Departments of 1 Reproductive Biology and 3 Virology, Parker Hughes Institute, 2657 Patton Road, St Paul; 2 Paradigm Pharmaceuticals, 2685 Patton Road, St Paul, MN 55113, USA
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Abstract |
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Keywords: HIV/AIDS, microbicide, spermicide, antiviral drugs, nucleoside analogues
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Introduction |
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Currently available spermicidal microbicides, such as nonoxynol-9 (N-9), octoxynol-9, sodium docusate, chlorhexidine, menfegol and benzalkonium chlorides, are cytotoxic to genital tract epithelial cells at spermicidal concentration.9 Frequent use of N-9, the most widely used vaginal contraceptive, has been associated with increased risks of vaginal irritation or ulceration.1113 Clinical studies have confirmed that detergent-type spermicides alter normal vaginal bacteria or flora, and lead to increased risks of contracting sexually transmitted diseases.1418 There is a growing concern that chemical irritation that disrupts the vaginal mucosa/flora might actually increase the risk of HIV transmission in sexually active women.19,20 N-9 also has a high contraceptive failure rate.21 Consequently, the development of new, safe, efficacious, non-detergent-type, dual-function vaginal spermicidal microbicides has become the focal point in translational anti-HIV microbicide research.9
For a microbicide to be an effective anti-HIV agent in genital tract secretions, it is essential that the drug is able to inactivate HIV replication in lymphocytes, epithelial cells and sperm, irrespective of the metabolic state of the cell. Inasmuch as trafficking HIV-infected mononuclear cells in semen contribute to the sexual transmission of HIV via cell-to-cell transmission of HIV,22,23 the anti-HIV microbicide should be metabolized with equal efficiency by seminal cells as well as genital tract epithelial cells of the cervico-vaginal region. Because of the dependence of nucleoside analogues such as zidovudine (ZDV) on intracellular thymidine kinase (TK) activation,24,25 we synthesized novel aryl methoxy alaninyl phosphate derivatives of 3'-azido-3'-deoxythymidine (ZDV, AZT), which undergo intracellular hydrolysis to yield monophosphate derivatives that are further phosphorylated by thymidylate kinase to give the bioactive triphosphate derivatives in a TK-independent fashion.2630 Aryl phosphate derivatives of 5-bromo-6-methoxy-zidovudine with an alanine methyl ester side chain and a methoxy substitution [compound WHI-05; 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl)-methoxyalaninyl phosphate] or a bromo substitution in the phenyl ring [compound WHI-07; 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate] were identified as the lead dual-function agents with potent anti-HIV and spermicidal activities (Figure 1). WHI-05 and WHI-07 displayed potent anti-HIV activity (IC50[RT] = 0.04 and 0.009 µM and IC50[p24] = 0.02 and 0.005 µM, respectively) when compared with N-9 (IC50[p24] = 2.2 µM). WHI-05 was active against both ZDV-sensitive and -resistant HIV-1 strains.28 The spermicidal EC50 values were 24 and 5 µM for WHI-05 and WHI-07, respectively, when compared with that of N-9 (EC50 = 81 µM). Structureactivity function studies of these novel ZDV derivatives established that the C-5 bromo and C-6 methoxy functionalization on the thymine ring, an alanine side chain and a bromo group on the C-5 position of the phenyl ring are essential for the potent anti-HIV as well as spermicidal activities of ZDV derivatives.26,31,32 Topological imaging and ultrastructural studies of WHI-05- and WHI-07-treated sperm by high-resolution, low-voltage scanning, as well as transmission electron microscopy, demonstrated that the spermicidal property of WHI-05 and WHI-07, unlike N-9, was not associated with membrane disruption.26,29
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Safety studies in cell systems |
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The MTT cell proliferation and viability assay was used to test the potential cytotoxicity of WHI-05 and WHI-07 in comparison with N-9 against normal human female ectocervical and endocervical epithelial cells.28,29,33 N-9 exhibited significant cytotoxicity to these cells at spermicidal concentration (mean IC50 values of 19 and 11 µM, respectively). By comparison, the mean IC50 values of the WHI-05 and WHI-07 dose survival curves were >1000 µM for ectocervical cells and >300 µM for endocervical cells. Unlike N-9, which was spermicidal only at cytotoxic concentrations [EC50 value: 81 µM; selectivity indices (SI): 0.23 and 0.13 for ectocervical and endocervical cells, respectively], WHI-05 and WHI-07 showed high SI against these cells (EC50 value: 24 µM for WHI-05; SI: >41 for ectocervical cells and >12 for endocervical cells; EC50 value: 5 µM for WHI-07; SI: >200 for ectocervical cells and >60 for endocervical cells). Thus, WHI-05 and WHI-07 were significantly less active against genital tract epithelial cells.
Genotoxicity/mutagenicity studies in cell systems
The effect of WHI-07 on genomic stability was tested in the yeast DEL recombination assay.34,35 This assay measures the frequency of intrachromosomal recombination between two partially deleted his3 alleles on chromosome XV. The his3 delta alleles share 400 bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3 delta alleles results in the deletion of the intervening LEU2 sequence (DEL) and the reversion to histidine prototrophy. Exposure of exponentially growing yeast cells to increasing concentrations of WHI-07 (0.01100 µM) was not found to increase the DEL recombination frequency of yeast cells.
Since the genotoxic stress response of human cells can be detected at the transcriptional level, we measured the activation of eight stress response specific promoters or regulatory elements (p53RE, GADD 45, FOS, HSP70, HMTIIa, NFBRE, GSTY and GRP78) in HepG2 cell lines using the CAT-Tox(L) assay.36,37 When eight separate cell lines carrying the promoterreporter constructs and control (HepG2) cells were simultaneously treated with increasing concentrations of WHI-07 (0.01100 µM) for 48 h, no significant activation of these reporter genes was evident as measured by ELISA. These studies in cell systems clearly established that WHI-07 does not induce primary DNA damage in human cells.
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Safety of systemic exposure in mice and non-human primates |
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WHI-05 and WHI-07 were administered to female cynomolgus monkeys either as a single intravenous (iv) injection or multiple (five) iv injections for 2 days. At doses of 20 mg/kg, WHI-05 and WHI-07 were non-toxic to cynomolgus monkeys (Tables 1 and 2). Blood for haematological (red blood cells, haemoglobin, white blood cells, packed cell volume (PCV), mean corpuscular volume, mean cell haemoglobin, mean cell haemoglobin concentration, red cell distribution width, mean platelet volume, Diff., platelets) and clinical chemistry [Na, K, Ca, Cl, HCO3, anion gap, glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), total bilirubin (TBIL), alanine aminotransferase (ALT), amylase (AMY), creatinine kinase (CK) and albumin (ALB)] determinations collected from cynomolgus monkeys before (day 1) and after (on days 2, 8, 13, 26 and 33 for WHI-05 and on days 2, 3, 5, 9, 17 and 24 for WHI-07) drug treatment did not reveal significant differences. Furthermore, toxicological grading performed post-treatment of monkeys showed no clinical evidence of significant toxicity (toxicity grades 0 to <2) (Tables 1 and 2).
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Mucosal safety studies in mice and rabbits |
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The rabbit vaginal irritation studies were performed by daily intravaginal administration of 1 mL of gel-microemulsion with and without 2% WHI-05 or WHI-07 compared with the standard 4% N-9 gel for 10 consecutive days. Histological evaluation of three distinct areas of the cervico-vaginal region showed no vaginal irritation or inflammation.28,29 None of the rabbits that received WHI-05 or WHI-07 gel intravaginally had vaginal erythema, oedema, exudate, leucocyte influx or epithelial disruption characteristic of inflammation (individual score 02 and total score <4; acceptable range) when compared with N-9 gel (individual scores 13; total score 9; marginal irritation). Thus, evidence obtained by histopathological and immunofluorescence studies indicated that unlike N-9 treatment, intravaginal application of WHI-05 or WHI-07 via a gel-microemulsion at a dose 1200 and 5700 times higher than their respective in vitro spermicidal EC50 values, and 6.1 x 104 and 5.7 x 106 times higher than their anti-HIV IC50 values, did not cause any membrane disruption or acute inflammatory response in the cervico-vaginal epithelial crypts.28,29
The systemic absorption of 2% WHI-05 and WHI-07 gel-microemulsion applied intravaginally was also investigated in NZW rabbits. Following intravaginal application of 2% WHI-05 and WHI-07, blood was collected at timed intervals for up to 4 h. Plasma WHI-05/07 and their major metabolites (ZDV, alaninyl-ZDV-monophosphate and ZDV-monophosphate) were analysed by a validated HPLC procedure with a detection limit of 25 pmol in plasma samples. Following intravaginal application of a 2% WHI-05 and WHI-07 gel-microemulsion, WHI-05/07 and their metabolites were undetectable (<25 pmol) in all blood samples throughout the 4 h sampling period.29 The concentration of WHI-05 and WHI-07 and their metabolites in the vaginal tissue 24 h after 10 days of daily intravaginal application of 2% WHI-05 or WHI-07 in a gel-microemulsion base was assayed after tissue homogenization, solvent extraction and analytical HPLC. In all rabbit vaginal tissues analysed (cervico-vagina, mid-vagina and uro-vagina), WHI-05/07 and their known metabolites were undetectable.28,29 These results indicated that WHI-05 and WHI-07 have very low capacity to be absorbed through the vaginal epithelium after repetitive intravaginal administration.
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Subchronic (13 weeks) toxicity studies in mice |
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The assay conditions consisted of gel-microemulsion with and without three increasing doses of WHI-05 and WHI-07. Female B6C3F1 mice in subgroups of 10 and 20 were treated with intravaginal applications of 0, 0.5, 1 or 2% WHI-05 or WHI-07 gel-microemulsion, 5 days per week, for 13 consecutive weeks.4447 On a molar basis, these concentrations of WHI-05 and WHI-07 dose are 1200 and 5700 times higher than their respective in vitro spermicidal EC50, and 6.1 x 104 and 5.7 x 106 times higher than their in vitro anti-HIV activity IC50. 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-05 or WHI-07 were similar to those of the gel-microemulsion controls. Table 4 summarizes the toxicity parameters for mice given increasing concentrations of intravaginal WHI-07 for 13 weeks.
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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, which were considered to be related to WHI-05 or WHI-07 exposure.4446 Statistically significant differences (P < 0.05) observed in the absolute organ weights of the WHI-07 treatment groups with respect to lung or liver were not considered biologically relevant because of the lack of a dose response and the absence of gross or microscopically detectable histological lesions in these organs. No treatment-related gross lesions were found at necropsy.4446
Microscopic examination of multiple tissues taken from the study animals did not reveal any treatment-related lesions. No organ-specific effects related to chemical exposure to 0.52% WHI-05 or WHI-07 via gel-microemulsion were evident in all mice evaluated. Incidental histological lesions included mild inflammation of the kidney, liver or urinary bladder, and focal haemorrhage of lung, which could normally be expected to be seen in the laboratory mice was distributed randomly throughout the study group. No histopathological lesions were observed in the ovarian and genital tract tissues of all WHI-05 and WHI-07 treated mice examined, which suggests a lack of toxicity to repeated intravaginal exposure to WHI-05 and WHI-07 via gel-microemulsion formulation.4446
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Reproductive performance after intravaginal exposure in mice |
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Table 5 summarizes the ovulation rates of placebo control versus 2% WHI-05- and WHI-07-treated CD-1 mice. In five independent experiments, the ovulation response was similar when PMSG/hCG-primed mice were given intravaginal gel-microemulsion with and without 2% WHI-05 or WHI-07 before and during superovulation for 5 consecutive days. All mice from the control and the WHI-07-treated groups ovulated. Also, the differences in the mean number of eggs recovered from the ampullae of their oviducts were not statistically significant (33.5 versus 35.4 and 29.8 for WHI-05 and WHI-07, respectively).
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Two year toxicity studies in mice |
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No significant difference in survival was observed among the groups of untreated control, placebo control and 2% WHI-07-treated mice at the end of the 2 year study (Table 9). At the completion of the 104 week study, the survival figures for the untreated control group, vehicle-only control group and 2% WHI-07-treated group females were 32/50 (64%), 40/50 (80%) and 31/50 (62%), respectively. There was no treatment-related effect on mortality rate. All animals survived beyond the first year treatment period (59 weeks) and >90% of mice from all three groups survived up to 85 weeks of the 2 year study. The spontaneous death rate in all groups was relatively low until week 90 of the study, when it rose in the untreated control and 2% WHI-07-treated groups in a similar manner. There were no statistically significant body weight differences among the groups of intravaginally exposed and untreated control mice at the completion of the 2 year intravaginal study. No compound-related clinical signs were observed.
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Analysis of 19 blood chemistry parameters for female B6C3F1 mice given no treatment or exposed intravaginally to a gel-microemulsion with and without 2.0% WHI-07 for 2 years revealed no significant treatment-related differences among the three groups, except for ALP and TBIL levels, which were slightly lower in the placebo control and 2% WHI-07-treated groups than in the untreated control group. These minor differences were not considered clinically significant, nor were they related to treatment with WHI-07. In general, the indicators tested for kidney function (BUN and CRE), liver function (TBIL, AST, ALT, ALP, CHO and TG), pancreas function (AMY, GLU), immunological function (G), nutritional status (TP), calcium (Ca), phosphorous (P) and plasma electrolytes (Na, K and Cl) were not affected adversely by repeated intravaginal exposure of gel-microemulsion with or without 2% WHI-07.
There were no statistically significant treatment-related differences in absolute and relative organ weights of brain, thymus, heart, lung, intestine, liver, pancreas, spleen, kidney, genital tract or urinary bladder among the three groups, or between vehicle-only control and 2% WHI-07 treatment groups that were considered to be related to WHI-07 exposure. A variety of non-neoplastic lesions were observed in both untreated and intravaginally treated groups.48 These lesions were considered to be incidental, related to ageing or procedural related to the intravaginal route of administration.
Light microscopic examination of brain, heart, intestine, lung, lymph node, pancreas, skeletal muscle, skin, spinal cord, stomach, thymus, cervico-vagina and urinary bladder specimens taken from the untreated control, vehicle-only control and 2% WHI-07-treated B6C3F1 mice demonstrated no significant increases in the severity index above that seen for the untreated group. The most frequent non-neoplastic lesions observed in all three groups included: fibro-osseous process in the bone, nephritis in the kidneys, hepatitis, lymphadenitis, ovarian cyst, ovarian inflammation, pancreatitis, cystitis, endometrial hyperplasia and metritis. A higher incidence of metritis of the uterus and inflammation of the ovaries was noted in vehicle-only control (28.832.5%) and 2% WHI-07-treated (18.637.5%) mice compared with untreated controls (7.110.8%). No significant differences in the increased total incidences of non-neoplastic lesions of the kidney (26.6% versus 25.5%), liver (35.5% versus 32.5%) and uterine specimens (64.4% versus 51.1%) were observed between the vehicle-only control and the 2% WHI-07-treated mice. There was a slight numerical increase in the incidence of moderate medullary hypoplasia (5/20) and moderate Type A (4/20) and Type B (3/20) subcapsular hyperplasia in the limited adrenal gland tissues obtained from the 2% WHI-07 treatment group versus the vehicle control group (2/16 and 1/45).
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Two year carcinogenicity studies in mice |
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In all three groups, tumours of the liver (15.520.9%), lung (11.620.9%), lymph node (2662.5%), ovaries (8.120%), spleen (4.628.8%), thymus (11.415.7%) and uterus (6.611.6%) were the most frequent, followed by urinary bladder (2.710.8%) and cervico-vagina (5.2%). The overall incidence of ovarian neoplastic lesions for untreated and vehicle control and 2% WHI-07-treated mice were 8.1%, 20% and 20%, respectively. Some of the more common findings included: lymphoma of the liver, lung, lymph node, ovary, pancreas, thymus, uterus, cervico-vagina and urinary bladder. Other spontaneous neoplastic lesions typical for the age and strain of the mouse were observed occasionally. All of the neoplastic lesions observed in the liver, lung, lymph node, ovaries, spleen and uterus were considered spontaneous for female B6C3F1 mice and did not exceed the incidences observed for the control group when compared with a historical database. Very low incidence of neoplastic lesions occurring sporadically in the various groups was also detected in adrenal, bone marrow, large and small intestine, kidney, pancreas, skeletal muscle, skin, spinal cord and stomach (2.27.1%).
Microscopic evaluations of mice that died during the second year indicated that the predominant cause of death in all groups was malignant lymphoma. At the end of the 2 year study, lymphocytic lymphoma accounted for 88.2%, 62.0% and 88.2% of tumour-bearing mice from untreated control, vehicle-only control and the 2% WHI-07 treatment group, respectively. Generalized lymphoma was frequently encountered in the liver, lung, lymph nodes, ovaries, thymus and uterus, and less frequently in other organs. The cumulative incidence of microscopic lesions in various organs showed that malignant lymphoma was the major cause of death in ageing female B6C3F1 mice, the incidence of which was unaffected by intravaginal treatment. Table 10 summarizes the total numbers of malignant tumours in female B6C3F1 mice killed or dying during the duration of the 2 year study. The incidental neoplastic lesions occurred with similar frequency in the untreated and 2% WHI-07-treated group and were slightly more pronounced in the vehicle-only group, although the differences were not statistically significant. The total number of malignant tumours as well as the type of malignant tumours in all three groups was comparable. Thus, long-term intravaginal administration of WHI-07 is not associated with systemic toxicity or increased carcinogenicity in mice.
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Summary |
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Acknowledgements |
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Footnotes |
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References |
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