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Secoverine selectively antagonizes muscarinic effects in various in vivo preparations
European Journal o f Pharmacology, 71 (1981) 165--168 © Elsevier/North-Holland Biomedical Press
165
Short communication S E C O V E R I N E S E L E C T I V E L Y A N T A G O N I Z E S M U S C A R I N I C E F F E C T S IN V A R I O U S IN VIVO P R E P A R A T I O N S JAN M.A. ZWAGEMAKERS and VOLKERT CLAASSEN Department o f Pharmacology, Duphar B. V,, P.O. Box 2, 1380 AA, Weesp, The Netherlands
J.M.A. ZWAGEMAKERS and V. CLAASSEN, Secoverine selectively antagonizes muscarinic effects in various in vivo preparations, European J. Pharmacol. 71 (1981) 165--168. Time-activity studies of secoverine and atropine were made with respect to mydriasis and oxotremorineinduced salivation, lacrimation and tremors. Marked differences were found in the anticholinergic activity relation between secoverine and atropine for various tissues. These differences remained present at all time intervals, which excludes a pharmacokinetic explanation. It may be concluded that secoverine possesses a different affinity for various muscarinic receptors. Muscarinic receptor heterogeneity Receptor-specific antagonists
Anticholinergic agents Organ specificity
1. I n t r o d u c t i o n We have studied a series o f s u b s t i t u t e d p h e n y l - e t h y l - a m i n o a l k y l k e t o n e s . One o f these c o m p o u n d s , 1-cyclohexyl-4- [ e t h y l ( p - m e t h o x y a-methylphenethyl)amino ] -1-butanone hyd r o c h l o r i d e (secoverine h y d r o c h l o r i d e , herea f t e r r e f e r r e d t o as secoverine) selectively and competitiycely blocks muscarinic r e c e p t o r s o n s m o o t h muscle ( Z w a g e m a k e r s and Claassen~ 1980). T h e c o m p o u n d is a c o m p e t i t i v e antagonist to c a r b a c h o l o n guinea-pig ileum and calf t r a c h e a muscle and to f u r t r e t h o n i u m on rat j e j u n u m . On the basis o f t h e m e a n d i f f e r e n c e o f the pA: values of secoverine and a t r o p i n e and in view o f the relative activity ( m e a s u r e d in 4 - p o i n t assays) o f the t w o c o m p o u n d s against cholinergic agonists in a series o f o t h e r s m o o t h muscle tissues, it m a y be c o n c l u d e d t h a t secoverine is 0.6 times as active as atropine. In vivo, secoverine s h o w e d a l m o s t the same relative activity as a t r o p i n e o n the intestinal tissue o f various species. S e c o v e r i n e was also effective in a n t a g o n i z i n g t h e central cholinergic effects o f o x o t r e m o r i n e . In c o n t r a s t , secoverine s h o w e d o n l y w e a k
Spasmolytic agents
Secoverine
m y d r i a t i c activity and little antagonism o f cholinergic-induced secretions. Such a diff e r e n c e in the anticholinergic activity o f secoverine on s m o o t h muscle c o m p a r e d w i t h o t h e r tissues has, besides its interesting therap e u t i c prospects, t h e o r e t i c a l interest in t h a t it c o u l d indicate c o n f o r m a t i o n a l d i f f e r e n c e s b e t w e e n various muscarinic r e c e p t o r s . As time-action relations are n o t necessarily identical for various organs, a w r o n g c h o i c e o f t h e time o f m e a s u r e m e n t o f the various anticholinergic effects m i g h t falsely indicate such a selectivity (Inch et al., 1973). F o r this reason we c o m p a r e d the antimuscarinic e f f e c t o f i n t r a v e n o u s l y (i.v.) a d m i n i s t e r e d secoverine with t h a t o f a t r o p i n e over a period f r o m o n e t o 4 0 m i n a f t e r drug administration. F o r e x p e r i m e n t a l c o n v e n i e n c e central anticholinergic activity was used in c o m p a r i s o n to the m y d r i a t i c , salivatory and l a c r i m a t o r y activities.
2. Materials and m e t h o d s M y d r i a t i c p o t e n c y at various time intervals after i.v. drug a d m i n i s t r a t i o n was tested in
166
J.M.A. ZWAGEMAKERS, V. CLAASSEN
female (Swiss CPB) mice (20-24g) using groups of 10 mice at each of three dose levels with a dose increment of 2.15. Pupil diameter was measured using a dissecting microscope with graduated ocular. After two initial readings, various doses of the test compounds were administered and pupil diameter was measured at different times after drug administration. The results were expressed as ED200 values, i.e. the mean dose that increased pupil diameter to 200%. Central and peripheral antimuscarinic activity was determined from the antagonism of the tremors and of salivation and lacrimation induced by oxotremorine in male (Swiss CPB) mice (18-25 g). The dose of oxotremorine sesquifumarate used was 160 p g . kg-' (1.5 times the ED90 for production of tremors, lacrimation and salivation in untreated animals). Secoverine and atropine were injected i.v. at various times before injecting oxotremorine i.v. Four groups of five mice were used with a dose increment of 2.15. The EDs0 values for inhibition of lacrimation, salivation
TABLE 1 Mydriatic activity in mice of intravenously injected secoverine hydrochloride and atropine sulphate after various time intervals. Time after dosing (min)
Dose mg • kg -1 to produce an increase in pupil diamter to 200% (ED200) Secoverine
2.5 5.0 10.0 20.0 40.0
2.0 2.6 3.7 >>3 >>3
Atropine 0.014 0.012 0.012 0.013 0.013
and tremors were calculated using the method of Horn (1956). The severity of tremors was not graded, and any dampness of the lips and the eyes was considered to indicate salivation and lacrimation respectively.
TABLE 2 Antagonism of oxotremorine-induced tremors, salivation and lacrimation in mice by intravenously injected secoverine hydrochloride and atropine sulphate after various time intervals. Time between administering antagonist and oxotremorine (min)
1.0 2.5 5.0 10.0 20.0 40.0
EDs0 values (95% confidence limits) mg• kg -1 Central Antagonism of tremors
Peripheral Antagonism of salivation
Antagonism of lacrimation
Secoverine
Atropine
Secoverine
Atropine
Secoverine
Atropine
3.48 (1.72-7.06) 4.30 (2.95-6.36) 4.22 (2.63-6.76) 3.48 (1.76-6.87) 6.81 (3.96-11.7) >10.0
1.71 (1.05-2.78) 1.71 (1.26-2.33) 1.62 (0.89-2.92) 0.88 (0.32-2.46) 1.90 (1.09-3.29) 1.21 (0.45-3.25)
>10
0.133 (0.066-0.270) 0.058 (0.043-0.079) 0.088 (0.042-0.184) 0.126 (0.078-0.205) 0.171 (0.105-0.278) 0.171 (0.101-0.291)
>10
0.100 (0.06-0.166) 0.058 (0.043-0.079) 0.058 (0.034-0.995) 0.068 (0.046-0.100) 0.147 (0.062-0.350) 0.147 (0.086-0.250)
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
SECOVERINE, A SELECTIVE MUSCARINIC ANTAGONIST 3. Results As is apparent from the results (table 1) secoverine had only a short-lasting, marginal, mydriatic activity in comparison with atropine during the whole time period studied, viz. 2.540 min after i.v. administration of the compound. The mydriatic activity of secoverine was less than 0.01 times that of atropine at all time intervals. The effects of secoverine and atropine against peripheral and central cholinergic actions of oxotremorine are depicted in table 2. It is clear that secoverine, during the time interval from 1 to 40 min after its i.v. administration up to a dose of 10 mg • kg -1 did not influence the cholinergic-induced lacrimation and salivation, whereas atropine proved to be very active in this respect. In contrast secoverine produced a marked antagonism of the oxotremorine-induced tremors during the time interval from 1 to 20 min after drug administration. Its activity declined somewhat more quickly than that of atropine. Comparison of the time-profile of the antagonism of central antimuscarinic (tremors) and of peripheral antimuscarinic (salivation and lacrimation) effects shows that the difference in potency between these two sites of action was present at all times studied.
4. Discussion Secoverine has been shown to be a highly active anticholinergic agent with an activity 0.6 times that of atropine on gastrointestinal smooth muscle. In the in vivo studies secoverine showed a swift onset of action and long-lasting reversible activity (Zwagemakers and Claassen, 1980). Secoverine showed a corresponding central anticholinergic effect. In contrast a weak activity of secoverine was found against cholinergic-induced secretions and the c o m p o u n d showed only minimal mydriatic effects. In the past, claims have been made about organ specific actions of antimuscarinic agents.
167
For instance, Biggs et al. (1972), in studies of diastereoisomeric esters of 3-tropanolol-l,3dimethyl-4-piperidinol and related compounds, claimed on the basis of response sensitivity that the muscarinic receptors in guinea-pig ileum differed from those in the circular muscle of the eye. However, Inch et al. (1973) criticized these conclusions as the mydriatic effect was measured long after the peak activity of the drugs under investigation. The relative sensitivity of various organs towards pharmacological agents may change with time and Inch et al. clearly demonstrated the importance in this c o n t e x t of making timeprofile studies. In the present studies we found that the relatively low anticholinergic responses of mydriasis, salivation and lacrimation were present during the entire time interval from one to f o r t y minutes after i.v. ,administration of secoverine. The organ-specific action of secoverine occurred irrespective of the time of measurement. It is unlikely that the difference in antimuscarinic activity in the organs tested was due to differences in drug kinetics. In both the central nervous system and intestinal smooth muscle secoverine showed a quick onset of action; there is no reason to expect that the compound should not reach other peripheral cholinergic receptor sites as easily. On the basis of the results obtained in our studies it is tempting to speculate about differences in the muscarinic receptors in the tissues investigated. Our results would suggest that the muscarinic receptors of the eye and exocrine glands are different from those located on smooth muscle and in the central nervous system. Many other investigators have found no differences between the muscarinic receptors in various tissues (Inch et al., 1973; Beld et al. 1975; Brimblecombe et al., 1971; Burgen et al., 1974, etc.). However, our ideas about subclasses of muscarinic receptors find support in the work of Barlow et al. (1976; 1980) who claimed the existence of different muscarinic receptors in guinea pig ileum and
168
atrium. The recent finding of another anticholinergic agent, pirenzepine, with an organ specific profile quite different from that of secoverine, supports the concept of heterogeneity of muscarinic receptors (Hammer et
a~., 1980). References Barlow, R.B., K.J. Berry, P.A.M. Glenton, N.M. Nikolaou and K.S. Soh, 1976, A comparison of affinity constants for muscarinic-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29°C and in ileum at 29°C and 37°C, Br. J. Pharmacol. 58,613. Barlow, R.B., K.V. Burston and A. Vis, 1980, Three types of muscarinic receptors, Br. J. Pharmacol. 68, 141P. Beld, A.J., S. v.d. Hoven, A.C. Wouterse and M.A.P. Zegers, 1975, Are muscarinic receptors in the central and peripheral nervous system different, European J. Pharmacol. 30, 360. Biggs, D.F., A.F. Casy and W.K. Jeffery, 1972, Stereochemical studies of antimuscarinic agents. Dia-
J.M.A. ZWAGEMAKERS, V. CLAASSEN stereoisomeric esters of 3-tropanol, 1,3-dimethyl4-piperidinol, and related compounds, J. Med. Chem. 15, 506. Brimblecombe, R.W., D.M. Green, T.D. Inch and P.B.J. Thompson, 1971, The significance of differences in the potency of enantiomers of antiacetylcholine drugs, J. Pharm. Pharmacol. 23, 745. Burgen, A.S.V., C.R. Hiley and J.M. Young, 1974, The properties of muscarinic receptors in mammalian cerebral cortex, Br. J. Pharmacol. 51,279. Hammer, R., C.P. Berrie, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature 283, 90. Horn, H.J., 1956, Simplified LDs0 (or EDs0) calculations, Biometrics 1 2 , 3 1 1 . Inch, T.D., D.M. Green and P.B.J. Thompson, 1973, The central and peripheral activities of anti-acetylcholine drugs. Some concepts of practical relevance, J. Pharm. Pharmacol. 25,359. Zwagemakers, J.M.A. and V. Claassen, 1980, Pharmacology of secoverine. A new spasmolytic agent with specific antimuscarinic properties. Part 1: antimuscarinic and spasmolytic effects, Arzneim. Forsch. 30, 1517.
165
Short communication S E C O V E R I N E S E L E C T I V E L Y A N T A G O N I Z E S M U S C A R I N I C E F F E C T S IN V A R I O U S IN VIVO P R E P A R A T I O N S JAN M.A. ZWAGEMAKERS and VOLKERT CLAASSEN Department o f Pharmacology, Duphar B. V,, P.O. Box 2, 1380 AA, Weesp, The Netherlands
J.M.A. ZWAGEMAKERS and V. CLAASSEN, Secoverine selectively antagonizes muscarinic effects in various in vivo preparations, European J. Pharmacol. 71 (1981) 165--168. Time-activity studies of secoverine and atropine were made with respect to mydriasis and oxotremorineinduced salivation, lacrimation and tremors. Marked differences were found in the anticholinergic activity relation between secoverine and atropine for various tissues. These differences remained present at all time intervals, which excludes a pharmacokinetic explanation. It may be concluded that secoverine possesses a different affinity for various muscarinic receptors. Muscarinic receptor heterogeneity Receptor-specific antagonists
Anticholinergic agents Organ specificity
1. I n t r o d u c t i o n We have studied a series o f s u b s t i t u t e d p h e n y l - e t h y l - a m i n o a l k y l k e t o n e s . One o f these c o m p o u n d s , 1-cyclohexyl-4- [ e t h y l ( p - m e t h o x y a-methylphenethyl)amino ] -1-butanone hyd r o c h l o r i d e (secoverine h y d r o c h l o r i d e , herea f t e r r e f e r r e d t o as secoverine) selectively and competitiycely blocks muscarinic r e c e p t o r s o n s m o o t h muscle ( Z w a g e m a k e r s and Claassen~ 1980). T h e c o m p o u n d is a c o m p e t i t i v e antagonist to c a r b a c h o l o n guinea-pig ileum and calf t r a c h e a muscle and to f u r t r e t h o n i u m on rat j e j u n u m . On the basis o f t h e m e a n d i f f e r e n c e o f the pA: values of secoverine and a t r o p i n e and in view o f the relative activity ( m e a s u r e d in 4 - p o i n t assays) o f the t w o c o m p o u n d s against cholinergic agonists in a series o f o t h e r s m o o t h muscle tissues, it m a y be c o n c l u d e d t h a t secoverine is 0.6 times as active as atropine. In vivo, secoverine s h o w e d a l m o s t the same relative activity as a t r o p i n e o n the intestinal tissue o f various species. S e c o v e r i n e was also effective in a n t a g o n i z i n g t h e central cholinergic effects o f o x o t r e m o r i n e . In c o n t r a s t , secoverine s h o w e d o n l y w e a k
Spasmolytic agents
Secoverine
m y d r i a t i c activity and little antagonism o f cholinergic-induced secretions. Such a diff e r e n c e in the anticholinergic activity o f secoverine on s m o o t h muscle c o m p a r e d w i t h o t h e r tissues has, besides its interesting therap e u t i c prospects, t h e o r e t i c a l interest in t h a t it c o u l d indicate c o n f o r m a t i o n a l d i f f e r e n c e s b e t w e e n various muscarinic r e c e p t o r s . As time-action relations are n o t necessarily identical for various organs, a w r o n g c h o i c e o f t h e time o f m e a s u r e m e n t o f the various anticholinergic effects m i g h t falsely indicate such a selectivity (Inch et al., 1973). F o r this reason we c o m p a r e d the antimuscarinic e f f e c t o f i n t r a v e n o u s l y (i.v.) a d m i n i s t e r e d secoverine with t h a t o f a t r o p i n e over a period f r o m o n e t o 4 0 m i n a f t e r drug administration. F o r e x p e r i m e n t a l c o n v e n i e n c e central anticholinergic activity was used in c o m p a r i s o n to the m y d r i a t i c , salivatory and l a c r i m a t o r y activities.
2. Materials and m e t h o d s M y d r i a t i c p o t e n c y at various time intervals after i.v. drug a d m i n i s t r a t i o n was tested in
166
J.M.A. ZWAGEMAKERS, V. CLAASSEN
female (Swiss CPB) mice (20-24g) using groups of 10 mice at each of three dose levels with a dose increment of 2.15. Pupil diameter was measured using a dissecting microscope with graduated ocular. After two initial readings, various doses of the test compounds were administered and pupil diameter was measured at different times after drug administration. The results were expressed as ED200 values, i.e. the mean dose that increased pupil diameter to 200%. Central and peripheral antimuscarinic activity was determined from the antagonism of the tremors and of salivation and lacrimation induced by oxotremorine in male (Swiss CPB) mice (18-25 g). The dose of oxotremorine sesquifumarate used was 160 p g . kg-' (1.5 times the ED90 for production of tremors, lacrimation and salivation in untreated animals). Secoverine and atropine were injected i.v. at various times before injecting oxotremorine i.v. Four groups of five mice were used with a dose increment of 2.15. The EDs0 values for inhibition of lacrimation, salivation
TABLE 1 Mydriatic activity in mice of intravenously injected secoverine hydrochloride and atropine sulphate after various time intervals. Time after dosing (min)
Dose mg • kg -1 to produce an increase in pupil diamter to 200% (ED200) Secoverine
2.5 5.0 10.0 20.0 40.0
2.0 2.6 3.7 >>3 >>3
Atropine 0.014 0.012 0.012 0.013 0.013
and tremors were calculated using the method of Horn (1956). The severity of tremors was not graded, and any dampness of the lips and the eyes was considered to indicate salivation and lacrimation respectively.
TABLE 2 Antagonism of oxotremorine-induced tremors, salivation and lacrimation in mice by intravenously injected secoverine hydrochloride and atropine sulphate after various time intervals. Time between administering antagonist and oxotremorine (min)
1.0 2.5 5.0 10.0 20.0 40.0
EDs0 values (95% confidence limits) mg• kg -1 Central Antagonism of tremors
Peripheral Antagonism of salivation
Antagonism of lacrimation
Secoverine
Atropine
Secoverine
Atropine
Secoverine
Atropine
3.48 (1.72-7.06) 4.30 (2.95-6.36) 4.22 (2.63-6.76) 3.48 (1.76-6.87) 6.81 (3.96-11.7) >10.0
1.71 (1.05-2.78) 1.71 (1.26-2.33) 1.62 (0.89-2.92) 0.88 (0.32-2.46) 1.90 (1.09-3.29) 1.21 (0.45-3.25)
>10
0.133 (0.066-0.270) 0.058 (0.043-0.079) 0.088 (0.042-0.184) 0.126 (0.078-0.205) 0.171 (0.105-0.278) 0.171 (0.101-0.291)
>10
0.100 (0.06-0.166) 0.058 (0.043-0.079) 0.058 (0.034-0.995) 0.068 (0.046-0.100) 0.147 (0.062-0.350) 0.147 (0.086-0.250)
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
SECOVERINE, A SELECTIVE MUSCARINIC ANTAGONIST 3. Results As is apparent from the results (table 1) secoverine had only a short-lasting, marginal, mydriatic activity in comparison with atropine during the whole time period studied, viz. 2.540 min after i.v. administration of the compound. The mydriatic activity of secoverine was less than 0.01 times that of atropine at all time intervals. The effects of secoverine and atropine against peripheral and central cholinergic actions of oxotremorine are depicted in table 2. It is clear that secoverine, during the time interval from 1 to 40 min after its i.v. administration up to a dose of 10 mg • kg -1 did not influence the cholinergic-induced lacrimation and salivation, whereas atropine proved to be very active in this respect. In contrast secoverine produced a marked antagonism of the oxotremorine-induced tremors during the time interval from 1 to 20 min after drug administration. Its activity declined somewhat more quickly than that of atropine. Comparison of the time-profile of the antagonism of central antimuscarinic (tremors) and of peripheral antimuscarinic (salivation and lacrimation) effects shows that the difference in potency between these two sites of action was present at all times studied.
4. Discussion Secoverine has been shown to be a highly active anticholinergic agent with an activity 0.6 times that of atropine on gastrointestinal smooth muscle. In the in vivo studies secoverine showed a swift onset of action and long-lasting reversible activity (Zwagemakers and Claassen, 1980). Secoverine showed a corresponding central anticholinergic effect. In contrast a weak activity of secoverine was found against cholinergic-induced secretions and the c o m p o u n d showed only minimal mydriatic effects. In the past, claims have been made about organ specific actions of antimuscarinic agents.
167
For instance, Biggs et al. (1972), in studies of diastereoisomeric esters of 3-tropanolol-l,3dimethyl-4-piperidinol and related compounds, claimed on the basis of response sensitivity that the muscarinic receptors in guinea-pig ileum differed from those in the circular muscle of the eye. However, Inch et al. (1973) criticized these conclusions as the mydriatic effect was measured long after the peak activity of the drugs under investigation. The relative sensitivity of various organs towards pharmacological agents may change with time and Inch et al. clearly demonstrated the importance in this c o n t e x t of making timeprofile studies. In the present studies we found that the relatively low anticholinergic responses of mydriasis, salivation and lacrimation were present during the entire time interval from one to f o r t y minutes after i.v. ,administration of secoverine. The organ-specific action of secoverine occurred irrespective of the time of measurement. It is unlikely that the difference in antimuscarinic activity in the organs tested was due to differences in drug kinetics. In both the central nervous system and intestinal smooth muscle secoverine showed a quick onset of action; there is no reason to expect that the compound should not reach other peripheral cholinergic receptor sites as easily. On the basis of the results obtained in our studies it is tempting to speculate about differences in the muscarinic receptors in the tissues investigated. Our results would suggest that the muscarinic receptors of the eye and exocrine glands are different from those located on smooth muscle and in the central nervous system. Many other investigators have found no differences between the muscarinic receptors in various tissues (Inch et al., 1973; Beld et al. 1975; Brimblecombe et al., 1971; Burgen et al., 1974, etc.). However, our ideas about subclasses of muscarinic receptors find support in the work of Barlow et al. (1976; 1980) who claimed the existence of different muscarinic receptors in guinea pig ileum and
168
atrium. The recent finding of another anticholinergic agent, pirenzepine, with an organ specific profile quite different from that of secoverine, supports the concept of heterogeneity of muscarinic receptors (Hammer et
a~., 1980). References Barlow, R.B., K.J. Berry, P.A.M. Glenton, N.M. Nikolaou and K.S. Soh, 1976, A comparison of affinity constants for muscarinic-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29°C and in ileum at 29°C and 37°C, Br. J. Pharmacol. 58,613. Barlow, R.B., K.V. Burston and A. Vis, 1980, Three types of muscarinic receptors, Br. J. Pharmacol. 68, 141P. Beld, A.J., S. v.d. Hoven, A.C. Wouterse and M.A.P. Zegers, 1975, Are muscarinic receptors in the central and peripheral nervous system different, European J. Pharmacol. 30, 360. Biggs, D.F., A.F. Casy and W.K. Jeffery, 1972, Stereochemical studies of antimuscarinic agents. Dia-
J.M.A. ZWAGEMAKERS, V. CLAASSEN stereoisomeric esters of 3-tropanol, 1,3-dimethyl4-piperidinol, and related compounds, J. Med. Chem. 15, 506. Brimblecombe, R.W., D.M. Green, T.D. Inch and P.B.J. Thompson, 1971, The significance of differences in the potency of enantiomers of antiacetylcholine drugs, J. Pharm. Pharmacol. 23, 745. Burgen, A.S.V., C.R. Hiley and J.M. Young, 1974, The properties of muscarinic receptors in mammalian cerebral cortex, Br. J. Pharmacol. 51,279. Hammer, R., C.P. Berrie, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature 283, 90. Horn, H.J., 1956, Simplified LDs0 (or EDs0) calculations, Biometrics 1 2 , 3 1 1 . Inch, T.D., D.M. Green and P.B.J. Thompson, 1973, The central and peripheral activities of anti-acetylcholine drugs. Some concepts of practical relevance, J. Pharm. Pharmacol. 25,359. Zwagemakers, J.M.A. and V. Claassen, 1980, Pharmacology of secoverine. A new spasmolytic agent with specific antimuscarinic properties. Part 1: antimuscarinic and spasmolytic effects, Arzneim. Forsch. 30, 1517.