Life Sciences, Vol. 60, Nos. 13/14, pp. 105j_lCM, 1597
`Cbpydght 0 1997 FWvier Science Inc.
`Printed in the USA. All rights resend
`0024-32nsp7 517.00 t .oo
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`PI1 s0024-3205(97)ooo47-7
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`DISCOVERY & DEVELOPMENT OF SELECTIVE M3 ANTAGONISTS FOR CLINICAL USE V.A. Alabaster Department of Discovery Biology Pfizer Central Research, Sandwich, Kent, CT 13 9NJ, U.K. Summary The treatment of airway obstructive disease may be improved by antimuscarinic agents which selectively block Ml and M3 receptors but do not inhibit prejunctional cholinergic autoreceptors which limit release of acetylcholine. Revatropate is a novel antimuscarinic agent which shows some SO-fold selectivity for MI and Ms receptors in guinea pig trachea and rabbit vas deferens over the M2 subtype in atria. This selectivity profile was seen in viva in anaesthetised guinea pigs and conscious dogs where bronchodilator activity was produced in the absence of any effect on heart rate. Revatropate, in contrast to the non-selective agent ipratropium, did not potentiate bronchoconstrictor responses induced by vagal nerve stimulation, indicating that inhibitory autoreceptors were still functional. Early clinical studies in COAD patients showed that inhaled revatropate was an effective bronchodilator which was well tolerated. Darifenacin differs from revatropate by showing selectivity for Ms receptors relative to both M2 and MI subtypes. [3H] darifenacin had 5-fold higher affinity for the human m3 relative to ml receptors while there was significantly reduced binding to m2, m4 and m5 receptors. The degree of selectivity in functional tissue preparations was even greater, with darifenacin showing lOO-fold selectivity for the ileum Ms receptors over Mz receptors in atria and 30-fold over MI receptors in rabbit vas deferens. Darifenacin was able to differentiate between M3 receptors in different tissues; although darifenacin was equipotent with atropine in the ileum and bladder, it was some IO-fold and 6-fold less potent at inhibiting muscarinic responses in the trachea and submandibular salivary gland respectively, relative to atropine. Studies in anaesthetised dogs confirmed this selectivity profile. Thus darifenacin inhibited responses of the gut and bladder to cholinergic stimulation without affecting heart rate. Salivary gland responses were inhibited at doses some 6-10 fold higher than those required to inhibit gut and bladder responses. Clinical studies are ongoing in urge incontinence and functional bowel disease which may confirm this selectivity profile. Key Words: muscarinic receptor subtypes,
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`pharmacology
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`M3
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`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1028 - Page 1
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`ELSEVIER
`antagonists, predinical
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`1054
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`Discovery of Selective M, Antagonists
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`Vol. 60, Nos. 13/14,1!97
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`The Datura genus of plants which contain a&opine and related anticholinergic alkaloids, have been used by man for several thousand years for a variety of medical and other purposes. Thus for example, inhalation of smoke from the herb Datura stramonium was recommended for the treatment of asthma in the seventeenth century (1). However, drying of secretions, tachycardia, urinary retention, blurred vision and central nervous system effects were all recognised complications associated with the use of atropine containing compounds and preparations. Muscarinic antagonists are still widely
`for example oxybutynin in urinary urge incontinence and dicyclomine in irritable bowel syndrome, but the clinical utility of these non-selective agents is still limited by adverse side effects. In 1976, Barlow’s studies of the relative activities of a series of muscarinic antagonists in the guinea pig ileum and the rabbit atrium indicated that responses in these two tissues were mediated by different receptors (2). Further work clearly established the heterogeneity of muscarinic receptors and they have been classified using pharmacological techniques into MI,
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`used,
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`MZ ,
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`M3 and M4 subtypes, and on genomic cloning into ml, m2, m3, m4 and m5 subtypes (3). Thus the possibility of designing sub-type selective agents to achieve efficacy with reduced side-effects was a feasible and exciting possibility. Muscarinic M3 receptors are located predominantly on smooth muscle and salivary glands, and it was considered that agents selective for this sub-class of receptors could have therapeutic utility in the treatment of incontinence, disorders of gastro-intestinal motility and as bronchodilators in respiratory disease. We therefore initiated projects at Pfizer to identify M3 selective antagonists and this paper describes the profile of 2 such agents, revatropate (UK-l 12,166) and darifenacin (UK- 88,525) (Fig. 1). Revatropate (UK-l 12,166) (R)-3-Quinuclidinyl (2&R,)-2- hydroxymethyl-4- (methylsulphinyl)-2- phenylbutanoate Darifenacin (UK-88,525) (S)-2-{ 1-[2-(2,3,- dihydrobenzofuran-5yl) ethyl]-3-pyrrolidinyl} -2,2_diphenylacetamide Fig. 1 Structural formulae of novel M3 antagonists.
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`Revatrouate:
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`- a drug for treatment
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`of COAD
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`Activation of cholinergic nerves is the major bronchoconstrictor neural mechanism in animal and human airways and there is evidence that this mechanism may be overactive in
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`Vol. 60, Nos. U/14,1997
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`Discovery of Selective M3 Antagonists
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`1055
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`vitro
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`profile of revatropate is shown in Table 1. Revatropate showed potent antagonism of M, and M3 receptors with some 50-fold selectivity over the Mz subtype. This was in contrast to ipratropium which was also potent, but non-selective ( IO) TABLE
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`I
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`muscarinic receptors M,, MZ, and M3 have been identified in human airways and their location and possible physiological function have been extensively reviewed (5). Stimulation of the vagus nerve releases acetylcloline (ACh) which activates muscarinic receptors on airway smooth muscle (M3) and submucosal gland (Mx) to induce bronchoconstriction and mucus secretion respectively. MI-receptors facilitate neurotransmission through parasympathetic ganglia and possibly enhance cholinergic reflexes. Additionally, there are also autoreceptors on cholinergic nerve terminals innervating bronchial and tracheal smooth muscle which inhibit ACh output when activated (6,7) which have been characterised as the M2 muscarinic subtype (5, 8). (In the guinea pig, there is some evidence that these’ autoreceptors may be more MJ-like rather than M2 (9)). Thus a rational approach to producing an improved agent to treat airways obstruction was to identify a compound which selectively inhibited M3 and Ml muscarinic receptors, but unlike non-selective agents such as ipratropium, did not interfere with the neurotransmitter negative feedback loop by blockade of Ml (or M&receptors. The in
`profile of revatropate and ipratropium. TISSUE/SUBTYPE Rabbit vas deferens Ml 9.26 (8.93-9.93) Guinea pig atria M2 7.25 (7.12-7.39) Guinea pig trachea M3 8.92 (8.67-9.24) REVATRC pA2 (95% conf. limits] ATE l/==Yr7 PA? slope (95% conf limits) 9.44 (9.16-9.86) I I 9.15 (8.91-9.45) 9.45 (9.24-9.73) 1.11 1.21 1.38 - n n=number of tissues This selectivity profile was also seen
`Thus in anaesthetised guinea pigs, revatropate antagonised ACh-induced Mj mediated bronchoconstrictor responses at some 80- fold lower doses than those required to inhibit ACh-induced Mz mediated bradycardia (10). Ipratropium inhibited both responses over a similar dose range. The functional involvement of pre-junctional muscarinic receptors on post-ganglionic airway cholinergic nerves that inhibit the release of ACh in lung responses to nerve stimulation has been demonstrated in animal experiments (5, 8, 1 I). For example, bronchoconstrictor responses to vagal nerve stimulation were potentiated by the Mz/Ma- selective antagonist methoctramine, indicating inhibition of the inhibitory feedback control on ACh release. Similar experiments were carried out in our laboratories in anaesthetised guinea pigs and cats with revatropate and ipratropium, comparing their effect on bronchoconstriction induced by vagal stimulation with responses to injected ACh (I 2). Ipratropium potently inhibited i.v. ACh-induced bronchoconstriction in both species (ID50 1.4Spg/kg in guinea pig, O.O8pg/kg in cat). Significantly higher doses were required to inhibit responses to vagal nerve
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`In vitro
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`in vivo.
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`1056 Discovery of SeIective M, Antagonists Vol. 60, Nos. l3/14, 1997 stimulation (ID+-lOpg/kg in guinea pig, 2.1pg/kg in cat). Additionally, low doses of ipratropium potentiated the bronchoconstriction responses to vagal-nerve stimulation by up to 150%. (See Fig 2). Revatropate also potently inhibited the bronchoconstrictor response to i.v. ACh (IDsa 0.94/q&g in guinea pig and 0.52 pg/kg in cat). However, in contrast to ipratropium, responses to vagai stimulation were similarly inhibited over the same dose range (Q. 1.7yg/kg in guinea pig, l.O3pg/kg in cat). Potentiation of the vagal response was not observed at any dose. The data from guinea pigs are shown in Fig 2. I I w 250- ti i ,: 9 200- 0 % K 6 150- F fj loo- < 0 z 9 z 50- 300 - r - $250 - < + z 2200 - B B 5150 - 0 F 5100 - 8 * f 50 - % n=5 OL., . . . . . . . . . . . ,,I. *1 fzT?& o&mm . . 0.01 0.1 1 10 0.01 0.1 1 10 IPRATROPIUM @g/kg i.v.) Revatropate (rgikg i.v.) -m- ACETYLCHOLINE IDso 1.45 f o.llFg/kg -I- ACETYLCHOLINE ID,@94 * 0.12pgikg -e- VAGAL STIMULATION IDS, >lO&kg -O- VAGAL STIMULATION 104.7 * 0,3&$q/kg Fig. 2 Selectivity of revatropate for post-junctional Ms receptors over pre-junctional autoreceptors - comparison with ipratropium in anaesthetised guinea pigs. The bronchodilator activity and lung selectivity of revatropate was confirmed in conscious dogs where revatropate, given orally and by metered-dose inhaler, antagonised nebulised methacholine-induced bronchospasm without affecting heart rate (13). The role of the autoreceptor on pre-junctional cholinergic nerve terminals in the airways in man is unknown, but indirect clinical pharmacology studies indicate their presence in non-asthmatics but absence in asthmatic subjects (14). It is possible, that while neuronal M2 receptors exert an important inhibitory control on the parasympathetic nerves supplying the airway smooth muscle in normal circumstances, the situation may be different in the disease state. Thus measurements of vagal-induced bronchoconstriction responses in guinea pig indicated that prejunctional autoreceptors are dysfunctional after exposure to viral airway infections, ozone or antigen inhalation (15). Inflammatory mediators and/or eosinophil degranulation produces were implicated in the loss of M2 function in these studies. The first clinical study evaluating the effect of revatropate in COAD patients was very encouraging. Forty two COAD patients were studied in a double-blind 3 treatment cross-over study, designed to compare the response to 32Opg revatropate, 8Opg ipratropium bromide and
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`Vol. 60, Nos. 13/14, 1997 Discovery of Selective M, Antagonists 1057 placebo given via Meter Dose Inhaler (16). The mean increase from pre-dose FBVi to peak- post dose FEV, was 0.34L (+27%) on revatropate, 0.36L (+29%) on ipratropium bromide and 0.15L (+12%) on placebo. Revatropate was well tolerated and no side effects were reported. Further studies are required to explore the dose-response relationships, and these may reveal advantages over non-selective agents. Darifenacin The functional smooth muscle responses induced by cholinergic nerve stimulation to the bladder and gut are mediated via M3 receptor activation (3). Darifenacin differs from revatropate by showing selectivity for the M3 subtype relative to all other muscarinic receptor subtypes and hence is anticipated to have an advantage over non-selective anti-muscarinic agents in the treatment of urge incontinence and functional bowel disease. The binding of [‘HI-darifenacin to the five cloned human muscarinic receptors (ml - m5) expressed in CHO cells was compared (17). [sH]-darifenacin was found to bind with S- fold higher affinity to m3 (Kn, 0.33nM) over ml (Kn, I .6nM) receptors. There was no specific binding to m2 receptors and specific binding to m4 and m5 receptors was insufficient to determine a Kn. Competition studies in cells expressing m3 and ml receptors using a range of muscarinic antagonists showed that darifenacin represents the first selective m3 ligand. A greater degree of selectivity for the M3 receptor subtype was observed in functional in
`profile of darifenacin and atropine. DAIUFENACIN ATROPINE TISSUE/SUBTYPE PA;?+ sem slope n PA;! f sem slope n Guinea pig ileum M3 9.44 + 0.07 1.16 6 9.4kO.07 0.96 6 Guinea pig trachea Ms 8.70 + 0.09 1.08 6 9.2OzkO.l 1.18 6 Guinea pig bladder M3 8.66 + 0.14 1.01 4 9.01 kO.09 0.86 4 Guinea pig atria M2 7.48 + 0.13 0.84 6 8.72kO.06 1.13 7 Rabbit vas deferens Mi 7.90 f 0.08 0.94 6 9.58 f 0.09 0.9 7 Guinea pig salivary gland 7.0 r 0.1 (pICsa) - 4 7.87~0.14(pIc50) - 4 M3 Functional responses to cholinergic activation of salivary glands am also mediated by the M3 muscarinic
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`studies. The pA2 values for darifenacin and atropine versus muscarinic activation in a variety of tissue preparations are shown in Table II. Atropine was essentially non-selective while darifenacin showed lOO-fold selectivity for ileum M3 receptors over M2 receptors in atria and 30-fold over Mi receptors in rabbit vas deferens (18, 19). TABLE II
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`In vitro
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`vitro
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`subtype.
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`However, although darifenacin was equipotent with atropine in the ileum and bladder preparations, darifenacin was some 6-fold less potent at inhibiting carbachol- induced “Rb efflux from the submandibular salivary gland and IO-fold less potent at
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`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1028 - Page 5
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`1058
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`Discovery of Selective M, Antagonists
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`Vol. 60, Nos. l3/14, 1997
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`I‘hus,
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`when compared with atropine, darifenacin showed a degree of selectivity for the ileum relative to other smooth muscle preparations studied and also relative to the salivary gland responses. Other muscarinic antagonists have also been reported to differentiate between M3 receptors expressed in different tissues, for example hexahydrosiladifenidol (20) and zamifenacin (21). The molecular basis for this selectivity is not understood, especially since only one M3 receptor has been identified from molecular sequence studies. However, the pharmacological data suggests that there may be some heterogeneity of conformation when expressed in the native tissues. It is possible that receptor configuration or antagonist binding are affected by tissue dependent factors such as signal transduction mechanisms, or the presence of other receptor subtypes in the tissue membrane. Thus, although it is well established that the functional receptor mediating contraction in the trachea, ileum and bladder is of the M3 subtype, the major population of muscarinic receptors in the smooth muscle of these tissues is of the M2 subtype (3). The role of MI receptors in these smooth muscle preparations is unknown, but it is conceivable that their presence and primed state (linked to adenylcylase) may modulate post receptor events in a tissue-dependent way. The availability of tritiated darifenacin should help to identify any differences in tissue binding and could provide useful data for receptor modelling and mutagenesis studies. Studies in anaesthetised dogs confirmed the potent M3 antagonist activity of darifenacin on gut and bladder and the clear selectivity over Mz cardiac muscarinic receptors (22). Thus darifenacin potently inhibited cholecystokinin (CCK-8) induced jejunal motility (ED5” 7.5pg kg-‘) whilst it was significantly weaker at inhibiting nerve induced salivary flow (EDsa 54pg kg-‘). Importantly, doses up to 25Opg kg-’ had no effect on heart rate. In contrast, atropine inhibited CCK-8 induced jejunal motility and salivary flow over the same dose range and these doses induced marked tachycardia. 250yl 100 50 0 1 ,J%_ The data are summarised in Fig 3. 150 + SI Motility - Salivation 0.01 0.1 1 IO 100 0.01 all 1 IO Dose (pg/kg/min) Dose (pg/kg/min) Fig.3 Effect of darifenacin and atropine on jejunal motility, salivation, heart rate and blood pressure in anaesthetised dogs.
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`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1028 - Page 6
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`inhibiting ACh induced contractions in trachea, relative to atropine.
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`Vol. 60, Nos. 13/14,1997
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`Discovery of Selective M, Antagonists
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`1059
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`Similar studies were carried out in anaesthetised dogs measuring contractions of the bladder to pelvic nerve stimulation (23). Darifenacin showed a 9-fold selectivity for bladder responses (ED50 6.8pg kg-‘) over salivary gland responses (ED50 58.3pg kg-‘) and had no effect on heart rate at doses up to 1OOpg kg-‘. Again this was in contrast to atropine which was non-selective over the dose range 1-4Opg kg“. Thus the in vitro profile of darifenacin and its clear selectivity for gut and bladder relative to effect on to salivary gland, translated to the in vivo situation. Darifenacin is currently in Phase II evaluation in urinary incontinence and functional bowel disease and early results are encouraging. A pilot study in patients with detrusor instability, assessed by continuous ambulatory monitoring, has shown that multiple doses of darifenacin were well tolerated and reduced total number, maximum amplitude, and duration of unstable bladder contractions (24). Ongoing and future research should help to rationalise these pharmacological findings and give a better understanding of how subtypes of muscarinic receptors function in different organs both physiologically and pathophysiologically. It may then be possible to design muscarinic antagonists with even greater receptor and tissue selectivity, providing therapeutic agents with further improvements in efficacy and toleration. Acknowledgements I would like to acknowledge all the people who were involved in the identification and development of revatropate and darifenacin at Sandwich, with special thanks to R. Wallis, A. Naylor, D. Newgreen, P. Quinn, B. Moore & R. Keir. Revatropate and darifenacin were synthesised in Pfizer laboratories by A. Stobie and A.R. Mackenzie respectively. References 1. B. GANDEVA, Postgrad. Med. J. 51 (Suppl. 7) 13-20 (1975). 2. R.B. BARLOW, K.J. BERRY, P.A.M. GLENTON, N.M. NIKOLAN & K.S. SOH, Br. J. Pharmacol58 613-620 (1976). 3. M.P. CAULFIELD, Pharmacol. Ther. 58 3 19-379 (1993). 4. N.J. GROSS and M.S. SKORODIN. Am. Rev. Respir. Dis. 129 856-870 (1984). 5. P.J. BARNES, Life Sci, 52 521-527 (1993). 6. I. WESSLER, D. HELLWIG and K. RACKE, Arch. Pharmacol. 342 387-393 (1990). 7. P.J. BARNES, P. MINETTE and J. MACLAGAN, Trends. Pharmacol. Sci, 9 412-416 ( 1988). 8. A.D. FRYER and J. MACLAGAN, Br. J. Pharmacol. 83 973-978 (1984). 9. H. KILBINGER, R.S. VON BARDELEBEN, H. SIEFKEN & D. WOLF, Life Sci. 56 981-987 (1995). 10. B.A. MOORE, R.F. KEIR, E.F. STUART, K.N. WRIGHT and A. STOBIE, Eur Resp J. 9 (Suppl. 23) 30s (1996). 11. R.F. KEIR, B.A. MOORE, V.A. ALABASTER, Br. J. Pharmacol. 102 337P (199 1). 12. R.F. KEIR, E.F. STUART, K.N. WRIGHT and B.A. MOORE, Eur Resp. J. 9 (Suppl. 23) 29s (1996). 13. I. MACHIN, P.A. MERNER, L.M. OAKES, M.R. SUTTON, I.R. WITHERDEN, Eur. Resp. J. 9 (Suppl. 23) 29s (1996).
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`1060 Discovery of Selective Mj Antagonists Vol. 60, Nos. 13/14,19!27 14. P.A.H. MINETTE, J. LAMMERS, C.M.S. DIXON, M.T. McCUSr’ER, 8~ P.J. BARNES, J. Appl. Physio167 2461-2465 (I 989). 15. A.D. FRYER and D.B. JACOBY, Life Sci. 52 529-536 (1993). 16. F.P.V. MAESEN, J.J. SMEETS, P. SMEETS, I. GORTER DE VRIES and M. HODGES. Eur Resp. J. 9 (Suppl. 23) 29s (I 996). 17. CM. SMITH and R.M. WALLIS, J. of Recep. and Signal Transd. Res. In press 18. R.M. WALLIS, R.A. BURGES, P.E. CROSS, A.R. MACKENZIE. D.T. NEWGREEN and P. QUINN, Pharmacol. Res. 31 PS4 (199.5). 19. D.T. NEWGREEN & A.M. NAYLOR, Br. J. Pharmacol. 117 107P (1996). 20. R.M. EGLEN, A.D. MICHEL, W.W. MONTGOMERY, E.A. KUNYSZ, C.A. MACHADO and R.L. WHITING, Br. J. Pharmacol. 99 637-642 ( 1990). 21. R.M. WALLIS, Life Sci. 56 861-868 (1995). 22. P. QUINN, P. MCINTYRE, W.D. MINER and R.M. WALLIS, Br. J. Pharmacol. 119 198P (1996). 23. D.T. NEWGREEN, C.W.P. ANDERSON, A.J. CARTER & A.M. NAY LOR, Neurourology and Urodyn. 14 555 ( 1995). 24. D.J. ROSARIO, B.R. LEAKER, D.J. SMITH and C.R. CHAPPLE, Neurourology and Urodyn. 14 5 (I 995).
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