Development and evaluation of a thermoreversible ovule formulation of stampidine,a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5-2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.     

A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice

For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field.     

Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide

BACKGROUND: The predominant mode of HIV-1 transmission is by heterosexual contact. The cervical/vaginal mucosa is the main port of HIV entry in women. A safe and effective topical microbicide against HIV is urgently needed to prevent sexual transmission. Hence, we evaluated griffithsin (GRFT), a 12.7 kDa carbohydrate-binding protein, both native and recombinant GRFT, potently inhibited both CXCR4-and CCR5-tropic HIV infection and transmission in vitro. METHODS: The antiviral efficacy of native and recombinant GRFT against CXCR4-and CCR5-tropic HIV and SHIV strains and SIVmac251 was evaluated by in vitro assays. We also evaluated the time course of antiviral activity and stability of GRFT in cervical/vaginal lavage as a function of pH 4-8. RESULTS: Griffithsin blocked CXCR4-and CCR5-tropic viruses at less than 1 nm concentrations and exhibited a high potency. GRFT was stable in cervical/vaginal lavage fluid and maintained a similar potency of anti-HIV activity. GRFT is not only a highly potent HIV entry inhibitor, but also prevents cell fusion and cell-to-cell transmission of HIV. CONCLUSIONS: The in vitro efficacy of GRFT revealed low cytotoxicity, high potency, rapid onset of antiviral activity and long-term stability in cervical/vaginal lavage. GRFT is an excellent candidate for anti-HIV microbicide development.     

Conceival,a novel noncontraceptive vaginal vehicle for lipophilic microbicides

The objective of this study was to develop a nontoxic and noncontraceptive vaginal drug delivery vehicle for lipophilic anti-human immunodeficiency virus (HIV) microbicides. Three representative poorly water-soluble novel broad-spectrum anti-HIV microbicides, PHI-113, PHI-346, and PHI-443, were evaluated in 11 different solvent systems. Based on their solubility profiles, a novel non-spermicidal self-emulsifying gel (viz Conceival) composed of pharmaceutical excipients, sorbitol, polyethylene glycol 400, polysorbate 80, microcrystalline cellulose, xanthan gum, and water was optimized. Conceival enhanced the solubility of these poorly water-soluble (<0.001 mg/mL) anti-HIV drugs by at least 150- to 270-fold. Conceival was evaluated in vivo in the New Zealand white rabbit model for the preservation of sperm function based on pregnancy outcome and the potential for vaginal irritation following single and multiple intravaginal applications, respectively. Conceival administered intravaginally immediately prior to artificial insemination with semen had no adverse effects on subsequent reproductive performance, neonatal survival, or pup development when compared with untreated control group. Histologic evaluation of vaginal tissues of rabbits exposed intravaginally to Conceival for 14 consecutive days revealed lack of epithelial, submucosal, and vascular changes at the gel application site (total irritation score <3 out of a possible 16). These findings indicate that Conceival has potential to become a clinically useful, safe noncontraceptive vaginal vehicle for lipophilic microbicides. Published: September 20, 2005     

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development

Background  We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates.
Methods  Vaginal and rectal microflora in Chinese rhesus macaques ( Macaca mulatta ) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization.
Results  Chinese rhesus macaques harbored abundant vaginal Lactobacillus , with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals.
Conclusions  The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus -based topical microbicides.     

Dimethyl methyl phosphonate induction of dominant lethal mutations in male mice

The reproductive toxicity of dimethyl methyl phosphonate (DMMP) was studied in the male B6C3F1 mouse. Male mice were treated with 0, 250, 500, 1000, and 2000 mg/kg DMMP by gavage 5 days per week for 13 weeks. After 4, 8 and 12 weeks of treatment the male mice were mated to untreated CD-1 female mice. At the two highest doses (1000 and 2000 mg/kg) the chemical caused a dominant lethal effect (early resorptions). Groups of male mice (at 1000 and 2000 mg/kg), mated after a 15-week recovery period without chemical dosing, had a resorption rate comparable to the control group. After 13 weeks of dosing, the male mice showed no histopathologic changes of the reproductive organs, no abnormalities in sperm concentration or sperm morphology, no evidence for hormone imbalance, no signs of general toxicity, and no effects on the fertilization rate. The male B6C3F1 mouse was less responsive than the male Fischer 344/N rat to the reproductive toxic effects of DMMP.     

Quantitative evaluation of steroid extrogens by study of the vaginal epithelium thickness and mitotic index in mice

The potency of estrone, estradiol, estriol, and equilenin, administered to mice subcutaneously or intravaginally, was quantitated by vaginal mitotic index and by epithelial thickness; results were compared with those previously obtained in the classical tests of rat vaginal cornification and uterotrophic activity in mice. Ovariectomized mice received .002-.7 mcg estrogens in oil sc, or .16-6250 pg in alcohol solution intravaginally. 19 hours later .1 mg colchicine was given to arrest cells in metaphase. 24 hours after estrogen treatment, vaginas were a positive log-dose response in both tests and by both routes. Estradiol by both routes increased vaginal thickness but without linear dose-response, increased vaginal mitosis with a less definite dose response, but generated a negative dose response in mitotic index. Equilenin had a positive but nonlinear effect by both routes in both tests. Comparing the activities of these estrogens by routes, estradiol was more active by subcutaneous than by intravaginal routes; estriol and equilenin were more active vaginally than subcutaneously. Estradiol was 3-7 times more active than estrone intravaginally and 25-45 times more active subcutaneously. Estriol was less active than estrone; equilenin was as active as estrone intravaginally, but less active subcutaneously. In comparison with the rat vaginal cornification or mouse uterotrophy tests, estradiol sc, estriol and equilenin sc and especially vaginally, are much more active.     

Routes of inoculation and the immune response to a resolving genital flavivirus infection in a novel murine model

The prolonged, abnormal immune response patterns produced by many sexually transmitted viruses have been intensively studied. Because normal antiviral immune responses in the vagina are less well-defined, we developed a resolving murine model using vaginal inoculation with the flavivirus, West Nile virus. Infection resulted in 12% mortality, with sterile protective immunity to vaginal or systemic re-challenge. B-cell numbers increased in the vaginal mucosa from day 1-7 after primary infection, while similar increases in B220(+), CD4(+) and CD8(+) lymphocytes in the draining lymph node were delayed by 48 h. By day 4 postinfection, a MHC-II(+) dendritic cell population became depleted from the stroma and formed aggregates below the basement membrane at points of demonstrable epithelial infection. In contrast, primary systemic or intradermal inoculation resulted in 80-90% mortality, but also conferred protective sterile immunity to vaginal West Nile virus re-challenge. Intravaginal and intradermal immunization elicited comparable, accelerated accumulation of larger B-cell numbers in the mucosa and draining lymph node upon intravaginal re-challenge than systemic immunization. However, accumulation of CD4(+) T cells in both sites in the intradermally immunized group was significantly greater than in intravaginally or systemically immunized mice. Accelerated accumulation of dendritic cells occurred at periodic sub-basement membrane sites in the absence of detectable virus 1 day after vaginal re-challenge, irrespective of the route of immunization. These data illustrate the diversity of possible effective immune responses to West Nile virus in the vaginal mucosa. They show primary vaginal inoculation produces effective immunity to flavivirus infection with lower mortality than other routes and suggest a local role for vaginal mucosal dendritic cells in both primary and secondary responses.     

Effect of virus dose and nonoxynol-9 on the genital transmission of SIV in rhesus macaques

One inoculation of cell-free SIVmac (50 TCID50) caused persistent viremia in nine of 13 female rhesus macaques inoculated intravaginally. Persistent viremia was produced in two of four male rhesus macaques by twice placing cell-free SIVMAC (50 TCID50) onto the skin and urethral os of the penis. Placing a spermicide containing nonoxynol-9 into the vaginal canal prior to repeated intravaginal inoculations of SIV prevented transmission of the virus in three of six female rhesus macaques.     

乳杆菌细胞壁成分对小鼠阴道组织β-防御素2诱导表达的影响

目的探讨乳杆菌细胞壁成分（Lactobacilluscell wall extract,LCWE）对小鼠阴道组织β-防御素2诱导表达的影响。方法将雌性ICR小鼠随机分成5组,实验组给予不同浓度的（5、20和100 mg/L）LCWE 200μL阴道接种处理;阴性对照组小鼠阴道接种生理盐水,阳性对照组小鼠阴道接种大肠埃希菌EPEC104菌液（3×109CFU/mL）,5 d后处死,取小鼠阴道组织,做阴道菌群分析评价,部分阴道组织提取总RNA,以β-actin为内参照,采用实时荧光定量PCR和Western blot方法检测小鼠阴道组织β-防御素2表达。结果 20 mg/L和100 mg/L浓度LCWE处理后,小鼠阴道菌群较生理盐水组的肠杆菌、葡萄球菌明显减少（P〈0.05）,乳杆菌无显著性变化（P〉0.05）。生理盐水组小鼠阴道组织β-防御素2 mRNA表达量极低,大肠埃希菌阳性对照组能显著诱导小鼠阴道组织β-防御素2 mRNA的表达;5 mg/L浓度LCWE处理组开始,β-防御素2表达量开始提高,在100 mg/L浓度LCWE组其表达量达到最高。Western blot结果显示：生理盐水组小鼠阴道组织未检测到β-防御素2蛋白表达,而100 mg/L浓度LCWE处理组和大肠埃希菌EPEC104组均出现特异性条带。结论 LCWE可上调小鼠阴道组织β-防御素2的表达且存在着剂量依赖关系。     

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background

Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, β-lactoglobulin (β-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.

Results

Maleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than β-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.

Conclusion

ML-OVA is a potent HIV fusion/entry inhibitor with the potential to be developed as an effective, safe and inexpensive anti-HIV microbicide.     

Progesterone increases susceptibility and decreases immune responses to genital herpes infection

Depo-provera, a long-acting progestational formulation, is widely used to facilitate infection of sexually transmitted diseases in animal models. We have previously reported that hormone treatments change susceptibility and immune responses to genital tract infections. In this study we compared the changes in susceptibility of mice to genital herpes simplex virus type 2 (HSV-2) after Depo-provera or a saline suspension of progesterone (P-sal). We found that following Depo-provera-treatment, mice had prolonged diestrus that lasted more than 4 weeks. This coincided with a 100-fold increase in susceptibility to genital HSV-2 compared to that of untreated mice. Mice given P-sal were in diestrous stage for 4 to 6 days before returning to irregular reproductive cycles. When these mice were infected at diestrus they showed a 10-fold increase in susceptibility compared to that of normal, untreated mice. P-sal-treated mice infected at estrus were susceptible to HSV-2, depending on the infectious dose. Normal, untreated mice in estrus were not susceptible to HSV-2, even at a high infectious dose of 10(7) PFU. In addition to alterations in susceptibility, Depo-provera treatment had inhibitory effects on immune responses to HSV-2. Mice immunized with HSV-2 protein (gB) and treated with Depo-provera showed significant lowering of local HSV-2-specific immunoglobulin G (IgG) and IgA in their vaginal washes. Mice immunized with an attenuated strain of HSV-2 2 weeks after Depo-provera treatment failed to develop protection when challenged intravaginally with wild-type HSV-2. In contrast, mice given progesterone and immunized at diestrus or estrus were completely protected from intravaginal challenge. These studies show that Depo-provera treatment changes susceptibility and local immune responses to genital HSV-2 infection. Animal models and vaccine strategies for sexually transmitted diseases need to consider the effect of hormone treatments on susceptibility and immune responses.     

Incomplete protection against simian immunodeficiency virus vaginal transmission in rhesus macaques by a topical antiviral agent revealed by repeat challenges

The rising prevalence of human immunodeficiency virus type 1 (HIV-1) infection in women, especially in resource-limited settings, accentuates the need for accessible, inexpensive, and female-controlled preexposure prophylaxis strategies to prevent mucosal transmission of the virus. While many compounds can inactivate HIV-1 in vitro, evaluation in animal models for mucosal transmission of virus may help identify which approaches will be effective in vivo. Macaques challenged intravaginally with pathogenic simian immunodeficiency virus (SIV(mac251)) provide a model to preclinically evaluate candidate microbicides. 2-Hydroxypropyl-beta-cyclodextrin (BCD) prevents HIV-1 and SIV infection of target cells at subtoxic doses in vitro. Consistent with these findings, intravaginal challenge of macaques with SIV(mac251) preincubated with BCD prevented mucosal transmission, as measured by plasma viremia and antiviral antibodies, through 10 weeks postchallenge. In an initial challenge, BCD applied topically prior to SIV(mac251) prevented intravaginal transmission of virus compared to controls (P < 0.0001). However, upon a second virus challenge following BCD pretreatment, the majority of the previously protected animals became infected. The mechanism through which animals become infected at a frequency similar to that of controls after prior exposure to BCD and SIV(mac251) in subsequent intravaginal virus challenges (P = 0.63), despite the potent antiviral properties of BCD, remains to be determined. These results highlight the unpredictability of antiviral compounds as topical microbicides and suggest that repeated exposures to candidate treatments should be considered for in vivo evaluation.     

New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.     

Formulation Development and Evaluation of Innovative Two-Polymer (SR-2P) Bioadhesive Vaginal Gel

The main objective of this investigation was to study the feasibility of developing a vaginal bioadhesive microbicide using a SRI’s proprietary two-polymer gel platform (SR-2P). Several formulations were prepared with different combinations of temperature-sensitive polymer (Pluronic® F-127) and mucoadhesive polymer (Noveon® AA-1), producing gels of different characteristics. Prototype polymeric gels were evaluated for pH, osmolality, buffering capacity, and viscosity under simulated vaginal semen dilutions, and bioadhesivity using ex vivo mini pig vaginal tissues and texture analyzer. The pH of the polymeric gel formulations ranged from 5.1 to 6.4; the osmolality varied from 13 to 173 mOsm. Absolute viscosity ranged from 513 to 3,780 cPs, and was significantly reduced (1.5- to 3-fold) upon incubation with simulated vaginal and semen fluid mixture. Among the tested gels (indicated in the middle row as a molar ratio of a mixture of Noveon vs. Pluronic), only SR-2P retained gel structure upon dilution with simulated fluids and mild simulated coital stress. The pH of the SR-2P gel was maintained at about 4.6 in simulated vaginal fluid and also showed high peak force of adhesion in mini pig vaginal tissue. Furthermore, SR-2P gel caused no or only minimal irritation in a mouse vaginal irritation model. The results of this preliminary study demonstrated the potential application of SR-2P gel as a vaginal microbicide vehicle for delivery of anti-HIV agents.

Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris

Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of homogeneously pure active PAP without batch-to-batch variation from its natural resource. Here, we report the expression of mature PAP (residues 23 to 284) with a C-terminal hexahistidine tag in the methylotrophic yeast Pichia pastoris, as a secreted soluble protein. The final yield of the secreted PAP is greater than 10 mg/L culture in shaker flasks. The secreted recombinant protein is not toxic to the yeast cells and has an apparent molecular mass of 33-kDa on SDS-PAGE gels. The in vitro enzymatic activity and cellular anti-HIV activity of recombinant PAP were of the same magnitude as those of the native PAP purified from P. americana. To our knowledge, this is the first large-scale expression and purification of soluble and biologically active recombinant mature PAP from yeast.     

Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives

Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.