AACR/NHRI Joint Conference: hepatitis and hepatocellular carcinoma: novel approaches

This international meeting, co-sponsored by the National Health Research Institutes of Taiwan (NHRI) and the AmericanAssociation for Cancer Research (AACR), was dedicated to two pioneering scientists, Juei-Low Sung (Koo Foundation, Sun Yat-Sen Cancer Center, Taipei, Taiwan) and Kwang-Juei Lo (Taipei Veterans General Hospital, Taipei, Taiwan). The meeting focused on various aspects of viral hepatitis and hepatocellular carcinoma (HCC), and the relation between the two. Topics ranged from epidemiological studies, identification of environmental and genetic risk factors, mechanisms of pathogenesis, and research presentations on standard and novel treatment modalities. Highlights included discussions on adefovir dipivoxil (Gilead Sciences), emtricitabine (FTC; Triangle Pharmaceuticals), clevudine (L-FMAU; Triangle Pharmaceuticals/Abbott/Bukwang Pharmaceutical Industries), entecavir (Bristol-Myers Squibb), and especially L-isomer nucleoside analogs, for use in hepatitis B virus (HBV) therapy. Mention was also made of the potential roles of histamine and mycophenolic acid in the treatment of hepatitis C virus (HCV) infections, and of polyphenols and thalidomide for HCC.     

Clevudine: a promising therapy for the treatment of chronic hepatitis B

Chronic hepatitis B virus (HBV) infection, affecting ～ 350 million people worldwide, is associated with significant morbidity and mortality. In the past 10 years, hepatitis B therapy research has led to a multitude of available antiviral therapies: IFN-α, pegylated IFN-α_2a, lamivudine, adefovir, entecavir, telbivudine and tenofovir. To further improve reductions in viral load and resistance profiles, development of new HBV therapeutic strategies has been an important focus. One such therapy is clevudine, an analogue of the β-L configuration. Clevudine is already licensed in Korea for anti-HBV therapy (Bukwang Pharmaceuticals, Seoul, Korea). Unique to clevudine is its ability to maintain antiviral activity following discontinuation of therapy. Typically, hepatitis B treatment requires continuous therapy to prevent reactivation. Sustained response is uncommon except in hepatitis B antigen (HBeAg)-positive patients who developed HBeAg seroconversion. This article reviews chronic HBV and its therapy options. Specifically, it describes clevudine's potent and sustained antiviral activity as observed in vitro and in vivo.     

DAPD (Emory University/Triangle Pharmaceuticals/Abbott Laboratories)

Triangle Pharmaceuticals is developing DAPD, a prodrug of the viral replication inhibitor dioxolane guanosine, as a potential therapy for HIV and HBV infection. Phase I/II dose range studies have commenced for HIV, and clinical development for HBV was to have commenced by late 1999 [319145], [319956]. Phase II trials are scheduled for the second quarter of 2001. The FDA has designated DAPD as a Fast Track product [365894]. DAPD is from a different nucleoside series to FTC and CS-92, which are also in development by Triangle. The compound may offer advantages over several nucleosides from other series that are already on the market because of its unique structure and pharmacological properties [247083]. Both DAPD and DXG are dioxolane purine nucleoside analogs [319660]. Preclinical data suggest DAPD may be of use in combination therapies for HIV-infected patients who are therapy-naive, in addition to patients who have previously received treatment and including those infected with drug-resistant strains of HIV-1 [341145], [341335]. Triangle licensed DAPD from Emory University [216900]. In June 1999, Triangle and Abbott Laboratories entered into an alliance for the development and marketing of six antiviral products, including DAPD [326824].     

Emtricitabine (Emory University/Glaxo Wellcome/Triangle Pharmaceuticals)

Triangle Pharmaceuticals has licensed the development rights to emtricitabine (FTC), an antiviral nucleoside analog that is structurally related to 3TC, from Emory University [207618]. It is in phase III trials for HIV [324921] and in phase I/II trials for hepatitis B (HBV) [305302]. In June 1999, Triangle and Abbott Laboratories entered into an alliance for the development and marketing of six antiviral products, including FTC [326824]. An NDA was expected to be filed in 2000 [324921]. US-05814639 has been issued covering FTC [300465]. In April 2000, a report appeared in the South African media which suggested that a Triangle-sponsored trial (FTC-302), commenced in August 1999, had been halted [361984]. A total of 470 patients had been enrolled on the trial, which is a comparison of Coviracil and lamivudine, both drugs being used in combination with d4T and nevirapine. Patients with high viral loads received efavirenz instead of nevirapine. There was a higher than expected incidence of liver toxicity in this study and, in two cases, this liver toxicity resulted in death. Initially, Triangle reported that the trial was ongoing, although no additional patients were being enrolled at the time. However, the company received notification from the Medicines Control Council (MCC) in South Africa informing it that study FTC-302 should be terminated. The company is in ongoing discussions with the MCC [361792,361793]. Subsequently, the FDA also issued a hold on the trial, which is being carried out in South Africa under a US IND, and indicated that as a result of factors leading to this decision, study FTC-302 may not provide adequate support as part of an NDA submission. The planned NDA filing may now be delayed until the second half of 2001 and Triangle will discuss with the FDA any additional data required for the NDA application [361984].     

Rapid enantiomeric quantification of an antiviral nucleoside agent (D,L-FMAU, 2'-fluoro-5-methyl-beta,D,L-arabinofurano-syluracil) by mass spectrometry

A novel mass spectrometric method is applied to rapid, accurate (<1%) quantification of chiral Clevudine (L-FMAU, 2'-fluoro-5-methyl-beta,L-arabinofuranosyluracil), a potent antiviral nucleoside agent against hepatitis B virus. Transition metal bound complex ions containing the chiral drug are generated by electrospray ionization mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture, allowing the determination of enantiomeric contamination in the drug.     

Clevudine for the treatment of chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for < or = 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-alpha and pegylated IFN-alpha2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural beta-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with < or = 9 log10 decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log10, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.     

Viramidine (Ribapharm)

Viramidine, an analog of ribavirin is a broad-spectrum antiviral under development by Ribapharm (previously the R&D division of ICN Pharmaceuticals) for the potential treatment of viral infections [378507]. In September 2000, phase I trials began in Europe. In December 2001, the company filed a US IND for the clinical development of viramidine as part of a combination therapy with interferon-afor the treatment of chronic hepatitis C virus (HCV) infection [435007], [441613]; a phase I trial was initiated in the US in late March 2002 [435598]. In November 2002, Ribapharm reported that it would start phase II trials of viramidine in the treatment of chronic HCV by the end of 2002 [469062].     

Clinical trial: a phase II, randomized study evaluating the safety, pharmacokinetics and anti-viral activity of clevudine for 12 weeks in patients with chronic hepatitis B

Background  Clevudine is a polymerase inhibitor that has the unusual feature of delayed viral rebound after therapy in some patients which may be related to its pharmacokinetics.
Aim  To characterize pharmacokinetic and pharmacodynamic profile of clevudine, a potent hepatitis B polymerase inhibitor.
Methods  A multicenter, randomized study comparing 10, 30 and 50 mg clevudine once daily for 12 weeks with 24 weeks off-treatment follow-up. Patients had chronic HBV infection, were nucleoside-naïve without co-infection. HBV viral load (VL) was assayed using Digene Hybrid Capture II with a lower limit of detection of 4700 copies/mL (940 IU/mL). Clevudine levels were measured using a liquid chromatography/mass spectrometery method.
Results  A total of 31 patients were enrolled into the 10 mg ( n  = 10), 30 mg ( n  = 11) and 50 mg ( n  = 10) groups, respectively. At week 12, the median VL change was −3.2, −3.7 and −4.2 log₁₀ copies/mL (−0.64, −0.74 and −0.84 log₁₀ IU/mL) in the 10, 30 and 50 mg groups, respectively ( P  = 0.012). At week 12, one of 10, five of 11 and two of 10 patients had VL below the assay lower limit of detection. Clevudine was well tolerated with no severe/serious adverse events. The mean plasma half-life of clevudine was 70 h and consequently is not the cause of the delayed viral rebound seen in some patients. Through modelling, 97% of the maximal treatment effect was reached with a 30 mg daily dose. Six patients had genomic changes without viral rebound.
Conclusion  Clevudine appears to be a potent and tolerable (over 12 weeks) anti-viral and the optimal dosage appears to be 30 mg once daily.     

Clevudine for the treatment of chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for ≤ 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-α and pegylated IFN-α2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural β-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with ≤ 9 log₁₀ decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log₁₀, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.     

TA-CD. Xenova

TA-CD (formerly IPC-1010), a cocaine conjugate that stimulates the production of antibodies against cocaine, is under development by Xenova (formerly Cantab) for the potential treatment of cocaine dependence [176912], [414867], [449997]. The vaccine was originally discovered at Stanford University and exclusively licensed to ImmuLogic Pharmaceuticals as IPC-1010 [176908]. It was renamed TA-CD when ownership was transferred to Cantab [463510], [463511]. By April 2002, a second phase IIa dose escalation trial was underway [445298], which was ongoing in September 2002 [463866].     

Bortezomib (millennium pharmaceuticals)

Bortezomib is a ubiquitin proteasome inhibitor under development by Millennium Pharmaceuticals (formerly LeukoSite Inc) for the potential treatment of various solid tumors [312219], [392555]. In the first quarter of 2001, Millennium initiated two phase II trials evaluating bortezomib for multiple myeloma (MM). A phase II trial in patients with chronic lymphocytic leukemia (CLL) was initiated in June 2001 [400636], [412848]. By November 2001, the agent was in a number of phase I trials and combination studies for various solid tumors, including prostate, pancreatic and colorectal carcinoma [412700], [429923], [435062], [452675]. In June 2002, bortezomib was awarded Fast Track status by the FDA [453557], and in the same month pivotal phase III trials evaluating bortezomib in MM were initiated in the US, Canada and Europe [454446].     

Sensitivity of L-(-)2,3-dideoxythiacytidine resistant hepatitis B virus to other antiviral nucleoside analogues.

L-(-)2',3'-Dideoxythiacytidine (L(-)SddC, Lamivudine) resistant hepatitis B virus (HBV) develops in patients after prolonged treatment. Point mutations detected in the viral genome from these patients have been shown to be responsible for L(-)SddC resistance. Therefore, new drugs active against L(-)SddC resistant HBV are needed. Using a transient transfection system, we studied the sensitivity of L(-)SddC resistant HBV to other anti-HBV nucleoside analogues. It was found that the L526M mutation alone caused greater resistance to penciclovir (PCV) than did the V553I mutation alone. Both mutations also caused the virus to be less sensitive to L(-)SddC and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU), although the degree of resistance was much less than that to PCV. The A546V mutation had no impact on the sensitivity to L(-)SddC, L-FMAU, and PCV. When these single mutations were coupled with the M550V/I mutation, all the double mutants were resistant to those drugs. Although 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine (L(-)Fd4C) was also less active, the IC50 of L(-)Fd4C against the L(-)SddC resistant mutant was at least fifty times lower than that against cell growth in culture. DNA polymerase associated with L(-)SddC resistant virions was also found to be less sensitive than that with wild-type HBV to those L-nucleoside triphosphates. All the L(-)SddC resistant mutants were still sensitive to 9-(2-phosphonylmethoxyethyl)-adenine (PMEA). These results suggest that different mutations in the HBV genome have a different impact on its sensitivity to those compounds, and L(-)SddC resistant HBV may also be resistant to PCV, L-FMAU, and L(-)Fd4C. A nucleoside analogue less toxic than PMEA could be developed against L(-)SddC resistant HBV.     

Entecavir (Bristol-Myers Squibb)

Bristol-Myers Squibb (BMS) is developing entecavir, a viral replication inhibitor, for the potential treatment of hepatitis B virus (HBV) infection [220240]. The compound is a cyclopentyl guanosine analog and is in phase II trials in the US [383065]. Entecavir was originally developed as SQ-34676 for the treatment of herpes simplex virus infections [221992], but displayed only moderate activity which eventually led to discontinuation of development for this indication. However, Bristol-Myers Squibb later discovered that entecavir was extremely potent against HBV (ED50 = 3.0 nM, compared with 200 nM for lamivudine) with relatively low toxicity (CC50 = 30,000 nM) [221986] and acted through inhibition of DNA polymerase [220240]. The triphosphate form is a potent HBV polymerase inhibitor in both woodchuck and duck models [306056]. By September 2000, a large-scale clinical trial was underway in China for HBV infection [400209] and by October 2000 phase I trials were ongoing in Japan [384751]. In March 2001 SG Cowen predicted sales of US$25 million in 2002, US$50 million in 2003 and US$75 million in 2004 [403751].     

Nystatin LF (Aronex/Abbott)

Nystatin LF (Nyotran) is a liposomal, intravenous nystatin formulation under development by Aronex, under license from the MD Anderson Cancer Research Center, as a systemic antifungal agent against strains including Aspergillus and Candida. Like amphotericin, nystatin is a polyene derivative that binds to ergosterol, a fungal cell membrane component, creating a pore in the membrane and thus killing the cell. Nystatin is an established antifungal agent, but is restricted to topical use as it is ineffective orally and severely toxic when administered iv [187583], [187589]. It has demonstrated good, broad in vitro antifungal activity against clinically relevant filamentous fungi [319465], including fungi resistant to fluconazole and amphotericin B products [264505], [2869821, [287790], 1289522]. The company is also conducting a phase III trial to evaluate its efficacy against cryptococcal meningitis [305531], [334156]. Aronex filed for approval of nystatin LF in Spain in December 1997 [272986] and expected to file an NDA in the US by the end of 1999 1311208], [342003]. However, in an effort to ensure that its US and European filings contained data from the phase III cryptococcal meningitis trial in its entirety, Aronex's marketing partner requested that all the 70-day data be gathered prior to unblinding this study. The filing had initially been based on interim data at the 14- and 21-day endpoint 1344887]. In September 2000, the company anticipated an NDA filing in the US in the fourth quarter of 2001 1382861], 1387947]. In December 1997, Aronex, together with Grupo Ferrer Internacional, filed an MAA in Spain seeking approval for Nyotran for the treatment of systemic fungal infections. Aronex intended to follow the filing with additional filings in other European countries 1272986]. In 1997, a commercialization agreement was signed with Ferrer for Spain and Portugal, with Aronex intending to form other such partnerships throughout Europe and Asia 1248346]. In November 1998, Aronex signed a licensing collaboration with Abbott Laboratories for the worldwide rights to nystatin LF [305531].     

Aripiprazole (Otsuka Pharmaceutical Co)

Otsuka Pharmaceuticals in collaboration with Bristol-Myers Squibb is developing aripiprazole, a dual dopamine autoreceptor agonist and postsynaptic D2 receptor antagonist, for the potential treatment of psychoses including schizophrenia [281327], [340364]. A regulatory filing for schizophrenia in the US was submitted at the end of 2001 [340364]. The compound entered phase III trials in Japan in 1995 [192966]. Although presynaptic dopamine autoreceptor agonists may be efficacious in the treatment of schizophrenia, they may also potentially increase the risk for exacerbation of psychosis through stimulation of postsynaptic dopaminergic receptors [245791], [350478], [350479]. However, earlier neuropharmacology studies have shown that aripiprazole can act as a presynaptic D2 agonist while displaying an antagonistic effect at the postsynaptic D2 receptors [281327], [337126], [350479], [424587], [424588]. In animal models, aripiprazole inhibits the apomorphine-induced stereotypy, without causing catalepsy [281327], [337126]. Moreover, in contrast to classical antipsychotics that produce disabling movement disorders, aripiprazole does not cause an upregulation of D2 receptors or an increase in expression of the c-fos mRNA in the striatum, in agreement with the low risk for extrapyramidal side effects (EPS) during aripiprazole treatment [245781], [262096], [350481], [350483]. Collectively, aripiprazole is an important atypical antipsychotic candidate with a favorable safety profile. Moreover, the mechanism of action of aripiprazole differentiates it from both typical and atypical antipsychotics and hence, may provide important leads for pharmacotherapy of schizophrenia and other psychotic disorders. In January 2000, Lehman Brothers predicted peak sales of aripiprazole could reach US $500 million [357788]. In February 2001, Credit Suisse First Boston predicted sales of US $403 million in 2005 [399484].     

Pleconaril Sanofi Synthélabo/ViroPharma

Pleconaril is an oral antiviral agent being developed by ViroPharma and Sanofi-Synthélabo (formerly Sterling Winthrop) for the potential treatment of several picornavirus-induced infections, including respiratory diseases and viral meningitis. A number of phase III clinical trials have been completed, and several others are ongoing [319499], [343187], [346302], [359231]. In early 1999, an NDA filing for viral meningitis was expected by the end of 1999 [313588], [319499]. However, an NDA had not been filed by February 2000 and, at this time, filing in the US was expected in 2000 for viral meningitis and 2001 for viral respiratory syndrome, while in Europe filing was expected in 2001 and 2002 for these indications, respectively [359231]. In July 2000, Salomon Smith Barney predicted a launch date of 2002 [387350]. In October 1999, Lehman Brothers predicted a 70% chance of the product reaching the market, with a possible launch date anticipated for 2001 and potential peak sales of US$50 million in 2009 [346267].     

Comparative evaluation of L-Fd4C and related nucleoside analogs as promising antiviral agents

As documented in the recent literature, there are more than 50 million people infected with HIV worldwide to date since the emergence of HIV and AIDS in the Western world in 1981. More importantly, about 7000 people die of AIDS daily with 2.5 and 2.6 millions total deaths in 1998 and 1999, respectively. On the other hand, human hepatitis B virus (HBV) is the leading cause of chronic hepatitis in the world. According to WHO executive summary, over 350 millions (approximately 5% of the world s population) people are chronically infected with HBV. There are about 1 million chronic HBV carriers in the United States. Although safe and effective vaccination for HBV is available for developing countries, there is still no effective treatment for the millions of chronically infected individuals. Consequently, long term infection with chronic HBV could lead to cirrhosis, and hepatocellular carcinoma. In light of these facts, it is evident that the discovery and development of novel antiviral agents for the treatment of HIV and HBV is an extremely important undertaking.The interest in L-nucleosides was spurred in recent years by the findings that L-nucleosides are generally endowed with lower host toxicity while maintaining good antiviral activity in comparison to their respective D-nucleosides. The recent FDA approval of Lamivudine [L-BCH 189 (3TC)] for the treatment of HIV and HBV further supports these notions. Since the discovery of Lamivudine, a large number of 2 ,3 -dideoxy (dd)- and 2 ,3 -didehydro-2 ,3 -dideoxy (D4)-L-nucleoside analogs have been synthesized and evaluated in hopes of identifying even better antiviral agents. As a result, 2 ,3 -Dideoxy-2 ,3 -didehydro-beta-L-fluorocytidine (beta-L-Fd4C) was found to be a promising new lead. The first synthesis and antiviral activity assessment of L-Fd4C were reported by Lin and Cheng et al. in 1996. Recent disclosures from several laboratories clearly demonstrated that L-Fd4C was the most potent anti-HBV agent reported to date (vs. 3TC, L-FddC, L-FMAU, etc.). In fact, L-Fd4C proved to be at least 10 times more potent than Lamivudine on HBV DNA synthesis in the hepatoma cell line HepG2 2.2.15. Compared with L-Fd4C, D-Fd4C showed similar anti-HIV activity yet reduced anti-HBV activity. 2 F-L-Fd4C exhibited excellent acid stability but reduced antiviral activity and cytotoxicity. Although L-Fd4C is converted intracellularly by cytoplasmic deoxycytidine kinase to its mono-, di- and triphosphate metabolites,43 the newly prepared bis(SATE)-L-Fd4CMP proved to be more potent against HBV yet less cytotoxic than L-Fd4C itself. The chemically synthesized L-Fd4CTP was found to be a poor substrate for human polymerase gamma. A recent report from Zhu and Cheng et al. indicated that L-Fd4C had no inhibitory effect on mitochondrial DNA synthesis at concentrations up to 10 microM. An in vivo study involving HBV-infected ducks showed that longer administration of L-Fd4C induced a sustained suppression of viremia (>95%) and of viral DNA synthesis in the liver. The same study also demonstrated that L-Fd4C is more potent than 3TC in vivo. In summary, on the basis of the data presented in this chapter, it is evident that L-Fd4C is endowed with exceptional anti-HBV activity (both in vitro and in vivo) as well as an acceptable toxicity profile, thus rendering it a very promising development candidate.     

Bexxar (Corixa/GlaxoSmithKline)

Bexxar (131I tositumomab) is a radiolabeled anti-CD20 monoclonal antibody for the treatment of relapsed and refractory follicular/low-grade and transformed non-Hodgkin's lymphoma. It has shown high response rates with durable complete remissions in patients who have received either prior chemotherapy or rituximab (Rituxan, MabThera; IDEC Pharmaceuticals Corp/Genentech/F Hoffmann La Roche). Complications include myelosuppression, secondary acute leukemia, myelodysplasia and hypothyroidism. The role of this promising new agent is being defined in phase II and III trials. In February 2001, ABN Amro Predicted launch in 2001 and sales of US $25 million rising to US $70 million in 2003 [422363]. Corixa and GlaxoSmithKline anticipate a launch in the US in 2002 [424619].