Discovery of substituted isoxazolecarbaldehydes as potent spermicides, acrosin inhibitors and mild anti-fungal agents

G. Gupta1,4, R.K. Jain1, J.P. Maikhuri1, P.K. Shukla2, M. Kumar2, A.K. Roy3, A. Patra3, V. Singh3 and S. Batra3

Divisions of 1Endocrinology, 2 Fermentation Technology and 3 Medicinal Chemistry, Central Drug Research Institute, Lucknow 226 001, India

4 To whom correspondence should be addressed. Email: gupta.gopal{at}rediffmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The continued endeavour to design novel, non-detergent molecules that can be useful as topical, prophylactic contraceptives has led to the discovery of substituted isoxazolecarbaldehydes as a new class of compounds exhibiting both spermicidal and acrosin inhibitory activities simultaneously. METHODS: Normal human semen samples were used to detect the spermicidal and acrosin inhibitory activities of the new compounds. Lactobacillus, HeLa and Candida cultures were used to determine the safety of compounds towards normal vaginal flora, their cytotoxicity and anti-fungal activity. Supravital staining and the hypo-osmotic swelling test (HOST) were used to detect the effect on sperm membrane integrity. Nonoxynol-9 (N-9) was used as a reference standard. RESULTS: The 5- and 3-substituted isoxazolecarbaldehydes showed significant spermicidal [minimum effective concentration (MEC)=0.005–2.5%] and acrosin inhibitory (IC50=3.9–58 x 10–4 mol/l) activities in several molecules along with weak fungicidal activity against Candida albicans. Lineweaver–Burk and Dixon plot analysis of a representative structure showed non-competitive inhibition of human acrosin enzyme, and the most potent acrosin inhibitors also considerably diminished the induction of the acrosome reaction by Ca2+ ionophore. Some compounds were found to be significantly safer than N-9 towards Lactobacillus acidophilus in vitro at their respective spermicidal MECs. In the cytotoxicity assay, the IC50 of these compounds towards the HeLa cell line was of the same order as N-9 (0.9–0.1 mmol/l); however, in contrast, the compounds exhibited only a moderate effect on sperm membrane integrity. CONCLUSIONS: This study indicates that 5- and 3-substituted isoxazolecarbaldehydes are ‘first generation’ multifunctional, spermicidal molecules that hold promise for development as topical contraceptives with useful associated activities that can add considerably to their effectiveness, safety and prophylaxis.

Key words: acrosin inhibition/anti-fungal/isoxazolecarbaldehydes/spermicide/topical microbicide


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
HIV/AIDS makes dual protection a must for contraceptives, which is only possible with topical microbicidal spermicides (World Health Organization, 2002Go). Oral contraceptives, intrauterine devices (IUDs), sterilization, injectables, implants, etc. do not offer any protection against sexually transmitted diseases (STDs) and AIDS. Condoms do provide dual protection if used correctly and consistently, but they are not women controlled. Thus, safe, effective, acceptable and self-administered topical preparations with both microbicidal and spermicidal activity are likely to have a major positive impact on reproductive health, especially in areas with a high prevalence of STDs including HIV (World Health Organization, 2002Go). Nonoxynol-9 (N-9), a non-ionic detergent with demonstrable in vitro toxicity to a number of bacteria and viruses including HIV-1 (Grimes and Cates, 1990Go), is widely used as an over-the-counter spermicide. However, studies have shown that frequent use of N-9 may cause vaginal irritation and ulceration (Rekart, 1992Go; Stafford et al., 1998Go) that may actually increase the risk of HIV-1 transmission (Stephenson, 2000Go). N-9 has been shown to cause a interleukin-1-induced pro-inflammatory response, involving NF-{kappa}B activation in cervicovaginal epithelium leading to recruitment of HIV-1 host cells and increased HIV-1 replication in infected cells (Fichorova et al., 2001Go). In addition, poor contraceptive efficacy of N-9 and its total failure as a microbicide has suddenly augmented the need for a new, better spermicidal molecule that is lacking detergent-type membrane toxicity and has clinical advantage over currently available vaginal preparations (D'Cruz and Uckun, 1999Go). Such spermicides will form an inevitable ingredient in all future microbicidal preparations.

Synthesis and evaluation of novel, non-detergent structures at this institute has led to the discovery of several prototypes with potent spermicidal activity. These include acrylophenones (Kumaria et al., 2002Go; Maikhuri et al., 2003Go), dithiocarbamates (Tripathi et al., 1996Go), quinines (Maikhuri et al., 2003Go), anysylidines, thymols and isoxazoles/isoxazolines (Srivastava et al., 1999Go). In isoxazoline and isoxazole derivatives reported by us previously, we have found a potent spermicidal response including anti-HIV activity in a few derivatives (Srivastava et al., 1999Go). In our continued efforts to discover spermicidal efficacy in new isoxazole derivatives, we undertook the evaluation of 5-, 4- and 3-substituted isoxazolecarbaldehydes (Patra et al., 2001Go; Roy and Batra, 2003aGo,bGo). It was discovered that a few of the derivatives belonging to 5- and 3-isoxazolecarbaldehydes show significant spermicidal activity. A spermicidal agent also possessing an acrosin inhibitory property can considerably increase the contraceptive efficacy of the compound besides concurrently reducing the application dose. On the other hand, protection against opportunistic fungal infections such as Candida, which is the most commonly encountered fungal pathogen in the human vagina, is highly desirable in topical preparations for vaginal use. In view of these considerations, these compounds were simultaneously evaluated for acrosin inhibition and anti-fungal activity. The details of our studies with respect to the biological evaluation as a spermicide, acrosin inhibitor and fungicide, the structure–activity relationship, cytotoxicity towards a human cervical cell line and sperm plasma membrane, and safety towards normal vaginal flora for these compounds are presented herein.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Substituted isoxazolecarbaldehydes
Suitably substituted isoxazolecarbaldehydes (28 compounds) were synthesized by the chemist authors in the Medicinal Chemistry Division of the Institute as per the molecular structures shown in Figure 1. All compounds were >99.5% pure [analytical high-performance liquid chromatography (HPLC)] and were characterized by mass spectroscopy, infrared spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, and by chemical analysis (Patra et al., 2001Go; Roy and Batra, 2003aGo,bGo) All other chemicals/biochemicals were procured from Sigma-Aldrich Inc., MO.



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Figure 1. The molecular structure of 5-, 4- and 3-substituted isoxazole carbaldehydes (1a–m=5-substituted; 2a–f, h, i and k=4-substituted; 3a–c, f, h and i=3-substituted).

 
Human spermatozoa
Fresh human semen samples obtained by masturbation into a sterile vial from healthy, young, fertile donors were liquefied for 45 min at 37°C and used for in vitro spermicidal and acrosin inhibitory assays. Samples having >60 x 106/ml sperm count with >65% motility and normal morphology were used in the study. Significant acrosomal defects were very rarely encountered in semen samples, and such samples were rejected.

Spermicidal test
The test compounds were dissolved in a minimum volume of dimethylsulphoxide (DMSO) and diluted with Kreb–Ringer bicarbonate (KRB) buffer (pH 7.4) to make a 1.0% (10 mg/ml) solution. The solutions were further diluted serially with KRB buffer. A spermicidal test was performed with each dilution starting from 1.0% till the minimum effective concentration (MEC) was arrived at, following the modified method of Sander and Cramer (1941)Go. Briefly, 0.05 ml of human semen was added to 0.25 ml of spermicidal compound solution and vortexed for 10 s. A drop was immediately placed on a microscope slide, covered with a cover glass and examined under a phase contrast microscope. The results were scored positive if 100% spermatozoa became immotile in 20 s. The MEC was determined in three individual semen samples from different donors.

Acrosin inhibition
Acrosin inhibitory activity was detected in the spermicidal compound solutions by adapting the method of Kennedy et al. (1989)Go. Briefly, 0.1 ml of liquefied human semen was carefully layered over 0.5 ml of 11% Ficoll solution containing NaCl (0.12 mol/l) and HEPES buffer (0.025 mol/l), pH 7.4 in a conical centrifuge tube and centrifuged at 1000 g for 30 min. After carefully removing the seminal plasma and Ficoll solution without disturbing the sperm pellet, 0.1 ml of test compound solution (0.1 ml of buffer in control tubes) was added and mixed. Benzamidine hydrochloride was used as positive control. Finally 0.8 ml of buffer–substrate solution containing N-{alpha}-benzoyl-DL-arginine p-nitroanilide (0.1%), Triton X-100 (0.01%), HEPES buffer (0.055 mol/l) and NaCl (0.055 mol/l) pH 8.0 was added, mixed thoroughly and incubated at 37°C for 3 h. At the end of the incubation period, 100 µl of benzamidine hydrochloride solution (0.5 mol/l) was added to stop the reaction and the tubes were centrifuged at 1000 g for 30 min. The optical density (OD) of the supernatant was read at 410 nm in a spectrophotometer.

Determination of type of inhibition
For determination of the type of inhibition, acrosin was partly purified from human sperm by following the method of Anderson et al. (1985)Go. Using different concentrations of substrate and compound, a Dixon plot (Figure 2) and Lineweaver–Burk plot (Figure 3) were drawn to determine the type of inhibition.



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Figure 2. Inhibition kinetics (Dixon plot) of human acrosin at different concentrations of compound 1m using substrate concentrations of 0.5 and 1.0 mg/ml. (Ki=2.28 x 10–3 mol/l)

 


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Figure 3. Inhibition kinetics (Lineweaver–Burk plot) of human acrosin at 0 and 0.5 mmol/l concentrations of compound 1m showing non-competitive acrosin inhibition (curves intersecting at the abscissa).

 
Effect on spontaneous and ionophore-induced acrosome reaction (AR)
AR was induced in motile human spermatozoa by the calcium ionophore A23187 (Sigma) by following the method of Liu and Baker (1998)Go. Briefly, motile sperm obtained by swim-up in Biggers–Whiten–Whittingham (BWW) containing 3 mg/ml bovine serum albumin (BSA) (Lee et al., 1987Go) were adjusted to a concentration of 10 x 106/ml. Sperm samples were treated separately with compounds 1d, 1m, 3c and 3f (experimental tubes) at the spermicidal MEC (Table I) and BWW (control and positive control tubes) for 30 min. After centrifugation, sperm samples in control tubes were treated with 0.1% DMSO in BWW and those in positive control and experimental tubes were treated with 10 µmol/l Ca2+ ionophore. All tubes were incubated for 1 h at 37°C in a 5% CO2/95% air atmosphere. The AR was assessed by staining with bismark brown and rose bengal (De Jonge et al., 1989Go). A total of 200 sperm were scored independently by two investigators at 1000 x for the number of acrosome-intact (pink acrosome) or acrosome-reacted (colourless acrosome) sperm. The experiment was conducted in three semen samples from different donors.


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Table I. Spermicidal potential of isoxazolecarbaldehydes in descending order of activity

 
Cytotoxicity towards a human cervical (HeLa) cell line by MTT assay
The MTT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assay for evaluation of cytotoxicity of isoxazolecarbaldehydes against the human cervical (HeLa) cell line (National Center for Cell Science, Pune, India) was adopted. Cells seeded at a density of 2.5 x 105 per well in 96-well plates were incubated in culture medium [Dulbecco's modified Eagle's medium (DMEM)] for 24 h at 37°C in an atmosphere of 5% CO2/95% air. After 24 h, culture medium was replaced with fresh medium containing 2-fold dilutions of the test compounds to yield dilutions of 1000–7.5 µmol/l. Positive control wells contained an equal concentration of N-9, whereas control wells contained 0.25% DMSO. After an incubation of 3 h, 10 µl of MTT solution (5 mg/ml) was added to each well. The formazan crystals formed inside the viable cells were solubilized in DMSO and the OD was read at 540 nm in a microplate reader (Microquant, Bio-Tek, USA).

Effect on sperm plasma membrane
Supravital staining with fluorescent dye (propidium iodide) and the hypo-osmotic swelling test (HOST) were used to assess the effect of new, non-detergent spermicides on sperm membrane permeability. A 0.2 ml aliquot of liquefied semen was treated with 1.0 ml of spermicide solution at the MEC (1.0 ml of buffer in control tubes) and incubated for 1 min at 37°C. The spermatozoa were pelleted by centrifugation (800 g for 5 min) and 0.5 ml of 0.1% propidium iodide solution [in phosphate-buffered saline (PBS)] was added to the pellet and mixed gently. The mixture was incubated for 15 min at 37°C. A wet mount for each compound was observed under the phase contrast microscope in normal light from a halogen lamp and the number of sperm visible in the field was recorded. The same field was again visualized under blue light from a mercury lamp using a B2A (Nikon) filter and the number of fluorescent (red) sperm heads was recorded. The same was repeated for other fields of view. The number of stained and unstained spermatozoa was recorded for each compound in three different semen samples.

The HOST of Jeyendran et al. (1992)Go was used to determine the effect on the physiological integrity of the sperm membrane. Human spermatozoa treated with spermicide solution (as in the supravital staining experiment) were pelleted, treated with hypo-osmotic solution and mixed gently. The suspension was incubated for 30 min at 37°C. A wet mount was prepared for each compound solution and observed under a phase contrast microscope, and spermatozoa with and without tail curling were counted and recorded in three different semen samples.

Effect on Lactobacillus acidophilus in vitro
The effect of compounds exhibiting potent spermicidal activity on Lactobacillus acidophilus was determined by following the method published earlier from this laboratory (Ojha et al., 2003Go). Briefly, Rogosa SL agar plates (7.5%; containing 0.132% acetic acid), prepared with (experimental) or without (control) the addition of spermicidal agents, were inoculated with L.acidophilus (~70 spores/10 cm2) and incubated at 37°C in 5%CO2 and 95% air for 72 h. The number and size of colonies were recorded at the end of the experiment. The average colony size (% of control) was multiplied by the colony number and divided by 100 to arrive at the data presented. The average colony size of control was taken as 100%. N-9 was used as reference control.

Anti-fungal activity
The species of fungi used in the present study were Candida albicans, Aspergillus fumigatus (patient isolates) and Candida parapsilosis (ATCC 22019). All the strains were maintained on Sabouraud dextrose agar. The minimum inhibitory concentration (MIC) of each compound was determined against the test fungi by using broth micro-dilution techniques as per guidelines M-27A of the National Committee for Clinical Laboratory Standards (1997)Go. MICs of a standard anti-fungal (fluconazole) and the new compounds were measured in a 96-well tissue culture plate (Cellstar Greiner Bio One, Germany) using RPMI 1640 medium buffered with MOPS. The starting inocula of test cultures were maintained at 1.0–5.0 x 103 c.f.u./ml. Microtitre plates were incubated at 35°C in a moist, dark chamber, and MICs were recorded spectrophotometrically (Softmax pro® 4.3, Versamax microplate reader, Molecular Devices) after 48 h for Candida spp. and 72 h for A.fumigatus.

Statistical analysis
The results were analysed by Student's t-test, and P-values <0.05 were considered as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Structure–activity relationship
The results of the detailed evaluation for spermicidal efficacy of these compounds (Table I) indicate that several derivatives elicit better or equipotent activity compared with N-9. Within this series of compounds, 5- and 3-isoxazolecarbaldehydes (structures 1 and 3) show a better biological response compared with the corresponding substituted 4-isoxazolecarbaldehydes (structure 2) (Table I). Even though the compounds devoid of substitution on the phenyl ring (1a and 3a) have significant spermicidal activity, still better activity was observed in compounds having a dichloro substitution on the phenyl ring (1l and 3f). Further analysis of the results shows that compounds belonging to series 3 having a flouro and bromo group (3i and 3h) on the phenyl ring were less potent than their chloro- substituted phenyl analogues (3f and 3c). In contrast, compounds of series 1 incorporating a fluoro and bromo on the phenyl ring (1h and 1i) were equipotent to their chloro analogues (1c and 1d). In addition, the compound of series 1 bearing the 4-nitro substitution (1l) was found to be more potent than its corresponding 3-nitro derivative (1j) while phenyl rings having hydrophobic groups such as methyl (1b) or benzyloxy (1k) were not desirable.

In the evaluation of these compounds for their acrosin inhibitory activity, it was apparent that compounds belonging to series 1 and 3 are active compared with the compounds of series 2. Here, the compounds bearing an unsubstituted phenyl ring were less active than the compounds having halogen substitutions. The halogen groups positioned at the meta- or ortho- position in the phenyl ring (1c–d, m and 3c–d, f–g) were the preferred substitutions for the bioactivity (Table II). Interestingly, compound 1m (bearing a para-nitro group on the phenyl ring) and 3f, which show significant spermicidal activity, were also found to possess substantial acrosin inhibitory activity.


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Table II. Acrosin inhibitory property of isoxazolecarbaldehydes in descending order of activity

 
In the evaluation for fungicidal activity in this series of compounds, it was observed that none of the compounds show a potent response compared with the standard anti-fungal drug fluconazole (Table III). Nevertheless, a few derivatives showing significant spermicidal activity (3f, 3c and 1l) also show reasonable fungicidal activity against C.albicans as compared with N-9.


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Table III. The anti-fungal activity profile of isoxazolecarbaldehydes (µg/ml)

 
The results of the safety towards normal vaginal flora (Lactobacillus) and the HeLa cell line of all compounds showing potent spermicidal and acrosin inhibitory activity are summarized in Figure 4 and Table IV, respectively. It was observed that the most active compounds 1l, 1m, 3c and 3f do not inhibit Lactobacillus growth and show a better profile as compared with N-9. In the cytotoxicity assay in vitro using the HeLa cell line, the new compounds exhibited almost the same order of safety as N-9 (Table IV). However, the results of HOST and the supravital staining test with propidium iodide indicate that isoxazolecarbaldehydes chiefly affected the physiological integrity of sperm plasma membrane (31.7–82.4% more HOST-negative above control) with limited damage to membrane structure (12.5–39.3% more propidium iodide-stained above control). Conversely, N-9 completely disrupted the structural integrity of the sperm plasma membrane, resulting in 100% HOST-negative and propidium iodide-stained cells (Figure 5).



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Figure 4. Inhibition of Lactobacillus acidophilus growth in vitro by isoxazolecarbaldehydes. Significant difference from control is indicated as {144738fx1}P<0.05; {144738fx2}P<0.01; blank histograms denote no colonies detected in 72 h.

 

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Table IV. Cytotoxicity of isoxazolecarbaldehydes towards HeLa cells in vitro (MTT assay) in order of increasing toxicity

 


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Figure 5. Effect of isoxazolecarbaldehydes on sperm plasma membrane integrity in vitro.

 
The IC50 for the acrosin inhibitory property of isoxazolecarbaldehydes is summarized in Table II. Eight compounds (1c, 1d, 1e, 1g, 1m, 3c, 3h and 3f) exhibited potent acrosin inhibitory activity that was higher than the positive control compound benzamidine hydrochloride (a well known trypsin inhibitor). Double reciprocal (Lineweaver–Burk) and Dixon plot analysis of 1m (representative compound showing significant spermicidal and acrosin inhibitory activities) has indicated that the two curves tend to meet at the abscissa as in a typical non-competitive type of acrosin inhibition (Figures 2 and 3). The Vmax is altered in the presence of the compound while the KM (Michaelis constant) remains the same (Figure 3). The Ki for 1m is ~2.28 x 10–3 mol/l (Figure 2). The most active acrosin inhibitory compounds (1d, 1m, 3c and 3f) also significantly inhibited the ionophore-induced AR in human sperm in vitro (Figure 6). An apparently lower AR than control (untreated) sperm in the case of compounds 1d and 3f may indicate suppression of spontaneous AR as well.



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Figure 6. Effect of isoxazolecarbaldehydes on spontaneous and ionophore-induced acrosome reaction in human sperm in vitro (IP = ionophore A23187). Significant difference from control + IP is denoted as {144738fx1}P<0.05; {144738fx2}P<0.01.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
STDs including AIDS pose a serious threat in those heterosexual contacts where the major concern is usually an unwanted pregnancy. Thus an effective and consistent use of a microbicide during every sexual act requires it to have an in-built contraceptive activity as well. With the failure of N-9 as a microbicidal spermicide due to its detergent (surfactant) type of cytotoxicity, there is an urgent need for a new effective, non-detergent spermicide that can replace N-9 (D'Cruz and Uckun, 1999Go). Besides ‘killing’ or ‘immobilizing’ the sperm, topical contraceptives can also act by inhibiting acrosomal enzymes essential for the penetration of spermatozoa into the oocyte such as acrosin and hyaluronidase. Studies carried out in the past established the contraceptive activity of such enzyme inhibitors in animals (Joyce and Zaneveld, 1985Go; Kaminski et al., 1985Go), but these molecules could not reach clinical evaluation stages. A recent study has shown the contraceptive potential of a promising non-spermicidal vaginal contraceptive agent, and one of the mechanisms proposed for its contraceptive activity is the inhibition of acrosomal (acrosin and hyaluronidase) enzymes (Zaneveld et al., 2002Go). While non-spermicidal, acrosomal enzyme inhibitors may have an edge over spermicides in their safety towards lactobacilli and vaginal/cervical cells, spermicidal agents may ensure better protection against unwanted pregnancy and STDs. It would be desirable to have a combination of the two activities to ensure both safety and contraceptive efficacy. Several 5- and 3-substituted isoxazolecarbaldehydes have shown the potential of both spermicidal and acrosin inhibitory activities in the same structure. Dixon and double-reciprocal plots of a representative molecule of the series indicate not only the non-competitive type of enzyme inhibition by isoxazolecarbaldehydes but also a very substantial acrosin inhibitory activity. The two activities put together are likely to augment the contraceptive potential of these molecules, thus increasing the contraceptive protection and decreasing the application dose. Such diverse activities are rarely encountered simultaneously in chemical structures and have only been reported earlier in substituted guanidinobenzoates (Bourinbaiar and Lee-Huang, 1995Go). While some studies have indicated that acrosin may not be required for fertilization (Baba et al., 1994Go; Adham et al., 1997Go), still it has been very well established that serine protease/acrosin inhibitors prevent sperm penetration into the zona (Liu and Baker, 1993Go; Takano et al., 1993Go), indicating the importance of sperm proteases in the fertilization process. Besides acrosin, mammalian sperm may also contain other trypsin-like proteases (Arboleda and Gerton, 1988Go; Akama et al., 1994Go; Tanii et al., 2001Go). Isoxazolecarbaldehydes not only inhibited human acrosin but also exhibited potent inhibitory activity towards pancreatic trypsin (Sigma; data not included) indicating strong serine protease inhibitory activity of the compounds. It is well known that sperm proteases are necessary for AR that is blocked by trypsin inhibitors (De Jonge et al., 1989Go; Liu and Baker, 1993Go; Llanos et al., 1993Go). In the present study, the most potent acrosin inhibitors effectively blocked ionophore-induced AR in human sperm. Ionophore-induced AR is known to be significantly diminished or absent in subfertile sperm (Cummins et al., 1991Go) and in sperm treated with acrosin inhibitors (Liu and Baker, 1993Go).

Candida albicans is the most commonly encountered fungal pathogen of the human vagina (Naglik et al., 2003Go). However, several other species of Candida have also been isolated from human vagina in patients suffering from candidal vaginitis (DeBernardis et al., 1989Go; Agatensi et al., 1991Go). Besides Candida, other fungal infections can also adversely affect female reproductive health. Aspergillus fumigatus spores introduced through the vagina cause abortion in pregnant animals (Venev, 1976Go). In view of this consideration, all compounds were also evaluated for their fungicidal activity. Though none of the compounds showed a very potent fungicidal activity, an appreciable fungicidal response was shown by a few active compounds in comparison with N-9, which may add considerably to the microbicidal value of these spermicides, especially in view of the fact that such compound would be used in vaginal preparations at concentrations well exceeding their MECs in vitro. Conversely, mild spermicidal effect has been reported in known fungicidal compounds (Foote, 2002Go).

Lactobacillus acidophilus is present in large numbers in the vagina of most normal women and is believed to play a protective role in the urogenital tract (Redondo-Lopez et al., 1990Go), guarding against infection by pathogens by a combination of competitive exclusion and inhibitor production (Reid et al., 1990Go). Lactobacilli sterically hinder the addition of pathogens to the urogenital epithelia and produce a number of antimicrobial substances such as lactic acid, which maintains a low vaginal pH, and H2O2 that is detrimental to many urogenital pathogens such as Escherichia coli (Hawes et al., 1996Go), G.vaginalis (Klebanoff et al., 1991Go) and HIV-1 (Klebanoff and Coombs 1991Go). The acidic pH itself acts as a natural defence against STDs and AIDS (Kempf et al., 1991Go; Mahmoud et al., 1995Go; Garg et al., 2001Go). However, during menstruation, the vagina becomes less acidic due to the presence of menstrual fluid and a diminished population of lactobacilli (Wagner and Ottesen, 1982Go), resulting in increased susceptibility to colonization by pathogenic microorganisms (Brzezinski et al., 2004Go). It is generally feared that use of vaginal products may further destroy the healthy flora. Of particular concern is N-9, the most widely used spermicide worldwide. In vitro studies have shown that N-9 is detrimental to Lactobacillus species, especially those producing H2O2 (Klebanoff, 1992Go; McGroarty et al., 1992Go; Tomecjec et al., 1992Go; Reid et al., 1996Go). The present study has identified several new spermicidal compounds that are significantly less toxic to L.acidophilus as compared with N-9. Such microbicidal preparations containing such compounds can safely be used even during menstruation when the requirement for protection against STDs is maximum. These compounds being non-detergent in nature would also lack the detergent type of membrane toxicity towards vaginal and cervical epithelium and hence are likely to be more vaginal eco-friendly than N-9. The non-surfactant nature of isoxazole compounds is clearly evident from the supravital staining and HOST results. The surfactant spermicide (N-9) totally damages the structural and physiological integrity of the sperm plasma membrane, whereas the new spermicides have a moderate effect on membrane structure and comparatively larger effect on membrane physiology. The effect on membrane physiology may be due to disturbances in ion and water channels of human spermatozoa by isoxazolecarbaldehydes.

The cytotoxicity assay using the HeLa cell line in vitro may indicate that the new spermicides have their IC50 in almost the same range as N-9. However, some of the new spermicides (1l, 1m and 1a) are significantly more potent (up to four times) than N-9 in their spermicidal property (Table I). Thus these compounds will be required to be used at a much lower concentration than N-9 in vaginal preparations and therefore will prove to be comparatively much safer. Also the associated acrosin inhibitory property of 1m (Table II) is likely to reduce its effective anti-fertility dose further. Additionally, the lack of a detergent type of membrane toxicity would not increase the susceptibility of vaginal/cervical epithelium to STDs and HIV in repeated use, as seen in the case of N-9 (Richardson et al., 2001Go).

Thus isoxazolecarbaldehydes present a new lead structure for potent spermicidal agents with complementary properties that add to their utility as safe, effective and prophylactic topical contraceptives. Multiple substitutions on the phenyl ring of this class of compounds may yield a molecule with ideal bioactivity that can replace surfactant spermicides such as N-9 in vaginal contraceptives, making them more safe and acceptable. Studies are underway in this direction.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
R.K.J., M.K.R., A.P., A.K.R. and V.S. gratefully acknowledge the financial support from the CSIR and DST, New Delhi in the form of research fellowships. This study was supported by grants from the Ministry of Health and Family Welfare and Department of Science and Technology, Government of India.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Adham IM, Nayernia K and Engel W (1997) Spermatozoa lacking acrosin protein show delayed fertilization. Mol Reprod Dev 46, 372–376.

Agatensi L, Franchi F, Mondello F, Bevilacqua RL, Ceddia T, DeBernardis F and Cassone A (1991) Vaginopathic proteolytic Candida species in outpatients attending a gynaecologyclinic. J Clin Pathol 44, 826–830.[Abstract]

Akama K, Terao K, Tanaka Y, Noguchi A, Yonizawa N, Nakano M and Tobita T (1994) Purification and characterization of a novel acrosin like enzyme from boar cauda epididymal sperm. J Biochem 116, 464–470.[Abstract]

Anderson RA, Oswald C, Leto S and Zaneveld LJD (1985) Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose induced acrosin inhibition. Arch Biochem Biophys 241, 509–520.[CrossRef][ISI][Medline]

Arboleda CE and Gerton GL (1988) Proacrosin/acrosin during guinea pig spermatogenesis. Dev Biol 125, 217–225.[CrossRef][ISI][Medline]

Baba T, Azuma S, Kashiwabara S and Toyoda Y (1994) Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization. J Biol Chem 269, 31845–31849.[Abstract/Free Full Text]

Bourinbaiar AS and Lee-Huang S (1995) Acrosin inhibitor 4'-acetamidophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive with anti-HIV activity. Contraception 51, 319–322.[CrossRef][ISI][Medline]

Brzezinski A, Stern T, Arbel R, Rahav G and Benita S (2004) Efficacy of a novel pH-buffering tampon in preserving the acidic vaginal pH during menstruation. Int J Gynecol Obstet 85, 298–300.[CrossRef][ISI][Medline]

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Submitted on November 4, 2004; resubmitted on March 22, 2005; accepted on March 31, 2005.





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