新型基因工程药物安全性评价方法探讨
|
摘要
近年来,各种新型基因工程药物不断涌现,但国内、外对此类药物安全性研究还缺乏完善的指导原则,在研究中常常遇到诸如敏感动物选择、剂量设计、给药期限、特异性观察指标的确定等新问题。本论文从敏感动物选择及特异性指标的检测方法考虑,以近期国内正在开发的基因治疗药物——重组溶瘤性腺病毒H101和H103;基因工程治疗性疫苗——重组人纽表位肽12(Recombination human erbB3
fragment483～540)及基因重组药物——重组白介素11(rhIL-11)为代表,对其进行研究,旨在为新型基因工程药物安全性评价及其临床研究提供安全性参数,也为我国相应法规的完善提供实验依据。
本研究选择豚鼠及恒河猴作为敏感动物,对H101和H103进行重复给药的毒性实验,采用PCR方法研究药物在组织内的分布。结果表明:H101及H103系—种毒性反应较小的抗肿瘤基因治疗药物,H101临床试验观察到的不良反应与临床前试验所见一致;H101及H103的抗体测定均为阳性,测定抗体滴度呈剂量—时间依赖关系;H101在豚鼠颈淋巴结有分布,而H103在动物任何脏器均无分布。证明豚鼠及恒河猴可用于腺病毒类医药产品或以腺病毒为载体的基因治疗的安全性评价,且豚鼠的敏感性不低于灵长类动物,其免疫系统的反应性比灵长类动物更敏感。这既为今后溶瘤病毒的毒理学研究提供了敏感动物模型及实验数据,也为以病毒为载体的基因治疗方法的安全性评价提供了参考。在对H103的组织分布研究中,采用5’核酸酶技术的Real-time定量PCR对重要脏器组织DNA样品的拷贝数进行确定,为溶瘤病毒或以病毒为载体的基因治疗产品的生物学分布研究提供参考。
重组人纽表位肽12是一种针对ErbB3抗原的治疗性肿瘤疫苗,用于治疗高表达ErbB2的乳腺癌病人。对其安全性评价研究中敏感动物的选择同样是至关重要的问题。自FVB/N转neu基因小鼠繁育成功以来,一直用于抗乳腺癌的药效学研究,是一种能反映药效的动物模型——敏感动物。本研究选择FVB/N转neu基因小鼠和食蟹猴进行长期毒性研究。结果表明:重组人纽表位肽12是一种安全系数较高的肿瘤治疗性疫苗,选择转基因动物及灵长类动物进行此类新药的毒理学研究,对综合评价其安全性提供临床参考更具有代表性。本研究为今后评价其它基因工程新药时选择敏感动物提供了可借鉴的模型。
由于大部分基因工程产品为源于人的蛋白质,一般认为此类药品对人CYP450s不会产生影响。因此,在对此类新药的安全性评价过程中往往忽视人或动物CYP450s酶系的变化。本研究选择重组人白细胞介素-11,研究其对大鼠CYP450s的影响,结果表明,大鼠皮下注射rhIL-11后,雌雄大鼠肝微粒体中CYP1A1、雌鼠肝CYP2A2比活性明显低于对照组,而肝微粒体蛋白含量、CYP450总量及CYP2E1比活性无明显变化。提示rhIL-11对雌雄大鼠肝CYP1A1、雌鼠肝CYP2A2比活性有一定的
上海医药工业研究院博士学位论文
抑制作用。因此临床上在使用rll比一!l与芳香烃尾物质(PH八)、少J不内脂钊元生素、
茶碱类和固醇一鹅物质·(及香豆素和争酮类物贡联合时,应考虑其相互作川本研究
提示应重视基川工程产品对CYr)45OsJ晦系的彩响。对指导“!台床合理用药,防止一,知生
反应,对预侧药物一药物间相互作用有重一要意义
关键词:
溶瘤性睬病毒H101
介素一11长期一澎匡
H 1 03
组织分布
基因工程疫苗重组人纽表位肤12重组人白细胞
PCR细胞色素P45O
Following the advances made in the field of biotechnology, many new kinds of biotechnology-derived drugs are being developed. However, only few guidelines for the safety evaluation of these new drugs are available, in China and internationally. As studies of these drugs progress a number of questions emerge, such as selecting sensitive animal species, dose levels to be used, duration of the administration period, which specific parameters should be evaluated etc.The purpose of this study was to select sensitive animal species and methods for investigating specific parameters which could provide an experimental basis for future Chinese guidelines of biotechnology-derived drugs.Repeat dose toxicity studies were performed to study the effects of the oncolytic adenoviruses H101 and H103. In these toxicity studies, guinea pigs and rhesus monkeys were chosen as experimental animals. At the end of the administration and recovery periods, major organs were collected from all animals. Both compounds were of low toxicity and the effects shown to be reversible. Furthermore, the preclinical results of H101 were in agreement with the adverse effects observed in clinical studies. Antibody detection in plasma samples was positive and time dependent for both compounds. H101 was distributed to cervical lymph nodes in guinea pigs as shown by PCR, while H103 did not show any tissue distribution by the PCR and RT-PCR techniques in rhesus monkeys.The present studies provide experimental data for toxicity evaluations of oncolytic adenoviruses and show that the guinea pig and rhesus monkey models can be used for safety evaluation of gene therapy involving virus carriers. Furthermore the guinea pig's immune system is more sensitive than that of primates.Toxicity studies of a biotechnology-derived vaccine, the recombinant human erbB3 fragment 483-540, were also performed. The erbB3 fragment 483-540 is a therapeutic cancer vaccine, aiming at the ErbB3 antigen and inducing a specific immunological response. It can be used to treat breast cancers highly expressing ErbB2. The FVB/N neu transgenic mice spontaneously develop breast cancer highly expressing ErbB2 and can thus be used as a model for pharmacodynamic studies of breast cancer therapies. The FVB/N neu transgenic mice and cynomolgus monkeys were chosen for long term toxicity studies. Effects of erbB3 on the immunological system were noted in the mice. Positive
and specific antibody reactions were observed in both species. Thus, transgenic mice and cynomolgus monkeys were shown to be useful experimental models for safety evaluation.Transgenic animals are expected to serve as models for safety evaluation of biotechnologically derived drugs in the future. Toxicity studies would benefit from the use of transgenic mice and monkeys since results would be more representative of those of clinical trials.Many biotechnology-derived drugs are based on human proteins and their effects on CYP450s have previously been ignored. Thus there are no guidelines regarding the toxicity effects of proteins on CYP450s. This thesis includes the study of the effects of rh-IL-11 on the hepatic CYP450s in rats. Rh-IL-11 is a multifunctional cytokine with hematopoietic and anti-inflammatory activities. It is mainly used to elevate the platelet count in cancer patients with chemotherapy-induced thrombocytopenia.No significant changes of the contents of microsomal proteins, the total CYP450s and the specific activity of CYP2E1 were observed after repeated rh-IL-11 administration for three months in rats. However, rh-IL-11 significantly decreased the specific activity of CYP2A2 in female rats and that of CYP1A1 in both male and female rats compared with the control group. Thus, the effects of the drug on the activities of CYP450s should be considered when it is co-administered with aromatic hydrocarbon drugs, such as cyclophosphamide, coumarin, testosterone and others. It should be emphasized that it is as important to evaluate the effects of biotechnology-derived drugs as of synthetic drugs on CYP450s to prevent second
fragment483～540)及基因重组药物——重组白介素11(rhIL-11)为代表,对其进行研究,旨在为新型基因工程药物安全性评价及其临床研究提供安全性参数,也为我国相应法规的完善提供实验依据。
本研究选择豚鼠及恒河猴作为敏感动物,对H101和H103进行重复给药的毒性实验,采用PCR方法研究药物在组织内的分布。结果表明:H101及H103系—种毒性反应较小的抗肿瘤基因治疗药物,H101临床试验观察到的不良反应与临床前试验所见一致;H101及H103的抗体测定均为阳性,测定抗体滴度呈剂量—时间依赖关系;H101在豚鼠颈淋巴结有分布,而H103在动物任何脏器均无分布。证明豚鼠及恒河猴可用于腺病毒类医药产品或以腺病毒为载体的基因治疗的安全性评价,且豚鼠的敏感性不低于灵长类动物,其免疫系统的反应性比灵长类动物更敏感。这既为今后溶瘤病毒的毒理学研究提供了敏感动物模型及实验数据,也为以病毒为载体的基因治疗方法的安全性评价提供了参考。在对H103的组织分布研究中,采用5’核酸酶技术的Real-time定量PCR对重要脏器组织DNA样品的拷贝数进行确定,为溶瘤病毒或以病毒为载体的基因治疗产品的生物学分布研究提供参考。
重组人纽表位肽12是一种针对ErbB3抗原的治疗性肿瘤疫苗,用于治疗高表达ErbB2的乳腺癌病人。对其安全性评价研究中敏感动物的选择同样是至关重要的问题。自FVB/N转neu基因小鼠繁育成功以来,一直用于抗乳腺癌的药效学研究,是一种能反映药效的动物模型——敏感动物。本研究选择FVB/N转neu基因小鼠和食蟹猴进行长期毒性研究。结果表明:重组人纽表位肽12是一种安全系数较高的肿瘤治疗性疫苗,选择转基因动物及灵长类动物进行此类新药的毒理学研究,对综合评价其安全性提供临床参考更具有代表性。本研究为今后评价其它基因工程新药时选择敏感动物提供了可借鉴的模型。
由于大部分基因工程产品为源于人的蛋白质,一般认为此类药品对人CYP450s不会产生影响。因此,在对此类新药的安全性评价过程中往往忽视人或动物CYP450s酶系的变化。本研究选择重组人白细胞介素-11,研究其对大鼠CYP450s的影响,结果表明,大鼠皮下注射rhIL-11后,雌雄大鼠肝微粒体中CYP1A1、雌鼠肝CYP2A2比活性明显低于对照组,而肝微粒体蛋白含量、CYP450总量及CYP2E1比活性无明显变化。提示rhIL-11对雌雄大鼠肝CYP1A1、雌鼠肝CYP2A2比活性有一定的
上海医药工业研究院博士学位论文
抑制作用。因此临床上在使用rll比一!l与芳香烃尾物质(PH八)、少J不内脂钊元生素、
茶碱类和固醇一鹅物质·(及香豆素和争酮类物贡联合时,应考虑其相互作川本研究
提示应重视基川工程产品对CYr)45OsJ晦系的彩响。对指导“!台床合理用药,防止一,知生
反应,对预侧药物一药物间相互作用有重一要意义
关键词:
溶瘤性睬病毒H101
介素一11长期一澎匡
H 1 03
组织分布
基因工程疫苗重组人纽表位肤12重组人白细胞
PCR细胞色素P45O
Following the advances made in the field of biotechnology, many new kinds of biotechnology-derived drugs are being developed. However, only few guidelines for the safety evaluation of these new drugs are available, in China and internationally. As studies of these drugs progress a number of questions emerge, such as selecting sensitive animal species, dose levels to be used, duration of the administration period, which specific parameters should be evaluated etc.The purpose of this study was to select sensitive animal species and methods for investigating specific parameters which could provide an experimental basis for future Chinese guidelines of biotechnology-derived drugs.Repeat dose toxicity studies were performed to study the effects of the oncolytic adenoviruses H101 and H103. In these toxicity studies, guinea pigs and rhesus monkeys were chosen as experimental animals. At the end of the administration and recovery periods, major organs were collected from all animals. Both compounds were of low toxicity and the effects shown to be reversible. Furthermore, the preclinical results of H101 were in agreement with the adverse effects observed in clinical studies. Antibody detection in plasma samples was positive and time dependent for both compounds. H101 was distributed to cervical lymph nodes in guinea pigs as shown by PCR, while H103 did not show any tissue distribution by the PCR and RT-PCR techniques in rhesus monkeys.The present studies provide experimental data for toxicity evaluations of oncolytic adenoviruses and show that the guinea pig and rhesus monkey models can be used for safety evaluation of gene therapy involving virus carriers. Furthermore the guinea pig's immune system is more sensitive than that of primates.Toxicity studies of a biotechnology-derived vaccine, the recombinant human erbB3 fragment 483-540, were also performed. The erbB3 fragment 483-540 is a therapeutic cancer vaccine, aiming at the ErbB3 antigen and inducing a specific immunological response. It can be used to treat breast cancers highly expressing ErbB2. The FVB/N neu transgenic mice spontaneously develop breast cancer highly expressing ErbB2 and can thus be used as a model for pharmacodynamic studies of breast cancer therapies. The FVB/N neu transgenic mice and cynomolgus monkeys were chosen for long term toxicity studies. Effects of erbB3 on the immunological system were noted in the mice. Positive
and specific antibody reactions were observed in both species. Thus, transgenic mice and cynomolgus monkeys were shown to be useful experimental models for safety evaluation.Transgenic animals are expected to serve as models for safety evaluation of biotechnologically derived drugs in the future. Toxicity studies would benefit from the use of transgenic mice and monkeys since results would be more representative of those of clinical trials.Many biotechnology-derived drugs are based on human proteins and their effects on CYP450s have previously been ignored. Thus there are no guidelines regarding the toxicity effects of proteins on CYP450s. This thesis includes the study of the effects of rh-IL-11 on the hepatic CYP450s in rats. Rh-IL-11 is a multifunctional cytokine with hematopoietic and anti-inflammatory activities. It is mainly used to elevate the platelet count in cancer patients with chemotherapy-induced thrombocytopenia.No significant changes of the contents of microsomal proteins, the total CYP450s and the specific activity of CYP2E1 were observed after repeated rh-IL-11 administration for three months in rats. However, rh-IL-11 significantly decreased the specific activity of CYP2A2 in female rats and that of CYP1A1 in both male and female rats compared with the control group. Thus, the effects of the drug on the activities of CYP450s should be considered when it is co-administered with aromatic hydrocarbon drugs, such as cyclophosphamide, coumarin, testosterone and others. It should be emphasized that it is as important to evaluate the effects of biotechnology-derived drugs as of synthetic drugs on CYP450s to prevent second
引文
1. 上海三维生物技术有限公司.溶瘤性重组腺病毒研究综述(H101).2000年,上海
2. Amold-Schild D, Hanan D, Spehner D, Schmid C, Rammensee HG, De La S, Schild H. Cutting edge: receptor-mediated endocytosis of HSP by professional antigen-presenting cells. J Immunol. 1999; 162: 3757-3760
3. Kirm DH, Mcconnick F. Replicating viruses as selective cancer therapeutics. Molecular Medicine Today Dec. 1996; 519-527
4. Mccormick F. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science. 1996; 274: 373
5. Yew PR, Berk AJ. Inhibition of p53 transactivation required for transformation by adenovirus early IB protein. Nature. 1992;357:82-85
6. 郭建成.热休克蛋白-肽复合物肿瘤疫苗.国外医学肿瘤学分册.1996;23(5):267-270
7. Srivastava PK. Heat shock proteins in immune response to cancer and in antigen presentation. Cancer Res. 1993; 62: 153 - 177
8. Binder R, Han PK, Srivastava PK. CD91: a receptor for hsp gp96. Nature Immunol.. 2. 151-155(2000)
9. 上海三维生物技术有限公司.重组溶瘤性腺病毒(H1O3)综述,2002年,上海 Kinn DH, et al. Replicating oncolytic viruses: an overview. Ecp. Opin. Invest. Drugs. 1996; 5(6): 753-762
10. Shao-Chun Wang, Lisha Zhang, Gabriel N. Hortobagyi, et al. Targeting HER2: Recent developments and future directions for breast cancer patients. Semin Oncol. 2001; 28: 21-29
11. Mary L. Disis and Kathy Schiffman. Cancer vaccines targeting the HER2/neu oncogenic protein. Semin Oncol. 2001; 18: 12-20
12. Glenn Dranoff. Interpreting cancer vaccine clinical trails. The Journal of Gene Medicine. 1999; 1: 80-83
13. Thoama Holbro, Roger R. Beerli, Francisca Maurer, Magdalena Koziczak, Carlos F, Barbas L. and Nancy E. Hynes. The ErbB2/ErbB3 heterodimer function as an oncongenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. PANAS. July 2003; 100(15): 8933-8938
14. Brockhoff G, Hesis P, Schlegel J, et al. Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells. Cytometry. 2001; 44: 338-348
15. 上海三维生物技术有限公司.溶瘤性重组腺病毒H101临床研究方案.2000年,上海
16. Kirm DH, et al. Replicating oncolytic viruses: an overview. Ecp. Opin. Invest. Drugs,
5(6): 753-62(1996)
17. Binder R, Han DK, Srivastava PK. CD91: a receptor for hsp gp96. Nature Immunol 2. 151-155(2000)
18 . Nielsen LL. RecomAbinant EI -deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and live histology in tumor xenograft models. Human Gene Therapy 9: 681-694(1998)
19. 颜呈准,刘瑞三主编.买验动物科学管理实用手册.云南科技出版社 1998年7月出版,33-35 20. Vitalis T. Z., Kern I., Crroome A., Behzad H., Hayashi S., Hogg J. C. The effect of latent adenovirus 5 infection on cigarette smoke induced lung inflammation. Eur Respir J. 1998; 11:664-669
21 . Adam CH, Johannes S, Williams A, et al Onyx-015, an E1B geneattenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nature Medicine, 1997; 3(6): 639
22. Arnold-Schild D., Hanau D., Spehner D., Schmid C, Rammensee H.G., De La S. and Schild H. Cutting edge: recepor-mediated endocytosis of hsp by professional antigen-presenting cells. J. Immunol. 1999;162:3757-3760
23. Castellino F., Boucher P. E., Eichelberg K., Mayhew M., Rothman J. E., Houghton A.N., Germain R. N. Receptor-mediated uptake of antigen/hsp complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J. Exp. Med. 2000; 191(11): 1957-1964
24. Wells A.D., Rai S.K., Salvato M. S., Band H., Malkovsky M. hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int. Immunol. 1998;10(5): 609-617
25. Svivastava PK. Heat shock proteins in immune response to cancer and in antigen presentation. Cancer Res. 1993; 62: 153 - 177
26. More SK., Rijli F, Appella E. Characterization of the mouse 84-KD heat shock protein gene family. DNA Cell BiolGene. 1990; 9(6): 387-400
27. Svivastava P.K. Experimentia. 1994; 50: 1054-1060
28. Moingeon P. Cancer Vaccines. Vaccine. 2001 ;19:1305-1326
29. Janetaki S. Palla D. Rosenhauer V, Lochs H., Lewis J.J. Svivastava P.K. Immunization of cancer patients with autologous cancer-derived hsp gp96 preparations: a pilot study. Int J Cancer. 2000; 88 232-238
30. Wilmott RW, et al. Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primate. Human Gene Therapy . 1996; 7: 301-318
31. Janetzki S, Palla D, Rosenhauer V, Lochs H, Lewis JJ, Srivastava P. K. Immunization of cancer patients with autologous cancer-derived hsp gp96 preparations: a pilot study. Int J Cancer. 2000; 88: 232-238
32 . Mackay, I.M., Arden K. E., Nitsche A.. 2002. Real-time PCR in virology. Nucl. Acids Res. 30: 1292.
33. Bela Bodey JR, Stuarte Siegel. Failure of Cancer Vaccines: The significant limitations of this approach to immunotherapy. Anticancer Research. 2000; 2: 2665-2676
34. Behjatilah Monzavi-Karbassi and Thomas Kieber-Emmons. Current concepts in cancer vaccine strategies. Biothchnique. 2001; 30:170-189
35. 上海泽生科技开发有限公司.重组人纽表位肽12研究综述.2003年8月
36. 上海泽生科技开发有限公司.重组人纽表位肽12药效学研究报告.2002年4月
37. Cheng Cheng. Perspectives on the use of oncolytic adenoviruses in cancer therapy. 2003 年上海国际生物技术和医药研讨会论文集.2003年4月,上海
38 . Yum L. Yip, Robyn L. Ward. Anti-ErbB-2 monoclonal antibodies and ErbB-2-directed vaccine. Cancer Immunother, 2002; 5: 569-587
39. David B. Agus, Robert W. Akita, William D. Fox. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell. 2002;8
40. Keith L, Knutson, Kathy Schiffiman and Mary L. Disis. Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity. J. Clin. Invest. 2001; 107:477-484
41. Nelson DR, Koymans L, Kamataki T, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 1996, 6:1-42
42. http.//drnelson. utmem. edu/homepage.html. David Nelson. Cytochrome P450s in humans. Last modified Nov. 24, 2000
43. 马璟,钱蓓丽,顾性初,张呈菊.人肝细胞色素P450含量及其同工酶[A]、2A6活性的测定.中国医药工业杂志,1999;30(10):449
44. Lowry OH, Rosebrough NJ, Farr AL et al. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951; 193:265
45. Omura T, Sato R. The carbon monoxide binding pigment of liver microsomes. J Biol Chem,1964;239:2379
46. 马璟,钱蓓丽,顾性初,杨炳华,王根宝等.人肝微粒体中CYP2C8/9、CYP2E1比活性测定中国药理学通报,2002;18(1):36-38
47. Hakkola J, Tanaka E, Pelkonen O. Developmental expression of cytochrome P450 enzymes in human liver. Pharmacology and Toxicology, 1998, 82:209
48. Yun CH, Shimada T, Guengerich FP. Purification and characterization of human liver microsomal cytochrome P-45-2A6, Mol. Pharmacol, 1991, 40: 679
49. Vieira IM, Sonnier & T. Cresteil. Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur. J. Biochem, 1996; 238: 476
2. Amold-Schild D, Hanan D, Spehner D, Schmid C, Rammensee HG, De La S, Schild H. Cutting edge: receptor-mediated endocytosis of HSP by professional antigen-presenting cells. J Immunol. 1999; 162: 3757-3760
3. Kirm DH, Mcconnick F. Replicating viruses as selective cancer therapeutics. Molecular Medicine Today Dec. 1996; 519-527
4. Mccormick F. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science. 1996; 274: 373
5. Yew PR, Berk AJ. Inhibition of p53 transactivation required for transformation by adenovirus early IB protein. Nature. 1992;357:82-85
6. 郭建成.热休克蛋白-肽复合物肿瘤疫苗.国外医学肿瘤学分册.1996;23(5):267-270
7. Srivastava PK. Heat shock proteins in immune response to cancer and in antigen presentation. Cancer Res. 1993; 62: 153 - 177
8. Binder R, Han PK, Srivastava PK. CD91: a receptor for hsp gp96. Nature Immunol.. 2. 151-155(2000)
9. 上海三维生物技术有限公司.重组溶瘤性腺病毒(H1O3)综述,2002年,上海 Kinn DH, et al. Replicating oncolytic viruses: an overview. Ecp. Opin. Invest. Drugs. 1996; 5(6): 753-762
10. Shao-Chun Wang, Lisha Zhang, Gabriel N. Hortobagyi, et al. Targeting HER2: Recent developments and future directions for breast cancer patients. Semin Oncol. 2001; 28: 21-29
11. Mary L. Disis and Kathy Schiffman. Cancer vaccines targeting the HER2/neu oncogenic protein. Semin Oncol. 2001; 18: 12-20
12. Glenn Dranoff. Interpreting cancer vaccine clinical trails. The Journal of Gene Medicine. 1999; 1: 80-83
13. Thoama Holbro, Roger R. Beerli, Francisca Maurer, Magdalena Koziczak, Carlos F, Barbas L. and Nancy E. Hynes. The ErbB2/ErbB3 heterodimer function as an oncongenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. PANAS. July 2003; 100(15): 8933-8938
14. Brockhoff G, Hesis P, Schlegel J, et al. Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells. Cytometry. 2001; 44: 338-348
15. 上海三维生物技术有限公司.溶瘤性重组腺病毒H101临床研究方案.2000年,上海
16. Kirm DH, et al. Replicating oncolytic viruses: an overview. Ecp. Opin. Invest. Drugs,
5(6): 753-62(1996)
17. Binder R, Han DK, Srivastava PK. CD91: a receptor for hsp gp96. Nature Immunol 2. 151-155(2000)
18 . Nielsen LL. RecomAbinant EI -deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and live histology in tumor xenograft models. Human Gene Therapy 9: 681-694(1998)
19. 颜呈准,刘瑞三主编.买验动物科学管理实用手册.云南科技出版社 1998年7月出版,33-35 20. Vitalis T. Z., Kern I., Crroome A., Behzad H., Hayashi S., Hogg J. C. The effect of latent adenovirus 5 infection on cigarette smoke induced lung inflammation. Eur Respir J. 1998; 11:664-669
21 . Adam CH, Johannes S, Williams A, et al Onyx-015, an E1B geneattenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nature Medicine, 1997; 3(6): 639
22. Arnold-Schild D., Hanau D., Spehner D., Schmid C, Rammensee H.G., De La S. and Schild H. Cutting edge: recepor-mediated endocytosis of hsp by professional antigen-presenting cells. J. Immunol. 1999;162:3757-3760
23. Castellino F., Boucher P. E., Eichelberg K., Mayhew M., Rothman J. E., Houghton A.N., Germain R. N. Receptor-mediated uptake of antigen/hsp complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J. Exp. Med. 2000; 191(11): 1957-1964
24. Wells A.D., Rai S.K., Salvato M. S., Band H., Malkovsky M. hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int. Immunol. 1998;10(5): 609-617
25. Svivastava PK. Heat shock proteins in immune response to cancer and in antigen presentation. Cancer Res. 1993; 62: 153 - 177
26. More SK., Rijli F, Appella E. Characterization of the mouse 84-KD heat shock protein gene family. DNA Cell BiolGene. 1990; 9(6): 387-400
27. Svivastava P.K. Experimentia. 1994; 50: 1054-1060
28. Moingeon P. Cancer Vaccines. Vaccine. 2001 ;19:1305-1326
29. Janetaki S. Palla D. Rosenhauer V, Lochs H., Lewis J.J. Svivastava P.K. Immunization of cancer patients with autologous cancer-derived hsp gp96 preparations: a pilot study. Int J Cancer. 2000; 88 232-238
30. Wilmott RW, et al. Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primate. Human Gene Therapy . 1996; 7: 301-318
31. Janetzki S, Palla D, Rosenhauer V, Lochs H, Lewis JJ, Srivastava P. K. Immunization of cancer patients with autologous cancer-derived hsp gp96 preparations: a pilot study. Int J Cancer. 2000; 88: 232-238
32 . Mackay, I.M., Arden K. E., Nitsche A.. 2002. Real-time PCR in virology. Nucl. Acids Res. 30: 1292.
33. Bela Bodey JR, Stuarte Siegel. Failure of Cancer Vaccines: The significant limitations of this approach to immunotherapy. Anticancer Research. 2000; 2: 2665-2676
34. Behjatilah Monzavi-Karbassi and Thomas Kieber-Emmons. Current concepts in cancer vaccine strategies. Biothchnique. 2001; 30:170-189
35. 上海泽生科技开发有限公司.重组人纽表位肽12研究综述.2003年8月
36. 上海泽生科技开发有限公司.重组人纽表位肽12药效学研究报告.2002年4月
37. Cheng Cheng. Perspectives on the use of oncolytic adenoviruses in cancer therapy. 2003 年上海国际生物技术和医药研讨会论文集.2003年4月,上海
38 . Yum L. Yip, Robyn L. Ward. Anti-ErbB-2 monoclonal antibodies and ErbB-2-directed vaccine. Cancer Immunother, 2002; 5: 569-587
39. David B. Agus, Robert W. Akita, William D. Fox. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell. 2002;8
40. Keith L, Knutson, Kathy Schiffiman and Mary L. Disis. Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity. J. Clin. Invest. 2001; 107:477-484
41. Nelson DR, Koymans L, Kamataki T, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 1996, 6:1-42
42. http.//drnelson. utmem. edu/homepage.html. David Nelson. Cytochrome P450s in humans. Last modified Nov. 24, 2000
43. 马璟,钱蓓丽,顾性初,张呈菊.人肝细胞色素P450含量及其同工酶[A]、2A6活性的测定.中国医药工业杂志,1999;30(10):449
44. Lowry OH, Rosebrough NJ, Farr AL et al. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951; 193:265
45. Omura T, Sato R. The carbon monoxide binding pigment of liver microsomes. J Biol Chem,1964;239:2379
46. 马璟,钱蓓丽,顾性初,杨炳华,王根宝等.人肝微粒体中CYP2C8/9、CYP2E1比活性测定中国药理学通报,2002;18(1):36-38
47. Hakkola J, Tanaka E, Pelkonen O. Developmental expression of cytochrome P450 enzymes in human liver. Pharmacology and Toxicology, 1998, 82:209
48. Yun CH, Shimada T, Guengerich FP. Purification and characterization of human liver microsomal cytochrome P-45-2A6, Mol. Pharmacol, 1991, 40: 679
49. Vieira IM, Sonnier & T. Cresteil. Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur. J. Biochem, 1996; 238: 476