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Preclinical Evaluation of a Dual-Acting Microbicidal Prodrug WHI-07 in Combination with Vanadocene Dithiocarbamate in the Female Reproductive Tract of Rabbit, Pig, and Cat

Drug Discovery Program, Paradigm Pharmaceuticals, LLC, St. Paul, MN 55113

Correspondence: Address correspondence to Osmond J. D’Cruz, Ph.D., Parker Hughes Clinics, 2657 Patton Road, St. Paul, MN 55113; e-mail:odcruz{at}ih.org

The mucosal safety of the combination antiretroviral spermicide, WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxy alaninyl phosphate] and vanadocene dithiocarbamate (VDDTC), was evaluated in 3 different animal models. Twenty-seven NZW rabbits in four subgroups were exposed intravaginally to a gel-microemulsion (GM) with and without three dose levels of WHI-07 plus VD-DTC (0.5 + 0.06%, 1.0 + 0.12% and 2.0 + 0.25%) or 4% nonoxynol-9 (N-9; Conceptrol^®) for 14 consecutive days. Ten nonestrus gilts (Duroc) in three subgroups received either a single or daily intravaginal application of GM with and without 2.0% WHI-07 plus 0.25% VDDTC or 2.0% benzalkonium chloride (BZK)-containing gel for 6 and 4 consecutive days, respectively. Five cats received a single intravaginal application of GM incorporating 2.0% WHI-07 plus 0.25% VDDTC. Genital tract histopathology was performed in the pig and rabbit at the end of dosing period but after 18 weeks post-dosing in the cat. Porcine cervicovaginal lavage (CVL) fluid was obtained for up to 72 hours after a single exposure and changes in the levels of inflammatory cytokines (IL-1β, IL-8, IFN-, and TNF-) were quantitated by a multiplexed chemiluminescence-based immunoassay. Rabbit vaginal tissues were evaluated for localized cellular inflammation and in situ apoptosis by immunohistochemical staining for CD45, nuclear factor (NF)-B, and terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick-end labeling (TUNEL) using confocal laser scanning microscopy (CLSM), respectively. Vanadium content in selected organs and body fluids from rabbits and pigs was determined by atomic absorption spectroscopy. When compared with 4% N-9 (total irritation score 13–14 out of a possible 16), none of the rabbits given WHI-07 plus VDDTC intravaginally, developed histological alterations such as epithelial erosion, edema, leukocyte influx or vascular congestion characteristic of inflammation (total irritation score 4–6). CD45 and NF-B immunoreactivity was limited to cells within the vascular lumen of both control and WHI-07 plus VDDTC-treated vaginal tissues. TUNEL assay revealed lack of increased apoptotic cells in vaginal mucosa exposed to increasing concentrations of WHI-07 plus VDDTC. Basal levels of proinflammatory cytokines (IL-1β, IL-8, IFN- and TNF-) in porcine CVL were unaffected by intravaginal exposure to WHI-07 plus VDDTC when compared with BZK used as a positive control. Endpoint histology of the reproductive tract from cats and pigs after a single or repeated intravaginal exposure to WHI-07 plus VDDTC, respectively, revealed lack of irritation/inflammation in the epithelium, subepithelium/lamina propria, vessels/perivascular tissues, and underlying/surrounding muscles. Vanadium was not preferentially incorporated into rabbit or porcine tissues and body fluids at levels above 1 µg/g. Based on comparative histologic data and surrogate markers for inflammation, repeated intravaginal administration of WHI-07 plus VDDTC via a gel-microemulsion did not result in vaginal irritation, mucosal toxicity, or systemic absorption of vanadium. Therefore, the combined use of WHI-07 and VDDTC via gel-microemulsion appears safe for topical use as a prophylactic anti-HIV microbicide.

Key Words: HIV/AIDS • gel-microemulsion • intravaginal • local tolerance microbicide • nucleoside prodrug • spermicide • vanadocene

Abbreviations: AIDS, acquired immunodeficiency syndrome • BZK, benzalkonium chloride • CCD, charge-coupled device • CLSM, confocal laser scanning microscopy • CVL, cervicovaginal lavage • DEL, intrachromosomal recombination resulting in deletion of intervening sequences • DNAse, deoxyribonuclease • ELISA, enzyme-linked immunosorbent assay • FDA, food and drug administration • FIV, feline immunodeficiency virus • GM, gel-microemulsion • H & E, hematoxylin and eosin • HIV-1, human immunodeficiency virus type-1 • IFN, interferon • IL, interleukin • MIP, macrophage inflammatory protein • N-9, nonoxynol-9 • NF-B, nuclear factor kappa B • PBS, phosphate-buffered saline • PI, propidium iodide • PVI, porcine vaginal irritation • RANTES, regulated on activation, normal T cell expressed and secreted • RT, reverse transcriptase • RVI, rabbit vaginal irritation • TK, thymidine kinase • TNF, tumor necrosis factor • TUNEL, terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick-end labeling • UV-VIS, ultraviolet-visible • VDDTC, vanadocene dithiocarbamate • WHI-07, 5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxy alaninyl phosphate • ZDV, zidovudine

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