Development of Novel Agents Active against Hepatitis B Virus

开发抗乙型肝炎病毒的新型药物

• 批准号: 8763559
• 负责人: Hiroaki Mitsuya
• 金额: $ 14.84万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763559
• 关键词: 2' -deoxyadenosine; Acute Hepatitis; Adverse effects; Alcohols; Antiviral Therapy; Biological Assay; Cardiomyopathies; Cell Line; Cells; Cessation of life; Chronic Hepatitis; Chronic Hepatitis B; Clinical; Clinical Trials; Complication; Cytarabine; DNA; DNA Polymerase Inhibitor; DNA biosynthesis; Data; Defect; Development; Didanosine; Dose; Double Stranded DNA Virus; Drug Controls; Drug resistance; Drug usage; Enzymes; Equipment and supply inventories; Exocrine pancreas; FDA approved; Failure; Family; Freezing; Generations; Genetic; HIV; HIV-1; Hepadnaviridae; Hepatitis B Vaccines; Hepatitis B Virus; Homosexuals; Hospitalization; In Vitro; Inhibitory Concentration 50; Lactic Acidosis; Lamivudine; Lead; Liver Cirrhosis; Liver Failure; Malignant Neoplasms; Measures; Mitochondria; Mitochondrial DNA; Myopathy; Neuropathy; New Agents; Nucleosides; Nucleotides; Pancytopenia; Patients; Pharmaceutical Preparations; Plasmids; Polymerase; Primary carcinoma of the liver cells; Public Health; Publishing; Reaction; Reporting; Residual state; Resistance; Reverse Transcriptase Inhibitors; Ribavirin; Sampling; Structure; System; Telbivudine; Tenofovir; Testing; Therapeutic; Time; Tobacco; Toxic effect; Toxicity Tests; Variant; Vidarabine; Virus; Virus Diseases; Zalcitabine; Zidovudine; abacavir; adefovir; anti-hepatitis B; antiretroviral therapy; chemotherapy; cytotoxicity; dideoxythymidine; drug testing; emtricitabine; entecavir; inhibitor/antagonist; insight; intravenous drug user; men; novel; nucleoside analog; response; viral DNA

1. Anti-HIV and anti-HBV activity of tested compounds Since published and our own data indicate that certain NRTIs are potent against HIV but not potent against HBV, and other NRTIs are, conversely, not potent against HIV-1 but highly potent against HBV, we surmised that we might find NRTIs that are not potent against HIV (therefore, they were put aside, frozen in the freezer, and "untouched' since then) but potent against HBV in our inventory that has been accumulated from 1980s to present. It is hoped that some of such NRTIs might serve as "lead compounds" to synthesize and identify anti-HBV candidates. In the present study, we comparatively determined anti-HIV and anti-HBV activity of selected nucleoside/nucleotide reverse transcriptase inhibitors (NRTI/NtRTIs). First, we chose 15 drugs, which are nucleoside/nucleotide reverse transcriptase inhibitors and are in clinical use or under clinical trials as anti-HIV-1 and/or anti-HBV NRTIs. They include zidovudine (AZT), emtricitabine (FTC), didanosine (ddI), sanilvudine (d4T) and abacavir (ABC), which are approved drugs as anti-HIV drug. Entecavir (ETV), lamivudine (3TC), tenofovir (TDF), adefovir pivoxil (ADV), and telbivudine (LdT), which are FDA-approved anti-HBV drugs, were laso examined. 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), zalcitabine (ddC), 2', 3'-dideoxyguanosine (ddG), 2', 3'-dideoxythymidine (ddT), are under or had been clinical trials as anti-HIV drugs. Among 15 drugs, 13 drugs had detectable activity against wild type HIV in MTT assay. Among such active drugs, EFdA exerted most potent activity, against HIV and Ed4T, a recently reported new generation anti-HIV NRTI, also exerted potent activity against HIV. TDF, ADV, and AZT also showed substantial activity against HIV with IC50 values of less than 100 nM. On the other hand, telbivudine (LdT), an FDA-approved HBV therapeutic, had no anti-HIV activity when we tested up to 10 microM. However, it is of note that the ETV had relatively poor anti-HIV activity with IC50 value of 375 nM. When we further conducted multiple anti-HIV assays using p24 assay, MAGI assay, and MTT assay with higher TCID50. In all assays, EFdA revealed the most potent anti-HIV activity with IC50 values ranging 0.8 to 3.3 microM, but ETV showed only poor anti-HIV activity in MAGI assay and MTT assay when high titer HIV was used. We then evaluated anti-HBV activity of selected inhibitors using 2.2.15 cell line by measuring changes of intracellular HBV-DNA level. We employed 2.2.15 cells for the assay and the real-time PCR to detect the level of viral DNA in the assay samples. Intracellular DNA containing HBV-DNA was isolated from cells after 12-days of assay, and CT (cycle threshold) values for each reaction was obtained. Standard reaction using diluted HBV-plasmid (C_AT) was also obtained simultaneously and CT values of each sample were converted to an HBV copy number using the standard curve. The well without the drug (control) had 1,670,000 copies of HBV, but in the presence of different concentration of ETV, the copy numbers decreased in a dose-response manner; with 1000 nM of ETV, the copy number decreased to 162,000 per well which is more than 1-log and significant decrease compared to that of control with 1,000 nM, 100 nM, 10 nM (p 0.005) and 1 nM (p 0.01). Most of the tested NRTI/NtRTIs gave dose response reductions in the viral DNA. For TDF, in the presence of 100 nM, viral DNA level in the cell decreased by more than 50% compared to that for no drug control. However, residual viral DNA was detected (515% compared to that for no drug control) even at the concentration of 10 microM and this finding was seen in all drugs tested. This residual DNA seems to represent the integrated HBV DNA. Lamivudine (3TC) had IC50 of 22.4 nM. TDF showed potent activity against both HIV and HBV with IC50 numbers less than 100 nM although its activity against HBV is 65-fold lower than that of ETV. ddI and ddC had anti-HBV activity in our assay system, but we could not detect activity of AZT against HBV in our system in which we employed drug concentration up to 10 microM. Ed4T showed essentially no activity against HBV, however, EFdA had moderate anti-HBV activity. 2. Cytotoxicity and mitochondrial toxicity of NRTI/NtRTIs. It is known that mitochondrial DNA (mtDNA) defects can be acquired by toxic agents such as tobacco, alcohol and drugs. NRTIs used in chemotherapy or antiviral therapy have been shown to induce mitochondrial toxicity not only anti-HIV NRTIs but also cytarabine, vidarabine, aciclovir, and ribavirin. These nucleoside analogs elicit complete mtDNA replication deficits due to their ability to inhibit polymerase-gamma the enzyme responsible for replication of mtDNA . Clinical features can be regarded as a complication of those seen in the genetic mitochondriocytopathy, such as myopathy, cardiomyopathy, neuropathy, lactic acidosis, exocrine pancreas failure, liver failure, and bone marrow failure. Some of the early generation NRTIs, such as ddC and d4T are known to reduce the mtDNA in vitro, we tried to establish the system to assess the mitochondrial toxicity of tested NRTIs. We employed Hep-G2 cells for the assay and the real-time PCR to detect the level of mtDNA in the assay samples. Intracellular DNA was isolated from cells after 12-days of assay, served for real-time PCR, and reduction of mtDNA has been compared from the cells with no drugs added. 3. The novel agents against anti-HIV and anti-HBV derivatives of EFdA. We also identified some derivatives of EFdA, YMS-01144 and YMS-01145 . They had very potent anti-HIV and anti-HBV activity with IC50 values for 0.15 and 0.21 nM, 0.37 nM and 0.39 nM, respectively. These values were as potent as EFdA for HIV and as potent as ETV for HBV. It is expected that considering the similarity of the structures of these derivatives, we can generate compounds that are potent against both HIV and HBV through redesign.

1. 测试化合物的抗 HIV 和抗 HBV 活性 自发表以来和我们自己的数据表明，某些 NRTI 对 HIV 有效，但对 HBV 无效，而其他 NRTI 则相反，对 HIV-1 无效，但对 HIV-1 有效。 HBV，我们推测，我们可能会发现 NRTI 对 HIV 无效（因此，它们被放在一边，冷冻在冰箱中，此后“未曾触及”），但在我们的库存中对 HBV 有效从 20 世纪 80 年代至今，我们已经积累了一些 NRTI 化合物，希望能够作为“先导化合物”来合成和鉴定抗 HBV 候选药物。在本研究中，我们比较测定了选定的抗 HIV 和抗 HBV 活性。核苷/核苷酸逆转录酶抑制剂（NRTI/NtRTIs）首先，我们选择了15种药物，它们是核苷/核苷酸逆转录酶抑制剂，作为抗HIV-1已在临床使用或正在进行临床试验。它们包括齐多夫定 (AZT)、恩曲他滨 (FTC)、去羟肌苷 (ddI)、沙尼夫定 (d4T) 和阿巴卡韦 (ABC)，这些药物均被批准作为抗 HIV 药物。对 FDA 批准的抗 HBV 药物恩替卡韦 (ETV)、拉米夫定 (3TC)、替诺福韦 (TDF)、阿德福韦酯 (ADV) 和替比夫定 (LdT) 进行了激光检查。 4'-乙炔基-2-氟-2'-脱氧腺苷 (EFdA)、2',3'-二脱氢-3'-脱氧-4'-乙炔基胸苷 (Ed4T)、扎西他滨 (ddC)、2',3'-二脱氧鸟苷(ddG)、2', 3'-二脱氧胸苷 (ddT) 正在或已经进行过临床试验抗艾滋病毒药物。在15种药物中，13种药物在MTT试验中可检测到抗野生型HIV活性。在这些活性药物中，EFdA 发挥了最有效的抗 HIV 活性，Ed4T（最近报道的新一代抗 HIV NRTI）也发挥了有效的抗 HIV 活性。 TDF、ADV 和 AZT 还表现出显着的抗 HIV 活性，IC50 值小于 100 nM。另一方面，FDA 批准的 HBV 治疗药物替比夫定 (LdT) 在我们测试浓度高达 10 microM 时没有抗 HIV 活性。但值得注意的是，ETV 的抗 HIV 活性相对较差，IC50 值为 375 nM。当我们进一步使用 p24 检测、MAGI 检测和具有较高 TCID50 的 MTT 检测进行多次抗 HIV 检测时。在所有测定中，EFdA 显示出最有效的抗 HIV 活性，IC50 值范围为 0.8 至 3.3 microM，但当使用高滴度 HIV 时，ETV 在 MAGI 测定和 MTT 测定中仅显示出较差的抗 HIV 活性。然后，我们通过测量细胞内 HBV-DNA 水平的变化，使用 2.2.15 细胞系评估了所选抑制剂的抗 HBV 活性。我们使用 2.2.15 细胞进行测定，并使用实时 PCR 来检测测定样品中的病毒 DNA 水平。检测 12 天后，从细胞中分离出含有 HBV-DNA 的细胞内 DNA，并获得每次反应的 CT（循环阈值）值。同时还获得了使用稀释的 HBV 质粒 (C_AT) 的标准反应，并使用标准曲线将每个样品的 CT 值转换为 HBV 拷贝数。未加药的孔（对照）有1,670,000个HBV拷贝，但在不同浓度的ETV存在下，拷贝数呈剂量反应性下降；使用 1000 nM ETV 时，拷贝数降至每孔 162,000 个，与 1,000 nM、100 nM、10 nM (p < 0.005) 和 1 nM (p < 0.01) 的对照相比，拷贝数显着下降，超过 1 个对数。大多数测试的 NRTI/NtRTI 都会降低病毒 DNA 的剂量反应。对于 TDF，在 100 nM 存在下，细胞中的病毒 DNA 水平与无药物对照相比下降了 50% 以上。然而，即使在 10 µM 的浓度下，也检测到残留病毒 DNA（与无药物对照相比为 515%），并且这一发现在所有测试的药物中均可见。这种残留的 DNA 似乎代表了整合的 HBV DNA。拉米夫定 (3TC) 的 IC50 为 22.4 nM。 TDF 对 HIV 和 HBV 均表现出有效的活性，IC50 值低于 100 nM，尽管其对 HBV 的活性比 ETV 低 65 倍。 ddI 和 ddC 在我们的测定系统中具有抗 HBV 活性，但在我们使用高达 10 microM 的药物浓度的系统中，我们无法检测到 AZT 抗 HBV 的活性。 Ed4T 基本上没有表现出抗 HBV 活性，但 EFdA 具有中等抗 HBV 活性。 2. NRTI/NtRTIs 的细胞毒性和线粒体毒性。众所周知，烟草、酒精和毒品等有毒物质会导致线粒体 DNA (mtDNA) 缺陷。化疗或抗病毒治疗中使用的 NRTI 已被证明不仅会引起抗 HIV NRTI 的线粒体毒性，而且还会引起阿糖胞苷、阿糖腺苷、阿昔洛韦和利巴韦林的线粒体毒性。这些核苷类似物由于能够抑制聚合酶-γ（负责 mtDNA 复制的酶）而导致 mtDNA 复制完全缺陷。临床特征可被视为遗传性线粒体细胞病的并发症，如肌病、心肌病、神经病、乳酸性酸中毒、外分泌胰腺衰竭、肝衰竭和骨髓衰竭。一些早期的 NRTIs，如 ddC 和 d4T 已知会在体外减少 mtDNA，我们试图建立一个系统来评估测试的 NRTIs 的线粒体毒性。我们使用 Hep-G2 细胞进行测定，并使用实时 PCR 来检测测定样品中 mtDNA 的水平。检测 12 天后，从细胞中分离出细胞内 DNA，用于实时 PCR，并与未添加药物的细胞中 mtDNA 的减少进行了比较。 3. EFdA 的抗 HIV 和抗 HBV 衍生物的新型药物。我们还鉴定了 EFdA、YMS-01144 和 YMS-01145 的一些衍生物。它们具有非常有效的抗 HIV 和抗 HBV 活性，IC50 值分别为 0.15 和 0.21 nM、0.37 nM 和 0.39 nM。这些值对于 HIV 而言与 EFdA 一样有效，对于 HBV 而言与 ETV 一样有效。考虑到这些衍生物结构的相似性，我们预计可以通过重新设计产生对 HIV 和 HBV 均有效的化合物。