Hum. Reprod. Advance Access published online on December 16, 2008
Human Reproduction, doi:10.1093/humrep/den415
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In vitro testing of rationally designed spermicides for selectively targeting human sperm in vagina to ensure safe contraception
1 Division of Endocrinology, Central Drug Research Institute, Lucknow 226 001, India 2 Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow 226 001, India 3 Division of Pharmaceutics, Central Drug Research Institute, Lucknow 226 001, India 4 Electron Microscopy Unit, Central Drug Research Institute, Lucknow 226 001, India
5 Correspondence address. Tel: +91-522-2612411 ext. 4396; Fax: +91-522-2624305; E-mail: gupta.gopal{at}rediffmail.com, ji_gupta{at}yahoo.co.in, www.cdriindia.org
BACKGROUND: Rational synthesis of novel structures resulted in two unique molecules (DSE-36 and DSE-37, disulphide esters of carbothioic acid) that killed sperm 25 times more strongly and with a precisely targeted action than nonoxynol-9 (N-9). We examine the effects of DSE-36 and DSE-37 on human spermatozoa versus HeLa cells to establish specificity and safety compared with N-9.
METHODS AND RESULTS: At spermicidal EC100 (20 µg/ml) DSE-36 and DSE-37 killed 100% sperm in <30 s (Sander–Cramer assay) and at EC50 induced apoptosis in sperm (Annexin-V-fluorescein isothiocyanate and JC-1 labelling and Flow Cytometry) in 3 h. However, at EC100 these molecules had no effect on HeLa cells by 24 h or on cell viability [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay], surface ultrastructure (scanning electron microscopy), Annexin-V and JC-1 labelling pattern and reactive oxygen species (ROS) generation. N-9, with a spermicidal EC100 of 500 µg/ml, decreased HeLa cell viability at 20 µg/ml in 24 h (P < 0.001), accompanied by acute damage to cell surface ultrastructural topography, induction of apoptosis and ROS generation. Unlike DSE-36 and DSE-37, N-9 also significantly induced mRNA levels (RT–PCR) of pro-inflammatory biomarkers (interleukin (IL)-1
, IL-6, IL-8, RANTES) in HeLa cells and increased IL-6 and IL-8 secretion (P < 0.001, enzyme-linked immunosorbent assay). Furthermore, DSE-36 and DSE-37 did not inhibit Lactobacillus growth at EC100 and exhibited mild microbicidal activity against Trichomonas vaginalis, while N-9 inhibited Lactobacillus and Trichomonas growth but had a lower prophylactic index.
CONCLUSIONS: The ability of these novel spermicides to kill sperm almost instantaneously at innocuously low concentration indicates their worth as improved active ingredients for vaginal contraceptive preparations compared with N-9.
Key words: DSE-36/spermicides/HeLa/contraception/nonoxynol-9
CDRI communication No. 7351. Submitted on April 2, 2008; resubmitted on August 5, 2008; accepted on September 16, 2008.