| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Article |
a Drug Discovery Program, Departments of Reproductive Biology,
b Chemistry, and
c Virology, Hughes Institute, St. Paul, Minnesota 55113
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Currently available over-the-counter prophylactic contraceptives for the prevention of HIV/AIDS are limited to membrane-active surfactants such as nonoxynol-9 (N-9), octoxynol-9, sodium docusate, or benzalkonium chloride [15,16]. Various formulations of N-9 are being recommended as vaginal microbicides despite the adverse effects of N-9 on cervicovaginal epithelium and vaginal microflora as well as the increased risk of development of opportunistic infections in the genitourinary tract [17–23]. Since the detergent-type microbicides have a high affinity for membrane lipids [24,25], they exhibit antiviral activity only at cytotoxic doses [26,27]. Furthermore, recent clinical trials have shown that vaginal contraceptive preparations containing N-9 have no effect on the transmission of HIV/AIDS and other sexually transmitted diseases when provided as part of a comprehensive program aimed at prevention of heterosexual transmission of HIV/AIDS [28,29]. Since the trafficking HIV-infected mononuclear cells in semen are the likely source for the sexual transmission of HIV, the infected seminal cells could easily come in direct contact with the target cells in the mucosa of the sexual partners after N-9-induced disruption of the cervicovaginal epithelial barrier, resulting in viral transmission [30–32]. Therefore, dual-function microbicides lacking detergent-type membrane toxicity would have advantages over the currently available vaginal microbicides.
In a systematic effort to develop a prophylactic contraceptive capable of preventing HIV transmission as well as providing fertility control, we have previously identified WHI-07, a novel-5-bromo 6-methoxy aryl phosphoramidate derivative of the anti-HIV drug, 3'-azido-3'-deoxythymidine (zidovudine [ZDV], or AZT), with potent anti-HIV and spermicidal activities [33]. Unlike ZDV, which is ineffective in thymidine kinase-negative cells, WHI-07 retained full activity in thimidine kinase-deficient cells, the main carriers of HIV in semen [34,35]. This study reports the structural as well as functional characteristics of novel analogues of WHI-07 that we synthesized by replacing the Ala side chain of WHI-07 with different amino acids containing nonpolar side chains. Notably, the replacement of the Ala side chain with Leu, Val, or Phe led to a complete loss of sperm-immobilizing activity (SIA), whereas replacement with Trp resulted in a 4-fold loss. No improvement in SIA could be achieved by replacement of the para-bromo substituent with other electron-withdrawing groups (namely, F, Cl, and NO2 groups) regardless of the choice of the amino acid side chain. Taken together, these results indicate that the Ala side chain as well as the para-bromo substituent on the phenyl moiety uniquely contribute to the potent spermicidal function of WHI-07. None of the amino acid side-chain replacements or para-position substituents in the phenyl ring resulted in cytotoxicity against normal human ectocervical or endocervical epithelial cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All of the anhydrous solvents and chemical reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) except for ZDV, which was obtained from Toronto Research Chemicals (ON, Canada). Proton (1H), carbon (13C), phosphorous (31P), and fluorine (19F) nuclear magnetic resonance (NMR) spectra were recorded on a Varian Oxford 300 MHz spectrometer (Varian Associates, Palo Alto, CA) using an automatic broadband probe. All NMR spectra were recorded in deuterated chloroform (CDCl3) at room temperature. The chemical shifts were recorded as 
values in parts per million (ppm) downfield from tetramethyl silane ( = 0.0 ppm) as internal standard or from the residual chloroform signal ( = 7.24 ppm for 1H NMR or = 77.0 ppm for 13C NMR). For 31P NMR, a solution of 1% phosphoric acid contained in a sealed capillary tube served as an internal standard, and the chemical shifts were recorded relative to this standard. In the case of 19F NMR, a fused capillary tube having 1% solution of trifluoroacetic acid in water was used as an internal standard. The multiplicity of the signals was designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. Ultraviolet (UV) spectra were recorded from a Beckman Model DU 7400 UV/visible spectrophotometer (Beckman Instruments, Fullerton, CA) using a cell path length of 1 cm. Fourier transform infrared (FT-IR) spectra were recorded using an FT-Nicolet Model Protege 460 instrument (Nicolet Instrument Corp., Madison, WI). The IR spectra of the liquid samples were run as undiluted liquids using KBr discs. Mass spectra analyses were performed using two different instruments. Some samples were analyzed using a Finnigan MAT 95 instrument (Madison, WI) after dissolving in chloroform. The source temperature was maintained at 200°C. The accelerating voltage was set at 5000 V, and the number of scans was approximately 70 for each sample. In addition, a Hewlett-Packard matrix-assisted laser desorption spectrometer Model G2025A (Wilmington, DE) was used in the molecular ion detection mode as a quick reference. The matrix used was cyano hydroxy cinnamic acid. Analytical HPLC was performed using a Hewlett-Packard 1100 series system consisting of a diode array detector, a quaternary pump, an automatic degasser, and a thermostat-controlled column compartment along with Chem station software (CambridgeSoft Corp., Cambridge, MA). In addition, an automatic injection assembly was used for repetitive analysis. A reverse-phase LiChrospher column (250 x 4 mm, Hewlett-Packard, RP-18) was used, and the analysis was performed using a isocratic flow consisting of water and acetonitrile (70%:30%). The flow rate was maintained at 1 ml/min throughout the analysis. Column chromatography was performed using silica gel.
Synthesis of WHI-07 and its Analogues
WHI-07 (Fig. 1) was synthesized in four steps by sequentially replacing the three chlorides in phosphorus oxychloride 1 with 4-bromophenol 2 (R2 = Br), L-alanine-methyl ester 3 (R1 = CH3), and ZDV to produce the 5'-phosphorylated ZDV 4, followed by the addition of bromo and methoxy groups across the double bond in the thymine moiety of 4 [35–37]. Accordingly, the new WHI-07 analogues were synthesized by 1) replacing L-alanine-methyl ester in the second step with the methyl ester derivatives of various amino acids such as Trp, Pro, Phe, Leu, Met, Val, or Gly (R1 = the side chain of Trp, Pro, Phe, Leu, Met, Val, or Gly) and/or 2) replacing 4-bromophenol in the first step with different phenols (R2 = H, F, Br, Cl, OCH3, or NO2) (Fig. 1). For the synthesis of Trp-substituted phosphoramidate derivatives, a modification was introduced in which after condensing the amino acid in methylene chloride solution, the resulting residue was solubilized in anhydrous tetrahydrofuran instead of ether. All 23 new WHI-07 analogues synthesized as shown in Figure 1 were characterized by 1H, 13C, and 31P NMR, FT-IR, UV-visible, mass spectra, and HPLC analyses. For each compound, the purity was > 98%.
|
In Vitro Sperm-Immobilizing Activity
All donor specimens were obtained after informed consent and in compliance with the guidelines of the Hughes Institute Institutional Review Board. To evaluate the spermicidal effects of ZDV derivatives, highly motile fraction of pooled donor sperm (n = 8) was prepared from liquefied normospermic semen by discontinuous (90–45%) gradient centrifugation using Enhance-S-Plus (Conception Technologies, San Diego, CA) cell isolation medium as previously described [38–40]. The resulting pellet was washed twice and resuspended in 1 ml of Biggers, Whitten, and Whittingham's medium (BWW; Irvine Scientific, Santa Ana, CA) containing 3% BSA (fraction V; Sigma Chemical Co., St. Louis, MO). Aliquots (1 ml) of the sperm suspension were centrifuged (500 x g, 5 min) and incubated at 45° for 90 min at 37°C in 5% CO2 atmosphere. The supernatant containing primarily motile sperm was aspirated carefully, washed once in BWW medium containing 25 mM Hepes and 0.3% BSA, and resuspended in the same medium. Pooled motile sperm (
10 x 106/ml) prepared from 3–5 donors were suspended in 1 ml of BWW-0.3% BSA in the presence and absence of serial 2-fold dilutions of test substance (1.9–500 µM) in 0.5% dimethyl sulfoxide (DMSO). The stock solutions of synthetic compounds were prepared in DMSO (100 mM) and diluted in medium to yield the desired concentrations. A corresponding volume of DMSO (0.5%) was added to control tubes. After 3-h incubation at 37°C, the percentage of motile sperm was evaluated by computer-assisted sperm motion analysis (CASA) [40–42]. The percentage motilities were compared with those of sham-treated control suspensions of motile sperm. The spermicidal activity of test compounds was expressed as the mean EC50 values (the final concentration of the compound in the medium that decreases the proportion of motile sperm by 50%) calculated from three independent experiments.
Sperm Kinematic Parameters
For CASA, 4 µl of each sperm suspension was loaded into a 20-µm Microcell (Conception Technologies) chamber at 37°C. Eight to ten fields per chamber were scanned for analysis using a Hamilton-Thorne Research (Danvers, MA) integrated visual optical system, version 10 instrument [40–42]. Each field was recorded for 30 sec. The computer calibrations were set at 30 frames at a frame rate of 30/sec. Other settings were as follows: minimum contrast, 8; minimum size, 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 µm/sec; threshold straightness at 80%; magnification factor, 1.95.
The sperm kinematic parameters that were determined included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head); straight-line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken); beat cross frequency (BCF, frequency of sperm head crossing sperm average path); the amplitude of lateral head displacement (ALH; the mean width of sperm head oscillation); straightness (STR = VSL/VAP x 100); and linearity (LIN = VSL/VCL x 100; departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled.
In Vitro Assay of Anti-HIV-1 Activity
The HIV-1 strain, HTLVIIIB, which was propagated in CCRF-CEM human leukemia cell line, was used for in vitro assays of the anti-HIV-1 activity of selected novel WHI-07 analogues. Cell-free supernatants of HTLVIIIB-infected CCRF-CEM cells were harvested, dispensed into 1-ml aliquots, and frozen at -70°C. Titration of stock virus was performed by examining its cytopathic effects in MT-2 cells [43]. Normal human peripheral blood mononuclear cells from HIV-negative donors were cultured for 72 h in RPMI 1640 supplemented with 20% (v:v) heat-inactivated fetal bovine serum, 3% interleukin-2, 2 mM L-glutamine, 25 mM Hepes, 2 g/L NaHCO3, 50 µg/ml gentamicin, and 4 µg/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1-h adsorption period at 37°C in a humidified 5% CO2 atmosphere. Subsequently, cells were cultured in 96-well microtiter plates (100 µl/well; 2 x 106 cells/ml) in the presence of various concentrations (0.001–100 µM) of spermicidal WHI-07 and its Trp-containing analogues (2a, 2b, and 2d). Aliquots of culture supernatants were removed from the wells on the seventh day after infection for p24 antigen assays, as previously described [43,44]. The p24 enzyme immunoassay applied was the unmodified kinetic assay commercially available from Coulter Corporation/Immunotech (Westbrook, ME). This assay utilizes a murine monoclonal antibody to HIV core protein coated onto microwell strips to which the antigen present in the test culture supernatant binds. Percentage viral inhibition was calculated by comparing the p24 values from the drug-treated infected cells with p24 values from untreated infected cells (i.e., virus controls). The effects of various treatments on cell viability were also examined. Noninfected peripheral blood mononuclear cells were treated with WHI-07 analogues for 7 days under identical experimental conditions. A microculture tetrazolium assay, using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide, was performed to quantitate cellular proliferation.
In Vitro Cytotoxicity Assay
The potential cytotoxicity of aryl phosphate derivatives of bromo-methoxy ZDV in comparison to N-9 against normal human ectocervical and endocervical epithelial cells (Clonetics Corporation, San Diego, CA) was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays (Boehringer Mannheim, Indianapolis, IN) [36,37,43]. Briefly, exponentially growing ectocervical and endocervical epithelial cells were seeded into 96-well plates at a density of 2 x 105 cells per well and incubated for 24 h at 37°C prior to drug exposure. On the day of treatment, culture medium was carefully aspirated from the wells and replaced with fresh medium containing drug concentrations ranging from 3.9 µM to 1000 µM. Triplicate wells were used for each treatment. The cells were incubated with the various compounds for 24 h at 37°C in a humidified 5% CO2 atmosphere. To each well, 10 µl of MTT (0.5 mg/ml final concentration) was added, and the plates were incubated at 37°C for 4 h to allow MTT to form formazan crystals by reacting with metabolically active cells. The formazan crystals were solubilized overnight at 37°C in a solution containing 10% SDS in 0.01 M HCl. The absorbance of each well was measured in a microtiter reader at 540 nm and a reference wavelength of 690 nm. To translate the OD540 values into the number of live cells in each well, the OD540 values were compared to those of standard OD540 versus cell number curves generated for each cell line. The percentage cell survival was calculated using the formula: % survival = live cell number [test]/live cell number [control] x 100. The results were expressed as IC50 values. The IC50 was defined as the concentration required for 50% reduction in cell survival. Two separate experiments were performed in triplicate to assess the potential cytotoxicity of spermicidal WHI-07 analogues versus N-9 against ectocervical and endocervical epithelial cells.
Statistical Analysis
Results are presented as the mean or mean ± SD values from independent measurements. Nonlinear regression analysis was used to find EC50 and IC50 values from the concentration-effect curves using GraphPad Prism (version 2.0) software (San Diego, CA). Correlations between EC50 values versus IC50 values and sperm motility versus VCL, VAP, and VSL were calculated using the Pearson's correlation coefficient; significance was set at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
WHI-07 is a potent spermicidal agent (EC50 = 5 µM). This novel agent is a phenyl phosphate derivative of ZDV, and the presence of bromo-methoxy functional groups in the thymine ring of its ZDV moiety is required for its SIA (Fig. 1). Although the phenyl phosphate moiety of WHI-07 is also very important for its activity, very little is known about the importance of esterification of the phosphate group with an Ala residue. In order to determine the importance of the Ala side chain of its phenyl phosphate moiety, we have synthesized new analogues of WHI-07 by replacing the Ala side chain (R1) with different amino acids containing nonpolar side chains, namely, Val, Leu, Met, Pro, Trp, Phe, and Gly. The para substituents (R2) on the phenyl moieties of these analogues included Br, Cl, F, NO2, and OMe groups. The assessment of the SIA of these compounds using CASA demonstrated that the choice of the amino acid side chain is a critical determinant for the SIA of the phenyl phosphate derivatives of ZDV. Notably, the replacement of the Ala side chain with Val, Leu, Pro, or Phe led to a complete loss of SIA with EC50 values of > 500 µM, whereas a replacement with Trp resulted in a 4-fold loss (EC50 = 20 µM; Fig. 2).
|
Ala Side Chain and the Bromo Substituent on the Aryl Moiety Contributed to Maximal SIA of Aryl Phosphate Derivatives of Bromo-Methoxy ZDV
No improvement in SIA could be achieved by replacement of the para-bromo substituent in the phenyl ring with other electron-withdrawing groups (F, Cl, NO2) regardless of the choice of the amino acid side chain (Fig. 3). However, many of the compounds with para substitution (R2) other than a bromine group exhibited SIA (Figs. 3 and 4). Thus, both the Ala side chain and the para-bromo substitution of the phenyl phosphate moiety, while neither was essential for the SIA of WHI-07, uniquely contributed to its potency. Specifically, both the unsubstituted and substituted compounds (1a–e) exhibited SIA with EC50 values ranging from 5 to 118 µM (Figs. 2, 3, and 4A). This property was also observed with the unsubstituted (H) and/or substituted aryl phosphate derivatives of bromo-methoxy ZDV containing Trp (2a, 2b, and 2d; Figs. 2, 3, and 4B), Pro (3b–d; Figs. 2, 3, and 4C), Meth (6a, 6b), and Gly (8a) as the amino acid ester (Fig. 3). In contrast, both the unsubstituted and para-substituted aryl phosphate derivatives of ZDV containing Phe (4a–d), Leu (5a–c), Val (7a, 7b), and the chloro-substituted derivative of Trp-containing side chain (2c), lacked SIA (Figs. 3 and 4B). Interestingly, while the para-bromo-substituted Ala compound WHI-07 was more potent than the unsubstituted Ala analogue (EC50: = 5 µM vs. 35 µM), the opposite was true for the Trp-containing (EC50: = 20 µM vs. 10 µM) and Pro-containing (EC50: = > 500 µM vs. 61 µM) compounds (Fig. 3). Among the Ala-containing WHI-07 analogues, the NO2-substituted compound was the least active. The potency order among the various compounds with various amino acid side chains and with or without para substituents on the phenyl ring was Br > F > OMe > H > NO2 for Ala-containing compounds, H > Br > NO2 > Cl for Trp-containing compounds, H > OMe > NO2 > Br for Pro-containing compounds, and OMe > H for Met-containing compounds.
|
The concentration-dependent SIA induced by WHI-07 analogues was associated with significant changes in the movement characteristics of the surviving sperm, particularly with respect to track speed (VCL), path velocity (VAP), and straight-line velocity (VSL). Figure 5 shows the concentration-dependent changes in sperm kinematic parameters obtained for representative spermicidal Ala (A), Trp (B), and Pro (C) analogues of WHI-07 with unsubstituted phenyl moiety. All three spermicidal analogues of WHI-07, irrespective of their amino acid variation, showed identical concentration-dependent changes in progressive motility accompanied by a parallel decline in VCL, VAP, and VCL (r2 = 0.889–0.936). The sperm motion parameters of control sperm showed no significant changes during the 180-min incubation period.
|
Diminished Anti-HIV Activity of Trp-Containing WHI-07 Analogues
Interestingly, while the para-bromo-substituted Ala compound WHI-07 was the most potent (IC50: = 0.005 µM), the opposite was true for the Trp-containing compounds (Table 1). The order of efficacy for Trp-containing WHI-07 analogues (2a, 2b, and 2d) was NO2 > H > Br. In an attempt to enhance the anti-HIV activity of 2a, we replaced the para-bromo substituent in the phenyl ring with a stronger electron-withdrawing para NO2 substituent (2d). Although compound 2d had improved anti-HIV activity in comparison to compound 2a, this activity was 8-fold less potent than that of WHI-07.
|
Aryl Phosphate Derivatives of Bromo-Methoxy ZDV Were Selectively Spermicidal
The MTT assay measuring cell proliferation and viability was used to test the in vitro cytotoxicity of novel spermicidal aryl phosphate derivatives of bromo-methoxy ZDV versus N-9 against confluent monolayers of normal human ectocervical and endocervical epithelial cells. Cells were exposed to these compounds at doses ranging from 3.9 µM to 1 mM for 24 h. Table 1 compares the mean IC50 (50% growth inhibitory calculated from the MTT assay-based concentration-response cell survival curves) values for these compounds with the mean EC50 values measured by CASA. In MTT assays, N-9 exhibited significant cytotoxicity to ectocervical epithelial and endocervical epithelial cells with mean IC50 values of 22 ± 8 µM and 16 ± 5 µM, respectively. By comparison, the IC50 values for spermicidal ZDV derivatives against normal human ectocervical and endocervical epithelial cells were > 300 µM (Table 1). Thus, N-9 was spermicidal only at cytotoxic concentrations (EC50 value: 81 µM; selectivity indices [SI]: 0.27 and 0.19 for ectocervical and endocervical epithelial cells, respectively), whereas even the most potent spermicidal derivatives of ZDV showed very little toxicity to epithelial cells. None of the ZDV derivatives were cytotoxic to female reproductive tract epithelial cells at spermicidal EC50 concentrations. Thus, none of the amino acid side-chain replacements or para-position substituents in the phenyl ring resulted in increased cytotoxicity against ectocervical or endocervical epithelial cells. Also, there was no correlation between EC50 values and IC50 values for ectocervical epithelial cells (r2 = 0.0011; n = 14) and endocervical epithelial cells (r2 = 0.0094; n = 14). The absence of a linear relationship between the IC50 and EC50 values for the 14 spermicidal compounds tested in our study illustrates that the SIA of this class of compounds was not caused by a nonspecific cytotoxic function (Fig. 6).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We compared the influence of both aliphatic and aromatic amino acid side chains on SIA of phosphoramidate derivatives of bromo-methoxy ZDV. Among the eight amino acid esters studied, Ala is the most effective phosphoramidate group. The hydrophobic methyl group on the Ala side chain may not confer advantage, since methyl group-containing Leu and Val analogues (5a–c and 7a, 7b) were devoid of SIA. The finding that these amino acids with larger hydrophobic side chains than Ala lacked SIA suggests that the spatial bulk of the amino acid side chain may be one of the determinants for SIA. The Met analogues (6a, 6b) were also less active than the Ala analogues, with SIA rather similar to that of the Pro derivatives (3b–d). Since the Pro analogues (3b–d) with blocked amino group retained SIA, although they were less active (12- to 16-fold) than the lead compound (1a), the SIA appears not to be dependent on the amino group either. This is also supported by the variable potency of Trp analogues (2a, 2b, and 2d) with enhanced steric bulk in the side chain, which were somewhat less active (2- to 8-fold) than the Ala lead compound (1a). The potency of SIA of Trp derivatives (2a, 2b, and 2d) can be attributed to the presence of an indole ring with an amino group that can facilitate faster intracellular entry, as well as to the presence of two bromo-methoxy substitutions in Trp analogues. Comparative structure-activity relationship studies of Ala- and Trp-containing analogues of WHI-07 unequivocally established the importance of Ala side chain and the contribution of an electron-withdrawing para-bromo substituent on the phenyl ring in addition to the bromo-methoxy functionalization for the potent, dual-function anti-HIV and spermicidal activities.
Our finding that aryl phosphate derivatives of bromo-methoxy ZDV inhibit sperm motility without affecting the female reproductive tract epithelial cells suggests that these membrane-permeable dual-function nucleoside analogues may provide the basis for a new strategy aimed at prevention of the sexual transmission of HIV while providing fertility control for women.
|
|
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, MD. 
2 Correspondence: Osmond J. D'Cruz, Hughes Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113. FAX: 651 697 0645; odcruz{at}ih.org
Accepted: September 7, 1999.
Received: May 19, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  O. J. D'Cruz and F. M. Uckun 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 Toxicol Pathol, December 1, 2007; 35(7): 910 - 927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. J. D'Cruz, B. Waurzyniak, and F. M. Uckun Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats Antimicrob. Agents Chemother., April 1, 2004; 48(4): 1082 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. J. D'Cruz, D. Erbeck, and F. M. Uckun Developmental Toxicology Studies of WHI-07, a Novel Nucleoside Analogue-Based Dual-Function Microbicide, Administered Intravaginally to Rabbits Toxicol Pathol, October 1, 2003; 31(6): 698 - 708. [Abstract] [PDF]
|
|
|
|
 |
|
 O. J. D'Cruz and F. M. Uckun Pre-clinical safety evaluation of novel nucleoside analogue-based dual-function microbicides (WHI-05 and WHI-07) J. Antimicrob. Chemother., December 1, 2002; 50(6): 793 - 803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. J. D'Cruz, T. K. Venkatachalam, C. Mao, S. Qazi, and F. M. Uckun Structural Requirements for Potent Anti-Human Immunodeficiency Virus (HIV) and Sperm-Immobilizing Activities of Cyclohexenyl Thiourea and Urea Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase Biol Reprod, December 1, 2002; 67(6): 1959 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. J. D'Cruz, B. Waurzyniak, and F. M. Uckun A 13-Week Subchronic Intravaginal Toxicity Study of the Novel Broad-Spectrum Anti-HIV and Spermicidal Agent, N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (PHI-346) in Mice Toxicol Pathol, October 1, 2002; 30(6): 687 - 695. [Abstract] [PDF]
|
|
|
|
 |
|
 O. J. D'Cruz and F. M. Uckun 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 Toxicol. Sci., April 1, 2001; 60(2): 373 - 378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun Thymidine Kinase-Independent Intracellular Delivery of Bioactive Nucleotides by Aryl Phosphate Derivatives of Bromo-Methoxy Zidovudine (Compounds WHI-05 and WHI-07) in Normal Human Female Genital Tract Epithelial Cells and Sperm Biol Reprod, January 1, 2001; 64(1): 51 - 59. [Abstract] [Full Text]
|
|
|
|
|
|
O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun Novel Thiourea Compounds as Dual-Function Microbicides Biol Reprod, July 1, 2000; 63(1): 196 - 205. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
