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Pharmacological studies on novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 and YM954
European Journal of Pharmacology, 187 (1990) 479-486
479
Elsevier EJP 51539
Pharmacological studies on novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 and YM954 Fumikazu Wanibuchi, Takako Konishi, Masatomi Harada, Michio Terai, Kazuyuki Hidaka, Toshinari Tamura, Shin-ichi Tsukamoto and Shinji Usuda Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, lbaraki 305, Japan
Received 30 May 1990, accepted 31 July 1990
We have investigated the pharmacological profiles of the novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 (2,8-dimethyl-3-methylene) and YM954 (2-ethyl-8-methyl-3-oxo). These compounds, like the putative M 1 agonists, RS86 and AF102B, inhibited [3H]pirenzepine binding to cerebral cortical membranes in the micromolar range and weakly inhibited [3H]quinuclidinyl benzylate binding to cerebellar membranes. Their ( - ) isomers had Hill coefficients lower than 1.0. (+)-YM796, (+)-YM954 and RS86, but not AF102B, stimulated phosphoinositide hydrolysis in hippocampal slices, an effect which is mainly linked to M~ receptors. (+)-YM796 (0.031 mg/kg p.o.) and (+)-YM954 (0.016 m g / k g p.o.) reversed the cognitive impairment in nucleus basalis magnocellularis-lesioned rats in a passive avoidance task more effectively than did RS86 and AF102B. Similar results were obtained in scopolamine-treated rats. Finally, (+)-YM796 was weaker than (+)-YM954 and RS86 in the induction of tremor, hypothermia and contraction of isolated ileum, which are mainly mediated by M 2 a n d / o r M 3 receptors. These results suggest that (+)-YM796, (_+)-YM954 and RS86 have M 1 agonistic activity in central nervous system and that (+)-YM796 has relatively weak M 2 and/or M 3 agonistic activity. Muscarinic acetylcholine receptor agonists; YM796; YM954; Muscarinic M1 receptors; Muscarinic M 2 receptors; Learning behaviour
I. Introduction A n u m b e r of reports (Bowen et al., 1976; Perry et al., 1977; Reisine et al., 1978) have shown that choline acetyltransferase (CHAT) activity, a marker of presynaptic cholinergic function, decreases in the brain of dementia of Alzheimer type ( D A T ) patients, with a marked loss of cholinergic neurons in the nucleus basalis of Mynert which provides innervation to the neocortex (Whitehouse et al., 1981; 1982). The significant correlation be-
Correspondence to: F. Wanibuchi, Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, lbaraki 305, Japan.
tween the severity of dementia and the reduction of C h A T activity in D A T patients suggests that the m e m o r y dysfunction in the patients is closely related to the cholinergic h y p o f u n c t i o n in the brain (Perry et al., 1978; Wilcock et al., 1982). The n u m b e r of postsynaptic muscarinic receptors, predominantly of the M a subtype, is suggested to be unchanged in D A T (Mash et al., 1985; A r a u j o et al., 1988). M a muscarinic receptors are enriched in cerebral cortex and hippocampus, b o t h of which are associated with m e m o r y and learning (Watson et al., 1986). Accumulating evidence suggests that M 1 agonists are possible therapeutic agents for D A T . A muscarinic agonist such as RS86 (Hollander et al., 1986), while it improves the cognitive deficit of D A T patients, also induces cardiovascu-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
480
#"'TjGH3 -
~'CH 2
YM796
/~
#----]I ~C2H5
~
~'~
~"O
Doses are expressed in terms of the free base. The drugs were dissolved in distilled water or in the appropriate buffer.
YM954
2.2. Animals"
O RS86
AF102B
Fig. 1. Chemical structures of YM796, YM954, RS86 and AFI02B. Asterisks denote asymmetric centers.
lar and abdominal side-effects which are considered to be mediated through the stimulation of M 2 a n d / o r M 3 muscarinic receptors. Therefore, selective Ma agonists would be expected to have an advantage over non-selective muscarinic agonists for the treatment of DAT regarding the avoidance of M z- a n d / o r M3-mediated side-effects. In the present studies, we have examined the in vivo and in vitro pharmacological profiles of YM796 (2,8-dimethyl-3-methylene-l-oxa-8-azaspiro[4.5]decane) and YM954 (2-ethyl-8-methyl-1oxa-8-azaspiro[4.5]decane-3-one) (fig. 1). Preliminary results of this work were reported elsewhere (Wanibuchi et al., 1989).
2. Materials and methods
2. l. Materials ( + )-YM796, ( + )-YM95& their optical isomers, RS86 (2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane1,3-dione hydrobromide, fig. 1) and AF102B (( _+)cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine hydrochloride, fig. 1) were synthesized in our laboratory. The following were obtained commercially: scopolamine hydrobromide (Tokyo Kasei Co., Ltd.), acetylcholine chloride (Dai-ichi Pharmaceutical Co., Ltd.), carbachol (Wako Pure Chemical Industries, Ltd.), arecoline (Tokyo Kasei Kogyo), [3H]pirenzepine (DuPont), [3H]quinuclidinyl benzylate (DuPont), myo-[3H]inositol (DuPont). Both YM compounds were synthesized as the fumarate, the maleate or the hydrochloride.
Male ICR mice (Japan Sic. Inc.), male Wistar rats (Japan Slc. Inc.) and male guinea pigs (Hitachi Animal Medical Research Institute) were used. The animals were given free access to food and water with a 12-h light-dark cycle (light on 7 : 00 19:00 h). Behavioral studies were performed between 9 : 00-17 : 00 h. 2.3. In vitro receptor binding assays The binding assays described here were conducted according to known methods; 0.06 nM [3H]quinuclidinyl benzylate (QNB) binding to M 2 and/or M 3 receptors in the cerebellum membrane of rats was determined by the methods of Yamamura and Snyder (1974) and 1.0 nM [3H]pirenzepine (PZ) binding to M~ receptors in the cerebral cortex membrane of rats, by the methods of Watson et al. (1982). The IC50 values, concentrations required to inhibit specific binding by 50%, were computed by logit-log analysis. 2.4. Phosphoinositide (PI) hydrolysis Rat hippocampal slices (350 × 350 ~m) were placed in vials and preincubated for 60 rain at 37 ° C in Krebs-Heinseleit bicarbonate buffer containing myo-[3H]inositol under 95% 02-5% CO2. After addition of muscarinic agonists and 10 mM LiC1, the slices were incubated further with high K + (19 raM) for 60 rain and the reaction was terminated by the addition of chloroformmethanol. The vials were centrifuged and the aqueous phase was removed for assay of [3H]inositolphosphates (InsPs) and the organic phase was taken for the assay of lipids (Costa et al., 1986; Court et al., 1986). The results are expressed as d.p.m. InsPs/(lipids + InsPs). 2.5. Nucleus basalis rnagnocellularis (NBM) lesion The rats (250-300 g) were anesthetized with sodium pentobarbital (50 m g / k g i.p.) and placed
481
in a stereotaxic apparatus (David K o p f Instruments) so that bregma and lambda were in the same horizontal plane. Bilateral electrolytic lesions of N B M (0.8 m m posterior to bregma, 2.5 m m lateral to the midline and 8.2 m m ventral to the surface of the skull) were produced by passing a DC current (2 mA, 30 s) through a monopolar electrode. Sham operations were made by the same surgical procedures without delivery of the current. Behavioral studies were performed at least 2 weeks after surgery. ChAT activity in the frontal cortex and hippocampus was measured after the behavioral studies by the methods of Fonnum (1975).
sic activity was determined as the m a x i m u m response relative to that obtained with acetylcholine in the same preparation.
3. Results
3.1. In vitro receptor binding assays As shown in table 1, ( + ) - Y M 7 9 6 and ( + ) YM954 inhibited [3H]pZ binding to the cerebral cortical membranes with IC50 values of 2.85 and 0.67 ~M, respectively, and [3H]QNB binding to the cerebellar membranes with values of 24.49 and 3.33 #M, respectively. The ratio of the IC50 values
2.6. Passive avoidance tasks with scopolaminetreated and NBM-lesioned rats TABLE 1
In the training session, each rat was placed in the lighted box of a two-compartment passive avoidance apparatus ( O ' H a r a Co., Ltd.). When the rat entered the dark compartment, a foot-shock (60-70 V AC) was applied for 1 s through the metal grid bars of the floor. Retention was assessed 24 h after the training session. The latency to enter the dark compartment was measured up to a maximum cut-off of 300 s. All of the compounds tested were administered p.o. 45 min before the training session. Scopolamine (1 m g / k g ) was i.p. injected in naive rats (210-280 g) 30 min before the session. 2.7. Tremor and hypothermia Tremor occurrence was checked 0 - 3 0 min after oral administration of drugs to mice (30-42 g). Rectal temperature was measured with a thermoprobe (MC-111, Omron) before and 30 min after administration. 2.8. Contraction of isolated ileum Contraction of isolated ileum of guinea pigs (400-700 g) was measured isometrically with transducers (Nihon Koden). ECs0 values, the concentrations producing 50% of the individual maxim u m response, were obtained graphically from the concentration-response relationship curves. Intrin-
Comparison of the affinity of YM796, YM954 and other muscarinic agonists for [3H]pirenzepine and [3H]QNB b binding sites in rat frontal cortex and cerebellum, respectively. IC50 values are expressed as the micromolar concentration (the means_+ S.E. of six experiments). Figures in parentheses show the Hill coefficient (NH). Drug
_+)-YM796
ICs0 (NH)
Selectivity a
[3H]Pirenzepin e [3H]QN B b
for M 1 receptor
2.85 _+0.04 (0.783 _+0.039) 6.95 _+0.50 (0.922 _+0.080) 2.12 _+0.05 (0.736 -+0.041)
24.49 _+1.87 8.60 (0.776_+0.064) 78.44 _+2.89 11.29 (0.980_+0.045) 11.42 _+0.15 5.39 (0.739-+0.029)
0.67 _+0.02 (0.928_+0.057) +)-YM954 0.79 _+0.01 (1.001 _+0.021) (-)-YM954 0.44 _+0.01 (0.883 _+0.026)
3.33 _+0.18 4.97 (0.757_+0.089) 8.02 _+0.27 10.15 (0.879_+0.075) 2.20 _+0.08 5.00 (0.750_+0.062)
RS86
0.59 +0.01 (0.805 ± 0.027) 0.70 _+0.06 (0.981 -+0.092)
4.63 _+0.09 (0.667_+0.042) 2.45 +0.04 (0.775-+0.037)
7.85
2.16 -+0.15 (0.525 _+0.033) 0.85 +0.05 (0.579+0.052)
0.37 -+0.03 (0.476_+0.014) 0.73 -+0.21 (0.612_+0.081)
0.17
+)-YM796 -)-YM796 _+)-YM954
AF102B Carbachol Arecoline
3.50
0.86
a Selectivity is shown by the ratio of the IC50 value for [3H]QNB binding to that for [3H]pirenzepine binding, b QNB, quinuclidinyl benzylate.
482
of [3H]QNB binding to those of [3H]pZ binding was 8.60 for ( + ) - Y M 7 9 6 and 4.97 for ( + )-YM954. These values were similar to those for RS86 and AF102B, and much greater than those for carbachol and arecoline. The Hill coefficients of ( - ) isomers of YM796 and YM954 were less than 1.0 whereas the corresponding ( + ) isomers had coefficients near unity, especially with [3H]PZ binding.
TABLE 2
3.2. Stimulation of PI hydrolysis
tions of 10-1000 t~M (fig. 2). (_+)-YM796 needed a higher concentration to exhibit similar effects. AF102B failed to stimulate hydrolysis at 10-1000 ~M. A full muscarinic agonist, carbachol (CCh), markedly stimulated hydrolysis at the concentration of 100/~M.
Both (_+)-YM954 and RS86 stimulated PI hydrolysis in hippocampal slices at the concentra-
Effects of (_+)-YM796, (_+)-YM954 and other putative M I agonists on scopolamine-induced amnesia in rats. Each value indicates the minimal effective dose (MED). Shock intensity in the training session was 70 V. Drug
(+)-YM796
(,+)-YM954
RS86
AF102B
MED ( m g / k g p.o.)
0.063
0.016
0.5
0.5
0.6
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Bilateral NBM lesioning markedly reduced the latency to enter the dark compartment 24 h after the training session, as compared with the sham operation (fig. 3). (_+)-YM796 significantly inhibited the reduction of latency at doses of 0.031 0.25 m g / k g p.o. (_+)-YM954 also inhibited, but somewhat less effectively, at doses of 0.016 and 0.063 m g / k g p.o. RS86 and AF102B also inhibited the reduction at doses of 0.13 1.0 and 0.5-4.0 m g / k g p.o., respectively. NBM lesioning decreased ChAT activity to about 70% in frontal cortex, as compared with the sham operation, but did not affect the activity in hippocampus (data not shown). Similar results were obtained with scopolamine-induced amnesia (table 2). ( + ) - Y M 7 9 6 , ( + ) - Y M 9 5 4 , RS86 and AF102B improved the scopolamine-induced deficit in learning behavior.
e-
TABLE 3
0.0 10"~10"~10-*10-a(M)
Cent CCh(±)gM954 Fig. 2. Effects of ( + ) - Y M 7 9 6 , (,+)-YM954 and other muscarinic agonists on K+-stimulated phosphoinositide hydrolysis in hippocampal slices. Each column indicates the mean-+ S.E. of triplicate determinations. CCh = carbachol. * P < 0.05, * * P < 0.01, * * * P < 0.001 compared with the corresponding control (Cont) using Student's t-test.
Effects of ( + ) - Y M 7 9 6 , (,+)-YM954 and other putative M 1 agonists on contraction of guinea pig isolated ileum. Each value was obtained graphically from four experiments. Drug
(+)-YM796
(,+)-YM954
RS86
AF102B
ECs0 (~tM)
8.6
2.3
0.90
6.1
Intrinsic activity a
0.96
0.97
1.02
0.51
The maximum response to acetylcholine = 1.00.
483
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(Dose, mg/kg, p.o.) Fig. 3. Effects of (4,)-YM796, (4-)-YM954 and other putative M l agonists on the amnesia induced by lesioning the nucleus basalis magnocellularis in rats. Ordinate indicates latency to enter the dark compartment 24 h after training. Each column indicates the mean 4- S.E. The number of rats is shown at the bottom of the columns. Shock intensity in the training session was 60 V. * P < 0.05, • * P < 0.01 compared with control (Cont) using Mann-Whitney U-test.
(4-)-YM796 and (_+)-YM954 were 8 and 32 times more potent, respectively, than the equally effective RS86 and AF102B at their minimal effective dose.
100
RS86 /13
C
~ (4-)YM954
/
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3.4. Tremor and hypothermia As shown in fig. 4, (4-)-YM796 failed to elicit tremor in mice even at a dose of 80 m g / k g p.o., whereas RS86 and (4-)-YM954 induced tremor at doses higher than 2.5 and 5.0 m g / k g p.o., respectively. AF102B at a dose of 80 m g / k g p.o. induced tremor in 38% of the mice. All of the compounds reduced rectal temperature in a dose-dependent manner (fig. 5). (4-)YM796 and AF102B significantly reduced the temperature at doses of 32-64 m g / k g p.o. but
~ 50 N o~
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o
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s
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Dose (mg/kg, p.o.) Fig. 4. Effects of ( _+)-YM796, ( 4- )-YM954 and other putative M l agonists on tremor in mice. Ordinate indicates the percentage of mice displaying tremor out of eight animals.
484 o .q v
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Dose (mg/kg, p.o.) Fig. 5. Effects of (+)-YM796, (+)-YM954 and other putative M1 agonists on rectal temperature in mice. Ordinate indicates the difference in temperature between before and 30 min after the drug administration. Each point represents the mean + S.E. for eight animals. * P < 0.001 compared with control using Scheffd's test.
their hypothermic activity was much weaker than that of RS86 and (_+)-YM954.
3.5. Contraction of isolated ileum Table 3 shows the effects of (_+)-YM796, (+_)YM954, RS86 and A F I 0 2 B on the contraction of guinea pig isolated ileum. RS86 was the most potent of the four compounds, followed by (_+)YM954. The remaining two compounds, ( + ) YM796 and AF102B, were much less potent.
4. Discussion ( _+)-YM796 and ( _+ )-YM954 inhibited [3H]pZ binding to the cerebral cortical membranes in the micro~nolar range to the same extent as did the reference c o m p o u n d s , RS86 (Palacios et al., 1986) and A F 1 0 2 B (Fisher et al., 1986), indicating that these two Y M c o m p o u n d s have an affinity to the M~ muscarinic receptor. These compounds, including the reference c o m p o u n d s , also inhibited [ 3 H ] Q N B binding to the cerebellar membranes. As there is essentially no M 1 receptor in the cerebellum (Giraldo et al., 1986), the binding site of [3H]QNB in the region is considered to be the
M 2 a n d / o r M 3 receptor. The ratio of IC50 values for [3H]QNB binding to those for [3H]pZ binding of ( _+)-YM796, (_+)-YM954, RS86 and A F 1 0 2 B binding was much greater than those for carbachol and arecoline, indicating that these c o m p o u n d s have a more selective affinity for the M 1 receptor than the classical agonists. PI hydrolysis has been suggested to be coupled with MI receptors as its second messenger system (Watson et al., 1984; H a r a d a et al., 1989). Since both ( ± ) - Y M 7 9 6 and ( + ) - Y M 9 5 4 , like RS86, stimulated PI hydrolysis, these Y M c o m p o u n d s are likely to have M 1 agonistic activity in brain tissue. A F I 0 2 B has been reported to produce a depolarizing response in isolated superior cervical ganglia of rabbits and thus has been suggested to be an M 1 agonist ( M o c h i d a et al., 1988). However, in agreement with the previous report (Fisher et al., 1987), we observed no stimulating effect of A F 1 0 2 B on PI hydrolysis. This implies that any M 1 agonistic activity of A F 1 0 2 B that exists in brain would be very weak as c o m p a r e d with that of Y M c o m p o u n d s and RS86. (_+)-YM796 and (_+)-YM954 ameliorated the learning deficit induced by scopolamine or N B M lesion in rats. The effective doses of both Y M c o m p o u n d s were smaller than those of RS86 and
485
AF102B. Scopolamine, an antimuscarinic drug, is well known to reduce learning ability in animals and humans. NBM provides a major cholinergic innervation to the neocortex and is thought to be homologous to the nucleus basalis of Mynert in humans (Salamone, 1986). Lesioning of NBM decreased ChAT activity in the rat frontal cortex to about 70% in the present studies, which was consistent with the results of Hepler et al. (1985). Muscarinic receptors in the cerebral cortex are predominantly of the M 1 subtype (Watson et al., 1986) and, as already described, ( _+)-YM796, ( + )YM954 and RS86 stimulated PI hydrolysis in the brain. Therefore, it is likely that the ameliorating effects of both YM compounds and RS86 are at least in part mediated by M1 receptors. AF102B improved the learning deficit both in the present studies and in that of Fisher et al. (1986). These effects might also be due to its weak M~ agonistic activity in the brain. (_+)-YM796 and AF102B were less potent to induce tremor and hypothermia than were ( + ) YM954 and RS86. Together with the finding that (_+)-YM796 but not AF102B stimulated P1 hydrolysis, it is of particular interest that ( _+)-YM796 induced neither tremor nor hypothermia at doses more than 10-fold higher than the minimal effective dose of RS86. In addition, (_+)-YM796 was weaker to induce contraction of guinea pig isolated ileum than were RS86 and (_+)-YM954. Tremor and hypothermia (Caulfield et al., 1983) and contraction of isolated ileum (Brow~ et al., 1980) by muscarinic agonists have been suggested as being associated with the muscarinic M 2 a n d / o r M 3 subtype. These results suggest that the M 2 a n d / o r M 3 agonistic activity of (_+)-YM796 is much weaker than that of RS86 and (_+)-YM954. Therefore, ( _+)-YM796 is expected to be devoid of serious cardiovascular effects through M 2 receptors. Binding studies using radiolabelled antagonists yielded displacement curves for muscarinic agonists with Hill coefficients less than 1.0, whereas muscarinic antagonists had much steeper curves (Birdsall et al., 1978; Hulme et al., 1978; Freedman et al., 1988). In the present studies, the displacement curves for ( - ) isomers of both YM compounds had lower Hill coefficients than those
of the corresponding ( + ) isomers. These results may indicate that the intrinsic activity of ( - ) isomers for the muscarinic receptors is more potent than that of ( + ) isomers. Further experiments are under way to clarify the pharmacological profiles of the optical isomers of YM796 and YM954. In conclusion, the present results suggest that (_+)-YM796, (_+)-YM954 and RS86 possess M t agonistic activity, and that ( + ) - Y M 7 9 6 has less potent M 2 a n d / o r M 3 agonistic activity in both central and peripheral tissues than (_+)-YM954 and RS86.
Acknowledgements The authors are grateful to Drs. H. Maeno and K. Koshiya for critical reading of the manuscript and to Mr. H. ltoh and Ms. R. Tsutsumi for their helpful suggestions and kind assisstance.
References Araujo, D.M., P.A. Lapchak, Y. Robitaille, S. Gauthier and R. Quirion, 1988, Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer's disease, J. Neurochem. 50, 1914. Birdsall, N.J.M., A.S.V. Burgen and E.C. Hulme, 1978, The binding of agonists to brain muscarinic receptors, Mol. Pharmacol. 14, 723. Bowen, D.M., C.B. Smith, P. White and A.N. Davison, 1976, Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain 99, 459. Brown, D.A., A. Forward and S. Marsh, 1980, Antagonist discrimination between ganglionic and ileal muscarinic receptors, Br. J. Pharmacol. 71, 362. Caulfield, M.P., G.A. Higgins and D.W. Straughan, 1983, Central administration of the muscarinic receptor subtypeselective antagonist pirenzepine selectively impairs passive avoidance learning in the mouse, J. Pharm. Pharmacol. 35, 131. Costa, L.G., G. Kaylor and S.D. Murphy, 1986, Carbacholand norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition, J. Pharmacol. Exp. Ther. 239, 32. Court, J,A., C.J. Fowler, J.M. Candy, P.R. Hoban and C.J. Smath, 1986, Raising the ambient potassium ion concentration enhances carbachol stimulated phosphoinositide hydrolysis in rat brain hippocampal and cerebral cortical miniprisms, Naunyn-Schmiedeb. Arch. Pharmacol. 334, 10. Fisher, A., R. Brandeis, Z. Pittel, I. Karton, M. Sapir, S. Dachir, A. Levy, F. Mizobe and E. Heldman, 1987,
486 AF102B: a new M1 agonist with potential application in Alzheimer's disease, Soc. Neurosci. Abstr. 13, 657. Fisher, A., E. Heldman, R. Bradeis, Z. Pittel, S. Dachir, A. Levy and I. Karton, 1986, AF102B: a novel putative M1 agonist reverses AF64A-induced cognitive impairments in rats, Soc. Neurosci. Abstr. 12, 702. Fonnum, F., 1975, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24, 407. Freedman, S.B., E.A. Harley and L.L. Iversen, 1988, Biochemical measurement of muscarinic receptor efficacy and its role in receptor regulation, Trends Pharmacol. Sci. (Suppl.) 3, 54. Giraldo, E., R. H a m m e r and H. Ladinsky, 1987, Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with A F - D X 116 and pirenzepine, Life Sci. 40, 833. Harada, M., K. Hidaka, M. Terai, S. Usuda, F. Wanibuchi, S. Tsukamoto and T. Tamura, 1989, Muscarinic receptor subtypes linked to phosphoinositide hydrolysis and acetylcholine release in rat brain, J. Neurochem. 52 (Suppl), $36A. Hepler, D.J., D.S. Olton, G.L. Wenk and J.T. Coyle, 1985, Lesions in nucleus basalis magnocellularis and medial septal area of rats produce qualitatively similar memory impairments, J. Neurosci. 5, 866. Hollander, E., M. Davidson, R.C. Mohs, T.B. Horvath, B.M. Davis, Z. Zemishlany and K.L. Davis, 1987, RS 86 in the treatment of Alzheimer's disease: cognitive and biological effects, Biol. Psychiat. 22, 1067. Hulme, E.C., N.J.M. Birdsall, A.S.V. Burgen and P. Mehta, 1978, The binding of antagonists to brain muscarinic receptors, Mol. Pharmacol. 14, 737. Mash, D.C., D.D. Flynn and L.T. Potter, 1985, Loss of M2 muscarinic receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation, Science 228, 1115. Mochida, S., F. Mizobe, A. Fisher, G. Kawanishi and H. Kobayashi, 1988, Dual synaptic effects of activating M1muscarinic receptors, in superior cervical ganglia of rabbits, Brain Res. 455, 9. Palacios, J.M., G. Bolliger, A. Closse, A. Ent, G. Gmelin and J. Malanowski, 1986, The pharmacological assessment of RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-l,3-dion hydrobromide); a potent specific muscarinic acetylcholine receptor agonist, European J. Pharmacol. 125, 45. Perry, E.K., R.H. Perry, P.H. Gibson, G. Blessed and B.E. Tomlinson, 1977, A cbolinergic connection between normal
aging and senile dementia in the h u m a n hippocampus, Neurosci. Lett. 6, 85. Perry, E.K., B.E. Tomlinson, G. Blessed, K. Bergmann, P.H. Gibson and R.H. Perry, 1978, Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia, Br. Med. J. 2, 1457. Reisine, T.D., H.I. Yamamura, E.D. Bird, E. Spokes and S.J. Enna, 1978, Pre- and postsynaptic neurochemical alterations in Alzheimer's disease, Brain Res. 159, 477. Salamone, J.D., 1986, Behavioral functions of nucleus basalis magnocellularis and its relationship to dementia, Trends Neurosci. 9, 256. Wanibuchi, F., S. Usuda, T. Konishi, M. Harada, M. Terai, K. Hidaka, S. Tsukamoto and T. Tamura, 1989, Characterization of novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives in biochemical and behavioral studies, Soc. Neurosci. Abstr. 15, 731. Watson, M., W.R. Roeske, T.W. Vickroy, T . L Smith, K. Akiyama, K. Gulya, S.P. Duckles, M. Serra, A. Adam, A. Nordberg, D.R. Gehlert, J.K. Wamsley and H.I. Yamamura, 1986, Biochemical and functional basis of putative muscarinic receptor subtypes and its implications, Trends Pharmacol. Sci. (Suppl. Subtypes of muscarinic receptors II), 46. Watson, M., W.R. Roeske and H.I. Yamamura, 1982, [3H]Pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rats cerebral cortex, Life Sci. 31, 2019. Watson, M., T.W. Vickroy, W.R. Roeske and H.I. Yamamura, 1984. Subclassification of muscarinic receptors based upon the selective antagonist pirenzepine, Trends Pharmacol. Sci. (Suppl. Subtypes of muscarinic receptors), 9. Whitehouse, P.J., D.L. Price, A.W. Clark, J.T. Coyle and M.R. Delong, 1981, Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol. 10, 122. Whitehouse, P.J., D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle and M.R. Delong, 1982, Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain, Science 215, 1237. Wilcock, G.K_ M.M. Esiri, D.M. Bowen and C.C.T. Smith. 1982, Alzheimer's disease; correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities, J. Neurol. Sci. 57, 407. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Natl. Acad. Sci. U.S.A. 71, 1725.
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Elsevier EJP 51539
Pharmacological studies on novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 and YM954 Fumikazu Wanibuchi, Takako Konishi, Masatomi Harada, Michio Terai, Kazuyuki Hidaka, Toshinari Tamura, Shin-ichi Tsukamoto and Shinji Usuda Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, lbaraki 305, Japan
Received 30 May 1990, accepted 31 July 1990
We have investigated the pharmacological profiles of the novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 (2,8-dimethyl-3-methylene) and YM954 (2-ethyl-8-methyl-3-oxo). These compounds, like the putative M 1 agonists, RS86 and AF102B, inhibited [3H]pirenzepine binding to cerebral cortical membranes in the micromolar range and weakly inhibited [3H]quinuclidinyl benzylate binding to cerebellar membranes. Their ( - ) isomers had Hill coefficients lower than 1.0. (+)-YM796, (+)-YM954 and RS86, but not AF102B, stimulated phosphoinositide hydrolysis in hippocampal slices, an effect which is mainly linked to M~ receptors. (+)-YM796 (0.031 mg/kg p.o.) and (+)-YM954 (0.016 m g / k g p.o.) reversed the cognitive impairment in nucleus basalis magnocellularis-lesioned rats in a passive avoidance task more effectively than did RS86 and AF102B. Similar results were obtained in scopolamine-treated rats. Finally, (+)-YM796 was weaker than (+)-YM954 and RS86 in the induction of tremor, hypothermia and contraction of isolated ileum, which are mainly mediated by M 2 a n d / o r M 3 receptors. These results suggest that (+)-YM796, (_+)-YM954 and RS86 have M 1 agonistic activity in central nervous system and that (+)-YM796 has relatively weak M 2 and/or M 3 agonistic activity. Muscarinic acetylcholine receptor agonists; YM796; YM954; Muscarinic M1 receptors; Muscarinic M 2 receptors; Learning behaviour
I. Introduction A n u m b e r of reports (Bowen et al., 1976; Perry et al., 1977; Reisine et al., 1978) have shown that choline acetyltransferase (CHAT) activity, a marker of presynaptic cholinergic function, decreases in the brain of dementia of Alzheimer type ( D A T ) patients, with a marked loss of cholinergic neurons in the nucleus basalis of Mynert which provides innervation to the neocortex (Whitehouse et al., 1981; 1982). The significant correlation be-
Correspondence to: F. Wanibuchi, Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, lbaraki 305, Japan.
tween the severity of dementia and the reduction of C h A T activity in D A T patients suggests that the m e m o r y dysfunction in the patients is closely related to the cholinergic h y p o f u n c t i o n in the brain (Perry et al., 1978; Wilcock et al., 1982). The n u m b e r of postsynaptic muscarinic receptors, predominantly of the M a subtype, is suggested to be unchanged in D A T (Mash et al., 1985; A r a u j o et al., 1988). M a muscarinic receptors are enriched in cerebral cortex and hippocampus, b o t h of which are associated with m e m o r y and learning (Watson et al., 1986). Accumulating evidence suggests that M 1 agonists are possible therapeutic agents for D A T . A muscarinic agonist such as RS86 (Hollander et al., 1986), while it improves the cognitive deficit of D A T patients, also induces cardiovascu-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
480
#"'TjGH3 -
~'CH 2
YM796
/~
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~
~'~
~"O
Doses are expressed in terms of the free base. The drugs were dissolved in distilled water or in the appropriate buffer.
YM954
2.2. Animals"
O RS86
AF102B
Fig. 1. Chemical structures of YM796, YM954, RS86 and AFI02B. Asterisks denote asymmetric centers.
lar and abdominal side-effects which are considered to be mediated through the stimulation of M 2 a n d / o r M 3 muscarinic receptors. Therefore, selective Ma agonists would be expected to have an advantage over non-selective muscarinic agonists for the treatment of DAT regarding the avoidance of M z- a n d / o r M3-mediated side-effects. In the present studies, we have examined the in vivo and in vitro pharmacological profiles of YM796 (2,8-dimethyl-3-methylene-l-oxa-8-azaspiro[4.5]decane) and YM954 (2-ethyl-8-methyl-1oxa-8-azaspiro[4.5]decane-3-one) (fig. 1). Preliminary results of this work were reported elsewhere (Wanibuchi et al., 1989).
2. Materials and methods
2. l. Materials ( + )-YM796, ( + )-YM95& their optical isomers, RS86 (2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane1,3-dione hydrobromide, fig. 1) and AF102B (( _+)cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine hydrochloride, fig. 1) were synthesized in our laboratory. The following were obtained commercially: scopolamine hydrobromide (Tokyo Kasei Co., Ltd.), acetylcholine chloride (Dai-ichi Pharmaceutical Co., Ltd.), carbachol (Wako Pure Chemical Industries, Ltd.), arecoline (Tokyo Kasei Kogyo), [3H]pirenzepine (DuPont), [3H]quinuclidinyl benzylate (DuPont), myo-[3H]inositol (DuPont). Both YM compounds were synthesized as the fumarate, the maleate or the hydrochloride.
Male ICR mice (Japan Sic. Inc.), male Wistar rats (Japan Slc. Inc.) and male guinea pigs (Hitachi Animal Medical Research Institute) were used. The animals were given free access to food and water with a 12-h light-dark cycle (light on 7 : 00 19:00 h). Behavioral studies were performed between 9 : 00-17 : 00 h. 2.3. In vitro receptor binding assays The binding assays described here were conducted according to known methods; 0.06 nM [3H]quinuclidinyl benzylate (QNB) binding to M 2 and/or M 3 receptors in the cerebellum membrane of rats was determined by the methods of Yamamura and Snyder (1974) and 1.0 nM [3H]pirenzepine (PZ) binding to M~ receptors in the cerebral cortex membrane of rats, by the methods of Watson et al. (1982). The IC50 values, concentrations required to inhibit specific binding by 50%, were computed by logit-log analysis. 2.4. Phosphoinositide (PI) hydrolysis Rat hippocampal slices (350 × 350 ~m) were placed in vials and preincubated for 60 rain at 37 ° C in Krebs-Heinseleit bicarbonate buffer containing myo-[3H]inositol under 95% 02-5% CO2. After addition of muscarinic agonists and 10 mM LiC1, the slices were incubated further with high K + (19 raM) for 60 rain and the reaction was terminated by the addition of chloroformmethanol. The vials were centrifuged and the aqueous phase was removed for assay of [3H]inositolphosphates (InsPs) and the organic phase was taken for the assay of lipids (Costa et al., 1986; Court et al., 1986). The results are expressed as d.p.m. InsPs/(lipids + InsPs). 2.5. Nucleus basalis rnagnocellularis (NBM) lesion The rats (250-300 g) were anesthetized with sodium pentobarbital (50 m g / k g i.p.) and placed
481
in a stereotaxic apparatus (David K o p f Instruments) so that bregma and lambda were in the same horizontal plane. Bilateral electrolytic lesions of N B M (0.8 m m posterior to bregma, 2.5 m m lateral to the midline and 8.2 m m ventral to the surface of the skull) were produced by passing a DC current (2 mA, 30 s) through a monopolar electrode. Sham operations were made by the same surgical procedures without delivery of the current. Behavioral studies were performed at least 2 weeks after surgery. ChAT activity in the frontal cortex and hippocampus was measured after the behavioral studies by the methods of Fonnum (1975).
sic activity was determined as the m a x i m u m response relative to that obtained with acetylcholine in the same preparation.
3. Results
3.1. In vitro receptor binding assays As shown in table 1, ( + ) - Y M 7 9 6 and ( + ) YM954 inhibited [3H]pZ binding to the cerebral cortical membranes with IC50 values of 2.85 and 0.67 ~M, respectively, and [3H]QNB binding to the cerebellar membranes with values of 24.49 and 3.33 #M, respectively. The ratio of the IC50 values
2.6. Passive avoidance tasks with scopolaminetreated and NBM-lesioned rats TABLE 1
In the training session, each rat was placed in the lighted box of a two-compartment passive avoidance apparatus ( O ' H a r a Co., Ltd.). When the rat entered the dark compartment, a foot-shock (60-70 V AC) was applied for 1 s through the metal grid bars of the floor. Retention was assessed 24 h after the training session. The latency to enter the dark compartment was measured up to a maximum cut-off of 300 s. All of the compounds tested were administered p.o. 45 min before the training session. Scopolamine (1 m g / k g ) was i.p. injected in naive rats (210-280 g) 30 min before the session. 2.7. Tremor and hypothermia Tremor occurrence was checked 0 - 3 0 min after oral administration of drugs to mice (30-42 g). Rectal temperature was measured with a thermoprobe (MC-111, Omron) before and 30 min after administration. 2.8. Contraction of isolated ileum Contraction of isolated ileum of guinea pigs (400-700 g) was measured isometrically with transducers (Nihon Koden). ECs0 values, the concentrations producing 50% of the individual maxim u m response, were obtained graphically from the concentration-response relationship curves. Intrin-
Comparison of the affinity of YM796, YM954 and other muscarinic agonists for [3H]pirenzepine and [3H]QNB b binding sites in rat frontal cortex and cerebellum, respectively. IC50 values are expressed as the micromolar concentration (the means_+ S.E. of six experiments). Figures in parentheses show the Hill coefficient (NH). Drug
_+)-YM796
ICs0 (NH)
Selectivity a
[3H]Pirenzepin e [3H]QN B b
for M 1 receptor
2.85 _+0.04 (0.783 _+0.039) 6.95 _+0.50 (0.922 _+0.080) 2.12 _+0.05 (0.736 -+0.041)
24.49 _+1.87 8.60 (0.776_+0.064) 78.44 _+2.89 11.29 (0.980_+0.045) 11.42 _+0.15 5.39 (0.739-+0.029)
0.67 _+0.02 (0.928_+0.057) +)-YM954 0.79 _+0.01 (1.001 _+0.021) (-)-YM954 0.44 _+0.01 (0.883 _+0.026)
3.33 _+0.18 4.97 (0.757_+0.089) 8.02 _+0.27 10.15 (0.879_+0.075) 2.20 _+0.08 5.00 (0.750_+0.062)
RS86
0.59 +0.01 (0.805 ± 0.027) 0.70 _+0.06 (0.981 -+0.092)
4.63 _+0.09 (0.667_+0.042) 2.45 +0.04 (0.775-+0.037)
7.85
2.16 -+0.15 (0.525 _+0.033) 0.85 +0.05 (0.579+0.052)
0.37 -+0.03 (0.476_+0.014) 0.73 -+0.21 (0.612_+0.081)
0.17
+)-YM796 -)-YM796 _+)-YM954
AF102B Carbachol Arecoline
3.50
0.86
a Selectivity is shown by the ratio of the IC50 value for [3H]QNB binding to that for [3H]pirenzepine binding, b QNB, quinuclidinyl benzylate.
482
of [3H]QNB binding to those of [3H]pZ binding was 8.60 for ( + ) - Y M 7 9 6 and 4.97 for ( + )-YM954. These values were similar to those for RS86 and AF102B, and much greater than those for carbachol and arecoline. The Hill coefficients of ( - ) isomers of YM796 and YM954 were less than 1.0 whereas the corresponding ( + ) isomers had coefficients near unity, especially with [3H]PZ binding.
TABLE 2
3.2. Stimulation of PI hydrolysis
tions of 10-1000 t~M (fig. 2). (_+)-YM796 needed a higher concentration to exhibit similar effects. AF102B failed to stimulate hydrolysis at 10-1000 ~M. A full muscarinic agonist, carbachol (CCh), markedly stimulated hydrolysis at the concentration of 100/~M.
Both (_+)-YM954 and RS86 stimulated PI hydrolysis in hippocampal slices at the concentra-
Effects of (_+)-YM796, (_+)-YM954 and other putative M I agonists on scopolamine-induced amnesia in rats. Each value indicates the minimal effective dose (MED). Shock intensity in the training session was 70 V. Drug
(+)-YM796
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MED ( m g / k g p.o.)
0.063
0.016
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Bilateral NBM lesioning markedly reduced the latency to enter the dark compartment 24 h after the training session, as compared with the sham operation (fig. 3). (_+)-YM796 significantly inhibited the reduction of latency at doses of 0.031 0.25 m g / k g p.o. (_+)-YM954 also inhibited, but somewhat less effectively, at doses of 0.016 and 0.063 m g / k g p.o. RS86 and AF102B also inhibited the reduction at doses of 0.13 1.0 and 0.5-4.0 m g / k g p.o., respectively. NBM lesioning decreased ChAT activity to about 70% in frontal cortex, as compared with the sham operation, but did not affect the activity in hippocampus (data not shown). Similar results were obtained with scopolamine-induced amnesia (table 2). ( + ) - Y M 7 9 6 , ( + ) - Y M 9 5 4 , RS86 and AF102B improved the scopolamine-induced deficit in learning behavior.
e-
TABLE 3
0.0 10"~10"~10-*10-a(M)
Cent CCh(±)gM954 Fig. 2. Effects of ( + ) - Y M 7 9 6 , (,+)-YM954 and other muscarinic agonists on K+-stimulated phosphoinositide hydrolysis in hippocampal slices. Each column indicates the mean-+ S.E. of triplicate determinations. CCh = carbachol. * P < 0.05, * * P < 0.01, * * * P < 0.001 compared with the corresponding control (Cont) using Student's t-test.
Effects of ( + ) - Y M 7 9 6 , (,+)-YM954 and other putative M 1 agonists on contraction of guinea pig isolated ileum. Each value was obtained graphically from four experiments. Drug
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RS86
AF102B
ECs0 (~tM)
8.6
2.3
0.90
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0.96
0.97
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(Dose, mg/kg, p.o.) Fig. 3. Effects of (4,)-YM796, (4-)-YM954 and other putative M l agonists on the amnesia induced by lesioning the nucleus basalis magnocellularis in rats. Ordinate indicates latency to enter the dark compartment 24 h after training. Each column indicates the mean 4- S.E. The number of rats is shown at the bottom of the columns. Shock intensity in the training session was 60 V. * P < 0.05, • * P < 0.01 compared with control (Cont) using Mann-Whitney U-test.
(4-)-YM796 and (_+)-YM954 were 8 and 32 times more potent, respectively, than the equally effective RS86 and AF102B at their minimal effective dose.
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3.4. Tremor and hypothermia As shown in fig. 4, (4-)-YM796 failed to elicit tremor in mice even at a dose of 80 m g / k g p.o., whereas RS86 and (4-)-YM954 induced tremor at doses higher than 2.5 and 5.0 m g / k g p.o., respectively. AF102B at a dose of 80 m g / k g p.o. induced tremor in 38% of the mice. All of the compounds reduced rectal temperature in a dose-dependent manner (fig. 5). (4-)YM796 and AF102B significantly reduced the temperature at doses of 32-64 m g / k g p.o. but
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their hypothermic activity was much weaker than that of RS86 and (_+)-YM954.
3.5. Contraction of isolated ileum Table 3 shows the effects of (_+)-YM796, (+_)YM954, RS86 and A F I 0 2 B on the contraction of guinea pig isolated ileum. RS86 was the most potent of the four compounds, followed by (_+)YM954. The remaining two compounds, ( + ) YM796 and AF102B, were much less potent.
4. Discussion ( _+)-YM796 and ( _+ )-YM954 inhibited [3H]pZ binding to the cerebral cortical membranes in the micro~nolar range to the same extent as did the reference c o m p o u n d s , RS86 (Palacios et al., 1986) and A F 1 0 2 B (Fisher et al., 1986), indicating that these two Y M c o m p o u n d s have an affinity to the M~ muscarinic receptor. These compounds, including the reference c o m p o u n d s , also inhibited [ 3 H ] Q N B binding to the cerebellar membranes. As there is essentially no M 1 receptor in the cerebellum (Giraldo et al., 1986), the binding site of [3H]QNB in the region is considered to be the
M 2 a n d / o r M 3 receptor. The ratio of IC50 values for [3H]QNB binding to those for [3H]pZ binding of ( _+)-YM796, (_+)-YM954, RS86 and A F 1 0 2 B binding was much greater than those for carbachol and arecoline, indicating that these c o m p o u n d s have a more selective affinity for the M 1 receptor than the classical agonists. PI hydrolysis has been suggested to be coupled with MI receptors as its second messenger system (Watson et al., 1984; H a r a d a et al., 1989). Since both ( ± ) - Y M 7 9 6 and ( + ) - Y M 9 5 4 , like RS86, stimulated PI hydrolysis, these Y M c o m p o u n d s are likely to have M 1 agonistic activity in brain tissue. A F I 0 2 B has been reported to produce a depolarizing response in isolated superior cervical ganglia of rabbits and thus has been suggested to be an M 1 agonist ( M o c h i d a et al., 1988). However, in agreement with the previous report (Fisher et al., 1987), we observed no stimulating effect of A F 1 0 2 B on PI hydrolysis. This implies that any M 1 agonistic activity of A F 1 0 2 B that exists in brain would be very weak as c o m p a r e d with that of Y M c o m p o u n d s and RS86. (_+)-YM796 and (_+)-YM954 ameliorated the learning deficit induced by scopolamine or N B M lesion in rats. The effective doses of both Y M c o m p o u n d s were smaller than those of RS86 and
485
AF102B. Scopolamine, an antimuscarinic drug, is well known to reduce learning ability in animals and humans. NBM provides a major cholinergic innervation to the neocortex and is thought to be homologous to the nucleus basalis of Mynert in humans (Salamone, 1986). Lesioning of NBM decreased ChAT activity in the rat frontal cortex to about 70% in the present studies, which was consistent with the results of Hepler et al. (1985). Muscarinic receptors in the cerebral cortex are predominantly of the M 1 subtype (Watson et al., 1986) and, as already described, ( _+)-YM796, ( + )YM954 and RS86 stimulated PI hydrolysis in the brain. Therefore, it is likely that the ameliorating effects of both YM compounds and RS86 are at least in part mediated by M1 receptors. AF102B improved the learning deficit both in the present studies and in that of Fisher et al. (1986). These effects might also be due to its weak M~ agonistic activity in the brain. (_+)-YM796 and AF102B were less potent to induce tremor and hypothermia than were ( + ) YM954 and RS86. Together with the finding that (_+)-YM796 but not AF102B stimulated P1 hydrolysis, it is of particular interest that ( _+)-YM796 induced neither tremor nor hypothermia at doses more than 10-fold higher than the minimal effective dose of RS86. In addition, (_+)-YM796 was weaker to induce contraction of guinea pig isolated ileum than were RS86 and (_+)-YM954. Tremor and hypothermia (Caulfield et al., 1983) and contraction of isolated ileum (Brow~ et al., 1980) by muscarinic agonists have been suggested as being associated with the muscarinic M 2 a n d / o r M 3 subtype. These results suggest that the M 2 a n d / o r M 3 agonistic activity of (_+)-YM796 is much weaker than that of RS86 and (_+)-YM954. Therefore, ( _+)-YM796 is expected to be devoid of serious cardiovascular effects through M 2 receptors. Binding studies using radiolabelled antagonists yielded displacement curves for muscarinic agonists with Hill coefficients less than 1.0, whereas muscarinic antagonists had much steeper curves (Birdsall et al., 1978; Hulme et al., 1978; Freedman et al., 1988). In the present studies, the displacement curves for ( - ) isomers of both YM compounds had lower Hill coefficients than those
of the corresponding ( + ) isomers. These results may indicate that the intrinsic activity of ( - ) isomers for the muscarinic receptors is more potent than that of ( + ) isomers. Further experiments are under way to clarify the pharmacological profiles of the optical isomers of YM796 and YM954. In conclusion, the present results suggest that (_+)-YM796, (_+)-YM954 and RS86 possess M t agonistic activity, and that ( + ) - Y M 7 9 6 has less potent M 2 a n d / o r M 3 agonistic activity in both central and peripheral tissues than (_+)-YM954 and RS86.
Acknowledgements The authors are grateful to Drs. H. Maeno and K. Koshiya for critical reading of the manuscript and to Mr. H. ltoh and Ms. R. Tsutsumi for their helpful suggestions and kind assisstance.
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