A randomized placebo-controlled, dose-finding study of oral LB80380 in HBeAg-positive patients with chronic hepatitis B

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1 Antiviral Therapy 11: A randomized placebo-controlled, dose-finding study of oral LB80380 in HBeAg-positive patients with chronic hepatitis B Man-Fung Yuen 1 *, John Kim 2, Chung Ryeol Kim 2, Vincent gai 1, John Chi-Hang Yuen 1, Changhee Min 2, Hyang Mi Kang 2, Beom Soo Shin 3, Sun Dong Yoo 3 and Ching-Lung Lai 1 * 1 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong 2 LG Life Sciences Ltd, Seoul, Korea 3 College of Pharmacy, Sungkyunkwan University, Seoul, Korea *Corresponding authors: Tel: ; Fax: : mfyuen@hkucc.hku.hk; hrmelcl@hkucc.hku.hk Background: LB80380 is potent antiviral agent against hepatitis B virus (HBV) in vitro and in the woodchuck model. It has an excellent preclinical safety profile including lower potential for renal toxicity than adefovir. It is effective against both wild-type and YMDD mutant HBV. LB80380 is converted to its parent drug, LB80331, after oral absorption, and further metabolized to its active form, LB Aims/Methods: This randomized placebo-controlled Phase I/II clinical study of LB80380 was conducted to assess the safety, antiviral activity and pharmacokinetics of its parent drug LB80331 and its active form LB80317 in 29 Asian adults with chronic hepatitis B positive for hepatitis B e antigen in four escalating dose groups (30, 60, 120 and 240 mg once per day) for 4 weeks with a 12-week follow-up period. Results: The mean maximum HBV DA reduction was 3.05, 4.20, 3.67 and 3.68 log 10 copies/ml for 30, 60, 120 and 240 mg per day, respectively. Viral dynamic analysis suggested a high degree of inhibition of HBV replication at doses of 60 mg or higher per day. LB80380 was well tolerated at all dose groups, and no dose-related clinical or laboratory adverse event was reported. Conclusion: LB80380 is shown to be a potent and safe antiviral agent for HBV. Marked HBV DA suppression was observed in all dose groups. The HBV DA suppression was approximately constant at doses of 60 mg and higher over the 28-day dosing period. The dose response of LB80380 will be evaluated further in large clinical studies. Introduction It is estimated that 400 million people worldwide have chronic hepatitis B (CHB) infection. f these, 25 40% will develop hepatocellular carcinoma and cirrhosisrelated complications [1]. Effective antiviral treatment is urgently needed. To date, four drugs have been approved for CHB. These include interferon-α (IF-α; conventional or pegylated IF), lamivudine (3TC), adefovir dipivoxil and entecavir (ETV). The latter three drugs are nucleos(t)ide analogues with negligible side effects and convenient daily oral dosing. However, prolonged use of 3TC is associated with increasing chance of emergence of 3TC-resistant hepatitis B virus (HBV) in the YMDD motif, namely, rtm204i and rtm204v [2]. Although adefovir dipivoxil is associated with a lower chance of emergence of adefovir-resistant HBV (rt236t) [3], there is the potential renal toxicity to consider with prolonged treatment [4]. f the three nucleo(t)side analogues, ETV is the most potent one. However, ETV has been shown to be carcinogenic in mice at dose exposures 3 40 times higher than those in humans [5]. Several newer nucleos(t)ide analogues are being developed and extensively tested in preclinical and clinical trials. The ideal nucleos(t)ide analogues should possess the following features: (i) marked viral suppression, preferably down to levels undetectable by PCR assays; (ii) lowest rate of emergence of drug-resistant HBV; (iii) effective action against all known forms of resistant HBV; (iv) no or low toxicity profile on long-term treatment; and (v) pharmacokinetics that allow once-daily dosing. Since more effective HBV DA reduction is associated with a significantly lower chance of emergence of drug-resistant HBV [6], highly potent and safe nucleos(t)ide analogues that are effective against both wild-type HBV and 3TC resistant HBV are required. LB80380 is an oral nucleotide prodrug and is chemically comparable with adefovir dipivoxil and tenofovir 2006 International Medical Press

2 M-F Yuen et al. disoproxil fumarate. LB80380 is rapidly converted to the parent drug LB80331 in the liver and intestine, probably by esterases. LB80331 is further metabolized to LB80317, a nucleotide analogue of guanosine monophosphate (Figure 1), by oxidases such as aldehyde oxidase or xanthine oxidase, a nucleotide analogue of guanosine monophosphate (Figure 1). After phosphorylation to the di- and tri-phosphate forms, the molecule inhibits viral replication following incorporation into viral DA. We report the safety, antiviral activity and pharmacokinetics of 4-week, escalating doses of LB80380 in a Phase I/II, double-blind, randomized, placebocontrolled study in patients with CHB. Patients and methods Study centre and approval The trial BVCL002 was approved by the Ethics Committee, Institutional Review Board, The University Hong Kong, Queen Mary Hospital, Hong Kong, and conducted under Good Clinical Practice standards with local regulatory authorization and an Investigational ew Drug application approved by the US Food and Drug Administration. All screened and recruited patients gave written informed consent. Study design Four escalating doses of LB80380 (30, 60, 120 and 240 mg once daily) were tested in consecutive cohorts. Each cohort comprised seven patients of whom six were randomized to receive LB80380 of the respective dose and one to receive placebo. The randomization was performed by computer allocation. Treatment was given for 4 weeks. Recruitment of each cohort was performed in a dose-escalating manner. Initiation of the next higher dose cohort was only allowed under two conditions: (i) ot more than one out of the six patients receiving the 4-week treatment in the preceding lower-dose cohort experienced dose-limiting toxicity, or (ii) if two out of the six patients developed dose-limiting toxicity during the 4-week treatment, two additional patients should complete the 4-week treatment at the same dose without dose-limiting toxicity. Dose-limiting toxicity was defined as a prothrombin time of more than 3 s above control; a serum albumin level less than 30 g/l; grade 3 or greater elevation of total bilirubin, creatinine or amylase; grade 4 alanine aminotransferase (ALT) elevation (>10 times baseline) with any evidence of hepatic insufficiency; or any other grade 4 clinical or laboratory toxicity considered by the investigators to be at least reasonably or possibly related to the study drug. Pharmacokinetics study was performed in all patients at baseline and during the apparent steady state between weeks 2 and 4 at the time points of pre-dose and 0.5, 1, 2, 3, 6, 8 and 24 h post-dose. Plasma concentrations of LB80331 and LB8017 were measured with a validated liquid chromatographic assay with tandem mass spectrometric detection (LC/MS/MS). The lower limits of quantitation were 5 and 10 ng/ml for LB80331 and LB80317, respectively. Intra- and inter-assay precision and accuracy variations were less than 10%. Blood samples for viral Figure 1. The chemical structure of LB80380 (the prodrug), LB80331 (the parent drug) and LB80317 (the active moiety) H 2 LB80380 (prodrug) P H 2 H H P LB (parent drug) H Further phosphorylation H 2 H H P LB (active moeity) International Medical Press

3 LB80380 in chronic hepatitis B load measurement were obtained at baseline and weekly through to week 8 and thereafter every other week through to week 16. Serum HBV DA was quantified using the CBAS Amplicor TM PCR assay for HBV DA (Roche Diagnostics, Branchburg, J, USA) with a lower limit of detection of 300 genome copies/ml. Patients Patients were recruited under the following inclusion criteria: adults of years of age; hepatitis B surface antigen (HBsAg)-positive for at least 6 months; hepatitis B e antigen (HBeAg) positive for at least 1 month; HBV DA level copies/ml measured by the CBAS Amplicor TM HBV Monitor test; and serum ALT level <5 times upper limit of normal (UL). Patients were not eligible for the study if they had any one of the following conditions: coinfection with hepatitis C or D virus or HIV; pregnancy or breastfeeding; history of receiving nucleos(t)ide analogues or any other treatment including immunomodulatory agents, for example, IF-α or corticosteroids for HBV within 6 months prior to study entry; history of receiving drugs with potential nephrotoxicity or hepatotoxicity within 6 months prior to study entry; evidence of decompensated liver disease; haemoglobin <9.0 g/dl; neutrophil count < /l; platelet count < /l; serum creatinine level >133 µmol/l; serum amylase >165 U/l (1.5 UL); serum lipase >304 U/l (1.5 UL); α-fetoprotein level >20 ng/ml with evidence of hepatocellular carcinoma by imaging; or history of severe allergic disease or illicit drug or alcohol abuse. A total of 66 patients were screened and 28 patients were recruited (all completed the 4-week dosing and fulfilled the whole study protocol). The remaining 38 were not enrolled because of the following reasons: patient withdrawal of consent (n=21); HBV DA levels < copies/ml at screening (n=7); ALT levels >5 UL (n=2); HBeAg negativity (n=1); HBV DA levels < copies/ml and HBeAg negativity (n=5); HBV DA levels < copies/ml and anti-hcv positivity (n=1); and anti-hiv positivity (n=1). Follow-up and monitoring Patients were followed up every week until week 8 and every 2 weeks until week 16. During every follow-up, clinical adverse events, liver and renal function tests and complete blood count, as well as HBV DA levels were monitored. Data analysis Statistical analysis The baseline characteristics were compared between different groups by Fisher s exact test for gender distribution (categorical variable) and Mann Whitney U test for age, ALT levels and HBV DA levels (non-parametric continuous variables). For per protocol efficacy analysis, one patient in the 60 mg group was not included due to a drug dispensing error resulting in the wrong dose being given; however, this patient was included for safety assessment. The antiviral activities of different LB80380 doses at week 4 were compared in terms of HBV DA reduction by the following two methods: (i) differences in the median logarithmic HBV DA reduction (tested by Mann Whitney U test) and (ii) proportions of patients with HBV DA reduction 2 logs (tested by Fisher s exact test). Pharmacokinetics Single-dose and steady-state LB80331 and LB80317 pharmacokinetics were analysed using a noncompartment model. The following pharmacokinetic parameters were evaluated: maximal plasma concentration (C max ), time to C max (T max ), area under the plasma concentration curve from zero to infinity (AUC inf ) or from zero to 24 h (AUC 24hr ) for single dose and steadystate, respectively, and apparent terminal half-life (t 1/2 ). Molar metabolic AUC ratio was calculated by dividing the AUC of LB80317 by the AUC of LB80331 adjusted for molecular weight difference (multiplying by ). Virological response and HBV DA reduction Virological response was defined as a 2-log or more reduction in HBV DA levels during the 4-week treatment period. Dose response in terms of HBV DA reduction was analysed in three ways: (i) descriptive statistics of HBV DA reduction, (ii) maximal effect (E max ) modelling, and (iii) viral dynamics analysis. The E max model equation is given as: dose E max E = _ ED 50 + dose In the equation, E is the maximal viral load reduction during the 4-week treatment period, ED 50 is the LB80380 dose producing a 50% reduction in HBV DA, and E max is the expected maximum effect as defined as complete loss of serum HBV DA. Viral dynamic model Viral dynamic modelling was used to characterize plasma viral load changes after the initiation of treatment. Previous reports indicated a biphasic response to antiviral therapy: a first-phase with rapid reduction in HBV DA corresponding to clearance of plasma virus and inhibition of HBV replication, and a prolonged second-phase reflecting net loss of HBV-infected cells [7]. For the viral dynamic analyses, two approaches were taken: (i) a Antiviral Therapy 11:8 979

4 M-F Yuen et al. Table 1. Demographic data, serum ALT and HBV DA levels at baseline 30 (n=6) 60 (n=6) 120 (n=6) 240 (n=6) Placebo (n=4) Age, median (range) 34.3 ( ) 31.2 ( ) 29.3 ( ) 23.9 ( ) 22.0 ( ) Sex, M:F 5:1 5:1 4:2 4:2 2:2 Weight, mean kg ±SD 64.1 ± ± ± ± ±6.7 Serum HBV DA, median (range) 8.47 ( ) 8.66 ( ) 8.71 ( ) 9.14 ( ) 9.08 ( ) ALT, median IU/ml (range) 79.5 (50 100) 68.7 (22 165) 34.5 (22 96) 27 (20 36) 25 (22 61) All patients are ethnic Chinese and seropositive for HBsAg and HBeAg. ALT, alanine aminotransferase; F, female; HBV, hepatitis B virus; M, male. model-independent approach assessing dose-related HBV DA reduction during week 1 and weeks 2 4, and (ii) a viral dynamic model utilizing the equation previously described [7]. The modelling equation is given as: (1 ε)uv 0 V(t) = V 0 e at + (e at e ut ) (u a) In this equation, V(t) is viral load at time (t), V 0 is viral load at baseline, a is the clearance rate constant of infected cells, u is the clearance rate constant of free virus, and ε is the degree of inhibition of viral replication. The model estimates of virus (u) and infected cell (a) clearance rate constants were used to calculate the half-life of virus and infected cells using the Winonlin program (Pharsight, Mountain View, CA, USA). Results Patient population The demographic data, serum ALT levels and HBV DA levels of each cohort are listed in Table 1. All patients were ethnic Chinese. There were no significant differences in any parameters among the groups, though the median ALT levels seemed to be lower in the groups receiving higher doses. Antiviral activity The median HBV DA changes over the 4-week dosing and 12-week post-dosing periods are depicted in Figure 2. The median maximal HBV DA changes were 3.05, 4.20, 3.67 and 3.68 log 10 copies/ml for 30, 60, 120 and 240 mg daily, respectively. These HBV DA reductions were significantly greater compared with those of placebo (change of the HBV DA 1.02 log 10 copies/ml) (Table 2). There were also significantly higher proportions of patients with 2 log 10 HBV DA reductions at week 4 in patients receiving various doses of LB80380 compared with the placebo group (Table 2). A total of five out of six patients receiving 60 mg daily and all patients receiving 120 mg daily (n=6) and 240 mg daily (n=6) had more than 2 log 10 HBV DA reduction at week 4. Safety LB80380 was well tolerated at all doses. o serious adverse events or dose-limiting toxicities related to drug treatment were observed in any cohort during the dosing and post-dosing periods. Treatment-emergent adverse events regardless of relation to the study drug are listed in Table 3. Although five out nine adverse events occurred in the group receiving 240 mg daily, most of the adverse events were mild, self-limiting and probably unrelated to the drug. verall, the safety profile of LB80380 was excellent and comparable with that of placebo. Two patients experienced elevated ALT levels during the dosing (n=1) and post-dosing (n=1) periods. ne patient receiving 60 mg daily had elevation of ALT levels from a baseline level of 37 U/l to 88 U/l at week 4, reaching a peak level of 93 U/l at week 5. The ALT decreased to 50 U/l (normal <53 U/l) by week 7. Another patient receiving 120 mg daily had elevation of ALT levels from a baseline level of 69 U/l to 233 U/l at week 12 (8 weeks after cessation of treatment), reaching a peak level of 643 U/l at week 16 (12 weeks Figure 2. Effect of LB80380 on serum HBV DA levels during and after treatment with mg using Roche CBAS Amplicor assay Median HBV DA reduction, log Time, week Placebo, n=4 30 mg, n=6 60 mg, n=6 120 mg, n=6 240 mg, n= International Medical Press

5 LB80380 in chronic hepatitis B Table 2. Changes of HBV DA levels during a 4-week treatment period with LB (n=6) 60 (n=6) 120 (n=6) 240 (n=6) Placebo (n=4) Median (range) maximum decrease 3.05 ( ) 4.20 ( ) 3.67 ( ) 3.68 ( ) 1.02 ( ) from baseline of HBV DA P-value for comparison with placebo* umber of patients with HBV DA 4 (66.7) 5 (83.3) 6 (100) 6 (100) 0 (0) reduction >2 logs at week 4, n (%) P-value for comparison to placebo *P-value obtained from Mann Whitney U test. P-value obtained from Fisher s exact test. Table 3. Adverse events reported in the dosing and postdosing periods Placebo Adverse events (n=6) (n=7) (n=6) (n=6) (n=4) Flu-like symptom Headache 1 Abdominal pain 1 ausea 1 Diarrhoea 1 Dizziness 1 Infection 1 after cessation of treatment). The ALT level decreased to 124 U/l by week 20. The hepatitis flare was probably due to the rebound of viral activity after cessation of LB The ALT level returned to normal after 3 months of 3TC treatment. The bilirubin levels and prothrombin times remained normal throughout for both patients. Pharmacokinetics LB80380 was rapidly absorbed after oral administration and converted to its parent drug form LB o LB80380 was detectable in plasma samples. The maximal concentration of LB80331 was attained at about h post-dose, and thereafter, the plasma concentration declined in a mono-exponential manner with an elimination half-life of about 2 3 h. LB80317, the active oxidative metabolite, was measured in plasma samples. Following single dose administration, it was only measurable at higher doses, but greater concentration of LB80317 was found during steady state. LB80317 has much longer elimination half-life (about 50 h) than that of LB80331, which is indicative of sustained exposure to the active metabolite with a once-a-day dose regimen. Figure 3. E max modelling of HBV DA reduction dose response data DA reduction, log Individual reduction Median reduction Model prediction E max model Figure 3 shows the relationship between different doses of LB80380 and serum HBV DA reduction in the E max model. The dose response data fitted the E max model equation well (r 2 =0.8043). There was marked increase in the serum viral load reduction when the dose was increased from 30 mg daily to 60 mg daily. Further incremental reduction was smaller at doses above 60 mg. The fitted curve indicates that near maximal viral load reduction occurred within the dose range of mg of LB80380 daily. Viral dynamics During the first week of treatment, all doses of LB80380 resulted in rapid reduction in serum HBV DA, corresponding to the first phase of clearance of viral load, as shown in Figure 2. Viral load was further decreased but at a slower rate during weeks 2 4, corresponding to the second phase of viral clearance. Doses of 60, 120 and 240 mg daily resulted in greater HBV DA reduction when compared with the 30 mg Antiviral Therapy 11:8 981

6 M-F Yuen et al. Figure 4. Serum HBV DA reductions from baseline by the response phase Serum HBV DA reduction, log dose in the first phase (Figure 4). The maximal viral suppression for the first phase reached a plateau when the dose was increased to more than 60 mg. There was no dose-proportionality observed in the second phase. Bi-exponential viral dynamic modelling was used to estimate the parameters for viral load reduction. The estimated half-life of free virions was about days. This was within the range of the previously reported values of about 1 day [8 10]. There was a substantial overlap among dose groups, suggesting that the estimated half-life was independent of dose. The estimated half-life of infected cells was about days. As with the half-life of free virions, no clear dose-proportionality was observed within the dose range tested. Compared with 10.4 days in the 30 mg group, slightly lower values for the estimated halflife were observed in the higher dose group above 60 mg. This relationship was consistent with the findings from the E max model. Discussion First phase Second phase During the 4-week treatment period, LB80380 induced a marked suppression of serum HBV DA in HBeAgpositive patients with CHB, with median viral load reduction of log 10 copies/ml at daily doses of mg. All the patients except three (21 out of 24, 88%) achieved at least 2-log 10 reductions in viral load during the 4-week period. f the three patients with <2 log 10 reductions, two were in the 30 mg group and one was in the 60 mg group. The degree of viral suppression after 4 weeks of LB80380 is comparable with that of telbivudine [11] and is superior to other anti-hbv agents currently available, namely 3TC at 2.3 logs [12], adefovir dipivoxil at 2 logs [13] and ETV at logs [12 14]. However, studies with different agents are necessary to directly compare these efficacies, which are also dependent on the dosages of different nucleos(t)ides used. With similar magnitude of HBV DA reduction at week 4 and similar time duration for HBV DA rebound after cessation of therapy (Figure 2) in patients taking 60, 120 and 240 mg daily, optimal viral suppression may be achieved within the dose range of mg daily. From the two distinct phases of viral clearance in the viral dynamic analysis (Figure 4), doses at or higher than 60 mg compared with 30 mg were associated with greater magnitude of HBV DA reduction in the first phase, the phase of clearance of free virions. The second phase of viral clearance (clearance of virus in infected hepatocytes) did not appear to be dose-dependent. A study with a larger number of patients is required to confirm this observation. In addition, it will be of great interest to examine the relationship in a larger population between the second phase of viral clearance and ALT levels in patients receiving LB The dose response relationship analysis using the E max model (Figure 3) confirmed that the maximal antiviral effect was already achieved with the 60 mg daily dose. The estimated mean half-lives of free virions and infected cells under the treatment of LB80380 at doses of mg are days and days, respectively. These are shorter than the published values for 3TC, adefovir dipivoxil, ETV and telbivudine [8 11]. However, the values for LB80380 should be interpreted cautiously due to the limited number of patients, difference in population of patients and different methods used to estimate half-life. LB80380 was well absorbed following oral administration and rapidly converted to its parent drug LB The C max of LB80331 was reached in about 2 h and declined monoexponentially with an apparent half-life of about 2 3 h. Following multiple oral administration, no further accumulation of LB80331 was observed. LB80331 is further metabolized to its active metabolite, LB In this study, LB80317 had a much longer elimination half-life of about 50 h. The long half-life of LB80317 supports a sustained exposure to active drug with once-daily dosing. In addition to its antiviral activity against wild-type HBV, LB80380 has also been shown to be effective against YMDD mutants, including rtm204i and rtm204v in both in vitro (data not shown) and in vivo studies [15]. Further trials of LB80380 are being conducted in patients with 3TC-resistant HBV. In conclusion, LB80380 was well-tolerated and safe during a 4-week treatment period using daily doses of mg. It was effective in reducing viral load by more than 3 log 10 after a 4-week course with doses of 60 mg daily or above. Further larger trials are warranted to assess its efficacy and safety International Medical Press

7 LB80380 in chronic hepatitis B Acknowledgements This study was sponsored by LG Life Sciences Ltd, Seoul, Korea. References 1. Lai CL, Ratziu V, Yuen MF, et al. Viral hepatitis B. Lancet 2003; 362: Lai CL, Dienstag J, Schiff E, et al. Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis 2003; 36: Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 2003; 125: Izzedine H, Launay-Vacher V, Deray G. Antiviral druginduced nephrotoxicity. Am J Kidney Dis 2005; 45: Baraclude Full US prescribing information. Bristol- Myers-Squibb Company. Accessed on Dec Yuen MF, Sablon E, Hui CK, Yuan HJ, Decraemer H, Lai CL. Factors associated with hepatitis B virus DA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001; 34: Tsiang M, Rooney JF, Toole JJ, Gibbs CS. Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy. Hepatology 1999; 29: owak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics in hepatitis B virus infection. Proc atl Acad Sci USA 1996; 93: Wolters LM, Hansen BE, iesters HG, DeHertogh D, de Man RA. Viral dynamics during and after entecavir therapy in patients with chronic hepatitis B. J Hepatol 2002; 37: Wolters LM, Hansen BE, iesters HG, Zeuzem S, Schalm SW, de Man RA. Viral dynamics in chronic hepatitis B patients during lamivudine therapy. Liver 2002; 22: Lai CL, Lim SG, Brown A, et al. A dose-finding study of once-daily oral telbivudine in HBeAg-positive patients with chronic hepatitis B virus infection. Hepatology 2004; 40: Lai CL, Rosmawati M, Lao J, et al. Entecavir is superior to lamivudine in reducing hepatitis B virus DA in patients with chronic hepatitis B infection. Gastroenterology 2002; 123: Marcellin P, Chang TT, Lim SG, et al. Adefovir Dipivoxil 437 Study Group. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. Engl J Med 2003; 348: de Man RA, Wolters LM, evens F, et al. Safety and efficacy of oral entecavir given for 28 days in patients with chronic hepatitis B virus infection. Hepatology 2001; 34: Lai CL, Han KH, Yoon SK, et al. Interim report for a phase II, multi-centre, dose-escalating study of LB80380/AA380 in hepatitis B patients with lamivudine-resistant YMDD mutant HBV. J Hepatol 2005; 42(Suppl 2):72A. Accepted for publication 19 July 2006 Antiviral Therapy 11:8 983

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