* Drug Discovery Program, Departments of Reproductive Biology and
Virology, Parker Hughes Institute, St. Paul, Minnesota 55113
Received November 6, 2000; accepted December 22, 2000
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ABSTRACT |
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Key Words: 13-week intravaginal; zidovudine; HIV/AIDS; microbicide.
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INTRODUCTION |
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WHI-07 is a derivative of the nucleoside analogue ZDV. Because WHI-07 is non-cytotoxic and is not absorbed systemically when given intravaginally (D'Cruz et al., 1999a), it is unlikely to have adverse effects on the general well being of women. However, because oral administration of the parent compound, ZDV, has been shown to result in reversible hematologic toxicity in humans and experimental animals (Pluda et al., 1991
; Richman et al., 1987
; Thompson et al., 1991
), it was prudent to test the potential local and systemic side effects of this dual-function ZDV derivative. It was anticipated that under the conditions of its intended use as an intravaginal/rectal microbicide, individuals would be exposed to WHI-07 on a short-term repeated use. Therefore, it was necessary to determine the effects of repeated intravaginal exposure to WHI-07. Because sexual transmission is the predominant mode of HIV transmission, WHI-07 was formulated for intravaginal use as a potential candidate anti-HIV spermicide for women who are at high risk of acquiring HIV by sexual transmission. In a preliminary study, we demonstrated that WHI-07 given intravaginally via a gel-microemulsion fomulation, for 13 weeks (90 days), did not cause significant or acute subchronic toxicity in mice (D'Cruz et al., 2000b
). The present preclinical study was designed to confirm and expand the preliminary findings of lack of toxicity with repeated intravaginal exposure of mice to WHI-07. Using a larger sample size, this study confirms and extends the preliminary study that intravaginal exposure of the experimental dual-function anti-HIV and contraceptive agent, WHI-07, for up to 13 weeks, lacks systemic toxicity.
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MATERIALS AND METHODS |
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Animals.
Eighty female B6C3F1 mice approximately 6 weeks-of-age were obtained from Charles River Laboratories (Wilmington, DE). They were assigned to groups of 5 per cage in polycarbonate cages, and were provided with corncob or wood chip bedding (Harlan Teklad, Madison WI). All assigned mice were uniquely identified with metal ear tags and ear notches. Tap water and laboratory diet (Teklad LM-485; Harlan Teklad) were available ad libitum. The animals were maintained in a room that was kept at 22 ± 2°C with relative humidity of 50 ± 10% and a 12:12-h light:dark cycle. Animal studies were approved by the Parker Hughes Institute Animal Care and Use Committee and all animal husbandry operations were conducted under current NIH Guidelines.
Thirteen-week toxicity study.
WHI-07 was dissolved in a submicron particle size (3080 nm) gel-microemulsion formulation at concentrations of 0.5, 1.0, and 2.0%. Eighty female B6C3F1 mice were allocated to 4 groups of 20. They were given 50 µl of intravaginal gel microemulsion containing 0, 0.5, 1.0, or 2.0% WHI-07, respectively. The treatment period was 5 days per week for 13 consecutive weeks (90 days). The control group received gel microemulsion alone. The gel microemulsions were prepared weekly and the intravaginal application was performed inside a microisolator. All animals were individually observed daily for signs of toxic effects. Body weights were obtained before exposure (day 0), weekly during exposure, and preceding sacrifice. At the end of the study, blood samples were collected from all 13-week case study mice for clinical pathology studies (hematology and clinical chemistry). Blood collected from all 20-mice/exposure groups was used for determination of each of the 13 hematology parameters. Adequate plasma samples were obtained from 10-mice/exposure group for determination of each of the 18 clinical chemistry parameters. Plasma samples obtained from 15 mice/group were available to perform at least 13 clinical chemistry parameters. Complete necrospies was performed on all treated and control animals.
Hematology parameters.
Hematology parameters were analyzed using a Abbot CELL-DYN 3200 multiparameter, automated hematology analyzer (Abbot Laboratories, Abbot Park, IL) which was standardized for mouse blood (D'Cruz et al., 2000b. This instrument uses flow cytometric techniques to generate the following hematologic measurements in potassium-EDTA anticoagulated whole blood: red blood cells (RBC; 104/µl), total and differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils as 103/µl or %), hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %), mean corpuscular volume (MCV; fl), mean cell hemoglobin (MCH; pg), mean cell hemoglobin concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelets (PLT; 104/µl), and mean platelet volume (MPV; fl).
Clinical chemistry profiles.
Biochemical analyses were performed using a Boehringer Mannheim Hitachi 911 analyzer (Indianapolis, IN). Blood samples collected following 13 weeks of intravaginal application from placebo and WHI-07-treated mice was used for the determination of plasma levels of total protein (TP), globulin (GLOB), albumin:globulin (ALB/G) ratio, blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (CHO), triglycerides (TG), aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), alkaline phosphatase (ALP), amylase (AMY), total bilirubin (TBIL), glucose (GLU), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and phosphorus (P), using reagents and methods provided by the manufacturer.
Necropsy and histopathology.
All mice from the control and WHI-07 treatment groups were sacrificed after 13 weeks of intravaginal exposure for complete necropsy and histopathological evaluation of all tissues. At necropsy, the absolute and relative weights of brain, thymus, heart, lungs, intestine, liver, pancreas, spleen, kidneys, and genital tract (including ovaries) of each animal were measured. The above mentioned organs and the bone (sternum and femur) and bone marrow, large and small intestine, skeletal muscle, skin, spinal cord, uterus, and urinary bladder were fixed in 10% buffered formalin solution, trimmed, embedded in paraffin, sectioned at 46 µm, and stained with hematoxylin and eosin.
Organ weights.
The brain, thymus, heart, lungs, intestine, liver, pancreas, spleen, kidneys, and genital tract from each of 20 per dose were weighed at necropsy. Organ weights were recorded as absolute weights and as a percentage of body weight.
Statistical analysis.
Group means and standard deviations were calculated from initial and terminal body weights, organ weights, hematology, and clinical chemistry parameters. Statistical significance of the treated group mean with that of the control group was analyzed by a one-way analysis of variance, followed by Dunnett's multiple comparison test, using GraphPad Instat software (San Diego, CA). Differences were considered statistically significant if p < 0.05.
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RESULTS |
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DISCUSSION |
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WHI-07 is a derivative of the nucleoside analogue ZDV. The anti-HIV efficacy of ZDV as an inhibitor of HIV-1 reverse transcriptase is dependent on cellular thymidine kinases (McGuigan et al., 1993). Inasmuch as the thymidine kinase activity is known to be low or lacking in monocyte/macrophage cells, the main carriers of HIV in semen, ZDV by itself is unlikely to have significant anti-HIV activity in genital tract secretions. WHI-07, which is an aryl phosphate pro-drug of ZDV, on the other hand, can inactivate HIV in cells that have low or deficient thymidine kinase activity. We recently reported that human female genital tract vaginal and cervical epithelial cells as well as sperm efficiently convert WHI-07 to bioactive ZDV metabolites despite their thymidine kinase deficiency (D'Cruz et al., 2001a
). Taking advantage of this drug delivery system, with particular emphasis on nonlymphocytic cell types, including sperm as the infectious cells in semen, we synthesized WHI-07 by the addition of bromo-methoxy functional groups on the thymine ring and a p-bromo substitution on the phenyl moiety yielding a potent dual-function anti-HIV and spermicidal agent (D'Cruz et al., 2000a
).
The anti-HIV activity of WHI-07 was 439-fold more potent than the detergent-type microbicide, N-9 (D'Cruz et al., 1999a). The spermicidal activity of WHI-07 was 14-fold more potent than N-9 (D'Cruz et al., 1999a
). Unlike N-9, the spermicidal activity of WHI-07 was shown not to be associated with cytotoxicity to reproductive tract epithelial cells (D'Cruz et al., 2000a
). Also, unlike N-9, repetitive intravaginal application of a 2% WHI-07 gel microemulsion did not damage the vaginal epithelium or cause local inflammation in the rabbit model (D'Cruz et al., 1999a
). WHI-07 is non-genotoxic in yeast DEL recombination assay and transcriptional activation of genotoxic stress-specific promoters in human hepatoma cells using the CAT-TOX(L) assay. The LD10 dose for WHI-07 when administered intravenously or intraperitoneally was >500 mg/kg for mice. In addition, female cynomolgus monkeys treated with 20 mg/kg of WHI-07 intravenously developed no grade 24 systemic toxicities when monitored for up to 24 days. Quantitative tissue adsorption and retention studies, using a validated HPLC procedure for WHI-07 and its metabolites, clearly demonstrated that WHI-07 lacks the capacity to be absorbed through vaginal epithelium and thus has low potential to produce systemic toxicity following intravaginal applications. Therefore, it does not appear to be toxic to the female reproductive tract (D'Cruz et al., 1999a
).
In the present study, the kidney, liver, and pancreas functions, as well as the nutritional status, were not affected adversely by repeated intravaginal WHI-07 exposure. In general, there was no correlation between treatment-related increases or decreases of clinical chemistry profiles. Also, there were no dose-dependent effects of WHI-07 on the absolute and relative organ weights. The minor fluctuations with platelet counts and organ weights of lung observed in WHI-07 dose groups were not considered of physiological significance, due to the lack of a dose-related toxicological effect and to the absence of any histopathological changes. Statistically significant variation in mean triglycerides in the 2% WHI-07-dose group was not considered biologically significant, because all values observed were within normal ranges reported in a previous study (D'Cruz et al., 2000b). Also, in the absence of a significant reduction in plasma levels of Na and K, the modest decline (i.e., <7% with respect to control value) in Cl levels was not indicative of alteration in any organ functioning.
In conclusion, under the conditions of its intended use, a 13-week intravaginal application of WHI-07 gel microemulsion in B6C3F1 mice with concentrations of up to 2.0% did not result in systemic toxicity, and no other specific target organs were identified. No statistically significant treatment-related effects on survival, body-weight gain, hematological and clinical chemistries, absolute or relative organ weights, or histopathology were noted. In previous studies, repeated intravaginal exposure of mice to WHI-07 for 13 weeks had no adverse effects on subsequent reproductive performance, neonatal survival, or pup development (D'Cruz et al., 2000b; D'Cruz and Uckun, 2001
). Therefore, the dual anti-HIV and spermicidal activities, together with lack of toxicity of intravaginal gel-microemulsion formulation of WHI-07, show unique clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk of acquiring HIV by heterosexual vaginal transmission.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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