MCC-478 Derivatives Anti-HBV In Vitro And Vivo

Hepatitis B virus (HBV) infection is a major health problem around the world. Among the 350 million people with chronic HBV infection, the risk of dying from HBV-related complications is between 15% to 25%. Several nucleoside analogs are under clinical development for use against hepatitis B virus. Lamivudine (3TC), adefovir (ADV) and entecavir (ETV) are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new safe and effective antiviral agents with novel mechanisms of action. MCC-478 is a 2-amino-6-arylthio-9- phosphonomethoxyethylpurine bis (2, 2, 2-trifluoroethyl) ester derivative. MCC-478 showed a substantially higher (ca.20-fold) and special anti-hepatitis B virus activity than that of lamivudine, despite no significant anti-human immunodeficiency virus (HIV) activity. A library of more than 100 derivatives of MCC-478 synthesized by parallel synthesis was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15.Through an iterative process of synthesis, lead optimization, and screening, three analogs (030605, 030705, 030703, 030717) with specific structure were identified as potent inhibitor of HBV replication. The aims of this study were to evaluate 1) the anti-HBV activities, inhibitory effects of correlated protein secretion, cytotoxicity and reduction of the mitochondrial DNA content of MCC-478 analogs in vitro; and to observed 2) anti-duck hepatitis B virus (DHBV) activities, drug toxicity and inflammatory changes of hepatic tissue of MCC-478 analogs in vivo.Anti-HBV activities of test compounds were evaluated in vitro using HBV-producing cell line HepG2.2.15. 2.2.15 cells (5×104 cells/well) were plated on 24-well plates. After incubation for 24h, the 2.2.15 cells were treated with medium containing different concentrations of test compounds (0.01uM,0.03uM, 0.1uM, 0.3uM, 1.0uM) and Adefovir Dipioxil (0.1uM, 0.3uM, 1.0uM, 3.0uM, 10.0uM) for 9 days. The drug-containing medium changed every other day, the media and monolayers were collected for test. Anti-HBV activities were evaluated by real-time quantitive PCR assay and Southern blot hybridization. Extracellilar HBV DNA analysis was evaluated by real-time quantitive PCR assay. Whereas, intracellular HBV DNA analysis was evaluated by Southern blot hybridization. HBV transcription analysis was evaluated by real-time quantitive RT-PCR assay. The inhibitory effects of HBeAg secretion were analyzed by ELISA method. The influence of different concentrations 030605 (0.1uM, 1.0uM, 10.0uM) to HBsAg and HBcAg were analyzed by Western blotting method. HepG2 cells (105 cells/ml) were plated in 96-well plates and treated with different concentrations of test compounds (10uM, 30uM, 100uM, 300uM, 1000uM) for 3 days. Then the cytotoxicity of test compounds was tested by MTT method. After HepG2 cells (5×104 cells/well) were plated in 12-well plate and treated with different concentrations of ddC and test compounds (0.1uM, 1.0uM, 10.0uM) for 8days, the mitochondrial DNA content was tested by Dot blot hybridization.Anti-duck hepatatitis B virus (DHBV) activities and toxicological profile of test compounds were investigated in vivo using the DHBV/duck animal model of chronic DHBV vertical infection. Twelve groups of five ducks were used. Two groups served as DHBV DNA(+) placebo control and DHBV DNA(-) placebo control, respectively. One group served as positive drug control (Adefovir Dipioxil 10mg/kg). Each test compound had three doses (50mg/kg, 10mg/kg, 2mg/kg) for therapy (one group of animals for each dose). Treatment was administered orally once daily for 4 weeks. Serum samples were taken for analysis prior to the initiation of treatment, at weeks 2, 4 of treatment, and 2 weeks after treatment. Liver biopsies were obtained at the end of therapy. Anti-duck hepatatitis B virus (DHBV) activities were tested by real-time quantitive PCR assay. The toxicological profile of MCC-478 analogs and liver inflammation changes were observed by optical and electron microscope.Test compounds 030705, 030605, 030703 and 030717 were discovered to have potent antiviral activity against extracellular HBV DNA in HepG2.2.15 cellsk, with 50% inhibitory concentration (IC50) of 0.82uM, 0.29uM, 0.61uM, 0.16uM, respectively. Comparison of the antiviral activity of test compounds with lamivudine (IC50=0.10uM) and Adefovir Dipioxil (IC50=0.58uM), demonstrated their superior potency in vitro. Intracellular HBV replicative intermediates (RI) were uniformly reduced when cells were treated with test compounds. Compared with Adefovir Dipioxil (IC50=0.48uM), 030705, 030605, 030703 and 030717 had the 50% inhibitory concentration of 0.93uM, 0.62uM, 0.15 uM, 0.44uM, against HBV replicative intermediates. There were no apparent inhibitory effects on HBV transcription. And no inhibitory effects of HBeAg secretion from the test compounds were observed in HepG2.2.15 cells (P>0.05). No direct influence were observed in the test compound 030605 to HBsAg and HBcAg. The concentrations of 030705, 030605, 030703 and 030717 causing 50% cytotoxicity (CC50) value were all more than 1000uM in HepG2 cells (2014uM, 1117uM, 3912uM, 1114 uM, respectively). And no apparent reductions of mitochondrial DNA content by test compounds were observed, whereas 2′, 3′-dideoxycytidine (ddC) as a positive control reduced mitochondrial DNA content to nearly half that of the untreated control.The groups of higher,moderate and lower doses of drug 030705 had 3 (60%), 2 (40%) and 0 (0%) ducks which DHBV DNA level fell 3-fold greater than that of pretreatment, respectively. Compared with control group, the group of higher doses had significant difference (X2=4.28, P<0.05). The groups of moderate and lower doses had no significant difference (X2=2.50, P>0.05). The groups of higher, moderate and lower doses of drug 030605 had 4 (80%), 3 (60%) and 1 (20%) ducks which DHBV DNA level fell 3-fold greater than that of pretreatment, respectively. Compared with control group, the groups of higher and moderate doses groups had significant difference (X2=6.67, P<0.01; X2=4.28, P<0.05). The groups of lower doses had no significant difference (X2=1.11, P>0.05). The groups of higher,moderate and lower doses of drug 030703 had 3 (60%), 2 (40%) and 1 (20%) ducks which DHBV DNA level fell 3-fold greater than that of pretreatment, respectively. Compared with control group, the group of higher doses had significant difference (X2=4.28, P<0.05). The groups of moderate and lower doses had no significant difference (X2=2.50, P>0.05;χ2=1.11, P>0.05). Three test compounds all had some antiviral activity in vitro. Antiviral activity of 030605 was superior to that of two other drugs, however DHBV DNA rebound was observed following the cessation of therapy in its group of higher doses.No pathological toxicity was observed in all drug-treating groups. Under electron microscope, alleviation of liver inflammation resulted from the treatment of all three drug. Among them, pharmic effects of 030605 was superior to that of Adefovir Dipioxil. And pharmic effects of two other drugs were similar to that of Adefovir Dipioxil. This implied the improvement of liver inflammation resulted from the inhibition of viral replication indrectly.Our preliminary study indicated that four novel nucleoside analogs 030705, 030605, 030703 and 030717 (in vivo) exhibited high efficient activity to inhibit HBV/DHBV replication with low toxicity in vitro and vivo, suggesting they may be new promising anti-HBV agent.