Evaluation of the antifertility effect of magainin-A in rabbits: in vitro and in vivo studies

Evaluation of the antifertility effect of magainin-A in rabbits: in vitro and in vivo studies

FERTILITY AND STERILITY威 VOL. 73, NO. 2, FEBRUARY 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. P...

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FERTILITY AND STERILITY威 VOL. 73, NO. 2, FEBRUARY 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

REPRODUCTIVE BIOLOGY

Evaluation of the antifertility effect of magainin-A in rabbits: in vitro and in vivo studies Venkata Rami K. Reddy, Ph.D.,* and Dhananjay D. Manjramkar, M.V.Sc.† Institute for Research in Reproduction, Mumbai, Parel, India

Objective: To evaluate the safety and contraceptive potential of magainin-A in rabbits. Design: Controlled laboratory study. Setting: Department of Immunology, Institute for Research in Reproduction, Mumbai, Parel, India. Animal(s): Forty-eight female New Zealand white rabbits. Intervention(s): The effect of magainin-A on sperm motility (in vitro and in vivo studies) and on vaginal epithelium (histologic study) was assessed along with serum and liver biochemical profiles. Main Outcome Measure(s): Suitability of magainin-A for contraceptive use. Result(s): Magainin-A arrested sperm motility, and 1 mg of magainin-A administered intravaginally blocked conception. No histopathologic abnormalities in the vaginal tissue or any changes in serum biochemical profiles were observed. Conclusion(s): Magainin-A may be used as an effective and safe intravaginal contraceptive compound that also has antibacterial properties. (Fertil Steril威 2000;73:353– 8. ©2000 by American Society for Reproductive Medicine.) Key Words: Magainin-A, rabbits, spermatozoa, fertility, vaginal contraception

Received February 17, 1999; revised and accepted September 21, 1999. Presented in part at the VIth International Congress of Andrology, May 25, 1997, Salzburg, Austria. Reprint requests: Venkata Rami K. Reddy, Ph.D., Immunology Division, Institute for Research in Reproduction, J.M. Street, Parel, Mumbai 400 012, India (FAX: 91-22-4139412; E-mail: [email protected] .nic.in). * Division of Immunology. † Department of Animal Maintenance. 0015-0282/00/$20.00 PII S0015-0282(99)00499-9

The pandemics of unwanted pregnancies and sexually transmitted diseases (STDs), lack of widespread use of condoms, and absence of successful vaccines make clear the need for a nonirritating, broad-spectrum vaginal microbicide (1–3). The spermicide nonoxynol-9 is commonly used in marketed vaginal contraceptive preparations (4). Numerous laboratory studies have shown that nonoxynol-9 can inactivate most STD pathogens (5). In clinical studies, however, the effect of nonoxynol-9 in preventing STDs and the transmission of human immunodeficiency virus remains controversial (6). Frequent use of a high dose of nonoxynol-9 has been reported to cause local lesions on the vaginal epithelium and burning sensation of the genital organs (7). At present, contraceptive methods that prevent STDs are urgently needed (8). Therefore, improvement of the safety and efficacy of vaginal contraceptives and anti-STD methods has become an area of high priority. More information is needed on these female-controlled methods.

Magainin-A is a potent broad-spectrum agent that has proved effective against a wide range of gram-negative, gram-positive, and antibiotic-resistant microorganisms (9), besides having antifertility activity in rats (8). The magainins are a class of simple proteins, having 23 amino acids, initially isolated from the skin of the African clawed frog, Xenopus laevis. Magainin analogues that demonstrate increased antimicrobial activity also have been synthesized (10). It would be interesting to develop a vaginal contraceptive with these compounds, which have many biologic properties. The objective of this study was to assess the safety and efficacy of magainin-A in rabbits as a vaginal contraceptive agent for future use in humans.

MATERIALS AND METHODS Semen Collection An artificial vagina for the collection of rabbit semen was constructed and used as described previously (11). Briefly, a doe was introduced to each buck of known fertility at the 353

time of semen collection. The prewarmed (45 ⫾ 2°C) artificial vagina was placed close to the penis, followed by penile insertion (thrust) into the artificial vagina and ejaculation.

Effect of Magainin-A on Fertility

Evaluation of Spermicidal Activity of Magainin-A

In the first set of experiments, a minimum of three does were used in each group. After isolation of ⱖ3 weeks to ensure that the does were not pseudopregnant, the animals were allowed to mate and were induced to ovulate 6 – 8 hours before the expected time of mating. Ovulation was induced by administration of hCG (200 IU).

The in vitro spermicidal experiments performed were essentially the same as described earlier (8). Briefly, known volumes of semen samples or spermatozoa free from seminal plasma (collected by the swim-up method) were mixed with various concentrations of magainin-A (0, 50, 100, 200, 400, 800, and 1,600 ␮g) (M7152; Sigma, St. Louis, MO) separately and then incubated at 37 ⫾ 1°C for different intervals (20 – 480 seconds). After mixing for 10 seconds, five fields under low-power magnification (⫻100) followed by five fields under high power (⫻400) were rapidly examined at the 20-second time point.

Magainin-A (1 mg/mL of sterile saline) was applied vaginally by means of a 3-mL disposable plastic syringe fitted with a 12.5-cm piece of polyethylene tubing with a 4-mm bore. Approximately 6 – 8 cm of the tubing was inserted into the vagina, and the formulation was pushed through the tubing with the syringe. The does were held in a supine position during the application of magainin-A. The tubing was removed, and the rabbits were kept in a supine position for an additional 5 minutes to allow distribution of the mixture in the vagina. Control animals were maintained simultaneously and received only the vehicle.

Sperm Revival Test

The does were subsequently mated with bucks of proved fertility on day 0 (group 1), day 1 (group 2), day 2 (group 3), and day 3 (group 4) after treatment. The vaginal lavage was aspirated as early as possible from the mated animals, and the effect of magainin-A on sperm motility was observed under the microscope. Pregnancy rates were calculated as the proportion of does that became pregnant and delivered. Newborn rabbits were maintained through lactation to weaning and were monitored accordingly for general health and survival.

In Vitro Studies

For all test samples showing 100% arrest of sperm motility at 20 seconds in the spermicidal assay, we added 500 ␮L of buffered glucose, mixed the samples thoroughly, and incubated them in a water bath at 37 ⫾ 1°C. At the end of 60 minutes, the mixture was observed again for any motile sperm. The compound was recorded as effective if the test indicated the absence of motile sperm. The sperm viability was checked with the trypan blue dye exclusion method. Effect of Magainin-A on Erythrocyte Hemolysis Erythrocyte hemolytic activity was measured in the presence of various concentrations of magainin-A (12). Briefly, 2.5 mL of diluted rabbit erythrocyte suspension (10% v/v) in isotonic phosphate-buffered saline (PBS) was added to 75 ⫻ 12-mm borosilicate test tubes containing a predetermined amount of dried peptide in duplicate. After gentle mixing and incubation for 10 minutes at 37°C, the tubes were centrifuged at 3,000 ⫻ g for 10 minutes. The supernatant was separated from cells and debris and diluted (if necessary), and the OD350 was measured by a spectrophotometer (Uv-160A; Schimadzu Corp., Kyoto, Japan). We observed 100% hemolysis with 0.1% Triton X-100.

In Vivo Studies Animals and Methods Mature female New Zealand white rabbits (n ⫽ 48; 8 –10 months old; 2.4 ⫾ 0.2 kg) exhibiting both vaginal and psychic signs of estrous were used in this study. The rabbits were housed in individual metal cages and fed high-fiber rabbit formula and water ad libitum. A constant room temperature of 23 ⫾ 1°C, 60% ⫾ 10% relative humidity, and a 12-hour dark-light cycle was maintained throughout the study. 354

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Effect of Magainin-A on Vaginal Epithelium (Histologic Study) In the second set of experiments, structural abnormalities in the vaginal epithelium were studied after repeated applications of magainin-A. A contraceptive dose of magainin-A was applied to a group of three animals for 5 consecutive days as described earlier, and the does were killed on day 6 after the fifth and final treatment. In controls, instead of magainin-A, 1 mL of vehicle was applied. At autopsy, the vagina was slit open ventrally between the urethral orifice and the fornices, and representative samples of the proximal portion (near the urethral orifice), middle portion, and distal portion were taken and fixed in Bouin’s solution for histologic evaluations. After fixation, the specimens were embedded in paraffin, sectioned at 5 ␮m, and stained routinely with hematoxylin and eosin. A piece of uterine horn and/or ovary was processed similarly to exclude pseudopregnancy. The vaginal sections were evaluated semiquantitatively. Four basic criteria were considered: epithelial ulceration, leukocyte infiltration, edema, and vascular congestion. The magnitude of vaginal changes was rated according to the published procedure (13). A score of 0 –11 (none to intense) was given to each of them and compared with those of controls. A total score between 0 and 8 was considered Vol. 73, No. 2, February 2000

acceptable, one of ⱖ11 was unacceptable, and an intermediate score of 9 or 10 was marginal (either acceptable or unacceptable). Quantification of Circulatory Levels of Magainin-A

FIGURE 1 Spermicidal action of magainin-A on rabbit spermatozoa in vitro. Dashed lines ⫽ rabbit sperm; solid lines ⫽ rabbit semen.

In the third set of experiments after intravaginal application of a contraceptive dose of magainin-A, 3– 4 mL of blood was collected six times within the first 24 hours (0, 30, 60, 180, 360, and 1,440 minutes). Serum was separated by centrifugation and kept at ⫺70°C until use. Magainin-A peptide was coupled through the NH2-terminal cysteine residue to keyhole limpet hemocyanin with use of the water-soluble heterobifunctional cross-linking reagent in maleimidobenzoyl sulfosuccinimide ester, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL). Rabbit antisera to the peptide– keyhole limpet hemocyanin conjugate were raised in New Zealand white rabbits. After confirming the sensitivity and specificity, we used the antibodies in an ELISA to measure the rate of absorption, retention time, and withdrawal of magainin-A from the circulation. The level of magainin-A in the serum was measured by ELISA. Briefly, a known concentration of magainin-A in sodium carbonate buffer (0.1 M, pH 9.6) was used to coat a polystyrene microtiter plate. After overnight incubation at 4°C, the plate was washed (three times) with PBS-T20. Different concentrations of magainin-A were added to the fixed concentration of anti–magainin-A antibodies (1: 25,000) in separate test tubes. After incubation for 1 hour at 37°C, a known amount of antigen-antibody mixture was added to each well of the microtiter plate. Subsequent incubations were performed sequentially for 30 minutes at room temperature, followed by washings with PBS between each step. After the secondary antibody (goat anti-rabbit horseradish peroxidase ) incubation, bound peroxidase was visualized with orthophenylene diamine in 0.05 M citrate phosphate buffer, pH 5.0, containing 0.05% H2O2, incubated in the dark at room temperature for exactly 20 minutes. The reaction was stopped with 2 N H2SO4, and the absorbance of the colored substrate produced was measured at 492 nm by an ELISA reader (ELX-800; Bio-tek Instruments, Inc., Winooski, VT). Effect of Magainin-A on Changes in Blood and Liver Indices (Biochemical Study) At the time of autopsy, a required quantity of liver tissue and 8 –10 mL of blood were collected from the rabbits used in the second experiment. The possible toxic systemic effects of the compound and its routine administration were assessed by checking body weight and blood analysis (total red blood cells [RBCs] hemoglobin levels). The RBCs were counted by a Newbauer hemocytometer, and hemoglobin levels were determined by a hemometer. Biochemical profiles such as total proteins (14), enzyme activity levels, FERTILITY & STERILITY威

Reddy. Magainin. Fertil Steril 2000.

alanine aminotransferase, and aspartate aminotransferase of the serum and liver were estimated (15).

Statistical Analysis Statistical analysis of the variance between control and experimental values was done by Student’s t-test (16).

RESULTS In Vitro Efficacy of Magainin-A on Sperm Motility The effect of magainin-A on sperm motility was found to be dose- and time-dependent (Fig. 1). Complete sperm (40 – 60 ⫻ 106/mL of saline) immobilization was observed with 150 –200 ␮g of magainin-A. The potency of magainin-A was found to be significantly decreased in the presence of seminal plasma compared with its activity in the absence of seminal plasma. The spermatozoa that were immobilized by magainin-A did not revive after incubation at 37 ⫾ 1°C in buffered glucose for 60 minutes.

Contraceptive Potential of Magainin-A in Rabbits Magainin-A inhibited motility of sperm at 100% in vivo at a concentration approximately 4 –5 times higher than the minimum inhibitory concentration in vitro. From preliminary studies, it was confirmed that 1 mg of magainin-A is 355

sufficient to immobilize all the sperm that are ejaculated in a single coitus, and hence this dose was selected for further fertility and toxicologic studies. A contraceptive dose of magainin-A applied intravaginally once before mating resulted in complete protection. The effect was seen until 24 hours because the does did not conceive when mated during this period. In addition to the control group, however, the does from groups 3 and 4 became pregnant when mated at 48 and 96 hours after treatment.

FIGURE 2 Circulatory levels of magainin-A in the serum after intravaginal administration of magainin-A, analyzed by ELISA. Each bar represents the mean of six determinations; error bars indicate SDs.

Magainin-A did not induce any changes in the duration of gestation (32 ⫾ 2 days, compared with 31 ⫾ 2 days in the treated group), number of pups delivered (4 – 6 pups in both groups), or weight of the pups (60 ⫾ 10 g in both groups). The growth of the pups was observed for 30 days and was found to be normal and similar in both groups.

Effect of Magainin-A on Structural Changes in the Vaginal Epithelium Light microscopic examination of pH-mediated changes of magainin-A on the vaginal epithelial cells indicated no abnormalities in the morphology of the cells when compared with controls (data not shown). Intravaginal administration of 1 mg of magainin-A for 5 consecutive days did not induce inflammatory reaction(s) in the epithelial and subendothelial connective tissue of the vagina. There was no edematous thickening of the submucosal layer or infiltration of polymorphonuclear leukocytes into the mucosa. The overall mean semiquantitative score of the microscopic analysis showed that the changes did not exceed the definition for acceptability (data not shown).

Serum Levels of Magainin-A in the Circulation of Rabbits The ELISA method was standardized to measure the magainin-A level in the blood circulation that was absorbed from the vaginal epithelium. The maximum amount of magainin-A administered was detected in the serum samples drawn 60 minutes after treatment. The retention time of magainin-A in the circulation declined rapidly thereafter and reached the control level by 24 hours (Fig. 2). These results may indicate no apparent systemic effects after magainin-A treatment in these rabbits.

Reddy. Magainin. Fertil Steril 2000.

gainin-A possesses spermicidal activity to a certain extent even at much lower doses. Earlier we reported that susceptibility of sperm to magainin-A varied with the species used (8), and the results of this study indicated that rabbit sperm are more sensitive to magainin-A than human sperm. We also noticed that seminal plasma altered the spermicidal efficacy of magainin-A by increasing the minimum concentration at which magainin-A was spermicidal. This probably is due to secretions of the accessory sex organs that protect the spermatozoa or the presence of proteases and proteolytic enzymes in the seminal plasma that modify the action of magainin-A, resulting in decreased activity, as suggested by Edelstein et al. (17).

Magainin-A did not show hemolytic activity when tested in vitro with different concentrations (10 –300 ␮g/mL heparinized blood) (Fig. 3). Total protein and the activity levels of transaminases (alanine and aspartate aminotransferase) in serum and liver did not deviate from the control values (Table 1).

The in vivo efficacy of magainin-A on fertility and reversibility showed that 1 mg of magainin-A is sufficient to arrest sperm motility by 100%. Sperm motility was inhibited whether mating occurred shortly after administration of magainin-A or was delayed by 24 hours. Tests detecting only immotile sperm in cervical fluids, however, may indicate that fertilization is unlikely but not that it is impossible.

DISCUSSION

In contrast, all of the does in groups 3 and 4 became pregnant, suggesting that the contraceptive effect of magainin-A is rapid and completely reversible. Furthermore, no change was seen in the length of gestation or the number of pups delivered (data not shown).

The present in vitro results clearly demonstrated that magainin-A is an effective spermicidal compound, and the effect was found to be dose- and time-dependent. Ma356

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FIGURE 3 Effect of magainin-A on erythrocyte hemolysis. Each bar represents the mean of three observations. Hemolysis of 100% was obtained with 5 ␮L of 0.1% Triton X-100.

viscosity appears to aid retention near a single cervix. Furthermore, sperm transport from the vagina to the fallopian tubes is slower in rabbits (3– 4 hours) than in humans (30 minutes) (18). We observed no histopathologic lesions in the vaginal epithelium after repeated treatment with magainin-A for 5 consecutive days. The levels of total proteins and the enzyme activities of transaminases, which mark the functional status of the tissue (18) under various physiologic situations, were measured after magainin-A treatment, and the values were found to be similar to those obtained from the normal does. These results suggest that magainin-A did not induce any noticeable abnormalities in the structure and function of the liver in these animals. However, studies at longer intervals with a high dose of magainin-A are mandatory before definite conclusions can be reached. For the evaluation of safety of new formulations, it is important to examine the concentrations achieved in serum after placement in the vagina and the potential systemic effects in addition to any local reactions. At present, there is no literature available on the toxicologic effects of magainin-A in an animal model. Hence, an ELISA method was standardized to measure the systemic magainin-A levels.

Reddy. Magainin. Fertil Steril 2000.

Although our antifertility data are encouraging, extrapolation of these studies to the human may be difficult for a number of reasons. Sperm sensitivity varies among species, and rabbits and women differ in their physiology of coitus, anatomy, and sperm transport. Rabbits ejaculate a smaller volume of semen, which is approximately fourfold more concentrated with respect to sperm. A gel plug is frequently ejaculated in the rabbit, which prevents backflow of semen away from each of the cervices, whereas in women, semen

Studies of the absorption of magainin-A through the vaginal epithelium into the circulation and the withdrawal time from the circulation suggested rapid absorption of magainin-A, with a detectable amount in the blood at 30 minutes. The major peak of magainin-A administered was detected in the serum sample drawn 60 minutes after treatment (Fig. 2). At 6 hours, there was still a detectable concentration; the level declined rapidly thereafter and reached a baseline level by 24 hours. This rapid absorption of magainin-A may be attributed to its direct entry into the sys-

TABLE 1 Biochemical indices of serum and liver in rabbits before and after repeated applications of magainin-A. Tissue Blood Control Before treatment After treatment Serum (mg/mL) Control Before treatment After treatment Liver (mg/g tissue) Control Before treatment After treatment

RBCs (⫻106/mm3)

Hemoglobin (g/dL)

Total proteins

Alanine aminotransferase*

Aspartate aminotransferase*

6.66 ⫾ 1.04 6.81 ⫾ 1.13 6.83 ⫾ 1.21

12.0 12.0 12.0

— — —

— — —

— — —

— — —

— — —

68.38 ⫾ 4.9 70.81 ⫾ 5.32 69.93 ⫾ 5.48

0.059 ⫾ 0.004 0.054 ⫾ 0.0030 0.054 ⫾ 0.004

0.051 ⫾ 0.03 0.053 ⫾ 0.004 0.051 ⫾ 0.004

— — —

— — —

123.76 ⫾ 11.39 117.84 ⫾ 14.32 116.63 ⫾ 9.10

0.098 ⫾ 0.006 0.088 ⫾ 0.005 0.092 ⫾ 0.006

0.73 ⫾ 0.006 0.080 ⫾ 0.005 0.076 ⫾ 0.006

Note: Each value is the mean ⫾ SD of six observations. Values are not statistically significant compared with controls. * Values are ␮mol of pyruvate formed per mg of protein per h. Reddy. Magainin. Fertil Steril 2000.

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temic circulation without passing through the liver. In general, these results may suggest that there are no apparent systemic effects after daily application of magainin-A for 5 days. It is well known that frog skin is the richest source of magainin-A (9). Magainin-A demonstrated potent antibacterial activity in a rat incised-wound model (19) and also facilitated wound closure and reduced inflammation. In the context of vaginal spermicidal activity, the property of reducing inflammation, rather than instigating it, would constitute a major advantage of magainin-A. In addition to its antibacterial properties, magainin-A also has antifungal and antiprotozoan activities. Magainin-A thus has a unique advantage that may greatly enhance its prophylactic capability against conventional STDs. We conclude that magainin-A is highly spermicidal with antimicrobial properties and is safe to use intravaginally. It appears to be a highly promising vaginal contraceptive.

4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Acknowledgments: The authors thank Dr. H. S. Juneja, Director, for giving us continued encouragement and guidance in carrying out this study, and Prasanna Chavan for typing the manuscript.

16. 17.

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