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
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Abstract |
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Key words: acrosin inhibition/anti-fungal/isoxazolecarbaldehydes/spermicide/topical microbicide
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Introduction |
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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., 2002; Maikhuri et al., 2003
), dithiocarbamates (Tripathi et al., 1996
), quinines (Maikhuri et al., 2003
), anysylidines, thymols and isoxazoles/isoxazolines (Srivastava et al., 1999
). 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., 1999
). 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., 2001
; Roy and Batra, 2003a
,b
). 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 structureactivity relationship, cytotoxicity towards a human cervical cell line and sperm plasma membrane, and safety towards normal vaginal flora for these compounds are presented herein.
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Materials and methods |
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Spermicidal test
The test compounds were dissolved in a minimum volume of dimethylsulphoxide (DMSO) and diluted with KrebRinger 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). 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). 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 buffersubstrate solution containing N-
-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). Using different concentrations of substrate and compound, a Dixon plot (Figure 2) and LineweaverBurk plot (Figure 3) were drawn to determine the type of inhibition.
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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) 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., 2003). 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). 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 1.05.0 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.
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Results |
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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 (1cd, m and 3cd, fg) 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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Discussion |
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Candida albicans is the most commonly encountered fungal pathogen of the human vagina (Naglik et al., 2003). However, several other species of Candida have also been isolated from human vagina in patients suffering from candidal vaginitis (DeBernardis et al., 1989
; Agatensi et al., 1991
). 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, 1976
). 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, 2002
).
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., 1990), guarding against infection by pathogens by a combination of competitive exclusion and inhibitor production (Reid et al., 1990
). 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., 1996
), G.vaginalis (Klebanoff et al., 1991
) and HIV-1 (Klebanoff and Coombs 1991
). The acidic pH itself acts as a natural defence against STDs and AIDS (Kempf et al., 1991
; Mahmoud et al., 1995
; Garg et al., 2001
). However, during menstruation, the vagina becomes less acidic due to the presence of menstrual fluid and a diminished population of lactobacilli (Wagner and Ottesen, 1982
), resulting in increased susceptibility to colonization by pathogenic microorganisms (Brzezinski et al., 2004
). 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, 1992
; McGroarty et al., 1992
; Tomecjec et al., 1992
; Reid et al., 1996
). 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., 2001).
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.
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Acknowledgements |
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Submitted on November 4, 2004; resubmitted on March 22, 2005; accepted on March 31, 2005.
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