Gen. Pharmac. Vol. 23, No. 5, pp. 805-809, 1992 Printed in Great Britain.All rights reserved
0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
ACTIONS OF TWO NOVEL PROSTAGLANDIN ANALOGS, SC-29169 AND SC-31391, ON GUINEA PIG AND HUMAN ISOLATED URINARY BLADDER ENZO POLI,t GIACOMOMACALUSO2 and CRISTINA POZZOLI l lInstitute of Pharmacology, University of Parma, School of Medicine, Via Gramsci 14, 43100 Parma and 2Department of Urology, Ospedale Maggiore di Parma, Parma, Italy [Tel. 0521-290-383] (Received 27 March 1992) Abstract--1. We examined the effects of two novel PGE a analogs, SC-29169 and SC-31391, on bladder muscle isolated preparations from guinea pig and man, in comparison with some naturally-occurring prostanoids and misoprostol. 2. In the guinea pig detrusor muscle, both prostaglandin analogs enhanced twitch responses elicited by field stimulation in the following order of potency: SC-31391 > SC-29169 > PGF2~I> PGE2, while a well-defined contractile effect was elicited only by SC-31391. 3. In the human detrusor muscle, nerve-mediated responses were not modified by prostaglandin analogs, as well as by PGF2~or PGE:, while a contractile effect was observed with the same compounds: PGF2~= SC-31391 > SC-29169 = PGE2= PGE I. 4. The selective EPl-agonist, misoprostol did not induce any effect, in both guinea pig and human bladder. 5. These data suggest that the effect of prostaglandins in the bladder muscle differs according to the animal species and that, in the human detrusor muscle, SC-31391 and SC-29169 probably stimulate FP receptors.
INTRODUCTION The involvement of prostaglandins (PGs) in the control of urinary bladder motility has been evidentiated both in animals (Andersson and Sj6gren, 1982; Downie and Karmazyn, 1984; Maggi et al., 1984) and in man (Andersson and Sj6gren, 1982; Mikhailidis et al., 1987). Endogenous PGF2~ and PGE2 delivered from vesical tissues following pharmacological (Jeremy et al., 1986; Andersson et al., 1980) or mechanical (Downie and Karmazyn, 1984) stimulation have been shown to increase detrusor muscle contractility and to facilitate bladder emptying (Andersson and Sj6gren, 1982). For these reasons, different authors speculated about a possible therapeutical use of PGs in patients affected by deranged motility of the bladder (Andersson and Sj6gren, 1982; Koonings et al., 1990). Since naturally-occurring PGs do not represent reliable clinical means, considering the fast degradation and the lack of selectivity, an increasing number of synthetic analogs became available for both pharmacological and preclinical studies. In the present paper, two novel PG analogs, the compounds marked SC-29169 and SC-31391, have been characterized for their ability to stimulate bladder muscle contractility. These compounds derive from the natural ¢icosanoid, PGE~ (Fig. 1). As it can be seen, SC-29169 is the (15,20)dimethyl-PGEl; SC-31391 is the 1-methoxy,14-ine-PGE~, in which the 17-20 portion of the moiety was replaced with a phenoxygroup. The attempt was made to obtain stable analogs, active also for the oral route (Dajani, personal communication).
The effect of these compounds was investigated o n human and guinea pig bladder muscle in vitro, in comparison with some natural PGs and misoprostol, a PGE~ derivative recently introduced in the clinical practice as an effective antiulcer drug (Steiner, 1987). This compound selectively stimulates a PG receptor subtype (EP0 in different tissues (Eglen and Whiting, 1988).
805
O
(±) OH: OH
OH SC-29169
O
O
(±)
SC-31391
Fig. I. Chemical structure of compounds $C-29169 and SC-31391.
ENZO POLl et al.
806 METHODS
Guinea pig urinary bladder As previously described (Poli et al., 1988), urinary bladders were excised from male guinea pigs (250-350 g body wt), longitudinally cut to obtain three strips (10 mm long and 1.5 mm thick) and set up into isolated organ chambers at 37°C, containing a modified Krebs Henseleit solution of the following composition (mM): NaCl 113, KC1 4.5, CaC12 2.5, KH2PO 4 1.2, MgSO 4 1.2, NaHCO 3 25 and glucose 11.5. A mixture of 02 (95%) and CO 2 (5%) was continuously bubbled into the chamber, obtaining a pH value of 7.4-7.5. The contractile activity was measured by means of an isometric transducer, developing a passive stretch of 1.5-2 g, connected to a pen writing poligraph (Basile, Milano). Electrical field stimulation was applied in some preparations to induce muscle contraction through the activation of the intrinsic excitatory neurons. A pair of ring platinum electrodes was positioned around the tissues and connected to an electronic stimulator (Lace, Pisa), and 5 sec trains of pulses (20 Hz, 0.5 msec, 120-150 mA) were delivered every 2 min. Human urinary bladder Fragments of detrusor muscle were taken from 15 male patients (age ranging between 42 and 68 years), undergoing prostatic surgery or bladder cancer. Longitudinal strips (2.5 x 0.4cm) were excised from macroscopically normal tissues and set up as previously described (Zappia et al., 1986). Preparations were then left to equilibrate for 120 min before the administration of drugs. Electrical field stimulation was applied in some preparations as described above for guinea pig. Evaluation of drug effects Compounds tested were administered into the organ chambers and left to act until the maximum effect was reached. The mean_+SEM of 4-6 observations was calculated and plotted.
To minimize desensitization phenomena, when concentration-response curves were constructed, PG-s were administered into the organ chamber by single doses every 45 min. The comparison of the activity of PGs was performed on the ECs0 values, calculated on the regression lines of the concentration-response curves. Student's t-test was applied, to compare different series of data; a level of significance greater than 95% (P < 0.05) was considered.
Drugs used Prostaglandins El and E2, tetrodotoxin, atropine, prazosin, bethanechol and acetylcholine (Sigma, St Louis); misoprostol, SC-29169, SC-31391 and prostaglandin F~ were a gift from Dr Tony Martinez (Searle, Chicago). RESULTS
Guinea pig urinary bladder L o n g i t u d i n a l strips o f guinea pig bladder were contracted by all the P G s tested. PGF2~, PGE2 a n d SC-29169 induced a slight contraction, starting from a c o n c e n t r a t i o n o f 1 0 - 7 M (Fig. 2, right side). Conversely, the c o m p o u n d SC-31391 induced a strong increase o f muscle tone (ECs0 _+ SEM: 1.5 _ 1.2 x 10-6 M) being nearly 50% as effective as b e t h a n e c h o l or acetylcholine, c o m p o u n d s able to maximally contract guinea pig bladder muscle (not shown). P G E j was as p o t e n t a n d effective as PGE~ (not shown). The contractile effect o f P G s was n o t modified by tetrodotoxin, atropine or prazosin ( 1 0 - 6 M ) . I n h i b i t o r y effects o n the basal tone were never observed, even w h e n s u b t h r e s h o l d c o n c e n t r a t i o n s were tested. W h e n field stimulation was applied, twitch responses were obtained. Pilot experiments revealed
100
10.
.I ? o
o !
I
I
I
8
7
6
5
7 Drugs
6
5
(~Log M )
Fig. 2. Guinea pig bladder: effect of some prostaglandins and synthetic analogs on the muscle tone (right) and on the twitch responses elicited by field stimulation (left). SC-31391 (El); SC-29169 (C)); PGF~ ( 0 ) ; PGE2 ( , ) . Values are the mean + SEM of 4-6 observations. In ordinata: percent of enhancement, calculated on the basal twitch response, elicited by field stimulation, taken as 100 (left), or contraction of the preparation, expressed in g of isometric tension (right); in abscissa: molar concentration of drugs.
Prostagiandins and vesicatlmotility
SC-29169 10"e
807
SC- 31391 ~i
Iml SC-31391
SC-29169 1 0 - e
10 - e
Fig. 3. Guinea pig bladder: original recordings showing the effect of prostaglandin analogs on field stimulation- (upper tracing) or acetylcholine-elicited contraction (lower tracing). O, Acetylcholine 10-5 M; *, wash-out. The concentration of drugs is expressed in moi/l.
that these responses are mainly due to the release of acetylcholine and ATP from excitatory neurons, as assessed also by other authors in the same tissue (Andersson et al., 1980; Peterson and Noronha-Blob, 1989). PGs enhanced twitch responses in a concentrationdependent manner (Fig. 2, left side). This facilitatory effect of PGs occurred at concentrations lower than those requested to enhance muscle tone. Compound SC-31391 was the most potent (EC50 + SEM: 9.3 +_ 1.4 x 10 -s M). SC-29169, in turn, was more potent than PGF2~ (ECs0 _+ SEM: 2.3 _+ 2.1 x 10 -7 M and 6.3 _+ 2.8 x 10 -7 M, respectively). No differences in the maximum effect was observed. PGE 2 (Fig. 2) and PGEI (not shown) were as potent as PGF2~ (ECs0 + SEM: 4.1 + 1.3 x 10 -7 and 5.4 +_ 2.1 x 10 -7 M, respectively), but less efficacious in terms of maximum response. Apparently, no crossdesensitization between PG analogs and PGF2~ or PGE2 was seen. Contractions elicited by exogenous acetylcholine were not modified by SC-29169, SC-31391 (Fig. 3) and PGF2~ (not shown), at the same concentrations able to potentiate field stimulation-evoked contractions. Misoprostol (10-s-10-~M) did not induce any contractile or enhancing effect.
Human urinary bladder Human detrusor muscle strips were contracted by low concentrations of PGs (3 x 10-1°-3 u 10 -s M) (Fig. 4). PGF2~ and SC-31391 were equiactive (ECs0 + SEM: 3.2 _+ 1.3 × 10 -9 M and 4.1 + 2.1 × 10 -9 M, respectively), while SC-29169 was less potent and efficacious (ECs0_+SEM: 7.3+2.1 × 10-gM). The response was not modified by tetrodotoxin, prazosin and atropine (10-6M), excluding the involvement
of nerve-mediated mechanisms or the stimulation of ~- or muscarinic-receptors. Desensitization of the preparations occurred after maximum concentrations of PGF2r This desensitization was rather specific for PGF2= , since the effect of acetylcholine was not modified (Fig. 5). Furthermore, cross-takyphylaxis between PGF2~ and SC-29169 or SC-31391 was observed (Fig. 5). Conversely, the two PG analogs induced a lesser degree of desensitization, in comparison with PGF2~. us.
A
U
O5-
!
~
I
8
!
7
|
6
Drugs (-Log M)
Fig. 4. Human bladder: contractile effect of PGF2~ (Q); SC-31391 ([-I) and SC-29169 (O). Values are the mean + SEM of 4-5 observations. In ordinata: g of isometric contraction; in abscissa: molar concentration of drugs.
ENZO POLl et al.
808 m A
7
~, 10-
ACh 10-5
I" ZX
PGF2o
0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
ACTIONS OF TWO NOVEL PROSTAGLANDIN ANALOGS, SC-29169 AND SC-31391, ON GUINEA PIG AND HUMAN ISOLATED URINARY BLADDER ENZO POLI,t GIACOMOMACALUSO2 and CRISTINA POZZOLI l lInstitute of Pharmacology, University of Parma, School of Medicine, Via Gramsci 14, 43100 Parma and 2Department of Urology, Ospedale Maggiore di Parma, Parma, Italy [Tel. 0521-290-383] (Received 27 March 1992) Abstract--1. We examined the effects of two novel PGE a analogs, SC-29169 and SC-31391, on bladder muscle isolated preparations from guinea pig and man, in comparison with some naturally-occurring prostanoids and misoprostol. 2. In the guinea pig detrusor muscle, both prostaglandin analogs enhanced twitch responses elicited by field stimulation in the following order of potency: SC-31391 > SC-29169 > PGF2~I> PGE2, while a well-defined contractile effect was elicited only by SC-31391. 3. In the human detrusor muscle, nerve-mediated responses were not modified by prostaglandin analogs, as well as by PGF2~or PGE:, while a contractile effect was observed with the same compounds: PGF2~= SC-31391 > SC-29169 = PGE2= PGE I. 4. The selective EPl-agonist, misoprostol did not induce any effect, in both guinea pig and human bladder. 5. These data suggest that the effect of prostaglandins in the bladder muscle differs according to the animal species and that, in the human detrusor muscle, SC-31391 and SC-29169 probably stimulate FP receptors.
INTRODUCTION The involvement of prostaglandins (PGs) in the control of urinary bladder motility has been evidentiated both in animals (Andersson and Sj6gren, 1982; Downie and Karmazyn, 1984; Maggi et al., 1984) and in man (Andersson and Sj6gren, 1982; Mikhailidis et al., 1987). Endogenous PGF2~ and PGE2 delivered from vesical tissues following pharmacological (Jeremy et al., 1986; Andersson et al., 1980) or mechanical (Downie and Karmazyn, 1984) stimulation have been shown to increase detrusor muscle contractility and to facilitate bladder emptying (Andersson and Sj6gren, 1982). For these reasons, different authors speculated about a possible therapeutical use of PGs in patients affected by deranged motility of the bladder (Andersson and Sj6gren, 1982; Koonings et al., 1990). Since naturally-occurring PGs do not represent reliable clinical means, considering the fast degradation and the lack of selectivity, an increasing number of synthetic analogs became available for both pharmacological and preclinical studies. In the present paper, two novel PG analogs, the compounds marked SC-29169 and SC-31391, have been characterized for their ability to stimulate bladder muscle contractility. These compounds derive from the natural ¢icosanoid, PGE~ (Fig. 1). As it can be seen, SC-29169 is the (15,20)dimethyl-PGEl; SC-31391 is the 1-methoxy,14-ine-PGE~, in which the 17-20 portion of the moiety was replaced with a phenoxygroup. The attempt was made to obtain stable analogs, active also for the oral route (Dajani, personal communication).
The effect of these compounds was investigated o n human and guinea pig bladder muscle in vitro, in comparison with some natural PGs and misoprostol, a PGE~ derivative recently introduced in the clinical practice as an effective antiulcer drug (Steiner, 1987). This compound selectively stimulates a PG receptor subtype (EP0 in different tissues (Eglen and Whiting, 1988).
805
O
(±) OH: OH
OH SC-29169
O
O
(±)
SC-31391
Fig. I. Chemical structure of compounds $C-29169 and SC-31391.
ENZO POLl et al.
806 METHODS
Guinea pig urinary bladder As previously described (Poli et al., 1988), urinary bladders were excised from male guinea pigs (250-350 g body wt), longitudinally cut to obtain three strips (10 mm long and 1.5 mm thick) and set up into isolated organ chambers at 37°C, containing a modified Krebs Henseleit solution of the following composition (mM): NaCl 113, KC1 4.5, CaC12 2.5, KH2PO 4 1.2, MgSO 4 1.2, NaHCO 3 25 and glucose 11.5. A mixture of 02 (95%) and CO 2 (5%) was continuously bubbled into the chamber, obtaining a pH value of 7.4-7.5. The contractile activity was measured by means of an isometric transducer, developing a passive stretch of 1.5-2 g, connected to a pen writing poligraph (Basile, Milano). Electrical field stimulation was applied in some preparations to induce muscle contraction through the activation of the intrinsic excitatory neurons. A pair of ring platinum electrodes was positioned around the tissues and connected to an electronic stimulator (Lace, Pisa), and 5 sec trains of pulses (20 Hz, 0.5 msec, 120-150 mA) were delivered every 2 min. Human urinary bladder Fragments of detrusor muscle were taken from 15 male patients (age ranging between 42 and 68 years), undergoing prostatic surgery or bladder cancer. Longitudinal strips (2.5 x 0.4cm) were excised from macroscopically normal tissues and set up as previously described (Zappia et al., 1986). Preparations were then left to equilibrate for 120 min before the administration of drugs. Electrical field stimulation was applied in some preparations as described above for guinea pig. Evaluation of drug effects Compounds tested were administered into the organ chambers and left to act until the maximum effect was reached. The mean_+SEM of 4-6 observations was calculated and plotted.
To minimize desensitization phenomena, when concentration-response curves were constructed, PG-s were administered into the organ chamber by single doses every 45 min. The comparison of the activity of PGs was performed on the ECs0 values, calculated on the regression lines of the concentration-response curves. Student's t-test was applied, to compare different series of data; a level of significance greater than 95% (P < 0.05) was considered.
Drugs used Prostaglandins El and E2, tetrodotoxin, atropine, prazosin, bethanechol and acetylcholine (Sigma, St Louis); misoprostol, SC-29169, SC-31391 and prostaglandin F~ were a gift from Dr Tony Martinez (Searle, Chicago). RESULTS
Guinea pig urinary bladder L o n g i t u d i n a l strips o f guinea pig bladder were contracted by all the P G s tested. PGF2~, PGE2 a n d SC-29169 induced a slight contraction, starting from a c o n c e n t r a t i o n o f 1 0 - 7 M (Fig. 2, right side). Conversely, the c o m p o u n d SC-31391 induced a strong increase o f muscle tone (ECs0 _+ SEM: 1.5 _ 1.2 x 10-6 M) being nearly 50% as effective as b e t h a n e c h o l or acetylcholine, c o m p o u n d s able to maximally contract guinea pig bladder muscle (not shown). P G E j was as p o t e n t a n d effective as PGE~ (not shown). The contractile effect o f P G s was n o t modified by tetrodotoxin, atropine or prazosin ( 1 0 - 6 M ) . I n h i b i t o r y effects o n the basal tone were never observed, even w h e n s u b t h r e s h o l d c o n c e n t r a t i o n s were tested. W h e n field stimulation was applied, twitch responses were obtained. Pilot experiments revealed
100
10.
.I ? o
o !
I
I
I
8
7
6
5
7 Drugs
6
5
(~Log M )
Fig. 2. Guinea pig bladder: effect of some prostaglandins and synthetic analogs on the muscle tone (right) and on the twitch responses elicited by field stimulation (left). SC-31391 (El); SC-29169 (C)); PGF~ ( 0 ) ; PGE2 ( , ) . Values are the mean + SEM of 4-6 observations. In ordinata: percent of enhancement, calculated on the basal twitch response, elicited by field stimulation, taken as 100 (left), or contraction of the preparation, expressed in g of isometric tension (right); in abscissa: molar concentration of drugs.
Prostagiandins and vesicatlmotility
SC-29169 10"e
807
SC- 31391 ~i
Iml SC-31391
SC-29169 1 0 - e
10 - e
Fig. 3. Guinea pig bladder: original recordings showing the effect of prostaglandin analogs on field stimulation- (upper tracing) or acetylcholine-elicited contraction (lower tracing). O, Acetylcholine 10-5 M; *, wash-out. The concentration of drugs is expressed in moi/l.
that these responses are mainly due to the release of acetylcholine and ATP from excitatory neurons, as assessed also by other authors in the same tissue (Andersson et al., 1980; Peterson and Noronha-Blob, 1989). PGs enhanced twitch responses in a concentrationdependent manner (Fig. 2, left side). This facilitatory effect of PGs occurred at concentrations lower than those requested to enhance muscle tone. Compound SC-31391 was the most potent (EC50 + SEM: 9.3 +_ 1.4 x 10 -s M). SC-29169, in turn, was more potent than PGF2~ (ECs0 _+ SEM: 2.3 _+ 2.1 x 10 -7 M and 6.3 _+ 2.8 x 10 -7 M, respectively). No differences in the maximum effect was observed. PGE 2 (Fig. 2) and PGEI (not shown) were as potent as PGF2~ (ECs0 + SEM: 4.1 + 1.3 x 10 -7 and 5.4 +_ 2.1 x 10 -7 M, respectively), but less efficacious in terms of maximum response. Apparently, no crossdesensitization between PG analogs and PGF2~ or PGE2 was seen. Contractions elicited by exogenous acetylcholine were not modified by SC-29169, SC-31391 (Fig. 3) and PGF2~ (not shown), at the same concentrations able to potentiate field stimulation-evoked contractions. Misoprostol (10-s-10-~M) did not induce any contractile or enhancing effect.
Human urinary bladder Human detrusor muscle strips were contracted by low concentrations of PGs (3 x 10-1°-3 u 10 -s M) (Fig. 4). PGF2~ and SC-31391 were equiactive (ECs0 + SEM: 3.2 _+ 1.3 × 10 -9 M and 4.1 + 2.1 × 10 -9 M, respectively), while SC-29169 was less potent and efficacious (ECs0_+SEM: 7.3+2.1 × 10-gM). The response was not modified by tetrodotoxin, prazosin and atropine (10-6M), excluding the involvement
of nerve-mediated mechanisms or the stimulation of ~- or muscarinic-receptors. Desensitization of the preparations occurred after maximum concentrations of PGF2r This desensitization was rather specific for PGF2= , since the effect of acetylcholine was not modified (Fig. 5). Furthermore, cross-takyphylaxis between PGF2~ and SC-29169 or SC-31391 was observed (Fig. 5). Conversely, the two PG analogs induced a lesser degree of desensitization, in comparison with PGF2~. us.
A
U
O5-
!
~
I
8
!
7
|
6
Drugs (-Log M)
Fig. 4. Human bladder: contractile effect of PGF2~ (Q); SC-31391 ([-I) and SC-29169 (O). Values are the mean + SEM of 4-5 observations. In ordinata: g of isometric contraction; in abscissa: molar concentration of drugs.
ENZO POLl et al.
808 m A
7
~, 10-
ACh 10-5
I" ZX
PGF2o
