96 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 Anti-HBV effect of nucleotide analogues on mouse model of chronic HBV infec

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1 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 95 Chinese Journal of Biotechnology January 25, 2013, 29(1): Chin J Biotech, All rights reserved 8 1,2* 2* 3 2, ,,,. 8., 2013, 29(1): Wang GJ, Wang G, Dong XY, et al. Assessment of anti-hbv effects of nucleotide analogues on mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8. Chin J Biotech, 2013, 29(1): : (raav8-1.3hbv) HBV raav8-1.3hbv 30 C57BL/6 HBV 27 6 (ETV) (LAM) 1 10 d 15 d ETV (1.0 mg/(kg d)) (0.1 mg/(kg d)) LAM (500 mg/(kg d)) (100 mg/(kg d)) HBV DNA HBeAg HBsAg 10 d HBV DNA (P 0.05) 15 d ETV LAM HBV DNA (P 0.05) HBeAg HBsAg ETV LAM HBV HBeAg HBsAg AAV8-1.3HBV HBV ETV LAM HBV HBV : raav8-1.3hbv Received: July 23, 2012; Accepted: October 27, 2012 Supported by: Special Key Program on Infectious Diseases of China (No. 2008ZX ), Major Program of New Drugs Creation and Technology (No. 2009ZX ). * These authors contributed equally to this study. Corresponding author: Xiaobing Wu. Tel: ; Fax: ; wuxb0168@vip.sina.com Hong Meng. Tel/Fax: ; menghlc@163.com (No. 2008ZX ) (No. 2009ZX )

2 96 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 Anti-HBV effect of nucleotide analogues on mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8 Guojing Wang 1,2, Gang Wang 2, Xiaoyan Dong 3, Wenhong Tian 2,4, Jie Yuchi 2, Guochao Wei 2, Hong Meng 1, and Xiaobing Wu 5 1 Institute of Basic Medical Science, Shandong Academy of Medical Science, College of Life Science, University of Jinan, Jinan , Shandong, China 2 Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing , China 3 Beijing Five Plus Molecular Medicine Institute, Beijing , China 4 School of Life Science, Jilin University, Changchun , Jilin, China 5 Beijing Yizhuang International Biomedical Investment & Management Co. Ltd., Beijing , China Abstract: We evaluated the anti-hbv effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (raav8-1.3hbv). Ninety percent (27 of 30 mice) of raav8-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kg d) or 0.1 mg/(kg d)) and LAM (500 mg/(kg d) or 100 mg/(kg d)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P<0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P<0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-hbv drug discovery. Keywords: raav8-1.3hbv, Hepatitis B virus, mouse model, nucleotide analogues, drug screening, drug evaluation (Hepatitis B virus HBV) DNA [1] 1/3 HBV HBV 3~4 25 ~40 HBV [2] HBV HBsAg 10 [3] HBsAg [4] [5] HBV [6] HBV HBV HBV

3 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 HBV (ayw ) (Recombinant adeno-associated virus type 8 carrying 1.3 copies of HBV genome (ayw), raav8-1.3hbv) C57BL/6 [7] HBV HBeAg HBsAg HBV DNA HBV HBV ( ) HBV DNA HBeAg HBsAg HBV ~6 C57BL/6 (HBV) (PCR- ) ( S ) HBeAg HBsAg HBeAg HBsAg Abbott ( H ) ( H ) 1.2 raav8-1.3hbv raav8-1.3hbv paav2neo-1.3hbv BHK μg/ml G d BHK21/AAV-1.3HBV rep2cap8 (MOI=1~5) BHK21/AAV-1.3HBV 72 h AAV [8] 1.3 raav8-1.3hbv 30 C57BL/ vg/ 200 μl/ d HBeAg HBsAg HBV DNA 1.4 PCR HBV DNA (HBV) (PCR) HBV DNA 1.5 ELISA HBeAg HBsAg HBeAg HBsAg HBeAg HBsAg OD cjb@im.ac.cn

4 98 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 40 Abbott HBeAg HBsAg HBeAg HBsAg HBeAg (S/CO) 1 HBsAg (miu/ml) HBV HBeAg HBsAg 42 ( 0 d) HBeAg HBsAg HBV DNA 30 raav8-1.3hbv HBV DNA IU/mL HBeAg HBsAg HBeAg HBsAg HBV DNA 27 (Untreated) 5 (NS) 4 ETV (1.0 mg/(kg d)) 5 ETV (0.1 mg/(kg d)) 4 LAM (500 mg/(kg d)) 5 LAM (100 mg/(kg d)) d ( 1~10 ) 1 15 d ( 11~25 ) t (0 d 10 d 25 d) NS HBV DNA t 1 Fig. 1 The schematic map of procedure for experiment. In the whole of experiment process, mouse model of HBV chronic infection was firstly established by injection of raav8-1.3hbv and determination of model. Then suitable models were chosen, different kinds of drugs were given and effects of drugs on model were assessed.

5 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 99 HBV DNA HBeAg HBsAg t (0 d 10 d 25 d) NS t SPSS raav8-1.3hbv 30 C57BL/ HBsAg 93.24±7.24 miu/ml miu/ml HBV DNA HBeAg HBsAg HBV DNA raav8-1.3hbv HBV ETV 10 d 15 d HBV DNA 1 ETV 10 d 30 HBeAg HBsAg HBeAg 30 HBsAg HBeAg HBsAg 42 HBV DNA HBeAg HBsAg 2 30 HBV DNA (lg (IU/mL)) ±0.34 HBeAg HBsAg 3 HBV DNA 11 HBeAg 2.00 S/CO HBsAg miu/ml 25 HBeAg 1.13 S/CO HBsAg 8.10 miu/ml 28 HBeAg 8.27 S/CO HBsAg miu/ml 27 HBeAg 13.22±1.78 S/CO 9.50 S/CO 2 HBV Fig. 2 Determination of mouse model of HBV chronic infection. Thirty mice were injected with raav8-1.3hbv. Forty-two days post-injection, HBsAg, HBeAg, and HBV DNA titer in sera were assayed. Mouse whose HBV DNA, HBsAg (40-fold diluted), and HBeAg (40-fold diluted) titer in sera was separately more than 5.00 (lg(iu/ml)), miu/ml, and 9.50 S/CO was determined as model. Twenty-seven mice were chose as models. The hollow circles indicated the mouse model of HBV chronic infection; the solid circles indicated the mice which were not determined as models. cjb@im.ac.cn

6 100 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 ETV HBV DNA (P 0.05) ETV ( 3) 5 d 0 d HBV DNA (lg (IU/mL)) 5.31± ±0.46 (P 0.05) 10 d 0 d 4.26±0.05 (P 0.05) 15 d 10 d HBV DNA 4.57±0.14 (P 0.05) HBV DNA ETV ( 1) ETV 5 d (P 0.01) 10 d (P 0.01) 15 d (P 0.01) ETV ( 3) 5 d 0 d HBV DNA (lg (IU/mL)) 5.36± ±0.18 (P 0.01) 10 d 0 d 1 ETV LAM raav8-1.3hbv HBV HBV DNA Table 1 HBV DNA titer in sera of HBV chronic infection mouse model mediated by raav8-1.3hbv at different time points treated with ETV or LAM Treatment Dose (mg/(kg d)) HBV DNA titer in sera ( lg(iu/ml)) 0 a 5 a 10 a 25 a Untreated 5.33± ± ± ±0.28 NS 5.39± ± ± ±0.14 ETV LAM ± ± ±0.05 *, 4.57± ± ±0.18 **, 3.75±0.21 **, 3.69±0.27 ** ± ± ±0.22 *, 5.10± ± ± ±0.32 *, 4.17±0.28 ** Twenty-seven mice models were divided into 6 groups. The two groups were set as untreated and normal saline (NS) control, respectively. The other four groups were separately treated with different doses of ETV or LAM. Different time points post-treatment (0, 5, and 10 d), HBV DNA titers in sera of mice were detected. HBV DNA titer was shown means±s.d (lg(iu/ml)). Data were analyzed using the independent-sample T test for comparison of different groups at the same time point or paired-sample T test for comparison of the same group at different time points. ETV, Entecavir; LAM, Lamivudine; NS, normal saline. a indicated the time treated by drug whose unit was d. * P<0.05,compared with NS controls at the same time point, from day 0 to day 25. ** P<0.01, compared with NS controls at the same time point, from day 0 to day 25. P<0.05, compared with day 0 value in the same group, from day 0 to day 10. P<0.01, compared with day 0 value in the same group, from day 0 to day 10. P<0.05, compared with day 10 value in the same group, at the day 25. P<0.01, compared with day 10 value in the same group, at the day 25.

7 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 raav8-1.3hbv HBV HBV DNA Fig. 3 HBV DNA titer in sera of the HBV chronic infectious mice models mediated by raav8-1.3hbv at different time points. Mice models of HBV chronic infection were continuously treated by drugs day by day for 10 days. Then drugs were withdrew and lasted for fifteen days. At different time points in the process, HBV DNA titer in sera was tested. 3.75±0.21 (P 0.01) ±0.27 raav8-1.3hbv HBV LAM 10 d 15 d HBV DNA 1 LAM 10 d LAM HBV DNA (P 0.05) LAM 5 d 0 d HBV DNA (lg (IU/mL)) 5.22± ±0.22 (P 0.05) 10 d 0 d 4.42±0.22 (P 0.01) 15 d 10 HBV DNA 5.10±0.49 (P 0.05) HBV DNA LAM ( 1) LAM 10 d (P 0.05) 15 d (P 0.01) LAM ( 3) 5 d 0 d HBV DNA (lg (IU/mL)) 5.61± ±0.40 (P 0.01) 10 d 0 d 4.32±0.32 (P 0.01) ±0.28 cjb@im.ac.cn

8 102 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 HBV DNA HBV DNA 2.3 HBeAg HBsAg ETV HBeAg HBeAg 4A 40 ETV 0 HBeAg 13.49±1.72 S/CO 10 d 13.28±1.87 S/CO 15 d 13.26±1.60 S/CO HBeAg ETV ±1.93 S/CO 10 d 13.05±1.74 S/CO 15 d 12.55±1.64 S/CO HBeAg HBeAg 4 raav8-1.3hbv HBV HBeAg HBsAg Fig. 4 HBeAg and HBsAg titer in the HBV chronic infectious mice models mediated by raav8-1.3hbv at different time points. (A) The HBeAg titer. (B) The HBsAg titer.

9 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 103 ETV HBsAg HBeAg 4B 40 ETV 0 HBsAg 94.43±6.48 miu/ml 10 d 94.10±5.67 miu/ml 15 d 91.76±7.71 miu/ml HBsAg ETV 0 HBsAg 90.02±7.42 miu/ml 10 d 90.01±6.47 miu/ml 15 d 91.01±6.94 miu/ml HBsAg HBsAg LAM HBeAg HBeAg 4A 40 LAM 0 HBeAg 14.22±0.91 S/CO 10 d 12.65±2.00 S/CO 15 d 13.43±1.41 S/CO HBeAg LAM 0 HBeAg 14.65±1.38 S/CO 10 d 14.29±1.82 S/CO 15 d 14.57±1.50 S/CO HBeAg LAM HBsAg HBeAg 4B 40 LAM 0 HBsAg 93.42±9.41 miu/ml 10 d 90.99±6.77 miu/ml 15 d 92.33±8.35 miu/ml HBsAg LAM 0 HBsAg 96.61±8.23 miu/ml 10 d 93.62±8.73 miu/ml 15 d 93.16±6.30 miu/ml HBsAg 3 HBV [9] HBV 1.3 HBV 8 (raav8-1.3hbv) C57BL/6 HBV HBV DNA HBeAg HBsAg HBV DNA raav8-1.3hbv HBV HBV DNA [10-11] HBV DNA HBV cjb@im.ac.cn

10 104 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 [12] HBV HBV [13-14] HBV DNA raav8-1.3hbv HBV (ETV LAM) HBV ETV LAM [15-16] [17-18] HBV pg RNA (Pregenomic RNA) DNA (Minusstrand DNA) RC DNA (Relaxed circular DNA) DSL DNA (Double-stranded linear DNA) [19] HBV HBV HBV DNA HBeAg HBsAg raav8-1.3hbv HBeAg HBsAg HBV DNA (lg (IU/mL)) HBV DNA (lg (IU/mL)) HBeAg HBsAg 3 HBeAg HBsAg 27 HBeAg (13.22±1.78 S/CO) HBsAg (93.24±7.24 miu/ml) raav8-1.3hbv HBV 90 raav8-1.3hbv HBV ETV LAM HBV DNA HBV DNA HBV DNA 10 d HBV DNA HBV DNA HBV DNA 5 d 10 d ETV HBV DNA LAM [20-21] 15 d 10 d HBV DNA [22] HBV DNA ELISA HBeAg HBsAg

11 王国婧等 / 用重组 8 型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果 105 pg RNA DNA HBV DNA HBV DNA HBeAg HBsAg HBV DNA HBeAg HBsAg [23-26] raav8-1.3hbv HBV HBV DNA HBeAg HBsAg HBV HBV REFERENCES [1] Jin Q. Medical Molecular Virology. Beijing: Science Press, 2001: (in Chinese)... :, 2001: [2] Zhuang H. Progress in epidemiology of hepatitis B virus. Chin J Fronti Med Sci, 2009, 1(2): (in Chinese)..., 2009, 1(2): [3] Zhao H, Si CW. Current study on epidemiology of viral hepatitis Bin China. Chin J Front Med Sci, 2008, 1(2): 1 3 (in Chinese).,.., 2008, 1(2): 1 3. [4] Ayoub WS, Keeffe EB. Review article: current antiviral therapy of chronic hepatitis B. Aliment Pharmacol Ther, 2011, 34(10): [5] Fung J, Lai CL, Seto WK, et al. Nucleoside/ nucleotide analogues in the treatment of chronic hepatitis B. J Antimicrob Chemother, 2011, 66(12): [6] Zoulim F. Hepatitis B virus resistance to antiviral drugs: where are we going? Liver Int, 2011, 31(Suppl 1): [7] Dong XY, Yuchi J, Wang G, et al. Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome(raav8-1.3hbv). Chin J Virol, 2010, 26(6): (in Chinese).,,,. 8., 2010, 26(6): [8] Wu XB, Dong XY, Wu ZJ, et al. A novel method for purification of recombinant adeno-associated virus vectors on a large scale. Chin Sci Bull, 2001, 46(6): [9] Li P, Yu YZ. The mouse model of HBV infection. Inter J Epidemiol Infect Dis, 2006, 33(6): (in Chinese).,.., 2006, 33(6): [10] Takashima H, Araki K, Miyazaki J, et al. Characterization of T-cell tolerance to hepatitis B virus (HBV) antigen in transgenic mice. Immunology, 1992, 75(3): [11] Akbar SK, Onji M. Hepatitis B virus (HBV)-transgenic mice as an investigative tool to study immunopathology during HBV infection. Int J Exp Pathol, 1998, 79(5): [12] Lok AS, Zoulim F, Locarnini S, et al. Antiviral drug-resisitant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology, 2007, 46(1): [13] Yang PL, Althage A, Chung J, et al. Hydrodynamic cjb@im.ac.cn

12 106 ISSN CN /Q Chin J Biotech January 25, 2013 Vol.29 No.1 injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci USA, 2002, 99(21): [14] Huang LR, Wu HL, Chen PJ, et al. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci USA, 2006, 103(47): [15] Yamanaka G, Wilson T, Innaimo S, et al. Metabolic studies on BMS , a new antiviral compound active against hepatitis B virus. Antimicrob Agents Chemother, 1999, 43(1): [16] Lai CL, Yuen MF. Profound suppression of hepatitis B virus replication with lamivudine. J Med Virol, 2000, 61(3): [17] Seifer M, Hamatake RK, Colonno RJ, et al. In vitro inhibition of hepadnavirus polymerases by the triphosphates of BMS and lobucavir. Antimicrob Agents Chemother, 1998, 42(12): [18] Doong SL, Tsai CH, Schinazi RF, et al. Inhibition of the replication of hepatitis B virus in vitro by 2', 3'-dideoxy-3'-thiacytidine and related analogues. Proc Natl Acad Sci USA, 1991, 88(19): [19] Fields BN, Knipe DM, Howley PM, et al. Fields virology. 3rd ed. Philadelphia: Lippincott-Raven Publisher, 1996: [20] Lai CL, Rosmawati M, Lao J, et al. Entecavir is superior to lamivudine in reducing hepatitis B virus DNA in patients with chronic hepatitis B infection. Gastroenterology, 2002, 123(6): [21] Shepherd J, Gospodarevskaya E, Frampton G, et al. Entecavir for the treatment of chronic hepatitis B infection. Health Technol Assess, 2009, 13(Suppl. 3): [22] Hong JH, Choi Y, Chun BK, et al. Current status of anti-hbv chemotherapy. Arch Pharm Res, 1998, 21(2): [23] Nagahata T, Araki K, Yamamura K, et al. Inhibition of intrahepatic hepatitis B virus replication by antiviral drugs in a novel transgenic mouse model. Antimicrob Agents Chemother, 1992, 36(9): [24] Morrey JD, Bailey KW, Korba BE, et al. Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral evaluation of lamivudine. Antiviral Res, 1999, 42(2): [25] Julander JG, Sidwell RW, Morrey JD. Characterizing antiviral activity of adefovir dipivoxil in transgenic mice expressing hepatitis B virus. Antiviral Res, 2002, 55(1): [26] Julander JG, Colonno RJ, Sidwell RW, et al. Characterization of antiviral activity of entecavir in transgenic mice expressing hepatitis B virus. Antiviral Res, 2003, 59(3): ( )

600 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 were significantly decreased, but the levels of IL-12 in two groups were significantly increa

600 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 were significantly decreased, but the levels of IL-12 in two groups were significantly increa 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 599 喘可治注射液联合细辛脑注射液治疗小儿毛细支气管炎的临床研究唐辉英, 佘定平, 马颐娇深圳市儿童医院呼吸科, 广东深圳 518026 摘要 : 目的探讨喘可治注射液联合