European Journal of Pharmacology, 187 (1990) 193-199
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Elsevier EJP 51484
In vivo characterisation of novel efficacious muscarinic receptor agonists S t e p h e n B. F r e e d m a n , E l i z a b e t h A. H a r l e y , R o s e m a r i e S. M a r w o o d a n d Shil Patel Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K.
Received I March 1990, revised MS received 6 June 1990, accepted 26 June 1990 Although a number of muscarinic agonists have been used in clinical trials for Alzheimer's Disease, many of these compounds are low in potency and have only limited intrinsic efficacy. The present study describes four non-quaternary oxadiazole based muscarinic agonists from a quinuclidine and a 1-azanorbornane series. These displayed up to 1000 fold higher affinity than arecoline and were efficacious muscarinic agonists at cortical receptors. All four compounds produced peripherally mediated salivation and centrally mediated hypothermia at doses 50-50000 fold lower than arecoline. The most potent was L-670,548, the methyl oxadiazole in the 1-azanorbornane series, which had an ED50 of 0.0016 mg/kg on the hypothermia model. This derivative was also the most potent compound in ex vivo binding studies (EDs0 0.0069 mg/kg) and showed excellent brain penetration (3.8% of the administered dose). These derivatives are the first non quaternary efficacious agonists which show good penetration into the CNS (central nervous system), and will prove useful tools in understanding the role of muscarinic receptors in CNS function. Muscarinic receptor agonists; Muscarinic receptors; Hypothermia; Salivation; Oxadiazoles; (Efficacy); (Central muscarinic activity); (Ex vivo binding) I. Introduction
Interest in muscarinic pharmacology has been stimulated in the last 10 years by the discovery of a number of neurochemical deficits in the brains of patients with Alzheimer's Disease. A major feature of the neuropathology is a selective degeneration of certain cholinergic pathways (Davies and Maloney, 1976). In particular a number of studies have demonstrated a decrease in acetylcholine content and choline acetyl transferase activity in the cerebral cortex of patients who have been diagnosed as having senile dementia (Perry et al., 1977). This has led to suggestions that a cholinergic replacement strategy might represent a valid therapeutic approach.
Correspondence to: S.B. Freedman, Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K.
A number of different strategies have been considered, including precursor loading with lecithin (Etienne, 1983), enhancement of neurotransmission with acetylcholinesterase inhibitors (Thal and Fuld, 1983) and the administration of directly acting muscarinic agonists. A number of muscarinic agonists have been tested including the natural products arecoline (Christie et al., 1981) and pilocarpine (Caine, 1980), and such synthetic agonists as RS-86 (Mouradian et al., 1988). The clinical results with all of these compounds have, however, been very disappointing. The lack of clinical efficacy in these trials may be due to a variety of reasons, including the poor bioavailability and short duration of action observed with compounds such as arecoline. A further consideration is that most of the compounds that have been evaluated have only limited intrinsic efficacy and are therefore not full muscarinic agonists (Freedman et al., 1988). The intrinsic efficacy of agonists is an important parameter in determining an agonist re-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
194 sponse (Kenakin, 1986). In tissues where the coupling efficiency varies compounds may show functional differences even in the absence of true receptor selectivity. In the cerebral cortex the muscarinic receptors linked to phosphatidylinositol (PI) turnover appear to lack an effective receptor reserve (Freedman, 1986). This results in many agonists being unable to produce a full stimulation of PI turnover. We have previously demonstrated that the only compounds with sufficient intrinsic efficacy to stimulate PI turnover are quaternary agonists such as carbachol and muscarine (Freedman et al., 1988). Although these compounds are relatively efficacious, the fact that they are quaternised means that they would not readily pass through the blood brain barrier. We have recently described a novel series of muscarinic agonists which utilise an oxadiazole moiety to replace the ester moiety of arecoline (Saunders et al., 1990). This series includes a number of non quaternised compounds having high affinity for muscarinic receptors. Several of these compounds which have high intrinsic efficacy for muscarinic receptors are able to maximally stimulate cortical PI turnover (Freedman et al., in press). In the present study we describe the in vivo profile of four of these compounds and compare their in vivo activity to the lead natural product arecoline.
ally 10 min) the cotton buds were weighed and the saliva produced estimated by weight. Where necessary results were analysed using non linear least squares regression analysis using RS1 (BBN Research Systems, Cambridge, Mass.) and a computerised iterative procedure written by Dr A. Richardson, NRC, Terlings Park.
2.3. Measurement of hypothermia Male BKTO mice (20-30 g) were housed individually in perspex cages at ambient temperature for at least 60 rain prior to experiment. Mice were restrained for up to 1 min every 20 min in a perspex restraining cage (Plus Labs, Lansing, Michigan) and rectal temperature determined with either a Jenway 2000 or a Sensotek BAT-12 thermometer. Measurement of temperature was estimated using either a rounded 2.5 mm diameter probe inserted 2.4 cm into the rectum or a rounded 1.5 mm probe inserted 1.6 cm into the rectum. In both cases the probe insertion was lubricated with liquid paraffin. In subsequent studies used to assess muscarinic agonists mice were routinely treated (unless otherwise indicated) with a low dose of the peripheral muscarinic antagonist Nmethyl-scopolamine (1 mg/kg). All test substances were administered by the i.p. route over a range typically of 0.0001-1.0 mg/kg. Where necessary results were analysed using the RS1 procedure described in 2.2.
2. Materials and methods
2.4. Ex vivo binding assay 2.1. Receptor binding studies Binding studies were performed as previously described (Freedman et al., 1988).
2.2. Measurement of salivation Male BKTO mice (20-30 g) were anaesthetised with 40 m g / k g Sagatal and allowed to stabilise over a 15 rain period. Following i.p. administration of test compound (typically 0.1-30 m g / k g ) salivation was estimated by periodic insertion (every 2 min) of a preweighed cotton bud into the buccal cavity. At the end of the test period (usu-
Mice identical to those used in the above studies were injected i.p. with 100/~1 of test compound dissolved in physiological saline. Following a 30 min period animals were killed, brains rapidly removed and homogenised in ice cold 20 mM HEPES buffer pH 7.4 containing 1 mM MgC12 (0.4 g wet weight/10 ml). Aliquots, 100 #1, of homogenate were subsequently assayed with 0.6 nM [3H]oxotremorine-M in a total volume of 1 ml. All incubations were performed in 20 mM HEPES buffer pH 7.4 containing 1 mM MgC12. Specific binding was determined by incubating parallel samples with 1 /zM atropine sulphate (2 /~M atropine). Following a 40 min incubation at
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30°C, samples were filtered over Whatman G F / C filters presoaked in 0.05% polyethyleneimine. Radioactivity was determined by liquid scintillation spectrometry. Binding parameters were determined by non-linear least squares regression analysis using RS1 as described in 2.2. To determine potency each compound was tested on at least three separate occasions using a minimum of four separate doses. The penetration of the compounds was then calculated by comparing the inhibition of binding observed, with a calibration curve of the test compound. This was then used to calculate the corresponding amount of compound present in whole mouse brain. This value was then compared with the original amounts administered to the animal and calculated as a percentage of the administered dose. All animals which displayed inhibition of specific binding by between 10 and 90% were included in this calculation. Three animals were tested at each dose which was typically 0.001-10 m g / k g unless otherwise indicated.
N
L-658,903
1_-660,863
N--~CH3
~-N~O'N L-670,548
L-670,207
Fig. 1. Novel oxadiazole muscarinic agonists.
2.5. Materials
3.1. Radioreceptor binding studies
Compounds and reagents for these studies were obtained from the following sources: RS-86 (2ethyl 8-methyl-2,8-diazaspiro-[4,5]decan-l,3-dion hydrobromide), Sandoz Ltd.; [3H]oxotremorine-M ([3H]Oxo-M) NET-671 84.9 Ci/mmol. All other reagents were obtained through Sigma, Dorset or Research Biochemicals Incorporated (Semat, UK). L-658,903, L-660,863, L-670,207 and L-670,548 were synthesised by L. Street and A. Macleod. The detailed syntheses will be published elsewhere (Saunders et al., 1990; Street et al., manuscript submitted).
All four oxadiazoles showed high affinity for the muscarinic binding assays. The two quinuclidines displayed 10 fold higher affinity than arecoline whereas the 1-azanorbornane derivatives were between 50 and 100 fold more potent (table 1). Three of the derivatives L-660,863, L-670,548 and L-670,207 displayed N M S / O x o - M binding ratios significantly higher than arecoline and were the first non quaternized muscarinic agonists able to produce a virtually full maximal stimulation of cortical PI turnover (Freedman et al., in press). 3.2. Salivation studies
3. Results
The four oxadiazole derivatives selected for detailed in vivo study were the methyl and amino oxadiazoles from a quinuclidine series (L-658,903 and L-660,863) and the corresponding methyl and amino oxadiazoles from an azanorbornane series (L-670,548 and L-670,207) (fig. 1).
Muscarinic agonists produce a range of physiological responses in the periphery including stimulation of M 3 muscarinic receptors in the salivary glands. Following i.p. injection of muscarinic agonists into mice, dose dependent increases in salivation were observed. The response was linear over 15 min and a maximal 300-500 mg of saliva was produced. Quantification was estimated over an experimental period of 10 min and the dose
196 TABLE 1 In vitro characterisation of novel muscarinic agonists. For detailed methods see materials and methods section and Freedman et al. (1988). Results are expressed as an apparent affinity constant (Kapp) which is corrected for ligand occupancy using the Cheng-Prussoft relationship (Cheng and Prussoff, 1973). Each dose response curve was typically performed in triplicate and consisted of 8-12 points. Values are the geometric means of at least three independent determinations. Numbers in parentheses indicate the low and high error value of the geometric mean. Studies were performed with 0.1 nM [3H]N-methylscopolamine ([3H]NMS) and 3.0 nM [3H]oxotremorine-M ([3H]Oxo-M) using tissue from rat cerebral cortex. Compound
Kap p (gM)
[ 3H]NMS
[ 3H]Oxo_M
NMS/Oxo-M ratio
Atropine Pilocarpine RS-86 Arecoline Carbachol
0.0010 (0.00094-0.0011) 4.0 (3.0-5.1) 5.0 (4.6-5.5) 6.2 (4.9-7.8) 22 (20-24)
0.00048 (0.00040-0.00057) 0.040 (0.036-0.044) 0.040 (0.036-0.046) 0.011 (0.0097-0.011) 0.0049 (0.0036-0.0061)
2.1 100 130 560 4 500
L-658,903 L-660,863 L-670,548 L-670,207
0.44 (0.42-0.45) 0.60 (0.55-0.66) 0.10 (0.090-0.12) 0.031 (0.027-0.040)
0.00096 (0.00082-0.0011) 0.00047 (0.00040-0.00054) 0.000090 (0.000074-0.00011) 0.000043 (0.000039-0.000055)
460 1300 1 100 720
between 500 and 10000 greater than arecoline. The most potent compound was the methyl oxadiazole from the 1-azanorbornane series L670,548 with a potency of 0.0014 mg/kg.
required to produce 100 mg of saliva determined. This was found previously to produce more consistent data (Freedman et al., 1989). Arecoline, RS-86 and pilocarpine produced a salivation response although RS-86 and pilocarpine were some 50 fold more potent with ED10o values of 0.73 and 1.0 m g / k g respectively (table 2). All of the novel oxadiazole muscarinic agonists dose dependently stimulated salivation although the potency was
3.3. Hypothermia studies As well as peripheral actions muscarinic receptors also mediate a variety of central actions in-
TABLE 2 Effect of muscarinic agonists upon salivation and core body temperature in mice. ED100: dose of agonist required to produce 100 mg of saliva during the experimental 10 re.in period. Number in parentheses represent the 95% confidence limits. EDso: dose of agonist required to produce 50% of the maximum fall in core body temperature. Values are expressed as the means + S.E.M. Each compound was examined at a minimum of five doses, each point from at least three animals. The data are from the means of at least three independent experiments. Compound
Salivation ED10o (mg/kg)
Hypothermia ED~0 (mg/kg)
Arecoline Pilocarpine RS-86
66 a 1.0 (0.72-1.4) 0.73 (0.41-1.1)
13 8.0 1.7
L-658,903 L-660,863 L-670,548 L-670,207
0.012 (0.0074-0.020) 0.0081 (0.0044-0.015) 0.0014 (0.00081-0.0025) 0.0018 (0.00078-0.0038)
+4.9 ± 3.3 _+0.42
0.026 ±0.0045 0.19 ±0.078 0.0016 + 0.00027 0.0063 ± 0.0017
Ratio hypothermia/salivation 0.20 8.0 2.3 2.1 23 1.1 3.5
a N o confidence limits could be determined for arecoline since toxic effects prevented testing at higher doses.
197 cluding the induction of a centrally mediated hypothermia (Freedman et al., 1989). The muscarinic agonist pilocarpine induced a dose dependent hypothermia which was slow in onset and reached a maximal fall in body temperature of approximately 6 ° C at between 20-40 min following i.p. administration. The duration of the effect was dependent on the dose but was typically 2-3 h. All three standard agonists produced good CNS mediated hypothermia with potency in the 1-15 m g / k g range (table 2). Interestingly RS-86 produced hypothermia at a similar dose range to that which the peripheral effects were observed, indicating good CNS penetration. The four novel oxadiazole agents all dose dependently produced hypothermia with potencies of between 0.001 and 0.2 m g / k g . These compounds were therefore between 8-1000 fold more potent than the standard agonists. In particular L-670,548 produced hypothermia at doses of 0.0016 m g / k g making this methyl oxadiazole one of the most potent CNS active muscarinic agonists known. Since these results were obtained in the presence of the peripheral muscarinic antagonist N methyl scopolamine, the agonists appear to be exerting a CNS action. When the doses inducing hypothermia were compared with those that produced salivation (table 2) it can be seen that the two methyl oxadiazoles (L-658,903 and L-670,548) induced these effects with similar ratios. In order to check the specificity of the novel muscarinic compounds the sensitivity of the responses to pretreatment with scopolamine was tested. Administration of 10 m g / k g scopolamine 20 min prior to testing completely blocked both the hypothermia and salivation induced by L670,207 and L-670,548.
3. 4. Ex vivo binding studies We have recently described a novel ex vivo binding assay which can be used to estimate the potency and penetration of muscarinic agents into the CNS (Freedman et al., 1989). In this assay RS-86 and pilocarpine displayed a potency of 11 and 23 m g / k g whereas arecoline was 10 fold weaker (table 3). This low potency of arecoline is due to the relatively labile ester linkage which
TABLE 3 Effects of novel oxadiazole agents upon ex vivo binding of [3H]oxotremorine-M. EDso is the dose of compound required to inhibit by 50% the specific binding of [3H]oxotremorine-M to whole brain mouse homogenates using 0.6 nM ligand. The numbers in parentheses refer to the low and high error values of the geometric mean. Penetration of each compound was estimated by use of a calibration curve of test compoundwhich was added exogenously to homogenates from saline treated animals. Using this curve the amount of compound present in the treated mice was estimated. The % penetration was related to the original dose of compound administered to the animal. Results are expressed as mean_+S.E.M. Each value is from three to five independent experiments and was from a minimum of three animals at each point on the dose response curve.
Compound
Ex vivo binding
Arecoline Pilocarpine RS-86
EDso (mg/kg) 220 (130-360) 23 (17-29) 11 (7.7-16)
L-658,903 L-660,863 L-670,548 L-670,207
0.21 (0.17-0.27) 0.64 (0.48-0.86) 0.0069 (0'.003-0.0074) 0.064 (0.038-0.11)
% penetration 0.0085 _+0.0064 0.31 _+0.064 1.5 _+0.18 1.9 0.13 3.8 0.24
_+0.32 _+0.038 5:1.5 +0.10
results in poor activity in vivo. In contrast the two quinuclidine oxadiazoles were up to 1000 fold more active than arecoline reflecting the more stable nature of the oxadiazole compared with the ester. This was further observed with the two 1-azanorbornane derivatives which were a further 10 and 100 fold more potent than the quinuclidines. The methyl oxadiazole from this series, L-670,548 was the most potent muscarinic agonist tested with a potency of 0.0069 m g / k g . The penetration of the test compounds were estimated by comparing the results of treated mice with calibration curves of the compound added exogenously to brain homogenates of saline treated animals. Compounds such as RS-86 which previously had been shown to induce peripheral and central effects at relatively similar doses showed excellent CNS penetration with a value of 1.5% of the administered dose. A similar result was observed with the two methyl oxadiazoles which showed relative penetrations of 1.9 and 3.8% for the quinuclidine and azanorbornane respectively. In contrast the two amino oxadiazoles showed
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about a 10 fold poorer CNS penetration although these values were similar to that seen with pilocarpine.
4. Discussion The four oxadiazoles described here possess some of the highest binding affinities of any known muscarinic agonists. In particular the two azanorbornane derivatives possessed 100 fold higher affinity than arecoline, confirming that the oxadiazole is a suitable ester replacement for binding at the muscarinic receptor. The smaller size of the azanorbornane compared with quinuclidine may account for the increased activity observed with L-670,548 and L-670,207. The high N M S / O x o - M binding ratios obtained with the novel oxadiazole derivatives suggests that these compounds are muscarinic agonists with high intrinsic efficacy. The induction of hypothermia and salivation by these compounds in these studies support this suggestion. The distinction between the oxadiazoles and arecoline was found to be more pronounced in the whole animal studies where the novel oxadiazoles were almost 50 000 more active. The relatively weak in vivo activity of arecoline in all three of the assays is a reflection of the very labile ester linkage present in this molecule. Oxadiazoles are presumably metabolically more stable and as a result display higher potency. These c o m p o u n d s are amongst the most potent muscarinic agonists ever described. Arecoline displayed relatively poor penetration in the ex vivo binding assay although this may be due in part to the reportedly short plasma half life of the compound. In the present design of these studies, compounds were evaluated for CNS penetration at a single time of 30 min. The two methyl oxadiazoles displayed apparent CNS penetrations of 1-4% of the administered dose. These values represent very good penetration since the total weight of the mouse brain as a proportion of total body weight is in the region of 2-5%. The only standard agonist with similarly good penetration in this assay was RS-86 which has been previously reported by Palacios and colleagues (Palacios et al., 1986) to penetrate well
into the CNS. The two amino oxadiazoles were apparently 10 fold weaker in their brain penetration, a finding which corresponded with their biological profile in the hypothermia and salivation studies where the methyl oxadiazoles generally produced central and peripheral effects at relatively similar doses. This was most apparent in the quinuclidine series. These findings correspond to the relative lipophilicity of the four compounds as estimated by log P measurements (data not presented). All of the measurements of brain penetration relied upon a number of assumptions including the important assumption that all of the measured activity corresponded to parent compound rather than an active metabolite. In previous studies we demonstrated that the penetration of [3H]quinuclidinyl benzilate administered to mice corresponded to that estimated in the ex vivo studies (Freedman et al., 1989). In the present study [a4C]L-660,863 (5.0 /~Ci) was administered i.v. to two 300 g male Sprague-Dawley rats. Following killing, 30 min later, 0.2% of the radioactivity was found to be present within the brain, a value that was close to that found in the ex vivo studies (0.13%). This result suggests that the results obtained with the quinuclidine L-660,863 in the ex vivo studies were reasonably accurate. In tissues the coupling efficiency of the receptors may vary considerably such that the effective receptor reserve may result in a compound being a full agonist in one tissue yet be an antagonist in another (Kenakin, 1986). Thus pilocarpine is a relatively full agonist on the superior cervical ganglion (Newberry and Priestley, 1987) and on PI induced turnover in parotid gland (Freedman, 1986) but has only limited ability to evoke a PI response in rat cerebral cortex. In contrast muscarine and carbachol are relatively full agonists on all three tissues. Indeed the only agonists with sufficient intrinsic efficacy to stimulate PI turnover in rat cerebral cortex were the quaternary agonists previously described. The four compounds described in this study are amongst the first non quaternary high efficacy agonists described which retain sufficient intrinsic efficacy to maximally stimulate PI turnover in rat cerebral cortex (Freedman et al., in press). The in
199 v i v o e x p e r i m e n t s c o n f i r m t h a t all of the a g o n i s t s a r e a b l e to p e n e t r a t e w e l l i n t o t h e b r a i n a n d are t h e r e f o r e a b l e to a c t i v a t e m u s c a r i n i c r e c e p t o r s w i t h i n the C N S . T h e s e a g o n i s t s m a y possess the a b i l i t y to fully a c t i v a t e m u s c a r i n i c r e c e p t o r s in t h o s e areas of t h e C N S w h e r e p e r h a p s the c o u p l i n g is less e f f i c i e n t t h a n elsewhere. O n e m i g h t h y p o t h e s i s e t h a t if the P I r e s p o n s e in c e r e b r a l c o r t e x is a s s o c i a t e d w i t h h i g h e r f u n c t i o n in this area, t h e n t h e s e c o m p o u n d s m a y b e t h e first des c r i b e d w i t h the a b i l i t y to b e a b l e to fully a c t i v a t e r e c e p t o r s in the c o r t e x f o l l o w i n g p e r i p h e r a l administration. These compounds may therefore be u s e f u l tools in o r d e r to fully test t h e r o l e o f m u s c a r i n i c r e c e p t o r s in the c o m p l e x p r o c e s s e s o f memory and cognition.
Acknowledgements The authors would like to thank Miss S. Farrow and Mrs S. Burton for manuscript preparation. Portions of this work were presented at the Subtypes of Muscarinic Receptor Meeting (IV) Wiesbaden, 20-22 July, 1989 (Saunders, J. and Freedman, S.B., 1989).
References Caine, E.D., 1980, Cholinomimetic treatment fails to improve memory disorder, N. Engl. J. Med. 303, 585. Cheng, K.C. and W.H. Prussoff, 1973, Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Christie, J.E., A. Shering, J. Ferguson and A.I.M. Glen, 1981, Physostigmine and arecoline: Effects of intravenous infusions in Alzheimer presenile dementia, Br. J. Psychiat. 138, 46. Davies, P. and A.J.F. Maloney, 1976, Selective loss of central cholinergic neurons in Alzheimer's Disease, Lancet 2, 1403. Etienne, P., 1983, Treatment of Alzheimer's Disease with
lecithin, in: Alzheimer's Disease, Ed. B. Reisberg (Free Press, New York). Freedman, S.B., 1986, Regional differences in muscarinic receptor linked inositol-phospholipid hydrolysis, Br. J. Pharmacol. 87, 29 p. Freedman, S.B., E.A. Harley and L.L. Iversen, 1988, Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo-M ratio as an index of efficacy at cortical muscarinic receptors, Br. J. Pharmacol 93, 437. Freedman, S.B., E.A. Harley and S. Patel, 1989, Direct measurement of muscarinic agents in the central nervous system of mice using ex vivo binding, European J. Pharmacol. 174, 253. Freedman, S.B., E.A. Harley, S. Patel, N. Newberry, M. Gilbert, A.T. McKnight, J.J. Maguire, N.T. Mudunkotuwa, R. Baker, L. Street, A. Macleod, J. Saunders and L.L. Iversen, A novel series of non quaternary oxadiazoles acting as full agonists at cortical muscarinic receptors, Br. J. Pharmacol. (in press). Kenakin, T.P., 1986, Receptor reserve as a tissue misnomer, Trends Pharmacol. Sci. 7, 93. Mouradian, M.M., E. Molar, J.A. Williams and T.N. Chase, 1988, No response to high-dose muscarinic agonist therapy in Alzheimer's Disease, Neurology 38, 606. Newberry, N.R. and T. Priestley, 1987, Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro, Br. J. Pharmacol. 92, 817. Palacios, J.M., G. Bolliger, A. Closse, A. Enz, G. Gmelin and J. Malanowsld, 1986, The pharmacological assessment of RS86 (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., P.H. Gibson, G. Blessed, R.H. Perry and B.E. Tomlinson, 1977, Neurotransmitter enzyme abnormalities in senile dementia. Choline acetyltransferase and glutamic acid decarboxylase activities in necropsy brain tissue, J. Neurol. Sci. 34, 247. Sannders, J., M. Cassidy, S.B. Freedman, E.A. Harley, L.L. Iversen, C. Kneen, A. Macleod, K. Merchant, R.J. Snow and R. Baker, 1990, Novel quinuclidine based ligands for the muscarinic cholinergic receptor, J. Med. Chem. 33, 1128. Thai, L.J. and P.A. Fuld, 1983, Memory enhancement with oral physostigmine in Alzheimer's Disease, N. Engl. J. Med. 308, 720.
193
Elsevier EJP 51484
In vivo characterisation of novel efficacious muscarinic receptor agonists S t e p h e n B. F r e e d m a n , E l i z a b e t h A. H a r l e y , R o s e m a r i e S. M a r w o o d a n d Shil Patel Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K.
Received I March 1990, revised MS received 6 June 1990, accepted 26 June 1990 Although a number of muscarinic agonists have been used in clinical trials for Alzheimer's Disease, many of these compounds are low in potency and have only limited intrinsic efficacy. The present study describes four non-quaternary oxadiazole based muscarinic agonists from a quinuclidine and a 1-azanorbornane series. These displayed up to 1000 fold higher affinity than arecoline and were efficacious muscarinic agonists at cortical receptors. All four compounds produced peripherally mediated salivation and centrally mediated hypothermia at doses 50-50000 fold lower than arecoline. The most potent was L-670,548, the methyl oxadiazole in the 1-azanorbornane series, which had an ED50 of 0.0016 mg/kg on the hypothermia model. This derivative was also the most potent compound in ex vivo binding studies (EDs0 0.0069 mg/kg) and showed excellent brain penetration (3.8% of the administered dose). These derivatives are the first non quaternary efficacious agonists which show good penetration into the CNS (central nervous system), and will prove useful tools in understanding the role of muscarinic receptors in CNS function. Muscarinic receptor agonists; Muscarinic receptors; Hypothermia; Salivation; Oxadiazoles; (Efficacy); (Central muscarinic activity); (Ex vivo binding) I. Introduction
Interest in muscarinic pharmacology has been stimulated in the last 10 years by the discovery of a number of neurochemical deficits in the brains of patients with Alzheimer's Disease. A major feature of the neuropathology is a selective degeneration of certain cholinergic pathways (Davies and Maloney, 1976). In particular a number of studies have demonstrated a decrease in acetylcholine content and choline acetyl transferase activity in the cerebral cortex of patients who have been diagnosed as having senile dementia (Perry et al., 1977). This has led to suggestions that a cholinergic replacement strategy might represent a valid therapeutic approach.
Correspondence to: S.B. Freedman, Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K.
A number of different strategies have been considered, including precursor loading with lecithin (Etienne, 1983), enhancement of neurotransmission with acetylcholinesterase inhibitors (Thal and Fuld, 1983) and the administration of directly acting muscarinic agonists. A number of muscarinic agonists have been tested including the natural products arecoline (Christie et al., 1981) and pilocarpine (Caine, 1980), and such synthetic agonists as RS-86 (Mouradian et al., 1988). The clinical results with all of these compounds have, however, been very disappointing. The lack of clinical efficacy in these trials may be due to a variety of reasons, including the poor bioavailability and short duration of action observed with compounds such as arecoline. A further consideration is that most of the compounds that have been evaluated have only limited intrinsic efficacy and are therefore not full muscarinic agonists (Freedman et al., 1988). The intrinsic efficacy of agonists is an important parameter in determining an agonist re-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
194 sponse (Kenakin, 1986). In tissues where the coupling efficiency varies compounds may show functional differences even in the absence of true receptor selectivity. In the cerebral cortex the muscarinic receptors linked to phosphatidylinositol (PI) turnover appear to lack an effective receptor reserve (Freedman, 1986). This results in many agonists being unable to produce a full stimulation of PI turnover. We have previously demonstrated that the only compounds with sufficient intrinsic efficacy to stimulate PI turnover are quaternary agonists such as carbachol and muscarine (Freedman et al., 1988). Although these compounds are relatively efficacious, the fact that they are quaternised means that they would not readily pass through the blood brain barrier. We have recently described a novel series of muscarinic agonists which utilise an oxadiazole moiety to replace the ester moiety of arecoline (Saunders et al., 1990). This series includes a number of non quaternised compounds having high affinity for muscarinic receptors. Several of these compounds which have high intrinsic efficacy for muscarinic receptors are able to maximally stimulate cortical PI turnover (Freedman et al., in press). In the present study we describe the in vivo profile of four of these compounds and compare their in vivo activity to the lead natural product arecoline.
ally 10 min) the cotton buds were weighed and the saliva produced estimated by weight. Where necessary results were analysed using non linear least squares regression analysis using RS1 (BBN Research Systems, Cambridge, Mass.) and a computerised iterative procedure written by Dr A. Richardson, NRC, Terlings Park.
2.3. Measurement of hypothermia Male BKTO mice (20-30 g) were housed individually in perspex cages at ambient temperature for at least 60 rain prior to experiment. Mice were restrained for up to 1 min every 20 min in a perspex restraining cage (Plus Labs, Lansing, Michigan) and rectal temperature determined with either a Jenway 2000 or a Sensotek BAT-12 thermometer. Measurement of temperature was estimated using either a rounded 2.5 mm diameter probe inserted 2.4 cm into the rectum or a rounded 1.5 mm probe inserted 1.6 cm into the rectum. In both cases the probe insertion was lubricated with liquid paraffin. In subsequent studies used to assess muscarinic agonists mice were routinely treated (unless otherwise indicated) with a low dose of the peripheral muscarinic antagonist Nmethyl-scopolamine (1 mg/kg). All test substances were administered by the i.p. route over a range typically of 0.0001-1.0 mg/kg. Where necessary results were analysed using the RS1 procedure described in 2.2.
2. Materials and methods
2.4. Ex vivo binding assay 2.1. Receptor binding studies Binding studies were performed as previously described (Freedman et al., 1988).
2.2. Measurement of salivation Male BKTO mice (20-30 g) were anaesthetised with 40 m g / k g Sagatal and allowed to stabilise over a 15 rain period. Following i.p. administration of test compound (typically 0.1-30 m g / k g ) salivation was estimated by periodic insertion (every 2 min) of a preweighed cotton bud into the buccal cavity. At the end of the test period (usu-
Mice identical to those used in the above studies were injected i.p. with 100/~1 of test compound dissolved in physiological saline. Following a 30 min period animals were killed, brains rapidly removed and homogenised in ice cold 20 mM HEPES buffer pH 7.4 containing 1 mM MgC12 (0.4 g wet weight/10 ml). Aliquots, 100 #1, of homogenate were subsequently assayed with 0.6 nM [3H]oxotremorine-M in a total volume of 1 ml. All incubations were performed in 20 mM HEPES buffer pH 7.4 containing 1 mM MgC12. Specific binding was determined by incubating parallel samples with 1 /zM atropine sulphate (2 /~M atropine). Following a 40 min incubation at
195
30°C, samples were filtered over Whatman G F / C filters presoaked in 0.05% polyethyleneimine. Radioactivity was determined by liquid scintillation spectrometry. Binding parameters were determined by non-linear least squares regression analysis using RS1 as described in 2.2. To determine potency each compound was tested on at least three separate occasions using a minimum of four separate doses. The penetration of the compounds was then calculated by comparing the inhibition of binding observed, with a calibration curve of the test compound. This was then used to calculate the corresponding amount of compound present in whole mouse brain. This value was then compared with the original amounts administered to the animal and calculated as a percentage of the administered dose. All animals which displayed inhibition of specific binding by between 10 and 90% were included in this calculation. Three animals were tested at each dose which was typically 0.001-10 m g / k g unless otherwise indicated.
N
L-658,903
1_-660,863
N--~CH3
~-N~O'N L-670,548
L-670,207
Fig. 1. Novel oxadiazole muscarinic agonists.
2.5. Materials
3.1. Radioreceptor binding studies
Compounds and reagents for these studies were obtained from the following sources: RS-86 (2ethyl 8-methyl-2,8-diazaspiro-[4,5]decan-l,3-dion hydrobromide), Sandoz Ltd.; [3H]oxotremorine-M ([3H]Oxo-M) NET-671 84.9 Ci/mmol. All other reagents were obtained through Sigma, Dorset or Research Biochemicals Incorporated (Semat, UK). L-658,903, L-660,863, L-670,207 and L-670,548 were synthesised by L. Street and A. Macleod. The detailed syntheses will be published elsewhere (Saunders et al., 1990; Street et al., manuscript submitted).
All four oxadiazoles showed high affinity for the muscarinic binding assays. The two quinuclidines displayed 10 fold higher affinity than arecoline whereas the 1-azanorbornane derivatives were between 50 and 100 fold more potent (table 1). Three of the derivatives L-660,863, L-670,548 and L-670,207 displayed N M S / O x o - M binding ratios significantly higher than arecoline and were the first non quaternized muscarinic agonists able to produce a virtually full maximal stimulation of cortical PI turnover (Freedman et al., in press). 3.2. Salivation studies
3. Results
The four oxadiazole derivatives selected for detailed in vivo study were the methyl and amino oxadiazoles from a quinuclidine series (L-658,903 and L-660,863) and the corresponding methyl and amino oxadiazoles from an azanorbornane series (L-670,548 and L-670,207) (fig. 1).
Muscarinic agonists produce a range of physiological responses in the periphery including stimulation of M 3 muscarinic receptors in the salivary glands. Following i.p. injection of muscarinic agonists into mice, dose dependent increases in salivation were observed. The response was linear over 15 min and a maximal 300-500 mg of saliva was produced. Quantification was estimated over an experimental period of 10 min and the dose
196 TABLE 1 In vitro characterisation of novel muscarinic agonists. For detailed methods see materials and methods section and Freedman et al. (1988). Results are expressed as an apparent affinity constant (Kapp) which is corrected for ligand occupancy using the Cheng-Prussoft relationship (Cheng and Prussoff, 1973). Each dose response curve was typically performed in triplicate and consisted of 8-12 points. Values are the geometric means of at least three independent determinations. Numbers in parentheses indicate the low and high error value of the geometric mean. Studies were performed with 0.1 nM [3H]N-methylscopolamine ([3H]NMS) and 3.0 nM [3H]oxotremorine-M ([3H]Oxo-M) using tissue from rat cerebral cortex. Compound
Kap p (gM)
[ 3H]NMS
[ 3H]Oxo_M
NMS/Oxo-M ratio
Atropine Pilocarpine RS-86 Arecoline Carbachol
0.0010 (0.00094-0.0011) 4.0 (3.0-5.1) 5.0 (4.6-5.5) 6.2 (4.9-7.8) 22 (20-24)
0.00048 (0.00040-0.00057) 0.040 (0.036-0.044) 0.040 (0.036-0.046) 0.011 (0.0097-0.011) 0.0049 (0.0036-0.0061)
2.1 100 130 560 4 500
L-658,903 L-660,863 L-670,548 L-670,207
0.44 (0.42-0.45) 0.60 (0.55-0.66) 0.10 (0.090-0.12) 0.031 (0.027-0.040)
0.00096 (0.00082-0.0011) 0.00047 (0.00040-0.00054) 0.000090 (0.000074-0.00011) 0.000043 (0.000039-0.000055)
460 1300 1 100 720
between 500 and 10000 greater than arecoline. The most potent compound was the methyl oxadiazole from the 1-azanorbornane series L670,548 with a potency of 0.0014 mg/kg.
required to produce 100 mg of saliva determined. This was found previously to produce more consistent data (Freedman et al., 1989). Arecoline, RS-86 and pilocarpine produced a salivation response although RS-86 and pilocarpine were some 50 fold more potent with ED10o values of 0.73 and 1.0 m g / k g respectively (table 2). All of the novel oxadiazole muscarinic agonists dose dependently stimulated salivation although the potency was
3.3. Hypothermia studies As well as peripheral actions muscarinic receptors also mediate a variety of central actions in-
TABLE 2 Effect of muscarinic agonists upon salivation and core body temperature in mice. ED100: dose of agonist required to produce 100 mg of saliva during the experimental 10 re.in period. Number in parentheses represent the 95% confidence limits. EDso: dose of agonist required to produce 50% of the maximum fall in core body temperature. Values are expressed as the means + S.E.M. Each compound was examined at a minimum of five doses, each point from at least three animals. The data are from the means of at least three independent experiments. Compound
Salivation ED10o (mg/kg)
Hypothermia ED~0 (mg/kg)
Arecoline Pilocarpine RS-86
66 a 1.0 (0.72-1.4) 0.73 (0.41-1.1)
13 8.0 1.7
L-658,903 L-660,863 L-670,548 L-670,207
0.012 (0.0074-0.020) 0.0081 (0.0044-0.015) 0.0014 (0.00081-0.0025) 0.0018 (0.00078-0.0038)
+4.9 ± 3.3 _+0.42
0.026 ±0.0045 0.19 ±0.078 0.0016 + 0.00027 0.0063 ± 0.0017
Ratio hypothermia/salivation 0.20 8.0 2.3 2.1 23 1.1 3.5
a N o confidence limits could be determined for arecoline since toxic effects prevented testing at higher doses.
197 cluding the induction of a centrally mediated hypothermia (Freedman et al., 1989). The muscarinic agonist pilocarpine induced a dose dependent hypothermia which was slow in onset and reached a maximal fall in body temperature of approximately 6 ° C at between 20-40 min following i.p. administration. The duration of the effect was dependent on the dose but was typically 2-3 h. All three standard agonists produced good CNS mediated hypothermia with potency in the 1-15 m g / k g range (table 2). Interestingly RS-86 produced hypothermia at a similar dose range to that which the peripheral effects were observed, indicating good CNS penetration. The four novel oxadiazole agents all dose dependently produced hypothermia with potencies of between 0.001 and 0.2 m g / k g . These compounds were therefore between 8-1000 fold more potent than the standard agonists. In particular L-670,548 produced hypothermia at doses of 0.0016 m g / k g making this methyl oxadiazole one of the most potent CNS active muscarinic agonists known. Since these results were obtained in the presence of the peripheral muscarinic antagonist N methyl scopolamine, the agonists appear to be exerting a CNS action. When the doses inducing hypothermia were compared with those that produced salivation (table 2) it can be seen that the two methyl oxadiazoles (L-658,903 and L-670,548) induced these effects with similar ratios. In order to check the specificity of the novel muscarinic compounds the sensitivity of the responses to pretreatment with scopolamine was tested. Administration of 10 m g / k g scopolamine 20 min prior to testing completely blocked both the hypothermia and salivation induced by L670,207 and L-670,548.
3. 4. Ex vivo binding studies We have recently described a novel ex vivo binding assay which can be used to estimate the potency and penetration of muscarinic agents into the CNS (Freedman et al., 1989). In this assay RS-86 and pilocarpine displayed a potency of 11 and 23 m g / k g whereas arecoline was 10 fold weaker (table 3). This low potency of arecoline is due to the relatively labile ester linkage which
TABLE 3 Effects of novel oxadiazole agents upon ex vivo binding of [3H]oxotremorine-M. EDso is the dose of compound required to inhibit by 50% the specific binding of [3H]oxotremorine-M to whole brain mouse homogenates using 0.6 nM ligand. The numbers in parentheses refer to the low and high error values of the geometric mean. Penetration of each compound was estimated by use of a calibration curve of test compoundwhich was added exogenously to homogenates from saline treated animals. Using this curve the amount of compound present in the treated mice was estimated. The % penetration was related to the original dose of compound administered to the animal. Results are expressed as mean_+S.E.M. Each value is from three to five independent experiments and was from a minimum of three animals at each point on the dose response curve.
Compound
Ex vivo binding
Arecoline Pilocarpine RS-86
EDso (mg/kg) 220 (130-360) 23 (17-29) 11 (7.7-16)
L-658,903 L-660,863 L-670,548 L-670,207
0.21 (0.17-0.27) 0.64 (0.48-0.86) 0.0069 (0'.003-0.0074) 0.064 (0.038-0.11)
% penetration 0.0085 _+0.0064 0.31 _+0.064 1.5 _+0.18 1.9 0.13 3.8 0.24
_+0.32 _+0.038 5:1.5 +0.10
results in poor activity in vivo. In contrast the two quinuclidine oxadiazoles were up to 1000 fold more active than arecoline reflecting the more stable nature of the oxadiazole compared with the ester. This was further observed with the two 1-azanorbornane derivatives which were a further 10 and 100 fold more potent than the quinuclidines. The methyl oxadiazole from this series, L-670,548 was the most potent muscarinic agonist tested with a potency of 0.0069 m g / k g . The penetration of the test compounds were estimated by comparing the results of treated mice with calibration curves of the compound added exogenously to brain homogenates of saline treated animals. Compounds such as RS-86 which previously had been shown to induce peripheral and central effects at relatively similar doses showed excellent CNS penetration with a value of 1.5% of the administered dose. A similar result was observed with the two methyl oxadiazoles which showed relative penetrations of 1.9 and 3.8% for the quinuclidine and azanorbornane respectively. In contrast the two amino oxadiazoles showed
198
about a 10 fold poorer CNS penetration although these values were similar to that seen with pilocarpine.
4. Discussion The four oxadiazoles described here possess some of the highest binding affinities of any known muscarinic agonists. In particular the two azanorbornane derivatives possessed 100 fold higher affinity than arecoline, confirming that the oxadiazole is a suitable ester replacement for binding at the muscarinic receptor. The smaller size of the azanorbornane compared with quinuclidine may account for the increased activity observed with L-670,548 and L-670,207. The high N M S / O x o - M binding ratios obtained with the novel oxadiazole derivatives suggests that these compounds are muscarinic agonists with high intrinsic efficacy. The induction of hypothermia and salivation by these compounds in these studies support this suggestion. The distinction between the oxadiazoles and arecoline was found to be more pronounced in the whole animal studies where the novel oxadiazoles were almost 50 000 more active. The relatively weak in vivo activity of arecoline in all three of the assays is a reflection of the very labile ester linkage present in this molecule. Oxadiazoles are presumably metabolically more stable and as a result display higher potency. These c o m p o u n d s are amongst the most potent muscarinic agonists ever described. Arecoline displayed relatively poor penetration in the ex vivo binding assay although this may be due in part to the reportedly short plasma half life of the compound. In the present design of these studies, compounds were evaluated for CNS penetration at a single time of 30 min. The two methyl oxadiazoles displayed apparent CNS penetrations of 1-4% of the administered dose. These values represent very good penetration since the total weight of the mouse brain as a proportion of total body weight is in the region of 2-5%. The only standard agonist with similarly good penetration in this assay was RS-86 which has been previously reported by Palacios and colleagues (Palacios et al., 1986) to penetrate well
into the CNS. The two amino oxadiazoles were apparently 10 fold weaker in their brain penetration, a finding which corresponded with their biological profile in the hypothermia and salivation studies where the methyl oxadiazoles generally produced central and peripheral effects at relatively similar doses. This was most apparent in the quinuclidine series. These findings correspond to the relative lipophilicity of the four compounds as estimated by log P measurements (data not presented). All of the measurements of brain penetration relied upon a number of assumptions including the important assumption that all of the measured activity corresponded to parent compound rather than an active metabolite. In previous studies we demonstrated that the penetration of [3H]quinuclidinyl benzilate administered to mice corresponded to that estimated in the ex vivo studies (Freedman et al., 1989). In the present study [a4C]L-660,863 (5.0 /~Ci) was administered i.v. to two 300 g male Sprague-Dawley rats. Following killing, 30 min later, 0.2% of the radioactivity was found to be present within the brain, a value that was close to that found in the ex vivo studies (0.13%). This result suggests that the results obtained with the quinuclidine L-660,863 in the ex vivo studies were reasonably accurate. In tissues the coupling efficiency of the receptors may vary considerably such that the effective receptor reserve may result in a compound being a full agonist in one tissue yet be an antagonist in another (Kenakin, 1986). Thus pilocarpine is a relatively full agonist on the superior cervical ganglion (Newberry and Priestley, 1987) and on PI induced turnover in parotid gland (Freedman, 1986) but has only limited ability to evoke a PI response in rat cerebral cortex. In contrast muscarine and carbachol are relatively full agonists on all three tissues. Indeed the only agonists with sufficient intrinsic efficacy to stimulate PI turnover in rat cerebral cortex were the quaternary agonists previously described. The four compounds described in this study are amongst the first non quaternary high efficacy agonists described which retain sufficient intrinsic efficacy to maximally stimulate PI turnover in rat cerebral cortex (Freedman et al., in press). The in
199 v i v o e x p e r i m e n t s c o n f i r m t h a t all of the a g o n i s t s a r e a b l e to p e n e t r a t e w e l l i n t o t h e b r a i n a n d are t h e r e f o r e a b l e to a c t i v a t e m u s c a r i n i c r e c e p t o r s w i t h i n the C N S . T h e s e a g o n i s t s m a y possess the a b i l i t y to fully a c t i v a t e m u s c a r i n i c r e c e p t o r s in t h o s e areas of t h e C N S w h e r e p e r h a p s the c o u p l i n g is less e f f i c i e n t t h a n elsewhere. O n e m i g h t h y p o t h e s i s e t h a t if the P I r e s p o n s e in c e r e b r a l c o r t e x is a s s o c i a t e d w i t h h i g h e r f u n c t i o n in this area, t h e n t h e s e c o m p o u n d s m a y b e t h e first des c r i b e d w i t h the a b i l i t y to b e a b l e to fully a c t i v a t e r e c e p t o r s in the c o r t e x f o l l o w i n g p e r i p h e r a l administration. These compounds may therefore be u s e f u l tools in o r d e r to fully test t h e r o l e o f m u s c a r i n i c r e c e p t o r s in the c o m p l e x p r o c e s s e s o f memory and cognition.
Acknowledgements The authors would like to thank Miss S. Farrow and Mrs S. Burton for manuscript preparation. Portions of this work were presented at the Subtypes of Muscarinic Receptor Meeting (IV) Wiesbaden, 20-22 July, 1989 (Saunders, J. and Freedman, S.B., 1989).
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