A 13-week subchronic intravaginal toxicity study of pokeweed antiviral protein in mice.

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a broad-spectrum antiviral agent. PAP shows unique clinical potential to become the active ingredient of a non-spermicidal microbicide because of its potent in vivo anti-HIV activity, non-interference with in vivo sperm functions, and lack of cytotoxicity to genital tract epithelial cells. Over 13 weeks the subchronic and reproductive toxicity potential of an intravaginally administered gel formulation of PAP was studied in mice to support its further development as a vaginal microbicide. Female B6C3F1 and CD-1 mice in subgroups of 20, were exposed intravaginally to a gel formulation containing 0, 0.025, 0.05, or 0.1% PAP, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations are 500- to 2000-times higher than the in vitro anti-HIV IC50 value. After 13 weeks of intravaginal treatment, B6C3F1 mice were evaluated for survival, body weight gain, and absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo-control and PAP-dosed female CD-1 mice were mated with untreated males in order to evaluate if PAP has any deleterious effects on reproductive performance. There were no treatment-related mortalities. Mean body weight gain was not reduced by PAP treatment during the dosing period. The hemogram and blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PAP for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in the PAP dose groups. Extensive histopathological examination of tissues showed no increase in treatment-related microscopic lesions in any of the three PAP dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PAP for 13 weeks showed no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%) or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of PAP at concentrations as high as 2000 times its in vitro anti-HIV IC50 value was not associated with local or systemic toxicity and did not adversely affect the reproductive performance of mice. PAP may be useful as an active ingredient of a safe vaginal microbicide for prevention of the sexual transmission of viruses, particularly of HIV-1.

Subchronic (13-week) toxicity studies of intravaginal administration of spermicidal vanadocene dithiocarbamate in mice.

Spermicidal organometallic complexes of vanadium(IV) with bis(cyclopentadienyl) rings or vanadocenes are a new class of experimental contraceptive agents. In a systematic search for vanadocenes with selective spermicidal activity, we identified vanadocene dithiocarbamate (VDDTC) as the most potent and stable spermicidal compound. In this study, groups of 10 B(6)C(3)F(1) and 20 female CD-1 mice were exposed intravaginally to a gel-microemulsion containing 0, 0.06, 0.12, and 0.25% VDDTC 5 days per week for 13 consecutive weeks. The doses of VDDTC used were nearly 1250- to 5000-fold higher than its in vitro spermicidal EC(50) value. After 13 weeks of intravaginal treatment, B(6)C(3)F(1) mice were evaluated for survival, body weight gain, absolute and relative organ weights, and systemic toxicity. Blood was analyzed for hematologic and clinical chemistry parameters. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Vanadium content in tissues was determined by atomic absorption spectroscopy. Placebo control and VDDTC-dosed female CD-1 mice were mated with untreated males to evaluate whether VDDTC has any deleterious effects on the reproductive performance. There were no treatment-related effects on survival and mean body weight and mean body weight gain during the dosing period. The blood chemistry or hemogram profiles did not reveal any toxicologically significant changes that could be attributed to VDDTC treatment. No clinically significant changes in absolute and relative organ weights were noted in VDDTC dose groups. Extensive histopathological examination of tissues revealed no treatment-related abnormalities in any of the three VDDTC dose groups. The vanadium content of all mouse tissue analyzed was <1 microg/g. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of VDDTC for 13 weeks had no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%), or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of VDDTC to yield effective spermicidal concentrations (<0.1%) in the vagina was not associated with systemic toxicity and did not adversely affect the reproductive performance in mice. VDDTC may have clinical utility as an active ingredient of non-detergent type, safe, vaginal spermicidal contraceptives.

Lack of adverse effects on fertility of female CD-1 mice exposed to repetitive intravaginal gel-microemulsion formulation of a dual-function anti-HIV agent: aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07).

5-bromo-6-methoxy-5,6-dihydro-3(')-azidothymidine-5(')-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent with anti-HIV activity. Its potential for reproductive toxicity was assessed in a series of experiments using CD-1 mice under the conditions of its intended use as an intravaginal microbicide. Female CD-1 mice were exposed intravaginally to a gel-microemulsion formulation containing 0%, 0.5%, 1.0% or 2.0% WHI-07 for up to 13 weeks. On a molar basis, these concentrations represent 1400-5700 times its in vitro spermicidal IC(50) and 1.4-5.7(x10(6)) times its in vitro anti-HIV IC(50). We examined the effects of intravaginally administered WHI-07 on: ovulation efficiency; in vivo fertilization and early embryonic, fetal development; and reproductive outcome, including neonatal survival and pup development. Compound WHI-07 was administered intravaginally during superovulation, organogenesis and prior to mating for 5 and 10 consecutive days and for 13 weeks, respectively. Mice were evaluated for ovulation efficiency and fertilization rate and cleavage 14 and 40 h after human chorionic gonadotropin (hCG) injection, respectively. Pregnant mice were administered 2% WHI-07 intravaginally during gestation days (GD) 6-15 and measures of teratogenicity were evaluated on GD 17. For short-term toxicity study, mice were given intravaginal treatment of gel-microemulsion containing 0%, 0.5%, 1.0% and 2.0% WHI-07 for 13 weeks and then mated with untreated males to evaluate potential reproductive and developmental effects. Repeated intravaginal exposure of mice to 2% WHI-07 had no adverse effects on ovulation response, mean number of eggs recovered or the percentage of eggs fertilized or cleaved. No evidence of reproductive toxicity, fetal toxicity or teratogenicity was found following repetitive intravaginal application of 2% WHI-07 during the period of organogenesis. Furthermore, repeated intravaginal exposure of mice to 0.5-2.0% WHI-07 for 13 weeks had no adverse effect on the subsequent reproductive capability, perinatal outcome or growth and development of the offspring. Compound WHI-07 shows unique clinical potential as a safe, dual-function vaginal contraceptive for curbing mucosal and perinatal HIV transmission.

GM-144, a novel lipophilic vaginal contraceptive gel-microemulsion.

In a systematic effort to develop a dual-function intravaginal spermicide as well as a drug delivery vehicle against sexually transmitted pathogens, a submicron particle size (30-80 nm), lipophilic and spermicidal gel-microemulsion (viz GM-144) containing the pharmaceutical excipients propylene glycol, Captex 300, Cremophor EL, Phospholipon 90G, Rhodigel, Pluronic F-68, and sodium benzoate was formulated. GM-144 completely immobilized sperm in human or rabbit semen in less than 30 seconds. Therefore, the in vivo contraceptive potency of intravaginally applied GM-144 was compared in the standard rabbit model to those of the detergent spermicide, nonoxynol-9 (N-9)-containing formulation. Eighty-four ovulated New Zealand White rabbits in subgroups of 28 were artificially inseminated with and without intravaginal administration of GM-144 or 2% N-9 (Gynol II) formulation and allowed to complete term pregnancy. GM-144 showed remarkable contraceptive activity in the rigorous rabbit model. When compared with control, intravaginal administration of GM-144 and Gynol II resulted in 75% and 70.8% inhibition of fertility (P <.0001 versus control, Fisher's exact test), respectively. Thus, GM-144 as a vaginal contraceptive was as effective as the commercially available N-9 gel. In the rabbit vaginal irritation test, none of the 6 rabbits given daily intravaginal application of spermicidal GM-144 for 10 days developed epithelial ulceration, edema, leukocyte influx, or vascular congestion characteristic of inflammation (total score = 5). Therefore, GM-144 has the potential to become a clinically useful safe vaginal contraceptive and a vehicle for formulating lipophilic drugs used in reducing the risk of heterosexual transmission of sexually transmitted diseases.

Novel thiourea compounds as dual-function microbicides.

Sexually active women represent the fastest growing human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we previously reported the synthesis of novel nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase with sperm-immobilizing activity (SIA). To gain further insight into the structure-function relationship controlling these two properties of NNIs, we have rationally designed and synthesized 30 novel thiourea compounds and examined them for dual-function, anti-HIV and spermicidal activity. Twelve of the 30 thiourea compounds exhibited potent anti-HIV activity in the nanomolar range (IC(50) = <1-9 nM). Nine of the 30 thiourea derivatives exhibited both anti-HIV and spermicidal activity. Among the phenyl ring-containing thioureas, the 2-fluoro (HI-240) -substituted and 2-chloro (HI-253) -substituted derivatives exhibited potent anti-HIV activity (IC(50) = <1 nM) with SIA (EC(50) = 70 microM and 147 microM). Among the alicyclic ring-containing thioureas, the 5-bromo (HI-346) and 5-chloro (HI-445) functionalized cyclohexenyl ring-substituted thioureas were the most potent dual-function spermicides (EC(50) = 42 and 57 microM), with anti-HIV activity at nanomolar range (IC(50) = 3 nM). Unlike nonoxynol-9 (N-9), none of the potent dual-function thiourea compounds were cytotoxic to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations. We conclude that as potent anti-HIV agents with SIA and reduced cytotoxicity when compared with N-9, the phenyl-substituted and cyclohexenyl-substituted thiourea derivatives, especially compounds HI-253 (N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea), and HI-445 (N-[2-(5-chloropyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea) show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Prophylactic contraceptives for HIV/AIDS.

The current pandemic of sexually transmitted human immunodeficiency virus (HIV) infection--the causative agent of acquired immunodeficiency syndrome (AIDS), has created an urgent need for a new type of contraceptive: one that is both a spermicide and a microbicide. Because most women at risk for HIV infection are of reproductive age (15-44 years), effective use of dual-function contraceptives is important to prevent HIV transmission and unintended pregnancies. In the absence of an effective prophylactic anti-HIV therapy or vaccine, new emphasis has been placed on the development of intravaginal microbicidal agents capable of reducing the transmission of HIV. Topical microbicidal spermicides would ideally provide a female-controlled method of self-protection against HIV as well as preventing pregnancy. However, several microbicides that are undergoing preclinical and human clinical trials contain detergent-type ingredients. The detergent-type spermicide, nonoxynol-9, the only recommended microbicide for protection against sexual transmission of HIV has been shown to cause lesions in vaginal and cervical epithelia leaving women more vulnerable to HIV infection. Therefore, a major challenge in microbicide research has been to design mechanism-based microbicides that are highly effective against pregnancy and HIV transmission while lacking detergent-type effects on epithelial cells and normal vaginal flora. We present an overview of current microbicide research and report on the identification and preclinical development of novel non-detergent spermicidal nucleoside and non-nucleoside inhibitors aimed at decreasing pregnancy and preventing sexual transmission of HIV.

Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities.

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.