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Differential effects of ibuprofen and naproxen sodium on menstrual prostaglandin release and on prostaglandin production in the rat uterine homogenate
Prostaglandins Leukotrienes and Medicine 13: 129-137, 1984
DIFFERENTIAL EFFECTS OF IBUPROFEN AND NAPROXEN SODIUM ON MENSTRUAL PROSTAGLANDIN RELEASE AND ON PROSTAGLANDIN PRODUCTION IN THE RAT UTERINE HOMOGENATE1 Andrea M. Powell and W. Y. Chan Department of Pharmacology Cornell University Medical College New York, New York 10021 (Reprint requests to WYC) ABSTRACT In two independent studies, ibuprofen and naproxen sodium were found to be equi-effective in alleviating dysmenorrheic symptoms. However, the effects of these drugs on menstrual PG release were found to be dissimilar. Ibuprofen primarily inhibited menstrual PGF2n release with little effect on PGE release, whereas, naproxen sodium inhibited both PGF20 and PGE releaze equally. To verify these results, we determined the inhzbitory potency, IC50, of ibuprofen and naproxen sodium on PGF20 and PGE2 synthesis in the rat uterine homogenate system. The preferential PGF2, inhibitory activity of ibuprofen was confirmed. These findings suggest that ibuprofen may, in addition to inhibiting fatty acid cyclooxygenase, also inhibit PGF2u reductase, or some and PGE synthesis diffother PG metabolic pathways which affect PGF erently. The significance of this different2* al PG syn$hesis inhibitory effect in dysmenorrheic therapy is discussed. INTRODUCTION In recent years, extensive clinical trials have established the effectiveness of nonsteroidal anti-inflammatory drugs (NSAIDS) in the treatment of primary dysmenorrhea (l-3). Although there were few direct comparative studies among the NSAIDs, individual drug trials showed that, with the exception of aspirin, the various NSAIDs tested so far were equi-effective. This clinical finding was interpreted by many as an indication that these agents alleviate dysmenorrhea principally by their common action as fatty acid cyclooxygenase inhibitors (4). %'his work was supported by research grants from NICHHD (HD-13064) and the NIGMS (GM-07547). 129
Recently, we completed two independent studies on dysmenorrhea, one with ibuprofen and the other with naproxen sodium, and the effects of these two agents on menstrual PG release (5,6). In accordance with other clinical trials, our results showed that ibuprofen and naproxen sodium were equally effective in relieving dysmenorrhea. It was therefore of great interest when we found that their effects on menstrual PG release were dissimilar. Because of the important clinical and pharmacologic implications of this differential effect between ibuprofen and naproxen sodium, we sought verification of this finding in a controlled biochemical system and determined the effects of these two agents on PG synthesis in the rat uterine homogenate. In this paper, we report the differential effects of ibuprofen and naproxen sodium on menstrual PG release in dysmenorrheic subjects and the results of our comparative study of the inhibitory activities of these two agents on uterine PG synthesis. METHODS AND MATERIALS Menstrual specimens Menstrual specimens were collected from two groups of dysmenorr-. heic subjects who volunteered for our ibuprofen trial and our naproxen sodium trial. Methods for specimen collection, menstrual PG extraction and clinical protocols were described in our published papers (5-7). In essence, the subject collected all of her tampon specimens for menstrual PG determination. Each subject was studied for at least three cycles. The protocol consisted of a control cycle followed by treatment cycles, either ibuprofen and placebo, or naproxen sodium and placebo, according to standard double-blind, cross-over procedure. Determination of PG synthesis innibitory activity The effects of ibuprofen and naproxen sodium on PGF2u and PGE biosynthesis from endogenous substrate were determined in non-pregnan$ rat uterine homogenates. Uterine horns from randomly chosen adult, virgin, Wistar albino rats were separated in matched-pair fashion, with one horn serving as control and the contralateral horn as the experimental sample. Four rats were used in each experiment. The horns were dissected out and immediately placed in ice-cold Krebs-Ringer bicarbonate solution (pH 7.4) for the control samples, or, ice-cold Krebs-Ringer bicarbonate solution with the appropriate concentration of the test drug for the experimental samples. After the wet weights were taken, the uterine horns were minced in 35 mls of the Krebs-Ringer bicarbonate solution or the Krebs-Ringer bicarbonate solution with drug, and homogenized while still on ice with a Polytron homogenizer (Brinkman Instruments) with two, 10 second bursts. The samples were then incubated in a bath shaker at 37'C for 60 minutes with continuous aeration with 95% 0 and 5% CO . At the end of the incubation period, an equal volume of z sopropyl az cohol was added. This stopped the enzyme activity and improved the efficiency of the PG extraction. The incubate was shaken for an additional five minutes and then centrifuged. The alcoholic supernatant was withdrawn and washed twice with an equal volume
130
of hexane to remove neutral fats and lipids. It was then acidified to pH 4 with 1 N HCl and extracted twice with chloroform. The chlorofo? phase was evaporated to a small volume with a rotary evaporator at 40 C under reduced pressure. The final drying step was carried out under nitrogen at atmospheric pressure. The residue was stored at -2O'C until assayed by RIA. The inhibitory activity of each drug was measured against its own matched-pair control and expressed as percent inhibition. At least three different concentrations were studied for each drug. The doseresponse curves for ibuprofen and naproxen sodium were plotted. Based on these curves, the IC 's, the concentrations of drug which gave 50% reduction of PG biosynti!O esis, for PGF2a and PGE2 were calculated and their 95% confidence limits computed. Assays
PG contents of the samples were measured by bioassays and/or RIAs. For bioassay, the rat fundal strip preparation as described by Vane (8) was used. The tissue was superfused with Krebs-Ringer bicarbonate solution at 37OC. Authentic PGF2c was used as the standard. Bracket assays were performed. A minimum of three bracket assays, each on a different fundal strip, were made on each sample. The mean value of the bioassays and the standard error of the mean were calculated. For RIAs, commercially prepared goat anti-PGF2, and either rabbit anti-PGE, A, B or goat anti-PGEl sera were used. The antiserum cross reactivities measured in our systems were: goat anti-PGF2ucrossreacts with PGE2 <3% with 6-keto-PGFlu <2.5% and has no detectable crossreactivity with TKB2. Goat anti-PGEl crossreacts with PGE2 and has'no detectable crossreactivity with PGF2c. Rabbit anti-PGE, A, B does not differentiate between PGEl and PGE2 and has no detectable crossreactivity with PGF2c. Crossreactivities with other PGs were not determined. The suppliers provided the data on crossreactivities with PGA, PGB and . These PGs are not present in the uterus in appreciable amounts, PGF the$Efore, crossreactivity with these PGs did not significantly affect our RIAs of PGF and PGE. However, PGE activity was assayed in PGE2 equivalents, s&e the antiserum did not2differentiate between PGEl and PGE2. For the RIAs of menstrual samples, the same PGE anti-serum was used to analyze all of the cycles from an individual subject. For the RIAs of PG synthesis inhibitory activity in uterine homogenates, the same PGE anti-serum was always used for each matched-pair, control and treatment. In the few cases where two different anti-PGE sera had to be used for the same series of matched-pair experiments, no significant differences were seen in the individual values of inhibitory potency. Each sample was determined in duplicate and at two different dilutions. The sensitivity of the assay was between 16 and 30 pg. The intraassay coefficient of variation was less than 10% and the interassay coefficient of variation was less than 15%. Parallel bioassays and RIAs for PGF o were carried out in a number of experiments for validation of the i? IA system.
131
MATERIALS Authentic PGF , PGE2, ibuprofen and the corresponding placebo were kindly supplie 2% by the Upjohn Co., Kalamazoo, MI. Naproxen sodium and the corresponding placebo were supplied by Syntex Research, Palo Alto, CA. Goat anti-PGF2, and anti-PGE sera were purchased from Research Products International, Mount Prospict, IL. Rabbit anti-PGE, A,B serum was urchased from Calbiochem-Behring Corp., LaJolla, CA. Multilabeled [';H ]-PGF2, and [~H]-PGE (loo-150 Ci/mmol) were purchased from New England Nuclear Corp., Bostzn, MA. RESULTS Effects of ibuprofen and naproxen sodium treatment on menstrual PG release The effects of ibuprofen and placebo treatment on menstrual PG release were determined by parallel bioassay and RIA in 4 subjects for a total of 18 cycles. The results are summarized in Table 1. Ibuprofen treatment, 400 mg q.i.d., but not placebo, caused a significant decrease in menstrual PG release, as determined by bioassay in PGFp equivalents. RLAs of the menstrual samples show that only the decrease in PGF release was significant (p < 0.01 by the paired 'It"test). The ef$" ect on PGE2 release was not statistically significant. In nearly all the samples measured, the PG activity determined by bioassay was greater than the sum total of PGF2a and PGE2 determined by RIA. Table 1.
Effects of Ibuprofen or Placebo Treatment on Menstrual PG Release
Total Menstrual PG Release Per Cycle in ng + S.E. Treatment Bioassay in PGF20 equivalents
RIA PGF2o
PGE2
PGF2a + PGE2
Control (4 patients, 4 cycles)
59.4 + 16.8
27.3 f 7.1
5.23 + 2.19
32.49 + 8.63
Ibuprofen (4 patients, 7 cycles)
16.7 t 2.8*
9.12 + 2.57* 3.42 + 1.54
12.54 -_ + 4.07
Placebo (4 patients, 7 cycles)
47.6 + 8.7
24.5 + 4.5
28.04 f 5.02
*
3.54 +_ 0.59
Difference in mean values of menstrual PG released during ibuprofen treatment cycles and either control or placebo cycles was highly significant, P < 0.01.
132
Naproxen sodium treatment 275 mg q.i.d. also markedly reduced menstrual PG release in dysmenorrheic subjects. However, unlike ibuand PGE2 profen, naproxen sodium therapy reduced both menstrual PGF release equally. Table 2 shows the effects of naproxen so&ium therapy on menstrual PG release in 12 subjects for a total of 36 cycles.
Table 2.
Effects of Naproxen Sodium or Placebo Treatment on Menstrual PG Release
Treatment
Total Menstrual PG Release Per Cycle in pg + S.E. RIA PGE2
pGF2a Control (12 patients, 12 cycles) Naproxen sodium (12 patients, 12 cycles) Placebo (12 patients, 12 cycles) *
23.19 + 4.75
5.09 + 1.30*
20.13 + 2.43
3.71 -+ 0.76
0.75 2 0.19*
3.22 + 0.48
Difference in menstrual PG released during naproxen sodium treatment cycles and either control or placebo cycles was highly significant, P < 0.01.
Although the effects of ibuprofen and naproxen sodium on menstrual PG release in these two trials were different, both therapies were equally effective in alleviating dysmenorrhea. The clinical results of these two drug trials have been published (5,6). PG synthesis inhibitory activity of ibuprofen and naproxen sodium in rat uterine homogenates Incubation of the rat uterine homogenates for 60 minutes in the presence of varied concentrations of ibuprofen or naproxen sodium resulted in a dose related inhibition of both PGF and PGE biosynthesis Table 3 shows the ICso 's and the calculated 95X2%onfidencg limits of ibuprofen and naproxen sodium for PGF and PGE2 biosynthesis in the rat uterine homogenate system. In th2* system, ibuprofen is more potent in inhibiting PGF synthesis than PGE synthesis. The ratio of the IC50-PGE2 : IClp-RF 2,_+ expresses the dzfference in the degree of preferential inhib ion o PGFB biosynthesis over PGE2 by ibuprofen and naproxen sodium. This is also shown in Table 3.
133
Table 3.
's of Ibuprofen and Naproxen Sodium in the Rat Uterine IC50 Homogenate System
Drug
- PGF2o IC50 (mean & 95% confidence limits)
- PGE2 IC50 (mean h 95% confidence limits)
IC50
IC50
- PGE2
- PGF2o
Ibuprofen (n = 11)
1.1 x 10-6M (0.7 - 1.7)
3.8 x lO+jM (2.5 - 5.8)
3.5
Naproxen Sodium (n = 13)
2.8 x 10-7M
2.6 x 10-7M
0.9
(2.2 - 3.6)
(1.7 - 4.0)
DISCUSSION NSAIDs are effective agents for the treatment of primary dysmenorrhea. The effectiveness of this class of drug is assumed to be the result of their common action, their ability to inhibit fatty acid cyclooxygenase. Inhibition of this enzyme which catalyzes the conversion of arachidonic acid to the endoperoxides, would logically result in a non-selective inhibition of both PGF* and PGE2 biosynthesis, We found that ibuprofen and naproxen sodium, the two most commonly used NSAIDs in dysmenorrheic therapy, each affects menstrual-PG release differently. When administered for the first three days of menses in comparable doses recommended for the treatment of rheumatoid arthritis, ibuprofen therapy suppressed primarily menstrual PGF release with little effect on PGE2 release, whereas, naproxen sodI* urn therapy suppressed both PGFp and PGE2 release equally. The percent suppression of menstrual PG release in the treatment cycles relative to the control cycles for ibuprofen was 66.6% for PGF20 and 34.6% for PGE2 (statistically not significant) and for naproxen sodium was 78.1% for PGF20 and 79.8% for PGE2. The different effects of ibuprofen on menstrual PGF2a and PGE release may suggest that the doses of ibuprofen were not sufficienZ , in comparison to the naproxen sodium trial, to produce a consistent and PGE2. However, ibuprofen suppression of both menstrual PGF therapy produced a greater percentoreduction in PGF20 release than in PGE2 release in six of the seven treatment cycles. This clearly indicates a greater sensitivity of uterine PGF2u than PGE2 to ibuprofen suppression. This preferential PGF2, inhibitory activity of ibuprofen was confirmed in a more quantitative study using rat uterine homogenates. In this study, we measured the PGF2o synthesis and the PGE2 synthesis inhibitory potencies of ibuprofen simultaneously, in the same homogenate preparation, and calculated the respective IC50 values. The
134
IC50 for PGE was nearly four times higher than the IC50 for PGF2 . Thus, ibupro$en appears to have a preferential effect on PGF2, syffthesis. Naproxen sodium did not display this preferential inhibition of PGF Its IC50-PGE2:IC50-PGF2o ratio was near unity. 2a' It should be pointed out that in each experiment, the IC50 determinations for PGF2c and PGE2 were always carried out simultaneously, in the same homogenate, and with the matched-pair controls incubated at the same time. The uterine horns used in these experiments were taken from rats chosen at random. Our own experience as well as that reported by Poyser and Scott (9) have shown that in nonpregnant uterine homogenates using endogenous substrate, without addition of cofactors, the productions of PGF2, and PGE2 were not significantly affected by the estrus cycle. Our use of the matched-pair control in each experiment further minimized the effect of the small cyclic variance of PG production on the IC50 determination. Our finding that ibuprofen preferentially inhibits uterine PGF20 biosynthesis over PGE2 biosynthesis, suggests that ibuprofen may, in addition to inhibiting fatty acid cyclooxygenase, also inhibit PGF2, reductase, which catalyzes the enzymatic conversion of PGH2 to PGF20. Also, it is well known that NSAIDs may affect PG synthesis differently in different tissues, suggesting that the PG generating system exists in multi-molecular forms (10). Our findings with ibuprofen are of particular interest because they may suggest a heterogeneity of PG synthesizing enzymes within the same tissue. One is tempted to speculate that two separate and distinct fatty acid cyclooxygenases may exist in the uterine PG system, one associated with PGF2o reductase and the other with PGE2 isomerase which can be affected individually by the NSAIDs. The idea of the existence of isomers in the PG synthetase complex is not new. As early as 1973, Maddox suggested the existence of two distinct forms of PG synthetase complex in sheep vesicular tissue (11). However, recently, Lysz and Needleman presented biochemical evidence for the existence of two distinct forms of fatty acid cyclooxygenase in the rabbit brain which can be differentially inhibited by the NSAIDs (12). It is also possible that ibuprofen might inhibit 9-keto-reductase which converts PGE2 to PGF2o. This would result in a preferential inhibition of PGF20. However, the inhibition of this pathway by ibuprofen does not appear to be a significant factor. Our preliminary study has not demonstrated an active conversion of 13HlPGE2 to i3H]PGF20 in the uterine homogenate system used'in our study. Of equal interest is our finding that, although subjects treated with naproxen sodium had a greater suppression of menstrual PG release than subjects treated with ibuprofen, there was no clear difference in clinical efficacy between the two drugs in our clinical trials (5,6). However, the majority of the patients treated with naproxen sodium, but not patients treated with ibuprofen, showed a reduction in their menstrual
135
fluid volume (5,6). The effect of naproxen sodium on the menstrual fluid volume probably is related to its ability to inhibit PGE2 release as well as PGF2d. It is possible that the apparent equi-effectiveness of ibuprofen and naproxen sodium in dysmenorrheic therapy may be the result of administering doses which are clinically supramaximal. It may not be necessary to suppress menstrual PG release to the degree attained by the trial dose of naproxen sodium or ibuprofen in order to ameliorate dysmenorrheic symptoms. Neither the normal range of menstrual PGs nor the optimal dose for these drugs in dysmenorrheic therapy has been determined. It has been shown that during menstruation, PGFZc,is a uterine stimulant while PGE2 can act as a uterine relaxant (13). A drug such as ibuprofen, which preferentially inhibits PGF2, over PGE2 may manipulate the menstrual PG profile more favorably. On the other hand, a drug such as naproxen, which inhibits both PGFzc,and PGE2 will also reduce menstrual fluid volume, an effect desirable in dysmenorrhea with menorrhagia. This consideration should be germane to drug choice and to the further development of dysmenorrheic therapy. ACKNOWLEDGMENTS The authors wish to acknowledge the skillful technical assistance of Ms. Donna Robertson, Ms. Maria Sergi and Ms. Elizabeth Zaff. They also wish to thank Thomas J. Vecchio, M.D., of The Upjohn Co., for his active support. REFERENCES 1.
Chan WY. Prostaglandin inhibitors and antagonists in dysmenorrhea therapy. p 209 in Dysmenorrhea. (MY Dawood ed) Williams and Wilkins, Baltimore, 1981.
2.
Jacobson J, Cavalli-Bjorkman K, Lundstrom V, Nilsson B, Norbeck M. Prostaglandin synthetase inhibitors and dysmenorrhea. A survey and personal clinical experience. Acta Obstetricia et Gynecologica Scandinavica Supplement 87: 73, 1979.
3.
Ylikorkala 0, Dawood MY. New concepts in dysmenorrhea, American Journal of Obstetrics and Gynecology 130: 833, 1978.
4.
Vane JR. Inhibition of prostaglandin biosynthesis as the mechanism of action of aspirin-like drugs. Advances in Biosciences 9: 395, 1973.
5.
Chan WY, Dawood MY, Fuchs F. Prostaglandins in primary dysmenorrhea, comparison of prophylactic and nonprophylactic treatment with ibuprofen and the use of oral contraceptives. American Journal of Medicine 70: 535, 1981.
136
6.
Chan WY, Fuchs F, Powell AM. Effects of naproxen sodium on menstrual prostaglandins and primary dysmenorrhea. Obstetrics and Gynecology 61: 285, 1983.
7.
Chan WY, Hill JC. Determination of menstrual prostaglandin levels in non-dysmenorrheic and dysmenorrheic subjects. Prostaglandins 15: 365, 1978.
a.
Vane JR. A sensitive method for the assay of 5-hydroxytryptamine. British Journal of Pharmacology 12: 344, 1957.
9.
Poyser NL, Scott FM. Prostaglandin and thromboxane production by the rat uterus and ovary -in vitro during the oestrous cycle. Journals of Reproduction & Fertility 60: 33, 1980.
10.
Vane JR. Prostaglandins and aspirin-like drugs. 7: 61, 1972.
11.
Maddox IS. The role of copper in prostaglandin synthesis. Biochemica et BYophysica Acta 306: 74, 1973.
12.
Lysz TW, Needleman P. Evidence for two distinct forms of fatty acid cyclooxygenase in brain. Journal of Neurochemistry 38: 1111, 1982.
13.
Bygdeman M, Bremme K, Gillespie A, Lundstrom V. Effects of the prostaglandins on the uterus. Prostaglandins and uterine contractility. Acta Obstetricia et Gynecologica Scandinavica Supplement 87: 33, 1979.
137
Hospital Practice
DIFFERENTIAL EFFECTS OF IBUPROFEN AND NAPROXEN SODIUM ON MENSTRUAL PROSTAGLANDIN RELEASE AND ON PROSTAGLANDIN PRODUCTION IN THE RAT UTERINE HOMOGENATE1 Andrea M. Powell and W. Y. Chan Department of Pharmacology Cornell University Medical College New York, New York 10021 (Reprint requests to WYC) ABSTRACT In two independent studies, ibuprofen and naproxen sodium were found to be equi-effective in alleviating dysmenorrheic symptoms. However, the effects of these drugs on menstrual PG release were found to be dissimilar. Ibuprofen primarily inhibited menstrual PGF2n release with little effect on PGE release, whereas, naproxen sodium inhibited both PGF20 and PGE releaze equally. To verify these results, we determined the inhzbitory potency, IC50, of ibuprofen and naproxen sodium on PGF20 and PGE2 synthesis in the rat uterine homogenate system. The preferential PGF2, inhibitory activity of ibuprofen was confirmed. These findings suggest that ibuprofen may, in addition to inhibiting fatty acid cyclooxygenase, also inhibit PGF2u reductase, or some and PGE synthesis diffother PG metabolic pathways which affect PGF erently. The significance of this different2* al PG syn$hesis inhibitory effect in dysmenorrheic therapy is discussed. INTRODUCTION In recent years, extensive clinical trials have established the effectiveness of nonsteroidal anti-inflammatory drugs (NSAIDS) in the treatment of primary dysmenorrhea (l-3). Although there were few direct comparative studies among the NSAIDs, individual drug trials showed that, with the exception of aspirin, the various NSAIDs tested so far were equi-effective. This clinical finding was interpreted by many as an indication that these agents alleviate dysmenorrhea principally by their common action as fatty acid cyclooxygenase inhibitors (4). %'his work was supported by research grants from NICHHD (HD-13064) and the NIGMS (GM-07547). 129
Recently, we completed two independent studies on dysmenorrhea, one with ibuprofen and the other with naproxen sodium, and the effects of these two agents on menstrual PG release (5,6). In accordance with other clinical trials, our results showed that ibuprofen and naproxen sodium were equally effective in relieving dysmenorrhea. It was therefore of great interest when we found that their effects on menstrual PG release were dissimilar. Because of the important clinical and pharmacologic implications of this differential effect between ibuprofen and naproxen sodium, we sought verification of this finding in a controlled biochemical system and determined the effects of these two agents on PG synthesis in the rat uterine homogenate. In this paper, we report the differential effects of ibuprofen and naproxen sodium on menstrual PG release in dysmenorrheic subjects and the results of our comparative study of the inhibitory activities of these two agents on uterine PG synthesis. METHODS AND MATERIALS Menstrual specimens Menstrual specimens were collected from two groups of dysmenorr-. heic subjects who volunteered for our ibuprofen trial and our naproxen sodium trial. Methods for specimen collection, menstrual PG extraction and clinical protocols were described in our published papers (5-7). In essence, the subject collected all of her tampon specimens for menstrual PG determination. Each subject was studied for at least three cycles. The protocol consisted of a control cycle followed by treatment cycles, either ibuprofen and placebo, or naproxen sodium and placebo, according to standard double-blind, cross-over procedure. Determination of PG synthesis innibitory activity The effects of ibuprofen and naproxen sodium on PGF2u and PGE biosynthesis from endogenous substrate were determined in non-pregnan$ rat uterine homogenates. Uterine horns from randomly chosen adult, virgin, Wistar albino rats were separated in matched-pair fashion, with one horn serving as control and the contralateral horn as the experimental sample. Four rats were used in each experiment. The horns were dissected out and immediately placed in ice-cold Krebs-Ringer bicarbonate solution (pH 7.4) for the control samples, or, ice-cold Krebs-Ringer bicarbonate solution with the appropriate concentration of the test drug for the experimental samples. After the wet weights were taken, the uterine horns were minced in 35 mls of the Krebs-Ringer bicarbonate solution or the Krebs-Ringer bicarbonate solution with drug, and homogenized while still on ice with a Polytron homogenizer (Brinkman Instruments) with two, 10 second bursts. The samples were then incubated in a bath shaker at 37'C for 60 minutes with continuous aeration with 95% 0 and 5% CO . At the end of the incubation period, an equal volume of z sopropyl az cohol was added. This stopped the enzyme activity and improved the efficiency of the PG extraction. The incubate was shaken for an additional five minutes and then centrifuged. The alcoholic supernatant was withdrawn and washed twice with an equal volume
130
of hexane to remove neutral fats and lipids. It was then acidified to pH 4 with 1 N HCl and extracted twice with chloroform. The chlorofo? phase was evaporated to a small volume with a rotary evaporator at 40 C under reduced pressure. The final drying step was carried out under nitrogen at atmospheric pressure. The residue was stored at -2O'C until assayed by RIA. The inhibitory activity of each drug was measured against its own matched-pair control and expressed as percent inhibition. At least three different concentrations were studied for each drug. The doseresponse curves for ibuprofen and naproxen sodium were plotted. Based on these curves, the IC 's, the concentrations of drug which gave 50% reduction of PG biosynti!O esis, for PGF2a and PGE2 were calculated and their 95% confidence limits computed. Assays
PG contents of the samples were measured by bioassays and/or RIAs. For bioassay, the rat fundal strip preparation as described by Vane (8) was used. The tissue was superfused with Krebs-Ringer bicarbonate solution at 37OC. Authentic PGF2c was used as the standard. Bracket assays were performed. A minimum of three bracket assays, each on a different fundal strip, were made on each sample. The mean value of the bioassays and the standard error of the mean were calculated. For RIAs, commercially prepared goat anti-PGF2, and either rabbit anti-PGE, A, B or goat anti-PGEl sera were used. The antiserum cross reactivities measured in our systems were: goat anti-PGF2ucrossreacts with PGE2 <3% with 6-keto-PGFlu <2.5% and has no detectable crossreactivity with TKB2. Goat anti-PGEl crossreacts with PGE2 and has'no detectable crossreactivity with PGF2c. Rabbit anti-PGE, A, B does not differentiate between PGEl and PGE2 and has no detectable crossreactivity with PGF2c. Crossreactivities with other PGs were not determined. The suppliers provided the data on crossreactivities with PGA, PGB and . These PGs are not present in the uterus in appreciable amounts, PGF the$Efore, crossreactivity with these PGs did not significantly affect our RIAs of PGF and PGE. However, PGE activity was assayed in PGE2 equivalents, s&e the antiserum did not2differentiate between PGEl and PGE2. For the RIAs of menstrual samples, the same PGE anti-serum was used to analyze all of the cycles from an individual subject. For the RIAs of PG synthesis inhibitory activity in uterine homogenates, the same PGE anti-serum was always used for each matched-pair, control and treatment. In the few cases where two different anti-PGE sera had to be used for the same series of matched-pair experiments, no significant differences were seen in the individual values of inhibitory potency. Each sample was determined in duplicate and at two different dilutions. The sensitivity of the assay was between 16 and 30 pg. The intraassay coefficient of variation was less than 10% and the interassay coefficient of variation was less than 15%. Parallel bioassays and RIAs for PGF o were carried out in a number of experiments for validation of the i? IA system.
131
MATERIALS Authentic PGF , PGE2, ibuprofen and the corresponding placebo were kindly supplie 2% by the Upjohn Co., Kalamazoo, MI. Naproxen sodium and the corresponding placebo were supplied by Syntex Research, Palo Alto, CA. Goat anti-PGF2, and anti-PGE sera were purchased from Research Products International, Mount Prospict, IL. Rabbit anti-PGE, A,B serum was urchased from Calbiochem-Behring Corp., LaJolla, CA. Multilabeled [';H ]-PGF2, and [~H]-PGE (loo-150 Ci/mmol) were purchased from New England Nuclear Corp., Bostzn, MA. RESULTS Effects of ibuprofen and naproxen sodium treatment on menstrual PG release The effects of ibuprofen and placebo treatment on menstrual PG release were determined by parallel bioassay and RIA in 4 subjects for a total of 18 cycles. The results are summarized in Table 1. Ibuprofen treatment, 400 mg q.i.d., but not placebo, caused a significant decrease in menstrual PG release, as determined by bioassay in PGFp equivalents. RLAs of the menstrual samples show that only the decrease in PGF release was significant (p < 0.01 by the paired 'It"test). The ef$" ect on PGE2 release was not statistically significant. In nearly all the samples measured, the PG activity determined by bioassay was greater than the sum total of PGF2a and PGE2 determined by RIA. Table 1.
Effects of Ibuprofen or Placebo Treatment on Menstrual PG Release
Total Menstrual PG Release Per Cycle in ng + S.E. Treatment Bioassay in PGF20 equivalents
RIA PGF2o
PGE2
PGF2a + PGE2
Control (4 patients, 4 cycles)
59.4 + 16.8
27.3 f 7.1
5.23 + 2.19
32.49 + 8.63
Ibuprofen (4 patients, 7 cycles)
16.7 t 2.8*
9.12 + 2.57* 3.42 + 1.54
12.54 -_ + 4.07
Placebo (4 patients, 7 cycles)
47.6 + 8.7
24.5 + 4.5
28.04 f 5.02
*
3.54 +_ 0.59
Difference in mean values of menstrual PG released during ibuprofen treatment cycles and either control or placebo cycles was highly significant, P < 0.01.
132
Naproxen sodium treatment 275 mg q.i.d. also markedly reduced menstrual PG release in dysmenorrheic subjects. However, unlike ibuand PGE2 profen, naproxen sodium therapy reduced both menstrual PGF release equally. Table 2 shows the effects of naproxen so&ium therapy on menstrual PG release in 12 subjects for a total of 36 cycles.
Table 2.
Effects of Naproxen Sodium or Placebo Treatment on Menstrual PG Release
Treatment
Total Menstrual PG Release Per Cycle in pg + S.E. RIA PGE2
pGF2a Control (12 patients, 12 cycles) Naproxen sodium (12 patients, 12 cycles) Placebo (12 patients, 12 cycles) *
23.19 + 4.75
5.09 + 1.30*
20.13 + 2.43
3.71 -+ 0.76
0.75 2 0.19*
3.22 + 0.48
Difference in menstrual PG released during naproxen sodium treatment cycles and either control or placebo cycles was highly significant, P < 0.01.
Although the effects of ibuprofen and naproxen sodium on menstrual PG release in these two trials were different, both therapies were equally effective in alleviating dysmenorrhea. The clinical results of these two drug trials have been published (5,6). PG synthesis inhibitory activity of ibuprofen and naproxen sodium in rat uterine homogenates Incubation of the rat uterine homogenates for 60 minutes in the presence of varied concentrations of ibuprofen or naproxen sodium resulted in a dose related inhibition of both PGF and PGE biosynthesis Table 3 shows the ICso 's and the calculated 95X2%onfidencg limits of ibuprofen and naproxen sodium for PGF and PGE2 biosynthesis in the rat uterine homogenate system. In th2* system, ibuprofen is more potent in inhibiting PGF synthesis than PGE synthesis. The ratio of the IC50-PGE2 : IClp-RF 2,_+ expresses the dzfference in the degree of preferential inhib ion o PGFB biosynthesis over PGE2 by ibuprofen and naproxen sodium. This is also shown in Table 3.
133
Table 3.
's of Ibuprofen and Naproxen Sodium in the Rat Uterine IC50 Homogenate System
Drug
- PGF2o IC50 (mean & 95% confidence limits)
- PGE2 IC50 (mean h 95% confidence limits)
IC50
IC50
- PGE2
- PGF2o
Ibuprofen (n = 11)
1.1 x 10-6M (0.7 - 1.7)
3.8 x lO+jM (2.5 - 5.8)
3.5
Naproxen Sodium (n = 13)
2.8 x 10-7M
2.6 x 10-7M
0.9
(2.2 - 3.6)
(1.7 - 4.0)
DISCUSSION NSAIDs are effective agents for the treatment of primary dysmenorrhea. The effectiveness of this class of drug is assumed to be the result of their common action, their ability to inhibit fatty acid cyclooxygenase. Inhibition of this enzyme which catalyzes the conversion of arachidonic acid to the endoperoxides, would logically result in a non-selective inhibition of both PGF* and PGE2 biosynthesis, We found that ibuprofen and naproxen sodium, the two most commonly used NSAIDs in dysmenorrheic therapy, each affects menstrual-PG release differently. When administered for the first three days of menses in comparable doses recommended for the treatment of rheumatoid arthritis, ibuprofen therapy suppressed primarily menstrual PGF release with little effect on PGE2 release, whereas, naproxen sodI* urn therapy suppressed both PGFp and PGE2 release equally. The percent suppression of menstrual PG release in the treatment cycles relative to the control cycles for ibuprofen was 66.6% for PGF20 and 34.6% for PGE2 (statistically not significant) and for naproxen sodium was 78.1% for PGF20 and 79.8% for PGE2. The different effects of ibuprofen on menstrual PGF2a and PGE release may suggest that the doses of ibuprofen were not sufficienZ , in comparison to the naproxen sodium trial, to produce a consistent and PGE2. However, ibuprofen suppression of both menstrual PGF therapy produced a greater percentoreduction in PGF20 release than in PGE2 release in six of the seven treatment cycles. This clearly indicates a greater sensitivity of uterine PGF2u than PGE2 to ibuprofen suppression. This preferential PGF2, inhibitory activity of ibuprofen was confirmed in a more quantitative study using rat uterine homogenates. In this study, we measured the PGF2o synthesis and the PGE2 synthesis inhibitory potencies of ibuprofen simultaneously, in the same homogenate preparation, and calculated the respective IC50 values. The
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IC50 for PGE was nearly four times higher than the IC50 for PGF2 . Thus, ibupro$en appears to have a preferential effect on PGF2, syffthesis. Naproxen sodium did not display this preferential inhibition of PGF Its IC50-PGE2:IC50-PGF2o ratio was near unity. 2a' It should be pointed out that in each experiment, the IC50 determinations for PGF2c and PGE2 were always carried out simultaneously, in the same homogenate, and with the matched-pair controls incubated at the same time. The uterine horns used in these experiments were taken from rats chosen at random. Our own experience as well as that reported by Poyser and Scott (9) have shown that in nonpregnant uterine homogenates using endogenous substrate, without addition of cofactors, the productions of PGF2, and PGE2 were not significantly affected by the estrus cycle. Our use of the matched-pair control in each experiment further minimized the effect of the small cyclic variance of PG production on the IC50 determination. Our finding that ibuprofen preferentially inhibits uterine PGF20 biosynthesis over PGE2 biosynthesis, suggests that ibuprofen may, in addition to inhibiting fatty acid cyclooxygenase, also inhibit PGF2, reductase, which catalyzes the enzymatic conversion of PGH2 to PGF20. Also, it is well known that NSAIDs may affect PG synthesis differently in different tissues, suggesting that the PG generating system exists in multi-molecular forms (10). Our findings with ibuprofen are of particular interest because they may suggest a heterogeneity of PG synthesizing enzymes within the same tissue. One is tempted to speculate that two separate and distinct fatty acid cyclooxygenases may exist in the uterine PG system, one associated with PGF2o reductase and the other with PGE2 isomerase which can be affected individually by the NSAIDs. The idea of the existence of isomers in the PG synthetase complex is not new. As early as 1973, Maddox suggested the existence of two distinct forms of PG synthetase complex in sheep vesicular tissue (11). However, recently, Lysz and Needleman presented biochemical evidence for the existence of two distinct forms of fatty acid cyclooxygenase in the rabbit brain which can be differentially inhibited by the NSAIDs (12). It is also possible that ibuprofen might inhibit 9-keto-reductase which converts PGE2 to PGF2o. This would result in a preferential inhibition of PGF20. However, the inhibition of this pathway by ibuprofen does not appear to be a significant factor. Our preliminary study has not demonstrated an active conversion of 13HlPGE2 to i3H]PGF20 in the uterine homogenate system used'in our study. Of equal interest is our finding that, although subjects treated with naproxen sodium had a greater suppression of menstrual PG release than subjects treated with ibuprofen, there was no clear difference in clinical efficacy between the two drugs in our clinical trials (5,6). However, the majority of the patients treated with naproxen sodium, but not patients treated with ibuprofen, showed a reduction in their menstrual
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fluid volume (5,6). The effect of naproxen sodium on the menstrual fluid volume probably is related to its ability to inhibit PGE2 release as well as PGF2d. It is possible that the apparent equi-effectiveness of ibuprofen and naproxen sodium in dysmenorrheic therapy may be the result of administering doses which are clinically supramaximal. It may not be necessary to suppress menstrual PG release to the degree attained by the trial dose of naproxen sodium or ibuprofen in order to ameliorate dysmenorrheic symptoms. Neither the normal range of menstrual PGs nor the optimal dose for these drugs in dysmenorrheic therapy has been determined. It has been shown that during menstruation, PGFZc,is a uterine stimulant while PGE2 can act as a uterine relaxant (13). A drug such as ibuprofen, which preferentially inhibits PGF2, over PGE2 may manipulate the menstrual PG profile more favorably. On the other hand, a drug such as naproxen, which inhibits both PGFzc,and PGE2 will also reduce menstrual fluid volume, an effect desirable in dysmenorrhea with menorrhagia. This consideration should be germane to drug choice and to the further development of dysmenorrheic therapy. ACKNOWLEDGMENTS The authors wish to acknowledge the skillful technical assistance of Ms. Donna Robertson, Ms. Maria Sergi and Ms. Elizabeth Zaff. They also wish to thank Thomas J. Vecchio, M.D., of The Upjohn Co., for his active support. REFERENCES 1.
Chan WY. Prostaglandin inhibitors and antagonists in dysmenorrhea therapy. p 209 in Dysmenorrhea. (MY Dawood ed) Williams and Wilkins, Baltimore, 1981.
2.
Jacobson J, Cavalli-Bjorkman K, Lundstrom V, Nilsson B, Norbeck M. Prostaglandin synthetase inhibitors and dysmenorrhea. A survey and personal clinical experience. Acta Obstetricia et Gynecologica Scandinavica Supplement 87: 73, 1979.
3.
Ylikorkala 0, Dawood MY. New concepts in dysmenorrhea, American Journal of Obstetrics and Gynecology 130: 833, 1978.
4.
Vane JR. Inhibition of prostaglandin biosynthesis as the mechanism of action of aspirin-like drugs. Advances in Biosciences 9: 395, 1973.
5.
Chan WY, Dawood MY, Fuchs F. Prostaglandins in primary dysmenorrhea, comparison of prophylactic and nonprophylactic treatment with ibuprofen and the use of oral contraceptives. American Journal of Medicine 70: 535, 1981.
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6.
Chan WY, Fuchs F, Powell AM. Effects of naproxen sodium on menstrual prostaglandins and primary dysmenorrhea. Obstetrics and Gynecology 61: 285, 1983.
7.
Chan WY, Hill JC. Determination of menstrual prostaglandin levels in non-dysmenorrheic and dysmenorrheic subjects. Prostaglandins 15: 365, 1978.
a.
Vane JR. A sensitive method for the assay of 5-hydroxytryptamine. British Journal of Pharmacology 12: 344, 1957.
9.
Poyser NL, Scott FM. Prostaglandin and thromboxane production by the rat uterus and ovary -in vitro during the oestrous cycle. Journals of Reproduction & Fertility 60: 33, 1980.
10.
Vane JR. Prostaglandins and aspirin-like drugs. 7: 61, 1972.
11.
Maddox IS. The role of copper in prostaglandin synthesis. Biochemica et BYophysica Acta 306: 74, 1973.
12.
Lysz TW, Needleman P. Evidence for two distinct forms of fatty acid cyclooxygenase in brain. Journal of Neurochemistry 38: 1111, 1982.
13.
Bygdeman M, Bremme K, Gillespie A, Lundstrom V. Effects of the prostaglandins on the uterus. Prostaglandins and uterine contractility. Acta Obstetricia et Gynecologica Scandinavica Supplement 87: 33, 1979.
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Hospital Practice