Discussion
Although lamivudine was approved for the treatment of patients with chronic hepatitis B, short-term treatment is usually insufficient to clear the virus. Moreover, long-term treatment is associated with the development of drug-resistant HBV in 14–75% of patients (2, 4, 15, 32). These lamivudine-resistant HBV harbor M552I or M552V mutations in the C domain of the polymerase gene. The L528M mutation frequently accompanies M552V and has recently been reported to accompany M552I occasionally (7–10, 12). To summarize three published reports, the clinical frequency of lamivudine-resistant mutants was 18.6% for M552I, 1.4% for M552V, 11.4% for L528M/M552I, and 64.3% for L528M/M552V (10, 11, 13). Given that mutations in the polymerase gene have been associated with changes in the replication competency of the virus (18, 19), we examined the influence, singly or in combination, of the B-domain mutation (L528M) and the two C-domain mutations (M552I and M552V) on replication ability. Compared with the wild-type HBV, the single C-domain mutants M552I and M552V had markedly decreased replication abilities (18). Of particular interest, the double mutants with both B- and C-domain mutations (L528M/M552I and L528M/M552V) replicated significantly better than did the single C-domain mutants, suggesting that the B-domain mutation L528M rescued the defective replication competence of the C-domain mutants. It was previously indicated that L528M could compensate for the impact of a mutation in the YMDD motif on viral replication in vitro (33). The B-domain L528 in the amino acid 521-537 helix is close enough to interact with M552 of the YMDD loop in a hypothetical molecular model of HBV RT (10). It could be speculated that the L528M mutation reduces the imbalance of conformation caused by the M552V and M552I mutations, thereby improving the replication competence of single C-domain mutants. Of the 11 compounds tested, entecavir, L-D4FC, L-FMAU (–)-FTC, lamivudine, and adefovir inhibited the replication of wild-type HBV effectively, with an EC50 less than 1 μM. These results are comparable with previous reports that demonstrated inhibition of HBV replication by 50% at concentrations of less than 0.1 μM in 2.2.15 cells (34–38). Because several different groups tested these compounds in different ways, it was difficult to compare their EC50 values, as they varied according to the assay used, cell type used, marker of viral replication selected, composition of the medium, and time in culture (39). We studied the effect of the 11 compounds simultaneously. Therefore, we could determine and compare their relative potencies. Entecavir, L-D4FC, L-FMAU, and (–)-FTC inhibited wild-type HBV replication two to 1,556 times more than lamivudine. Similar results were obtained in 2.2.15 cells and pSM2-transfected HuH7 cells. In these cells, entecavir was 237–2,200 times more potent than lamivudine. In general, the more potent the antiviral agents used to suppress viral replication, the less likely the virus is to develop drug-resistant mutations, because mutations arise as replication errors (40–43). Therefore, in the absence of toxicological considerations of these experimental agents, entecavir, L-D4FC, and L-FMAU are potentially useful “first line” drugs for the treatment of HBV. However, it must be noted that in vitro sensitivities do not always match the sensitivities of virus infection in humans in vivo. Therefore, the ultimate test is their effectiveness in patients with chronic hepatitis B. While adefovir, entecavir (+)-FTC (–)-FTC, L-D4FC, and L-FMAU are effective (EC50 < 10 μM) against some of the five lamivudine-resistant HBV mutants, only adefovir and entecavir are effective against all five mutants. As previously described, adefovir might be a good treatment option in those patients who have failed lamivudine therapy because of drug-resistant HBV (25). In fact, it was recently demonstrated that adefovir resulted in a rapid and sustained reduction in HBV DNA levels associated with improvement in liver function in patients who failed to respond to lamivudine therapy (44). In addition, based on our data, entecavir could be an option for the treatment of lamivudine-resistant mutants. However, it must be noted that the EC50 values for lamivudine-resistant mutants were 2 to 778 times higher than those for the wild-type HBV. Therefore, the dose of entecavir necessary to treat lamivudine-resistant mutants would be considerably higher than that used for wild-type HBV. L-D4FC and L-FMAU were effective against single B- or C-domain mutants L528M and M552V with an EC50 of less than 2 μM, but were ineffective against the double mutant L528M/M552V, with an EC50 exceeding 10 μM. In addition, the EC50 of entecavir for the double mutants was about 500 times higher than that for the single B-domain mutant L528M, and 90 times higher than that for the single C-domain mutants. The addition of the B-domain mutation L528M to C-domain mutants may contribute to increased levels of resistance to entecavir, L-D4FC, and L-FMAU but does not seem to increase the level of resistance to adefovir, suggesting that the resistance pattern for adefovir is unique and different from those of entecavir, L-D4FC, and L-FMAU. Although further studies are needed to address the cytotoxicity of these drugs in humans, the doses of the compounds used in our study are far below the toxic doses reported by others. Using 2.2.15 cells, no apparent cytotoxicity was noted at concentrations greater than 1,000, 150, and 30 μM for lamivudine, adefovir, and entecavir, respectively (34, 45, 46). For L-D4FC, the concentration required to inhibit 50% of HepG2 growth was estimated to be 20 μM (35). Unfortunately, L-D4FC is cytotoxic in various cells and with prolonged treatment has been shown to increase lactic acid production in HepG2 (47). L-FMAU (±)-FTC, and (–)-FTC did not show any cytotoxicity up to 200 μM in 2.2.15 cells (36–38). In addition, with the exception of entecavir, in vivo studies have shown that these compounds are not associated with overt toxicity at high doses (>100 mg/kg). In duck hepatitis B virus–infected ducklings, treatment with 15–30 mg/kg of adefovir or 40 mg/kg of L-FMAU produced no toxic side effects (46, 48). In the woodchuck model, 0.5–0.1 mg/kg of entecavir, 1–4 mg/kg of L-D4FC, or 20–30 mg/kg of (–)-FTC inhibited replication of woodchuck hepatitis virus without associated toxicity (49–52). As learned from the treatment of HIV, it is likely that combinations of HBV drugs should be used to maximize suppression of replication and consequently decrease the probability of the emergence of a drug-resistant virus (40, 53, 54). This approach would permit the use of lower doses of the antiviral agents and, therefore, reduce the likelihood of side effects. It also seems advantageous to combine adefovir with lamivudine, entecavir, L-D4FC, L-FMAU, or (–)-FTC in an effort to better suppress HBV replication and delay the development of resistance. Although entecavir, L-D4FC, and L-FMAU had the lowest EC50, a combination of these three drugs may be compromised, as they have a similar cross-resistance profile. Further studies are necessary to determine the potential synergistic interaction of compounds in combination therapy.
Acknowledgments
This study was supported by a Research Grant for Immunology, Allergy, and Organ Transplantation from the Ministry of Health and Welfare, Japan. S.K. Ono is a Research Fellow of the Japan Society for the Promotion of Science. R.F. Schinazi is supported in part by the Department of Veterans Affairs and NIH grant RO1-AI-41980. We thank Mitsuko Tsubouchi for technical assistance. Neither this study nor the authors received grant support from the pharmaceutical companies whose products are examined.
Top
Abstract
Introduction
Methods
Results
Discussion
References
References
World Health Organization warns of growing “crisis of suffering.” http://www.who.ch/whr/1997/presse.htm. Gordon, D. & Walsh, J.H. (1998). Hepatitis drugs win approval. Gastroenterology. 116, 235236 Honkoop, P., de Man, R.A., Zondervan, P.E., & Schalm, S.W. (1997). Histological improvement in patients with chronic hepatitis B virus infection treated with lamivudine. Liver. 17, 103106 [PubMed] Lai, C.L. et al. (1998). A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N. Engl. J. Med. 339, 6168 [PubMed][Full Text] Omata, M. (1998). Treatment of chronic hepatitis B infection. N. Engl. J. Med. 339, 114115 [PubMed][Full Text] Lai, C.L. (1999). Antiviral therapy for hepatitis B and C in Asians. J. Gastroenterol. Hepatol. 14(Suppl.), S19S21 [PubMed] Bartholomew, M.M. et al. (1997). Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet. 349, 2022 [PubMed][Full Text] Ling, R. et al. (1996). Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine. Hepatology. 24, 711713 [PubMed][Full Text] Tipples, G.A. et al. (1996). Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology. 24, 714717 [PubMed][Full Text] Allen, M.I. et al. (1998). Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology. 27, 16701677 [PubMed][Full Text] Chayama, K. et al. (1998). Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology. 27, 17111716 [PubMed][Full Text] Yeh, C.T., Chien, R.N., Chu, C.M., & Liaw, Y.F. (2000). Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy. Hepatology. 31, 13181326 [PubMed][Full Text] Benhamou, Y. et al. (1999). Long-term incidence of hepatitis B virus resistance to lamivudine in human immunodeficiency virus-infected patients. Hepatology. 30, 13021306 [PubMed][Full Text] Bartholomeusz, A., Schinazi, R.F., & Locarnini, S.A. (1998). Significance of mutations in the hepatitis B virus polymerase selected by nucleoside analogues and implications for controlling chronic disease. Viral Hepatitis Reviews. 4, 167187 Lau, D.T.-Y. et al. (2000). Long-term lamivudine therapy for chronic hepatitis B. Antivir. Ther. 5(Suppl. 1), 43 (Abstr.) Poch, O., Sauvaget, I., Delarue, M., & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 8, 38673874 [PubMed] Kamer, G. & Argos, P. (1984). Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 12, 72697282 [ Free Full text in PMC] Ono-Nita, S.K. et al. (1999). YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: a study by in vitro full-length viral DNA transfection. Hepatology. 29, 939945 [PubMed][Full Text] Melegari, M., Scaglioni, P.P., & Wands, J.R. (1998). Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology. 27, 628633 [PubMed][Full Text] Jacobo-Molina, A. et al. (1993). Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc. Natl. Acad. Sci. USA. 90, 63206324 [ Free Full text in PMC] Seigneres, B. et al. (2000). Evolution of hepatitis B virus polymerase gene sequence during famciclovir therapy for chronic hepatitis B. J. Infect. Dis. 181, 12211233 [PubMed][Full Text] Liaw, Y.F., Chien, R.N., Yeh, C.T., Tsai, S.L., & Chu, C.M. (1999). Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology. 30, 567572 [PubMed][Full Text] Ben-Ari, Z., Pappo, O., Zemel, R., Mor, E., & Tur-Kaspa, R. (1999). Association of lamivudine resistance in recurrent hepatitis B after liver transplantation with advanced hepatic fibrosis. Transplantation. 68, 232236 [PubMed] Bessesen, M., Ives, D., Condreay, L., Lawrence, S., & Sherman, K.E. (1999). Chronic active hepatitis B exacerbations in human immunodeficiency virus-infected patients following development of resistance to or withdrawal of lamivudine. Clin. Infect. Dis. 28, 10321035 [PubMed] Ono-Nita, S.K. et al. (1999). Susceptibility of lamivudine-resistant hepatitis B virus to other reverse transcriptase inhibitors. J. Clin. Invest. 103, 16351640 [PubMed][Free Full Text] Merchant, Z. et al. (1997). BMS-200475, a novel carbocyclic 2′-deoxyguanosine analog with potent and selective anti-hepatitis B virus activity in vitro. Bioorg. Med. Chem. Lett. 7, 127132 Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., & Sato, J. (1982). Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 42, 38583863 [PubMed] Sells, M.A., Chen, M.L., & Acs, G. (1987). Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA. 84, 10051009 [ Free Full text in PMC] Togo, G. et al. (1996). A transforming growth factor beta type II receptor gene mutation common in sporadic cecum cancer with microsatellite instability. Cancer Res. 56, 56205623 [PubMed] Günther, S. et al. (1995). A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J. Virol. 69, 54375444 [ Free Full text in PMC] Yokosuka, O., Omata, M., Imazeki, F., Okuda, K., & Summers, J. (1985). Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA. Hepatology. 5, 728734 [PubMed] Dienstag, J.L. et al. (1999). Lamivudine as initial treatment for chronic hepatitis B in the United States. N. Engl. J. Med. 341, 12561263 [PubMed][Full Text] Fu, L. & Cheng, Y.C. (1998). Role of additional mutations outside the YMDD motif of hepatitis B virus polymerase in L(-)SddC (3TC) resistance. Biochem. Pharmacol. 55, 15671572 [PubMed][Full Text] Innaimo, S.F. et al. (1997). Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus. Antimicrob. Agents Chemother. 41, 14441448 [ Free Full text in PMC] Zhu, Y.L., Dutschman, D.E., Liu, S.H., Bridges, E.G., & Cheng, Y.C. (1998). Anti-hepatitis B virus activity and metabolism of 2′,3′-dideoxy-2′,3′-didehydro-beta-L(-)-5-fluorocytidine. Antimicrob. Agents Chemother. 42, 18051810 [PubMed][Free Full Text] Balakrishna Pai, S., Liu, S.H., Zhu, Y.L., Chu, C.K., & Cheng, Y.C. (1996). Inhibition of hepatitis B virus by a novel L-nucleoside, 2′-fluoro-5-methyl-beta-L-arabinofuranosyl uracil. Antimicrob. Agents Chemother. 40, 380386 [ Free Full text in PMC] Jansen, R.W., Johnson, L.C., & Averett, D.R. (1993). High-capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay. Antimicrob. Agents Chemother. 37, 441447 [ Free Full text in PMC] Furman, P.A. et al. (1992). The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine. Antimicrob. Agents Chemother. 36, 26862692 [ Free Full text in PMC] Hirsch, M.S. et al. (1998). Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. International AIDS Society—USA Panel. JAMA. 279, 19841991 [PubMed][Full Text] Condra, J.H. (1998). Resisting resistance: maximizing the durability of antiretroviral therapy. Ann. Intern. Med. 128, 951954 [PubMed][Full Text] Coffin, J.M. (1995). HIV population dynamics in viv implications for genetic variation, pathogenesis, and therapy. Science. 267, 483489 [PubMed] Tsiang, M., Rooney, J.F., Toole, J.J., & Gibbs, C.S. (1999). Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy. Hepatology. 29, 18631869 [PubMed][Full Text] Havlir, D.V. & Richman, D.D. (1996). Viral dynamics of HIV: implications for drug development and therapeutic strategies. Ann. Intern. Med. 124, 984994 [PubMed][Full Text] Peters, M. et al. (2000). Adefovir dipivoxil treatment of hepatitis B virus disease in patients failing lamivudine therapy. Antivir. Ther. 5(Suppl. 1), 45 Kruining, J., Heijtink, R.A., & Schalm, S.W. (1995). Antiviral agents in hepatitis B virus transfected cell lines: inhibitory and cytotoxic effect related to time of treatment. J. Hepatol. 22, 263267 [PubMed][Full Text] Heijtink, R.A. et al. (1993). Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection. Antiviral Res. 21, 141153 [PubMed] Shi, J. et al. (1999). Synthesis and biological evaluation of 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (D4FC) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase. J. Med. Chem. 42, 859867 [PubMed][Full Text] Aguesse-Germon, S. et al. (1998). Inhibitory effect of 2′-fluoro-5-methyl-beta-L-arabinofuranosyl-uracil on duck hepatitis B virus replication. Antimicrob. Agents Chemother. 42, 369376 [PubMed][Free Full Text] Genovesi, E.V. et al. (1998). Efficacy of the carbocyclic 2′-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection [erratum 1999, 43:726]. Antimicrob. Agents Chemother. 42, 32093217 [PubMed][Free Full Text] Le Guerhier, F. et al. (1999). 2′,3′-dideoxy-2′,3′-didehydro-b-L-5-fluorocytidine (b-L-FD4C) exhibits a more potent antiviral effect than lamivudine in chronically WHV infected woodchucks. Hepatology. 30, 344A (Abstr.) Cullen, J.M. et al. (1997). In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks. Antimicrob. Agents Chemother. 41, 20762082 [ Free Full text in PMC] Korba, B.E., Schinazi, R.F., Cote, P., Tennant, B.C., & Gerin, J.L. (2000). Effect of oral administration of emtricitabine on woodchuck hepatitis virus replication in chronically infected woodchucks. Antimicrob. Agents Chemother. 44, 17571760 [PubMed][Free Full Text] Balzarini, J. (1999). Suppression of resistance to drugs targeted to human immunodeficiency virus reverse transcriptase by combination therapy. Biochem. Pharmacol. 58, 127 [PubMed][Full Text] Schinazi, R.F. 1991. Combined therapeutic modalities for viral infections: rationale and clinical potential. In Synergism and antagonism in chemotherapy. T.-C. Chou and D.C. Rideout, editors. Academic Press. Orlando, Florida, USA. 110–181.
|