壳聚糖碘液体外抗性传播疾病病原体活性的研究
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摘要
性传播疾病(Sexually Transmitted Disease,STD)是以性接触为主要传播方式的一组疾病,国际上将30多种通过性行为或类似性行为引起的感染性疾病列入性病范畴。性传播疾病是危害人类最严重、发病最广泛的一种传染病,同时也是全球性生殖健康和卫生防疫的难题。迄今为止,在对各种STD的治疗方面虽然已经取得可喜的成绩,可是对性传播疾病中容易反复染病的细菌性病原体和一些具有高度变异性的病毒性病原体仍然没有行之有效的治疗方法[1]。所以开发研制高效、价廉和安全的杀微生物剂就成为预防性传播疾病的最重要途径。杀微生物剂的研究开发已被联合国艾滋病规划署(UNAIDS)和世界卫生组织(WHO)定为全球优先发展的策略。杀微生物剂是指性生活前应用于阴道和宫颈或直肠内,能够有效杀灭或中和HIV-1等病毒和细菌类病原体的人工合成的或天然的物质。在过去的十几年里,杀微生物剂的研究和发展已经取得非常显著的成绩,但是到目前为止,还没有一种安全有效的杀微生物剂上市,正在研制的临床前和进入临床的杀微生物剂有60多种,截止到2009年10月,在研究的杀微生物剂中有9种进展到临床试验阶段[2,3]。
为了寻找新的预防性传播疾病的有效制剂,我们采用MTT比色法、时间过程实验及预作用实验等方法对壳聚糖碘液进行了研究。在对壳聚糖碘液的稳定性实验中,54℃恒温箱内放置14天后检测到有效碘浓度为4.536g/L,与新配制的溶液浓度4.897 g/L相比有效碘碘浓度变化仅为7.3%,说明壳聚糖碘液具有较好的稳定性;壳聚糖碘液对白色念珠菌的MIC50为0.25 mg/ml,对HSV的EC50为0.025 mg/ml,治疗指数TI50为24,说明对白色念珠菌和HSV都有较强的抑制能力。此外,实验结果还表明,壳聚糖碘液与人单纯疱疹病毒(HSV)的预作用时间越长,HSV的滴度越小,而在病毒感染细胞2小时内加入壳聚糖碘液,对HSV有较强的抑制作用,对照药物ACV为合成核苷类抗病毒药物,主要作用于病毒DNA的复制阶段,在时间过程实验中ACV的对HSV的作用时间为病毒感染细胞6小时左右,而在预作用实验中,20分钟内加入ACV对病毒的滴度影响不是很大,由此可以推测壳聚糖碘液的作用位点在病毒的吸附阶段,并且可以与病毒直接发生作用,从而达到抑制病毒活性的效果。
综上所述,壳聚糖碘液对白色念珠菌和人单纯疱疹病毒都有较强的抑制作用,为进一步将其作为杀微生物剂的研究提供了依据。
Sexually transmitted diseases (STD), of which main mode of transmission is sexual contact, have received widely concern in recent years. Internationally, more than 30 kinds of infectious diseases caused by sexual behavior or similar sexual behavior belonged to STD. STD with the most extensive incidence not only do the most serious harm to us human, but also are a global issue to reproductive health and epidemic prevention. So far, though we have gained some encouraging achievements on the treatment of various STD, the most serious STD has received no effective way of treatment due to the high complexity and variability of virus and other exually transmitted disease pathogens [1]. The word microbicides refers to a new type of product being developed that people could use vaginally or rectally to protect themselves from HIV and possibly other sexually transmitted infections.Therefore, developing highly efficient, inexpensive and safe microbicides has become the most important way to prevent STD. The United Nations AIDS programme (UNAIDS) and World Health Organization (WHO) has regarded the research of microbicides as a global priority in development strategies. Over the past 10 years, this field has made significant achievements, but so far, no safe and effective microbicides were listed. To be specific, more than 60 kinds of microbicides have been entering into pre-clinical and clinical trials, and up to October 2009, 9 of them have developed into clinical trials [2, 3].
In order to find novel and effective pharmaceutics to prevent SID, this paper studied the chitosan-iodine against Candida albicans and herpes simplex virus (HSV) through MTT assay and time course experiments and pre-function test. To study the stability of chitosan-iodine, of which the effective iodine concentration was 4.897 g/L, it was put inside the thermostat at a constant temperature of 54℃for 14 days. Then, the detected concentration turned to 4.536 g/L, and only changed 7.3%, showing the better stability of the chitosan-iodinen. As for the inhibition by chitosan-iodine, the MIC50 for Candida albicans was 0.25 mg/mL. For the HSV, the EC50 was 0.025 mg/mL, and the therapeutic index TI50 was 24. In addition, the results also showed that the longer the pre-function test of chitosan-iodine with human herpes simple virus (HSV), the smaller HSV title was. If the chitosan-iodine was added within 2 hours when virus infected the cells, it has a strong inhibitory effect against HSV. Comparative drug ACV, which acts on viral DNA replication phase, is synthetically nucleoside antiviral drugs. In the time course experiments, ACV started to act when HSV have infected cells for 6 hours, while in the pre-function test, there has no large impact on the virus titer after adding ACV within 20 minutes. It would be speculated that the action sites of the chitosan-iodine is in the virus adsorption stage, and it could interact with the virus directly so as to achieve the effect of inhibition on virus activity.
In conclusion, chitosan-iodine has a strong inhibitory effect against Candida albicans and HSV, and it provides a theory basis to further study it as microbicides.
为了寻找新的预防性传播疾病的有效制剂,我们采用MTT比色法、时间过程实验及预作用实验等方法对壳聚糖碘液进行了研究。在对壳聚糖碘液的稳定性实验中,54℃恒温箱内放置14天后检测到有效碘浓度为4.536g/L,与新配制的溶液浓度4.897 g/L相比有效碘碘浓度变化仅为7.3%,说明壳聚糖碘液具有较好的稳定性;壳聚糖碘液对白色念珠菌的MIC50为0.25 mg/ml,对HSV的EC50为0.025 mg/ml,治疗指数TI50为24,说明对白色念珠菌和HSV都有较强的抑制能力。此外,实验结果还表明,壳聚糖碘液与人单纯疱疹病毒(HSV)的预作用时间越长,HSV的滴度越小,而在病毒感染细胞2小时内加入壳聚糖碘液,对HSV有较强的抑制作用,对照药物ACV为合成核苷类抗病毒药物,主要作用于病毒DNA的复制阶段,在时间过程实验中ACV的对HSV的作用时间为病毒感染细胞6小时左右,而在预作用实验中,20分钟内加入ACV对病毒的滴度影响不是很大,由此可以推测壳聚糖碘液的作用位点在病毒的吸附阶段,并且可以与病毒直接发生作用,从而达到抑制病毒活性的效果。
综上所述,壳聚糖碘液对白色念珠菌和人单纯疱疹病毒都有较强的抑制作用,为进一步将其作为杀微生物剂的研究提供了依据。
Sexually transmitted diseases (STD), of which main mode of transmission is sexual contact, have received widely concern in recent years. Internationally, more than 30 kinds of infectious diseases caused by sexual behavior or similar sexual behavior belonged to STD. STD with the most extensive incidence not only do the most serious harm to us human, but also are a global issue to reproductive health and epidemic prevention. So far, though we have gained some encouraging achievements on the treatment of various STD, the most serious STD has received no effective way of treatment due to the high complexity and variability of virus and other exually transmitted disease pathogens [1]. The word microbicides refers to a new type of product being developed that people could use vaginally or rectally to protect themselves from HIV and possibly other sexually transmitted infections.Therefore, developing highly efficient, inexpensive and safe microbicides has become the most important way to prevent STD. The United Nations AIDS programme (UNAIDS) and World Health Organization (WHO) has regarded the research of microbicides as a global priority in development strategies. Over the past 10 years, this field has made significant achievements, but so far, no safe and effective microbicides were listed. To be specific, more than 60 kinds of microbicides have been entering into pre-clinical and clinical trials, and up to October 2009, 9 of them have developed into clinical trials [2, 3].
In order to find novel and effective pharmaceutics to prevent SID, this paper studied the chitosan-iodine against Candida albicans and herpes simplex virus (HSV) through MTT assay and time course experiments and pre-function test. To study the stability of chitosan-iodine, of which the effective iodine concentration was 4.897 g/L, it was put inside the thermostat at a constant temperature of 54℃for 14 days. Then, the detected concentration turned to 4.536 g/L, and only changed 7.3%, showing the better stability of the chitosan-iodinen. As for the inhibition by chitosan-iodine, the MIC50 for Candida albicans was 0.25 mg/mL. For the HSV, the EC50 was 0.025 mg/mL, and the therapeutic index TI50 was 24. In addition, the results also showed that the longer the pre-function test of chitosan-iodine with human herpes simple virus (HSV), the smaller HSV title was. If the chitosan-iodine was added within 2 hours when virus infected the cells, it has a strong inhibitory effect against HSV. Comparative drug ACV, which acts on viral DNA replication phase, is synthetically nucleoside antiviral drugs. In the time course experiments, ACV started to act when HSV have infected cells for 6 hours, while in the pre-function test, there has no large impact on the virus titer after adding ACV within 20 minutes. It would be speculated that the action sites of the chitosan-iodine is in the virus adsorption stage, and it could interact with the virus directly so as to achieve the effect of inhibition on virus activity.
In conclusion, chitosan-iodine has a strong inhibitory effect against Candida albicans and HSV, and it provides a theory basis to further study it as microbicides.
引文
[1] Zinkernagel RM. The challenges of an HIV vaccine enterprise [J]. Science, 2004, 303: 1294-1295.
[2] Microbicide and prep candidates in ongoing clinical trials summary as of october 2009, http://www.microbicide.org/cs/clinical.
[3] HIV/STI Prevention Research and Development: October 2009 Pipeline Update of Microbicide and PrEP Candidates, http://www.microbicide.org/cs/pipeline.
[4] WHO. Global strategy for the prevention and control of sexually transmitted infection, 2006-2015: breaking the chain of transmission[R].
[5] Alliance for Microbicide Development. http://www.microbicide.org/cs/about_microbicides
[6] Klasse PJ, Shattock R, Moore JP. Antiretroviral drug-based microbicides to prevent HIV-1 sexual transmission[J]. Annu RevMed, 2008, 59: 455-471.
[7] Shattock RJ, Moore JP. Inhibiting sexual transmission of HIV-1 infection.[J]. Nat Rev Microbiol. 2003, Oct, 1(1): 25-34.
[8] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections[J]. Nat Rev Drug Discov, 2002, Dec, 1(12): 977-985.
[9] UNAIDS/WHO. AIDS epidemie update: December 2005 [M]. Geneva: UNAIDS/WHO, 2005: 1.
[10] United Nations. HIV/AIDS: Developing new fronts against an extraordinary crisis. Genewa. WWW.un-ngls.org/document/pdf/roundup/RU117-AIDS. pdf- 2004-12-01: 2
[11]邵健,扬宇民,撩秉洳等.甲壳素和壳聚糖在生物医学方面的应用[J].南通医学院学报1997,17(1):145-146.
[12]张澄波,梅学文,都奉业.脱己酰壳多糖对肿瘤厦免疲系统作用的研究[J].中国实验临床免疲学杂志, 1992,4(1):1-4.
[13]王芳宇,杨露青,周艺等.壳聚糖碘液杀菌效果的实验观察[J].衡阳医学院学报, 2000, 28 (5): 442-443.
[14]高怀生,黄是是,张世达等.碘壳聚糖生物敷料的制备[J].中国药学杂志, 1996, 31(5): 280~282.
[15]王科兵,肖激文,王芳宇.壳聚糖碘液对乙型肝炎病毒血清指标物灭活效果观察[J].中国消毒学杂志, 2004, 21(3): 244-245.
[16]白天玺,张庆华.现代口腔念珠菌药学[M] .北京:人民军医出版社.1995.237-238.
[17]郑之北,刘伟光,吴亦栋等.儿童脑脊液中人疱疹病毒的基因芯片检测[J].中华传染病杂志, 2007, 11: 32.
[18]张伟.宫颈单纯疱疹病毒感染检测分析[J].中原医刊, 2005, 12: 23.
[19]周南进,谢勇,李弼民等.壳聚糖抗幽门螺杆菌的实验研究[J].中国生化药物杂志, 2003, 24(2): 71-73.
[20]何淑雅,王芳宇,李邦良等.壳聚糖碘液的制备及抗真菌活性研究[J].南华大学学报, 2001, 29(3): 227-229.
[21]叶亚明.紫外分光光度法测定碘化钾溶液含量[J].海峡药学, 2(X)3, 15(l): 34-35.
[22]崔胜云,池善女,刘立春等.碘化壳聚糖的制备及其抗菌活性的研究[J].中国生化药物杂志, 2005, 26(3): 138-140
[1] Alliance for Microbicide Development. http://www.microbicide.org/cs/about_microbicides
[2] Klasse PJ, Shattock R, Moore JP. Antiretroviral drug-based microbicides to prevent HIV-1 sexual transmission[J]. Annu RevMed, 2008, 59: 455-471.
[3] Shattock RJ, Moore JP. Inhibiting sexual transmission of HIV-1 infection.[J]. Nat Rev Microbiol. 2003, Oct, 1(1): 25-34.
[4] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections[J]. Nat Rev Drug Discov, 2002, Dec, 1(12) : 977-985
[5]贲昆龙.杀微生物剂与性传播疾病的预防[J].中国性病艾滋病防治, 2002, 7(1) : 59-61.
[6] Microbicide and prep candidates in ongoing clinical trials summary as of october 2009. http://www.microbicide.org/cs/clinical
[7] HIV/STI Prevention Research and Development: October 2009 Pipeline Update of Microbicide and PrEP Candidates. http://www.microbicide.org/cs/pipeline
[8] Global Investment in Microbicide R&D http://www.microbicide.org/cs/investments_expenditures
[9]王睿睿,葛争鸣,马丽等.具有避孕作用的杀微生物剂[J].中国新药杂志, 2005, 14(3): 274-277. (Wang RR, Ge ZM, Ma L, et al. Microbicides with contraceptive action[J]. Chinese Journal of New Drugs, 2005, 14(3): 274-277.
[10] Van Herrewege Y, Michiels J, Van Roey J, et al. In vitro evaluation of nonnuceloside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virus microbicides[J]. Antimicrob Agents Chemother, 2004, 48 (1): 337-339.
[11] L.Van Damme, A. Wright, K. Depraetere, et al. A phase I study of a novel potential intravaginal microbicide, PRO 2000, in healthy sexually inactive women[J]. Sex Transm Infect, 2000 April, 76(2): 126–130.
[12] Joshi Smita, Dutta Soma, Bell Beverly, et al. Phase I safety study of 0.5% PRO2000 vaginal Gel among HIV un-infected women in Pune[J]. AIDS Research and Therapy, 2006, 3: 4.
[13] J. Gerardo García-Lerma, Ron A Otten, Shoukat H Qari, et al. Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir[J]. PloS Med, 2008, February, 5(2): e28.
[14] N. A. Margot, J. M. Waters, and M. D. Miller, et al. In Vitro Human, Immunodeficiency Virus Type 1 Resistance Selections with Combinations of Tenofovir and Emtricitabine or Abacavir and Lamivudine[J]. Antimicrob Agents Chemother, 2006, December, 50(12): 4087–4095
[15] Leah Tong, Truc K. Phan, Kelly L. Robinson, et al. Effects of Human Immunodeficiency Virus Protease Inhibitors on the Intestinal Absorption of Tenofovir Disoproxil Fumarate In Vitro[J]. Antimicrob Agents Chemother, 2007 October, 51(10): 3498–3504.
[16] James J Kohler, Seyed H Hosseini, Amy Hoying-Brandt, et al. Tenofovir renal toxicity targets mitochondria of renal proximal tubules[J]. Lab Invest, 2009 May, 89(5): 513–519.
[17] Jennifer J. Kiser, Courtney V. Fletcher, Patricia M. Flynn, et al. Pharmacokinetics of Antiretroviral Regimens Containing Tenofovir Disoproxil Fumarate and Atazanavir-Ritonavir in Adolescents and Young Adults with Human Immunodeficiency Virus Infection[J]. Antimicrob Agents Chemother, 2008, February, 52(2): 631–637.
[18] lard Whiteford SL, Matecka D, O’Rear JJ, et al. Recommendations for the nonclinical development of topical microbcides for prevention of HIV trasmission:An update[J]. J Acquir Immune Defic Syndr, 2004, 36(l): 541- 552.
[19]张冬梅,曾忠铭,兰建萍.表面杀微生物剂的研究进展与中药的机会分析[J].中国药业, 2006, 15(7): 31-33. (Zhang DM, Zeng ZM, Lan JP,et al. Research progress and opportunity analysis of traditional chinese medicine on topical microbicides[J]. China Pharmaceuticals, 2006, 15(7): 31-33.
[20] Jan Balzarini, Lut Van Damme. Microbicide drug candidates to prevent HIVinfection[J]. Lancet, 2007, 369: 787-797.
[21]刘叔文,李卫华.预防HIV性传播的杀微生物剂[J] .中国计划生育杂志, 2008, 5: 317-320.
[22] Stone A.,Jiang S. Microbicides: stoppingHIV at the gate [J]. Lancet, 2006, 368: 431-433.
[23] Margaret E. Bentley, PhD, Andrew M. Fullem, MSPH, et al. Acceptability of a Microbicide Among Women and Their Partners in a 4-Country Phase I Trial American[J]. Journal of Public Health, 2004, 94: 1159–1164.
[24] Mantell JE, Myer L, Carballo-Dieguez A, et al. Microbicide acceptability research:current approaches and future directions[J]. Soc Sci Med, 2005, 60(2): 319-330.
[2] Microbicide and prep candidates in ongoing clinical trials summary as of october 2009, http://www.microbicide.org/cs/clinical.
[3] HIV/STI Prevention Research and Development: October 2009 Pipeline Update of Microbicide and PrEP Candidates, http://www.microbicide.org/cs/pipeline.
[4] WHO. Global strategy for the prevention and control of sexually transmitted infection, 2006-2015: breaking the chain of transmission[R].
[5] Alliance for Microbicide Development. http://www.microbicide.org/cs/about_microbicides
[6] Klasse PJ, Shattock R, Moore JP. Antiretroviral drug-based microbicides to prevent HIV-1 sexual transmission[J]. Annu RevMed, 2008, 59: 455-471.
[7] Shattock RJ, Moore JP. Inhibiting sexual transmission of HIV-1 infection.[J]. Nat Rev Microbiol. 2003, Oct, 1(1): 25-34.
[8] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections[J]. Nat Rev Drug Discov, 2002, Dec, 1(12): 977-985.
[9] UNAIDS/WHO. AIDS epidemie update: December 2005 [M]. Geneva: UNAIDS/WHO, 2005: 1.
[10] United Nations. HIV/AIDS: Developing new fronts against an extraordinary crisis. Genewa. WWW.un-ngls.org/document/pdf/roundup/RU117-AIDS. pdf- 2004-12-01: 2
[11]邵健,扬宇民,撩秉洳等.甲壳素和壳聚糖在生物医学方面的应用[J].南通医学院学报1997,17(1):145-146.
[12]张澄波,梅学文,都奉业.脱己酰壳多糖对肿瘤厦免疲系统作用的研究[J].中国实验临床免疲学杂志, 1992,4(1):1-4.
[13]王芳宇,杨露青,周艺等.壳聚糖碘液杀菌效果的实验观察[J].衡阳医学院学报, 2000, 28 (5): 442-443.
[14]高怀生,黄是是,张世达等.碘壳聚糖生物敷料的制备[J].中国药学杂志, 1996, 31(5): 280~282.
[15]王科兵,肖激文,王芳宇.壳聚糖碘液对乙型肝炎病毒血清指标物灭活效果观察[J].中国消毒学杂志, 2004, 21(3): 244-245.
[16]白天玺,张庆华.现代口腔念珠菌药学[M] .北京:人民军医出版社.1995.237-238.
[17]郑之北,刘伟光,吴亦栋等.儿童脑脊液中人疱疹病毒的基因芯片检测[J].中华传染病杂志, 2007, 11: 32.
[18]张伟.宫颈单纯疱疹病毒感染检测分析[J].中原医刊, 2005, 12: 23.
[19]周南进,谢勇,李弼民等.壳聚糖抗幽门螺杆菌的实验研究[J].中国生化药物杂志, 2003, 24(2): 71-73.
[20]何淑雅,王芳宇,李邦良等.壳聚糖碘液的制备及抗真菌活性研究[J].南华大学学报, 2001, 29(3): 227-229.
[21]叶亚明.紫外分光光度法测定碘化钾溶液含量[J].海峡药学, 2(X)3, 15(l): 34-35.
[22]崔胜云,池善女,刘立春等.碘化壳聚糖的制备及其抗菌活性的研究[J].中国生化药物杂志, 2005, 26(3): 138-140
[1] Alliance for Microbicide Development. http://www.microbicide.org/cs/about_microbicides
[2] Klasse PJ, Shattock R, Moore JP. Antiretroviral drug-based microbicides to prevent HIV-1 sexual transmission[J]. Annu RevMed, 2008, 59: 455-471.
[3] Shattock RJ, Moore JP. Inhibiting sexual transmission of HIV-1 infection.[J]. Nat Rev Microbiol. 2003, Oct, 1(1): 25-34.
[4] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections[J]. Nat Rev Drug Discov, 2002, Dec, 1(12) : 977-985
[5]贲昆龙.杀微生物剂与性传播疾病的预防[J].中国性病艾滋病防治, 2002, 7(1) : 59-61.
[6] Microbicide and prep candidates in ongoing clinical trials summary as of october 2009. http://www.microbicide.org/cs/clinical
[7] HIV/STI Prevention Research and Development: October 2009 Pipeline Update of Microbicide and PrEP Candidates. http://www.microbicide.org/cs/pipeline
[8] Global Investment in Microbicide R&D http://www.microbicide.org/cs/investments_expenditures
[9]王睿睿,葛争鸣,马丽等.具有避孕作用的杀微生物剂[J].中国新药杂志, 2005, 14(3): 274-277. (Wang RR, Ge ZM, Ma L, et al. Microbicides with contraceptive action[J]. Chinese Journal of New Drugs, 2005, 14(3): 274-277.
[10] Van Herrewege Y, Michiels J, Van Roey J, et al. In vitro evaluation of nonnuceloside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virus microbicides[J]. Antimicrob Agents Chemother, 2004, 48 (1): 337-339.
[11] L.Van Damme, A. Wright, K. Depraetere, et al. A phase I study of a novel potential intravaginal microbicide, PRO 2000, in healthy sexually inactive women[J]. Sex Transm Infect, 2000 April, 76(2): 126–130.
[12] Joshi Smita, Dutta Soma, Bell Beverly, et al. Phase I safety study of 0.5% PRO2000 vaginal Gel among HIV un-infected women in Pune[J]. AIDS Research and Therapy, 2006, 3: 4.
[13] J. Gerardo García-Lerma, Ron A Otten, Shoukat H Qari, et al. Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir[J]. PloS Med, 2008, February, 5(2): e28.
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