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A prostaglandin analogue which potently relaxes human uterus but not gut muscle
European Journal of Pharmacology, 81 (1982) 141-143
141
Elsevier Biomedical Press
Short communication A PROSTAGLANDIN ANALOGUE WHICH POTENTLY RELAXES HUMAN UTERUS BUT NOT GUT MUSCLE GARETH J. SANGER *, ANNE JACKSON and ALAN BENNETT **
Department of Surgery, King's College Hospital Medical School, London SE5 8RX, U.K. Received 27 January 1982, accepted 13 April 1982
G.J. SANGER, A. JACKSON and A. BENNETT, A prostaglandin analogue which potently relaxes human uterus but not gut muscle, European J. Pharmacol. 81 (1982) 141-143. A prostaglandin analogue, (-+)-5-(6-carboxyhexyl)-l-(3-cyclohexyl-3-hydroxypropyl) hydantoin was tested on strips of human isolated myometrium and gastro-intestinal muscle. The analogue usually caused marked dose-dependent myometrial relaxation and inhibition of spontaneous activity, with a threshold concentration sometimes of 10 p g / m l and a maximum effect at 10 ng/mi. In contrast 1-10000 ng/ml had no effect on longitudinal muscle strips of humam stomach or colon. Prostaglandin analogue
Gastrointestinal muscle
Myometrium
1. Introduction
Many prostaglandin (PG) analogues have been tested as abortifacients. Some, such as 15(S)-15methyl PGE2, 15(S)-15-methyl PGF2~, and 16,16dimethyl PGE 2 administered by various routes, show greater activity on the myometrium in vivo than does the parent compound, and have less gastro-intestinal activity (Wiqvist et al., 1975). Similarly, with animals in vivo, certain prostaglandin analogues Mfect the uterus more than other tissues (Loge et al., 1978; Oshima et al., 1978). These results may mean that prostaglandin receptors in the myometrium differ from those in the gut, or that other factors may be involved in vivo, such as differences in pharmacokinetics, the route of administration, and the interaction of the analogue with responses to nerve stimulation and hormones. We have now tested a PG analogue, ( ---)-5-(6carboxyhexyl)- 1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C; fig. 1), on human isolated
Human smooth muscle
0
/~r~C02 0
H
OH
Fig. 1. The structure of BW245C.
myometrium and gastro,intestinal muscle. These tissues usually relax in response to PGI 2 (Omini et al., 1978; Bennett and Sanger, 1979, 1980; Bennett et al., 1981; Sanger et al., 1981; Wilhelmsson et al., 1981), but contract strongly in response to epoxymethano analogues of PGH 2 (Sanger and Bennett, 1979; Bennett et al., 1981; Sanger et al., 1981). We now report that BW 245C showed greater potency than PGI 2 on human myometrial strips, but lacked activity on gastro-intestinal muscle tone.
2. Materials and methods * Present address: Beecham Pharmaceuticals, Harlow, Essex,
U.K. ** To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
Macroscopically normal specimens of human myometrium, stomach and transverse and sigmoid
142
were obtained at operation for benign or malignant disease. Uterus was resected for menorrhagia (5 cases), menorrhagia and prolapse, pelvic endometriosis, irregular periods and back pain, and prolapse (1 case each). Subsequent examination showed benign leiomata (3 cases), no abnormality (2 cases), endometrial haemorrhage and necrosis, myometrial foci of endometriosis, cervical inflammation, and cervicitis with ulceration (1 case each). The phase of the menstrual cycle in which the myometrium was taken was classified histologically as either proliferative or secretory. Gastric muscle was removed for prepyloric ulceration, oesophageal or gastric carcinoma (1 case each) and colon was removed for rectal carcinoma or faecal incontinence (2 and 1 cases). The uterine tissue was from patients aged 31-44 (median 40) years, and the gut tissue was from 4 female and 2 male patients aged 19-79 (median 65) years. The tissue was either used immediately (3 uteri, 2 gut) or stored overnight (6 uteri, 4 gut) at 4°C in Krebs solution equilibrated with 5% CO 2 in 02. Strips of myometrium approximately 4 × 4 m m thick and 2 - 3 cm long were cut from the uterine body, approximately parallel to the longitudinal axis. Strips of gastro-intestinal muscle 4 - 5 m m wide and 2 - 3 cm long, were cut parallel to the longitudinal muscle fibres (the taenia was used as the colonic longitudinal muscle) after cutting away the mucosa and submucosa. Each strip was suspended under a load of 1 g in a tissue bath containing Krebs solution (NaC1 118.5; CaC12 2.5; K H 2 P O 4 1.2; KCI 4.7; MgSO 4 1.2; N a H C O 3 2.5; dextrose 11.7 m M at 37°C and bubbled with 5% CO 2 in 02. Isotonic responses were magnified 8-16 times. BW245C was dissolved in ethanol (5 m g / m l ) and stored at - 2 0 ° C . Fresh solutions, prepared from this by diluting with Krebs solution, were kept at 4°C until 10-15 min before the experiment, and then allowed to achieve room temperature. When spontaneous myometrial contractions, or acetylcholine-induced gut contractions were consistent, BW245C or its vehicle controls were added (contact times respectively 15 and 10 min for the two types of muscle).
ij B'W
B-W 0"001
a, o 0 A 1A1A
C
BW 1
C
BW 10
"i-
Fig. 2. Top trace: BW 245C (BW) reproducibly inhibited
myometrial spontaneous activity and reduced the muscle tone. acetylcholine (A) in contact with the tissue for 1 min contracted gastric muscle, and PGI 2 1 ~g/ml (I) caused relaxation. However, BW245C (BW) at a concentration 10000 times higher than that maximally inhibiting the myometrium had no effect. The numbers below A and BW represent/~g/ml. Vehicle controls (C) did not affect the tissues. Horizontal calibration 30 min, vertical calibration 5 cm. B o t t o m trace:
3. Results
BW245C caused marked, dose-dependent myometrial relaxation a n d / o r inhibition of spontaneous activity (fig. 2), except in one specimen of myometrium (cervicitis with ulceration, proliferative phase) in which BW245C up to 10 n g / m l had no effect. Sometimes the threshold concentration was as low as 0.01 n g / m l , and the maximally effective concentration was 10 n g / m l (n = 8; 4 proliferative and 4 secretory). Overall, the results were similar with myometrium from either the proliferative or the secretory phase. In contrast, BW245C 1 n g / m l to 10 t t g / m l had no effect on stomach or colonic longitudinal muscle tone (both n = 3 ) , whereas sodium P G I 2 1/~g/ml usually caused marked relaxation of the gastric tissues.
4. Discussion
P G I 2 potently relaxes human gastric tissues, in agreement with our earlier experiments in which
143 c o n c e n t r a t i o n s as low as 5 0 - 2 0 0 n g / m l relaxed gastric strips a n d 1 - 1 0 # g / m l i n h i b i t e d colonic strips (Bennett et al., 1981). P G I 2 also relaxes h u m a n m y o m e t r i u m , with threshold c o n c e n t r a tions of 10 n g / m l to 1 # g / m l (Sanger et al., 1981). C o m p a r e d with P G I 2 , B W 2 4 5 C therefore seems m u c h less active o n isolated gastrointestinal muscle, b u t m u c h m o r e p o t e n t on h u m a n m y o m e t r i a l strips. R e c e p t o r s for P G I 2 a n d / o r BW245C m a y therefore differ b e t w e e n these two tissues, a c o n c e p t t h a t could b e e x a m i n e d further using other P G I 2 analogues. T h e extent to which these findings reflect in vivo activity of the a n a l o g u e is not known. A l t h o u g h P G I 2 relaxes isolated m y o m e t r i u m , it seems to have little or n o effect on m y o m e t r i a l m o t i l i t y in vivo (L. M/ik/ir~iinen a n d O. Y l i k o r k a l a , p e r s o n a l c o m m u n i c a t i o n ) . O t h e r p r o s t a n o i d s also have different in vitro a n d in vivo effects, a n d studies on i s o l a t e d m y o m e t r i u m m a y b e of little value for p r e d i c t i n g the activity of these c o m p o u n d s in patients. Massele a n d Senior (1981) r e a c h e d this conclusion, a n d showed that a l t e r a t i o n o f the b a t h ing fluid f r o m K r e b s solution at 37°C to D e J a l o n fluid at 32°C c h a n g e d the responses to P G E 1 or P G E 2 f r o m r e l a x a t i o n to contraction. It r e m a i n s to b e seen whether o t h e r changes to the b a t h i n g fluid, such as the a d d i t i o n of h o r m o n e s , increases the similarity b e t w e e n in vitro a n d in vivo responses. However, if BW245C were to cause selective m y o m e t r i a l r e l a x a t i o n in vivo, the d r u g might be useful in t h r e a t e n e d a b o r t i o n or in p a i n f u l uterine spasm.
Acknowledgement We thank Wellcome Research Laboratories for BW245C and PGI 2.
References Bennett, A., C.N. Hensby, G.J. Sanger and I.F. Stamford, 1981, Metabolites of arachidonic acid formed by human gastroinestinal tissues, and their actions on the muscle layers, Br. J. Pharmacol. 74, 435. Bennett, A. and G.J. Sanger, 1979, Prostacyclin and prostaglandin E 2 selectively antagonise responses of human isolated myometrium to excitatory prostaglandins, J. Physiol. 292, 36P. Bennett, A. and G.J. Sanger, 1980, Prostacyclin relaxes the longitudinal muscle of human isolated stomach and antagonizes contractions to some prostanoids, J. Physiol. 298, 45P. Loge, O., E. Schillinger and W. Losert, 1978, Sulprostone (CP-34, 089, ZK57671), a prostaglandin E 2 analogue with potent antifertility effects and improved selectivity for uterine smooth muscles, Naunyn-Schmiedeb. Arch. Pharmacol. 302 (Suppl.), R28. Masselle, A.Y. and J. Senior, 1981, Responses of the human non-pregnant myometrium in vitro to prostaglandins and bradykinin, Br. J. Clin. Pharmacol. 12, 285P. Omini, C., R. Pasargiklian, G.C. Folco, M. Fano and F. Berti, 1978, Pharmacological activity of PGI 2 and its metabolite 6-oxo-PGFl,~ on human uterus and fallopian tubes, Prostaglandins 15, 1045. Oshima, K., K. Matsumoto, T. Tsuda, K. Shibata and M. Hayashi, 1978, Decreased smooth muscle side effects with 16,16-dimethyl-trans-A2-PGEI methyl ester in Japanese Monkey (Macaca Fuscata Fuscata), Prostaglandins 15, 473. Sanger, G.J. and A. Bennett, 1979, Fenamates may antagonise the actions of prostaglandin endoperoxides in human myometrium, Br. J. Ciin. Pharmacol. 8, 479. Sanger, G.J., C.N. Hensby, I.F. Stamford and A. Bennett, 1981, Identification of arachidonic acid metabolites extracted from human uterus, and their effects on the isolated myometrium, J. Pharm. Pharmacol. 33, 607. Wilhelmsson, L., M. Wikland and N. Wiqvist, 1981, PGH2, TxA 2 and PGI 2 have potent and differentiated actions on human uterine contractility, Prostaglandins 21,277. Wiqvist, N., J.N. Martin, M. Bygdeman and K. Gr6en, 1975, Prostaglandin analogues and uterotonic potency. A comparative study of seven compounds, Prostaglandins 9, 255.
141
Elsevier Biomedical Press
Short communication A PROSTAGLANDIN ANALOGUE WHICH POTENTLY RELAXES HUMAN UTERUS BUT NOT GUT MUSCLE GARETH J. SANGER *, ANNE JACKSON and ALAN BENNETT **
Department of Surgery, King's College Hospital Medical School, London SE5 8RX, U.K. Received 27 January 1982, accepted 13 April 1982
G.J. SANGER, A. JACKSON and A. BENNETT, A prostaglandin analogue which potently relaxes human uterus but not gut muscle, European J. Pharmacol. 81 (1982) 141-143. A prostaglandin analogue, (-+)-5-(6-carboxyhexyl)-l-(3-cyclohexyl-3-hydroxypropyl) hydantoin was tested on strips of human isolated myometrium and gastro-intestinal muscle. The analogue usually caused marked dose-dependent myometrial relaxation and inhibition of spontaneous activity, with a threshold concentration sometimes of 10 p g / m l and a maximum effect at 10 ng/mi. In contrast 1-10000 ng/ml had no effect on longitudinal muscle strips of humam stomach or colon. Prostaglandin analogue
Gastrointestinal muscle
Myometrium
1. Introduction
Many prostaglandin (PG) analogues have been tested as abortifacients. Some, such as 15(S)-15methyl PGE2, 15(S)-15-methyl PGF2~, and 16,16dimethyl PGE 2 administered by various routes, show greater activity on the myometrium in vivo than does the parent compound, and have less gastro-intestinal activity (Wiqvist et al., 1975). Similarly, with animals in vivo, certain prostaglandin analogues Mfect the uterus more than other tissues (Loge et al., 1978; Oshima et al., 1978). These results may mean that prostaglandin receptors in the myometrium differ from those in the gut, or that other factors may be involved in vivo, such as differences in pharmacokinetics, the route of administration, and the interaction of the analogue with responses to nerve stimulation and hormones. We have now tested a PG analogue, ( ---)-5-(6carboxyhexyl)- 1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C; fig. 1), on human isolated
Human smooth muscle
0
/~r~C02 0
H
OH
Fig. 1. The structure of BW245C.
myometrium and gastro,intestinal muscle. These tissues usually relax in response to PGI 2 (Omini et al., 1978; Bennett and Sanger, 1979, 1980; Bennett et al., 1981; Sanger et al., 1981; Wilhelmsson et al., 1981), but contract strongly in response to epoxymethano analogues of PGH 2 (Sanger and Bennett, 1979; Bennett et al., 1981; Sanger et al., 1981). We now report that BW 245C showed greater potency than PGI 2 on human myometrial strips, but lacked activity on gastro-intestinal muscle tone.
2. Materials and methods * Present address: Beecham Pharmaceuticals, Harlow, Essex,
U.K. ** To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
Macroscopically normal specimens of human myometrium, stomach and transverse and sigmoid
142
were obtained at operation for benign or malignant disease. Uterus was resected for menorrhagia (5 cases), menorrhagia and prolapse, pelvic endometriosis, irregular periods and back pain, and prolapse (1 case each). Subsequent examination showed benign leiomata (3 cases), no abnormality (2 cases), endometrial haemorrhage and necrosis, myometrial foci of endometriosis, cervical inflammation, and cervicitis with ulceration (1 case each). The phase of the menstrual cycle in which the myometrium was taken was classified histologically as either proliferative or secretory. Gastric muscle was removed for prepyloric ulceration, oesophageal or gastric carcinoma (1 case each) and colon was removed for rectal carcinoma or faecal incontinence (2 and 1 cases). The uterine tissue was from patients aged 31-44 (median 40) years, and the gut tissue was from 4 female and 2 male patients aged 19-79 (median 65) years. The tissue was either used immediately (3 uteri, 2 gut) or stored overnight (6 uteri, 4 gut) at 4°C in Krebs solution equilibrated with 5% CO 2 in 02. Strips of myometrium approximately 4 × 4 m m thick and 2 - 3 cm long were cut from the uterine body, approximately parallel to the longitudinal axis. Strips of gastro-intestinal muscle 4 - 5 m m wide and 2 - 3 cm long, were cut parallel to the longitudinal muscle fibres (the taenia was used as the colonic longitudinal muscle) after cutting away the mucosa and submucosa. Each strip was suspended under a load of 1 g in a tissue bath containing Krebs solution (NaC1 118.5; CaC12 2.5; K H 2 P O 4 1.2; KCI 4.7; MgSO 4 1.2; N a H C O 3 2.5; dextrose 11.7 m M at 37°C and bubbled with 5% CO 2 in 02. Isotonic responses were magnified 8-16 times. BW245C was dissolved in ethanol (5 m g / m l ) and stored at - 2 0 ° C . Fresh solutions, prepared from this by diluting with Krebs solution, were kept at 4°C until 10-15 min before the experiment, and then allowed to achieve room temperature. When spontaneous myometrial contractions, or acetylcholine-induced gut contractions were consistent, BW245C or its vehicle controls were added (contact times respectively 15 and 10 min for the two types of muscle).
ij B'W
B-W 0"001
a, o 0 A 1A1A
C
BW 1
C
BW 10
"i-
Fig. 2. Top trace: BW 245C (BW) reproducibly inhibited
myometrial spontaneous activity and reduced the muscle tone. acetylcholine (A) in contact with the tissue for 1 min contracted gastric muscle, and PGI 2 1 ~g/ml (I) caused relaxation. However, BW245C (BW) at a concentration 10000 times higher than that maximally inhibiting the myometrium had no effect. The numbers below A and BW represent/~g/ml. Vehicle controls (C) did not affect the tissues. Horizontal calibration 30 min, vertical calibration 5 cm. B o t t o m trace:
3. Results
BW245C caused marked, dose-dependent myometrial relaxation a n d / o r inhibition of spontaneous activity (fig. 2), except in one specimen of myometrium (cervicitis with ulceration, proliferative phase) in which BW245C up to 10 n g / m l had no effect. Sometimes the threshold concentration was as low as 0.01 n g / m l , and the maximally effective concentration was 10 n g / m l (n = 8; 4 proliferative and 4 secretory). Overall, the results were similar with myometrium from either the proliferative or the secretory phase. In contrast, BW245C 1 n g / m l to 10 t t g / m l had no effect on stomach or colonic longitudinal muscle tone (both n = 3 ) , whereas sodium P G I 2 1/~g/ml usually caused marked relaxation of the gastric tissues.
4. Discussion
P G I 2 potently relaxes human gastric tissues, in agreement with our earlier experiments in which
143 c o n c e n t r a t i o n s as low as 5 0 - 2 0 0 n g / m l relaxed gastric strips a n d 1 - 1 0 # g / m l i n h i b i t e d colonic strips (Bennett et al., 1981). P G I 2 also relaxes h u m a n m y o m e t r i u m , with threshold c o n c e n t r a tions of 10 n g / m l to 1 # g / m l (Sanger et al., 1981). C o m p a r e d with P G I 2 , B W 2 4 5 C therefore seems m u c h less active o n isolated gastrointestinal muscle, b u t m u c h m o r e p o t e n t on h u m a n m y o m e t r i a l strips. R e c e p t o r s for P G I 2 a n d / o r BW245C m a y therefore differ b e t w e e n these two tissues, a c o n c e p t t h a t could b e e x a m i n e d further using other P G I 2 analogues. T h e extent to which these findings reflect in vivo activity of the a n a l o g u e is not known. A l t h o u g h P G I 2 relaxes isolated m y o m e t r i u m , it seems to have little or n o effect on m y o m e t r i a l m o t i l i t y in vivo (L. M/ik/ir~iinen a n d O. Y l i k o r k a l a , p e r s o n a l c o m m u n i c a t i o n ) . O t h e r p r o s t a n o i d s also have different in vitro a n d in vivo effects, a n d studies on i s o l a t e d m y o m e t r i u m m a y b e of little value for p r e d i c t i n g the activity of these c o m p o u n d s in patients. Massele a n d Senior (1981) r e a c h e d this conclusion, a n d showed that a l t e r a t i o n o f the b a t h ing fluid f r o m K r e b s solution at 37°C to D e J a l o n fluid at 32°C c h a n g e d the responses to P G E 1 or P G E 2 f r o m r e l a x a t i o n to contraction. It r e m a i n s to b e seen whether o t h e r changes to the b a t h i n g fluid, such as the a d d i t i o n of h o r m o n e s , increases the similarity b e t w e e n in vitro a n d in vivo responses. However, if BW245C were to cause selective m y o m e t r i a l r e l a x a t i o n in vivo, the d r u g might be useful in t h r e a t e n e d a b o r t i o n or in p a i n f u l uterine spasm.
Acknowledgement We thank Wellcome Research Laboratories for BW245C and PGI 2.
References Bennett, A., C.N. Hensby, G.J. Sanger and I.F. Stamford, 1981, Metabolites of arachidonic acid formed by human gastroinestinal tissues, and their actions on the muscle layers, Br. J. Pharmacol. 74, 435. Bennett, A. and G.J. Sanger, 1979, Prostacyclin and prostaglandin E 2 selectively antagonise responses of human isolated myometrium to excitatory prostaglandins, J. Physiol. 292, 36P. Bennett, A. and G.J. Sanger, 1980, Prostacyclin relaxes the longitudinal muscle of human isolated stomach and antagonizes contractions to some prostanoids, J. Physiol. 298, 45P. Loge, O., E. Schillinger and W. Losert, 1978, Sulprostone (CP-34, 089, ZK57671), a prostaglandin E 2 analogue with potent antifertility effects and improved selectivity for uterine smooth muscles, Naunyn-Schmiedeb. Arch. Pharmacol. 302 (Suppl.), R28. Masselle, A.Y. and J. Senior, 1981, Responses of the human non-pregnant myometrium in vitro to prostaglandins and bradykinin, Br. J. Clin. Pharmacol. 12, 285P. Omini, C., R. Pasargiklian, G.C. Folco, M. Fano and F. Berti, 1978, Pharmacological activity of PGI 2 and its metabolite 6-oxo-PGFl,~ on human uterus and fallopian tubes, Prostaglandins 15, 1045. Oshima, K., K. Matsumoto, T. Tsuda, K. Shibata and M. Hayashi, 1978, Decreased smooth muscle side effects with 16,16-dimethyl-trans-A2-PGEI methyl ester in Japanese Monkey (Macaca Fuscata Fuscata), Prostaglandins 15, 473. Sanger, G.J. and A. Bennett, 1979, Fenamates may antagonise the actions of prostaglandin endoperoxides in human myometrium, Br. J. Ciin. Pharmacol. 8, 479. Sanger, G.J., C.N. Hensby, I.F. Stamford and A. Bennett, 1981, Identification of arachidonic acid metabolites extracted from human uterus, and their effects on the isolated myometrium, J. Pharm. Pharmacol. 33, 607. Wilhelmsson, L., M. Wikland and N. Wiqvist, 1981, PGH2, TxA 2 and PGI 2 have potent and differentiated actions on human uterine contractility, Prostaglandins 21,277. Wiqvist, N., J.N. Martin, M. Bygdeman and K. Gr6en, 1975, Prostaglandin analogues and uterotonic potency. A comparative study of seven compounds, Prostaglandins 9, 255.