Toxicological Sciences 60, 373-378 (2001)
Copyright © 2001 by the Society of Toxicology
SAFETY EVALUATION |
Short-Term (13-week) Toxicity Study of 5-Bromo-6-methoxy-5,6- dihydro-3'-azidothymidine-5'-(p-bromophenyl) Methoxyalaninyl Phosphate (WHI-07), a Novel Anti-HIV and Contraceptive Agent, in B6C3F1 Mice
* 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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The zidovudine derivative, WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate), is a dual-function spermicidal agent with potent anti-HIV activity. In this study, groups of 20 female B6C3F1 mice were exposed intravaginally to a gel microemulsion containing 0, 0.5, 1.0, or 2.0% WHI-07, 5 days per week for 13 consecutive weeks. On a molar basis, these concentrations of WHI-07 are 1400- to 5700-times higher than its spermicidal EC50 and 1.4 x 106 to 5.7 x 106 times higher than its in vitro anti-HIV IC50. After 13 weeks of intravaginal treatment, mice were evaluated for toxicity. The endpoints that were used for evaluation included survival, body weight, hematologic and clinical chemistry profiles, absolute and relative organ weights, and histopathology. No effects related to WHI-07 treatments were observed on survival, mean body weight, and mean body-weight gain. Repeated intravaginal exposure of mice to WHI-07 for 13 weeks had no toxicologically significant effect on organ weights, and did not cause any adverse changes in hematology parameters or blood chemistry profiles. Extensive histopathologic examination of tissues showed no lesions of pathologic significance. Thus, intravaginal application of WHI-07, for up to 13 weeks, does not cause systemic toxicity.
Key Words: 13-week intravaginal; zidovudine; HIV/AIDS; microbicide.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexual transmission of HIV-1, the causative agent of AIDS, continues to be the predominant mode of the epidemic spread of HIV/AIDS. The emergence of AIDS as a disease spread through sexual intercourse has prompted the search for new, effective, safe, and female-controlled vaginal microbicides for curbing mucosal viral transmission (Uckun and D'Cruz, 1999
; Quinn et al., 2000). In a systematic effort to develop a virucidal contraceptive capable of reducing the risk of HIV infection during sexual activity, as well as to provide a fertility control, we have identified novel aryl phosphate derivatives of 3'-azido-3'-deoxythymidine (zidovudine, ZDV), which exhibit potent anti-HIV and contraceptive activity (D'Cruz et al., 1998, 1999b; Jan et al., 1999). Our preclinical studies of repetitive intravaginal exposure of gel formulations of WHI-07 in mice and rabbits indicates that this novel contraceptive agent was non-cytotoxic to reproductive-tract epithelial cells and lacks inflammation-inducing properties characteristic of currently used, detergent-type microbicidal spermicides such as nonoxynol-9 (N-9) (D'Cruz et al., 1998, 1999a). Furthermore, female cynomolgus monkeys treated with 20 mg/kg of WHI-07 intravenously developed no grade 2–4 systemic toxicities when monitored for up to 24 days. These studies indicated that WHI-07 shows unique clinical potential to become the active ingredient of a new female-controlled topical, microbicidal vaginal contraceptive for women who are at high risk of acquiring HIV/AIDS by heterosexual vaginal transmission.
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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemical synthesis and characterization of WHI-07.
WHI-07 (Fig. 1
) was synthesized by the phosphorochloridate chemistry in 4 steps, according to our earlier published procedure (D'Cruz et al., 1999a, Jan et al., 1999). The purity was >98%, as determined by the proton (1H), carbon (13C), phosphorous (31P) nuclear magnetic resonance spectra and Fourier transform infra-red spectra.
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Gel-microemulsion formulation of WHI-07.
Due to the lipophilic nature of WHI-07, a microemulsion-based formulation strategy was developed. A submicron-particle size (30–80 nm), lipophilic microemulsion containing the pharmaceutical excipients, Captex 300, Cremophor EL, phospholipon 90G, propylene glycol, PEG-200, and sodium benzoate, was formulated (D'Cruz et al., 2001b. This microemulsion-based system with high solubilizing capacity for WHI-07 was identified through systemic mapping of ternary phase diagrams, and drug solubilization study (D'Cruz et al., 2001b. Various polymeric gels were screened to produce a gel with desirable viscosity. Polymer suspensions of seaspan and viscarin carrageenan were selected as additives to the microemulsion-based system to obtain a gel with desirable viscosity containing 0.5–2.0% WHI-07 with high thickening capability and compatibility with microemulsion. The gel polymers did not cause drug precipitation or alter the microemulsion particle size. The gel microemulsion was found to be very stable at ambient temperature. Particle size determination was made using Nicomp Model 380 laser diode source (Particle Sizing Systems, Santa Barbara, CA). Measurements of drug concentrations were carried out by the Hewlett -Packard 1100 series HPLC system (Wilmington, DE), and the Beckman DU7500 UV-visible spectrophotometer (Fullerton, CA). Viscosity measurements were made using the Brookfield Viscometer (Brookfield Engineering Laboratories, Spoughton, MA).
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 (30–80 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 4–6 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal Observations and Measurements
Groups of 20 female B6C3F1 mice were treated with intravaginal applications of 0, 0.5, 1, or 2% WHI-07 gel microemulsion, 5 days per week, for 13 consecutive weeks. On a molar basis, these concentrations of WHI-07 are 1400 to 5700 times higher than its in vitro spermicidal EC50 (drug concentration inhibiting sperm motility by 50%) and 1.4 x 106 to 5.7 x 106 times higher than its in vitro anti-HIV activity IC50 (drug concentration inhibiting HIV-p24 antigen production by 50%). Mortality did not occur in any mouse group and all animals were clinically healthy at the end of the study. Mean body weight gain and final mean body weight of the mouse group exposed to increasing doses of WHI-07 were similar to those of the gel-microemulsion controls (Fig. 2
).
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Hematological Findings
Complete blood-cell counts in the mice revealed no biologically significant differences between WHI-07-dosed and vehicle control mice (Table 1
). The values of hematologic parameters for red cells (total count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean cell hemoglobin, mean cell hemoglobin concentration, and red cell distribution width) and platelets (total count and mean platelet volume) were within normal limits. A slight increase in platelet counts was observed in the highest-dose group (p < 0.05). The white blood cell counts and differentials were similar.
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Blood Chemistry Findings
Analysis of blood chemistry parameters for female mice given 0%, 0.5%, 1.0%, or 2.0% WHI-07 intravaginally for 13 weeks revealed no significant treatment-related differences between WHI-07-dosed and vehicle control groups (Table 2
). The functions of kidney (BUN and CRE), liver (TBIL, AST, ALT, ALP, CHO, and TG), pancreas (AMY, GLU), and immune system (GLOB); nutritional status (TP); and calcium (Ca), phosphorous (P), and plasma electrolytes (Na, and K) were not affected adversely by repeated intravaginal exposure of WHI-07. The mean triglyceride value was increased in the WHI-07 treatment group; however, a significant increase (p < 0.05) was observed only in the highest-dose group. Mean Cl levels decreased by 5 to 7 mEq/l in all WHI-07 treatment groups (p < 0.01), while mean levels of Na, K, and P were unchanged. Although the decreased plasma Cl values observed for individual mice in 0.5% and 2.0% WHI-07 groups fell outside the 95% confidence interval, no single mouse had multiple variables in plasma electrolytes outside this interval.
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Necropsy/Organ Weights
Table 3
summarizes the terminal body weight and terminal absolute and relative organ weights of vehicle control and WHI-07-treated groups, respectively, at the conclusion of the 13-week study. There were no statistically significant treatment-related differences in absolute and relative organ weights of brain, thymus, heart, lung, intestine, liver, pancreas, spleen, kidney, or genital tract between vehicle control and treatment groups that were considered related to WHI-07 exposure. Statistically significant differences (p < 0.05) observed in the absolute organ weights of 1.0% WHI-07 treatment group with respect to lung were not considered biologically relevant because of the lack of a dose response and absence of gross or microscopically detectable histologic lesions in these organs. No treatment-related gross lesions were found at necropsy.
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Histopathology
Microscopic examination of specimens taken from the study animals did not reveal any treatment-related lesions (Table 4
). No organ-specific effects related to chemical exposure to 0.5% to 2% WHI-07 via gel microemulsion were evident in all mice evaluated. Incidental histologic lesions included mild inflammation of the kidney, liver or urinary bladder and focal hemorrhage of lung that could normally be expected to be seen in the laboratory mice was distributed randomly throughout the study group. An ovarian cyst was detected in one mouse that had received gel microemulsion only. No histopathological lesions were observed in the ovarian and genital-tract tissues of any WHI-07-treated mice examined, which suggests lack of toxicity to repeated intravaginal exposure to WHI-07 via gel-microemulsion formulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, intravaginal administration of WHI-07, a potent, dual-function anti-HIV and spermicidal agent, given to female B6C3F1 mice via a gel-microemulsion formulation for 13 weeks caused no detectable adverse effects on survival, mean body-weight gain, mean body weight, hematological and clinical chemistries, absolute or relative organ weights, or histopathology.
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 2–4 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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This work was supported by grant RO1 HD 37357 (O.J.D.) from the National Institutes of Health, National Institute of Child Health and Human Development, Bethesda, MD.
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NOTES |
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1 To whom correspondence should be addressed at the Parker Hughes Institute, 2665 Long Lake Road, Suite 300, St. Paul, MN 55113. Fax: (651) 697-0645. E-mail: odcruz{at}ih.org.
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