Designed Chemical Intervention with Thiols for Prophylactic Contraception

Citation: Sharma M, Kumar L, Jain A, Verma V, Sharma V, et al. ( Designed Chemical Intervention with Thiols for Prophylactic Contraception Gopal Gupta 0 Monika Sharma 0 Lokesh Kumar 0 Ashish Jain 0 Vikas Verma 0 Vikas Sharma 0 Bhavana Kushwaha 0 Nand Lal 0 Lalit Kumar 0 Tara Rawat 0 Anil K. Dwivedi 0 Jagdamba P. Maikhuri 0 Vishnu L. Sharma 0 Suresh Yenugu, University of Hyderabad, India 0 1 Division of Endocrinology, CSIR-Central Drug Research Institute , Lucknow , India , 2 Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute , Lucknow , India , 3 Division of Pharmaceutics, CSIR-Central Drug Research Institute , Lucknow , India Unlike somatic cells, sperm have several-fold more available-thiols that are susceptible to redox-active agents. The present study explains the mechanism behind the instant sperm-immobilizing and trichomonacidal activities of pyrrolidinium pyrrolidine-1-carbodithioate (PPC), a novel thiol agent rationally created for prophylactic contraception by minor chemical modifications of some known thiol drugs. PPC, and its three derivatives (with potential active-site blocked by alkylation), were synthesized and evaluated against live human sperm and metronidazole-susceptible and resistant Trichomonas vaginalis, in vitro. Sperm hexokinase activity was evaluated by coupled enzyme assay. PPC irreversibly immobilized 100% human sperm in ,30 seconds and totally eliminated Trichomonas vaginalis more efficiently than nonoxynol-9 and metronidazole. It significantly inhibited (P,0.001) thiol-sensitive sperm hexokinase. However, the molecule completely lost all its biological activities once its thiol group was blocked by alkylation. PPC was subsequently formulated into a mucoadhesive vaginal film using GRaS excipients and evaluated for spermicidal and microbicidal activities (in vitro), and contraceptive efficacy in rabbits. PPC remained fully active in quick-dissolving, mucoadhesive vaginal-film formulation, and these PPC-films significantly reduced pregnancy and fertility rates in rabbits. The films released ,90% of PPC in simulated vaginal fluid (pH 4.2) at 37uC in 5 minutes, in vitro. We have thus discovered a common target (reactive thiols) on chieflyanaerobic, redox-sensitive cells like sperm and Trichomonas, which is susceptible to designed chemical interference for prophylactic contraception. The active thiol in PPC inactivates sperm and Trichomonas via interference with crucial sulfhydryl-disulfide based reactions, e.g. hexokinase activation in human sperm. In comparison to non-specific surfactant action of OTC spermicide nonoxynol-9, the action of thiol-active PPC is apparently much more specific, potent and safe. PPC presents a proof-of-concept for prophylactic contraception via manipulation of thiols in vagina for selective targeting of sperm and Trichomonas, and qualifies as a promising lead for the development of dually protective vaginal-contraceptive. - Funding: Authors acknowledge the grant of research fellowships by the University Grants Commission, New Delhi (LK, VS, NL), the Indian Council of Medical Research, New Delhi (VV, AJ) and the Council of Scientific and Industrial Research, New Delhi (LK), India. VV is enrolled with the Integral University, Lucknow, India, for PhD. This study was supported by grants from the CSIR Network Project (PROGRAM) and Ministry of Health and Family Welfare, Government of India. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The transmission of fertile spermatozoa and sexually transmitted disease (STD) pathogens during heterosexual contacts may lead to frequent unwanted pregnancies (mostly ending in abortions) [1], and infections. Trichomoniasis, the most prevalent non-viral STD, predisposes women to viral STDs, HIV/AIDS and cervical cancer; and newborns to pre-term delivery, low birth weight and high mortality rate [2]. Prophylactic contraceptives targeting both sperm and Trichomonas could be an ideal strategy to prevent the heterosexual spread of trichomoniasis since contraception is desired during majority of sexual acts. Unfortunately metronidazole, (the FDA-approved drug against Trichomonas vaginalis) lacks contraceptive activity, has insufficient intra-vaginal efficacy [3] and proves ineffective against resistant Trichomonas [4]. Some non-imidazoles have shown intra-vaginal potency against metronidazole-resistant Trichomonas infection [5], but are devoid of contraceptive activity. Nonoxynol-9 (a non-ionic detergent), which forms the active ingredient in most OTC spermicides, kills sperm and STD pathogens (including Trichomonas) by its non-specific, surfactant action. However, clinical trials have shown that repeated use of N-9 containing vaginal products could harm the vaginal mucosa and increase susceptibility to STDs, including HIV [6,7]. According to an estimate, human sperm contain .55 nmoles of reactive thiols per 108 cells which are ,30 times more than those on erythrocytes [8]. The significance of thiols in sperm cell motility/function is evident from the fact that asthenozoospermic infertile men have significantly less thiols on sperm than normozoospermic men [9]. It has already been well established that the motility and metabolism of sperm can be inhibited substantially by agents having affinity for sulfhydryls, the effect being reversible only negligibly in some cases by cysteine and glutathione [10]. Equally important, T. vaginalis lacks glutathione (the intracellular redox buffer), glutathione dependent peroxidase, and catalase, and therefore it relies heavily on cysteine (which constitutes .70% of cells total thiol pool) for protection against redox-stress, making it extremely susceptible to sulfhydryl-manipulating agents [11]. Thus, exploiting thiols as a common target on both sperm and Trichomonas we designed several dually active, nonsurfactant molecular prototypes for prophylactic contraception [1217]. However, a perfect balance of the two activities could not be achieved optimally. Nevertheless, our recent efforts in this direction has yielded a valuable series of dually-active molecules and the most promising structure (pyrrolidinium pyrrolidine-1carbodithioate, PPC) instantly inactivated 100% human sperm more efficiently and specifically than N-9, and completely eliminated Trichomonas vaginalis (resistant and susceptible strains) more potently than metronidazole, in vitro [18]. The human sperm permanently paralyzed by PPC had considerably reduced numbers of free thiols [18]. We now present data to pinpoint the active site on the molecule and its potential interference with the activity of a thiol-sensitive, rate-limiting key enzyme of the sperm energy metabolism. Yet, the microbicidal and contraceptive relevance of such molecules depend on (...truncated)