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Regular Article |
b Drug Discovery Program, Departments of Reproductive Biology,
c Chemistry, and
d Virology, Parker Hughes Institute, St. Paul, Minnesota 55113
ABSTRACT
The compounds WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [3H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.
female reproductive tract, gamete biology kinases, sperm, vagina
INTRODUCTION
Infection by human immunodeficiency virus (HIV)—the causative agent of acquired immunodeficiency syndrome (AIDS)—is the fourth leading cause of death among women of reproductive age [1]. Currently, heterosexual transmission of HIV is the dominant mode of the epidemic spread of HIV. Worldwide, it accounts for 90% of all HIV infections in women [2, 3]. The ability of mononuclear cells isolated from semen of HIV-positive men to infect lymphocytes and cervical epithelial cells in vitro supports the notion that trafficking HIV-infected mononuclear cells in semen contribute to the sexual transmission of HIV [4–6]. Consequently, the development of intravaginal microbicides with anti-HIV activity for curbing the mucosal and perinatal HIV transmission has become an area of intense investigation in contemporary translational HIV research [7, 8].
Currently three classes of drugs are in use for treatment of HIV-infected individuals. These drugs include the nucleoside analogues that inhibit reverse transcriptase (RT), non-nucleoside inhibitors of RT, and the more recently introduced protease inhibitors [9, 10]. The RT inhibitors of viral replication have been proposed by the WHO as candidates for intravaginal microbicides to inhibit HIV replication in mucosal cells [11, 12]. However, such an approach to inhibit HIV replication in the genital tract has not yet been tested mainly due to the limitation of the parent drugs. Zidovudine (ZDV, azidothymidine, AZT), the first anti-HIV drug of choice, is anabolized as a thymidine analogue through the action of thymidine kinase (TK), thymidylate kinase, and nucleoside diphosphokinase that sequentially catalyze the conversion of ZDV to ZDV 5'-monophosphate (ZDV-MP), ZDV 5'-diphosphate, and ZDV 5'-triphosphate nucleotides [13, 14]. The nucleotide triphosphate analogue acts as a selective RT inhibitor and DNA chain terminator [15]. Also, ZDV-MP may play a role in inhibiting viral replication through inhibition of RNase H activity of HIV RT [16]. Of the aforementioned cellular kinases involved in ZDV metabolism, TK has low cellular activity that is highly regulated [17]. Low TK activity with consequent reduction in the ability to phosphorylate ZDV has been implicated in the failure of antiviral nucleoside analogues to inhibit HIV replication particularly in monocytes-macrophages [18, 19].
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. Because trafficking HIV-infected mononuclear cells in semen contribute to the sexual transmission of HIV via a cell-to-cell transmission of HIV [4–6], the anti-HIV drug should be metabolized with equal efficiency by seminal cells as well as genital tract epithelial cells of the cervicovaginal region. A major limitation of nucleoside analogues such as ZDV as potential microbicides is the decreased capability of HIV-infected TK-deficient monocytes-macrophages in semen to convert ZDV to its active triphosphate metabolites [20–22].
In a systematic effort to develop a microbicide contraceptive capable of preventing sexual transmission of HIV as well as providing fertility control, we previously reported the synthesis of novel aryl phosphate derivatives of bromomethoxy ZDV (compounds WHI-05 and WHI-07), as potential dual-function anti-HIV and contraceptive agents [23–27]. These drugs were designed to bypass the TK pathway. They have been previously shown to be active against ZDV-resistant HIV isolates and inhibit HIV replication in TK-deficient cells [24, 25]. Unlike ZDV, our lead compound WHI-05 was active against both ZDV-sensitive and ZDV-resistant strains of HIV-1, and the anti-HIV activity of our second lead compound WHI-07 was 10-fold more potent than that of ZDV [24, 25].
The aim of the present study is to investigate the intracellular activation of WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) in normal human vaginal, ectocervical, and endocervical epithelial cells as well as sperm. Here, we demonstrate for the first time, the metabolic disposition of WHI-05 and WHI-07 in epithelial cells obtained from human cervicovaginal tissues. The successful intracellular delivery of bioactive nucleotides by the prodrugs despite low or lack of TK activity is particularly promising for curbing vaginal transmission of HIV by leukocytes and sperm as the infectious cells in the semen.
MATERIALS AND METHODS
Cells
Normal human primary vaginal, ectocervical, and endocervical epithelial cells were obtained from Clonetics Corporation (San Diego, CA) and were grown in modified small airway epithelial cell basal medium supplemented with 30 µg/ml bovine pituitary extract, 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, 0.1 µg/ml retinoic acid, 0.5 g/ml fatty acid-free BSA, 10 µg/ml transferrin, 6.5 ng/ml triiodothyronine, 0.5 µg/ml epinephrine, 0.5 ng/ml human epidermal growth factor, and 5% fetal bovine serum (FBS) [28]. The NALM-6 cells were obtained from the American Tissue Culture Collection (Rockville, MD) and were grown in RPMI-1640 medium supplemented with 10% FBS.
Synthesis of WHI-05 and WHI-07
The compounds WHI-05 and WHI-07 (Fig. 1) were synthesized according to our published procedures [24, 25].
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Cellular Metabolism Studies
Normal human vaginal, ectocervical, and endocervical epithelial cells were seeded at a density of 5 x 105 cells/well in 6-well plates for overnight incubation at 37°C under a humidified 5% CO2 atmosphere. The medium was then replaced with the same culture medium containing WHI-05 or WHI-07. In experiments designed to detect the concentration-response effect of prodrugs, the cell monolayers were incubated with increasing concentrations of (i.e., 5, 10, 25, 50 µM) of WHI-05 or WHI-07 for 24 h. In experiments designed to detect the time-course of prodrug metabolism, the cells were incubated with a fixed concentration (10 µM) of WHI-05 and WHI-07 or vehicle (0.1% dimethylsulfoxide ;obDMSO;cb) for 1, 2, 3, 6, and 24 h. Cells were harvested, centrifuged, washed with cold PBS twice, and then extracted by the addition of 0.5 ml of 60% cold methanol. After overnight storage at -20°C, the cells were centrifuged at 15 000 x g for 10 min, and 100-µl aliquots of the supernatant were used for metabolite(s) detection by HPLC [29]. Three independent experiments were performed to detect ZDV metabolites derived from WHI-05 and WHI-07 in normal human vaginal, ectocervical, and endocervical epithelial cells. For each experiment, the primary cells obtained were from a different donor. The viability of epithelial cells following drug treatment was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-based colorimetric assay as described previously [27, 28].
Intracellular metabolism of WHI-05 and WHI-07 was also examined using the highly motile fraction of pooled donor sperm obtained from fresh donor semen (n = 5) by discontinuous (90–45%) centrifugation using Enhance-S-Plus cell isolation media (Conception Technologies, San Diego, CA) followed by the swim-up method as described previously [23–25]. Sperm were obtained from healthy volunteers after informed consent in compliance with the guidelines of the Parker Hughes Institute Institutional Review Board. Motile sperm (20 x 106) were resuspended in 1 ml of Biggers, Whitten, and Whittingham medium containing 0.3% BSA in the presence and absence of 10 µM and 100 µM WHI-05, WHI-07 or vehicle (0.1% DMSO). After 15, 30, and 60 min of incubation, the sperm suspension was washed twice in Tyrode's salt solution, and the final sperm pellet was extracted with 60% cold methanol as described earlier. The percentage of motile sperm following drug treatment was evaluated by computer-assisted sperm motion analysis using a Hamilton Thorne Integrated Visual Optical System version 10.9i instrument (Hamilton Thorne Research, Danvers, MA) as described previously [23, 28].
High-Performance Liquid Chromatographic Analysis
Separation of metabolites was performed on a 250 x 4.6-mm Zorbax SB-C18 column by using a Hewlett-Packard 1100-series HPLC system (Wilmington, DE), containing a quaternary pump, an auto sampler, an electronic degasser, a diode-array detector, and a computer with a Chemstation software program (CambridgeSoft Corp., Cambridge, MA) for data analysis. The mobile phase consisted of a mixture of acetonitrile and 10 mM ammonium phosphate (pH 3.7). The gradient was run at a flow rate of 1 ml per min from 5% to 18% acetonitrile over 8 min, kept at 18% from 8 to 12 min, and finished with a linear gradient from 18% to 100% in the next 20 min. Under these chromatographic conditions, the retention times for alaninyl ZDV monophosphate (Ala-ZDV-MP) and ZDV-MP were 10 min and 7 min, respectively. The detection wavelength was set at 278 nm. The identity of the metabolite(s) was confirmed by comparing the HPLC retention times, UV spectra, and mass spectra of the native metabolites with those of synthetic molecules, as described elsewhere [29]. The quantity of each metabolite was calculated based on the integrated peak area of the chromatogram for each metabolite using an external standard. These values were then normalized based on the number of cells used in the extraction process and expressed in pmol/106 cells. The HPLC methodology had a detection limit of approximately 2 pmol/106 cells for Ala-ZDV-MP and ZDV-MP.
Synthesis of Fluorescent Thymidine Analogue
Coupling of 5-dimethylamino-1-naphthalene-sulfonyl-chloride (DANS) to the 5 position of 5-amino-2-deoxyuridine (AUdR) was performed by a Schiff base reaction [30]. In brief, 50 mmol/L DANS was incubated with 25 mmol/L AUdR for 1 h at room temperature in the dark. The fluorescent thymidine analogue obtained was purified by one-dimensional thin layer chromatography on silica gel plates as described previously [30].
Thymidine Kinase Assays
Flow cytometric assay Exponentially growing NALM-6 pre-B leukemia cell line and confluent monolayers of normal human vaginal, ectocervical, and endocervical epithelial cells (5 x 105) were exposed to 5 µM of the fluorescent thymidine analogue, AUdR-DANS, for 1 h in serum-free medium. Thereafter, adherent cells were harvested by trypsinization, and both the adherent and nonadherent cells were washed by centrifugation and resuspended in PBS. The highly motile fraction of sperm (107/ml) prepared from normal donor semen was also exposed to 5 µM of AUdR-DANS, for 1 h in Biggers, Whitten and Whittingham medium-0.3% medium and washed with PBS. Fluorescence from intracellular phosphorylation of AUdR-DANS was measured in living cells using a fluorescence-activated cell sorter-Vantage flow cytometer (Becton Dickinson, Mountain View, CA) as previously described [20]. Excitation was near UV below 420 nm; emission of the analogue was measured at 500 nm. Analysis was performed on 10 000 gated events. Three separate experiments were performed in triplicate to assess the in situ TK activity in live cells.
Radiolabeled assay Cytoplasmic extracts of exponentially growing normal human endocervical epithelial cells and NALM-6 cells that were used as a positive control were prepared as previously described [31]. Briefly, cells (30 x 106) were washed with PBS, and pelleted by centrifugation at 200 x g. Cell lysis was performed using a buffer consisting of 10 mM Tris-HCl (pH 7.5), 250 mM sucrose, 160 mM KCl, 1.5 mM MgCl2, 3 mM ß-mercaptoethanol, 0.8 mg/ml digitonin, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Cell lysates were spun at 100 000 x g for 60 min, and the supernatant was used as a source of TK. Thymidine kinase activity in vitro was measured by the conversion of radioactive thymidine to thymidine monophosphate as described previously [31]. The reaction mixture consisted of 25 µl supernatant and 75 µl reaction buffer. The reaction buffer consisted of 0.1 M Tris maleate (pH 6.5), 20 mM MgCl2, 25 mM KCl, 10 mM ATP, 7 mM ß-mercaptoethanol, 2 µM thymidine, and 4 µCi of [3H]thymidine (5 Ci/mM; Amersham Corp., Arlington Heights, IL). After 30 min incubation at 37°C, 25-µl aliquots were spotted onto Whatman (Clifton, NJ) DEAE-81 cellulose filter discs, washed with 4 mM ammonium formate, and the radioactivity on the discs was determined by scintillometry. Thymidine kinase activity was normalized based on the number of cells used as well as the total protein concentration of the cell extract that was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Five independent experiments were performed in triplicate to measure the TK activity.
RESULTS
Low Levels of TK Activity in Genital Tract Epithelial Cells and Sperm
It has been reported that the phosphorylation pattern of ZDV, which is dependent on cellular TK, is different in different cell lines [20, 21]. Therefore, we tested the ability of normal human vaginal, ectocervical, and endocervical epithelial cells, as well as motile sperm to phosphorylate the fluorescent nucleoside, AUdR-DANS in culture. The AUdR-DANS is phosphorylated by TK in situ in living cells [30]. The flow cytometric signal of the accumulated fluorescence is used to determine the TK activity of cells. Following treatment of vaginal and cervical epithelial cells in culture as well as the motile fraction of sperm with the DANS nucleoside, the fluorescence of in situ-phosphorylated product in living cells was assessed by flow cytometry. The NALM-6 cells, a pre-B leukemia cell line, was used as a positive control. Figure 2 shows the fluorescence histograms obtained for NALM-6 cells, and vaginal, ectocervical, and endocervical epithelial cells, respectively. The intracellular phosphorylation of the thymidine analogue, AUdR-DANS, by NALM-6 cells caused a right shift of the histogram (trace 2), indicative of TK activity in these cells, whereas only background fluorescence was evident with normal female vaginal, ectocervical, and endocervical epithelial cells, indicative of low or lack of TK activity. Similarly, no fluorescence was detectable following incubation of motile sperm with AUdR-DANS.
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This was further confirmed by radiolabeled studies based on the ability of cytoplasmic extracts of NALM-6, endocervical epithelial cells and motile sperm to convert [3H]thymidine to thymidine monophosphate (Fig. 3). In five independent experiments, NALM-6 cells showed high TK activity (mean cpm: 67 820/107 cells), whereas the TK activity of endocervical epithelial cells was very low (mean cpm: 865/107 cells). The relative TK activity was even lower in motile sperm (mean cpm: 171 cpm/107 sperm).
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Intracellular Ala-ZDV-MP and ZDV-MP Formation from WHI-05 and WHI-07 in Female Genital Tract Epithelial Cells
The exposure of genital tract epithelial cells to WHI-05 or WHI-07, which had no effect on cell viability, led to a concentration-dependent intracellular formation of Ala-ZDV-MP and ZDV-MP as the major metabolites (Fig. 4). The extent of intracellular formation of Ala-ZDV-MP was similar in all three epithelial cell lines studied; however, incubation of cells with WHI-07 consistently produced relatively higher levels of the metabolite Ala-ZDV-MP than with WHI-05 (Fig. 4, A–C). At 50 µM, the respective mean concentrations of Ala-ZDV-MP levels measured in vaginal, ectocervical, and endocervical epithelial cells were 490, 641, and 1268 pmol/106 cells for WHI-05 and 1127, 2373, and 2338 pmol/106 cells for WHI-07. At 50 µM, the intracellular levels of Ala-ZDV-MP observed were nearly two- to eightfold greater than at 10 µM concentration of WHI-05 and WHI-07.
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The extent of concentration-dependent intracellular formation of ZDV-MP was also similar in all three cell lines (Fig. 4, D–F). At 50 µM, the respective mean intracellular levels of ZDV-MP in vaginal, ectocervical, and endocervical epithelial cells were 198, 245, and 1228 pmol/106 cells for WHI-05 and 316, 208, and 975 pmol/106 cells for WHI-07. At 50 µM, the levels of ZDV-MP detected were 8- to 31-fold greater when compared at 10 µM concentration of WHI-05 and WHI-07.
The time-dependent intracellular formation of Ala-ZDV-MP in genital tract epithelial cells incubated with a fixed concentration (10 µM) of WHI-05 and WHI-07 is shown in Figure 5, A–C. The extent of intracellular formation of Ala-ZDV-MP was similar in vaginal and cervical epithelial cells. Both WHI-05 and WHI-07 were rapidly converted to Ala-ZDV-MP in these cells, which was detectable within 1 h of exposure, reached a maximum at 3 h, and declined gradually thereafter. At 10 µM of WHI-05 and WHI-07, the maximum amounts of Ala-ZDV-MP detected in vaginal, ectocervical, and endocervical epithelial cells were 1594, 1347, and 2180 pmol/106 cells, and 1085, 2094, and 1960 pmol/106 cells, respectively.
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Figure 6 shows the time-course of intracellular formation of ZDV-MP in vaginal and endocervical epithelial cells. Both WHI-05 and WHI-07 were rapidly converted to ZDV-MP in vaginal (Fig. 6A) and endocervical (Fig. 6B) epithelial cells. At 10 µM of WHI-05 and WHI-07, the maximum ZDV-MP levels detectable in these cells were 89 and 120 pmol/106 cells, respectively. In vaginal epithelial cells exposed to 10 µM concentrations of WHI-05 and WHI-07, ZDV-MP levels measured at 3 h constituted 5.2% and 6% of the total phosphorylated metabolites, respectively. The ZDV-MP was still detectable at the end of the 24-h incubation period. Whereas, in ectocervical epithelial cells exposed to 10 µM concentration of WHI-05 and WHI-07, ZDV-MP levels (60 and 27 pmol/106 cells, respectively) were detectable only at 24 h.
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Intracellular Ala-ZDV-MP Formation in Sperm
Exposure of the highly motile fraction of sperm to either 10 µM and 100 µM WHI-05 and WHI-07 resulted in time-dependent sperm motility loss that was also associated with intracellular formation of Ala-ZDV-MP. A complete spermicidal effect was observed within 30 min and 60 min of incubation with WHI-07 and WHI-05, respectively. Due to the loss of sperm viability following exposure to WHI-05 and WHI-07, the levels of Ala-ZDV-MP and ZDV-MP observed for sperm were 4- to 37-fold lower when compared to genital tract epithelial cells. At 60 min of incubation with 10 µM WHI-05 and WHI-07, the mean intracellular levels of Ala-ZDV-MP were 23 and 26 pmol/106 sperm, respectively (Table 1). The corresponding values obtained with 100 µM WHI-05 and WHI-07 were 46 and 40 pmol/106 sperm, respectively. The ZDV-MP was barely detectable (
2 pmol/106 sperm) (data not shown).
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DISCUSSION
In this study, we have assessed the intracellular metabolism of two dual-function aryl phosphate derivatives of ZDV with potent anti-HIV and spermicidal activity in genital tract epithelial cells and sperm. Our data show that despite their low TK activity, genital tract epithelial cells as well as sperm convert WHI-05 and WHI-07 to Ala-ZDV-MP and ZDV-MP. The Ala-ZDV-MP was found to be the predominant metabolite. The efficient metabolism of WHI-05 and WHI-07 in TK-deficient cells is particularly promising for curbing vaginal transmission of HIV by leukocytes and sperm as the infectious cells in the semen.
Cellular pharmacology of antiviral nucleoside analogues, including ZDV and other phosphotriester drugs of ZDV, has been studied primarily in cells noted for antiviral activity such as primary peripheral blood mononuclear cells, bone marrow cells, and established lymphoid cell lines, and more recently in placental cells [20–22, 32–35]. While these cells are adequate for evaluating the anabolic processes of these drugs, their intracellular fate in these cells cannot be generalized because the phosphorylation pattern of ZDV is different in different cell lines [20–22]. Because sexual transmission of HIV is primarily a cell-cell-mediated event between the HIV-infected macrophage-monocytes in the semen and genital tract epithelial cells, the latter are an appropriate in vitro system to study drug metabolism. Therefore, we investigated the time- and concentration-dependent intracellular formation of ZDV monophosphates in normal female genital tract epithelial cells as potential cellular targets for WHI-05 and WHI-07 for the prevention of heterosexual transmission of HIV.
The ability of ZDV to inhibit proviral DNA synthesis in various host cells depends upon the level of activity of cellular kinases, which are dependent on the activation state of the target cells as well as the cell cycle [20–22]. We used exponentially growing epithelial cells to detect TK activity by flow cytometry and radiolabeled methods. According to the radiolabeled assay, TK activity was very low in endocervical epithelial cells, being only 1.3% of that in the NALM-6 cells. The relative TK activity was even lower in motile sperm (<0.3% of that in NALM-6 cells). This enzyme, responsible for the conversion of ZDV into ZDV-MP, represents an essential step for the further activation of the ZDV to ZDV-TP [36]. Such marked reduction in TK activity in genital tract epithelial cells and sperm suggest that the parent nucleoside analogues are unlikely to have significant anti-HIV activity in genital tract secretions.
Despite the low or lack of TK activity, both vaginal and cervical epithelial cells were able to form Ala-ZDV-MP and ZDV-MP from WHI-05 and WHI-07. The intracellular levels of major ZDV monophosphates peaked at 3 h and decreased subsequently during the 24-h incubation period. We also found slightly lower levels of Ala-ZDV-MP and ZDV-MP in vaginal epithelial cells when compared with cervical epithelial cells. In both time- and concentration-dependent studies with WHI-05 and WHI-07, the endocervical epithelial cells consistently showed higher levels of Ala-ZDV-MP as well as ZDV-MP when compared with vaginal and ectocervical epithelial cells. Incubation of motile sperm with WHI-05 and WHI-07 also resulted in consistent increases in the intracellular levels of Ala-ZDV-MP. Due to their low cytoplasmic volume as well as to the spermicidal effect of WHI-05 and WHI-07, the levels of Ala-ZDV-MP measured were approximately 4- to 37-fold lower when compared to epithelial cells from healthy female genital tract. Nevertheless, motile sperm were able to form Ala-ZDV-MP within minutes of exposure to WHI-05 and WHI-07. Together, these studies provide the first analysis of the metabolic activation of phosphotriester prodrugs in female genital tract epithelial cells as well as sperm.
In addition to the HIV-infected mononuclear cells, recent studies form several laboratories indicate that HIV can be carried by human sperm as well [37, 38]. The HIV-1 particles bind and enter normal human sperm, through a receptor, galactosyl-alkyl-acylglycerol, an alternative to CD4 [39]. Three kinds of viral particles have been detected in the seminal fluid as well as in the space between the plasma membrane and cytoplasmic organelles or the midpiece mitochondrial region [40]. Sperm from HIV-1 seropositive men contain antigenically identifiable HIV-1 particles and HIV nucleic acid has been detected by polymerase chain reaction assays [41]. Such sperm are morphologically normal, motile, and can fertilize oocytes and transfer HIV-1 to the resulting embryos [42]. Therefore, the antiviral and spermicidal action of WHI-05 and WHI-07 is particularly relevant in genital tract secretions because of their ability to bypass the dependence of TK-mediated activation especially in leukocytes and germ cells in semen-associated virus.
Based on the primary metabolites obtained and from the known decomposition pathway of aryl phosphoramidate derivatives [29, 43, 44], we propose a novel scheme for intracellular activation of WHI-05 and WHI-07 (Fig. 7). In this scheme, the first step involves the attack on the bromomethoxy functional groups of WHI-05 and WHI-07 by cellular thiols to regenerate the double bond of the thymine ring. This pathway is suggested due to the fact that earlier investigators demonstrated that bromomethoxy-substituted uracil derivatives underwent this reaction either by substitution or via elimination. In the second step, WHI-05 and WHI-07 are converted to their corresponding free acids by a carboxyesterase. This reaction is followed by internal nucleophilic attack on the phosphoramidate by the anion and subsequent release of para-substituted phenols. The unstable cyclic mixed anhydride species undergoes rapid hydrolysis to generate the corresponding phosphomonoester amidate that can then be processed by endogenous phosphodiesterases and phosphatases to the 5'-monophosphate and alanine phosphate.
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In summary, the primary steps in the metabolism of two potent anti-HIV and spermicidal aryl phosphate derivatives of ZDV (WHI-05 and WHI-07) have been studied in normal female genital tract epithelial cells and sperm. Both WHI-05 and WHI-07 are rapidly converted to ZDV monophosphates, Ala-ZDV-MP, and ZDV-MP, as the major metabolites. The initial metabolic conversion of phosphotriester prodrugs to ZDV monophosphates appears to be independent of TK because the intracellular activity of this enzyme was very low. The successful intracellular delivery of bioactive nucleotides by the prodrugs despite low or lack of TK activity is particularly promising for curbing vaginal transmission of HIV by leukocytes and sperm as the infectious cells in the semen.
FOOTNOTES
1 This work was supported in part by grant RO1 HD37357 (O.J.D.) from the National Institutes of Health, National Institute of Child Health and Human Development, Bethesda, Maryland. 
2 Correspondence: Osmond J. D'Cruz, Parker Hughes Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113. FAX: 651 697 0645; odcruz{at}ih.org
Accepted: August 15, 2000.
Received: May 24, 2000.
REFERENCES
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