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Some pharmacological properties of a new antitumor drug, CPT-11, in isolated muscle preparations
Gen. Pharmac. Vol. 20, No. 6, pp. 763-766, 1989 Printed in Great Britain. All rights reserved
0306-3623/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
SOME PHARMACOLOGICAL PROPERTIES OF A NEW ANTITUMOR D R U G , CPT-11, IN ISOLATED MUSCLE PREPARATIONS I. TAKAYANAGI, K. KOXKE, M. TAGAWA a n d E. MITSUHASHI Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan (Tel. (0474)72-I 141)
(Received 7 February 1989) Abstract--1. Pharmacological properties of a new antitumor drug, CPT-11, were studied in some muscle preparations. 2. CPT-11 induced contraction of guinea-pig ileal and tracheal preparations, which was blocked by atropine (10 -6 M). 3. CPT-11 potentiated the contractile responses of guinea-pig ileum to acetylcholine, nicotine, serotonin and BaCl 2. 4. The chronic and inotropic effects induced by isoprenaline were depressed by CPT-I 1. 5. These results suggest that CPT-I1 has an acetylcholine action.
INTRODUCTION CPT- 11, for which the chemical structure is s h o w n in Fig. 1, is a new derivative o f c a m p t o t h e c i n , a p l a n t alkaloid developed in Japan. W a n g et al. (1987) reported t h a t the a n t i t u m o r activity o b t a i n e d from CPT-11 is c o m p a r a b l e to those o f adriamycin, cyc l o p h o s p h a m i d e a n d 5-fluorouracil a n d concluded t h a t CPT-11 is a good c a n d i d a t e for clinical trials. F u r t h e r m o r e , the antileukemic activity o f CPT-11 ~against L1210 was f o u n d to be m u c h higher t h a n t h a t o f adriamycin a n d the acute toxicity o f CPT-11 'was also f o u n d to be extremely low, particularly in the case of oral a d m i n i s t r a t i o n (Nitta et al., 1987). However, p h a r m a c o l o g i c a l properties o f CPT-11 o n ,muscle p r e p a r a t i o n s were n o t studied. MATERIALS AND METHODS
Ileum, trachea and right atrium from guinea-pig Male guinea-pigs (250-350 g) of Hartley strain were killed by a blow on the neck and the ileum, trachea and heart were isolated. A piece (about 3 cm) of the ileum was suspended in a 20 ml organ bath filled with a physiological solution (NaC1 154, KCI 5.6, MgCI 2 2.1, CaCI: 2.2, NaHCO 3 5.9 and glucose 2.8 mM), gassed with a mixture of 95% 02 and 5% CO 2 (carbogen) and kept at 32°C. Responses to agonist were recorded isotonically under a tension of 0.5 g. The tracheal preparation was made by the method of Takagi and Takayanagi (1958) and mounted in a similar organ bath as described. Responses to a drug were recorded isotonically under a tension of 1.0 g. An isolated heart was placed in the physiological solution gassed with carbogen and kept at
N
N
37°C. All other tissues were cut away until nothing was left except the fight atria. The preparation was suspended in the physiological solution at 37°C, through which a brisk stream of carhogen was blown. Chronotropic and inotropic effects were recorded isometrically under a resting tension of 0.5 g (Takayanagi and Koike, 1985).
Vas deferens from rat Male Wistar strain rats (250-300 g) were killed by a blow on the head and the vasa deferentia were isolated. A piece (2-3 cm) of vas deferens was suspended in the similar organ bath as described. Responses to the drug were recorded isotonically under a tension of 0.5 g. Bath temperature used was 32°C. Thoracic aorta from rabbit Male rabbits (2.0-3.5 kg) were killed by bleeding from the neck. About 5 cm of thoracic aorta was dissected out close to the arch and cut helically. The aortic strips were then stripped of their endothelial cells by mechanical rubbing. A piece (about 2 mm in width and 3 cm in length) of aortic preparation was mounted in the similar bath as described. Responses to a drug were recorded isotonically under a tension of 2.0 g. Bath temperature used was 37°C. Uterus from rat Female Wistar rats (200-300 g) treated with estradiol benzate (0.2 mg/kg, s.c.) at 24 and 48 hr before sacrifice, female Wistar rats (300-350 g) castrated one week before sacrifice and also female Wistar rats in the 17th and 18th day of pregnancy were used. They were killed by a blow on the head and pieces (about 3 cm) of uterus were isolated. They were suspended in the similar organ bath as described. Responses to a drug were recorded isotonically under a tension of 0.5 g. Bath temperature used was 32°C.
N
0
HOgf -']0 Fig. 1. A chemical structure of CPT-I 1. 763 OP 20/6--D
HCI • sl.-~O
I. TAKAYANAGIet al.
764 100
150
50
100 o= -o
g c~ 50 -7
-6
-5
-4
Log[CPT-11]
Fig. 2. A concentration-response curve of CPT- l 1 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of CPT-11. Each point is presented as a mean with SE of 8 experiments.
In order to estimate antagonistic potency of a drug, the preparations were equilbrated with an antagonist for 5 min, after determination of control concentration-response curves of the agonist. Drugs used. CPT-I1 (Fig. 1) was kindly supplied from Daiichi Pharmaceutical (Tokyo, Japan). Other drugs were obtained commercially, all in powder form. RESULTS
Guinea-pig ileum (nicotinic and muscarinic cholinoceptors and histamine receptors) CPT-11 induced a c o n t r a c t i o n o f ileum in a conc e n t r a t i o n d e p e n d e n t m a n n e r , which was completely inhibited by a t r o p i n e (10 -6 M). The m a x i m u m cont r a c t i o n by CPT-11 was a b o u t 2 1 % o f t h a t induced by acetylcholine (Fig. 2). The contractile response to BaC12 (10 -3 M ) was p o t e n t i a t e d by CPT-11. Concent r a t i o n - r e s p o n s e curves o f acetylcholine a n d nicotine were also p o t e n t i a t e d by CPT-11, whereas the highest c o n c e n t r a t i o n (10 -4 M) used to inhibit the curves for b o t h the agonists (Figs 3 a n d 4). The effect o f CPT-11
~
-7
Rat vas deferens (¢t-adrenoceptors) CPT-11 (10-7-10 -4 M ) did n o t influence the spont a n e o u s m o v e m e n t o f vas deferens n o r the contractile responses o f vas deferens to n o r e p i n e p h r i n e (10-7-10-4 M).
100
100 "
~
,
t
,
-9
-8
-7
-6
,
,
-5
-4
-4
on the response to serotonin was also similar. The responses to lower concentrations (10-8-3 x 10 -7 M ) of histamine were potentiated by CPT-I1 (10-7-10-4M) but those to higher concentrations (10-6-10-5M) of histamine were not practically influenced. The highest concentration (10-4M) of CPT-11 used, inhibited the contractile response to h i s t a m i n e (Fig. 5).
150
o
-5
Fig. 4. Concentration-response curves of nicotine in the absence and presence of CPT-11 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of nicotine. Each point is presented as a mean with SE of 8 experiments. O: nicotine, alone, O: with CPT-II, 10 -7 M, A: with CPT-11, 10 -6 M, A: with CPT-1 l, l0 -5 M and I1: with CPT-I1, 10-4M.
150
5o
-6
Log[Serotonin)
~
-
5o
o -'3
Log[Acet, yl cho]ine]
Fig. 3. Concentration-response curves of acetylcholine in the absence and presence of CPT- 11 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of acetylcholine. Each point is presented as a mean with SE of 8 experiments. O: acetylcholine, alone, O: with CPT-11, 10-7 M, A: with CPT-I 1, 10-6 M, A: with CPT-I1, 10-SM and II: with CPT-I1, 10-4M.
-9
-8
-7
-6
-5
-4
Log[Histamine]
Fig. 5. Concentration-response curves of histamine in the absence and presence of CPT-ll tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to histamine and abscissa: log concentration (M) of histamine. Each point is presented as a mean with SE of 8 experiments. O: histamine, alone, (3: with CPT-11, 10 -7 M, A: with CPT-I1, 10-6 M, A: with CPT-I 1, 10-SM and II: with CPT-11, 10-4M.
Antitumor drug CPT-I 1
765 100
50
~
5o
-3
-50 -S
-50
-100
Log[ Isoproterenol ]
Log[CPT-11]
Fig. 6. A concentration-response curve of CPT-I1 on spontaneous inotropic response of the guinea-pig fight atrium. Ordinate: percent of the spontaneous tension and abscissa: log concentration (M) of CPT-I1. Each point is presented as a mean with SE of 5 experiments.
Rabbit thoracic aorta prostaglandin receptors)
(ot-adrenoceptors and
CPT-11 (10-7-10-4M) did not spontaneous movement of thoracic concentration response curves of (10-s-10-SM) and prostagiandin 3 x 10 -s M).
influence the aorta nor the norepinephrine F2= (3 × 10-7-
Guinea-pig right atrium (~-adrenoceptors) CPT-11 (more than 3 x l0 -5 M) decreased spontaneous chronotropic and inotropic actions (Figs 6 and 7). Isoprenaline 0 0 - 9 - 3 x l0 -7 M) increased the chronotropic and inotropic actions in a concentration dependent manner. The chronotropic and inotropic responses induced by isoprenaline were not influenced by CPT-11 0 0 -7 and l0 -6 M), whereas the higher concentrations (10 -5 and l0 -4 M) of CPT-I 1 parallelly shifted the concentration-response curves of isoprenaline to the bottom suggesting a physiological antagonism (Fig. 8).
Fig. 8. Concentration-ehronotropic response curves of isoprenaline in the absence and presence of CPT-II tested in the guinea-pig right atrium. Ordinate: percent of a maximal heart rate induced by isoprenaline and abscissa: log concentration (M) of isoprenaline. Each point is presented as a mean with SE of 6 experiments. 0 : isoprenaline, alone, O: with CPT-I1, 10-7M, &: with CPT-11, 10-6M, A: with CPT-11, 10-SM and I1: with CPT-11, 10-4M.
Guinea-pig trachea ([3-adrenoceptors and histamine
receptors) CPT-I1 (10-7-10 -5 M) induced the contractile response, the maximum of which was about 18% of the maximum response to histamine. The contractile responses to histamine (10-8-10 -4 M) were slightly (but not significantly) potentiated by CPT-11 (10-7-10-5M) but not by 10-4M of CPT-I1. The relaxation responses to isoprenaline (10-9-3 × 10-TM) were slightly inhibited by CPT-11 (10-7-10 -s M) but not by 10-4M of CPT-I 1.
Rat uterus (oxytocin receptors) Spontaneous contractile response of uteri from both estradiol-treated and castrated rats were not influenced by CPT-11 (less than 10 -5 M) but inhib-
150 50
100 0
a
----
-7
-3 50
-50
0
-7
-100 Log[CPT-11 ]
Fig. 7. A concentration-response curve of CPT-]I on spontaneous chronotropic response of the 8uinea-pig right atrium. Ordinate: percent of spontaneous heart rate and abscissa: log concentration (M) of CPT-II. Each point is
presented as a mean with SE of 9 experiments.
-'6
-'s
-'4
:3
Log[CPT-11 ]
Fig. 9. Concentration-response curves of CPT-11 tested in the uteri from ¢stradiol-treated (O) and castrated (O) rats. Ordinate: percent of spontaneous contraction and abscissa: log concentration (M) of CPT- 11. Each point is presented as a mean with SE of 6 experiments.
I. TAKAYANAGIet al.
766 150
100
50
0
,
-z
-~
*
-'5
-'4
-3
Log[CPT-11]
Fig. 10. Concentration-response curves of CPT- 11 on spontaneous contraction (O) and oxytocin-treated contraction (O) of the uteri from 15-17 day pregnant rats. Abscissa: percent of spontaneous contraction of percent of oxytocin (10-4 unit/ml)-induced contraction and abscissa: log concentration (M) of CPT-11. Each point is presented as a mean SE of 6 experiments.
ited by 10-4M of CPT-11 (Fig. 9). Spontaneous movement and oxytocin (10 -4 unit/ml)-induced contraction of uterus of pregnant rats were not influenced by CPT-11 (10-7-10 -4 U). DISCUSSION CPT- 11 induced contraction of guinea-pig ileal and tracheal preparations was completely inhibited by 10-6M of atropine, a sufficient concentration to block a maximal response to acetylcholine. These results indicated that CPT-11 has a muscarinic agonistic action. CPT-11 potentiated not only the response to acetylcholine but also those to nicotine, serotinin and BaC12 which were reported to contract the guinea-pig ileum, at least partially, through the acetylcholine release from cholinergic nerves (Gershon, 1967; Day and Vane, 1963; Brownlee and Harry, 1963). Furthermore, CPT-I1 was recently found to have acetylcholine action (unpublished observation). These results suggest that CPT-11 may contract the smooth muscle preparations with acetylcholine accumulated through an inhibition of enzymatic breakdown of acetylcholine but not through muscarinic acetylcholinoceptor activation. This view is supported by the present findings that the chronotropic and inotropic actions of atrium increased by isoprenaline, and the relaxation response of trachea to isoprenaline were inhibited by 10-SM
of CPT-11, which did not influence responses to the agonists except isoprenaline in the present study, suggesting a physiological antagonism. The present results indicate that CPT-11 does not interact with drug receptors, such as nicotonic and muscarinic cholinoceptors, c¢- and /7-adrenoceptors, prostaglandin receptors and oxytocin receptors. CPT-11 (more than 3 x 10 -5 M) inhibited the spontaneous movement in the uteri of the estradiol-treated and castrated rats but not that of uterus from pregnant rats. We cannot explain this discrepancy between the uteri from pregnant and nonpregnant rats, but these phenomena were observed in the experiments with other drugs (Takayanagi et al., 1981, 1988, 1989). Furthermore, CPT-11 (10 -4 M) inhibited the concentration-response curves of some agonists noncompetitively, therefore, CPT- 11 might have nonspecific action at its higher concentrations.
REFERENCES
Brownlee G. and Harry J. (1963) Some pharmacological properties of the circular and longitudinal muscle strips from the guinea-pig isolated ileum. Br. J. Pharmac. 21, 544-554. Day M. and Vane J. R. (1963) An analysis of the direct and indirect actions of drugs on the isolated guinea-pig ileum. Br. J. Pharmac. 20, 150-170. Gershon M. D. (1967) Effects of tetrodotoxin on innervated smooth muscle preparations. Br. J. Pharmac. 29, 259-279. Nitta K., Yokokura T., Sawada S., Kunimoto T., Tanaka T., Uehara N., Baba H., Takeuchi M., Miyasaka T. and Mutai M. (1987) Antitumor activity of novel derivatives of camptotheein. Jpn. J. Cancer Chemother. 14, 850-857 (In Japanese). Takagi K. and Takayanagi I. (1958) Chemico-pharmacotogical studies on antispasmodic action XV. Non-specific antispasmodic action on tracheal muscle. Chem. Pharm. Bull. 11, 379-383. Takayanagi I. and Koike K. (1985) A beta-adrenoceptor blocking agent, befunolol as a partial agonist in isolated organ. Gen. Pharmac. 16, 265-267. Takayanagi I., Fukae M., Suzuki M. and Shidama S. (1981) Effects of clenbuterol on various organs. Oyoyakuri 22, 345-353 (In Japanese). Takayanagi I., Konno F., Sato Y. and Uno H. (1988) Pharmacological properties of a new centrally acting muscle relaxant (NC-1200) in isolated muscle preparations. Gen. Pharmac. 19, 117-121. Takayanagi I., Hisayama T., Iwase M., Sakuma N. and Nagai H. (1989) Pharmacological properties of tiropramide, an antispasmodic drug. Gen. Pharmac. 20, 335-339. Wang Y., Inoue K., Shibata H., Itoh Y., Chen S. C. and Ogawa M. (1987) Preclinical evaluation of a new camptothecin derivative, CPT-I 1, on the subrenal capsule assay. Jpn. J. Cancer Chemother. 14, 1264-1267 (In Japanese).
0306-3623/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
SOME PHARMACOLOGICAL PROPERTIES OF A NEW ANTITUMOR D R U G , CPT-11, IN ISOLATED MUSCLE PREPARATIONS I. TAKAYANAGI, K. KOXKE, M. TAGAWA a n d E. MITSUHASHI Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan (Tel. (0474)72-I 141)
(Received 7 February 1989) Abstract--1. Pharmacological properties of a new antitumor drug, CPT-11, were studied in some muscle preparations. 2. CPT-11 induced contraction of guinea-pig ileal and tracheal preparations, which was blocked by atropine (10 -6 M). 3. CPT-11 potentiated the contractile responses of guinea-pig ileum to acetylcholine, nicotine, serotonin and BaCl 2. 4. The chronic and inotropic effects induced by isoprenaline were depressed by CPT-I 1. 5. These results suggest that CPT-I1 has an acetylcholine action.
INTRODUCTION CPT- 11, for which the chemical structure is s h o w n in Fig. 1, is a new derivative o f c a m p t o t h e c i n , a p l a n t alkaloid developed in Japan. W a n g et al. (1987) reported t h a t the a n t i t u m o r activity o b t a i n e d from CPT-11 is c o m p a r a b l e to those o f adriamycin, cyc l o p h o s p h a m i d e a n d 5-fluorouracil a n d concluded t h a t CPT-11 is a good c a n d i d a t e for clinical trials. F u r t h e r m o r e , the antileukemic activity o f CPT-11 ~against L1210 was f o u n d to be m u c h higher t h a n t h a t o f adriamycin a n d the acute toxicity o f CPT-11 'was also f o u n d to be extremely low, particularly in the case of oral a d m i n i s t r a t i o n (Nitta et al., 1987). However, p h a r m a c o l o g i c a l properties o f CPT-11 o n ,muscle p r e p a r a t i o n s were n o t studied. MATERIALS AND METHODS
Ileum, trachea and right atrium from guinea-pig Male guinea-pigs (250-350 g) of Hartley strain were killed by a blow on the neck and the ileum, trachea and heart were isolated. A piece (about 3 cm) of the ileum was suspended in a 20 ml organ bath filled with a physiological solution (NaC1 154, KCI 5.6, MgCI 2 2.1, CaCI: 2.2, NaHCO 3 5.9 and glucose 2.8 mM), gassed with a mixture of 95% 02 and 5% CO 2 (carbogen) and kept at 32°C. Responses to agonist were recorded isotonically under a tension of 0.5 g. The tracheal preparation was made by the method of Takagi and Takayanagi (1958) and mounted in a similar organ bath as described. Responses to a drug were recorded isotonically under a tension of 1.0 g. An isolated heart was placed in the physiological solution gassed with carbogen and kept at
N
N
37°C. All other tissues were cut away until nothing was left except the fight atria. The preparation was suspended in the physiological solution at 37°C, through which a brisk stream of carhogen was blown. Chronotropic and inotropic effects were recorded isometrically under a resting tension of 0.5 g (Takayanagi and Koike, 1985).
Vas deferens from rat Male Wistar strain rats (250-300 g) were killed by a blow on the head and the vasa deferentia were isolated. A piece (2-3 cm) of vas deferens was suspended in the similar organ bath as described. Responses to the drug were recorded isotonically under a tension of 0.5 g. Bath temperature used was 32°C. Thoracic aorta from rabbit Male rabbits (2.0-3.5 kg) were killed by bleeding from the neck. About 5 cm of thoracic aorta was dissected out close to the arch and cut helically. The aortic strips were then stripped of their endothelial cells by mechanical rubbing. A piece (about 2 mm in width and 3 cm in length) of aortic preparation was mounted in the similar bath as described. Responses to a drug were recorded isotonically under a tension of 2.0 g. Bath temperature used was 37°C. Uterus from rat Female Wistar rats (200-300 g) treated with estradiol benzate (0.2 mg/kg, s.c.) at 24 and 48 hr before sacrifice, female Wistar rats (300-350 g) castrated one week before sacrifice and also female Wistar rats in the 17th and 18th day of pregnancy were used. They were killed by a blow on the head and pieces (about 3 cm) of uterus were isolated. They were suspended in the similar organ bath as described. Responses to a drug were recorded isotonically under a tension of 0.5 g. Bath temperature used was 32°C.
N
0
HOgf -']0 Fig. 1. A chemical structure of CPT-I 1. 763 OP 20/6--D
HCI • sl.-~O
I. TAKAYANAGIet al.
764 100
150
50
100 o= -o
g c~ 50 -7
-6
-5
-4
Log[CPT-11]
Fig. 2. A concentration-response curve of CPT- l 1 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of CPT-11. Each point is presented as a mean with SE of 8 experiments.
In order to estimate antagonistic potency of a drug, the preparations were equilbrated with an antagonist for 5 min, after determination of control concentration-response curves of the agonist. Drugs used. CPT-I1 (Fig. 1) was kindly supplied from Daiichi Pharmaceutical (Tokyo, Japan). Other drugs were obtained commercially, all in powder form. RESULTS
Guinea-pig ileum (nicotinic and muscarinic cholinoceptors and histamine receptors) CPT-11 induced a c o n t r a c t i o n o f ileum in a conc e n t r a t i o n d e p e n d e n t m a n n e r , which was completely inhibited by a t r o p i n e (10 -6 M). The m a x i m u m cont r a c t i o n by CPT-11 was a b o u t 2 1 % o f t h a t induced by acetylcholine (Fig. 2). The contractile response to BaC12 (10 -3 M ) was p o t e n t i a t e d by CPT-11. Concent r a t i o n - r e s p o n s e curves o f acetylcholine a n d nicotine were also p o t e n t i a t e d by CPT-11, whereas the highest c o n c e n t r a t i o n (10 -4 M) used to inhibit the curves for b o t h the agonists (Figs 3 a n d 4). The effect o f CPT-11
~
-7
Rat vas deferens (¢t-adrenoceptors) CPT-11 (10-7-10 -4 M ) did n o t influence the spont a n e o u s m o v e m e n t o f vas deferens n o r the contractile responses o f vas deferens to n o r e p i n e p h r i n e (10-7-10-4 M).
100
100 "
~
,
t
,
-9
-8
-7
-6
,
,
-5
-4
-4
on the response to serotonin was also similar. The responses to lower concentrations (10-8-3 x 10 -7 M ) of histamine were potentiated by CPT-I1 (10-7-10-4M) but those to higher concentrations (10-6-10-5M) of histamine were not practically influenced. The highest concentration (10-4M) of CPT-11 used, inhibited the contractile response to h i s t a m i n e (Fig. 5).
150
o
-5
Fig. 4. Concentration-response curves of nicotine in the absence and presence of CPT-11 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of nicotine. Each point is presented as a mean with SE of 8 experiments. O: nicotine, alone, O: with CPT-II, 10 -7 M, A: with CPT-11, 10 -6 M, A: with CPT-1 l, l0 -5 M and I1: with CPT-I1, 10-4M.
150
5o
-6
Log[Serotonin)
~
-
5o
o -'3
Log[Acet, yl cho]ine]
Fig. 3. Concentration-response curves of acetylcholine in the absence and presence of CPT- 11 tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to acetylcholine and abscissa: log concentration (M) of acetylcholine. Each point is presented as a mean with SE of 8 experiments. O: acetylcholine, alone, O: with CPT-11, 10-7 M, A: with CPT-I 1, 10-6 M, A: with CPT-I1, 10-SM and II: with CPT-I1, 10-4M.
-9
-8
-7
-6
-5
-4
Log[Histamine]
Fig. 5. Concentration-response curves of histamine in the absence and presence of CPT-ll tested in the guinea-pig ileum. Ordinate: percent of a maximal contractile response to histamine and abscissa: log concentration (M) of histamine. Each point is presented as a mean with SE of 8 experiments. O: histamine, alone, (3: with CPT-11, 10 -7 M, A: with CPT-I1, 10-6 M, A: with CPT-I 1, 10-SM and II: with CPT-11, 10-4M.
Antitumor drug CPT-I 1
765 100
50
~
5o
-3
-50 -S
-50
-100
Log[ Isoproterenol ]
Log[CPT-11]
Fig. 6. A concentration-response curve of CPT-I1 on spontaneous inotropic response of the guinea-pig fight atrium. Ordinate: percent of the spontaneous tension and abscissa: log concentration (M) of CPT-I1. Each point is presented as a mean with SE of 5 experiments.
Rabbit thoracic aorta prostaglandin receptors)
(ot-adrenoceptors and
CPT-11 (10-7-10-4M) did not spontaneous movement of thoracic concentration response curves of (10-s-10-SM) and prostagiandin 3 x 10 -s M).
influence the aorta nor the norepinephrine F2= (3 × 10-7-
Guinea-pig right atrium (~-adrenoceptors) CPT-11 (more than 3 x l0 -5 M) decreased spontaneous chronotropic and inotropic actions (Figs 6 and 7). Isoprenaline 0 0 - 9 - 3 x l0 -7 M) increased the chronotropic and inotropic actions in a concentration dependent manner. The chronotropic and inotropic responses induced by isoprenaline were not influenced by CPT-11 0 0 -7 and l0 -6 M), whereas the higher concentrations (10 -5 and l0 -4 M) of CPT-I 1 parallelly shifted the concentration-response curves of isoprenaline to the bottom suggesting a physiological antagonism (Fig. 8).
Fig. 8. Concentration-ehronotropic response curves of isoprenaline in the absence and presence of CPT-II tested in the guinea-pig right atrium. Ordinate: percent of a maximal heart rate induced by isoprenaline and abscissa: log concentration (M) of isoprenaline. Each point is presented as a mean with SE of 6 experiments. 0 : isoprenaline, alone, O: with CPT-I1, 10-7M, &: with CPT-11, 10-6M, A: with CPT-11, 10-SM and I1: with CPT-11, 10-4M.
Guinea-pig trachea ([3-adrenoceptors and histamine
receptors) CPT-I1 (10-7-10 -5 M) induced the contractile response, the maximum of which was about 18% of the maximum response to histamine. The contractile responses to histamine (10-8-10 -4 M) were slightly (but not significantly) potentiated by CPT-11 (10-7-10-5M) but not by 10-4M of CPT-I1. The relaxation responses to isoprenaline (10-9-3 × 10-TM) were slightly inhibited by CPT-11 (10-7-10 -s M) but not by 10-4M of CPT-I 1.
Rat uterus (oxytocin receptors) Spontaneous contractile response of uteri from both estradiol-treated and castrated rats were not influenced by CPT-11 (less than 10 -5 M) but inhib-
150 50
100 0
a
----
-7
-3 50
-50
0
-7
-100 Log[CPT-11 ]
Fig. 7. A concentration-response curve of CPT-]I on spontaneous chronotropic response of the 8uinea-pig right atrium. Ordinate: percent of spontaneous heart rate and abscissa: log concentration (M) of CPT-II. Each point is
presented as a mean with SE of 9 experiments.
-'6
-'s
-'4
:3
Log[CPT-11 ]
Fig. 9. Concentration-response curves of CPT-11 tested in the uteri from ¢stradiol-treated (O) and castrated (O) rats. Ordinate: percent of spontaneous contraction and abscissa: log concentration (M) of CPT- 11. Each point is presented as a mean with SE of 6 experiments.
I. TAKAYANAGIet al.
766 150
100
50
0
,
-z
-~
*
-'5
-'4
-3
Log[CPT-11]
Fig. 10. Concentration-response curves of CPT- 11 on spontaneous contraction (O) and oxytocin-treated contraction (O) of the uteri from 15-17 day pregnant rats. Abscissa: percent of spontaneous contraction of percent of oxytocin (10-4 unit/ml)-induced contraction and abscissa: log concentration (M) of CPT-11. Each point is presented as a mean SE of 6 experiments.
ited by 10-4M of CPT-11 (Fig. 9). Spontaneous movement and oxytocin (10 -4 unit/ml)-induced contraction of uterus of pregnant rats were not influenced by CPT-11 (10-7-10 -4 U). DISCUSSION CPT- 11 induced contraction of guinea-pig ileal and tracheal preparations was completely inhibited by 10-6M of atropine, a sufficient concentration to block a maximal response to acetylcholine. These results indicated that CPT-11 has a muscarinic agonistic action. CPT-11 potentiated not only the response to acetylcholine but also those to nicotine, serotinin and BaC12 which were reported to contract the guinea-pig ileum, at least partially, through the acetylcholine release from cholinergic nerves (Gershon, 1967; Day and Vane, 1963; Brownlee and Harry, 1963). Furthermore, CPT-I1 was recently found to have acetylcholine action (unpublished observation). These results suggest that CPT-11 may contract the smooth muscle preparations with acetylcholine accumulated through an inhibition of enzymatic breakdown of acetylcholine but not through muscarinic acetylcholinoceptor activation. This view is supported by the present findings that the chronotropic and inotropic actions of atrium increased by isoprenaline, and the relaxation response of trachea to isoprenaline were inhibited by 10-SM
of CPT-11, which did not influence responses to the agonists except isoprenaline in the present study, suggesting a physiological antagonism. The present results indicate that CPT-11 does not interact with drug receptors, such as nicotonic and muscarinic cholinoceptors, c¢- and /7-adrenoceptors, prostaglandin receptors and oxytocin receptors. CPT-11 (more than 3 x 10 -5 M) inhibited the spontaneous movement in the uteri of the estradiol-treated and castrated rats but not that of uterus from pregnant rats. We cannot explain this discrepancy between the uteri from pregnant and nonpregnant rats, but these phenomena were observed in the experiments with other drugs (Takayanagi et al., 1981, 1988, 1989). Furthermore, CPT-11 (10 -4 M) inhibited the concentration-response curves of some agonists noncompetitively, therefore, CPT- 11 might have nonspecific action at its higher concentrations.
REFERENCES
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