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a Drug Discovery Program,
b Departments of Reproductive Biology,
c Chemistry, and
d Virology, Wayne Hughes Institute, St. Paul, Minnesota 55113
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we show that aryl phosphate derivatives of 5-bromo-6-methoxy-AZT are spermicidal and that they maintain potent anti-HIV activities. We have investigated further as to their effects on sperm membrane ultrastructure, interaction of sperm with human and zona-free hamster eggs, local toxicity to the cervicovaginal epithelium, and in vivo contraceptive efficacy of the lead compound, WHI-07, in a mouse model. Our data suggest that AZT derivatives that have 5-bromo-6-methoxy functional groups on the thymine ring may be useful as topical microbicides capable of preventing the sexual transmission of HIV as well as providing fertility control for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We synthesized three new analogues of AZT (WHI-01, -02, and -03) with 5-bromo and 6-methoxy groups on the thymine ring and azido/amino substitution in the C-3 position of the pentose ring using published procedures [21]. The chemical compositions of these three AZT analogues are shown in Figure 1. Similarly, 9 novel aryl phosphate derivatives of AZT (WHI compounds 04, 05, 06, 07, 08, 09, 10, 11, and 12) were prepared using phosphorochloridate chemistry [22, 23]. In brief, reaction of appropriately substituted phenols with phosphorous oxy chloride in the presence of triethylamine yielded the phosphorodichloridate derivative. Condensation with AZT in anhydrous tetrahydrofuran containing N-methylimidazole as base yielded the target aryl phosphoramidate derivative of AZT. Further modifications in the structure were achieved by reaction with bromine in anhydrous methanol to obtain the desired target compounds (Fig. 2). For each compound, the purity was > 95%, as determined by the proton (1H) nuclear magnetic resonance spectra (Varian 300 MHz; Varian Associates, Palo Alto, CA) and HPLC. For each compound, the characterization included carbon (13C) NMR, Fourier transform infrared spectra (FT-Nicolet model Protege 460; Nicolet Corporation, Madison, WI), UV-visible spectra (Beckman model DE 7400 spectrophotometer; Beckman Instruments, Fullerton, CA), HPLC (Hewlett Packard model 1100 series; Hewlett-Packard Co., Wilmington, DE), and mass spectra (Hewlett Packard Matrix Assisted Laser Desorption Time-of-Flight spectrometer model G2025A) analysis. Azidothymidine/zidovudine was purchased from Aldrich Chemical Co. (Milwaukee, WI).
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HIV-1 Replication Assay
To evaluate anti-HIV-1 activities of AZT, WHI compounds, and N-9, peripheral blood mononuclear cells (PBMC) from HIV-1-negative donors were cultured for 72 h in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 20% (v:v) heat-inactivated fetal calf serum, 5% interleukin-2, 2 mM 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. Subsequently, cells were cultured in 96-well microtiter plates (100 µl/well; 2 x 106/ml, triplicate wells) in the presence and absence of various concentrations (0.001–100 µM) of drugs for 7 days. Aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen enzyme immunoassay as an indication of viral replication [24, 25]. Percentage inhibition of virus replication was calculated by comparing the p24 antigen values from the test drug-treated infected cells with p24 antigen values from untreated infected cells. The anti-HIV activity of tested compounds was expressed as the IC50 values (the final concentration of the compound in culture medium that decreases the replication of HIV-1 by 50%). Cell viability was quantified by a colorimetric assay monitoring the ability of viable cells to reduce 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide.
Sperm Immobilization Assay (SIA)
To evaluate the spermicidal effects of AZT and WHI compounds 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, and 12 in comparison with the detergent spermicide N-9 (IGEPAL CO-630; Rhone Poulenc, Cranbury, NJ), a highly motile fraction of pooled donor sperm (n = 10) was prepared by discontinuous (90–45%) Percoll gradient (Conception Technologies, San Diego, CA) centrifugation and the "swim-up" method as previously described [26]. All donor specimens were obtained after informed consent and in compliance with the guidelines of the Wayne Hughes Institute Institutional Review Board. Motile sperm (
10 x 106/ml) were suspended in 0.5 ml of Biggers, Whitten, and Whittingam's medium (BWW) containing 25 mM Hepes (Irvine Scientific, Santa Ana, CA) and 0.3% BSA (fraction V; Sigma Chemical Co., St. Louis, MO) in the presence and absence of serial 2-fold dilutions of test substance (300-4.6 µM) in 1% dimethyl sulfoxide (DMSO). The stock solutions of synthetic WHI compounds were prepared in DMSO (10 mg/ml) and diluted in DMSO to yield the desired concentrations. A corresponding volume of DMSO (1%) was added to control tubes. N-9 was diluted in BWW-0.3% BSA (pH 7.4) to yield the desired concentrations (300-4.6 µM). After 3 h of incubation at 37°C, the percentage of motile sperm was evaluated by computer-assisted sperm motion analysis (CASA) as described previously [27]. 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 EC50 values (the final concentration of the compound in medium that decreases the proportion of motile sperm by 50%).
To test the effect of duration of incubation on SIA in the presence of WHI compounds (01, 03, 05, and 07), a motile fraction of sperm (107/ml) was incubated at 37°C in 0.5 ml of BWW-0.3% BSA in the presence of 200 µM WHI-01, or 100 µM each of WHI-03, WHI-05, WHI-07, and N-9 or 1% DMSO alone. At timed intervals, duplicate aliquots (4-µl) were transferred to two 20-µm Microcell (Conception Technologies, San Diego, CA) chambers, and sperm motility was assessed by CASA. For WHI-01, CASA was performed every 20 min for 150 min; for N-9, every 15 min for 120 min; for WHI-05, every 10 min for 70 min; and for WHI-03 and WHI-07, every 5 min for 40 min.
The possible effects of seminal plasma on SIA of WHI compounds were studied either in the presence of 10% cell-free seminal plasma in the assay medium or by direct addition (200 µM) of WHI-03, WHI-05, and WHI-07 to dilutions (1:2–1:6) of liquefied donor semen (n = 3) in PBS and incubation at 37°C. After 3 h of incubation, duplicate aliquots (4-µl) were used for CASA.
To assess the persistence of SIA after removal of WHI compounds, pooled motile sperm (5 x 106) were added to 0.5 ml of assay medium in the presence (100 µM) or absence of WHI-01, WHI-05, and WHI-07 in 1% DMSO. After incubation for 30 min at 37°C, duplicate aliquots were used for sperm motility assessment using CASA. The remaining sperm suspension was washed by the addition of fresh assay medium and centrifugation (500 x g for 5 min). This supernatant was discarded, and the pellet was resuspended in fresh medium (without WHI compounds) to the original volume and reincubated. After 30 min at 37°C, duplicate aliquots were reassessed for sperm motion parameters by CASA. The results were expressed as the mean of two assessments and were compared to the sperm motion parameters of similarly processed sperm suspensions of motile sperm suspended in medium containing DMSO-only controls.
Sperm Kinematic Parameters
For CASA, 4 µl each of sperm suspension was loaded into two 20-µm Microcell chambers placed onto a counting chamber at 37°C. At least 5–8 fields per chamber were scanned for analysis using a Hamilton Thorne Integrated Visual Optical System (IVOS) version 10 instrument (Hamilton Thorne Research Inc., Danvers, MA). Each field was recorded for 30 sec. The Hamilton Thorne computer calibrations were set at 30 frames at a frame rate of 30 images/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 and threshold straightness at 80%; magnification factor, 1.95. The performance of the analyzer was periodically checked using the playback function.
The attributes of sperm kinematic parameters evaluated 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), and the derivatives, straightness (STR = VSL divided by VAP x 100), linearity (LIN = VSL divided by 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.
Membrane Integrity Studies
The percentages of sperm with an intact acrosome were evaluated by fluorescence microscopy (Olympus BX-60 model; Olympus Corp., Lake Success, NY) after fixation and ethanol permeabilization of the sperm pellets and subsequent staining with fluorescein (FITC)-conjugated Pisum sativum lectin (Sigma). In positive control sperm, the acrosome reaction was induced by incubating (3 h at 37°C) the sperm suspension with 10 µM calcium ionophore (CaI) A23187 (Sigma). Motile sperm (5 x 106/ml) were incubated for 4 h at 37°C in the presence and absence of 100 µM each of WHI-01, WHI-03, WHI-05, WHI-07, or N-9 followed by the addition of propidium iodide (PI; 10 µg/ml) and analyzed by flow cytometry. The percentage of sperm positive for PI was determined by flow cytometry using a fluorescence-activated cell sorting Vantage flow cytometer (Becton Dickinson and Co., Mountain View, CA). The sperm in the swim-up fractions were identified by their characteristic forward and 90° angle light-scattering properties [28]. All analyses were done using the 488-nm excitation from an krypton/argon laser with a 635-nm band pass filter for PI emission. The percentage of sperm positive for PI staining was determined using cutoff signals for membrane-intact motile sperm.
Confocal Laser Scanning Microscopy
Ethanol-permeabilized and air-dried sperm smears were stained sequentially with the three fluorescent markers, FITC-Pisum sativum, TOTO-3 iodide, and Nile red (Molecular Probes, Eugene, OR) because their targets are different (acrosome, nucleus, and plasma membrane of permeabilized sperm, respectively). Samples were examined under a Bio-Rad MRC-1024 Laser Scanning Confocal Microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a krypton/argon mixed gas laser (excitation lines 488, 568, and 647 nm) and mounted on a Nikon Eclipse E800 (Garden City, NY) series upright microscope. The fluorescence emissions of fluorescein, TOTO-3 iodide, and Nile red from the acrosomal region, nucleus, and the plasma membrane of sperm after ethanol permeabilization were simultaneously detected using the 598/40 nm, 522 DF32, and 680 DF32 emission/filter, respectively. Confocal images were obtained using a Nikon x60 (NA 1.35) objective lens and Kalman collection filter. Digitized images were saved on a Jaz disk (Iomega Corporation, Roy, UT) and processed with the Adobe Photoshop software (Adobe Systems, Mountain View, CA). Final images were printed using a Fujix Pictography 3000 (Fuji Photo Film Co., Tokyo, Japan) color printer.
High-Resolution Low-Voltage Scanning Electron Microscopy (HR-LVSEM)
HR-LVSEM was utilized for topographical imaging of different membrane domains over the sperm head as described previously [29, 30]. Aliquots (20 x 106) of motile sperm were incubated with DMSO alone (1%), or 100 µM each of WHI-05 or WHI-07 or 10 µM CaI in DMSO for 3 h at 37°C. Washed sperm suspensions were placed on 0.1% poly-L-lysine-coated glass chips and allowed to adhere to the glass over a 60-min incubation period on ice. The supernatants were decanted, and adherent cells were fixed in 1% paraformaldehyde and 1% glutaraldehyde in 0.14 M sodium cacodylate buffer for 3 h. To preserve plasma membrane integrity, sperm were postfixed in 1% osmium tetroxide (OsO4) containing 0.1% ruthenium red in 0.14 M cacodylate buffer for 1 h at 4°C. All samples were dehydrated through an ascending ethanol series, critical point dried, and coated with approximately 2 nm of platinum using ion beam sputtering with argon (4 mA at 10 kV; Ion Tech Ltd., Middlesex, England). All samples were examined in a Hitachi S-900 SEM (Hitachi Instruments, Gaithersburg, MD) at an accelerating voltage of 2 kV. Sperm were observed under low magnification (x2000–5000), and representative sperm were photographed under intermediate magnification (x18 000–25 000). In each specimen evaluated, at least 200 sperm were scanned for the intactness of sperm acrosomal region.
Transmission Electron Microscopy (TEM)
For TEM, treated sperm were fixed in 3% glutaraldehyde in 5% sucrose in 0.2 M cacodylate buffer for 3 h at room temperature. The samples were postfixed in 1% OsO4, 5% sucrose, and 0.1% ruthenium red for 2 h at 4°C. The samples were dehydrated in ascending ethanol series and embedded in Spurr's epoxy. Thin sections (90 nm) were prepared on a Reichert-Jung (Nossloch, Germany) Ultracut E and stained with uranyl acetate and lead citrate. The grids were examined using a JEOL-1200 EX II electron microscope (JEOL, Paris, France) at a accelerating voltage of 60 kV. Sperm were observed under low (x25 000) and high (x100 000) magnification, and representative sperm were photographed. In each specimen evaluated, 100–200 sperm were evaluated for the intactness of sperm head membranes.
Homologous Sperm-Zona Binding Assay
Specifically, human eggs that failed to fertilize in vitro were separately inseminated with a mixture of equal numbers of SYBR 14 (green)- and SYTO 17 (blue)-labeled sperm, the latter pretreated with three concentrations (25, 50, and 100 µM) of the tested compounds. After coincubation, the number of tightly bound green (SYBR-14) and blue (SYTO-17) sperm were assessed by confocal laser scanning microscopy.
Frozen human eggs (n = 46) were thawed and rinsed three times in BWW-0.3% BSA, and then three times in BWW-3.5% BSA, before addition of sperm suspension. The process of egg recovery, freezing, thawing, and manipulation in vitro invariably resulted in cumulus-free, nonviable eggs. To investigate the effect of WHI-05 and WHI-07 on the binding of human sperm to human zona, the motile fraction of capacitated sperm was divided into two aliquots of 107/ml and labeled with two cell-permeant DNA-specific dyes (SYBR-14 and SYTO-17). The first aliquot was incubated with 2.5 µM SYBR-14 (Molecular Probes), and the second was incubated with 5 µM SYTO-17. After 30 min of incubation, sperm motility was assessed by CASA. Motile sperm with intensely stained green or blue nuclei were washed thrice in BWW, and one million motile sperm labeled with SYBR-14 (green; control) and SYTO-17 (blue; control or test) were added to a dish containing human zona (n = 2) under mineral oil and coincubated for 4 h. After extensive washing of eggs to dislodge any loosely adherent sperm, the eggs were mounted on glass slides and examined under a Bio-Rad MRC-1024 Laser Scanning Confocal Microscope as described above. Using fluorescence imaging, the fluorescence emission of SYBR-14 and SYTO-17 localized on the sperm head was simultaneously recorded using 598/40 nm and 680 DF32 emission/filter, respectively. Confocal images of Z-sections were obtained using a Nikon x20 (NA 1.4) objective and Kalman collection filter. Digitized images were processed as described above. The number of tightly bound green- and blue-colored sperm nuclei adherent to each human egg was counted from the Z-sections of fluorescence images of each egg on all focal planes and was expressed as a binding ratio.
Heterologous Sperm-Egg Binding and Penetration Assay
The fertilizing capacity of human sperm exposed to WHI-05 and WHI-07 was evaluated using the zona-free hamster egg penetration assay [31], with modifications. Motile fractions of sperm (n = 4) obtained by 90–45% Percoll gradient centrifugation and wash were resuspended in BWW-3.5% BSA medium (pH 7.4) and divided into aliquots of 20 x 106 sperm/ml and allowed to capacitate for 24 h. Subsequently, sperm were treated at 37°C for 3 h with 25 µM or 100 µM concentrations of WHI-05 and WHI-07 (in 0.5% DMSO) under capacitating conditions. After exposure to drugs, the control and WHI-treated sperm suspensions were washed in BWW-3.5% BSA medium. Sperm motility following capacitation and washing in BWW-3.5% BSA medium was assessed by CASA.
Frozen hamster eggs (Charles River Laboratories, Wilmington, MA) were thawed and transferred to 35-mm glass-bottom microwells (Mat Tek Corporation, Ashland, MA) in BWW medium. To remove zona, the washed eggs were treated with 0.05% trypsin (Irvine Scientific) in BWW medium for 20 min. Then 15 to 45 zona-free eggs per test were transferred into 100 µl of BWW-3.5% BSA medium under mineral oil. Control and test sperm suspensions (2 x 106) in < 100 µl of BWW-3.5% BSA medium were added, and the gametes were coincubated for 4 h at 37°C in 5% CO2 in air. After incubation, the eggs were washed three times with BWW medium to remove the loosely associated sperm. The eggs were fixed and stained as previously described [32]. Bound and decondensed fluorescent sperm were viewed with a Olympus model BX-60 epifluorescent microscope equipped with an excitation filter BP 360–370 and DM 400 barrier filter BA 420. The number of sperm bound per egg, the number of swollen sperm heads per egg, and the percentage of penetrated eggs were determined for control and test sperm. An egg was considered penetrated when it contained at least one swollen sperm head in the cytoplasm.
In Vivo Fertility Trials
Sexually mature adult female CD-1 mice were superovulated by a i.p. injection of 5 IU of eCG (Gestyl; Diosynth B.V., Oss, Holland) followed by an i.p. injection of 5 IU of hCG (Steris Laboratories, Phoenix, AZ) 46–48 h later. Mice were randomly assigned to one of the two treatment groups (27 or 24 per group): 1) vehicle controls received cream base (Taro Pharmaceuticals, Hawthorne, NJ) with 1% DMSO; 2) the test group received cream base with 1% WHI-07 in 1% DMSO. These treatments were given intravaginally (50 µl) prior to artificial insemination. Caudae epididymal sperm were obtained from proven breeder adult CD-1 males [33] and suspended in a modified Krebs-Ringer-bicarbonate medium (M2; Gibco-BRL) supplemented with pyruvate, lactate, and glucose. Sperm were suspended in M2 medium-5% BSA for 1 h prior to use. For each experiment, cauda epididymal sperm pooled from 25 male mice and adjusted to 1–2 x 106 motile sperm/50 µl were used for insemination. A 50-µl volume per mouse was ejected through a 1-ml syringe with a blunted 18-gauge needle. Preparations were analyzed for sperm concentration, motility, and sperm motion parameters by CASA using software designed for oval sperm head morphology (Hamilton Thorne). On Day 8, individual females representing the control and test group were killed, and their uteri were examined for the presence or absence of embryos. A total of three independent fertility experiments were performed.
In Vivo Local Toxicity Studies
Thirty adult female CD-1 mice were randomly divided into two groups (A and B). Each group was further divided into three subgroups of 5 mice. Mice from group A were treated for 5 days and mice from group B for 20 days via daily intravaginal application of a cream base (Taro Pharmaceuticals) supplemented with 1% DMSO alone (control), 5% WHI-07 in 1% DMSO, or 5% N-9. After 5 and 20 days, respectively, mice were killed by cervical dislocation, and tissues from the genitourinary tract were fixed in 10% buffered formalin. To determine the degree of inflammation and membrane integrity of squamous epithelia, conventional paraffin-embedded sections (6-µm) were prepared and stained with hematoxylin and eosin and observed under x300 magnification with a Leica (Milton Keynes, Buckinghamshire, UK) microscope interfaced with an image analysis system. The images were captured using the ImagePro Plus program (Media Cybernetics, Silver Spring, MD) in conjunction with a 3CCD camera (DAGE-MTI Inc., Michigan City, KS), and images were transferred to Adobe Photoshop software for printout. The four stages of the estrous cycle were determined histologically.
Indirect Immunofluorescence Assay (IFA)
IFA and confocal microscopy were used to identify the neutrophil infiltrates in the squamous epithelia of cervicovaginal sections of control, WHI-07-treated, and N-9-treated mice. Cervicovaginal tissue sections were deparaffinized, hydrated through graded ethanol, and immunostained with a rat monoclonal antibody (mAb; Cederlane Laboratories Ltd., Westbury, NY) directed against mouse neutrophils (clone 5120-26.1-110). After antigen retrieval by heating (93°C for 10 min in 10 mM sodium citrate buffer, pH 6.0), sections were preincubated with 10% normal goat serum in PBS and treated with optimal dilution (5 µg/ml) of the mAb followed by FITC-conjugated goat anti-rat IgG (Pierce Chemical Co., Rockford, IL). Each incubation step lasted 60 min, with 5 min PBS washes between each step. The slides were counterstained with PI (1 µg/ml), washed in deionized water, mounted in anti-fade (Bio-Rad), and visualized by laser confocal microscopy as described above. Negative controls were carried out by replacing the primary mAb with PBS or by using rat IgG2 as control mAb.
Statistical Analysis
Results are presented as mean or mean ± SD. Comparisons between WHI-01-, 05-, or 07-treated sperm and control sperm motility, acrosomal loss, and sperm binding to eggs were analyzed by a paired, two-tailed Student's t-test. Linear regression analysis was used to find correlated values between two measured parameters, and t-test was used to find the significance of differences between two independent correlations. A p value of < 0.05 was considered significant. Nonlinear regression analysis was used to find IC50 and EC50 values from the concentration effect curves using GraphPad Software (San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of several AZT analogues and derivatives on human sperm function and HIV replication in HIV-infected human PBMC were examined. Exposure of the highly motile fraction of human sperm to AZT, which inhibited HIV-1 replication in human PBMC in vitro with an IC50 value of 0.006 µM, did not affect sperm motility even at concentrations as high as 300 µM (Fig. 1). Further, sperm motion kinematics using CASA (Hamilton Thorne version 10) confirmed that AZT treatment did not alter the sperm motion parameters, such as the progressive motility, track speed, path velocity, straight-line velocity, straightness of the swimming pattern, linearity of the sperm tracks, beat-cross frequency, and the amplitude of lateral sperm head displacement. Introduction of a bromo at the 5-position (R2) and methoxy at the 6-position (R3) on the thymine base ring of AZT to yield 5-bromo-6-methoxy-5,6-dihydro-AZT (compound WHI-01) resulted in gain of significant spermicidal (as determined by sperm motility assay) function (EC50 = 104 µM) without decreasing the anti-HIV activity (Fig. 1). Replacement of the azide group (R1) in the pentose ring with an NH2 group (compound WHI-03) substantially enhanced the spermicidal activity (EC50 = 12 µM), but it also caused a 50-fold loss in the anti-HIV activity. The addition of bromo and methoxy functional groups was essential for the spermicidal activity of WHI-03 since 3'-amino-3'-deoxythymidine (compound WHI-02), which was used as a control, was not spermicidal (EC50 > 300 µM) (Fig. 1).
Aryl Phosphate Derivatives of 5-Bromo-6-Methoxy-AZT Exhibited Potent Anti-HIV and Spermicidal Activities
Next, novel aryl phosphate derivatives of compound WHI-01 were synthesized by phosphorochloridate chemistry and examined for their anti-HIV efficacy in cell culture and spermicidal activity by CASA (Figs. 2 and 3). Compound WHI-04 with the unsubstituted aryl moiety at the R4 position elicited 3-fold greater spermicidal activity than WHI-01. Introduction of a p-methoxy (compound WHI-05, EC50 = 29 µM), p-fluoro (compound WHI-06, EC50 = 15 µM), or p-bromo (compound WHI-07, EC50 = 6 µM) substituent at the R4 position in the aryl moiety further enhanced the spermicidal activity with an order of potentiation p-bromo > p-fluoro > p-methoxy. Interestingly, the p-bromo substitution at the R4 position of the aryl moiety was also associated with a significant gain in anti-HIV function. The p-methoxy-substituted aryl phosphate derivative WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl) methoxyalaninyl phosphate) had an EC50 value of 29 µM in sperm motility assays and an IC50 value of 0.05 µM in HIV replication assays. The lead compound, WHI-07, with a p-bromo substitution (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate), had an EC50 value of 6 µM in sperm motility assays (Fig. 2), which is consistent with a one-log higher potency than that of the detergent spermicide N-9 (EC50 = 81 µM). WHI-07 displayed a potent anti-HIV activity with an IC50 value of 0.005 µM in HIV replication assays, which was virtually identical to that of AZT (IC50 = 0.006 µM) and 439-fold more potent than that of N-9 (IC50 = 2.195 µM) when tested side by side under identical experimental conditions (Fig. 2).
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The removal of the azido group of the pentose ring (WHI-11 and WHI-12) was associated with a substantial loss of the anti-HIV activity and attenuation of the spermicidal activity for these novel dual-function AZT derivatives (Fig. 2). The 5-bromo-6-methoxy functional groups in the thymine ring appeared to be essential for the spermicidal activity of the p-methoxy-, p-fluoro-, or p-bromo-substituted aryl phosphate derivatives of AZT, since no spermicidal activity was observed in their absence (i.e., the EC50 values for compounds WHI-08, WHI-09, and WHI-10 were all > 300 µM) (Fig. 2). In contrast, the anti-HIV activity of the aryl phosphate derivatives of AZT did not require the bromo-methoxy functional groups in the thymine ring.
Kinetics of Sperm Immobilization
The comparative concentration-dependent effects of AZT, three analogues of AZT, and four aryl phosphate derivatives of AZT on sperm motility were analyzed by CASA (Fig. 3A). At a concentration > 25 µM, WHI-07 abrogated the motility of > 95% of the treated sperm, whereas AZT and its derivatives, WHI-02, WHI-08, and WHI-09 without the 5-bromo 6-methoxy functional groups, were inactive even at 300 µM. Also, the kinetics of sperm immobilization was fast with spermicidal AZT derivatives when compared to that with N-9 at 100 µM (Fig. 3B). There was a linear relationship between incubation time and progressive sperm motility by CASA. Regression analysis of progressive motility against duration of incubation time for WHI-05 and WHI-07 showed positive correlation coefficients of 0.88 (p < 0.005) and 0.84 (p < 0.003). The corresponding times required for 50% motility loss of progressively motile sperm exposed to WHI-05 and WHI-07 (at 100 µM concentration) were 17 min (95% confidence interval: 12–26 min) and 6 min (95% confidence interval: 4–11 min), respectively. Complete sperm immobilization was achieved after 120 min of exposure to N-9, 60 min exposure to WHI-05, and 30 min of exposure to WHI-07. By comparison, the sperm motility in control samples remained stable (96 ± 2.5% compared to baseline) during the 150-min monitoring period.
Spermicidal AZT Derivatives Affected Sperm Kinematics
The time-dependent sperm motility loss induced by WHI-01, WHI-05, and WHI-07 was associated with significant changes in the movement characteristics of the surviving sperm, particularly with respect to the track speed (VCL), path velocity (VAP), and straight-line velocity (VSL). The representative sperm kinematic parameters observed for WHI-07-treated sperm versus time are shown in Figure 4. The decreases in VSL and VCL were similar in magnitude. Therefore, linearity (LIN) of the sperm tracks were not altered from 5 to 20 min in the presence of WHI-07, from 10 to 60 min in the presence of WHI-05, and from 20 to 150 min in the presence of WHI-01. Similarly, the decreases in VSL and VAP were similar in magnitude. Therefore, straightness (STR) of the swimming pattern was not altered either. Also, the beat-cross frequency (BCF) and the amplitude of lateral sperm head displacement (ALH) were relatively uniform as the proportion of motile sperm declined with exposure time. The sperm motion parameters of control sperm showed insignificant changes during the 150-min exposure.
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Spermicidal Activity of AZT Derivatives Was Irreversible
In order to determine whether the SIA of WHI-05 or WHI-07 was reversible, sperm exposed to 100 µM of either compound for 30 min were washed and resuspended in fresh sperm motility assay medium, and sperm motility was reassessed by CASA (Table 1). No recovery in sperm motility was observed, indicating that the drug-induced sperm immobilization was irreversible. Direct addition of compounds WHI-05 and WHI-07 to semen diluted in PBS resulted in complete loss of motility as assessed by CASA (data not shown). Thus, the spermicidal activity was not affected by seminal plasma.
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Lack of Membrane Disruption by Spermicidal AZT Derivatives
Because the great majority of spermicidal compounds are believed to immobilize sperm as a result of a detergent-type action on the sperm plasma membrane [34–36], we also tested the effects of WHI-05 and WHI-07 on sperm plasma membrane permeability by flow cytometric analysis of PI-stained sperm and acrosome integrity by examination of FITC-lectin-, TOTO-3 iodide-, and Nile red-stained sperm using confocal laser scanning microscopy. Despite complete immobilization of sperm in the presence of WHI-05 and WHI-07, < 3% of sperm were permeable to PI after 4 h of incubation with these compounds. In acrosome integrity studies, WHI-05- and WHI-07-treated sperm (similar to sham-treated sperm) remained acrosome intact (97 ± 2% and 93 ± 5.6%, respectively) after 3 h of incubation at a concentration of 100 µM despite a complete loss of motility.
Examination of FITC-lectin-, TOTO-3-, and Nile red-stained sperm by confocal microscopy revealed an intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3 (blue), and membrane staining (red) with Nile red, respectively (Fig. 5, upper panels). In acrosome-intact sperm, more than half of the sperm head (the acrosomal region) exhibited a uniform, bright green fluorescence in sperm exposed to vehicle (i.e., 1% DMSO) alone (Fig. 5A), 100 µM WHI-05 (Fig. 5B), and 100 µM WHI-07 (Fig. 5C) for 3 h. By comparison, in sperm exposed to 100 µM of N-9 for 3 h under identical conditions, green fluorescence was absent due to disruption of membrane integrity and acrosomal loss (Fig. 5D), consistent with previous SEM observations [34]. Thus, the spermicidal activity of the dual-function AZT derivatives was not accompanied by a loss of membrane integrity.
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Topographical imaging of drug-treated sperm heads by HR-LVSEM revealed intact acrosomes (Fig. 5, middle panels) with smooth contiguous surfaces in sperm exposed to vehicle (Fig. 5A') or 100 µM WHI-05 (Fig. 5B'), whereas sperm treated with WHI-07 (Fig. 5C') revealed signs of a mild acrosomal membrane ruffling. By comparison, sperm exposed to 10 µM CaI A23187, as a positive control, revealed characteristic blebbing or vesiculation, fenestration, and loss of plasma and acrosomal membranes (Fig. 5D').
TEM of the tangential sections of sperm head confirmed the intactness of the plasma membrane and outer and inner acrosomal membranes of sperm exposed to vehicle (Fig. 5A''), WHI-05 (Fig. 5B''), and WHI-07 (Fig. 5C'') but not to the CaI A23187 (Fig. 5D'') for 3 h (Fig. 5, bottom panels). Taken together, these results demonstrate that the spermicidal effects of WHI-05 or WHI-07 were not caused by a detergent-type action resulting in disruption of the sperm plasma membrane within the acrosomal region of the sperm head. These features of aryl phosphate derivatives of bromo-methoxy-AZT differ from those of N-9 [34], the most widely used prototype detergent spermicide.
Inhibitory Effects of WHI-05 and WHI-07 on Homologous Sperm-Zona Binding Assay
The human sperm-zona recognition and binding assay appears to predict the in vitro and in vivo fertilization outcome [37, 38]. Therefore, the inhibitory effects of WHI-05 and WHI-07 on sperm-egg interaction were next assessed by laser scanning confocal microscopy using intact human zona and two-colored sperm separately labeled with cell-permeant DNA-specific dyes, SYBR 14 (green) and SYTO 17 (blue), in a homologous sperm-zona binding assay. Due to the variable efficiency of sperm binding to frozen human zona, the sperm-zona binding ratio was used to assess sperm-zona binding capacity. The ratios of the number of green and blue sperm that were bound to control and test human zonae showed marked differences. The mean sperm-zona binding ratio for control eggs coincubated with SYBR 14- and SYTO 17-labeled control sperm was 1.07. In contrast, the mean sperm-zona binding ratios for eggs coincubated with control (SYBR 14) and 25, 50, and 100 µM WHI-07-treated sperm (SYTO 17) were 0.088, 0.039, and 0.009, respectively. The corresponding mean ratios for sperm treated with 25, 50, and 100 µM of WHI-05 were 0.18, 0.07, and 0.10, respectively. Figure 6 shows the representative sperm-zona binding patterns of green and blue sperm to a control egg (Fig. 6A) and the test eggs in which the blue sperm were pretreated with either 100 µM of WHI-05 (Fig. 6B) or 25 µM of WHI-07 (Fig. 6C) prior to coincubation with green-labeled sperm. Despite the variable number of sperm binding to individual zona, a clear reduction in the number of blue-colored sperm bound to test eggs is apparent in comparison to control eggs.
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Inhibitory Effects of WHI-05 and WHI-07 on Heterologous Sperm-Zona-Free Hamster Egg Binding and Penetration Assay
The heterologous zona-free hamster egg penetration assay appears to correlate with the in vivo fertilizing capacity of human sperm as well as the ability of human sperm to fertilize intact human eggs in vitro [39, 40]. Therefore, the inhibitory effects of WHI-05 and WHI-07 on sperm-egg interaction were further confirmed using the zona-free hamster egg penetration assay. As expected, preincubation of capacitated sperm with either compound resulted in significant inhibition (p < 0.001) of sperm binding to zona-free eggs (Table 2). Similarly, the penetration rate of human sperm to zona-free hamster eggs following exposure to increasing concentrations of WHI-05 or WHI-07 showed marked inhibition (41–100% inhibition when compared with control). Thus, pretreatment of human sperm with spermicidal aryl phosphate derivatives of bromo-methoxy-AZT resulted in a concentration-dependent loss of the ability to bind and penetrate zona-free hamster eggs as well as inhibition of binding to human eggs.
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In Vivo Contraceptive Efficacy of WHI-07
Because fertilization in vivo is dependent on successful sperm transport through the female genital tract [41], we next determined whether exposure of sperm to the lead compound, WHI-07, in vivo affected the subsequent fertility outcome. Hormonally primed adult female Swiss (CD-1) mice were artificially inseminated with motile epididymal sperm via the cervix with and without prior intravaginal application of 1% WHI-07 in a cream base. Females were examined 8 days later for the presence or absence of embryos in uteri. The results of three fertility trials are summarized in Table 3. In mice receiving WHI-07 intravaginally prior to artificial insemination, the percentage of fertility rate was drastically reduced in comparison to that in controls (7.6% vs. 38.4%; p < 0.001), which demonstrates a substantially impaired ability of WHI-07-exposed sperm to reach the site of fertilization.
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Lack of Local Toxicity of WHI-07
Next, the local tissue alterations and inflammatory response to repetitive intravaginal applications of N-9 versus WHI-07 in mice were compared. Two groups of 15 adult female Swiss (CD-1) mice were treated for 5 (group A) or 20 (group B) consecutive days with either 5% N-9, 5% WHI-07, or control vehicle in a cream base, and the cervicovaginal tissue sections were examined for histopathological changes and influx of inflammatory cells (Fig. 7). The cervicovaginal region consisted of a stratified squamous epithelium overlying a vascular submucosa. The thickness of the epithelium varied depending on the stage of the estrous cycle. Similar to what was observed in the 10 vehicle alone-treated control mice, none of the 10 mice treated with WHI-07 for 5 days or 20 days revealed any inflammatory response or membrane disruption of the squamous epithelia (Fig. 7A and Table 4). By contrast, disruption of the epithelial lining, and an inflammatory response with influx of neutrophils in the squamous epithelia of cervicovaginal crypts, were evident in 9 of 10 mice given N-9 intravaginally (Fig. 7C and Table 4), consistent with previously published observations in rats [19].
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Two-color laser scanning confocal fluorescence images of the cervicovaginal epithelia of WHI-07- and N-9-treated sections with a monoclonal antibody specific to mouse neutrophils revealed absence of neutrophils in the stratified squamous epithelial crypts of WHI-07-treated specimens (Fig. 7B) and intense positive staining (green color) in the squamous epithelia of N-9-treated tissue section (Fig. 7D). Thus, evidence obtained by histopathologic and immunofluorescence studies indicates that unlike N-9 treatment, intravaginal application of WHI-07 in a cream base does not cause any membrane disruption or an acute inflammatory response in the cervicovaginal epithelial crypts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Physiological fertilization depends on the ability of the ejaculated sperm to swim, bind the zona pellucida, and penetrate the egg, all of which abilities are primarily dependent on sperm motility [41, 42]. The mechanisms by which spermicidal AZT derivatives affect sperm motility are not fully understood; however, their spermicidal activity differed from that of detergent-type spermicides. The fact that spermicidal AZT derivatives were not permeable to PI, as well as the intactness of the plasma membrane and acrosomal membranes observed after several hours of exposure to these drugs, suggested that these spermicidal AZT derivatives immobilize sperm without membrane disruption as confirmed at the ultrastructural level using HR-LVSEM, which reveals the true surface of a cell in its exposed state [29, 30]. These properties of AZT derivatives differ from those of currently used spermicides, N-9 and gramicidin, which exert their effects via a detergent-like ability to damage the sperm membrane, perturb its conformation, and destroy its semipermeable nature, thereby impairing the sperm motility and egg-fertilizing functions [34–36]. Because of their nonspecific membrane-disruptive properties, such vaginal spermicides have been shown to damage the cervicovaginal epithelium [17–19], as well, which may lead to a lower degree of protection from STD [20]. A vaginal contraceptive that does not elicit any of the nonspecific membrane toxicity characteristic of detergent-type contraceptives would be desirable.
The human sperm-zona recognition and binding assays and the zona-free hamster egg binding and penetration assays are useful in predicting the in vivo fertilizing capacity of human sperm [37–40]. We investigated the effects of dual-function AZT derivatives, WHI-05 and WHI-07, on the membrane fusogenic potential of sperm in the hamster egg penetration assay. Both these AZT derivatives that affected sperm motility also inhibited binding and fertilization of zona-free hamster eggs in a concentration-dependent manner. Furthermore, using a novel two-colored DNA-stained sperm-zona binding competition assay and confocal microscopy, we also demonstrated the concentration-dependent capacity of WHI-05 and WHI-07 treatment to inhibit binding of sperm to human zona as compared with that of control sperm using the same human zona. The use of cell-permeant DNA specific fluorescent dyes SYBR 14 and SYTO 17 to bind zona enhanced the specificity of the human sperm-zona binding test in comparison to conventional two-colored sperm-zona binding assays that employ nonspecific fluorescein- and rhodamine-labeled protein tags to detect fluorescent sperm binding to zona [43, 44].
Fertilization in vivo depends upon successful sperm transport through the female genital tract. Results of our in vivo contraceptive efficacy studies in the mouse model demonstrated that prior exposure of artificially inseminated epididymal sperm to WHI-07 via cervix significantly affected sperm fertilizing ability in vivo in hormonally primed female mice, indicative of the inability of WHI-07-exposed sperm to reach the site of fertilization. The results of this pilot study indicate that WHI-07 would be an attractive candidate for further contraceptive efficacy trials in larger animal models.
Experiments designed to test the comparative local toxicity of N-9 versus WHI-07 clearly demonstrated that, unlike what occurs with the intravaginally applied N-9, repetitive intravaginal application of WHI-07 did not induce an inflammatory response by detergent-type disruption. Evidence obtained by histopathologic and immunofluorescence studies suggests that intravaginal application of N-9 caused marked underlying acute inflammatory response in the cervicovaginal epithelial crypts. The infiltrating leukocytes were identified to be neutrophils. These histopathological features of N-9 observed in this study are similar to those observed in the rat model [19]. That repetitive intravaginal infusion of WHI-07 does not cause a detergent-type membrane injury or an acute inflammatory response in the cervicovaginal epithelium, as demonstrated by our studies, may prove clinically advantageous. Future preclinical studies will examine intravaginally applied WHI-07 for potential systemic effects, especially those on female genital tract and reproductive function as well as early embryonic development and fetal morphogenesis.
The development of novel aryl phosphate derivatives of bromo-methoxy-AZT with potent spermicidal and anti-HIV activities represents a significant step forward in the development of a new virucidal spermicide for curbing heterosexual HIV transmission. The promising results reported herein warrant the further development of these dual-function AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk for acquiring HIV by heterosexual transmission. These dual-function AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: April 14, 1998.
Received: February 24, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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