of OTCA (below recommended dosages) does not play a role in the decompensation of cirrhosis. As expected, alcohol is an important contributor to decompensation in alcoholic cirrhosis. Partially supported by a grant from McNeil Pharmaceuticals.

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Poster 1185: FIRST PHASE HCV KINETICS IN INTERFERON AND RIBAVIRIN COMBINATION THERAPY IS AN USEFUL PREDICTOR OF TREATMENT RESPONSE IN CHRONIC HEPATITIS C PATIENTS

Akiko Makiyama, Yoshito Itoh, Tomoaki Nakajima, Atsushi Umemura, Youuichi Harano, Kouji Kunimoto, Mika Okita, Mika Nakayama, Koujirou Mori, Junko Yamaoka, Hideki Fujii, Kanji Yanaguchi, Norihito Yamauchi, Masahito Minami, Takeshi Okanoue, Kyoto Prefectural University, Graduate School of Medicine, Kyoto, Japan

Aim
In chronic hepatitis C patients, treatment response for interferon (IFN) and ribavirin (Rib) combination therapy can be predicted by the disappearance of serum HCV RNA from the serum 4 weeks after the start of the combination therapy, or by the viral decline during the second phase. Recently, several reports indicated that HCV viral load and viral decline after the first 24 hrs of the combination therapy (first phase viral kinetics) could also predict the response of IFN or IFN and Rib combination therapy. In this study, we investigated HCV kinetics during the combination therapy and analyzed whether or serum HCV viral load and viral decline after the first 24 hrs are useful in the prediction of treatment response as compared with the disappearance of HCV RNA from the sera after the first 4 weeks of treatment.

Patients and Method
Sixty-fifth chronic hepatitis C patients were treated with IFNalpha2b and Rib for 24 weeks. HCV viral load was determined at 11 points during the treatment protocol. Sustained viral responders (SVR) are defined as the patients showing negative serum HCV RNA at 6 months after the end of treatment. Non-responders (NR) were positive for serum HCV RNA after treatment.

Results
In 65 patients, 49 were genotype 1 (SVR 9, NR 40) and 16 were genotype 2 (SVR 13, NR 3). In genotype 1 patients, the mean half-life of HCV RNA in the first phase was significantly (p=0.0191) shorter in SVR (0.24 days) than in NR (0.47 days), although the significance was not so obvious in comparison with that in the second phase (SVR; 3.17 days and NR; 13.15 days: p=0.0024). Among the 49 genotype 1 patients, 1) 41 patients were positive for serum HCV RNA after the first 4 weeks of treatment, and SVR could not be achieved in 37 patients of them (NPV: negative predictive value=90.2%, PPV: positive predictive value=62.5%), 2) 42 patients showed serum HCV RNA level higher than 5.2 KIU/ml after the first 24 hrs of the therapy, and SVR could not be achieved in 38 patients of them (NPV=90.4%, PPV=71.4%), 3) the decreased ratio of in serum HCV RNA (24h/0h) were larger than 0.101 in 34 patients, and SVR could not be achieved in 31 patients of them (NPV=91.2%, PPV=40.0%), In genotype 2 patients, HCV dynamics were not significantly different between SVR and NR. Among the 16 genotype 2 patients, 1) all 10 patients with negative serum HCV RNA after 4 weeks of treatment achieved SVR (PPV=100%, NPV=50.0%), 2) all 12 patients with lower than 66 KIU/ml of serum HCV RNA after the first 24 hrs of treatment achieved SVR (PPV=100%, PPV=75.0%), 3) all 11 patients with the decreased ratio of serum HCV RNA (24h/0h) less than 0.102 achieved SVR (PPV=100%, NPV=60.0%).

Conclusion
1) In genotype 1 patients, HCV kinetics within the first 24 hrs of the combination therapy in an useful predictor of NR with the probability over 90% (NPV).
2) In genotype 2 patients, HCV kinetics within the first 24 hrs of the combination therapy is an accurate predictor of SVR (PPV).
3) HCV viral load and viral decline ratio within the first 24 hrs of the IFN and Rib combination therapy are good predictors of NR in genotype 1 and SVR in genotype 2 patients, which were comparable to the predictive value of the disappearance of HCV RNA from the sera after the first 4 weeks of treatment.

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Poster 1186: PEGINTERFERON ALFA-2a (40KD) (40KD) (PEGASYS?) PLUS AMANTADINE (AMA) OR RIBAVIRIN (RBV) IN TREATMENT-NAÏVE PATIENTS WITH CHRONIC HEPATITIS C (CHC)

G Ideo for the Hepatitis Italian Multicenter Pegasys Amantadine Cooperative Trial group (G Antonucci, A Attili, S Babudieri, A Chirianni, A Craxì, G Di Perri, A Francavilla A Mangia, A Picciotto, M Rizzetto, G Ruggiero, F Suter, S Tripi, M Sarracino)

Background
Pegylated interferons (PEG-IFN) plus RBV are currently the standard of care for most patients with CHC. The usefulness of amantadine (AMA) as an adjunct to PEG-IFN in treating treatment-naïve patients with CHC is controversial.

Aim
To compare the efficacy and tolerability of combination regimens of PEGASY? with RBV or AMA in a large randomized multicenter (49 centers) trial.

Patients and Methods
A total of 734 HCV RNA positive treatment-naïve patients with elevated ALT levels and biopsy-proven CHC have been randomized to peginterferon alfa-2a (40KD) 180 ?g once-weekly in combination with either RBV (1 or 1.2 g/day) or AMA (200 mg/day) for 48 weeks with a 24-week follow-up period.

Results
Demographic data of the 565 patients enrolled to date: M/F ratio 2:1, mean age 45.3 years, mean weight 73.1 kg, mean ALT 123.5 IU/L, 31.4% of patients have cirrhosis, and 59.6% were infected by HCV genotype 1 or 4. The mean baseline HCV-RNA was 650,000 IU/mL. The virological response is reported in the Table.

Conclusions
As expected, a more favorable efficacy trend was observed in the PEGASYS?® + RBV arm than the PEGASYS?® + AMA arm. This trend was more evident in the difficult-to-treat patients (genotype 1, 4).


G = genotype, NA = not applicable

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Poster 1187: THE PREDICTIVE VALUE OF CORE AG TESTING IN THE MANAGEMENT OF PATIENTS RECEIVING PEG-IFN/RIBAVIRIN TREATMENT - RELEVANCE FOR THE EARLY IDENTIFICATION OF NON-RESPONDERS AND RELAPSERS

Pierre Pradat, Marianne Maynard, Hotel-Dieu, Lyon, France; Pascale Berthillon, INSERM U271, Lyon, France; Gaston Picchio, Ortho Clinical Diagnostics, Raritan, NJ; Hans Tillmann, Johannes Wiegand, Medizinische Hochschule, Hannover, Germany; Nicolas Voirin, Hotel-Dieu, Lyon, France; Michael Manns, Medizinische Hochschule, Hannover, Germany; Maria Buti, Juan-Ignacio Esteban, Hospital General Vall d'Hebron, Barcelona, Spain; Michele Martinot, Patrick Marcellin, INSERM U483, Paris, France; Christian Trépo, INSERM U271, Hotel Dieu, Lyon, France

Background
Treatment of chronic HCV infection with Peg-IFN/Ribavirin (Rib) leads to an overall sustained virological response (SVR) in 56% of the cases. However, treatment (Tx) is expensive and carries significant side effects thus, early interruption should be considered in those patients who will not benefit from this therapeutic approach. Two different criteria, one based on a quantitative threshold of 30,000 IU/mL (Berg et al.), and one based on a > 2 log drop in RNA concentration (Fried et al.) have been proposed to identify at 12 weeks post-treatment initiation those individuals who will become virological non-responders (NR). Both approaches provide an overall negative predictive value (NPV) ranging from 97-100%, and are capable of detecting approximately 50% of NR patients at this time-point.

Aims
A new quantitative marker of HCV viremia based on the detection of the core Ag of the virus has recently become commercially available in Europe. Applying a criteria based on a quantitative threshold, we retrospectively determined the utility of this test in the management of patients treated with Peg-IFN/Rib.

Methods
A total of 128 Peg-IFN/Rib treated patients (57 NR and 71 SVR as defined by RNA testing 6 months after end of Tx) were studied. Serum samples from week 4 (W4) and W12 time-points were available from 114 and 128 patients respectively, for retrospective core Ag quantification. Genotype data was available from 97 patients. Core Ag was quantified using the trak-C assay (Ortho Clinical Diagnostics, Raritan, NJ). A core Ag virological response at 4W or 12W was defined as concentration <1.5 pg/mL (approximately equivalent to 12,000 IU/mL HCV RNA).

Results
At 4W for all genotypes, the PPV and NPV were 81.4% and 84.1% respectively, while at 12W they were 69.6% and 100% respectively. For genotype 1/4 infected patients, the PPV and NPV at 4W were 65.4% and 92.3% respectively, while at W12 they were 50% and 100% respectively. For genotypes 2 and 3 infected patients, the PPV and NPV at 4W were 93.9% and 100% respectively, while at 12W they were 88.9% and 100 % respectively. Applying this criteria, we were able to identify 26/57 (45.6%) NR patients at 12W. Finally, a positive core Ag result at 4W was more frequent among NR patients who achieved a transient virological response (<1.5 pg/mL) at week 12 than among SVR (41.2% vs. 11.1%, p=0.04).

Conclusion
The use of the core Ag assay in conjunction with the proposed quantitative threshold, would have allowed for the unequivocal identification at 12W of virological non-responders. The core assay is a new reliable alternative for the early identification of virological non-response.

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Poster 1188: SITE OF PEGYLATION AND PEG MOLECULE SIZE DIRECTLY ATTENUATE INTERFERON-ALPHA ANTI-VIRAL SPECIFIC ACTIVITY THROUGH THE JAK/STAT SIGNALING PATHWAY

Michael J Grace, Susan Cannon-Carlson, Sheri Bradshaw, Constance Cullen, Jeffrey Chapman, Jeffrey Spond, Seoju Lee, David Wylie, Stephen R Indelicato, Marcio Voloch, Ronald W Bordens, Schering-Plough Research Institute, Union, NJ

PEG Intron (12 kD-linear, pegylated-IFN-a2b) and Pegasys (40 kD-branched, pegylated-IFN-a2a) are mixtures of positional isomers that have been mono-pegylated at specific sites on the core interferon alpha molecule. The two pegylated interferon alphas differ principally in the size of the attached polyethylene-glycol molecule, the distribution of positional pegylation isomers, and the relative anti-viral and anti-proliferation specific activities. To understand how the site of pegylation and the size of the polyethylene-glycol molecule affect the anti-viral specific activity, we prepared purified, homogeneous positional pegylation isomers of IFN-a2b for biological analysis. The positional isomers studied were mono-pegylated at Cys1, His34, Lys31, Lys83, Lys121, Lys131, and Lys134; each positional isomer was also pegylated with a 5 kD, 12 kD, 20 kD, or 30 kD linear polyethylene-glycol molecule. All positional isomers were confirmed, before and after biological analysis, to have no free IFN-a2b present. Anti-viral protection was determined using an FS-71 cell/EMC virus, cytopathic-protection assay. Interferon alpha signaling through the JAK/STAT pathway was determined by measuring STAT molecule translocation from the cytoplasm to the nucleus in Huh-7 cells. The site of pegylation dramatically impacted both the STAT translocation and the anti-viral protection activity relative to IFN-a2b control. The highest residual STAT translocation and anti-viral activity was observed with the 5 kD- to 30 kD His34 positional isomers. The lowest residual translocation and anti-viral activity was observed with the 5 kD- to 30 kD Cys1 positional isomers. The Lys positional isomers demonstrated intermediate STAT translocation and anti-viral activity between the His34 and the Cys1 isomers, with a general order of activity Lys134>Lys83~Lys131~Lys121>Lys31. It was noteworthy that the 30 kD Lys31 positional isomer demonstrated about 6% anti-viral activity relative to 12 kD His34 positional isomer in this study. Pegasys, which has been reported to have about 3% anti-viral protection activity relative to PEG Intron, has a 40 kD Lys31 as a major constituent positional isomer, while PEG Intron has the 12 kD His34 positional isomer as a ~50% constituent. The high activity associated with the His34 position was unexpected. However, we confirmed that the increased activity associated with the His34 positional isomer was not due to any free IFN-a2b in either activity assay. In summary, the correlation of anti-viral activity with STAT translocation activity for the positional isomers suggests that the decreased anti-viral activity was due to reduced JAK/STAT signaling. The trend of decreasing activity associated with increasing peg-molecule molecular weight, and thus increasing Stoke’s radius, suggests that a putative mechanism-of-action may reside with the interaction and binding of IFN-a to the IFNAR1/IFNAR2 heterodimeric receptor. Additional direct-binding studies with the positional isomers and the IFN-a receptor are warranted to confirm this hypothesis.

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Poster 1189: MECHANISMS OF RIBAVIRIN EARLY ANTIVIRAL EFFECT IN CHRONIC HEPATITIS C: IS IT RELATED TO RIBAVIRIN MUTAGENIC PROPERTIES INDUCING "ERROR CATASTROPHE"

Jean-Michel Pawlotsky, Rozenn Brillet, Christophe Hezode, Daniel Dhumeaux, Hôpital Henri Mondor - Universite Paris XII, Creteil, France

Introduction
We have recently shown that ribavirin monotherapy exerts a significant, moderate and transient antiviral effect in a significant proportion of patients with chronic hepatitis C. This effect seems to be partly responsible for the improved efficacy of the combination of tiw standard IFN-alpha and ribavirin versus tiw IFN-alpha monotherapy by increasing the incidence of the initial response, whereas its influence on the Peg-IFN-ribavirin combination remains unknown. The mechanisms underlying ribavirin antiviral effect are unknown. Ribavirin was recently shown in vitro to be an RNA virus mutagen. As a result, ribavirin could potentially exert its antiviral action by accelerating mutation incorporation into newly synthesized genomes, thus inducing lethal mutations leading to "error catastrophe" (i.e. the generation of nonviable viral quasispecies populations).

Objective
To determine whether or not ribavirin has mutagenic properties in vivo that could explain its antiviral effect in HCV-infected patients.

Methods
Four patients with chronic hepatitis C receiving ribavirin monotherapy, 1.0-1.2 g qd for 24 weeks experienced a significant viral load decrease (-0.5 to -1.0 log HCV RNA IU/ml) at days 2-3 of ribavirin monotherapy, with a viral load that returned to pretreatment levels after 12 to 96 hours. The full-length NS3 protease gene was PCR-amplified in all of them, together with the full-length NS5A transcriptional activation domain in two of them, at four time points: 14 days prior to therapy (d-14), at the beginning of therapy (d0), at day 2 of therapy, i.e. at the HBV DNA load nadir (d+2), and 14 days after treatment initiation (d+14). 20 clones per studied region per time point per patient were generated and sequenced. We calculated the overall accumulation of genomic mutations over time, as well as the accumulation of synonymous mutations per synonymous site that better reflects random mutation accumulation on which ribavirin is hypothesized to act, regardless of any conservatory constraint on the protein structure.

Results
Overall, 480 sequences were generated and used for analysis, including 320 full-length NS3 protease gene and 160 full-length NS5A transcriptional activation domain gene sequences. In three patients, the accumulation of mutations in the NS3 region significantly decelerated during ribavirin monotherapy compared to the pretreatment period. In one of them, the NS5A transcriptional activation domain was also studied and the accumulation of mutations significantly accelerated in this region, suggesting mutations do not accumulate homogeneously over the HCV genome. In the remaining patient, the accumulation of mutations significantly accelerated under ribavirin treatment in both NS3 and NS5A genes.

Conclusions
Our data suggest an unlikely role for ribavirin mutagenic properties in its early (and probably only) antiviral effect in HCV-infected patients in vivo. Ribavirin dosages are currently being performed and the rates of mutation will be interpreted according to ribavirin pharmacokinetics.

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Poster 1190: DIABETES MELLITUS OF NEW ONSET DURING PEGYLATED INTERFERON AND RIBAVIRIN THERAPY FOR CHRONIC HEPATITIS C: OBSERVATIONS FROM THE WIN-R TRIAL

Furqaan Ahmed, Ira M Jacobson, Weill Medical College of Cornell University, New York, NY; Thomas Hargrave, Eastbay Center for Digestive Health, Oakland, CA; Eric Lawitz, Brooke Army Medical Center, San Antonio, TX; Barry Migicovsky, Gastroenterology Consultants, Pembroke Pines, FL; Vishnu Reddy, Internal Medicine Specialists, Orlando, FL; Norah Terrault, University of California San Francisco, San Francisco, CA; Robert S Brown Jr, Columbia-Presbyterian Medical Center, New York, NY; Clifford Brass, Schering-Plough Research Institute, Kenilworth, NJ


Background

Diabetes is associated with chronic hepatitis C (CHC) infection and has been reported to occur with interferon therapy for CHC. Aim: To assess the frequency and clinical presentation of new onset diabetes developing during hepatitis C (HCV) therapy with pegylated interferon and ribavirin.

Methods
Using data from the WIN-R Trial, a U.S. multi-center study comparing fixed (800 mg) versus weight-based (800-1400 mg) daily dosing of ribavirin in combination with peginterferon alfa-2b 1.5 mcg/kg/week, we identified newly diagnosed diabetics from patients with serious adverse events (SAE). Demographic data and data on HCV infection, and the diagnosis and management of diabetes were collected.

Results
Approximately 4800 patients have received at least one dose of pegylated interferon and ribavirin in this study. Of these patients, five patients with new onset diabetes during therapy were identified from the database of serious adverse events (SAE). Four patients were male and the patients ranged in age from 20-55 years (mean- 44 years). Two patients were African-American, two were Caucasian and the race of one patient was not known. All patients were HCV genotype 1. On liver biopsy, three patients were Metavir stage 0-2 and two patients were Metavir stage 3-4. Steatosis was present in only one patient. Three patients received fixed dose ribavirin and the remaining two patients received weight based ribavirin. Of the four patients who were symptomatic at the time of diabetes diagnosis, three patients experienced polyuria and polydipsia, two patients complained of blurry vision, two had lost weight, and one patient experienced fatigue. Two patients had a family history of diabetes, and none of the patients were on steroids or were consuming alcohol. The episodes of hyperglycemia occurred after a mean period of 18 weeks of therapy in four patients. In the fifth patient, hyperglycemia occurred four weeks after completion of 48 weeks of therapy. All patients were hospitalized. Fasting blood glucose in two patients ranged from 373-417 mg/dL and random blood glucose in the remaining three patients ranged from 400 to greater than 1000 mg/dL. HbA1c values were available in three patients and ranged between 7.2-10% (mean- 8.4). Islet cell auto-antibodies were not measured in any patient. Two patients had diabetic ketoacidosis. All patients were started on insulin and oral hypoglycemic agents during hospitalization and the hyperglycemia was controlled in all patients by the time of discharge from the hospital. Of the four patients who developed diabetes during therapy, three patients discontinued pegylated interferon and ribavirin therapy at the time of the adverse event and one patient continued therapy for 16 weeks to complete 48 weeks of therapy.

Conclusions
(1) New onset diabetes of sufficient severity to warrant hospitalization occurred in 0.1% of patients on pegylated interferon and ribavirin therapy in this study.

(2) Severe hyperglycemia and diabetic ketoacidosis may be features of new onset diabetes developing during pegylated interferon and ribavirin therapy.

(3) The total incidence of diabetes may have been underestimated in this study because only those reported as SAEs were evaluated.

(4) Physicians treating patients for chronic hepatitis C should be vigilant in monitoring for symptoms of hyperglycemia during treatment.

(5) Further studies are needed to evaluate the safety of continuing interferon and ribavirin therapy after the diagnosis of new onset diabetes.

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