In Vitro Evaluation Of Anti-HIV-1 Activity For Three Types Of Microbicide Candidates And Recipe San Huang San

By today it remains difficult to prevent and treat AIDS (Acquired Immunodeficiency Syndrome), thus it is very important to develop an efficient, low-cost and safe microbicide to prevent sexual HIV (Human Immunodeficiency Virus) transmission and to look for new medicine for AIDS treatment. Microbicide is an agent that inactivates or inhibits sexually transmitted pathogens ,especially HIV-1, and is a topically applied agent before intercourse. At present, 23 microbicide candidates have entered clinical trails. It is estimated that at least one microbicide will be on the market as early as 2007. In order to find microbicide candidates, using 3 in vitro assays including inhibition of syncytial formation, MTT colorimetric assay and quantitative enzyme-linked immunosorbent assay (ELISA), we detected anti-HIV activities of polysaccharides sulfate or non-sulfate and rare earth compounds. The inhibition of HIV-1 _â…¢B induced CPE (cytopathic effect) was used to measure the anti-HIV activities of all agents. The CPE was assessed by counting the number of syncytia formation under microscope. HIV-1R5 strains are major virus for HIV-1 transmission, therefore, detecting anti-HIV-l_Ada-M (R5 use virus) activities of a compound is very important. We used p24 ELISA method which can objectively reflect amount of HIV-1 p24 antigen in cell culture supernatant to measure percentage of inhibition of HIV-1_Ada-M p24 expression by a compound. The cytotoxicities of all agents were tested by MTT colorimetric assay. This assay is based on the MTT formazan formation by mitochondrial enzymes in viable host cells. The blue color of the MTT formazan is objectively depended on the number of viable cells. Furthermore, the time-of-addition experiments were performed in order to dertermine the steps of the replicative cycle of HIV-1 possibly inhibited by the compounds. The inhibiton of the compounds on cell-cell fusion was assayed by coculturing 1×10⁴ chronically infected H9/ HIV-1_â…¢B cells with 3×10⁴ uninfected C8166 cells, which can determine whether the compounds may inhibit the fusion of infected cells with uninfected cells. On basis of the results of above investigation, we tested anti-HSV- â…¡ (type â…¡ human herpes simplex virus) activities of some compounds byprotective assay of HSV- II infected Vero cells. Additionally, we also utilized above assays to measure in vitro anti-HIV-1 activity of recipe San Huang San capsule used for AIDS treatment.The data demonstrated that Ql and DN1 among polysaccharides had high anti-HIV-1 me activities, and their effective concentration (EC50) were 8.664 and 2.542 ug/ml, respectively, and their therapeutic index (TI) were 301 and 1458, respectively, However, the others had very weak anti-HIV-1 hib activities. A panel of polysaccharide sulfate (Q4-1, Q4-2, 728A, 728B, 728C, 728C-1/728D, 728E, 728F, 821 A, 821D, 82IE) had very strong anti-HIV-1 mB activities, and their EC50 were 1.878, 2.171, 0.5851, 0.582, 0.882, 0.831, 0.677, 0.566, 0.5431, 0.598, 1.145 and 0.629 ug/ml, respectively, and their TI were 4214, 2648, 10597, 15964, 8818, 3795, 9653, 8623, 10449, 11885, 8734, 13954, respectively; The anti-HIV activities of 821A,821D and 821E were tested by p24 ELISA assay for R5 strain (HrV-lAda.M) and their EC50 were 13.01, 1.295, 2.635 ug/ml, respectively, and their TI were 385, 1931 and 1712,respectively.Among rare earth compounds we tested, LN43 and LN43-2 possesed highanti-HIV-li]IB activities, and their EC50 were 13.7 and 7.23 ug/ml, respectively, and their TI were 221 and 273 , respectively; However, they had low anti-HIV-lAda-M activities, and their EC50 were 332.0uM and 282.0uM, respectively, and their TI were 22 and 18, respectively.Recipe San Huang San capsule not only had low anti-HIV-1 hib activity, but also did very weak inhibitory activity on and- HIV-lAda-M ? For HlV-lms its EC50 and TI were 54ug/ml and 28, respectively, and for HIV-lAda-M its EC50 and TI were 286ug/ml and 6.16, respectively.In order to explore the active mechanism of 821A ,LN43 and LN43-2, we performed the time-of-addition experiment and inhibition of cell fusion, the results showed that they might target to early stage (binding/fusion) of HIV-1 replicate cycle.Furthermore, EC50 of 821A, 821D, 821E, Q4-1 and Q4-2 in anti-HSV activity detected by infected cell protective assay were 85.62, 94.7, 74.3, 69.9 and 13.98 ug/ml, respectively, and their TI were 114, 25, 33, 103 and 106, respectively; These results demonstrated that 821 A, Q4-1 and Q4-2 had high anti-HSV activities.In conclusion, among the compounds detected, Ql, DN1, Q4-1, Q4-2, 728A,728B, 728C, 728C-1, 728D, 728E, 728F, 821 A, 821D, 821E, LN43, LN43-2 showed strong anti-HIV activities, and 821A, Q4-1, Q4-1, 821D and 821E had anti-HSV activities. These results suggest that they may be potential candidates for microbicides development. Furthermore, recipe San Huang San capsule for AIDS treatment has low anti-viral activities, and it is consistent with initially clinical trial.