Novel disulphide esters of carbothioic acid as potent, non-detergent spermicides with low toxicity to Lactobacillus and HeLa cells in vitro

R.K.Jain 2 3 J.P.Maikhuri 2 3 S.T.V.S.Kiran Kumar 1 2 V.L.Sharma 1 2 A.K.Dwivedi 0 2 K.Mitra 2 4 V.K.Bajpai 2 4 G.Gupta 2 3 0 Division of Pharmaceutics 1 Division of Medicinal Chemistry 2 PO Box 173, Lucknow 226001, Uttar Pradesh , India 3 Division of Endocrinology 4 Electron Microscopy Unit, Central Drug Research Institute , Lucknow, Uttar Pradesh , India BACKGROUND: The design, synthesis, characterization and evaluation of a novel series of non-detergent spermicides has led to the discovery of two unique molecules (DSE-36 and DSE-37) that were ~25 times more potent spermicides than nonoxynol-9 (N-9). METHODS: Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer Assay), the effect on sperm-membrane integrity [hypo-osmotic swelling test (HOST)], supravital staining and scanning electron microscopy (SEM) and the induction of apoptosis [fluorescein isothiocyanate (FITC) Annexin-V and JC-1 labelling using flow cytometry] by the new class of compounds. HeLa and Lactobacillus cultures were used to assess the cytotoxicity of compounds and their compatibility to normal vaginal flora, respectively. RESULTS: Compounds DSE-36 and DSE-37 exhibited a strong spermicidal activity [minimum effective concentration (MEC) = 0.002%], which was ~25 times more potent than that of N-9 and Sapindus saponins (MEC = 0.05%). As compared with surfactants, DSE-36 and DSE-37 were found to be safer at MEC towards the growth and survival of Lactobacilli and HeLa cells in vitro and to have a milder effect on sperm plasma membrane. At EC50 both induced apoptosis in sperm cells as characterized by increased labelling with Annexin-V and decreased polarization of sperm mitochondria. CONCLUSION: Preliminary studies have revealed that in sharp contrast to the non-specific surfactant action of N-9, DSE-36 and DSE-37 have a highly potent, mechanism-based, detrimental action on human sperm. The unique ability of these non-detergent molecules to selectively kill sperm and spare Lactobacilli and HeLa cells at MEC values much lower than that required for N-9 indicates their potential as superior ingredients for formulation into microbicidal contraceptives. Introduction The continued high rates of unintended pregnancies and the relentless expansion of the sexually transmitted disease/human immunodeficiency virus (STD/HIV) epidemic, especially in less-developed countries, warrant the development of novel strategies to help individuals avoid these risks (Doncel, 2006). Condoms can provide adequate protection against pregnancy as well as STDs and HIV but is not woman-controlled. In addition to the risk of unwanted pregnancy, women are also at a higher risk than men of acquiring HIV after unprotected sex (Saracco et al., 1993). Despite this, female partners in many relationships do not control pregnancy or STD risk and may benefit from discrete methods other than condoms (Howett and Kuhl, 2005). Microbicidal contraceptives offer a suitable alternative to condoms as the most viable, women-controlled method for dual protection. However, recent clinical trials have shown that nonoxynol-9 (N-9), the most promising spermicidalmicrobicide, not only failed to offer any protection against STDs and HIV but actually increased the incidence of these diseases in the users (Stephenson, 2000; Van Damme et al., 2002). This was mainly attributed to the surfactant (detergent) nature of the compound that caused vaginal inflammation/ lesions after repeated use resulting in increased susceptibility to STDs, including HIV (Fichorova et al., 2001). The WHO has cautioned the users of N-9 containing vaginal preparations especially those who are at high risk of acquiring HIV (WHO/ CONRAD, 2001). Thus, an urgent need has emerged to identify a suitable, safe, non-detergent and potent spermicide that can replace N-9 in vaginal contraceptive preparations/devices. This need is also augmented by the fact that 8090% of new HIV infections are caused by heterosexual contacts (Catalone et al., 2005), especially those contacts in which protective DSE-36 (M.W. 696) DSE-37 (M.W. 724) Nonoxynol-9 (M.W. 616) measures (if any employed) are limited only to pregnancy protection. Hence, unwanted pregnancy may be a greater concern than STDs and HIV during such contacts (Gupta et al., 2005). Consequently, vaginal preparations with potent spermicidal as well as microbicidal properties may be used with more compliance in such relationships than pure microbicides. Therefore, a potent contraceptive activity needs to be integrated essentially in most of the microbicidal preparations so as to ensure its consistent use during the majority of coital acts (Gupta, 2005). Keeping in view the ill effects of N-9, the design, synthesis and evaluation of many non-detergent spermicidal/microbicidal molecules was taken up at the Central Drug Research Institute (Srivastava et al., 1999; Kumaria et al., 2002; Maikhuri et al., 2003; Gupta et al., 2005; Kiran Kumar et al., 2006a,b). Recently, a novel series of non-detergent disulphide esters of dialkylaminocarbothioic acid was discovered and yielded several very promising spermicidal molecules (Dwivedi et al., unpublished). Some of these can potentially replace N-9 in contraceptive preparations. We report here the biological activity, preliminary safety data and basic mechanism of action of two very potent and promising molecules from this series (Figure 1). These compounds have been studied in comparison with N-9 and Sapindus saponins to bring out the salient advantages of these molecules over surfactant spermicides. Materials and methods Human spermatozoa Fresh human semen samples obtained by masturbation into a sterile vial from healthy, young, fertile donors were liquefied for 45 min at 37C and used for in vitro spermicidal and mechanism of action assays. Samples having >65 106/ml sperm count with >70% motility and normal sperm morphology were used in the study. The ethical approval for this study was obtained from the Institutes ethics committee. DSE-36 and -37 DSE-36 and -37 were prepared as tartarate salts by the chemist authors (V.L.S., S.T.V.S.K.K. and A.K.D.) in the Process and Medicinal Chemistry Division of the Institute, as per the molecular structures shown in Figure 1. All compounds were >99.5% pure [analytical high-performance liquid chromatography (HPLC)] and were characterized by mass, infrared and nuclear magnetic resonance (NMR) spectroscopy and also by elemental analysis. The compounds were hygroscopic and extremely soluble in water (saline). Spermicidal test The test compounds were dissolved in physiological saline to make a 1.0% (10 mg/ml) solution and diluted serially up to 0.001%. A spermicidal test was performed with each dilution starting from 1.0% till the minimum effective concentration (MEC) was arrived at, following the modified method of Sander and Cramer (1941). Briefly, 0.05 ml of human semen was added to 0.25 ml of spermicidal compound solution and vortexe (...truncated)