BOR Papers in Press. Published on April 30, 2003 as DOI:10.1095/biolreprod.102.014043

Thermoreversible Gel Formulation Containing Sodium Lauryl Sulfate as a Potential Contraceptive device

Running title: New contraceptive device

Key Words: Pregnancy, female reproductive tract, vagina, cervix, fertilization

Caroline Haineault, Pierrette Gourde, Sylvie Perron, André Désormeaux, Jocelyne Piret, Rabeea F. Omar, Roland R. Tremblay and Michel.G. Bergeron

Centre de Recherche en Infectiologie, Université Laval, Québec, Québec, Canada G1V 4G2

Send all correspondence to: Michel G. Bergeron, MD FRCP Centre de Recherche en Infectiologie, RC 709 Centre Hospitalier Universitaire de Québec, Pavillon CHUL 2705 Boul. Laurier, Ste-Foy, Québec Canada, G1V 4G2 Tel: (418) 654-2705 Fax: (418) 654-2715 Email: [email protected]

Abstract

The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited in a 5

concentration-dependent manner the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility, as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their

10

physiologically-relevant-buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits prior to their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with non-lubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential

15

candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.

3 Introduction

It is estimated that 75 million human unintented pregnancies occur annually, worldwide. Among these, 50 million are terminated by abortion, 20 million of which are made under unsafe 20

practices (WHO, 1999). Unintented pregnancy represents an important public health concern in the United States as well since 1.5 million cases occur each year, half of which ending in abortion [1]. The most widely used vaginal spermicides on the market contain nonoxynol-9, as the active ingredient, but several other compounds have also been used or proposed such as propanolol [2], chlorhexidine [3, 4], magainins [5], gramicidin, gossypol [6] and menfegol [4, 7]. Nonoxynol-9 has

25

been used for more than 30 years in over-the-counter gels, foams, creams, sponges, films and foaming tablets designed to kill sperm. Nonoxynol-9 is a nonionic surfactant which acts by indiscriminately solubilizing membrane lipids. This compound exerts its effect via a detergent-like ability to damage the sperm membrane, perturbs its conformation, and destroys its semi-permeable nature, thereby impairing the sperm motility and egg fertilizing functions [8, 9].

30 Since the onset of acquired immunodeficiency syndrome (AIDS) epidemic, there is an urgent need for a dual protection capable of preventing the transmission of human immunodeficiency virus (HIV) by heterosexual vaginal transmission as well as providing fertility control for women who are at high risk for acquiring HIV. Nonoxynol-9 has been demonstrated to 35

inactivate the infectivity of enveloped viruses, such as HIV-1 and herpes viruses, and other sexually transmitted pathogens, such as Neisseria gonorrhoeae and Chlamydia trachomatis in vitro [6, 1014]. Until now, nonoxynol-9 was the only topical vaginal spermicide evaluated in clinical trials to protect women against HIV and other sexually transmitted pathogens. However, because of its membrane-disruptive properties, the frequent use of nonoxynol-9 has been shown to damage the

4 40

cervicovaginal epithelium [4, 15-17], cause an acute tissue inflammatory response [18, 19], alter the vaginal microflora leading to an increased risk of developing opportunistic infections in the genitourinary tract [20], and even enhance the risk of acquiring HIV by heterosexual vaginal transmission [21-23]. Results from clinical trials have recently shown that nonoxynol-9 can no longer be deemed a potential HIV prevention method [24, 25].

45 The Food and Drug Administration (FDA) has recently issued a final rule stating that nonoxynol-9 in over-the-counter products is not generally recognized as safe and effective for its intended use or is misbranded. The experts found no evidence that nonoxynol-9-lubricated condoms provided any more protection against pregnancy or sexually transmitted infections than 50

condoms lubricated with silicone, used as a lubricant for the majority of condoms available in developing countries. Since nonoxynol-9 may cause some adverse effects, the experts recommended

that

such

condoms

should

no

longer

by promoted.

Therefore,

new

spermicidal/microbicidal formulations, without toxicity for the vaginal mucosa, are now requested to replace nonoxynol-9 in male latex condoms to increase their effectiveness. 55 During the past several years, we have developed a thermoreversible gel formulation containing sodium lauryl sulfate (SLS) that could prevent the transmission of HIV and other sexually transmitted pathogens once applied to the vaginal and cervical mucosa of women. We have demonstrated that the polymer formulation acts by itself as a physical barrier against 60

enveloped viruses, such as herpes viruses and possibly HIV-1, through a trapping process preventing infection of susceptible cells in vitro [26]. Sodium lauryl sulfate, an anionic surfactant with protein denaturant potency, inhibits the infectivity of HIV-1 [27, 28] and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to susceptible cultured cell lines [29-31]. Sodium lauryl sulfate

5 has also been reported to inhibit the infectivity of the nonenveloped human papillomavirus in 65

experimental xenografts of human foreskin epithelium [32]. In vivo studies showed that the gel formulation containing SLS was effective to protect mice following HSV-2 intravaginal challenge and that the formulation was well tolerated after repeated intravaginal administrations to rabbits [33]. The gel formulation containing SLS was also well tolerated when applied intravaginally to women twice daily for 14 days [34, 35].

70 In the present study, we have evaluated the effects of SLS on sperm functions. We have first examined the effect of SLS on the activity of hyaluronidase (sheep testicular hyaluronidase) since this enzyme is localized on the sperm surface and is therefore readily accessible to SLS action. We have also tested the effects of SLS and of the gel formulation containing SLS on human sperm 75

motility in vitro. We have performed studies on the acid-buffering capacity of the gel formulation containing SLS because it has been demonstrated that an acidic pH environment may kill sperm as well as HIV-1 and other sexually transmitted pathogens. The acid-buffering capacity of gel formulations containing SLS was compared with that of Advantage 24™ which is a widely used topical vaginal spermicide. We have also examined the contraceptive efficacy of the gel

80

formulation containing SLS in a rabbit model since this model is widely used to assess the contraceptive effectiveness of spermicidal formulations. Finally, because the gel formulation containing SLS could be a potential candidate to replace nonoxynol-9 in latex condoms to increase their effectiveness, we have determined the compatibility of this formulation with non-lubricated latex condoms.

85

6 85

Materials and Methods

Chemicals. Sodium lauryl sulfate and Tergitol NP-9 (nonoxynol-9) were obtained from Sigma Chemical Co. (St Louis, Mo.).

90

Preparation of gel formulations. Polymers composed of polyoxypropylene and polyoxyethylene exhibit an interesting property referred to as reverse thermal gelation in which aqueous solutions are liquid at or below ambient temperature and form gel at higher (body) temperature [36]. The liquid to gel phase transition temperature is dependent of the polymer concentration and of the ingredients incorporated in the aqueous solution. Therefore, the viscosity of the formulation can be

95

adjusted by increasing or decreasing the polymer concentration allowing a variety of advantageous topical applications. Gel formulations were prepared by suspending appropriate amounts of the polymer (30%, w/w) and SLS (if present; 2%, w/w) in citrate buffer (0.05, 0.1, 0.2 or 0.4 M at pH 3.5) and had liquid to gel phase transition temperatures of 25°C. The different formulations were stirred under agitation overnight at 4ºC to ensure complete dissolution.

100 Hyaluronidase activity. The effect of SLS on sheep testicular hyaluronidase (type III; Sigma Chemical Co.) activity has been evaluated in vitro. Hyaluronidase (EC 3.2.1.35) activity was quantified using hyaluronic acid (from bovine vitreous humor; Sigma Chemical Co.) as substrate by the method of Aronson and Davidson [38]. Briefly, the hyaluronidase (7.2 U) was preincubated 105

with increasing SLS concentrations (50 to 1180 µg/mL) in acetate buffer (0.1 M sodium acetate, 0.15 M NaCl at pH 5.5) for 10 min at 37°C before starting the enzyme reaction by the addition of hyaluronic acid (0.3 mg/mL). The assay was carried out for 20 minutes at ambient temperature. The concentration of N-acetylglucosamine resulting from the enzyme reaction was measured

7 colorimetrically at a wavelength of 545 nm as previously described [39]. The concentration of SLS 110

which inhibits of the enzyme activity was determined.

Human sperm motility. The effect of SLS on human sperm motility has been compared with that of nonoxynol-9 (positive control) and of 0.9% NaCl (sham-treated control). The method was derived from that previously described by Sander and Cramer [40]. In brief, semen was collected 115

by self-masturbation from healthy volunteers after obtaining their consent at the Andrology Laboratory of the Laval University Hospital Center. The sperm immobilizing activity was tested by mixing 50 µL of freshly ejaculated semen with 250 µL of the test agent solutions (i.e., 144 to 433 µg/mL SLS or 100 µg/mL nonoxynol-9 in 0.9% NaCl). The percentage of motile spermatozoa was determined with a Hamilton Thorne sperm motility analyzer (Hamilton Thorne Research, Beverly,

120

Mass.). Five microliter of sperm suspension were deposited on a 20 µm MicroCell chamber (Conception Technologies, San Diego, Calif.) prewarmed at 37°C just prior to and 30 seconds after adding the test agent solutions. The chamber was then placed on the warmed stage (37°C) of the motility analyzer, and five to eight fields per chamber were selected and scanned automatically to evaluate the percentage of motile sperm. The concentration of SLS which inhibits 50% of human

125

sperm motility (minimum effective concentration; MEC) was determined. Compounds that have a MEC of 2 mg/mL or more were considered to have no significant spermatozoa immobilizing activity and were not considered as a spermicidal agent [40]. In order to assess whether the incorporation of SLS in the gel formulation could cause a sequestration of the surfactant in the polymer micelles and reduce therefore its efficacy on human sperm motility, we have evaluated the

130

spermicidal activity of SLS (2%, w/w), the gel formulation (30%, w/w), the gel formulation (30%, w/w) containing SLS (2%, w/w) prepared in Ham F-10 solution (Canadian Life Technologies, Burlington, Ontario, Canada) or in citrate buffer (0.05 M, pH 3.5) were also tested. Briefly, 30 µL

8 of the ejaculate was combined with 90 µL of the test agent solutions and incubated for 2 minutes at 37ºC. The percentage of motile spermatozoa was determined as described above. 135 Buffering capacity of gel formulations. The acid-buffering capacity of gel formulations (30%, w/w) containing SLS (2%, w/w) prepared in citrate buffers of increasing molarities (0.05, 0.1, 0.2 or 0.4 M at pH 3.5) has been compared with that of the commercial Advantage 24™ (Robert Pharmaceutical, Oakville, Ontario, Canada), also known as Advantage S™ in US. In this set of 140

experiments, one gram of each gel formulation or of Advantage 24™ was diluted with 10 mL of 0.9% NaCl (normal saline) as described previously [41]. Sodium hydroxide (1.0 N) was added in 20 µL increment under constant stirring. The pH was measured with a Beckman’s Epoxy-Calomel pH electrode (Beckman Instruments Inc., Fullerton, Calif.) 30 sec after each volume addition. This procedure was repeated until the pH rose above 10.0. Titrations were performed in triplicate for

145

each formulation. Titration curves were used to calculate the amount of NaOH required to bring the pH of each formulation solution to 5.0 (which corresponds to a measure of the buffering capacity of the formulation at the maximal desirable vaginal pH). The ability of a vaginal formulation to buffer the pH of semen below 6.0 is essential for good spermicidal activity. The sperm-buffering capacity of gel formulations (30%, w/w) containing SLS (2%, w/w) in citrate buffers of increasing

150

molarities was compared with that of Advantage 24™. Because some of the tested formulations demonstrated good buffering capacity, we used large volumes of semen in our experiments (formulations-to-semen ratios from 1:1 to 1:4) to better characterize the acid-buffering capacity. The formulations were mixed directly with semen according to these different proportions and the resulting pH was measured as described above.

155

9 Contraceptive efficacy in rabbits. The contraceptive efficacy of the gel formulation (30%, w/w) containing SLS (2%, w/w) prepared in citrate buffer (0.05 M, pH 3.5) was evaluated in rabbits. Rabbit semen is readily obtainable from sexually-mature bucks using a «teaser» female and an artificial vagina similar to that previously described by Castle et al. [42]. Semen samples from three 160

male New Zealand rabbits (Charles River Laboratories Inc., St. Constant, Québec, Canada) were collected, pooled and used within 1 h. The pooled rabbit ejaculate was analyzed for concentration and motility of spermatozoa with the Hamilton Thorne sperm motility analyzer (Hamilton Thorne Research). Seminal plasma was obtained by centrifuging the semen at 800 x g for 5 min and filtering the supernatant on a 0.45 µm membrane. The seminal plasma was stored at –80°C until

165

use.

Two groups of female New Zealand rabbits (weight 2.5 to 3.5 kg; Charles River Laboratories Inc.) were induced ovulators by injecting 100 IU human chorionic gonadotropin (hCG; 0.1 ml; Profasi® HP, Serono, Oakville, Ontario, Canada) in the marginal ear vein. Four mL 170

of the gel formulation containing SLS was deposited at 18 to 19 cm into the vagina of the first group of rabbits using a 10 mL male luer lock plastic syringe connected to an urethral catheter (MED-RX® 14 french x 16”, Benlan Inc., Oakville, Ontario, Canada) incorporated in a laparoscop pre-lubricated with K-Y jelly (Johnson & Johnson Medical Inc., Arlington, Tx). The second group of animals was left untreated to provide a negative control. After 2 min, 0.5 mL of collected semen

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diluted with seminal plasma to a final concentration of 2 x 106 sperm/mL (106 sperm correspond to the 50 fertilizing dose50 or 50 times the amount of sperm required to obtain 50% fertility, which represents a litter size half of a normal litter) was introduced 12.5 cm into the rabbit vagina using a 14 french urethral catheter. This way, we had a constant semen (0.5 ml) to gel formulation containing SLS (4 ml) volume ratio in all treated animals. Rabbits were maintained sleepy using

10 180

isoflurane throughout the administration of the gel formulation and artificial insemination periods. After 8 to 10 days, the female rabbits were euthanized by intravenous injection of euthanyl (120 mg/kg/dose), and reproductive tracts were removed. The number of embryos within the uterine horns and the number of corpora lutea in the ovaries were counted. The number of corpora lutea, which appears as vascularized nodules on the ovaries, indicates the number of ovulated eggs. Thus,

185

the ratio of the numbers of embryos to corpus lutea provides a measure of fertility that corrects for occasionally infertile or nonovulatory females [43].

Condom compatibility. The compatibility of the gel formulation (30%, w/w) alone or containing SLS (2%, w/w) prepared in citrate buffer (0.05 M, pH 3.5) with non-lubricated latex condoms 190

(LifeStyles® non-lubricated, Ansell Inc., Dothan, Al.) has been evaluated and compared with those of untreated (negative control) and Johnson’s® baby oil (Johnson & Johnson Inc., Montreal, Quebec, Canada) treated (positive control) latex condoms [44, 45]. The study was performed according to ASTM norm #D3492-89 [Standard Specification for Rubber Contraceptives (Condoms)]. In brief, latex condoms were unrolled, placed on an analytical balance (model AG245,

195

Mettler Toledo, Greifensee, Switzerland) and a fixed amount (0.5 ± 0.01 g) of gel formulations or baby oil was applied in the middle. Condoms were rolled, placed in separate plastic bags, sealed and incubated for 2 or 7 days in an oven at 70°C in order to test for the accelerated aging of the condoms in accordance with Specification D573 Test Method for Rubber – Deterioration in an Air Oven, as described in the ASTM protocol cited above. Twenty latex condoms were used for each

200

group (i.e., 10 for the 2 days incubation and another 10 for the 7 days incubation). Condoms were then unrolled and a 20 mm width ring was cut using a razor blade at approximately 80 mm from the open end. Samples were then wiped with isopropanol 99% and air dried. Thickness of latex rings was evaluated using a micrometer. The breaking force and the distance between the center of

11 rollers at break for latex rings were measured using an Instron tensiometer model#5565 (Instron 205

Corp., Canton, Mass.) with a 500 N tensil head tester and a speed of 500 mm/min. Roller grips (2.5 cm in length and 1 cm in diameter) were used. Latex rings were placed over rollers and elongation was started. The tensile strength and elongation at break were calculated according to the following equations:

210

T = F/(2*W*D) Where T = tensile strengh (MPa) F = breaking force (N) W = width of ring (20 mm)

215

D = single wall thickness (mm)

E = 100*((2*D+G-C)/C) Where E = elongation at break (%) 220

D = distance between center of rollers at break (mm) G = circumference of one roller (mm) C = circumference of the specimen (mm)

Statistical analysis. The significance of the differences between (i) the tensile strength and (ii) the 225

elongation at break for non-lubricated latex condoms treated with the different formulations and untreated latex condoms (controls) were evaluated by an unpaired Student t test. All statistical

12 analyses were performed with a computer package (Statview + SE Software; Abacus Concepts, Berkeley, Calif.). A p value of less than 0.05 was considered statistically significant.

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Results

Hyaluronidase activity: Figure 1 shows the effect of SLS on the activity of sheep testicular hyaluronidase in vitro. Sodium lauryl sulfate decreased, in a concentration-dependent manner, the enzyme activity. The concentration of SLS which inhibits 50% of the enzyme activity was 235

approximately 800 µg/ml, whereas an almost complete inhibition was seen at 1180 µg/mL.

Human sperm motility: Figure 2 shows the effect of SLS on human sperm motility in vitro. Sodium lauryl sulfate decreased, in a concentration-dependent manner, the motility of human sperm after an exposure of 30 sec at 37°C. The MECvalue was 279.2 ± 11.9 µg/mL. No reduction 240

of the MEC value (253.2 ± 39.4 µg/mL) was observed when the incubation period of semen with SLS was increased to 10 min indicating that the action of SLS is rapid. Nonoxynol-9 at a concentration of 100 µg/mL reduced human sperm motility to 66.8 ± 3.72 % of control values. The concentration of SLS required to achieve a similar effect was approximately 2-times greater than that of nonoxynol-9. The effects of SLS, the gel formulation and the gel formulation containing

245

SLS prepared in Ham F-10 solution or in citrate buffer on human sperm motility have also been investigated. Table 1 shows that Ham F-10 solution (negative control) did not affect human sperm motility whereas treatment of semen with citrate buffer (0.05 M, pH 3.5) caused a complete inhibition. As expected, SLS at a concentration of 2% (20 mg/mL), either in Ham F-10 solution or in citrate buffer, completely inhibited sperm motility. The gel formulation prepared in Ham F-10

250

solution partly reduced human sperm motility to approximately 40% of control values suggesting that the viscosity of the gel formulation or the polymer by itself may alter the motility of spermatozoa. The addition of SLS (2%) to this formulation completely inhibited human sperm

14 motility. The gel formulation prepared in citrate buffer, either in the absence or in the presence of SLS, completely inhibited human sperm motility. 255 Acid-buffering capacity. The acid-buffering capacity of gel formulations containing SLS prepared in citrate buffers of increasing molarities (0.05, 0.1, 0.2 or 0.4 M at pH 3.5) has been compared with that of Advantage 24™. A relevant buffering capacity for a given formulation was defined as the amount of NaOH required to bring the pH of the formulation from its initial value to 5.0 (the 260

desirable vaginal pH; see Discussion). The initial pH values (i.e., for 1 g of formulation in 10 mL NaCl 0.9%) for the gel formulations containing SLS prepared in 0.05, 0.1, 0.2 and 0.4 M citrate buffers were 3.681, 3.608, 3.578 and 3.625, respectively, whereas that of Advantage 24 was 3.698. Figure 3 shows that the buffering capacity of gel formulations containing SLS was directly related to the molarity of the citrate buffer. Indeed, the amounts of NaOH required to bring 1 g

265

equivalent of gel formulations prepared in 0.05, 0.1, 0.2 and 0.4 M citrate buffers to pH 5.00 were 0.035, 0.072, 0.145 and 0.256 milliequivalent, respectively. On the other hand, the amount of NaOH required to bring 1 g equivalent of Advantage 24 to pH 5.00 was 0.080 milliequivalent.

The ability of a vaginal formulation to buffer the pH of semen below 6.0 is essential for 270

good spermicidal activity. Table 2 shows the physiologically-relevant buffering activity of the gel formulations containing SLS prepared in citrate buffers of increasing molarities estimated by determining the pH after direct addition of whole human semen to the undiluted gel formulations in varying proportions (w/w). Human semen has a pH of 7.2 to 8.0 with good buffering properties. Results from titration measurements showed that, as expected, a gel formulation prepared in a

275

citrate buffer of high molarity (0.4 M) was more effective to acidify the ejaculate when compared with a buffer of lower molarity (0.05 M). For instance, when one part of gel formulation was mixed

15 with two parts of semen, the pH remained below 5.0 when using 0.4 M citrate buffer, but increased to 7.0 with 0.05 M citrate. It was calculated from the data that the minimum formulation to semen ratio required to bring the pH to 5.0 was 1:2.12 and 1:0.22 for the gel formulations prepared in 0.4 280

M and in 0.05 M citrate buffers, respectively. As expected from the data obtained by titration with NaOH, the gel formulation containing SLS prepared in 0.1 M citrate buffer had acid-buffering capacity almost similar to that of Advantage 24. The minimum formulation to semen ratio required to obtain a pH of 5.0 was 1:0.43 and 1:0.56 for the gel formulation containing SLS prepared in 0.1 M citrate buffer formulation and Advantage 24, respectively.

285 Contraceptive efficacy. The contraceptive efficacy of the gel formulation containing SLS was examined in a rabbit model. Table 3 shows that 36 out of 47 total oocytes found in the ovaries of untreated control female rabbits were fertilized. The four untreated control rabbits became pregnant with a mean of 9 ± 3.1 embryos per rabbit. In contrast, no fertilized oocytes were observed in the 290

87 total oocytes found in the ovaries of the seven female rabbits treated with the gel formulation containing SLS. These results suggest that the formulation was completely contraceptive in this animal model.

Condom compatibility. Figure 4 shows the effect of gel formulations containing or not containing 295

SLS on the tensile strength (Panel A) and elongation at break (Panel B) of non-lubricated latex condoms after 2 and 7 days incubation at 70°C to test for accelerated aging. Results clearly showed that the application of the gel formulations alone or containing SLS on latex condoms did not affect the tensile strength and elongation at break even after 7 days incubation when compared with untreated controls. In contrast, treatment of latex condoms with baby oil (positive control)

300

significantly reduced both their elongation at break and tensile strength when compared with

16 untreated controls. These results showed that the formulation containing SLS is fully compatible with male latex condoms.

17 Discussion

305

Since the onset of AIDS epidemic, new strategies aimed at preventing the sexual transmission of HIV while providing fertility control for women at high risk of acquiring HIV are needed. In the past several years, we have demonstrated that a gel formulation containing SLS, an anionic surfactant with protein denaturant potency, could be a potential candidate for use as a topical vaginal microbicidal formulation. In the present study, we have examined the effects of SLS

310

on human sperm functions in vitro. We have also evaluated the contraceptive efficacy of a gel formulation containing SLS in a rabbit model. Finally, we have determined the compatibility of this gel formulation with non-lubricated latex condoms.

Sperm hyaluronidase has been implicated in sperm penetration of the extracellular matrix of 315

the cumulus oophorus and may play a crucial role in gamete interaction and fertility in humans. The measurement of the activity of hyaluronidase extracted from semen samples provides a reliable predictor of the in vitro fertilizing potential of human sperm [46]. The hyaluronidase enzyme is present on the surface of the plasma membrane overlying the acrosome and on the inner acrosomal membrane [37] and is readily accessible to SLS action. Results showed that SLS inhibits, in a

320

concentration-dependent manner, the activity of sheep testicular hyaluronidase. Hyaluronidases from testes and spermatozoa are immunologically and enzymatically identical. Enzymatic properties of some spermatozoal hyaluronidases from different species are indistinguishable [47] and the sensitivities of testicular and spermatozoal hyaluronidases to different types of inhibitors are very similar [48]. Therefore, results obtained with bovine testicular hyaluronidase are suspected

325

to be applicable to the spermatozoal enzyme. The ability of SLS to inhibit the enzyme activity is probably related to its potential to denature soluble proteins. Indeed, SLS affects the secondary and

18 higher structures of soluble proteins mainly by disrupting the hydrogen bonds and other interactions that maintain these structures [49]. The effect of SLS incorporated in the gel formulation was not evaluated because of technical problems due to the high viscosity of the 330

formulation.

Sodium lauryl sulfate possesses good spermicidal activity as evidenced by its ability to inhibit human sperm motility. A complete inhibition of human sperm motility, as evaluated by the 30-sec Sander-Cramer test, was obtained at a concentration of 430 µg/mL. Cholic acid (a 335

component of Protectaid vaginal sponge), another anionic surface-active agent, was also reported to produce an inhibition of human sperm motility which was concentration- and time-dependent [50, 51]. Anionic surfactants, such as SLS, first bind and unfold, and extract proteins before solubilizing membrane lipids. These effects greatly depend on the concentration of surfactants used and especially on their CMCs (i.e., 2.30 mM for SLS in 50 mM Na+). Kragh-Hansen et al. have

340

reported that detergents with strongly hydrophilic heads like SLS only very slowly solubilize liposomal membranes [52]. Sodium lauryl sulfate specifically interacts with the protein component of Ca2+-ATPase membranes, leading to cooperative unfolding and extraction of the protein before solubilization of lipids. This effect results from combination of hydrophobic and predominantly ionic interactions. Using liposomes, it was shown that SLS extracts membrane lipids from the outer

345

bilayer leaflet, which becomes partly depleted of phospholipids. Thereafter, a reorganization and a redistribution of phospholipid from the inner to the outer leaflet occur. The vesicles slowly open up, leading to fragmentation and finally to solubilization into mixed micelles [52, 53].

Human sperm motility was also partly inhibited by the gel formulation prepared in Ham F350

10 solution. This effect can be ascribed to the high viscosity of the gel formulation which could

19 impede spermatozoon movement. In addition, the micelles formed by the polymer possesses an affinity for lipid membranes and could bind to the spermatozoon surface. In this context, previous studies from our laboratory have shown that liposomes formed aggregates with the micelles of polymer [54] and that the enveloped herpes simplex virus was entrapped within the micelles of gel 355

or coated by the polymer [26].

An healthy vagina has a normal pH ranging from 3.5 to 4.5 [55], primarily because of the lactic acid produced by lactobacilli present in the normal vaginal flora. Spermatozoa, HIV and others sexually transmitted pathogens are known to be inactivated at such low pH [56-62]. In this 360

context, our data have shown that human sperm motility was completely inhibited in citrate buffer (0.05 M, pH 3.5). However, the acidic pH of the vaginal environment can be disturbed by several factors. Indeed, when semen enters the vagina, the pH rises to above 6.0 because of the slighly alkaline pH (7.2 - 8.0) of the ejaculate. In addition, vaginal infections are often associated with an increase of the vaginal pH above 6.0 [63]. Transmission of HIV or other sexually transmitted

365

pathogens are most likely to occur during these periods of increased vaginal pH [57]. Thus, it would be advantageous to develop an acid-buffering vaginal formulation that maintains the vaginal pH acidic when ejaculate is present in the vagina or during vaginal infection in order to obtain good contraceptive and microbicidal properties. The acid-buffering capacity of gel formulations containing SLS increased with the molarity of the citrate buffers used for their preparations. Gel

370

formulation prepared in 0.1 M citrate buffer and Advantage 24™ had almost similar acid-buffering capacity than the Aci-Jel [41], a vaginal gel under clinical investigation [64]. Indeed, the amount of NaOH required to bring 1 g equivalent of the Aci-Jel to pH 5.00 was 0.076 milliequivalent compared with 0.072 and 0.080 milliequivalents for the gel formulation prepared in 0.1 M citrate buffer and Advantage 24™, respectively. An even higher acid-buffering capacity was obtained

20 375

with gel formulations prepared in 0.2 and 0.4 M citrate buffers which required the addition of 0.145 and 0.156 milliequivalent of NaOH, respectively, to bring the pH to 5.0. Furthermore, experiments in which semen was mixed with undiluted gel formulations containing SLS confirmed their physiologically-relevant acid-buffering potential.

380

The contraceptive efficacy of the gel formulation containing SLS has also been evaluated in a rabbit model. The intravaginal application of the formulation 2 min prior to artificial insemination of female rabbits with freshly ejaculated semen was found to impede egg fertilization. Even if the dilution of semen with the gel formulation will be lower in women (approximately 2-fold) than that used in rabbits (8-fold), we may suggest that the contraceptive efficacy of the gel formulation

385

containing SLS observed in rabbits is expected to be contraceptive in humans for the following reasons: i) we showed that the rabbit sperm was a little bit more resistant to SLS than human sperm (IC50, 322.66 µg/mL versus 279.2 µg/mL); ii) the rabbit ejaculate used to inseminate the female rabbit was 100-fold more concentrated than that of humans (human ejaculate contains no more than 1 FD50 [65]); iii) the rate of sperm transport from the vagina to the fallopian tubes is slower in

390

rabbits (3 to 4 hours) than in humans (30 min).

Previous studies from our laboratory have also been performed to evaluate the tolerance and toxicity of a gel formulation (30%, w/w) containing SLS (2.5%, w/w) when applied intravaginally once daily for two weeks to rabbits [33]. Results showed that a mild irritation was observed after 395

application of the formulation, but without ulcerations or necrosis of tissues. Histologic examination revealed a slight loss of integrity of epithelial cells and accumulation of leukocytes and erythrocytes in the vaginal submucosae. The formulation was also not toxic to the uterine horns, uterus, ovaries, and urinary bladder. Overall, macroscopic and histopathologic examinations

21 indicated that the gel formulation containing SLS was well tolerated. The gel formulation 400

containing SLS was also well tolerated when applied intravaginally to women twice daily for 14 days [34, 35]. It is clear that more experiments will be needed to evaluate the tolerance of the woman vagina to the gel formulation containing SLS for longer periods of time.

The male condom has a relatively low contraceptive effectiveness (86% typical use; [66]), 405

which has led to the recommendation for a dual protection consisting of the concomitant use of the condom and a highly effective contraceptive method. Our data showed that the treatment of nonlubricated latex condoms with the gel formulation containing SLS for 2 and 7 days at 70°C resulted in no difference in the elongation at break and tensile strength when compared with untreated condoms. These data suggest that the gel formulation containing SLS could be used in combination

410

with latex condoms to obtain a maximal protection against pregnancies as well as HIV and other sexually transmitted pathogens. The composition of the gel formulation containing SLS could be adapted to optimize the contraceptive/HIV protection in women (eg. by increasing the molarity of the citrate buffer used for its preparation).

415

In conclusion, our results showed that SLS was effective to inhibit the activity of sheep testicular hyaluronidase and human sperm motility. The gel formulation containing SLS demonstrated a complete contraceptive efficacy in a rabbit model. The acid-buffering capacity of the formulation could be optimized by increasing the molarity of the citrate buffers used for its preparation. Taken together, these data demonstrated that, in addition to its previously reported

420

microbicidal efficacy against HIV, herpes simplex viruses and human papillomavirus, the gel formulation containing SLS could also be a potential candidate for use as a vaginal spermicide for the prevention of unwanted pregnancies. In addition, because of its compatibility with non-

22 lubricated latex condoms, the gel formulation containing SLS could be used in combination with the male condom to obtain a maximal protection against pregnancies and sexually transmitted 425

infections.

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31 Figure Legends

Figure 1: Effect of SLS on the activity of sheep testicular hyaluronidase in vitro. The enzyme was preincubated in acetate buffer (0.1 M sodium acetate, 0.15 M NaCl at pH 5.5) containing increasing SLS concentrations or not (control) before the addition of hyaluronic acid, as substrate. Incubations were carried out for 20 min at room temperature. The concentration of N-acetylglucosamine was measured colorimetrically at a wavelength of 545 nm. Blanks for which the enzyme was added at the end of the incubation period were run in parallel. Results are mean (± standard deviation) of three independent experiments.

Figure 2: Effect of SLS on human sperm motility in vitro. The percentage of motile spermatozoa in fresh human semen was determined with a Hamilton Thorne sperm motility analyzer (Hamilton Thorne Research) just prior to and 30 sec after mixing with increasing concentrations of SLS in 0.9% NaCl at 37°C. Values represent the mean (± standard deviation) obtained from three separate fresh semen samples.

Figure 3: Buffering capacity of gel formulations (30%, w/w) containing SLS (2%, w/w) prepared in 0.05 M ( ), 0.1 M (¨), 0.2 M (•) or 0.4 M ( ) citrate buffers (at pH 3.5) compared to Advantage 24 ( ). One gram of each gel formulation or of Advantage 24™ was diluted with 10 mL of 0.9% NaCl. Sodium hydroxide (1.0 N) was then added in 20 µL increment under constant stirring and the pH was measured. The procedure was repeated until the pH rose above 10.0. Results are mean (± standard deviation) of 3 independent experiments.

32 Figure 4: Effects of gel formulations containing or not containing SLS on the tensile strength (A) and elongation at break (B) of non-lubricated latex condoms. Untreated and baby oil-treated latex condoms were used as negative and positive controls, respectively. A fixed amount of formulations (0.5 ± 0.01 g) was applied in the middle of unrolled condoms. Condoms were rolled, placed in separate plastic bags and incubated for 2 or 7 days in an oven at 70°C to test for the accelerated aging. The tensile strength and the elongation at break of 20 mm width latex rings cut from these condoms were evaluated on a tensiometer with a 500 N tensil head tester and a speed of 500 mm/min. Results are the mean (± standard deviation) of values obtained for 10 different nonlubricated latex condoms for each group. * p < 0.05 when compared with the negative control.

33 Table 1: Effects of SLS, gel or gel containing SLS in Ham F-10 solution or in citrate buffer (0.05 M, pH 3.5) on human sperm motility.

Treatment

Motility (% of control)

None

100 ± 0

Ham F-10 solution

113 ± 33

Citrate buffer (0.05 M, pH 3.5)

0±0

SLS (2%, w/w) in Ham F-10 solution

0±0

SLS (2%, w/w) in citrate buffer (0.05 M, pH 3.5)

0±0

Gel (30%, w/w) in Ham F-10 solution

41 ± 24

Gel (30%, w/w) in citrate buffer (0.05 M, pH 3.5)

0±0

Gel (30%, w/w) + SLS (2%, w/w) in Ham F-10 solution

0±0

Gel (30%,w/w) + SLS (2%, w/w) in citrate buffer (0.05 M, pH 3.5)

0±0

Semen was collected by self masturbation from healthy volunteers after obtaining consent. The average sperm concentration was 152.6 x 106 cells/mL, and the average initial percentage of motile spermatozoa was 57% (range 43 - 65). Results are mean (± standard deviation) obtained from three separate fresh semen samples.

Table 2: Buffering activity of gel formulations containing SLS prepared in citrate buffers of increasing molarities and of Advantage 24™ when mixed with fresh human semen in different proportions.

Formulation-to-semen ratio (w/w)

Gel formulation containing SLS in

Advantage 24™

___________________________________________________________________ citrate buffer

citrate buffer

citrate buffer

citrate buffer

(0.05 M, pH 3.5)

(0.1 M, pH 3.5)

(0.2 M, pH 3.5)

(0.4 M, pH 3.5)

4.19 ± 0.02

4.13 ± 0.02

4.14 ± 0.02

4.09 ± 0.05

4.08 ± 0.03

1:1

6.48 ± 0.10

5.58 ± 0.15

4.84 ± 0.10

4.51 ± 0.01

5.35 ± 0.13

1:2

7.04 ± 0.01

6.41 ± 0.01

5.50 ± 0.03

4.92 ± 0.02

5.98 ± 0.04

1:4

7.30 ± 0.01

6.93 ± 0.08

6.17 ± 0.13

5.60 ± 0.02

6.58 ± 0.01

Formulation only

Values represent the mean (± standard deviation) obtained from three separate fresh human semen samples. The pH of semen only was 7.70 ± 0.06.

Table 3: Contraceptive efficacy of the gel formulation containing SLS in female rabbits.

Treatment

No. of fertile rabbits/

Total no. of fertilized oocytes/

Fertilization

No. of inseminated rabbits

total no. of recovered oocytes

(%)

None

4/4

36/47

76

Gel (30%)-SLS (2%)

0/7

0/87

0

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4