Mitsubishi-Tokyo and Lilly Announce License Agreement for Novel Anti B －MCC-478

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December 21, 2000 Mitsubishi-Tokyo Pharmaceuticals, Inc. Eli Lilly and Company Mitsubishi-Tokyo and Lilly Announce License Agreement for Novel Antihepatitis B Compound

Mitsubishi-Tokyo Pharmaceuticals, Inc. and Eli Lilly and Company (NYSE: LLY and Toky 4857) announced today that they have signed a license agreement for MCC-478, a novel antiviral agent for the treatment of hepatitis B virus (HBV) infection. The agreement gives Lilly exclusive rights to develop and market this compound worldwide, excluding Japan.

Infection with hepatitis B virus, a DNA virus, can lead to cirrhosis, hepatocellular carcinoma, liver failure and death. Currently, an estimated 350 million people worldwide are chronically infected with the virus, which is known to be the ninth leading cause of death globally.

Although a safe and effective vaccine is available for the prevention of infection with HBV, there is still a need for antiviral therapy for the large number of hepatitis B virus carriers worldwide. Current approved drug therapies are not always effective due to the emergence of drug-resistant strains or because of significant adverse side effects. Thus, an innovative drug for the treatment of chronic hepatitis B virus infection is needed. MCC-478, a nucleotide analog prodrug with potent and selective activity against HBV developed by Mitsubishi-Tokyo, may offer the first available curative treatment for hepatitis B virus infection. In addition, the course of therapy for this molecule may be shorter than for current therapies. MCC-478 is being tested in Phase I human clinical trials. "We believe Mitsubishi-Tokyo has developed a novel compound that may offer a potential breakthrough for the treatment of hepatitis B," said Gail H. Cassell, Ph.D., vice president of infectious disease discovery research and clinical investigation for Lilly. "Our agreement with Mitsubishi-Tokyo gives us an opportunity to further expand our efforts in this important medical area."

"We believe rapid development and commercialization of MCC-478 by Lilly will contribute toward remarkable advancement in the treatment of hepatitis B in the world market. A reliable partner with accumulated experience in this area has been sought since Mitsubishi-Tokyo considers MCC-478 has the outstanding therapeutic potential amongst the sub-target areas of current strategy," said Akihiro Tobe, Ph.D., president of the research and development division of Mitsubishi-Tokyo Pharmaceuticals, Inc.

Lilly, a leading innovation-driven corporation, is developing a growing portfolio of best-in-class pharmaceutical products by applying the latest research from its own worldwide laboratories and from collaborations with eminent scientific organizations. Headquartered in Indianapolis, Ind., Lilly provides answers - through medicines and information - for some of the world's most urgent medical needs. Additional information about Lilly is available at www.lilly.com.

Mitsubishi-Tokyo Pharmaceuticals, Inc. (2-6, Nihonbashi-Honcho 2-chome, Chuo-ku, Tokyo, Japan, Ryuichi Tomizawa, President), a wholly owned subsidiary of Mitsubishi Chemical Corporation (headquartered in 5-2, Marunouchi 2-chome, Chiyoda-ku, Tokyo, Kanji Shono, President), puts its focus on development of innovative drugs that are globally competitive to become a company with vivid presence in the world.

This press release contains forward-looking statements about the potential of the investigational compound MCC-478 in treating hepatitis B virus infection that reflects management's current beliefs. However, as with any pharmaceutical under development, there are risks and uncertainties in the process of development and regulatory review. There are no guarantees that future clinical trials will confirm the preliminary results referred to in this release or that the product will receive regulatory approvals or prove to be commercially successful. For further discussion of these and other risks and uncertainties, see Lilly's filings with the United States Securities and Exchange Commission.

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Eli Lilly and Company (LLY) And Mitsubishi-Tokyo Announce License Agreement For Novel Antihepatitis B Compound

INDIANAPOLIS, Dec. 21 /PRNewswire/ -- Eli Lilly and Company (NYSE: LLY and Toky 4857) and Mitsubishi-Tokyo Pharmaceuticals, Inc., announced today that they have signed a license agreement for MCC-478, a novel antiviral agent for the treatment of hepatitis B virus (HBV) infection. The agreement gives Lilly exclusive rights to develop and market this compound worldwide, excluding Japan.

Infection with hepatitis B virus, a DNA virus, can lead to cirrhosis, hepatocellular carcinoma, liver failure and death. Currently, an estimated 350 million people worldwide are chronically infected with the virus, which is known to be the ninth leading cause of death globally.

Although a safe and effective vaccine is available for the prevention of infection with HBV, there is still a need for antiviral therapy for the large number of hepatitis B virus carriers worldwide. Current approved drug therapies are not always effective due to the emergence of drug-resistant strains or because of significant adverse side effects. Thus, an innovative drug for the treatment of chronic hepatitis B virus infection is needed. MCC- 478, a nucleotide analog prodrug with potent and selective activity against HBV, may offer the first available curative treatment for hepatitis B virus infection. In addition, the course of therapy for this molecule may be shorter than for current therapies. MCC-478 is being tested in Phase I human clinical trials.

"We believe Mitsubishi-Tokyo has developed a novel compound that may offer a potential breakthrough for the treatment of hepatitis B," said Gail H. Cassell, Ph.D., vice president of infectious diseases discovery research and clinical investigation for Lilly. "Our agreement with Mitsubishi-Tokyo gives us an opportunity to further expand our efforts in this important medical area."

"We believe rapid development and commercialization of MCC-478 by Lilly will contribute toward remarkable advancement in the treatment of hepatitis B in the world market," said Akihiro Tobe, Ph.D., president of the research and development division of Mitsubishi-Tokyo Pharmaceuticals, Inc. "A reliable partner with accumulated experience in this area has been sought because we believe MCC-478 has outstanding therapeutic potential amongst the sub-target areas of the current strategy."

Mitsubishi-Tokyo Pharmaceuticals, Inc. (2-6, Nihonbashi-Honcho 2-chome, Chuo-ku, Tokyo, Japan, Ryuichi Tomizawa, President), a wholly owned subsidiary of Mitsubishi Chemical Corporation (headquartered in 5-2, Marunouchi 2-chome, Chiyoda-ku, Tokyo, Kanji Shono, President), puts its focus on the development of innovative drugs that are globally competitive to become a company with vivid presence in the world.

Lilly, a leading innovation-driven corporation, is developing a growing portfolio of best-in-class pharmaceutical products by applying the latest research from its own worldwide laboratories and from collaborations with eminent scientific organizations. Headquartered in Indianapolis, Ind., Lilly provides answers -- through medicines and information -- for some of the world's most urgent medical needs. Additional information about Lilly is available at www.lilly.com .

This press release contains forward-looking statements about the potential of the investigational compound MCC-478 in treating hepatitis B virus infection that reflect management's current beliefs. However, as with any pharmaceutical under development, there are risks and uncertainties in the process of development and regulatory review. There are no guarantees that future clinical trials will confirm the preliminary results referred to in this release or that the product will receive regulatory approvals or prove to be commercially successful. For further discussion of these and other risks and uncertainties, see Lilly's filings with the United States Securities and Exchange Commission. SOURCE Eli Lilly and Company

-0- 12/21/2000

/CONTACT: Terra L. Fox of Lilly, 317-276-5795; or Shigemasa Kobayashi of Mitsubishi, 03-3241-7905/

/Company News On-Call: http://www.prnewswire.com/comp/126236.html or fax, 800-758-5804, ext. 126236/

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New compound could beat wild-type and lamivudine-resistant hepatitis BHepatitis B Therapy September 9, 2002

by Sonia Nichols, senior medical writer - A novel nucleoside analogue could be the answer to overcoming resistance to standard therapy in patients infected with hepatitis B virus (HBV).

The agent is a derivative of phosphonomethoxyethyl purine and is known as MCC-478 (LY582563). The members of a research team in Japan at the University of Tokyo think MCC-478 could be a treatment alternative for those individuals who develop resistance to lamivudine, a drug commonly used for treating HBV.

Researchers have tested MCC-478 in HBV-infected human hepatoma cells, analyzing its effectiveness against wild-type virus and lamivudine-resistant mutants.

"The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478," Suzane Kioko Ono-Nita and colleagues acknowledged.

The 50% effective concentration (EC(50)) of MCC-478 for treating wild-type virus was 0.027 micromolar and was considered 20 times more potent than lamivudine. Though MCC-478 was also effective against lamivudine mutants, considerably more of it was required for achieving an EC(50) (Novel nucleoside analogue MCC-478 (LY582563) is effective against wild-type or lamivudine-resistant hepatitis B virus, Antimicrobial Agents and Chemotherapy, August 2002;46(8):2602-2605).

Given the number of new patients who develop lamivudine resistant HBV infections after being on long-term therapy, the analog could eventually be considered a viable treatment alternative for them.

"MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV," Ono-Nita and colleagues concluded.

The corresponding author for this study is Naoya Kato, Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655. E-mail: [email protected].

Key points reported in this study include:

* The novel nucleoside analogue MCC-478 was more efficient against wild-type hepatitis B virus (HBV) than lamivudine

* MCC-478 was effective for knocking down lamivudine-resistant HBV mutant infections, though higher doses of the agent were required

* MCC-478 is a potential treatment for wild-type and lamivudine resistant HBV infections This article was prepared by Hepatitis Weekly editors from staff and other reports.

©Copyright 2002, via NewsRx.com & NewsRx.net Click here for additional articles on Infectious Disease

return to OBGYN.net Headline News ... (2001 archives)

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Novel Nucleoside Analogue MCC-478 (LY582563) Is Effective against Wild-Type or Lamivudine-Resistant Hepatitis B Virus Suzane Kioko Ono-Nita,1,2 Naoya Kato,1* Yasushi Shiratori,1 Flair José Carrilho,2 and Masao Omata1

Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan1 Department of Gastroenterology, University of São Paulo School of Medicine, São Paulo CEP 05403-000, Brazil2

*Corresponding author. Mailing address: Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-3815-5411, ext. 37198. Fax: 81-3-3814-0021. E-mail: [email protected].

Received 2002 January 2; Revised 2002 February 11; Accepted 2002 May 13.

This article has been cited by other articles in PMC. Abstract The emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC50) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC50s were 0.027 μM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 μM for M552I, 3.3 μM for M552V, and 2.0 μM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.

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Introduction Hepatitis B is a serious disease, and according to a 1997 World Health Organization report (http://www.who.ch/whr/1997/presse.htm), it is among the 10 leading killer diseases. Interferon treatment is partially effective for chronic hepatitis B but has dose-limiting side effects (9, 19, 28). Recently, lamivudine became a standard oral therapy for hepatitis B treatment (6), and clinical data show that it rapidly reduces hepatitis B virus (HBV) DNA levels, is well tolerated, and improves liver histology (8, 12, 14, 20).

Short-term lamivudine therapy is not sufficient to clear the virus. However, long-term therapy is associated with the emergence of the lamivudine-resistant viruses M552I and M552V (the M552V mutation is often accompanied by a second L528M mutation). These viruses have amino acid substitutions in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of viral DNA polymerase (1, 16, 26). The emergence rates of lamivudine-resistant HBV have ranged from 14 to 46% after 1 year to as high as 67 to 75% after 3 to 4 years of continuous therapy (6, 12; D. T. Y. Lau, M. F. Khokhar, M. G. Ghany, E. Doo, D. Herion, D. E. Kleiner, Y. Park, P. Schmid, T. J. Liang, and J. H. Hoofnagle, Abstr. 10th Int. Symp. Vir. Hepatitis Liver Dis., abstr. 076, 2000). Although the natural history of patients with lamivudine-resistant HBV is not yet well defined, this virus has been associated with an acute exacerbation of liver disease (15), advanced hepatic fibrosis and necroinflammatory process in patients receiving liver transplantation (2), and severe hepatitis in patients coinfected with human immunodeficiency virus (3). Thus, the need for new antiviral agents for hepatitis B treatment is becoming clear.

MCC-478 {LY582563, 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-(4-methoxyphenylthio)purine bis(2,2,2-trifluoroethyl) ester}, a novel nucleoside analogue, was synthesized as a derivative of adefovir. In this study, we evaluated the susceptibility of wild-type HBV and lamivudine-resistant mutants to MCC-478 by a transient transfection of full-length HBV DNA into human hepatoma cells.

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MATERIALS AND METHODS Cells. The human hepatoma cell line HuH-7 (18) was obtained from the Human Science Research Resource Bank (Osaka, Japan) and was cultured in Dulbecco's modified Eagle's medium (Invitrogen, Groningen, the Netherlands) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 atmosphere.

HBV DNA. HBV DNA was amplified and cloned as described previously (22). In short, HBV DNA was extracted from the serum of a patient with hepatitis B e antigen-positive cirrhosis. Then, HBV DNA was amplified by PCR according to the method described by Günther et al. (7). The PCR product was digested with ScaI and then cloned into pBluescript II SK+ (Stratagene, La Jolla, Calif.). Three types of lamivudine-resistant mutants were created by substituting nucleotides to change the codon for methionine in the YMDD motif to isoleucine (M552I) or valine (M552V) and codon 528 for leucine in the B-domain motif to methionine (L528M/M552V) by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described previously (23).

Transfection of full-length HBV DNA into HuH-7 cells. HuH-7 cells (80 to 90% confluent in 60-mm-diameter dishes) were transfected with 0.9 μg of HBV DNA by using Effectene transfection reagent (Qiagen, Hilden, Germany) and incubated for 24 h. Then, cells were exposed to 0 to 10 μM of MCC-478 (a gift from Mitsubishi Pharma Corporation, Osaka, Japan) (Fig. 1) for 3 days. Cells were rinsed three times with ice-cold phosphate-buffered saline and then harvested. Transfection efficiency was monitored by cotransfecting 0.1 μg of β-galactosidase expression plasmid pCMVβ (Clontech Laboratories Inc., Palo Alto, Calif.). HuH-7 cell extracts were assayed for β-galactosidase as described previously (22). Experiments were performed at least in duplicate.

[upload=jpg]uploadimages/200410/2004102612161150.jpg[/upload]

FIG 1. Chemical structures of MCC-478 (A) and adefovir (B).

Isolation of HBV DNA from transfected cells. HBV DNA was purified from intracellular core particles by a method described by Günther et al. (7) with minor modifications. Briefly, cells were suspended in 500 μl of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1% NP-40; transferred to an Eppendorf tube; vortexed; and allowed to stand on ice for 15 min. Nuclei were pelleted by centrifugation at 4°C and 15,000 × g for 1 min. The supernatant was transferred to a new tube, the MgCl2 concentration was adjusted to 10 mM, and the DNA was digested with 100 μg of DNase I/ml for 30 min at 37°C. To stop the reaction, EDTA was added to a final concentration of 25 mM. Then, 0.5 mg of proteinase K/ml and 1% sodium dodecyl sulfate were added and samples were incubated at 50°C for 4 h. Phenol-chloroform (1:1) extraction was performed, and the nucleic acids were ethanol precipitated along with a glycogen carrier.

Southern blot hybridization of HBV DNA. HBV DNA was resolved on 1.5% agarose gel, transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Piscataway, N.J.) by Southern blotting, and hybridized with an alkaline-phosphatase-labeled full-length HBV DNA probe generated with the Gene Images AlkPhos Direct labeling system (Amersham Pharmacia Biotech). Chemiluminescent detection was performed with CDP-Star (Amersham Pharmacia Biotech) and analyzed by using a LAS1000 image analyzer (Fuji Photo Film, Tokyo, Japan).

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RESULTS Wild-type HBV is susceptible to MCC-478. To assess the effect of MCC-478 on wild-type HBV replication in vitro, HuH-7 cells transfected with wild-type HBV DNA were incubated with different concentrations (0 to 10 μM) of MCC-478. Southern blot hybridization of DNA extracts showed a single-stranded band (representative of the HBV replicative intermediates) in the drug-free sample (Fig. 2). This band diminished with increasing concentrations of MCC-478, indicating that wild-type HBV was susceptible to MCC-478. We determined the inhibition of wild-type HBV DNA synthesis by using 0, 0.01, 0.1, 1, and 10 μM concentrations of MCC-478 and then calculated the effective concentration that inhibited HBV replication by 50% (EC50) (Table 1). Comparing the EC50s, MCC-478 was about 20 times more potent than lamivudine (Table 1).

Lamivudine-resistant HBV is susceptible to MCC-478. To analyze the in vitro antiviral effect of MCC-478 on lamivudine-resistant HBV, HuH-7 cells transfected with M552I, M552V, and L528M/M552V HBV mutants were incubated with different concentrations (0 to 10 μM) of MCC-478. Southern blot hybridization of DNA extracts showed a single-stranded band (representative of the HBV replicative intermediates) in the drug-free samples (Fig. 2). This band diminished with increasing concentrations of MCC-478 in the M552I, M552V, and L528M/M552V samples, indicating that all three lamivudine-resistant HBV mutants were susceptible to the drug. We determined the inhibition of lamivudine-resistant HBV DNA synthesis by using 0, 1, 3, 8.7, and 10 μM concentrations of MCC-478 and calculated the EC50s (Table 1). Although MCC-478 inhibited the replication of all three lamivudine-resistant mutants, a much higher dose of the drug (about 100 times more) was necessary to inhibit the replication of mutants than was required for wild-type HBV inhibition (Table 1).

[upload=jpg]uploadimages/200410/20041026121746101.jpg[/upload]

FIG 2. Susceptibility of wild-type HBV and lamivudine-resistant mutants to MCC-478. (A) Southern blot hybridization analysis of HBV replication. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with full-length HBV DNA with indicated concentrations of MCC-478. Arrow indicates the single-stranded band. OC, open circular HBV DNA; DS, double-stranded HBV DNA; SS, single-stranded HBV DNA. (B) Diagram comparing the susceptibility (means ± standard deviations) of wild-type HBV and M552I, M552V, and L528M/M552V mutants to MCC-478 at the indicated concentrations. Experiments were performed two to four times. Single-stranded bands were quantified using a LAS1000 fluorescent image analyzer (Fuji Photo Film) and normalized for transfection efficiency based on β-galactosidase activity. Intensities are given on the left of the graphs. The intensity of the single-stranded band of control (0 μM) was defined as 100. The discovery of lamivudine was a breakthrough for the treatment of hepatitis B. Clinical trials show that a 1-year course of lamivudine antiviral therapy results in the suppression of viral replication and substantial histological improvement in patients with chronic hepatitis B (12). Moreover, a recent study revealed that a 2-year course of lamivudine therapy is well tolerated and more efficacious than a 1-year course (14). In fact, a 2-year course resulted in incremental HBeAg seroconversion from 17% at week 52 to 27% at week 104 (14). However, 38% of patients receiving a 2-year course developed drug-resistant mutant HBV compared to 14% of those receiving a 1-year course (12, 14). The longer treatment with lamivudine is continued, the more frequently drug-resistant HBV appears.

Lamivudine-resistant viruses harbor substitutions from methionine (M) to isoleucine (I) or valine (V) in the YMDD motif in the C domain of the polymerase, and the M552V mutant contains a substitution from leucine (L) to M in the B domain (L528M) (1, 16, 26). L528M has been associated with famciclovir resistance (17, 25). It was previously shown that the L528M mutation cooperates with the M552V mutation, increasing HBV replication and drug resistance to entecavir, L-D4FC, and L-FMAU (21).

The in vitro full-length HBV DNA transfection system used in this study is suitable for evaluating antiviral agents, especially those that inhibit the DNA polymerase of this virus (21-23). This system is advantageous, since it facilitates the testing of various antiviral agents against different HBV mutants. Using this system, we demonstrated that adefovir could be a good treatment option for patients who fail lamivudine therapy due to the emergence of resistant virus (23). In fact, adefovir dipivoxil (the oral prodrug of adefovir) was shown to be effective against lamivudine-resistant mutants in vivo (24). Therefore, in this study, we tried to evaluate a novel reverse transcriptase inhibitor, MCC-478, against wild-type and lamivudine-resistant HBV mutants by using this system. MCC-478 is effective against not only wild-type HBV but also lamivudine-resistant mutants. Moreover, MCC-478 was equally effective against M552I, M552V, and L528M/M552V. However, it must be noted that the dose required to inhibit the replication of the lamivudine-resistant mutants was 100 times higher than that for the wild-type virus.

By using this system, the effects of 15 reverse transcriptase inhibitors (including MCC-478) against wild-type and lamivudine-resistant HBV mutants were previously evaluated (21, 23). Of 15 antiviral agents, 8 were effective against wild-type HBV. Comparing the EC50s of the eight antiviral agents, MCC-478 (0.027 μM) was the second most potent next to entecavir (0.00036 μM) (21). Only four (adefovir, lobucavir, entecavir, and MCC-478) were effective against all three types of lamivudine-resistant HBV, suggesting that lamivudine-resistant mutants are cross resistant to other nucleoside analogues. Because clinical studies of lobucavir were suspended due to oncogenicity in rodents (Bristol-Myers Squibb, New York, N.Y. [http://www.bms.com/news/press/data/fg_press_release_824.html]), MCC-478 has been one of three valuable candidates used to treat lamivudine-resistant HBV infection so far. It is also worth noting that the EC50s of MCC-478 (2.6 μM for M552I, 3.3 μM for M552V, and 2.0 μM for L528M/M552V) against lamivudine-resistant HBV were lower than those of adefovir (4.5 μM for M552I, 4.9 μM for M552V, and 2.2 μM for L528M/M552V) (23).

Although results from clinical trials of MCC-478 are not yet available, human study data are accumulating on adefovir. A recent phase III clinical study showed that no HBV mutations associated with adefovir resistance could be identified at week 48 (C. E. Westland, H. Yang, W. E. Delany IV, C. S. Gibbs, M. D. Miller, R. Fallis, J. Fry, C. L. Brosgart, H. Namini, and S. Xiong, Abstr. 52nd Ann. Meet. Am. Assoc. Study Liver Dis., abstr. 1099, 2001). It is possible that MCC-478 resistance is also difficult to develop because MCC-478 and adefovir had similar drug resistance profiles in our study. The most common toxicity associated with adefovir is nephrotoxicity manifested by an elevation in serum creatinine levels, which occurs in 35% of patients treated with 120 mg of adefovir/day (10). Moreover, dose-related decreases in serum carnitine levels have been noted in treated patients due to renal excretion of carnitine, although clinical significance of this biochemical change is uncertain (5). Therefore, 10- and 30-mg/day doses for hepatitis B treatment are being evaluated in clinical trials with adefovir. MCC-478 toxicity in humans should be carefully examined, although MCC-478 had no cytotoxicity up to 1,000 μM in HB611 cells (27), a human hepatoma-derived cell line in which the wild-type HBV genome is integrated and continuously produces HBV (N. Kamiya, A. Kubota, Y. Iwase, K. Sekiya, M. Ubasawa, and S. Yuasa, submitted for publication).

Optimal antiviral therapy should provide sufficient suppression of HBV replication. Treatment strategies that are safe and suitable for long-term use and suppress the emergence of drug-resistant strains are urgently needed. As shown in human immunodeficiency virus treatment, the goal may be reached by using combinations of anti-HBV drugs. It was previously shown that a combination of lamivudine and penciclovir has synergistic effects against HBV in vitro (4, 11). In clinical trials, hepatitis B patients treated with lamivudine in combination with famciclovir (the oral form of penciclovir) showed better inhibition of viral replication than patients treated with lamivudine alone (13). These results suggest that the combination of lamivudine with adefovir, entecavir, and MCC-478 are advantageous because their drug resistance profiles differ from that for lamivudine. Our study indicates that MCC-478 is a potential antiviral agent for hepatitis B treatment and deserves further investigation.

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Acknowledgments This study was supported by Health Sciences Research Grants for Medical Frontier Strategy Research from the Ministry of Health, Labor, and Welfare of Japan and by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

We thank the Mitsubishi Pharma Corporation for the donation of MCC-478. We also thank Mitsuko Tsubouchi for technical assistance.

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