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Meloxicam inhibits rabbit ovulation☆
Contraception 63 (2001) 329 –333
Original research article
Meloxicam inhibits rabbit ovulation夞 A.S. Salhaba,*, M.N. Gharaibeha, M.S. Shomafb, B.I. Amroc a
Department of Pharmacology, University of Jordan, Amman, Jordan Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan c Faculty of Pharmacy, University of Jordan, Amman, Jordan
b
Abstract The nonsteroidal antiinflammatory, selective cyclo-oxygenase-2 (COX-2) inhibitor, meloxicam, was tested to assess its effect on rabbit ovulation. Meloxicam in different doses was administered intraperitoneally (ip) to adult female Californian rabbits at 2, 5, 8, and 24 h postcoitus with sperm-positive rabbits. Rabbits were killed on Day 10 of gestation. Meloxicam produced significant inhibition of ovulation in rabbits. This inhibition of ovulation by meloxicam was dose- and time-dependent. Ovulation in rabbits was completely inhibited by a single ip administration of meloxicam (20 mg/kg) when the drug was administered at 2 and 5 h postcoitus, whereas neither ovulation nor implantation were inhibited (pregnancy rate 75%) by the same dose administered 24 h postcoitus (approximately 14 h post ovulation). Further, ovulation was completely inhibited by 10 mg/kg of meloxicam when the drug was administered at 5 or 8 h postcoitus, but there was less inhibition of ovulation when 10 mg/kg of the drug was administered at 2 or 24 h postcoitus (pregnancy rate 25 and 80%, respectively). Corpora lutea, maternal plasma progesterone, ovary fresh weight, and maternal body weight gain were affected by meloxicam treatment. Histopathological findings observed in the ovaries of treated rabbits included microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to severe hemorrhage within the follicles which lost ova. These results show that ovulation can be inhibited in rabbits by meloxicam. Further studies are needed to assess the value of selective COX-2 inhibitors as potential nonhormonal contraceptive agents. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Meloxicam; Cyclo-oxygenase-2 inhibitor (COX-2); Ovulation Antiinflammatory agents; Indomethacin
1. Introduction Ovulation is accompanied by induction of prostaglandin (PG) synthesis as a consequence of the luteinizing hormone (LH) surge [1,2]. Cyclo-oxygenase (COX) plays a key regulatory role in PG synthesis and occurs in both the constitutive (COX-1) and the inducible (COX-2) isoforms [3,4]. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is the major isoform of the inflammatory reaction [5,6]. Further, COX-2 isoform is induced in the ovary and is necessary for the successful rupture of the follicle, probably directly mediating the generation or activation of proteolytic enzymes that are necessary for the release of ova [7–9]. Espey hypothesized in 1982 that the actual rupture of the ovarian surface is brought about by an inflammatory process [10]. COX-2 null mice show multiple failures in reproduc夞 This work was supported by grant no. 558/99 –2000 from the Deanship of Scientific Research at the University of Jordan. * Corresponding author. Tel.: ⫹9626-5355000 (Ext. 2465/2464); fax: ⫹9626-5355500. E-mail address: [email protected] (A.S. Salhab).
tive function, including ovulation, fertilization, implantation, and decidualizaition [11–13]. Meanwhile, the COX-1 mutant mice were fertile, but homozygous matings tend to yield dead pups for unknown reasons [14]. In a related area of research, indomethacin, a nonspecific COX inhibitor, inhibits ovulation in a number of animal species, such as rats, mice, rabbits, sheep, and monkeys [10,15–20]. The antifertility effect of indomethacin is likely to be mediated by COX-2 [21]. Meloxicam is a new nonsteroidal antiinflammatory (NSAID) that appears to have a greater selective inhibitory activity against the inducible COX-2 isoform, than against the constitutive isoform COX-1. Inhibition of COX-1 is associated with gastrointestinal, renal, and platelet aggregation adverse effects. Therefore, meloxicam and other COX-2 selective inhibitors are promoted for their safer profile of side effects. The objective of the present study was to investigate the effects of meloxicam, as an example of selective COX-2 inhibitor, on the fertility of female rabbits. The potential of antiovulatory activity of meloxicam was studied in regard to
0010-7824/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 1 0 - 7 8 2 4 ( 0 1 ) 0 0 2 0 7 - 4
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A.S. Salhab et al. / Contraception 63 (2001) 329 –333
Table 1 The effects of meloxicam administered before ovulation on maternal body weight and pregnancy rate Treatment (mg/kg) Control Indomethacin 20 Meloxicam 20 10 5 2.5
Maternal body weight (g)
Pregnancy (%)
12/13
92.3
Initial
Final
2297 ⫾ 333
2480 ⫾ 352
2020 ⫾ 170
2250 ⫾ 165
11.3
0/3
0.0
2403 ⫾ 323 2484 ⫾ 334 2316 ⫾ 316 2388 ⫾ 252
2479 ⫾ 373 2509 ⫾ 328 2578 ⫾ 303 2548 ⫾ 103
3.15 1.0 11.1 6.7
0/16 1/12 2/10 3/5
0.0 8.3 20 60
dose and temporal relationship. The present study is the first to investigate such effects.
2. Materials and methods 2.1. Drugs Meloxicam (1 g) in a powder form (Batch no. 8250381) was donated by Boehringer Ingelheim Pharma KG (Biberach an der Riss, Germany). Additional meloxicam (100 g; Batch no. 9700009015) was obtained from Advanced Pharmaceutical Industries Co. Ltd. (Amman, Jordan). Purity was checked by HPLC and UV spectrum against the authentic sample that was obtained from Boehringer Ingelheim. Indomethacin powder (Lot 67 H 1069) was purchased from Sigma Chemical Co. (St. Louis, MO) The different dilutions of meloxicam were freshly prepared in warm 0.15 M NaOH solution (pH 8.5) as recommended by Boehringer Ingelheim researchers (personal communications), whereas indomethacin was dissolved in 0.1 M Sorrenson’s phosphate buffer (pH 8.0) according to Espey et al. [10]. 2.2. Rabbit experiments Adult male and female Californian rabbits, purchased from a local farm, were individually housed in stainless steel cages and kept for 2 weeks for acclimatization before being used in experiments. Rabbits were provided with pelleted diet and tap water ad libitum. Female rabbits were introduced to male cages and kept for 1 h for mating. Vaginal smears were obtained to observe sperm under light microscope. The day of positive mating was considered as Day 0 of pregnancy. Meloxicam, indomethacin, or control vehicle solutions were administered intraperitoneally (ip) to rabbits as a single dose. Meloxicam dilutions of 20, 10, 5, and 2.5 mg/kg or vehicle were administered ip to spermpositive rabbits at 2, 5, 8, or 24 h postcoitus. Indomethacin (20 mg/kg) was used in this study as positive control and
Gain (%)
Number of pregnant rabbits/Number of treated rabbits
7.97
was administered ip to three sperm-positive rabbits at 5 h postcoitus [10]. On Day 10 of gestation, rabbits were laparotomized under ether anesthesia. Heart blood samples (10 ml) were collected for progesterone determination, and ovaries and uteri were dissected out and examined for fetuses or resorbing sites. The collected tissues were kept in formalin for histopathological examination. Ovulation was estimated by counting fresh corpora lutea [22]. The significance of difference was determined by the Student’s t test, considering a value of p ⱕ 0.05 as significant.
3. Results 3.1. Control rabbits A total of 13 sperm-positive rabbits were used as control in this investigation. Control rabbits received either meloxicam or indomethacin vehicle. The control results were pooled because there was no significant difference in pregnancy outcomes among controls. Only one of the 13 spermpositive rabbits was nonpregnant, yielding a pregnancy rate of 92.3%. Meanwhile, the maternal body weight gain among the control group was 7.97% during the 10 days of gestation period (Table 1). 3.2. Indomethacin-treated rabbits Indomethacin was used in this investigation as a positive control. None of the rabbits receiving indomethacin developed pregnancy. Maternal body weight gain was 11.3% during the 10 days observation period (Table 1). 3.3. Meloxicam-treated rabbits The highest ip meloxicam dose (20 mg/kg) used in this study is equivalent to 1/16 of the oral LD50 value (320 mg/kg) for rabbits [23]. No fatalities occurred among rabbits treated with meloxicam. Furthermore, there were no apparent toxic effects in rabbits during the course of the study.
A.S. Salhab et al. / Contraception 63 (2001) 329 –333 Table 2 The effects of meloxicam on pregnancy rate administered at different times Meloxicam dose (mg/kg)
Time of treatment (h postcoitus)
Number of pregnant rabbits/Number of treated rabbits
Pregnancy (%)
20
2 5 24 2 5 8 24 5 8 5
0/11 0/5 3/4 1/4 0/4 0/4 4/5 1/6 3/4 3/5
0.0 0.0 75.0 25.0 0.0 0.0 80.0 16.7 75.0 60.0
10
5 2.5
3.4. Drug study Ovulation in rabbits is an inducible event, and it occurs 8 –10 h postcoitus. Therefore, for meloxicam to be effective in preventing ovulation, it has to be available in sufficient concentration at the proper time during PG synthesis. Meloxicam in four different concentrations, 20, 10, 5, and 2.5 mg/kg, was administered ip as single doses to sperm-positive rabbits at three different times (2, 5, and 8 h) during the ovulation course. Furthermore, to include/exclude antiovulatory effect of meloxicam, the drug was administered 14 h post ovulation (24 h postcoitus). Table 1 summarizes the results of the effect of different meloxicam concentrations on the rabbit pregnancy rate. None of the 16 rabbits treated with 20 mg/kg was pregnant. The pregnancy rate was correlated inversely with meloxicam concentration. The pregnancy rate among rabbits receiving 20, 10, 5, or 2.5 mg/kg meloxciam were 0.0, 8.3, 20, and 60%, respectively.
331
Table 2 summarizes the effect of meloxicam dosing time on pregnancy rate. Meloxicam dose (20 mg/kg) prevented pregnancy when rabbits received treatment either at 2 or 5 h postcoitus. Meanwhile, little effect was exhibited on pregnancy rate when meloxicam treatment occurred at 24 h postcoitus. The pregnancy rate of 5 mg/kg meloxicam administered at 5 or 8 h postcoitus was 16.7 and 75%, respectively, whereas a 60% pregnancy rate was obtained in rabbits treated with 2.5 mg/kg at 5 h postcoitus. Table 3 summarizes the effect of meloxicam and indomethacin on the number of fetuses, ovary fresh weight, number of corpora lutea, and maternal plasma progesterone. The mean number of fetuses of the control group was about six fetuses; one rabbit of the control group yielded no fetuses. None of the rabbits receiving the 20 mg/kg dose in the preovulation period yielded any fetus. When administered during the postovulation period (24 h postcoitus), the same dose produced comparable number of fetuses to that of the control group. Results show that meloxicam treatment had no effect on ovary fresh weight of rabbits receiving different doses and at different times. Meanwhile, an appreciable decrease in the number of corpora lutea and the plasma progesterone level were observed in rabbits receiving 20 mg/kg treatment in the preovulation period. Lesser effects on the number of corpora lutea or maternal plasma progesterone were observed in the remaining meloxicam treatment groups when compared to control. 3.5. Histopathological results The histopathologic findings observed in ovaries of treated rabbits were mainly gross and microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to hemorrhage within the follicles that lost the ova (Fig. 1). These
Table 3 The effects of different doses of meloxicam on number of fetuses, ovaries weight, corpora lutea, and maternal plasma progesterone Treatment (mg/kg)
Number of rabbits
(Number of fetuses per dam) M ⫾ SD
Ovary weight (mg %)
Corpora lutea (M ⫾ SD)
Plasma progesterone ng/mL (number of analysed samples)
Control
13
(4, 0, 5, 10, 8, 10, 6, 8, 6, 6, 1, 5, 7) 5.85 ⫾ 2.99
26.6 ⫾ 9.4
6.31 ⫾ 2.84
11.1 ⫾ 1.51 (7)
(0,0,0)
24.8 ⫾ 12.9
4.0 ⫾ 1.73
8.7 ⫾ 4.0 (3)
23.0 ⫾ 10.9 31 ⫾ 10.3 21.6 ⫾ 13.6 24.1 ⫾ 10
4.73 ⫾ 3.71 5.17 ⫾ 3.61 4.33 ⫾ 3.33 6.0 ⫾ 2.24
6.78 ⫾ 5.85 (5) 9.16 ⫾ 3.21 (5) 6.37 ⫾ 3.33 (6) 11.2 ⫾ 1.2 (3)
6.75 ⫾ 2.99
9.03 ⫾ 3.6 (4)
24.7 ⫾ 8.9
5.0 ⫾ 4.2)
Indomethacin 20 Meloxicam Preovulation 20 10 5 2.5 Postovulation 20
10
3
16 12 10 5 4
5
(0, (0, (0, (0,
0, 1, 3, 0,
0, 0, 0, 5,
0, 0, 0, 1,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0) 0, 0, .0, 0, 0, 0, 0, 0) 0, 2, 0, 0, 0) 6)
(0, 9, 12, 6) 26.2 ⫾ 11 6.75 ⫾ 5.1 (0, 3, 7, 7, 11) 5.6 ⫾ 4.2
7.86 ⫾ 2.34 (5)
332
A.S. Salhab et al. / Contraception 63 (2001) 329 –333
Fig. 1. Severely dilated and hemorrhagic graffian follicles (arrow) from meloxicam 20 mg/kg-treated rabbit (Hematoxylin and eosin, 200⫻).
features were not seen in control rabbits (Fig. 2). In addition, the remaining follicles showed atrophy and atresia. However, the unruptured ova in treated and control rabbits looked similar. Corpora amelacia in treated rabbits showed central hemorrhages in most of them, whereas this was only seen occasionally in control rabbits.
4. Discussion The results of the present study indicate that the administration of meloxicam to sperm-positive rabbits exhibited significant inhibition in ovulation as reflected by a low rate of pregnancy. Ovulation inhibition in treated rabbits was dose- and time-dependent. A high dose of 20 mg/kg produced complete inhibition of pregnancy when given at any time before ovulation. The lowest effective dose of meloxicam, which produced complete inhibition of pregnancy, was 10 mg/kg when administered 5 h postcoitus. Lower doses of 5 and 2.5 mg/kg produced partial inhibition of pregnancy. However, when the drug was given 24 h postcoitus (14 h
post-ovulation), none of the studied doses produced a pronounced inhibitory effect on pregnancy. This indicates that meloxicam as a single dose has a major effect on ovulation and rules out other reproductive effects including fertilization, decidualization, and implantation. Earlier suggestive findings were reported when rabbits were treated with indomethacin [3]. The researchers detected two peaks (2 h and 5 h) of increased PG synthesis after human chorionic gonadotropin administration. Indomethacin is a typical nonselective PG synthesis inhibitor. We can assume that the inhibitory action of indomethacin on pregnancy is most probably due to COX-2 inhibition. In humans, indomethacin, as well as other nonselective COX inhibitors, were reported to cause ovulation failure and even infertility with no apparent effects on hormonal or menstrual status [24 – 26]. Like other NSAID agents, indomethacin causes significant gastrointestinal and renal side effects. Unlike indomethacin, meloxicam, being a selective COX-2 inhibitor, has less side effects on the gastrointestinal tract and kidney and has no effect on platelet aggregation. Meloxicam is more tolerable in patients than are other nonselective COX inhibitors, such as indomethacin, piroxicam, diclofenac, and ibuprofen [27]. COX-2 is induced in the ovary during the ovulation process. It was found to be necessary for successful rupture of the follicle, probably directly mediating the generation or activation of proteolytic enzymes necessary for the release of ova [9]. Because meloxicam is a selective inhibitor of COX-2 and found effective in preventing ovulation, we postulate the possibility of using meloxicam or any other selective COX-2 inhibitor as an alternative contraceptive agent in women. This warrants further clinical investigation. Acknowledgments The authors acknowledge the technical assistance of Miss Fyrial Mubarak, Mr. Mohammed Saber for animal care, and Mrs. Wafa Al-Shaer for excellent secretarial assistance. References
Fig. 2. Mature graffian follicles containing ova (arrow) from control rabbit (H and E, 200⫻).
[1] LeMaire WJ, Marsh JM. Interrelationships between prostaglandins, cyclic AMP and steroids in ovulation. J Reprod Fertil 1975;22: (Suppl)53–74. [2] Armstrong DT. Prostaglandins and follicular functions. J Reprod Fertil 1981;62:283–91. [3] Kujubu DS, Fletcher BS, Varnum BS, Lim RW, Herschman HRJ. T1 S1O, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 1991;266:12866 –72. [4] Xie WL, Chipman JG, Robertson DL, Erickson, RL, Simmons DL. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 1991;88:2692– 6. [5] Mitchell JA, Akarasereenot P, Thiemermann C, Flower R, Vane JR. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of
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constitutive and inducible cyclooxygenase. Proc Natl Acad Sci USA 1993;90:11693–7. Dewitt DL, Meadle EA, Smith WL. PGH synthase isoenzyme selectivity: the potential for safer nonsteroidal antiinflammatory drugs. Review. Am J Med 1993;95(Suppl):40 – 4. Richards JS, Fitzpatric SL, Clemenes JW, Morris JK, Aliston T, Sirois J. Ovarian cell differentiation: a cascade of multiple hormones, cellular signals and regulated genes. Rec Prog Hormone Res 1995; 50:223–54. Richards J. Hormonal control of gene expression in the ovary. Endocr Rev 1994;15:725–51. Tsafriri A. Ovulation as a tissue remodelling process. Proteolysis and cumulus expansion. Adv Exp Med Biol 1995;377:121– 40. Espey LL, Stein VI, Dumitrescu J. Survey of antiinflammatory agents and related drugs as inhibitors of ovulation in the rabbit. Fertil Steril 1982;38:238 – 47. Dinchuk JE, Car BD, Focht RJ, et al. Renal abnormalitis and an altered inflammatory response in mice lacking cyclooxyginase-2. Nature 1995;378:406 –9. Lim H, Dey SK. Prostaglndin E-2 receptor subtype EP2 gene expression in the mouse uterus coincides with differentiation of the luminal epithelium for implantation. Endocrinology 1997;138:4599 – 606. Lim H, Paria BC, Das Sk, et al. Multiple female reproductive failures in cyclooxygenase-2 deficient mice. Cell 1997;91:197–208. Langenbach R, Morham SG, Tiano HF, et al. Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 1995;83: 482–92. Orczyk GP, Behrman HR. Ovulation blockade by aspirin or indomethacin. In vivo evidence for role of prostaglandin in gonadotrophin secretion. Prostaglandins 1972;1:3–20.
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[16] Armstrong DT, Grinwich DL. Blockade of spontaneous and LHinduced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1972;1:21–36. [17] Lau 1F, Saksena SK, Chang MC. Pregnancy blockade by indomethacin, an inhibitor of prostaglandin synthesis: its reversal by prostaglandins and progesterone in mice. Prostaglandins 1972;4:794 – 803. [18] Akinlosotu BA, Verna OP. Detection of ovulation in goats by blood prostaglandins concentration. Am J Vet Res 1983;44:1339 – 43. [19] Jaszczak SE. Anovulatory luteal phase in primates. Contraception 1983;27:505–14. [20] Vane JR. Botting RM. New insight into the mode of action of antiinflammatory drugs. Inflamm Res 1995;44:1–10. [21] Emery P. Clinical aspects of COX-2 inhibitors. Drugs Today 1999; 35:267–74. [22] Kaufman RA, Savoy, Moore RT, Subramanian MG, Moghissi KS. Cocaine inhibits mating-induced, but not human chorionic gonadotropin-stimulated ovulation in the rabbit. Biol Reprod 1992;46:641–7. [23] Lehmann NA, Baumeister M, Lutzen L, Wiegleb J. Meloxicam: a toxicological overview. Inflammopharmacology 1996;4:105–23. [24] Smith G, Roberts R, Hall C, Nuki G. Reversible ovulatory failure associated with the development of luteinized unruptured follicles in women with inflammatory arthritis taking non-steroidal anti-inflammatory drugs. Br J Rheumatol 1996;35:458 – 62. [25] Akil M, Amos RS, Stewart P. Infertility may sometimes be associated with NSAID consumption. Br J Rheumatol 1996;35:76 – 8 [26] Athanasiou S, Bourne TH, Khalid A, et al. Effects of indomethacin on follicular structure, vascularity, and function over the periovulatory period in women. Fert Steril 1996;65:556 – 60 [27] Manuel D. Meloxicam clinical data on a preferential cyclooxygenase-2 inhibitor. J Clin Rheumatol 1998;4:S32–S39.
Original research article
Meloxicam inhibits rabbit ovulation夞 A.S. Salhaba,*, M.N. Gharaibeha, M.S. Shomafb, B.I. Amroc a
Department of Pharmacology, University of Jordan, Amman, Jordan Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan c Faculty of Pharmacy, University of Jordan, Amman, Jordan
b
Abstract The nonsteroidal antiinflammatory, selective cyclo-oxygenase-2 (COX-2) inhibitor, meloxicam, was tested to assess its effect on rabbit ovulation. Meloxicam in different doses was administered intraperitoneally (ip) to adult female Californian rabbits at 2, 5, 8, and 24 h postcoitus with sperm-positive rabbits. Rabbits were killed on Day 10 of gestation. Meloxicam produced significant inhibition of ovulation in rabbits. This inhibition of ovulation by meloxicam was dose- and time-dependent. Ovulation in rabbits was completely inhibited by a single ip administration of meloxicam (20 mg/kg) when the drug was administered at 2 and 5 h postcoitus, whereas neither ovulation nor implantation were inhibited (pregnancy rate 75%) by the same dose administered 24 h postcoitus (approximately 14 h post ovulation). Further, ovulation was completely inhibited by 10 mg/kg of meloxicam when the drug was administered at 5 or 8 h postcoitus, but there was less inhibition of ovulation when 10 mg/kg of the drug was administered at 2 or 24 h postcoitus (pregnancy rate 25 and 80%, respectively). Corpora lutea, maternal plasma progesterone, ovary fresh weight, and maternal body weight gain were affected by meloxicam treatment. Histopathological findings observed in the ovaries of treated rabbits included microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to severe hemorrhage within the follicles which lost ova. These results show that ovulation can be inhibited in rabbits by meloxicam. Further studies are needed to assess the value of selective COX-2 inhibitors as potential nonhormonal contraceptive agents. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Meloxicam; Cyclo-oxygenase-2 inhibitor (COX-2); Ovulation Antiinflammatory agents; Indomethacin
1. Introduction Ovulation is accompanied by induction of prostaglandin (PG) synthesis as a consequence of the luteinizing hormone (LH) surge [1,2]. Cyclo-oxygenase (COX) plays a key regulatory role in PG synthesis and occurs in both the constitutive (COX-1) and the inducible (COX-2) isoforms [3,4]. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is the major isoform of the inflammatory reaction [5,6]. Further, COX-2 isoform is induced in the ovary and is necessary for the successful rupture of the follicle, probably directly mediating the generation or activation of proteolytic enzymes that are necessary for the release of ova [7–9]. Espey hypothesized in 1982 that the actual rupture of the ovarian surface is brought about by an inflammatory process [10]. COX-2 null mice show multiple failures in reproduc夞 This work was supported by grant no. 558/99 –2000 from the Deanship of Scientific Research at the University of Jordan. * Corresponding author. Tel.: ⫹9626-5355000 (Ext. 2465/2464); fax: ⫹9626-5355500. E-mail address: [email protected] (A.S. Salhab).
tive function, including ovulation, fertilization, implantation, and decidualizaition [11–13]. Meanwhile, the COX-1 mutant mice were fertile, but homozygous matings tend to yield dead pups for unknown reasons [14]. In a related area of research, indomethacin, a nonspecific COX inhibitor, inhibits ovulation in a number of animal species, such as rats, mice, rabbits, sheep, and monkeys [10,15–20]. The antifertility effect of indomethacin is likely to be mediated by COX-2 [21]. Meloxicam is a new nonsteroidal antiinflammatory (NSAID) that appears to have a greater selective inhibitory activity against the inducible COX-2 isoform, than against the constitutive isoform COX-1. Inhibition of COX-1 is associated with gastrointestinal, renal, and platelet aggregation adverse effects. Therefore, meloxicam and other COX-2 selective inhibitors are promoted for their safer profile of side effects. The objective of the present study was to investigate the effects of meloxicam, as an example of selective COX-2 inhibitor, on the fertility of female rabbits. The potential of antiovulatory activity of meloxicam was studied in regard to
0010-7824/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 1 0 - 7 8 2 4 ( 0 1 ) 0 0 2 0 7 - 4
330
A.S. Salhab et al. / Contraception 63 (2001) 329 –333
Table 1 The effects of meloxicam administered before ovulation on maternal body weight and pregnancy rate Treatment (mg/kg) Control Indomethacin 20 Meloxicam 20 10 5 2.5
Maternal body weight (g)
Pregnancy (%)
12/13
92.3
Initial
Final
2297 ⫾ 333
2480 ⫾ 352
2020 ⫾ 170
2250 ⫾ 165
11.3
0/3
0.0
2403 ⫾ 323 2484 ⫾ 334 2316 ⫾ 316 2388 ⫾ 252
2479 ⫾ 373 2509 ⫾ 328 2578 ⫾ 303 2548 ⫾ 103
3.15 1.0 11.1 6.7
0/16 1/12 2/10 3/5
0.0 8.3 20 60
dose and temporal relationship. The present study is the first to investigate such effects.
2. Materials and methods 2.1. Drugs Meloxicam (1 g) in a powder form (Batch no. 8250381) was donated by Boehringer Ingelheim Pharma KG (Biberach an der Riss, Germany). Additional meloxicam (100 g; Batch no. 9700009015) was obtained from Advanced Pharmaceutical Industries Co. Ltd. (Amman, Jordan). Purity was checked by HPLC and UV spectrum against the authentic sample that was obtained from Boehringer Ingelheim. Indomethacin powder (Lot 67 H 1069) was purchased from Sigma Chemical Co. (St. Louis, MO) The different dilutions of meloxicam were freshly prepared in warm 0.15 M NaOH solution (pH 8.5) as recommended by Boehringer Ingelheim researchers (personal communications), whereas indomethacin was dissolved in 0.1 M Sorrenson’s phosphate buffer (pH 8.0) according to Espey et al. [10]. 2.2. Rabbit experiments Adult male and female Californian rabbits, purchased from a local farm, were individually housed in stainless steel cages and kept for 2 weeks for acclimatization before being used in experiments. Rabbits were provided with pelleted diet and tap water ad libitum. Female rabbits were introduced to male cages and kept for 1 h for mating. Vaginal smears were obtained to observe sperm under light microscope. The day of positive mating was considered as Day 0 of pregnancy. Meloxicam, indomethacin, or control vehicle solutions were administered intraperitoneally (ip) to rabbits as a single dose. Meloxicam dilutions of 20, 10, 5, and 2.5 mg/kg or vehicle were administered ip to spermpositive rabbits at 2, 5, 8, or 24 h postcoitus. Indomethacin (20 mg/kg) was used in this study as positive control and
Gain (%)
Number of pregnant rabbits/Number of treated rabbits
7.97
was administered ip to three sperm-positive rabbits at 5 h postcoitus [10]. On Day 10 of gestation, rabbits were laparotomized under ether anesthesia. Heart blood samples (10 ml) were collected for progesterone determination, and ovaries and uteri were dissected out and examined for fetuses or resorbing sites. The collected tissues were kept in formalin for histopathological examination. Ovulation was estimated by counting fresh corpora lutea [22]. The significance of difference was determined by the Student’s t test, considering a value of p ⱕ 0.05 as significant.
3. Results 3.1. Control rabbits A total of 13 sperm-positive rabbits were used as control in this investigation. Control rabbits received either meloxicam or indomethacin vehicle. The control results were pooled because there was no significant difference in pregnancy outcomes among controls. Only one of the 13 spermpositive rabbits was nonpregnant, yielding a pregnancy rate of 92.3%. Meanwhile, the maternal body weight gain among the control group was 7.97% during the 10 days of gestation period (Table 1). 3.2. Indomethacin-treated rabbits Indomethacin was used in this investigation as a positive control. None of the rabbits receiving indomethacin developed pregnancy. Maternal body weight gain was 11.3% during the 10 days observation period (Table 1). 3.3. Meloxicam-treated rabbits The highest ip meloxicam dose (20 mg/kg) used in this study is equivalent to 1/16 of the oral LD50 value (320 mg/kg) for rabbits [23]. No fatalities occurred among rabbits treated with meloxicam. Furthermore, there were no apparent toxic effects in rabbits during the course of the study.
A.S. Salhab et al. / Contraception 63 (2001) 329 –333 Table 2 The effects of meloxicam on pregnancy rate administered at different times Meloxicam dose (mg/kg)
Time of treatment (h postcoitus)
Number of pregnant rabbits/Number of treated rabbits
Pregnancy (%)
20
2 5 24 2 5 8 24 5 8 5
0/11 0/5 3/4 1/4 0/4 0/4 4/5 1/6 3/4 3/5
0.0 0.0 75.0 25.0 0.0 0.0 80.0 16.7 75.0 60.0
10
5 2.5
3.4. Drug study Ovulation in rabbits is an inducible event, and it occurs 8 –10 h postcoitus. Therefore, for meloxicam to be effective in preventing ovulation, it has to be available in sufficient concentration at the proper time during PG synthesis. Meloxicam in four different concentrations, 20, 10, 5, and 2.5 mg/kg, was administered ip as single doses to sperm-positive rabbits at three different times (2, 5, and 8 h) during the ovulation course. Furthermore, to include/exclude antiovulatory effect of meloxicam, the drug was administered 14 h post ovulation (24 h postcoitus). Table 1 summarizes the results of the effect of different meloxicam concentrations on the rabbit pregnancy rate. None of the 16 rabbits treated with 20 mg/kg was pregnant. The pregnancy rate was correlated inversely with meloxicam concentration. The pregnancy rate among rabbits receiving 20, 10, 5, or 2.5 mg/kg meloxciam were 0.0, 8.3, 20, and 60%, respectively.
331
Table 2 summarizes the effect of meloxicam dosing time on pregnancy rate. Meloxicam dose (20 mg/kg) prevented pregnancy when rabbits received treatment either at 2 or 5 h postcoitus. Meanwhile, little effect was exhibited on pregnancy rate when meloxicam treatment occurred at 24 h postcoitus. The pregnancy rate of 5 mg/kg meloxicam administered at 5 or 8 h postcoitus was 16.7 and 75%, respectively, whereas a 60% pregnancy rate was obtained in rabbits treated with 2.5 mg/kg at 5 h postcoitus. Table 3 summarizes the effect of meloxicam and indomethacin on the number of fetuses, ovary fresh weight, number of corpora lutea, and maternal plasma progesterone. The mean number of fetuses of the control group was about six fetuses; one rabbit of the control group yielded no fetuses. None of the rabbits receiving the 20 mg/kg dose in the preovulation period yielded any fetus. When administered during the postovulation period (24 h postcoitus), the same dose produced comparable number of fetuses to that of the control group. Results show that meloxicam treatment had no effect on ovary fresh weight of rabbits receiving different doses and at different times. Meanwhile, an appreciable decrease in the number of corpora lutea and the plasma progesterone level were observed in rabbits receiving 20 mg/kg treatment in the preovulation period. Lesser effects on the number of corpora lutea or maternal plasma progesterone were observed in the remaining meloxicam treatment groups when compared to control. 3.5. Histopathological results The histopathologic findings observed in ovaries of treated rabbits were mainly gross and microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to hemorrhage within the follicles that lost the ova (Fig. 1). These
Table 3 The effects of different doses of meloxicam on number of fetuses, ovaries weight, corpora lutea, and maternal plasma progesterone Treatment (mg/kg)
Number of rabbits
(Number of fetuses per dam) M ⫾ SD
Ovary weight (mg %)
Corpora lutea (M ⫾ SD)
Plasma progesterone ng/mL (number of analysed samples)
Control
13
(4, 0, 5, 10, 8, 10, 6, 8, 6, 6, 1, 5, 7) 5.85 ⫾ 2.99
26.6 ⫾ 9.4
6.31 ⫾ 2.84
11.1 ⫾ 1.51 (7)
(0,0,0)
24.8 ⫾ 12.9
4.0 ⫾ 1.73
8.7 ⫾ 4.0 (3)
23.0 ⫾ 10.9 31 ⫾ 10.3 21.6 ⫾ 13.6 24.1 ⫾ 10
4.73 ⫾ 3.71 5.17 ⫾ 3.61 4.33 ⫾ 3.33 6.0 ⫾ 2.24
6.78 ⫾ 5.85 (5) 9.16 ⫾ 3.21 (5) 6.37 ⫾ 3.33 (6) 11.2 ⫾ 1.2 (3)
6.75 ⫾ 2.99
9.03 ⫾ 3.6 (4)
24.7 ⫾ 8.9
5.0 ⫾ 4.2)
Indomethacin 20 Meloxicam Preovulation 20 10 5 2.5 Postovulation 20
10
3
16 12 10 5 4
5
(0, (0, (0, (0,
0, 1, 3, 0,
0, 0, 0, 5,
0, 0, 0, 1,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0) 0, 0, .0, 0, 0, 0, 0, 0) 0, 2, 0, 0, 0) 6)
(0, 9, 12, 6) 26.2 ⫾ 11 6.75 ⫾ 5.1 (0, 3, 7, 7, 11) 5.6 ⫾ 4.2
7.86 ⫾ 2.34 (5)
332
A.S. Salhab et al. / Contraception 63 (2001) 329 –333
Fig. 1. Severely dilated and hemorrhagic graffian follicles (arrow) from meloxicam 20 mg/kg-treated rabbit (Hematoxylin and eosin, 200⫻).
features were not seen in control rabbits (Fig. 2). In addition, the remaining follicles showed atrophy and atresia. However, the unruptured ova in treated and control rabbits looked similar. Corpora amelacia in treated rabbits showed central hemorrhages in most of them, whereas this was only seen occasionally in control rabbits.
4. Discussion The results of the present study indicate that the administration of meloxicam to sperm-positive rabbits exhibited significant inhibition in ovulation as reflected by a low rate of pregnancy. Ovulation inhibition in treated rabbits was dose- and time-dependent. A high dose of 20 mg/kg produced complete inhibition of pregnancy when given at any time before ovulation. The lowest effective dose of meloxicam, which produced complete inhibition of pregnancy, was 10 mg/kg when administered 5 h postcoitus. Lower doses of 5 and 2.5 mg/kg produced partial inhibition of pregnancy. However, when the drug was given 24 h postcoitus (14 h
post-ovulation), none of the studied doses produced a pronounced inhibitory effect on pregnancy. This indicates that meloxicam as a single dose has a major effect on ovulation and rules out other reproductive effects including fertilization, decidualization, and implantation. Earlier suggestive findings were reported when rabbits were treated with indomethacin [3]. The researchers detected two peaks (2 h and 5 h) of increased PG synthesis after human chorionic gonadotropin administration. Indomethacin is a typical nonselective PG synthesis inhibitor. We can assume that the inhibitory action of indomethacin on pregnancy is most probably due to COX-2 inhibition. In humans, indomethacin, as well as other nonselective COX inhibitors, were reported to cause ovulation failure and even infertility with no apparent effects on hormonal or menstrual status [24 – 26]. Like other NSAID agents, indomethacin causes significant gastrointestinal and renal side effects. Unlike indomethacin, meloxicam, being a selective COX-2 inhibitor, has less side effects on the gastrointestinal tract and kidney and has no effect on platelet aggregation. Meloxicam is more tolerable in patients than are other nonselective COX inhibitors, such as indomethacin, piroxicam, diclofenac, and ibuprofen [27]. COX-2 is induced in the ovary during the ovulation process. It was found to be necessary for successful rupture of the follicle, probably directly mediating the generation or activation of proteolytic enzymes necessary for the release of ova [9]. Because meloxicam is a selective inhibitor of COX-2 and found effective in preventing ovulation, we postulate the possibility of using meloxicam or any other selective COX-2 inhibitor as an alternative contraceptive agent in women. This warrants further clinical investigation. Acknowledgments The authors acknowledge the technical assistance of Miss Fyrial Mubarak, Mr. Mohammed Saber for animal care, and Mrs. Wafa Al-Shaer for excellent secretarial assistance. References
Fig. 2. Mature graffian follicles containing ova (arrow) from control rabbit (H and E, 200⫻).
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