THE ANTAGONISM BY ANTI-INFLAMMATORY ANALGESICS OF PROSTAGLANDIN F2,-INDUCED CONTRACTIONS OF HUMAN AND RABBIT MYOMETRIUM -IN VITRO.

Ian D. Smith, Diana M. Temple* and Rodney P. Shearman Department of Obstetrics and Gynaecology and Department of Pharmacology,* University of Sydney, New South Wales, 2006, Australia.

ABSTRACT

The anti-inflammatory analgesic drugs, aspirin, indomethacin, phenylbutazone, mefenamic acid, ibuprofen and flurbiprofen are shown to inhibit in a dose-dependent manner the force of cont;action of isolated human pregnant myometrial strips which have been stimulated to contract by adding prostaglandin (PG) F2a to the tissue bath. These drugs and also flufenamic acid and salicin show a similar antagonism of the action of PGF2, with isolated rabbit non-pregnant myometrium. The ratio of the inhibitory concentration -in vitro to the maximum plasma level after a normal dose -in vivo suggests that phenylbutazone and possibly ibuprofen may be capable of inhibiting human uterine contractions in vivo. Patients who were treated with aspirin during induction of abortion using PGF2, during the second trimester of pregnancy showed no significant change in the induction-abortion interval compared with patients not taking aspirin.

This study was supported by a grant from the National Health and Medical Research Council.

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INTRODUCTION

Anti-inflammatory analgesics are known to inhibit the biosynthesis of prostaglandins (1,2,3) and have been shown to prolong gestation and parturition in primates (4,5) and rats (6,7,8,9) as a result of such inhibition.

Aspirin, indomethacin

and mefenamic acid have been shown to significantly prolong, in a similar manner, the interval to abortion following the intraamniotic infusion of hypertonic saline (10,11,12,13). Fenamates, aspirin, phenylbutazone and indomethacin have further been shown to antagonise PGF2o-induced contractions of smooth muscle (14,15,16). Although analgesic and psychotropic drugs are widely used for the control of side-effects in patients receiving PGF2cl for the induction of middle trimester abortion, there have been few studies of the effect of such concimitant medication upon the ensuing interval to abortion (17,18). This paper describes the -in vitro effects of antiinflammatory analgesics upon PGF2o-induced contractions in human and rabbit myometrial strips, carried out as part of a more comprehensive study of the clinical and -in vitro effects of analgesic and psychotropic drugs upon PGF2o-induced uterine activity. It further describes the effect of medication with aspirin upon the interval to abortion following the intra-amniotic injection of PGF2o for the termination of middle trimester pregnancy.

MATERIALS AND METHODS

Isolated human myometrium:

Small segments of human myometrium were

obtained from patients undergoing hysterotomy during the middle trimester of pregnancy.

42

Longitudinal strips from this tissue were set

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up in a 15 ml. organ bath, with the upper end connected to a lever through which contractions were measured isotonically by a transducer on a Heathkit pen recorder.

The myometrial strips were immersed in

Ringer-Locke solution (NaCl 9 g/l, KCL 0.42 g/l, CaC12.2H20 0.31 g/l, NcHc03 0.5 g/l and glucose 1 g/l) at 37', through which a mixture of 5% carbon dioxide in oxygen was bubbled continuously.

PGF2, was

added to the bath in a dose of 500 ng in order to produce strong, regular contractions and the preparation was allowed to stabilise for 40 - 60 minutes before further treatment. Each drug was dissolved in isotonic saline or Ringer-Locke solution, and aspirin, mefenamic and flufenamic acids were dissolved as their sodium salts.

The cumulate dose technique of van Rossum (19)

was used, so that incremental amounts of drug solution were added at 20 minute intervals without washing out the previous addition, and the drug concentrations were chosen so that no more than a total volume of 1.5 ml was added to the 15 ml organ bath in any one experiment.

The initial drug concentrations were selected to resemble, as

far as possible, human plasma concentrations after normal doses. one drug was tested on any one isolated myometrial preparation.

Only The

responses were measured in terms of the initial PGF20-induced amplitude of contraction of the muscle prior to the addition of any other drug. Acidic drugs were administered as sodium salts and the pH of the bath contents did not vary significantly from 7.3. Isolated rabbit myometrium Both uterine horns were dissected from freshly-killed, mature, non-pregnant rabbits, and myometrial strips were set up as described above.

Each drug was tested on at least four preparations of rabbit

myometrium, and individual log concentration-response relationships determined for each preparation.

The mean percentage inhibition values

for each dose level were graphed for each drug, and log concentrationresponse curves were constructed, from which the ID50 (mean concentration which depressed the amplitude of contraction by 50% from its initial value) could be measured.

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Salicin was tested because it has analgesic action, and its aglycone (salicyl alcohol) has been used in the past in its unrefined form from'viburnum prunifolium as a uterine sedative. The possibility of metabolism of the drugs after addition to the organ bath is remote, but the stability of salicin to hydrolysis was tested by measuring its concentration by ultra-violet spectroscopy in Ringer-Locke solution, suitably diluted, before and after incubating with myometrium at 37' for 1 hour.

Specificity Specificity was tested using both single and cumulative doses of salicin and mefenamic acid on the isolated rabbit myometrium. Contractions of the muscle were induced with both acetylcholine and oxytocin, and concentrations of salicin and mefenamic acid equivalent to the ID50 and ID90 concentrations for PGF2o-induced contractions were added.

The cumulative dose technique was used only with oxytocin-

induced contractions. Clinical Studies All patients were between 12 and 20 weeks pregnant and were admitted to hospital at S-9 a.m. on the morning of treatment.

At 6 p.m.

30 mg of PGF2, was injected transabdominally into the amniotic sac, followed, if necessary, by doses of 15 mg at 24 and 42 hours. Approximately 30 minutes before PGF2o injection, all of the patients were premeditated with meperidine hydrochloride 100 mg and metoclopramide 10 mg by intramuscular injection.

This medication was

repeated at intervals of 3 hours throughout the subsequent interval to abortion.

NO

other medication was received during the induction-

abortion interval except in a randomly selected group which also received oral medication with aspirin 600 mg three-hourly.

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Drugs: The following drugs were received as gifts:

PGF2u (as the

tromethamine salt) from the Upjohn Company; aspirin from Reckitt and Colman; indomethacin from Merck, Sharpe and Dohme; phenylbutasone from Geigy Pharmaceuticals; mefenamic and flufenamic acids from Parke, Davis & Co.; ibuprofen and flurbiprofen from Boots.

Salicin was pur-

chased from Koch-Light.

RESULTS AND DISCUSSION Isolated Myometrial preparations The -in vitro preparations did not consistently contract spontaneously but the contractions induced by a single addition of 33 ng/ml of PGF2, became strong and regular in amplitude and rate within 30 minutes, and remained so, in control preparations, for at least 3 hours, although the baseline tone sometimes fell slightly, Lower concentrations of PGF2a did not produce maximal amplitude of contraction.

Typically, the contractile force was equivalent to 2 g.

and the frequency of contraction approximately 1 per minute, although there was considerable variation between muscle preparations. The addition of analgesic drugs in cumulative concentrations resulted in a concentration-dependent fall in amplitude of contraction. In some cases the rate of contraction was also reduced but this parameter was less reliable than amplitude. Log concentration response curves for the inhibition of contraction of human myometrium by anti-inflammatory analgesic drugs are shown in Figure 1; corresponding results for rabbit myometrium are shown in Figures 2 and 3.

With the exception of salicin (Figure 31,

which was inert at concentrations effective with rabbit tissue, all these drugs depressed PGF20-induced contractions of both human and rabbit myometrium.

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Standard errors of the mean percentage inhibition

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for each concentration of each drug, which were calculated for all the results and were usually approximately 5%, have been omitted from Figures 1 and 2 for the sake of clarity, but have been included in Figure 3. Salicin was found to be the most potent inhibitor in this series when tested on the isolated rabbit myometrium.

It proved to

be stable to hydrolysis under the conditions of the experiment, since its ultraviolet spectrum was not affected by incubation in RingerLocke solution in the presence of rabbit myometrium for 1 hour. Its potency -in vitro with rabbit myometrium suggests that further investigation is warranted. However, -in vitro studies with human middle trimester myometrial strips (7 preparations) indicated that a concentration of 5 x 10B3M was still less than the ID50. Figure 3 indicates that, at concentrations active on rabbit tissue, the curve from pregnant human myometrium was not significantly different from controls.

The inhibitory concentrations of all the drugs tested,

with the exception of salicin, were similar on human pregnant and rabbit non-pregnant myometrium, as previously demonstrated (18). Table 1 shows the relative potencies of the inhibitors of PGF20-induced contractions, expressed as the molar dose required to produce 50 percent depression of the amplitude of contraction (ID50). Each result represents the mean of at least 4 determinations with isolated rabbit myometrium, but owing to the limited availability of human middle trimester myometrium, the results for each drug on this tissue were obtained from 1 to 7 preparations.

With the exception of

salicin, the potency of each drug was similar on both tissues. Table II shows the relationship between the plasma concentration of these drugs after single doses in humans, as recorded in the literature, and their effective concentration as inhibitors -in vitro.

46

A drug whose maximum plasma concentration exceeds or approxi-

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mately equals the ID50 might be expected to inhibit PGF2o-induced myometrial contractions _in vivo. In the human, of the drugs tested, phenylbutazone might be in this category, although its high degree of protein binding reduces the effective plasma concentration by about 90%.

A comparison between human plasma levels and effective concen-

trations on the rabbit preparation -in vitro is valid only as an indication that phenylpropionic acid derivatives (ibuprofen, flurbiprofen etc.), possibly the fenamates as well as phenylbutazone, should be further studied as possible prostaglandin antagonists. The present -in vitro results confirm the earlier suggestion (23) of the antagonism of PGF2,-induced contractions of myometrium by fenamates -in vivo and extend reports of antagonism of PGF2o-induced contractions of bronchial, tracheal and pulmonary venous smooth muscle by fenamates and other anti-inflammatory analgesics in vitro (14,15,16). Specificity After the addition of single doses of salicin, equivalent to the ID50 and IDgO for PGF2o-induced contractions, to the isolated rabbit myometrium, both oxytocin and acetylcholine gave apparently unimpaired responses.

With mefenamic acid, however, the ID50 and IDgO treatment

produced sustained hypertonic spasm when oxytocin was added and an impaired response (15 per cent reduction in amplitude of contraction) to acetycholine. Using the cumulative dose technique (191, oxytocin-induced contractions of rabbit myometrium (oxytocin concentration of 7 x 10m4 units/ml) were found to be sensitive to salicin, the ID50 being 5 x 10m7M or less than half that for PGF2o-induced contractions. Following complete inhibition of oxytocin-induced contractions by salicin, the addition of a second dose of oxytocin restored contractions with an amplitude between 30 and 100 per cent of the original (8 experiments).

Mefenamic acid also inhibited oxytocin-induced contractions,

with an ID50 apparently identical to that for PGFgo-induced contrac-

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tions.

Contractions could not be restored following inhibition by

mefenamic acid.

Induction-abortion interval Regular medication with aspirin had no significant effect upon the interval to abortion following the intra-amniotic injection of PGF2cl,although in both nulliparous and parous patients the interval was reduced (Table III).

TABLE III

The effect of medication with aspirin upon the inductionabortion interval following the intra-amniotic administration of PGF2o. Number of patients is shown in parentheses.

Medication (three hourly)

Induction-abortion interval (Hours, mean f S.E.) Nulliparous patients

Parous patients

Routine (meperidine 100 mg + metoclopramide 10 mg)

27.7 + 1.9 (60)

23.5 + 2.7 (32)

Routine + aspirin 600 mg

25.6 f 4.3 (10)

17.2 + 4.1 (5)

These results support the evidence in Table II for the peak plasma level of aspirin not reaching the effective concentration for inhibition of PGF 2a-induced myometrial contractions.

Further, since

medication with aspirin at similar or lower dosage levels has been shown to prolong significantly the induction-abortion (I-A) interval following the intra-amniotic instillation of hypertonic saline (11) as well as the duration of pregnancy and spontaneous labor at term (4), the results indicate that endogenous prostaglandin synthesis is not of significance with regard to the duration of the I-A interval following

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the intra-amniotic

administration

of PGF2,.

Indeed, the slightly

shorter duration of I-A interval associated with aspirin medication, in both nulliparous and parous patients, may be associated with an inhibition of the 15-hydroxy dehydrogenation of the exogenous PGF2o: plasma levels of aspirin are of the order of 10m4M (peak level = 3-O x 10e4>, a concentration that has been shown, in vitro, to significantly depress the oxidation of the 15 (S) hydroxy group by 15-hydroxy prostaglandin dehydrogenase (24) and thus, potentially, to delay the inactivation of exogenous PGF2,.

Further studies of the

effect of medication with aspirin upon the induction of abortion with PGF2, are in progress. At present, however, it is obvious that aspirin can be used for analgesia during the induction of middle trimester abortion with PGF20 without any prolongation of the interval to abortion.

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REFERENCES

1.

Flower, R., R. Gryglewski, K. Herbacynska-Cedro and J.R. Vane. Effects of anti-inflammatory drugs on prostaglandin biosynthesis. Nature New Biol., 238:104, 1972.

2.

Vane, J.R. Inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs. Nature New Biol., 231~232, 1971.

3.

Vane, J.R. Inhibition of prostaglandin biosynthesis as the mechanism of action for aspirin-like drugs. Adv. Biosci., 9:395, 1973.

4.

Lewis, R.B. and J.C. Schulman. Influence of acetysalicyclic acid, an inhibitor of prostaglandin synthesis, on the duration of human gestation and labour. Lancet, 2: 1159, 1973.

5.

Novy, M.J., M.J. Cook and L. Manaugh. Indomethacin block of normal onset of parturition in primates. Am.J. Obstet. Gynecol., 118: 412, 1974.

6.

Aspirin and indomethacin prolong parturition in Aiken, J.W. rats: evidence that prostaglandins contribute to expulsion of foetus. Nature, 240: 21, 1972.

7.

Chester, R., M. Dukes, S.R. Slater and A.L. Walpole. Delay of parturition in the rat by anti-inflammatory agents which inhibit the biosynthesis of prostaglandins. Nature, 240: 37, 1972.

8.

Csapo, A.I., E.F. Csapo, E. Fay, M.R. Henzl and G. Salau. The delay of spontaneous labour in the rat model. Prostaglandins, 3: 827, 1973.

9.

Waltman, R., V. Tricomi, E.H. Shabanah and R. Arenas. The effect of anti-inflammatory drugs on parturition parameters in the rat. Prostaglandins, 4: 93, 1973.

10.

Waltman, R., V. Tricomi and A.B. Palav. Mid-trimester hypertonic saline-induced abortion: effect of indomethacin on inductionabortion time. Am. J. Obstet. Gynecol., 114: 829, 1972.

11.

Waltman, R., V. Tricomi and A. Palav. Aspirin and indomethacin: effect on instillation-abortion time of mid-trimester hypertonic saline induced abortion. Prostaglandins, 3: 47, 1973.

12.

Waltman, R. and V. Tricomi. hypertonic saline abortion.

50

Pentazocine, mefenamic acid and Lancet, 2: 468, 1974.

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13.

A. Kivikoski, M.O. Csapo, A.I., M.R. Henzl, H.L. K'aihol'a, Pulkkinen. Suppression of uterine activity and abortion by inhibition of prostaglandin synthesis. Prostaglandins, 7: 39, 1974.

14.

Collier, H.O.J. and W.J.K. Sweatman. Antagonism by fenamates of prostaglandin F2a and of slow reacting substance on human bronchial muscle. Nature, 219: 864, 1968.

15.

Farmer, J.B., D.G. Farrar and J. Wilson. Antagonism of tone and prostaglandin-mediated responses in a tracheal preparation by indomethacin and SC-19220. Br. J. Pharmac., 52: 559, 1974.

16.

Burka, J.F. and P. Eyre. Studies of prostaglandins and prostaglanclinantagonists on bovine pulmonary vein -in vitro. Prostaglandins, 6: 333, 1974.

17.

Brenner, W.E., J.R. Dingfelder, C.H. Hendricks and L. Staurovsky. Induction of therapeutic abortion with a single dose of intraamniotically administered prostaglandin F2a. Prostaglandins, 4: 485, 1973.

18.

Smith, .I.D. and D.M. Temple. The influence of analgesic drugs on the actions of prostaglandin F2clon the human uterus -in vivo and human and rabbit myometrial strips -in vitro. Prostaglanclins, 4: 469, 1973.

19.

Van Rossum, J.H. Cumulative dose-response curves. 11. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. Int. Pharmacodyn., 143: 299, 1963.

20.

Fenamates in medicine. Glazko, A.J. Supplement to Annals of Physical Medicine (Hume Kendall, P., editor) Bailliere, Tidal1 and Cassel, London, 1966.

21.

Mills, R.F.N., S.S. Adams, E.E. Cliffe, W. Dickinson and J.S. Nicholson. The metabolism of ibuprofen. Xenobiotica 3: 589, 1973.

22.

Chalmers, I.M., B.J. Cathcart, E.B. Kumar, W.C. Dick and W.W. Buchanan. Clinico-pharmacological studies and clinical evaluation of flurbiprofen, a new non-steroidal anti-inflammatory agent. Ann. Rheum. Dis., 31: 319, 1972.

23.

Levy, B. and H.R. Lindner. Selective blockade of the vasodepressor response to prostaglandin F2a in the anaesthetized rabbit. Br. J. Pharmac., 43: 236, 1971.

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24.

52

Hansen, H.S. Inhibition by indomethacin and aspirin of 15-hydroxyprostaglandin dehydrogenase -in vitro. Prostaglandins, 8: 95, 1974.

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'TABLE I

Relative potencies of some anti-inflammatory analgesic drugs as antagonists of PGF2, -induced contractions of isolated myometrial strips, expressed as the dose in moles/litre required to depress the amplitude of contraction of the muscle to 50% of its initial value (ID50). Figures in parentheses represent number of preparations studied.

Drug

ID50 human pregnant myometrium CM) x 10-4 (3)

ID50 rabbit non-pregnant myometrium CM)

Aspirin

4.5

Indomethacin

4*3 x 10-5 (1)

4.0

Phenylbutazone

3.4

x 10-5 (3)

9*4 x 10-5 (5)

Mefenamic Acid

l-2 x 10-4 (3)

7.6 x lO-5 (7)

Flufenamic Acid

3*6 x lO-4 (4) x 10-6 (5)

5-o x 10-5 (5)

Ibuprofen

5-7 x 10-5 (3)

4.5 x 10-5 (5)

Flurbiprofen

2-5 x lO-5 (1)

2.9 x lO-5 (5)

Salicin

>l

1*2 x 10-6 (4)

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x 10-3 (7)

53

10

22

Flufenamic acid

Ibuprofen

Salicin

3

10

Mefenamic acid

Flurbiprofen

55

Aspirin

1.8

Indomethacin

(22)

(21)

(20)

(20)

(1)

(1)

(1)

>300

6-O

11.7

28.9

81

15.5

0*34

7-O

9*3

14-o

18.3

67

l-4

29-o

10.3

150

rabbit myometrium

human myometrium

(Pg/ml)

(reference)

ID50 (pg/ml) against PGF2,induced contractions

Phenylbutazone

Drug

Peak plasma levels in man

o-5

1.9

0.35

0.68

o-12

14.5

0.43

2*4

o-71

o-55

O-8

1*3

5*2

Ratio peak plasma level ID50 __ . human rabbit

The relationship between peak plasma concentrations of anti-inflammatory analgesic drugs and inhibitory concentrations in vitro.

TABLE II

Figure 1:

0

20

40

60

80

loo

DOSES)

acid

Log dose response curves for anti-inflammatory drugs, from experiments with isolated human myometrial strips which had been stimulated by the addition of PGF2, (33 ng/ml) to give regular contractions representing 100% response.

(IN CUWlATlM

MOLAR CONCENlRATlON

3 Mhnamic

2 Aspirin

1 klicin

PROSTAGLANDINS

Figure 2:

56

Log dose response curves for anti-inflammatory analgesic drugs from experiments with isolated rabbit myometrium, pre-stimulated -in vitro by PGFza.

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Figure 3:

0

’ CONCENTRATION

(IN CUMUCATIVE DOSES)

MOM

10-6

10-5

The response to salicin of isolated human and rabbit myometrium, stimulated to contract by pre -addition of PGF2,. Vertical bars represent standard errors of the mean responses.

10-7

RABBIT

HUMAN

NON-PREGNANT

PREGNANT

The antagonism by anti-inflammatory analgesics of prostaglandin f 2 alpha-induced contractions of human and rabbit myometrium in vitro.

The anti-inflammatory analgesic drugs, aspirin, indomethacin, phenylbutazone, mefenamic acid ibuprofen and flurbiprofen are shown to inhibit in a dose...
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