Hepatitis B Virus Drug Candidate MCC478 Toxicity Studies Early New Esters Antibody

Viral hepatitis type B is caused by hepatitis B virus (HBV). It is a serious hazard to human health with a high incidence and is difficult to cure and easy to recur which has drug resistance.Recently, the main treatment for viral hepatitis type B is pharmacotherapy, drugs including the active substances of natural polysaccharides, interferon (IFN) alpha and nucleotide analogues (NUCs). The NUCs have a widely application, eg. Anti-HBV drugs lamivudine, adefovir dipivoxil, entecavir entecavir, tenofovir disoproxil, etc. Lamivudine is in need of long-term administration, but is easy to develop the drug resistance; adefovir dipivoxil is the most commonly anti-HBV drugs, the cells do not need phosphorylation, there is no cross-resistance with lamivudine, and itself is not easy to develop drug resistance,but it has a dose-limiting-dependent renal toxicity; tenofovir disoproxil is currently the most effective anti-HBV drugs, it has a patent protection in China, has not been listed,it is low bioavailability, and high cost. Adefovir dipivoxil and tenofovir disoproxil are active ester prodrug, and rapidly hydrolyzed in plasma after into the body, they have the poor chemical stability.The MCC478ester is a new type of chemical structure of nucleotides anti-HBV drugs, and it has a highly HBV selective inhibition against lamivudine resistant HBV strains (YMDD mutation) and the liver. The MCC478ester of the antiviral activity is100times o the MCC478’s. MCC478ester will form the toxic metabolic intermediates, but the complete hydrolysis of the nucleotide will avoid the toxicity of metabolic intermediates. In addition, through the formation of fumaric acid salt to improve the stability and water solubility.It is known that the pharmacology, pharmacokinetics, pharmacy and efficacy superiority of the MCC478ester, then the study will investigate the toxicity and the secure of it.The contents and the results of the researches are showed as follow:Derek software was to perform the toxicology prediction analysis, the results are that the MCC478ester has carcinogenicity, inhibition of cholinesterase, neurotoxicity, and acute toxic nephropathy, skin sensitization, white blood cells and immune cumulative toxicity, chromosomal damage, and toxicity of metabolic intermediates predicted may occur.MCC478, MCC478new ester had no liver toxicity in rats, dogs, and humans.MCC478new ester in vitro cytotoxicity test results are as follows:the impact of the the MCC478new esters on HepG2cells and HK-2cells, the cell growth of the control group, and the stretching is good, the treatment group occurred in varying degrees of cell wrinkle contraction, cell vacuolization, the death rate of HepG2cells and HK-2cells increased, and was dose-dependent.Follows the result of inhibition of cell proliferation by MTT assay determination MCC478esters:MCC478ester, and MCC478could inhibit cell proliferation, with IC50values were:MCC478ester113.68μM MCC478for638.20μM; the MCC478active product PMPA monoester and toxic metabolic intermediates were able to inhibit cell proliferation, the inhibitory rate did not reach50%. Therefore, the order of toxicity was MCC478> monoester> PMPA.Ames fluctuation test of the MCC478ester mutagenicity results are as follows: cyclophosphamide and sodium azide as a plus S9and without S9under the conditions of positive control, the results with the solvent control group had a significant difference that in this experimental system is authentic. With and without S9, there was no significantly difference between the MCC478and MCC478ester, and control group, suggesting that the MCC478and MCC478esters had no mutagenesis in the TA100strains.In the in vivo acute toxicity up and down method:adefovir dipivoxil orally in rats exposed to LD50=1296mg/kg (♀),95%CI (760～2000mg/kg); the MCC478ester in rats exposed to LD50>2000mg/kg (♀); acute toxicity grading standards are the toxic substances, and between, the MCC478ester LD50greater than adefovir dipivoxil, indicating that the acute toxicity of the MCC478ester was lower than adefovir dipivoxil. Manifestations of toxicity after exposure:listlessness, piloerection; corner of the eye secretions, perianal filthy, diarrhea, opisthotonos. To observe the MCC478new ester repeat toxicity characteristics gavage MCC478ester14days of repeated exposure toxicity test, were randomly divided into the solvent control group, the MCC478new esters of low-dose35mg/kg, in doses of70mg/kg, high dose140mg/kg, positive control adefovir100mg/kg group. The results showed that:As the dose increasing, the body weight of animals which had a treatment of MCC478ester reduced compared with the control group, and there was no significantly difference; and the food intake of low-dose group was slightly higher than control group, the food intake of the high dose group compared with control group reduced, but were not statistically different, implying MCC478ester had no significant effect on food intake in rats.MCC478new esters of low three dose groups with the dose increased, liver coefficient showed a downward trend, while the positive control group of adefovir dipivoxil in liver coefficient slightly higher than the solvent control group and there was no significant difference. The kidneys coefficient of MCC478esters of high dose group increased as well as the positive control group and the difference was statistically significant. Histopathological findings:the MCC478esters of low-dose of35mg/kg group, the dose of70mg/kg group, high dose of140mg/kg group, both show a normal kidney structure; adefovir100mg/kg positive control group showed the renal proximal tubule epithelial cells have a mild injury.35mg/kg of low-dose group, middle dose group70mg/kg show the normal structure of liver cells and liver tissue;140mg/kg of high-dose group show mild damage of the liver cell swelling; adefovir100mg/kg of the positive control group showed liver tissue normal structure of low-dose group35mg/kg dose group,70mg/kg to140mg/kg of the high dose group show normal small intestinal structure; adefovir100mg/kg of the positive control group showed small intestinal mucosal layer atrophy and intestinal cell damage. Lymphoid tissue the structure of each dose group was not injury.The conclusions of the study are showed as follows:1. MCC478esters have high antiviral activity;2. There was low acute toxicity of MCC478ester, but a liver toxicity in chronic treatment of high dose;3. The sensitive and specific biological markers suggest MCC478ester may have some renal toxicity;4. The reference drug Adefovir was manifested with some renal toxicity, which is in agreement with literatural reports and suggesting the validation of the test systems.5. Both MCC478and MCC478ester have no mutagenicity with or without S9;6. There were a good agreement between the in vitro and in vivo test results and the prediction of the Derek software.7. The toxicological profiles of MCC478ester was comparable with or superior to these for MCC478, which suggested the drugablility of the MCC478ester with aspect to toxicology.