Theaflavin Derivatives As A Microbicide Candidate For Preventing Sexual Transmission Of HIV

BACKGROUND:The AIDS epidemic becomes more and more serious in the world. Human immunodeficiency virus (HIV) through Sexual transmission currently is the primary means of AIDS epidemic. More than 90%of new HIV infections are spreading through unprotected intercourse in whole world, and women are especially vulnerable. Many HIV prevention approaches are used to decrease the risk of HIV transmission and infection, including abstinence, monogamy and regular use of condoms. However, these are not the most effective ways for women to combat HIV in regions of highest HIV prevalence, where socio-economic and cultural factors play strong roles. Therefore, development of topical microbicides that prevent sexual transmission of HIV has become a top global health priority and important research interests.In recent years, tremendous progress has been made in understanding the HIV-1 entry process, in which the fusion of the viral and cellular membranes results in the subsequent delivery of the viral genome into the host cell. The process of HIV fusing with target cells, mediated by the HIV envelope glycoprotein subunit gp120 and transmembrane subunit gp41, is an important step for drug intervention. The membrane fusion events leading to HIV entry into the target cells are initiated by binding of gp120 to CD4 receptor and subsequently to a coreceptor, CXCR4 or CCR5. Consequently, gp41 undergoes conformational changes, resulting in the fusion between the viral and cellular membranes or between the membrances of HIV-infected and uninfected cells. The mechanistic insight gained from these studies has led to the possibility to formulate new approaches for therapeutic and preventive interventions. It is one of the main strategies to develop anti-HIV microbicide to block the essential step in virus life cycle and to control their duplication and spreading.With their wide variety of sources, structural diversity, effective and low toxicity characteristics, the natural sources of HIV entry inhibitors become hot spots of recent research and development of new anti-HIV-1 drug. Theaflavin derivatives are the major components of tea polyphenols in brewed black tea, which have the benzotropolone structure. At present time, a total of 28 kinds of types of theaflavins have been found and identified, which consist of four main compounds:theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B) and theaflavin-3-3'-digallate (TF3). Now many studies from dometic and abroad confirmed their anti-tumor, anti-inflammatory, anti-virus, anti-oxidative, anti-antibacterial anti-proliferative effects. Therefore, theaflavin derivatives as natural product have high potential to develop. We previously demonstrated that these four compounds could effectively inhibit HIV-1 fusion/entry by interacting with gp41 pocket region and blocking the gp41 core formation. Using computer-aided molecular docking analyses, TF3 may bind to the highly conserved hydrophobic pocket on trimeric coiled-coil formed by the N-terminal heptad repeats (NHR) of gp41.Tea as health beverage was widely used over centuries, for it was abundantly available and highly safe. Since it has no adverse effect and toxic effect, theaflavins can be used as a "microbicide" for prevention of transmission of HIV infection. In addition, studies have shown that theaflavins are anti-inflammatory compounds. An ideal microbicide should have four basic natures, which are safe, effective, easy to use and inexpensive. The properties of theaflavin derivatives indicate that they could be developed as a second-generation microbicide for prevention of sexual transmission of HIV with their clear inhibitory activities against HIV and significant advantages.Though individual theaflavin derivative compounds showed potent anti-HIV, the pure theaflavin compound is unstable and difficult to purify. However, compared to individual theaflavin derivatives, we found that a mixture of theaflavin derivatives can lead to more stability at pH values lower than 6. This property is particularly advantageous for developing it as a vaginal microbicide, as it is well known that healthy women maintain acidic pH in the range of 4.0 to 5.0 in the vaginal microenvironment, which enhances the natural defense mechanism. Therefore, theaflavins can be developed as a cheap, safe and stable microbicide candidate for the prevention of sexually transmitted HIV-1.OBJECTIVE:To study the anti-HIV effects and the mechanisms of action of a theaflavin derivative mixture, in which the content of the above four compounds reached to 90%. Meanwhile,2% TF gel was prepared and the safety assessment of 2% TF gel was done from the cellular level and animal level. This study establishes foundation for the eventual successful development of a safe and effective and stable anti-HIV microbicide candidate for the prevention of HIV-1.sexually transmitionMETHODS.1. Theaflavin derivatives mixture (TF) was chosen to investigate because the pure compound was unstable in neutral or alkaline solution and difficult to separate and purity. Contents of TF were determined by reversed phase-high performance liquid chromatography (RP-HPLC).2. The inhibitory activity of TF on p24 antigen production of laboratory-adapted HIV-1 strains and drug-resistant strains was measured by ELISA assay. The cytotoxicity of TF on human immune cells was determined using XTT method.3. Time-of-addition assay and HIV envelope-mediated cell-cell fusion assay were used to determine the active site of TF in the HIV replication cycle.4. The inhibitory activities of those compounds on HIV gp41 six-helix bundle formation were screened by enzyme linked immunosorbent assay(ELISA) and futher identified by native-polyacrylamide gel electrophoresis (N-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC). The inhibitory activity of TF on the binding of gpl20 to CD4 was determined using ELISA assay. The inhibitory activity of TF against HIV-1 reverse transcriptase was also determined using a commercial kit.5. PAP251-286 was a synthetic peptide derived from prostatic acidic phosphatase (PAP) sequence. The effect of TF on PAP-derived amyloid fibril formation was observe by transmission electron microscopy.6. The stability of TF under different pH conditions was compared in citrate-phosphate buffer solutions.7. Based on single factor test, the optimum prescription is optimized by the index of accumulated amount and viscosity for TF gels using orthogonal design with three factors such as Carbopol(?) 974P concentration, glycerin, propylene glycol and three levels. The preliminary stability of TF gel was studied by stability test, including heat experiments, cold experiment, centrifugal experiments.8. The cytotoxicity of TF on immortalized human vaginal cells (VK2/E6E7, Ectl/E6E7 and Endl/E6E7) was measured by the XTT assay. Skin irritation testing and rabbit vaginal irritation test were used to evaluate safety associated with TF gel application. PCNA immunostaining was performed to evaluate the inflammation status of vaginal tissue sections. The levels of proinflammatory (IL-1β, IL-6, IL-8 and TNF-α) and immuno-regulatory cytokines (IL-10 and GM-CSF) in CVLs were measured by using ELISA assay. The in vivo absorption of TF after vaginal application of TF gel was assessed by RP-HPLC.RESULTS:1. Four major theaflavins derivatives, which were theaflavins (TF1), theaflavins-3-gallate (TF2A), theaflavins-3'-gallate (TF2B), and theaflavins-3-3'-digallate (TF3), were identified in the mixture. These four compounds accounted for 90%of the TF weight.2. TF displayed greater inhibitory activity on a number of lab-adapted HIV-1 strains, without distinction of X4 and R5 strains. TF also showed highly potent in inhibiting infection by drug resistant HIV-1 strains. TF showed low cytotoxicity on the human immune cells by cell cytotoxicity experiment. The results suggest that TF has a broad spectrum of antiviral activity.3. The results of time-of-addition assay showed that TF began to lose inhibition activity on p24 production when it was added to virus-cell mixture 2 h post-infection. It suggested TF inhibits HIV infection in the early life cycle of virus. The fusion between H9/HIV-1ⅢB cells and MT-2 cells significantly decreased when TF was added to cells in a dose-dependent manner with an IC50 of 1.788μg/ml. It is indicated that TF can inhibit HIV-1 entry by blocking HIV-1 Env-mediated membrane fusion.4. TF had an obvious inhibitory activity on the formation of HIV gp41 six-helix bundle, with the IC50 of 10.07μg/ml. We then confirmed the inhibitory activity of TF on the formation of six-helix bundle by using two biophysical methods, native-polyacrylamide gel electrophoresis (N-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC). Peptides could be separated by N-PAGE, according to their molecular weight and electrical properties. TF was able to inhibit the formation of HIV gp41 six-helix bundle significantly. Therefore, it was further proved that TF had the inhibitory activities on HIV gp41 six-helix bundle formation, corresponding to the results of ELISA. The SE-HPLC method was used to detect the inhibitory activity of TF on the formation of HIV gp41 six-helix bundle based on the differences of peak areas of six-helix bundle. TF could reduce the peak area of six-helix bundle significantly, corresponding to the former results of ELISA and N-PAGE. Combining together, it showed that TF could target HIV gp41 and inhibit the formation of six-helix bundle, and might play a role in blocking HIV entry into target cells. Binding of gp120 to CD4 is thought to be the initial step of HIV entry. TF shows a remarkable ability to inhibit the binding of gp120 with sCD4 in a dose-dependent manner with an IC50 of 1.22μg/ml. However, TF inhibits HIV-1 reverse transcriptase activity at high concentration. The IC50 of TF on RT activity is 34.88μg/ml, which is 30-300-fold higher than the effect of TF on gp416-HB formation and gp120-CD4 interaction. 5. Peptides in human semen, like PAP251-286, termed semen-derived enhancer of viral infection (SEVI), can enhance HIV infection potently when it formed amyloid fibrils. Therefore, SEVI was regarded as the main factor for promoting HIV-1 sexual transmission. PAP251-286 at a concentration of 8 mg/ml could form larger, typical fibrillary structures and was highly stable over a time period of 36 h. TF at a concentration of 2 mg/ml formed small aggregates. When PAP251-286 (8 mg/ml) was incubated with TF (2 mg/ml), no fibrils were seen, even 36 h later, by transmission electron microscope. This result showed that TF could interfere with the amyloid formation of SEVI, which effectively abrogates semen-mediated enhancement of HIV-1 infection.6. No notable change in peak areas of TF was observed during 24 h when TF was incubated in buffer solutions at pH 3-6. In contrast, TF was unstable at pH 7 and above, showing decrease of TF concentration at pH 7,8, and 9, respectively, after 4 h incubation. Most TF was degraded 24 h after treatment under pH 7-9. Therefore, the stability tests indicated that TF was stable at acid condition with pH value less than 6.7. The formulation containing 2%TF was considered most feasible for preclinical and clinical studies. TF gel at 4%concentration could not be absorbed for a long time because of the high concentration of active pharmaceutical effective ingredients. The best prescription optimized with orthogonal design contained 1 %Carbopol(?)974P,0.75%glycerin,15%propylene glycol. The pH of the final gel formulation was at range 4.1-4.7. The in vitro release profiles of TF gel were best described by the Higuchi model. TF gel is stabe at room temperature for X days tested.8. TF had low cytotoxicity on vaginal and cervical epithelial cells with a selection index (SI) more than 100. Application of TF gel did not cause skin irritation. The histopathological examination of cervicovaginal tissues was performed after repeated intravaginal application of TF gel. The results were compared with N-9 gel and placebo gel. TF gel was administered intravaginally once daily to three female rabbits (1 ml/day) for 14 days. TF gel has no vaginal mucosa irritation on rabbit. No significant difference in histopathological examination was observed between placebo and TF-treated groups which retained structural integrity of the vaginal epithelium. However, N-9 was clearly identified as the most irritating compound with multifocal damage of the vaginal mucosa, which showing congestion, edema, inflammatory infiltration, and epithelial disruption. The score of N-9-treated animals belongs to very acute irritation. Following the intravaginal administration of TF gel to rabbits (1 ml/day), the levels of various proinflammatory (IL-1β, IL-6, IL-8 and TNF-a) and immunoregulatory cytokines (IL-10 and GM-CSF) were not statistically different compared with placebo controls. In contrast, N-9 gel induced an obvious inflammatory reaction. CVL cytokine levels agreed with cumulative mucosal irritation scores after multiple applications (P<0.01).No significant differences in PCNA positivity were noted in the epithelium or stromal cells of placebo-and TF-treated tissues. However, PCNA expression was increased in epithelium or stromal cells after N-9 gel administered vaginally. No systemic absorption was detected in plasma after a single vaginal administration of TF gel. However, TF at high concentration could be detected in the CVLs even after 1 h. Most importantly, there is still a detectable concentration of TF components in the CVLs even after 6 h.