Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zaneveld, L. J.D.
Right arrow Articles by Cooper, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zaneveld, L. J.D.
Right arrow Articles by Cooper, M.
Agricola
Right arrow Articles by Zaneveld, L. J.D.
Right arrow Articles by Cooper, M.
Biology of Reproduction 66, 886-894 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Efficacy and Safety of a New Vaginal Contraceptive Antimicrobial Formulation Containing High Molecular Weight Poly(Sodium 4-Styrenesulfonate)1

Lourens J.D. Zaneveld2,a, Donald P. Wallerb, Robert A. Andersona, Calvin Chany IIa, William F. Rencherc, Kenneth Feathergilla, Xiao-Hui Diaoa, Gustavo F. Doncelc, Betsy Heroldd, and Morris Coopere

a Program for the Topical Prevention of Conception and Disease, Department of Obstetrics and Gynecology, Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612 b Program for the Topical Prevention of Conception and Disease, Department of Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, Chicago, Illinois 60612 c Contraceptive Research and Development Program, Eastern Virginia Medical School, Norfolk, Virginia 23507 d Department of Pediatric Infectious Diseases, Mount Sinai Medical Center, New York, New York 10029 e Department of Medical Microbiology and Immunology, Southern Illinois University, Springfield, Illinois 62794


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC50 = 5.3 µg/ml) and acrosin (IC50 = 0.3 µg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 µg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC50= 16 µg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC50 = 1.3 and 1.0 µg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC50 < 1.0 gel/ml) and Chlamydia trachomatis (IC50 = 1.2 µg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.

fertilization, sperm, sperm motility and transport, toxicology, vagina


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acquired immunodeficiency syndrome (AIDS) has caused a worldwide crisis. An estimated 5.8 million new infections by the human immunodeficiency virus (HIV) occurred in 1998, approximately 16 000 each day [1]. By the end of 1998, more than 43 million people had AIDS or were infected with the HIV virus. Presently, the majority of HIV infections occur through heterosexual contact. Women are particularly at risk. In 1998, 43% of the AIDS cases or HIV infections were in women [1]. Prevalences of other sexually transmitted diseases (STDs) are also on the rise. In 1995, more than 333 million new cases of Chlamydia and Trichomonas infections, gonorrhea, and syphilis occurred worldwide [2]. Not only are these other STDs a serious problem by themselves but they also increase the risk of HIV infection [3]. At the same time, the world population continues its steep rise [4]. The tragedy, suffering, and costs associated with infectious diseases and overpopulation are staggering.

For women, only those techniques preventing contact of semen with the genital tract or methods that inactivate HIV and STD pathogens and spermatozoa after ejaculation can prevent both infection and unplanned pregnancies. Condoms, both male and female, provide a physical barrier to the seminal contents, but because the use of these devices has been limited they have not had a major impact on disease prevention. In many cultures, it is difficult for woman to negotiate condom use. Thus, it is important to develop woman-controlled methodologies, i.e., prophylactic vaginal products, such as gels, suppositories, and foams, that are microbicidal and spermicidal.

Vaginal contraceptive products have been available for many years and usually contain the membrane-cytotoxic detergent/surfactant nonoxynol-9 as the active ingredient [5, 6]. However, frequent use of nonoxynol-9 products can cause irritation and inflammation of the vagina [7–9]. Nonoxynol-9 is toxic to vaginal and cervical cells [10], increases the permeability of vaginal tissue [11], and can inactivate lactobacilli [12, 13]. Lactobacilli produce lactic acid and hydrogen peroxide that serve to maintain the acidic pH of the vagina (approximately 3.5–5.0). At this pH, a number of STD-causing organisms, HIV, and spermatozoa are inactivated [14–16]. Disturbance of the vaginal microbial milieu can lead to vaginal infections, which in turn increase the chance of HIV/STD transmission [17].

Vaginal contraceptive products that contain nonoxynol-9 have high efficacy in preventing vaginal but not rectal herpes infections in the mouse model [18, 19] but appear to be of limited effectiveness clinically in preventing gonococcal and chlamydial infections [20]. So far, nonoxynol-9 products have been ineffective in preventing HIV transmission [21, 22]. The most recent data with one of these vaginal nonoxynol-9 products, Advantage S (Columbia Laboratories, Aventura, FL), suggest that the compound may even enhance HIV transmission, at least when used frequently [23], an effect also suggested previously [21]. It is important to identify and evaluate new contraceptive antimicrobial agents that can be used vaginally in effective doses without inactivating lactobacilli or causing overt vaginal irritation or other toxicity.

Many of the new vaginal antimicrobial compounds receiving attention during the past decade are cytotoxic, similar to nonoxynol-9 [24, 25]. Such cytotoxic agents are also likely to cause vaginal irritation and to inactivate the normal vaginal flora when used frequently at clinically effective doses.

In our search for agents that have noncytotoxic modes of action, we hypothesized that spermatozoa and several of the STD-causing microbes share common mechanisms whereby they bind to and enter into host cells. Thus, certain compounds may inhibit the functional activity of both types of organisms. Sulfated/sulfonated polymers and/or polysaccharides appeared to be compounds that fall into this category because some have been reported to inhibit STD-causing microbes [26–29], whereas others are known to be contraceptive [30, 31]. Our subsequent studies suggested that several of these compounds, including poly(sodium 4-styrenesulfonate; PSS) and cellulose sulfate, have dual activities [32, 33]. Contraceptive antimicrobial agents are of particular clinical interest because consumer preference studies suggest that most women worldwide prefer a vaginal prophylactic product to be both antimicrobial and contraceptive [34] (L. Fransen, European Commission, HIV/AIDS Programme in Developing Countries, News Release, 1998). Such dual activity can also be achieved by mixing nonoxynol-9 with a noncontraceptive sulfated polymer/polysaccharide [35]. However, it is more desirable for a single noncytotoxic agent to possess both properties because the addition of nonoxynol-9 will enhance toxic effects and may lead to formulation difficulties.

PSS comes in a variety of molecular masses, starting at about 4000 daltons. A high molecular mass (>500 000 daltons) compound was selected by the Program for the Topical Prevention of Conception and Disease (TOPCAD, Chicago, IL) for evaluation of its activity towards spermatozoa and STD-causing microbes. TOPCAD is a university-based program focusing on the discovery and development of vaginal antimicrobial contraceptive compounds and formulations. The high molecular mass was deemed desirable because it makes absorption by the vagina extremely unlikely [36], essentially eliminating any chance of systemic toxicity. Other forms of PSS (molecular mass unknown) have been reported to inhibit HIV [37, 38], inactivate the respiratory syncytial and influenza A viruses [39], and prevent vaginal herpes infection in the mouse [40].

Our original data on PSS were obtained with commercial material manufactured utilizing chlorinated hydrocarbons for the sulfonation of a long chain polystyrene to produce high molecular mass material [32]. However, residues of toxic chlorinated hydrocarbons may remain in the polymer preparation, making it undesirable for clinical use, and may affect test outcome. Therefore, TOPCAD developed an alternative method of synthesis for high molecular mass PSS utilizing a water-based polymerization of 4-styrenesulfonate that eliminates toxic residues in the preparation. This product is referred to as T-PSS. Initial tests with T-PSS showed inhibition of herpes simplex viruses (HSV-1 and HSV-2), Neisseria gonorrhea, and Chlamydia trachomatis [41].

These preliminary results encouraged the further evaluation of T-PSS for its sperm inhibitory, contraceptive, and antimicrobial properties. Studies focused on the compound in gel because this is the method whereby T-PSS will be used vaginally, but information was also obtained on the compound alone (drug substance) and used for comparative purposes to assess whether the compound retained full effectiveness in the formulation. The present experiments revealed that T-PSS (alone or in a gel) 1) is highly contraceptive without affecting sperm transport, 2) is not cytotoxic to spermatozoa, 3) inhibits hyaluronidase, acrosin, and cervical mucus penetration of spermatozoa, 4) causes the loss of the acrosomes from spermatozoa, and 5) inactivates HIV and HSV (both laboratory and clinical strains), gonococci, and chlamydia without being cytotoxic to the host cells. These results support the hypothesis that T-PSS does not act on a cytotoxic basis but rather inhibits the functional activity of both spermatozoa and certain STD-causing microbes. In addition, PSS retains its activity in gel formulations. The lack of cytotoxicity should make T-PSS safe for vaginal use; it had no effect on lactobacilli and was considered safe based on in vitro and animal toxicity studies. Our observations suggest that T-PSS is a novel vaginal contraceptive antimicrobial agent and that the gel form should be evaluated for its safety and efficacy in women.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

T-PSS (lot TC97001) was manufactured under Good Manufacturing Procedure (GMP) conditions by TOPCAD using free-radical polymerization of sodium styrene sulfonate in water (unpublished results). The polymer has a peak molecular mass of 864 kDa and an average molecular mass of 751 kDa. T-PSS is highly soluble in water (>330 mg/ml). A gel formulation (lot 312/118) containing 5% (50 mg/ml) T-PSS was manufactured under GMP conditions by Advanced Care Products (North Brunswick, NJ), utilizing hydroxyethyl cellulose, glycerin, propylene glycol, benzoic acid, methylparaben, and NaOH as a base material in a proprietary mixture. This formulation was used as the standard test gel for most experiments. Gel with 0% (placebo), 1%, and 2% T-PSS were also manufactured with additional hydroxyethyl cellulose to control for viscosity (75 000–80 000 cps). A 5% T-PSS gel was also prepared using K-Y Jelly (Advanced Care Products, Raritan, NJ) as base.

Contraception, Sperm Transport, and Sperm Function Inhibition Studies

All animal studies were approved by the Institutional Animal Care and Use Committee and performed according to the Guide for Care and Use of Laboratory Animals.

Contraception Details of the rabbit source and type, maintenance, hormone treatment to induce ovulation, artificial insemination, and collection of oocytes were previously presented [32, 42]. Spermatozoa were obtained from New Zealand White rabbits using an artificial vagina made in the laboratory, washed by mild centrifugation, and resuspended in phosphate-free Tyrode albumin lactate pyruvate (TALP) medium without albumin. The suspension was adjusted with TALP medium to a concentration of 62 x 106 spermatozoa/ml. Two experiments were performed. When the contraceptive effect of the T-PSS gel or its placebo was tested, the formulation (0.75 ml) was placed vaginally with a plastic insemination tube past the pelvic bone, followed 15 min later by insemination with 0.5 ml of washed spermatozoa (total: 31 x 106) in the same vaginal location. When the direct effect of the T-PSS drug substance on spermatozoa was to be determined, the gametes were incubated in a 9:1 (v/v) ratio with a 10-fold concentration of T-PSS, dissolved in TALP (0.45 ml of the sperm suspension mixed with 0.05 ml of the compound) for 15 min, and the mixture inseminated into the vagina of a hormone-treated female rabbit (without formulation in the vagina). In both experiments, the rabbits were killed with Sleep-away (Fort Dodge Laboratories, Fort Dodge, IA) approximately 28–34 h after insemination, and the oocytes were flushed from the oviducts and examined microscopically for cleavage (embryo formation). Data are reported as the percentage of fertilized oocytes recovered for each rabbit.

Effect on sperm transport Spermatozoa were obtained with an artificial vagina, washed by centrifugation, and resuspended in TALP buffer to a concentration of 400 x 106 spermatozoa/ml, and 0.5 ml of this final suspension was artificially inseminated into hormone-treated female rabbits as described in the previous section. The high number of spermatozoa was required to obtain sufficient recovery in the uterus and oviducts for reliable measurement. The T-PSS was formulated both in the hydroxycellulose-based gel and in K-Y Jelly. The same gels without T-PSS were used as placebos. Fifteen minutes prior to insemination, 0.75 ml of either the T-PSS gel or the placebo gel were placed vaginally in each rabbit. Approximately 6.5 h after insemination, the vagina from each rabbit was aspirated with 1.4 ml of modified TALP buffer by placing an insemination tube lubricated with K-Y Jelly into the vagina, past the pelvic bone. The buffer was expelled and then aspirated back into the insemination tube. After collection of the vaginal contents, the rabbits were killed with Sleep-away, and an incision was made in the abdomen along the midline. The uteri and cervical junctions were located. The right uterus was cut at its junction with the vagina. The vagina was proximally flushed by placing the insemination tube into the cervical os, repeatedly expelling 1.4 ml of TALP buffer, and drawing the buffer back into the insemination tube. The perfusate was then combined with the first vaginal aspirate. This procedure was then repeated.

Both uteri were excised from the reproductive tract, approximately 1.3 cm proximal to the utero-oviductal junction and at the uterocervical junction. The outside of each uterus was rinsed, and each uterine lumen was flushed with 2.25 ml of TALP buffer using a 3-ml syringe attached to a needle and a short piece of firm 1.5-mm (inside diameter) polyethylene tubing. The buffer was allowed to drain from both uteri into a small plastic funnel that was placed into a plastic culture tube. Buffer that remained in the uterus was extruded by forcing the thumb and forefinger along each uterus. The small portions of the uteri that remained attached to the oviducts were flushed with 0.2 ml of TALP buffer. The washings were pooled with the other uterine washings. Subsequently, each oviduct was perfused proximally with 1.8 ml of TALP buffer, and the oviductal perfusates were pooled.

Sperm counts from vaginal washes were made under bright-field microscopy with a Neubauer hemocytometer (400x) after appropriate dilution. Uterine and oviductal sperm counts were determined similarly before and after concentration by centrifugation, when appropriate.

Sperm function inhibition The use of human semen donors was approved by the Institutional Review Board and informed consent was obtained before participation.

Inhibition of sperm motility, hyaluronidase, and acrosin and the dispersal of the sperm acrosome were evaluated as detailed elsewhere [32, 42]. To determine the effect of test material on the ability of spermatozoa to penetrate cervical mucus, one end of a Penetrak tube (bovine cervical mucus; BioChem ImmunoSystems, Allentown, PA) was immersed for 30 min into a solution containing the test agent. The tube was removed from the test solution, and 50 µl of donor human semen, diluted with Baker buffer (27.4 mM Na2HPO4, 0.6 mM KH2PO4, and 167 mM glucose, pH 8.1) to a final concentration of 60 x 106 sperm/ml (minimum initial percentage of motile spermatozoa was 50%), was added to the test solution. The Penetrak tube was reimmersed into the test solution containing the sperm mixture, and the system was allowed to incubate at room temperature for 1 h. The tube was removed and examined microscopically to determine the length of the tube that was traversed by the most advanced motile spermatozoa. This distance was compared with that achieved by the most advanced spermatozoa from a sample containing no test substance (control). Migration distance achieved by the spermatozoa contained in the test solution was reported as a percentage of the distance achieved by the control sperm.

Statistical analyses Hyaluronidase and acrosin activities were expressed as means and SEMs. Data were best-fit to curves generated with TableCurve 2D software (SPSS Statistical Software, Chicago, IL), from which the IC50 of enzyme inhibition and coefficients of determination of the fitted curves were calculated [43]. Percentage data collected from the acrosomal loss, sperm immobilization, and contraception studies were subjected to arcsine transformation [44] before further analysis. In the contraception studies, the number of rabbits rather than the number of oocytes recovered was used as the sample size. Data were back-transformed for presentation and, depending on the assay, presented either as average percentage of maximal acrosomal loss, percentage of motile spermatozoa, or percentage of fertilization, with 90% confidence limits. Sperm transport data (sperm count) were subjected to logarithmic transformation [44] prior to further analysis. Values were expressed as log (total sperm) recovered in each area of the reproductive tract and as the back-transformed averages, with 90% confidence limits. Differences in acrosomal loss, contraception, and sperm transport in the presence and absence of test agent were compared with the Student t-test using the transformed data.

Antimicrobial Activity

HIV-1 inhibition assays, originally using the laboratory strain IIIB propagated in a T4-lymphoblastoid line and clinical isolates obtained from the University of Alabama at Birmingham, were performed using MT2 host cells and human periferal blood mononuclear cells (PBMCs) as described elsewhere [32]. The non-syncytium-inducing clinical isolates, RoJo and TEKI, were incubated with serial dilutions of the agents and PBMCs for 6 h. Incubation was terminated by centrifugation (350 x g, 10 min, 4°C) and replacing 75% of the medium. Cultures were then continued for 6 days, and virus replication was assessed by evaluation of reverse transcriptase activity in the supernatant. Techniques of the herpes, chlamydial, and gonococcal assays were published previously [41]. The HSV strains used in a plaque reduction assay were HSV-1 strain 17, HSV-2 strain 333, and clinical isolates designated as BBKC, MMA [45], DT2 [46], and H1 (obtained from a genital lesion of an HIV-positive male with persistent, extensive mucosal disease). Infectivity assays with C. trachomatis utilized serotype E-UW-5/CS on HeLa cells. The gonococcal assays evaluated the effect on N. gonorrhea MS11 growth on agar. The inhibitory effect of test agents towards lactobacillus growth was evaluated using Lactobacillus gasseri as detailed previously [32, 42]. Dose-response studies were performed with each microbe (unless the highest dose tested was without inhibitory effect).

Data on the antimicrobial activity were subjected to logarithmic transformation [44] before further analysis. Values are presented as the back-transformed average titers, with 90% confidence limits. Differences in C. trachomatis titers among treatment groups were evaluated with the Newmann-Keul multiple range test [47]. IC50 values and concentrations of test agent yielding 3-log reductions in infectivity (99.9% inhibition of microbial titer) were calculated from curves fit to the data with the TableCurve 2D software.

Safety Studies

All procedures were performed in compliance with Good Laboratory Practice (GLP) regulations at Biologic Safety Research Laboratory (Maywood, IL) under the direction of one of the investigators (D.W.). The bacterial reverse mutation assay was performed at BioReliance (Rockville, MD).

Bacterial reverse mutation assay (Ames test) The mutagenic potential of T-PSS was evaluated by the plate incorporation method [48] after dissolution in water at a maximum concentration of 5.0 mg/ml. Salmonella typhimurium (strains TA98, TA1000, TA1535, and TA1537) and Escherichia coli (strain WP2 uvrA) were used in the presence and absence of Aroclor-induced rat liver S9. The maximum dose tested was 5000 µg/plate.

Limit test for acute oral toxicity Acute oral toxicity of T-PSS was determined by the limit test [49]. T-PSS was prepared as a 33% solution in distilled water and was administered by oral gavage as a single dose of 5.0 g/kg body weight to five adult male and five adult female rats (Sprague-Dawley). The animals were observed for pharmacotoxic signs and mortality during a 14-day postadministration observation period. A gross necropsy examination was performed on all animals at the end of the 14-day observation period.

Primary dermal irritation The test gels containing 10% T-PSS and placebo gel were applied topically (0.5 ml/site) to one intact and one abraded test site per rabbit [49]. Each group consisted of six mature male New Zealand White rabbits. The test site was occluded by covering each site with a gauze pad and overwrapping the site with 4 mil (100 µm) plastic for 24 h following application of the gel. Dermal irritation was scored according to the Draize scoring system [50] at 24 and 72 h after gel application.

Penile mucosal irritation The test gel containing 10% T-PSS or placebo gel (0.2 ml) were applied directly to the penis of six mature male New Zealand White rabbits hourly for 4 h on three consecutive days. Three rabbits served as a sham control group. All animals were observed for erythema and eschar formation prior to the application of the test material and at 1, 24, and 48 h after the last application. The penises were dissected at the base of the pelvis (leaving the sheath intact), evaluated for gross pathology, and fixed in buffered formalin. Sections from the fixed penises were blocked, sectioned, stained with hematoxylin and eosin, and evaluated for microscopic pathologic changes.

Dermal sensitization Dermal sensitivity was performed utilizing a modified Buehler procedure [49]. T-PSS (10% in gel; 0.5 ml) or the placebo gel was placed in a Hilltop-style occlusive chamber applied to the shaved left trunk of 10 male albino guinea pigs. Each chamber was then overwrapped around the entire trunk with Micropore tape and further secured with a wrap of surgical tape. The animals were unwrapped after a 6-h exposure period. The induction phase consisted of treating the animals once weekly for 3 wk with the gels, and skin was scored for irritation 24 and 48 h after each application. Two weeks after the final induction dose, each animal received a challenge dose at a naive site located on the right trunk (6-h exposure period). Skins were scored for irritation 24 and 48 h after the challenge application. Using the same regimen, 0.4 ml of 2.5% {alpha}-hexylcinnamaldehyde, 85% (Aldrich Chemical Co., St. Louis, MO) was administered to 10 male guinea pigs to serve as a positive control.

Fourteen-day subacute/vaginal irritation Vaginal irritation testing was performed with 10% T-PSS gel and placebo gel in New Zealand White rabbits and ovariectomized Sprague-Dawley rats utilizing a procedure modified from that of Gad and Chengelis [49]. Thirty mature females of each species were divided into three test groups. One group consisted of untreated control animals. Animals in the other two groups were treated vaginally for 14 consecutive days with either T-PSS gel or placebo gel (1 ml for rabbits and 0.1 ml for rats). All animals were weighed on the first day of dosing and every seventh day thereafter. The animals were observed at least twice daily for moribundity/mortality. Approximately 0.5 and 4 h after dosing, observations were also made for vaginal bleeding and discharges, appearance, behavior, and pharmacotoxic signs. Detailed physical observations were performed daily. The animals were necropsied 24 h after the final vaginal dose of gel. The adrenal gland (left and right), cervix, heart, kidney, liver, lung, lymph node (mediastinal), ovary, oviduct, pancreas, spleen, urinary bladder, urethra, uterus, vagina, and vulva were excised, fixed, and examined histopathologically. The adrenal glands, spleen, liver, and kidneys were weighed before fixation.

Blood was collected at necropsy. Hematological measurements included hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and platelet count. Clinical chemistry tests were conducted for glucose, blood urea nitrogen (BUN), chloride, sodium, total serum protein, potassium, calcium, albumin, globulin, albumin:globulin ratio, blood creatinine, cholesterol, inorganic phosphate, total bilirubin, creatine kinase, serum glutamic-pyruvic transaminase, and serum glutamic-oxaloacetic transminase. Blood coagulation tests included prothrombin time, activated partial thrombin time, and fibrinogen concentration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Contraceptive Effect

T-PSS was an effective contraceptive in the rabbit model when the drug substance was mixed with spermatozoa before artificial insemination and when gel with 2% or 5% T-PSS was placed vaginally before artificial insemination (Table 1). The placebo or gel with 1% T-PSS had no or only minimal contraceptive effects.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Contraceptive properties of T-PSS gel

Lack of Effect on Sperm Migration

The contraceptive properties of the T-PSS gel were not due to an inhibitory effect on sperm migration (Fig. 1). Vaginal placement of the 5% test gel or the placebo gel in rabbits, followed by artificial insemination, resulted in the recovery of essentially the same number of spermatozoa from the vagina and oviducts in animals of both treatment groups. However, a relatively small but significant decrease (22% in the log transformed number of recovered spermatozoa) was noted in the number of spermatozoa recovered from the uterus.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1. Effect of 5% T-PSS gel on sperm migration in the rabbit. Approximately 15 min prior to insemination, each rabbit was treated vaginally with 0.75 ml of a 5% (w/w) T-PSS formulation (solid bars) or a formulation that contained no T-PSS (open bars). Semen for each experiment was collected from three New Zealand White rabbits and was pooled. Spermatozoa were washed in modified TALP buffer, and the suspension was adjusted to a sperm concentration of 400 x 106 spermatozoa/ml. The insemination volume was 0.5 ml. Approximately 6.5 h after insemination, spermatozoa were flushed from the vagina with TALP buffer, the rabbits killed, and the uteri and oviducts were excised and flushed

Sperm Function Inhibition

T-PSS at 10 mg/ml had no cytotoxic effects on spermatozoa as assessed by percentage of motile spermatozoa (Table 2). No agglutination of spermatozoa was observed in these tests. Test gel with 5% T-PSS or the placebo gel also exhibited no cytotoxic effects on spermatozoa at the highest doses tested (200 mg gel/ml; n = 3).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effect of T-PSS drug substance on sperm function indicators

T-PSS effectively inhibited hyaluronidase and acrosin (Table 2). Inhibition of both enzymes was irreversible. The compound retained its enzyme inhibitory properties in test gel formulation as assessed at single concentrations (selected from the dose-response curve obtained with the T-PSS drug substance). When 60 µg/ml of the 5% T-PSS gel was used (i.e., 3 µg/ml T-PSS), hyaluronidase was inhibited by 57% (n = 3), and when 200 µg/ml of the T-PSS gel was used (i.e., 10 µg/ml T-PSS), acrosin was inhibited by 63% (n = 3). The placebo gel had no effect on either enzyme at 200 µg/ml (highest dose tested).

The compound (tested at a single concentration) induced the loss of acrosomes from human spermatozoa (Table 2). Maximal acrosomal loss caused by ionophore A23187 under the same experimental conditions was taken as the maximal loss that could be induced [32]. The acrosomal loss-inducing properties of T-PSS were retained after formulating the compound. The test gel with 5% T-PSS caused 71% maximal ionophore-induced loss at 10 µg/ml gel (i.e., 0.5 µg/ml T-PSS), whereas the placebo had a <10% stimulatory effect at the same gel concentration (n = 4).

T-PSS had some inhibitory effect on the penetration of human spermatozoa into cervical mucus (Table 2). Furthermore, 1 mg/ml of the 5% T-PSS gel (i.e., 0.05 mg/ml T-PSS) reduced sperm entry into cervical mucus to 29.6% ± 3.9% of the value for the untreated control group (n = 10). However, the placebo gel also had an effect on sperm penetration of cervical mucus; 1 mg/ml of placebo reduced sperm penetration to 57.0% ± 4.4% of the value for the untreated control (n = 10), most likely explaining the apparent increased effectiveness of the T-PSS gel in inhibiting cervical mucus penetration as compared with the drug substance.

Antimicrobial Activity

Test gel with 5% T-PSS was highly effective against all STD-causing microbes tested, including HIV (Table 3). The placebo gel had no effect on these microbes at the highest doses tested (200 µg gel/ml). The T-PSS gel and the placebo gel had no cytotoxic effects on the host cells at the highest doses tested.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Antimicrobial properties of 5% T-PSS gel

Additional studies, i.e., not reported previously [41], were performed with T-PSS alone (not in gel). The compound was an effective inhibitor of HIV-1 IIIB, a lymphocytotropic strain, with an IC50 of 2.9 µg/ml and 3-log reduction (IC99.9) at 8.9 µg/ml. T-PSS displayed broad inhibitory activity against different laboratory strains, including the monocytotropic ADA and Bal and clinical isolates RoJo and TEKI, with IC50 values ranging from 0.3 to 7.9 µg/ml. The compound also possessed high inhibitory activity against clinical HSV-2 isolates. The IC50 values against MMA, BBKC, DT2, and H1 were 0.18, 0.16, 0.10, and 0.12 µg/ml, respectively.

The T-PSS drug substance (5 mg/ml), the 5% T-PSS gel (100 mg gel/ml), and the placebo (100 mg gel/ml) had no effect on lactobacillus growth (Table 3).

Safety/ Toxicity Studies

A maximum dose of 5 mg T-PSS per plate was tested in the in vitro mutagenicity assay (bacterial reverse mutation assay). No positive response was obtained even at the highest dose (Table 4). In the acute toxicity studies, all rats survived treatment with 5.0 g/kg body weight T-PSS in apparent good health and exhibited normal weight gains during the treatment period (Table 4). No pharmacotoxic signs were observed. At necropsy, no gross changes were observed in any of the animals. Thus, the acute oral LD50 of T-PSS is greater than 5.0 g/kg.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Safety/toxicity studies with T-PSS

In the dermal irritation studies, no edema was observed in any of the rabbits at the 24- and 72-h observation points with gel containing 10% T-PSS or placebo gel (Table 4). Slight erythema (grade 1) was observed in two of six animals treated with the T-PSS gel but not with the placebo gel. Both gels were classified as nonirritating to the skin. Penile irritation studies in the rabbit revealed no histopathological changes attributed to either the 10% T-PSS gel or the placebo gel, so both gels were classified as nonirritating to the penile mucosa (Table 4).

No dermal sensitization in the guinea pig occurred following treatment with a challenge dose of gel containing 10% T-PSS or with the placebo gel or in untreated control animals (Table 4). By contrast, a positive response was obtained following treatment with a challenge dose of hexylcinnamaldehyde, a known sensitizer. Based on these observations, neither the T-PSS gel nor the placebo gel were considered dermal sensitizing agents.

Subacute vaginal irritation studies were performed in the rabbit and rat. No differences in mean body weight were observed among the treatment groups (gel containing 10% T-PSS, the placebo gel, or untreated controls) in either the rat or the rabbit (Table 4). Also, no differences were observed among treatment groups in 1) hematological parameters, blood chemistry, and blood coagulation parameters, 2) gross observations of the organs at necropsy, and 3) organ weights. No histopathological alterations that could be attributed to either the T-PSS gel or the placebo gel were observed in the tissues. Thus, neither the T-PSS gel nor the placebo gel produced systemic toxicity in the rabbit or rat following vaginal application for a 14-day period. The rabbit vagina was also studied for histopathological changes. The regional and composite group averages were classified as minimal for the untreated control and the placebo gel groups and as mild for the T-PSS gel group; thus, both the T-PSS gel and the placebo gel were deemed acceptable for clinical trial.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Antimicrobial contraceptive products may be used to decrease or eliminate transmission of HIV and other STD-causing microbes and to prevent unplanned pregnancies. Unfortunately, presently marketed vaginal contraceptive products that contain nonoxynol-9 do not appear to be sufficiently effective in regard to their anti-HIV/STD activity [20–23], may cause vaginal inflammation and irritation if used frequently [7–9], and can inactivate the natural protective vaginal lactobacilli [12, 13]. One strategy worth pursuing is the development of new vaginal formulations with active ingredients that are not cytotoxic but that inhibit the functional activity (host cell binding and entry) of spermatozoa and STD-causing microbes. Initial studies suggested that PSS, an anionic polymer, may be such an ingredient [32].

PSS can in general be found in two forms: a soluble non-cross-linked form and a water-insoluble form in which repeating chains of the polymer are cross-linked to form a polymer matrix. The latter form is used clinically as a cation exchange resin for the treatment of hyperkalemia [51]. High oral doses (15–60 g/day) are administered without side effects other than severe ion depletion from overdoses. The cross-linked polymer is not of practical use for a vaginal formulation because of its insolubility. The soluble form of PSS is not marketed clinically, although it is used cosmetically as a skin firming product (Flexan 30; National Starch & Chemical Co., Bridgewater, NJ). This form is usually synthesized by a method that can result in contamination with toxic substances (e.g., chlorinated hydrocarbons), making the resultant product not applicable for clinical purposes. To avoid such contaminants, a water-based synthesis method was developed. The resultant material, called T-PSS, has a molecular mass of >500 000 daltons and is highly water soluble. The high molecular mass was hypothesized to effectively eliminate vaginal absorption. In support of this hypothesis, no systemic toxicity was found after vaginal application of gel with 10% T-PSS to the rabbit or rat for 14 consecutive days. The water solubility of T-PSS makes the compound easy to formulate. The choice of vehicle was important because its components (excipients) should not interfere with the properties of the active ingredient and must allow adequate bioavailability. A gel formulation for T-PSS was selected because this dosage form appears to be preferred by the majority of women [34].

T-PSS gel was highly contraceptive when 0.75 ml gel containing 2% or 5% T-PSS was placed vaginally in the rabbit before artificial insemination (depositing 15 mg and 37.5 mg of T-PSS, respectively). This finding suggests that the gel spreads adequately and relatively quickly through the vagina after deposition. Little or no contraceptive activity was observed when 0.75 ml of gel with 1% T-PSS (depositing 7.5 mg T-PSS) or the placebo were tested under the same conditions. Because the compound itself was completely contraceptive when mixed directly with spermatozoa at a concentration of 1 mg/ml before vaginal insemination of the mixture, it may appear that some contraceptive activity was lost. However, after vaginal placement, the gel presumably spread over a large portion of the vagina, effectively reducing the amount of T-PSS present at a particular site. Although 7.5 mg of T-PSS was deposited in the vagina by the 1% T-PSS gel, less may have been available in the area of contact with the spermatozoa. A concentration of 62 x 106 sperm/ml was used for these studies because initial experiments had shown that conception was consistently obtained when 0.5 ml was placed vaginally in the rabbit. Higher concentrations also produce conception but would reduce the sensitivity of the assay. In addition, the average sperm concentration of the human ejaculate is usually less than 62 x 106 sperm/ml [52].

The contraceptive properties of the test gel do not appear to be associated with an effect on sperm migration because the same number of spermatozoa were recovered from the oviducts of animals treated with both the test gel and the placebo gel. Although a slightly lower number of spermatozoa was found in the uteri of the test animals, fertilization potential is determined by the spermatozoa in the oviducts. The lack of effect on sperm migration suggests that the gel does not act by preventing sperm motility but rather acts by interfering with the sperm's fertilizing capacity.

Neither T-PSS alone nor the T-PSS gel caused a decrease in the percentage of motile spermatozoa, confirming the above supposition, i.e., that the contraceptive properties of the gel are not due to a cytotoxic effect on spermatozoa. T-PSS alone or in gel, but not the placebo gel, inhibited hyaluronidase and acrosin, enzymes with putative functions in the fertilization process [53]. Inhibitors of these enzymes are known to be contraceptive when placed vaginally [54, 55]. An effect of PSS of unknown molecular mass on the binding of sperm proacrosin to fucoidin, a sulfated polysaccharide, was previously reported [56]. Furthermore, both T-PSS alone and the T-PSS gel caused the dispersion of the acrosomes from spermatozoa. Because the acrosome is also important for successful penetration by the spermatozoon of the layers surrounding the egg [53], the induction of a premature loss of the acrosome in the vagina may have added to or been the cause of the contraceptive effects of T-PSS.

The 5% T-PSS gel had an inhibitory effect on the entry and migration of human spermatozoa into and through cervical mucus in the Penetrak test. Although the T-PSS itself also inhibited penetration of cervical mucus by spermatozoa, the gel appeared to be more active than would have been the case if the effect was only due to the active ingredient. Because the placebo gel also had a significant penetration inhibition effect, the gel's viscosity or other base properties most likely added to the ability of the active ingredient to prevent sperm-cervical mucus interaction. The rabbit has a relatively open cervix, so cervical mucus-blocking agents probably have only a minimal effect or no effect on sperm migration. No effect of the T-PSS gel on sperm transport in the rabbit was noted (see above). However, the human cervix is effectively blocked by the presence of mucus. If the in vitro data hold true in vivo, T-PSS will be contraceptive in the human by preventing sperm-egg interaction and by decreasing sperm entry and penetration into cervical mucus.

The T-PSS gel effectively inhibited the infectivity of HIV, HSV-1, HSV-2, and C. trachomatis and the multiplication of N. gonorrhea. By contrast, the placebo gel had no effect on these microbes; thus, the antimicrobial properties appear to be due entirely to the T-PSS and not to any of the vehicle components. T-PSS does not seem to lose any of its antimicrobial effectiveness after being formulated in the gel, based on comparisons of the gel data with the previously published results on the drug substance [41] and with the HIV inhibition results obtained with the drug substance in the present study. In the present experiments, T-PSS also had high inhibitory activity against a number of clinical and laboratory strains of HIV and against clinical isolates of HSV.

T-PSS (alone or in gel) had no effect on lactobacilli, which are important components of the normal protective vaginal microflora. In contrast to STD-causing microbes, lactobacilli do not infect cells, i.e., they do not bind to and enter target cells. Therefore, these microbes lack the invasion mechanisms that are probably the target for the action of T-PSS. T-PSS alone or in gel form also had no cytotoxic effects on the host cells in the microbial infection assays. Comparative studies with HSV have shown that nonoxynol-9 has lower antimicrobial activity than T-PSS and is very cytotoxic to the host cells, leading to the conclusion that T-PSS has a 10 000-fold higher selectivity index (50% cytotoxic dose/50% effective dose; CD50/ED50) than nonoxynol-9 [41].

The mechanism of antimicrobial action of T-PSS is presently not well understood. Glycosaminoglycans (GAGs), specifically heparan sulfate (HS), appear to be important for the adherence of such diverse pathogens as N. gonorrhea, C. trachomatis, and HSV to cell surfaces. Cell surface GAGs, including HS, also seem to have an important role in HIV binding/entry into host cells. Polysulfated polymers and polysaccharides can interfere with microbial infectivity, possibly by mimicking HS and preventing cell surface binding and cell entry [26, 29, 57, 58]. Experiments conducted to examine the mechanism(s) of T-PSS antiviral activity indicated that the compound is as effective as heparin (a known inhibitor of HSV binding to cell surface HS) in preventing absorption of HSV to host cells when the cells were pretreated with compound [41]. GAGs also appear to have an important role in the fertilization process (especially the acrosome reaction), and a high molecular mass (but not low molecular mass) sulfated polysaccharide (dextran sulfate) was reported to inhibit fertilization in vitro [59–62]. Thus, an interaction with cell surface GAGs may be one of the common mechanisms whereby T-PSS inhibits both the infectivity of microbes and the fertilization of spermatozoa.

Besides GAG interactions, other mechanisms are likely to play a role in the antimicrobial and contraceptive activity of T-PSS. T-PSS but not heparin inhibited viral binding when the virus was pretreated with compound and the mixture was subsequently diluted to noninhibitory concentrations of T-PSS [41]. This finding suggests that T-PSS has other than heparin-like inhibition properties and either inactivates the virus or irreversibly binds to HSV glycoproteins. In addition, T-PSS inhibits postbinding steps, including infection mediated by intracellular HSV and cell-cell spread [41]. Furthermore, the present experiments show that T-PSS inhibits hyaluronidase and acrosin, a proteinase. Besides their role in the fertilization process, hyaluronidase and proteinases also function in the infectivity of a number of pathogenic microbes, including HIV [63–67]. These observations suggest that the overall anti-STD/HIV and antifertility activity of T-PSS may involve multiple mechanisms.

Safety is an important concern to be addressed before a compound/formulation is selected for clinical trial. The tests reported presently are typical of those required by the U.S. Food and Drug Administration (FDA) before a clinical trial is allowed. Our results show that T-PSS has a very high safety profile. The drug substance was nonmutagenic in the standard Ames test and had very low oral toxicity. Gel containing a high concentration of T-PSS (10%) had no or minimal toxic effects in the penile irritation, skin irritation, and dermal sensitization tests. Vaginal application of 10% T-PSS gel for 14 consecutive days to both rabbits and rats caused no systemic changes, as indicated by a lack of effect on hematological, blood chemistry, and blood coagulation parameters, or on organ weights and histology. Vaginal irritation was mild after the 14-day application of 10% T-PSS gel and was well within FDA guidelines for acceptability. The low vaginal irritation parallels the lack of cytotoxicity of T-PSS. These toxicity studies suggest that a gel containing up to 100 mg/ml (10%) T-PSS can be used safely.

A desperate need exists for a vaginal formulation that prevents heterosexual transmission of HIV and other STD-causing pathogens. The formulation should also be contraceptive, as several surveys have shown. Such a formulation should have a very high safety profile, should have no effect on the normal vaginal microbial flora, e.g., lactobacilli, and should not cause vaginal irritation. Based on the present data, the T-PSS gel appears to possess these properties, making it an attractive candidate for clinical evaluation.


   FOOTNOTES
 
First decision: 19 May 2001.

1 Support for this project was provided by the CICCR Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School (contracts CIG-96-01 and CIG-00-48), the Rockefeller Foundation (grant RF95021), and the National Institute for Allergy and Infectious Diseases (grant PO1 A137940). The views expressed by the authors do not necessarily reflect the views of the funding agencies. Back

2 Correspondence: L. Zaneveld, Section of Ob/Gyn Research, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. FAX: 312 942 2771; lzanevel{at}rush.edu Back

Accepted: October 31, 2001.

Received: April 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fauci AS. The AIDS epidemic. N Engl J Med 1999; 341:1046-1050[Free Full Text]
  2. Gerbase AC, Rowley JT, Heymann DHL, Berkley SFB, Piot P. Global prevalence and incidence estimates of selected curable STDs. Sex Transm Infect 1998; 74:(suppl 1):S12-S16
  3. Laga M, Manoka A, Kivuvu M. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS 1993; 7:95-102[Medline]
  4. United Nations. Revision of the Official United Nations Population Estimates and Projections. World Population Nearing 6 Billion Projected Close to 9 Billion by 2050. New York: Population Division of the Department of Economic and Social Affairs, United Nations; 1998
  5. Trussell J. Contraceptive efficacy of barrier contraceptives. In: Mauck CK, Cordero M, Gabelnick HL, Spieler JM, Rivera R (eds.), Barrier Contraceptives, Current Status and Future Prospects. New York: Wiley-Liss; 1994: 17–52
  6. Zaneveld LJD. Vaginal contraception since 1984: chemical agents and barrier devices. In: PFA Van Look, G Perez-Palacios (eds.), Contraceptive Research and Development 1984 to 1994. Delhi: Oxford University Press; 1994: 69–90
  7. Niruthisard S, Roddy RE, Chutivongse S. The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis 1991; 18:176-179[Medline]
  8. Roddy RE, Cordero M, Cordero C, Fortney JA. A dosing study of nonoxynol-9 and genital irritation. Int J STD AIDS 1993; 4:165-170[Medline]
  9. Stafford MK, Ward H, Flanagan A, Rosenstein IJ, Taylor-Robinson D, Smith R, Weber J, Kitchen VS. Safety study of nonoxynol-9 as a vaginal microbicide: evidence of adverse effects. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 17:327-331[Medline]
  10. Patton DL, Wang SK, Kuo CC. In vitro activity of nonoxynol-9 on HeLa 229 cells and primary monkey cervical epithelial cells infected with Chlamydia trachomatis. Antimicrob Agents Chemother 1992; 36:1478-1482[Abstract/Free Full Text]
  11. Acaturk F, Robinson JR. Effect of the spermicide, nonoxynol-9, and vaginal permeability in normal and ovariectomized rabbits. Pharmacol Res 1996; 13:950-951[CrossRef]
  12. Rosenstein IJ, Stafford MK, Kitchen VS, Ward H, Weber JN, Taylor-Robinson D. Effect on normal vaginal flora of three intravaginal microbial agents potentially active against human immunodeficiency virus type 1. J Infect Dis 1998; 177:1386-1390[Medline]
  13. Richardson BA, Martin HL, Stevens CE, Hillier SL, Mwatha AK, Chohan BH, Nyange PM, Mandaliya K, Ndinya-Achola J, Kreiss JK. Use of nonoxynol-9 and changes in vaginal lactobacilli. J Infect Dis 1998; 178:441-445[Medline]
  14. Voeller B, Anderson DJ. Heterosexual transmission of HIV. J Am Med Assoc 1992; 267:1917-1918
  15. Mahmoud EA, Svensson LO, Olsson SE, Mardh PA. Antichlamydial activity of vaginal secretion. Am J Obstet Gynecol 1995; 172:1268-1272[CrossRef][Medline]
  16. Garg S, Anderson RA, Chany CJ, Waller DP, Diao XH, Vermani K, Zaneveld LJD. Properties of a new acid-buffering bioadhesive vaginal formulation. Contraception 2001; 64:67-75[CrossRef][Medline]
  17. Martin HL, Richardson BA, Nyange PM, Lavreys L, Hillier SL, Chohan B, Mandaliya K, Ndinya-Achola JO, Bwayo J, Kreiss J. Vaginal lactobacilli, microbial flora and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 1999; 180:1863-1868[CrossRef][Medline]
  18. Whaley KJ, Barratt RA, Zeitlin L, Hoen TE, Cone RA. Nonoxynol-9 protects mice against vaginal transmission of the genital herpes infections. J Infect Dis 1993; 168:1009-1011[Medline]
  19. Phillips DM, Zacharapoulos R. Nonoxynol-9 enhances rectal infection by herpes simplex virus in mice. Contraception 1998; 57:341-348[CrossRef][Medline]
  20. Roddy RE, Schulz KF, Cates W. Microbicides, meta-analysis, and the N-9 question. Sex Transm Dis 1998; 25:151-153[Medline]
  21. Kreiss J, Ngugi E, Holmes K, Nidinya-Achola J, Waiyaki P, Roberts PL, Ruminjo I, Sajabi R, Kimata J, Fleming RTR, Anzala A, Holton D, Plummer F. Efficacy of nonoxynol-9 contraceptive sponge use in preventing heterosexual acquisition of HIV in Nairobi prostitutes. J Am Med Assoc 1992; 268:477-482[Abstract]
  22. Roddy RE, Zekeng L, Ryan KA, Tamoufe U, Weir SS, Wong EL. A controlled trial of nonoxynol-9 film to reduce male-to-female transmission of sexually transmitted diseases. N Engl J Med 1998; 339::504-510[Abstract/Free Full Text]
  23. Stephenson J. Widely used spermicide may increase, not decrease, risk of HIV transmission. J Am Med Assoc 2000; 284:949[Free Full Text]
  24. Zaneveld LJD, Anderson DJ, Whaley KJ. Barrier methods. In: Harrison PF, Rosenfield A (eds.), Contraceptive Research and Development. Looking to the Future. Washington, DC: National Academic Press; 1996: 430–473
  25. Stone AB. Vaginal microbicides against HIV: current state of the art. Int J Pharm Med 1997; 11:81-83
  26. Bagasra O, Lischner HW. Activity of dextran sulfate and other polyanionic polysaccharides against human immunodeficiency virus. J Infect Dis 1988; 158:1084-1087[Medline]
  27. Zaretzky FR, Pearce-Pratt R, Phillips DM. Sulfated polyanions block Chlamydia trachomatis infection of cervix-derived human epithelia. Infect Immun 1995; 63:3520-3526[Abstract]
  28. Pearce-Pratt R, Phillips DM. Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1. Biol Reprod 1996; 54:173-182[Abstract]
  29. Herold BC, Siston A, Bremer J, Kirkpatrick R, Wilbanks G, Fugedi P, Peto C, Cooper M. Sulfated carbohydrate compounds prevent microbial adherence by sexually transmitted disease pathogens. Antimicrob Agents Chemother 1997; 41:2776-2780[Abstract]
  30. Homm RE, Foldesy RG, Hahn DW. ORF 13904, a new long-acting vaginal contraceptive. Contraception 1985; 32:267-274[CrossRef][Medline]
  31. Buchner P, Herbrand W, Rockl W. Uber eine neue kontrazeptionelle methode auf biologischer grundlage. Dtsch Med Wochenschr 1953; 78:907-911
  32. Anderson RA, Feathergill K, Diao X, Cooper M, Kirkpatrick R, Spear P, Waller DP, Chany C, Doncel GF, Herold B, Zaneveld LJD. Evaluation of poly (styrene-4-sulfonate) as a preventive agent for conception and sexually transmitted diseases. J Androl 2000; 21:862-875[Abstract]
  33. Rencher W, Doncel G, Anderson RA, Chany C, Zaneveld LJD, Waller DP, Cooper M, Herold B. Development of a new contraceptive/antimicrobial agent. In: Proceedings of the Microbicides 2000 Conference; 2000; Washington, DC. Abstract (p. 26)
  34. Hardy E, De Padua KS, Jimenez AL, Zaneveld LJD. Women's preferences for vaginal antimicrobial contraceptives. II. Contraception 1998; 58:239-244[CrossRef][Medline]
  35. Maguire RA, Zacharopoulos V, Phillips DM. Carrageenan-based nonoxynol-9 spermicides for prevention of sexually transmitted infections. Sex Transm Dis 1998; 25:494-500[Medline]
  36. Inamori T, Ghanem AH, Higuchi WI, Srinivasan V. Macromolecule transport in and effective pore size of ethanol pretreated human epidermal membrane. Int J Pharm 1994; 105:113-123[CrossRef]
  37. Mohan P, Schols D, Baba M, De Clerq E. Sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors. Antiviral Res 1992; 18:139-150[CrossRef][Medline]
  38. Tan GT, Wickramasinghe A, Verman S, Hughes SH, Pezzuto JM, Baba M, Mohan P. Sulfonic acid polymers are potent inhibitors of HIV-1 induced cytophathenicity and the reverse transcriptases of both HIV-1 and HIV-2. Biochim Biophys Acta 1993; 1181:183-188[Medline]
  39. Ikeda S, Neyts J, Verman S, Wickramsinghe A, Mohan P, De Clercq E. In vitro and in vivo inhibition of ortho- and paramyxovirus infection by a new class of sulfonic acid polymers interacting with virus-cell binding and/or fusion. Antimicrob Agents Chemother 1994; 38::256-259[Abstract/Free Full Text]
  40. Zeitlin L, Whaley KJ, Hegarty TA, Moench TR, Cone RA. Tests of vaginal microbicides in the mouse genital herpes model. Contraception 1997; 56:329-335[CrossRef][Medline]
  41. Herold BC, Bourne N, Marcellino D, Kirkpatrick R, Strauss DM, Zaneveld LJD, Waller DP, Anderson RA, Chany C, Cooper MD, Stanberry L. Poly (sodium 4- styrenesulfonate): an effective candidate topical anti-microbial for the prevention of sexually transmitted diseases. J Infect Dis 2000; 181:770-773[CrossRef][Medline]
  42. Anderson RA, Feathergill K, Kirkpatrick R, Zaneveld LJD, Coleman KT, Spear PG, Cooper MD, Waller DP, Thoene JG. Characterization of cysteamine as a potential contraceptive anti-HIV agent. J Androl 1998; 13:398-408[Abstract/Free Full Text]
  43. Anderson RA, Feathergill KA, Drisdel RC, Rawlins RG, Mack SR, Zaneveld LJD. Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome. J Androl 1994; 15:61-70[Abstract/Free Full Text]
  44. Sokal R, Rohlf FJ. Biometry, 2nd ed. San Franciso: WH Freeman; 1981: 400–453
  45. Terhune SS, Coleman KT, Sekulovich R, Burke RL, Spear PG. Limited variability of glycoprotein gene sequences and neutralizing targets in herpes simplex virus type 2 isolates and stability on passage in cell culture. J Infect Dis 1998; 178:8-15[Medline]
  46. Oram R, Marcellino D, Strauss D, Gustafson E, Talarico CL, Root AK, Fingeroth JD, Crumpacker C, Herold BC. Characterization of an acyclovir resistant herpes simplex type 2 strain isolated from a premature neonate. J Infect Dis 2000; 181:1455-1461
  47. Woolf CM. Principles of Biometry. Princeton, NJ: Van Nostrand Co.; 1968: 83–115
  48. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983; 113:173-215[CrossRef][Medline]
  49. Gad SC, Chengelis CP. Acute Toxicology Testing. San Diego, CA: Academic Press; 1998
  50. Draize JH. Appraisal of the Safety of Chemicals in Foods Drugs and Cosmetics. Topeka, KS: Association of Food and Drug Officials of the United States; 1965
  51. Meyer I. Sodium polystyrene sulfonate: a cation exchange resin used in treating hyperkalemia. Med Rev ANNA 1993; 20:93-95
  52. Comhaire FH. Conventional methods of semen analysis. In: Comhaire FH (ed.), Male Infertility. Clinical Investigation, Cause Evaluation and Treatment. London: Chapman & Hall Medical; 1966: 143–153
  53. Zaneveld LJD, De Jonge CJ. Mammalian sperm acrosomal enzymes and the acrosome reaction. In: Dunbar B, O'Rand MO (eds.), A Comparative Overview of Mammalian Fertilization. New York: Plenum; 1991: 63–79
  54. Joyce CL, Zaneveld LJD. Vaginal contraceptive activity of hyaluronidase and cyclooxygenase (prostaglandin synthetase) inhibitors in the rabbit. Fertil Steril 1985; 44:426-428[Medline]
  55. Kaminski JM, Nuzzo NA, Bauer L, Waller DP, Zaneveld LJD. Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits. Contraception 1985; 31:183-189
  56. Williams RM, Jones R. Specific binding of sulfated polymers to ram sperm proacrosin. FEBS Lett 1990; 270:168-172[CrossRef][Medline]
  57. Rostand KS, Esko JD. Microbial adherence to and invasion through proteoglycans. Infect Immun 1997; 65:1-8[Medline]
  58. Baba M, Schols E, De Clercque E, Pauwels R, Ngy M, Gyoryi-Edelenyi J, Low M, Sandor G. Novel sulfated polymers as highly potent and selective inhibitors of human immunodeficiency virus replication and giant cell formation. Antimicrob Agents Chemother 1990; 34::134-138[Abstract/Free Full Text]
  59. Miller DJ, Hunter AG. Effect of osmolality and glycosaminoglycans on motility, capacitation, acrosome reaction, and in vitro fertilizability of bovine ejaculated sperm. J Dairy Sci 1986; 69:2915-2924
  60. Lee CN, Clayton MK, Bushmeyer SM, First NL, Ax RL. Glycosaminoglycans in ewe reproductive tracts and their influence on acrosome reactions in bovine spermatozoa in vitro. J Anim Sci 1986; 63::861-867
  61. Parrish JJ, Susko-Parrish JL, Handrow RR, Ax RL, First NL. Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa. Gamete Res 1989; 24:403-413[CrossRef][Medline]
  62. Meizel S, Turner KO. Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm. J Exp Zool 1986; 237:137-139[CrossRef][Medline]
  63. Rosso G. Vaginal hyaluronidase activity in some pathological conditions. III. Cervico-vaginitis. Minerva Ginecol 1975; 27:537-544[Medline]
  64. Fitzgerald TJ, Repesh LA. The hyaluronidase associated with Treponema pallidum facilitates treponemal dissemination. Infect Immun 1987; 55:1023-1028[Abstract/Free Full Text]
  65. O'Reilly TM, Ghatti AR. Proteases of the pathogenic Neisseriae: possible role in infection. Microbios 1986; 45:113-129[Medline]
  66. Hattori T, Koito A, Takatsuki K, Kido H, Katunum N. Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection. FEBS Lett 1989; 248:48-52[CrossRef][Medline]
  67. Bourinbaiar AS, Lee-Huang S. Acrosin inhibitor, 4'-acetamidophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive with anti-HIV activity. Contraception 1995; 51:319-322[CrossRef][Medline]
  68. Sander FV, Cramer SD. A practical method for testing the spermicidal action of chemical contraceptives. Hum Fertil 1941; 6:134-153
  69. Aronson NN, Davidson EA. Lysosomal hyaluronidase from rat liver. I. Preparation. J Biol Chem 1967; 242:437-440[Abstract/Free Full Text]
  70. Anderson RA, Beyler SA, Mack SR, Zaneveld LJD. Characterization of a high-molecular weight form of human acrosin. Comparison with human pancreatic trypsin. Biochem J 1981; 199:307-316[Medline]
  71. De Jonge CJ, Mack SR, Zaneveld LJD. Inhibition of the human sperm acrosome reaction by proteinase inhibitors. Gamete Res 1989; 10::232-239
  72. Resnick L, Busso ME, Duncan RC. Anti-HIV screening technology. In: Heterosexual Transmission of AIDS. New York: Alan R. Liss; 1990: 311–325



This article has been cited by other articles:


Home page
 Antimicrob. Agents Chemother.Home page
D. Pinna, P. Oreste, T. Coradin, A. Kajaste-Rudnitski, S. Ghezzi, G. Zoppetti, A. Rotola, R. Argnani, G. Poli, R. Manservigi, et al.
Inhibition of Herpes Simplex Virus Types 1 and 2 In Vitro Infection by Sulfated Derivatives of Escherichia coli K5 Polysaccharide
Antimicrob. Agents Chemother., September 1, 2008; 52(9): 3078 - 3084.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
P. M. M. Mesquita, S. S. Wilson, P. Manlow, L. Fischetti, M. J. Keller, B. C. Herold, and R. J. Shattock
Candidate Microbicide PPCM Blocks Human Immunodeficiency Virus Type 1 Infection in Cell and Tissue Cultures and Prevents Genital Herpes in a Murine Model
J. Virol., July 1, 2008; 82(13): 6576 - 6584.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
A. Bugatti, C. Urbinati, C. Ravelli, E. De Clercq, S. Liekens, and M. Rusnati
Heparin-Mimicking Sulfonic Acid Polymers as Multitarget Inhibitors of Human Immunodeficiency Virus Type 1 Tat and gp120 Proteins
Antimicrob. Agents Chemother., July 1, 2007; 51(7): 2337 - 2345.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
R.K. Jain, J.P. Maikhuri, S.T.V.S. Kiran Kumar, V.L. Sharma, A.K. Dwivedi, K. Mitra, V.K. Bajpai, and G. Gupta
Novel disulphide esters of carbothioic acid as potent, non-detergent spermicides with low toxicity to Lactobacillus and HeLa cells in vitro
Hum. Reprod., March 1, 2007; 22(3): 708 - 716.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
G. F. Doncel
Exploiting common targets in human fertilization and HIV infection: development of novel contraceptive microbicides
Hum. Reprod. Update, March 1, 2006; 12(2): 103 - 117.