I am at the 40^th Annual Meeting of the European Association for the Study of the Liver, April 13-17 in Paris, France. When peginterferon was in the developmental stages there was more interest in attending this yearly conference to follow the data and study results. Now, there is less interest in attending this conference, certainly by Americans. There are however several interesting presentations on new drugs in development for HCV & HBV. This report will preview for you the abstracts and I will follow with more data during & after the conference.

CONTENTS: NM283; Viramidine; Albuferon; a-GLUCOSIDASES INHIBITORS

This is the furthest along new drug for HCV and the data looks promising. For non-responders this drug may provide a needed boost in combination with peginterferon. The two drugs together with ribavirin or Viramidine may provide just enough viral potency to achieve undetectable HCV RNA for non-responders to peginterferon plus ribavirin.

N. Afdhal1, M. Rodriguez-Torres2, E. Lawitz3, E. Godofsky4, G. Chao5, B. Fielman5, S. Knox5, N. Brown5

Background: Only 40-50% of HCV-1 infected hepatitis C patients respond to pegylated interferon (peg-IFNa) plus ribavirin therapy. NM283 is a novel nucleoside analog that as monotherapy reduced serum HCV RNA by a mean 1.2 log10 (94%) in a recent 15-day trial in HCV-1 patients, most of whom had previously failed antiviral therapy for HCV infection. NM107, the parent compound of NM283, and interferon a exhibit synergistic antiviral effects against BVDV models in vitro, prompting the current investigation of NM283 combined with peg-IFNa in treatment naive patients with chronic hepatitis C.

Methods: A multicenter, open-label phase IIa trial is evaluating whether NM283 plus peg-IFNa-2b has enhanced antiviral activity compared to NM283 alone in HCV-1 infected patients. Key entry criteria are: HCV RNA >5 log10 IU/ml, ALT < 5 _ ULN, compensated liver disease, treatment naive. Eligible patients are randomized 2:3 to NM283 or peg-IFNa + NM283 (combinationRx). NM283 is dosed orally QD for 24 weeks in both groups, escalating to 800 mg/day over the first week and then continuing at that dose. The group randomized to combinationRx receives peg-IFNa-2b (1.0 µg/kg weekly) starting on Day 8.

Results: Presently 19 of 30 planned patients (age 22-65, 68% males) are enrolled and have received 10 or 12 weeks of treatment. Tolerance of both treatments has been satisfactory. Mean HCV RNA reductions (log10 IU/ml) from baseline to the last patient visit are 1.0 for the NM283 monotherapy group and 3.2 for the combination-Rx group. Eleven of twelve combination-Rx patients treated for at least 10 weeks have had substantial HCV RNA reductions (range 1.2-6.2 log10), and 8 of 12 patients have achieved >2 log10 decrease in HCV RNA, which has been associated with sustained response to current standard Rx. Presently 4 patients are PCR-negative (<10 IU/ml). 12-week early virological response data will be presented at the meeting.

Conclusions: NM283 combined with peg-IFNa shows consistent, rapid and marked anti-HCV activity in patients with HCV-1 infection, a historically difficult-to-treat group. Initial EVR rates with combination-Rx are encouraging and support continued investigation of NM283 and NM283 + peg-IFNa, which may offer improved efficacy and tolerability for patients with HCV-1 infection.

ENHANCED ANTIVIRAL ACTIVITY OF NM107, ALONE OR IN COMBINATION WITH INTERFERON a

V. Bichko1, M. Tausek1, L. Qu1, M. LaColla1, S. Bergelson1, C. Pierra2, S. Benzaria2, D. Storer2, G. Gosselin2, J.P. Sommadossi1, D. Standring1

Background: NM107 is 2¢-C-methylcytosine, a ribonucleoside analogue that inhibits flavi- and pestivirus replication in vitro. NM283, an orally bioavailable prodrug of NM107, is currently in phase II clinical development for the treatment of chronic hepatitis C and was shown to reduce serum hepatitis C virus (HCV) RNA levels in both chronically infected chimpanzees and in patients with chronic hepatitis C. The objective of this study was to evaluate the antiviral activity of NM107 in vitro in combination with recombinant human interferons a-2b (Intron A) or b (Avonex).

Methods: The antiviral activities of NM107, interferon a-2b, interferon b and combinations thereof were studied in vitro using infection with bovine viral diarrhea virus (BVDV), a pestivirus related to HCV. Several virus strains from both cytopathic (cp) and noncytopathic (ncp) biotypes were used in de novo and/or persistently infected Madin-Darby bovine kidney (MDBK) cells.

Results: In vitro studies on BVDV NS5B polymerase indicate that NM107 triphosphate is a specific chain terminator of BVDV RNA synthesis. In cell-based assays, NM107 is a potent inhibitor of BVDV propagation (EC90 0.87±0.18 µM) and could eradicate a persistent BVDV infection after 28 days of treatment. Interferon a-2b inhibited BVDV modestly in vitro (EC90 32.5±18.2 IU/ml). Combination of 4 µM NM107 and 2000 IU/ml interferon a-2b exhibited synergistic antiviral activity. The reduction in virus titer was 4.56 log10, 2.4 log10 higher than the calculated logarithmic additive effect (2.16 log10). Enhancements of antiviral activity of over 4 log10 were seen for combinations of interferon a-2b and NM107 with the NY1 strain of BVDV. However interferon b did not inhibit BVDV and had no effect on the antiviral potency of NM107.

Conclusions: NM107 is a promising antiviral agent that acts synergistically with interferon a-2b in vitro. Clinical studies of NM283 in combination with interferon a are ongoing.

VIROLOGIC RESPONSE AND SAFETY OUTCOMES IN THERAPY-NAIVE PATIENTS TREATED FOR CHRONIC HEPATITIS C WITH VIRAMIDINE IN COMBINATION WITH PEGYLATED INTERFERON a-2a

R.G. Gish1, D. Nelson2, S. Arora3, M.W. Fried4, K.R. Reddy5, Y. Xu6, B. Murphy6, Study Group6

Introduction: Dose-limiting anemia can be a prominent adverse event of therapy with pegylated interferon and ribavirin. This dose-ranging study examined whether viramidine, a liver-targeting prodrug of ribavirin, may be a safer alternative when used in combination with pegylated interferon a-2a (PEG-IFN).

Methods: of 180 HCV therapy-naive patients enrolled in the study, 171 patients received full-dose viramidine (400 mg: n = 47; 600 mg: n = 43; 800 mg: n = 44) versus ribavirin 1000-1200 mg/d (n = 37) in combination with PEG-IFN 180 µg/wk SC. Patients were predominantly male (64%), Caucasian (76%), and genotype 1 (72%), with a median HCV RNA of 6.5 log10 copies/ml. Analyses assessed the incidence of anemia (hemoglobin <10 g/dl) and HCV RNA levels (Bayer TMA assay; sensitivity to 5 IU/ml) among patients without dose reduction due to anemia to evaluate the intrinsic activity of viramidine versus ribavirin without the confounder of dose modification.

Results: Among patients with no dose modification due to anemia at end of treatment (EOT), no significant differences were noted between viramidine (400, 600, and 800 mg BID) versus ribavirin in the proportion of patients with undetectable HCV RNA levels (55%, 63%, 55%, and 62%, respectively). Rates of anemia at EOT for the viramidine 400, 600, and 800 mg groups were 0%, 2%, and 11%, respectively, versus 27% for the ribavirin arm. Based on evaluable patients at EOT experiencing a decline in hemoglobin of at least 25%, the rate in the ribavirin group (48%) was higher versus the rate in the viramidine groups (400 mg: 14%; 600 mg: 18%; 800 mg: 15%). Other types of adverse events were similar between treatment arms.

Conclusions: At EOT in this Phase 2 study, viramidine demonstrated antiviral activity comparable to that of ribavirin when used in combination with pegylated interferon a-2a among patients with no dose modification due to anemia. Patients in the viramidine arms also showed a significantly lower incidence of anemia. Data lock for the Phase 2 study will occur by December 1, 2004, and sustained virologic response rates and safety outcomes will be presented at the 2005 EASL meeting.

C-C. Lin, C. Xu, N. Zhu, L-T. Yeh

Background: The combination of ribavirin and pegylated interferon-a is the standard treatment for chronic hepatitis C. However ribavirin may cause hemolytic anemia. Viramidine is a prodrug of ribavirin with excellent liver-targeting properties in monkeys. Improved safety has also been shown with viramidine in a one-month toxicity study in monkeys.

Aim: To evaluate absorption, metabolism and excretion of [14C]viramidine in humans following a single oral administration.

Methods: Six healthy adult male subjects ³50 years of age participated in the study. Following an overnight fast, each volunteer received a single oral dose of [14C]viramidine given as three 200 mg capsules with a total radioactivity of 52.2 microcuries. Plasma, red blood cell (RBC), urine and fecal samples were collected and analyzed for radioactivity. Plasma levels of viramidine and ribavirin were determined by a validated LC-MS/MS method. A HPLC procedure was used to evaluate the metabolic profile in urine.

Results: Viramidine was rapidly absorbed and extensively converted to ribavirin with a Tmax of 1.5, 2 and 3.5 hours for viramidine in plasma, ribavirin in plasma and total radioactivity in plasma, respectively. Viramidine and ribavirin accounted for only 5.5% and 59% of plasma AUC radioactivity, respectively, indicating extensive conversion of viramidine to ribavirin, followed by further metabolism of ribavirin. The drug was largely trapped in RBCs with a RBC to plasma radioactivity AUC ratio of 82.5. Viramidine and ribavirin accounted for 1.0% and ~99% of RBC AUC radioactivity, respectively. Excretion of total radioactivity in urine and feces accounted for 50.9% and 26.1% of the dose, respectively. The amount of unchanged viramidine (3.4% of dose) and ribavirin (10% of dose) in urine was small after oral administration of viramidine (0-336 h). Urine metabolic profile (0-24 h) indicated viramidine was excreted in urine, primarily as TCONH2 (64.1%), TCOOH (17.0%) and ribavirin (15.7%) with a small amount of unchanged viramidine (3.2%).

Conclusions: In humans, following oral dosing, viramidine was rapidly absorbed and extensively converted to ribavirin, followed by further metabolism. Ribavirin was largely trapped in RBC and excreted primarily into urine as TCONH2 with smaller amounts as TCOOH, ribavirin and viramidine.

A PHASE 2 STUDY TO ASSESS ANTIVIRAL RESPONSE, SAFETY, AND PHARMACOKINETICS OF ALBUFERONTM IN IFN-a NAIVE SUBJECTS WITH GENOTYPE 1 CHRONIC HEPATITIS C

V. Bain1, K. Kaita2, E. Yoshida3, M. Swain4, J. Heathcote5, J. McHutchison6, A. Neumann7, P. Bagchi8, B. Osborn8, P. Cronin8, L. Novello8, W. Freimuth8, M. Subramanian8

Background: AlbuferonTM is a novel recombinant protein consisting of IFN-a genetically fused to human serum albumin. The resulting polypeptide combines in one molecule the antiviral properties of IFN-a with the long serum half-life of albumin. This Phase 2, randomized, dose-ranging study was conducted in IFN-a naive, genotype 1 HCV subjects.

Methods: Subjects were enrolled in parallel into 3 dose cohorts (200 mcg, 450 mcg and 670 mcg). Within each dose group, 10 subjects were enrolled to receive 2 SC injections of Albuferon 14 days apart. Two additional dose cohorts of 900 mcg and 1200 mcg are ongoing.

Results: 32 subjects are currently enrolled. Mean baseline viral load was 6.63 log10 IU/ml and mean BMI was 28.2. Albuferon was well tolerated. Adverse events were transient, most were mild to moderate, and were not dose related. The most common adverse events were headache (52%), arthralgia (48%), and myalgia (45%). There was 1 SAE (acute colitis that has resolved). Reversible neutropenia (ANC < 750) occurred in 2 subjects and did not preclude repeat dosing. Cmax was dose-proportional, with a 17-30% drug accumulation after the second dose. The median terminal half-life was 141 hours and consistent with previous data from IFNa experienced subjects. VR4 response (>2 log HCV RNA reduction at week 4) and mean HCV RNA change (log10 IU/ml) at week 4 were as follows: 200 mcg, 2/10 (1.8); 450 mcg, 7/10 (2.5); 670 mcg, 3/9 (1.7). Reductions in ALT levels concomitant with or parallel to viral load reductions were observed in all subjects with an elevated ALT at baseline. Mathematical modeling of viral kinetics demonstrates a biphasic response, with a mean first phase decline of 0.85 (range 0-2.3) log during the first 1-4 days, followed by a second phase decline of mean 0.3 (range 0-1.0) log/week. 12 patients showed a second phase slope greater than 0.3 log/week after the first and second injections until day 28.

Conclusions: Albuferon was safe, well tolerated and showed robust antiviral activity after two doses in IFN-a naive, genotype 1 HCV subjects. Dosing every 2-4 weeks is supported by the pharmacokinetics and anti-viral response.

a-GLUCOSIDASES INHIBITORS: HCV entry inhibitor

a-GLUCOSIDASES INHIBITORS IMPAIR HCV PSEUDOPARTICLES MORPHOGENESIS, PREVENT VIRAL SECRETION AND ENTRY INTO HEPATOMA CELLS

C. Chapel1, I. Vuillermoz1, B. Bartosch2, F-L. Cosset2, N. Zitzmann3, R.A. Dweek3, J. Dubuisson4, C. Trépo1, F. Zoulim1, D. Durantel1

Background and Aims: The morphogenesis of HCV belongs to steps of the viral cycle that have not yet been targeted by antiviral strategies. Using the bovine viral diarrhoea virus, a pestivirus related to HCV, as a model, we have previously shown that a-glucosidase inhibitors derived from glucose could inhibit viral morphogenesis in cellulo via the perturbation of the N-glycosylation pathway and the folding of envelope glycoprotein. Due to the heavy N-glycosylation of HCV glycoproteins, it was hypothesised that such inhibitors would also affect HCV morphogenesis.

Methods: With the lack of an efficient and reliable culture system able to produce and secrete HCV virions, we use two complementary approaches to study the effect of a-glucosidase inhibitors on HCV morphogenesis, secretion and entry. First, we constructed various baculoviruses loaded with HCV structural proteins and used them to produce virus like particles (VLPs) in Sf-9 cells in order to study the effect of these compounds on the folding and assembly of HCV glycoproteins, on VLPs production and binding/internalisation properties. Second, we used infectious HCV pseudotyped retroviral particles (HCVpp) harboring unmodified gpE1 and gpE2 to determine the effect of these inhibitors on viral secretion and entry.

Results: With the first model, we have shown that, in presence of a-glucosidase inhibitors, gpE1 and gpE2 synthesised and retained in the ER i) contained unprocessed triglycosylated N-glycans, ii) were impaired in their interaction with calnexin and iii) were at least partially misfolded. Furthermore, we found that glycoproteins incorporated into VLPs had a higher molecular mass, suggesting the presence of triglycosylated N-glycans. Moreover we observed that VLPs produced in the presence of drug had modified binding and internalisation properties to hepatoma cells. Using the second model, we found i) that the production and secretion of HCVpp was impaired in presence of a-glucosidase inhibitors, ii) that HCVpp contained misfolded and misassembled viral glycoprotein, and iii) that HCVpp entry into target cells was impaired after treatment with drugs.

Conclusions: These two approaches have allowed us to demonstrate the mechanism of the antiviral effect of a-glucosidase inhibitors on HCV glycoprotein folding and assembly, morphogenesis, viral secretion, and viral infectivity.