0 Pharmacology & Toxicology 1997, 81, 169-172
`Printed in Denmark . AN rights reserved
`
`Copyright 0
`
`ISSN 0901-9928
`
`Antimuscarinic Potency and Bladder Selectivity of
`PNU-200577, a Major Metabolite of Tolterodine
`
`Lisbeth Nilvebrant’, Per-Goran Gillberg’ and Bengt Spa&
`‘Medical Department Urology and *Department of Pharmacology, Pharmacia & Upjohn AB, Uppsala, Sweden
`(Received December 4, 1996; Accepted April 24, 1997)
`
`Abstract: PNU-200577 (labcode DD 01 [(R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)-3-phenylpropanami-
`ne) is a major pharmacologically active metabolite of tolterodine, a new muscarinic receptor antagonist intended for the
`treatment of an overactive bladder. In virro, PNU-200577 produced a competitive and concentration-dependent inhibition
`of carbachol-induced contraction of guinea-pig isolated urinary bladder strips (KB=0.84 nM; pAz=9. 14). In vivo, PNU-
`200577 was significantly more potent at inhibiting acetylcholine-induced urinary bladder contraction than electrically
`induced salivation in the anaesthetised cat (IDSo 15 and 40 nmol . kg-l, respectively; P<O.Ol). In radioligand binding
`studies carried out in homogenates of guinea-pig tissues and Chinese hamster ovary cell lines expressing human muscarinic
`ml-m5 receptors, PNU-200577 was not selective for any muscarinic receptor subtype. Thus, PNU-200577 is similar to
`tolterodine in terms of antimuscarinic potency, functional selectivity for the urinary bladder in vivo and absence of selec-
`tivity for muscarinic receptor subtypes in vitro. The results of this study clearly indicate that PNU-200577 contributes to
`the therapeutic action of tolterodine, in view of its high antimuscarinlc potency, similar serum concentration and lower
`degree of protein binding.
`
`Contraction of the human urinary bladder is known to be
`mediated predominantly through stimulation of muscarinic
`receptors (Anderson 1993) and, thus, the pharmacological
`treatment of urinary urge incontinence is based on muscar-
`inic receptor antagonists (Anderson 1988; Wein et al.
`1994). Tolterodine is a new, potent competitive muscarinic
`receptor antagonist that has been developed for the treat-
`ment of urinary urge incontinence and other symptoms re-
`lated to an overactive bladder. In vitro, tolterodine exhibits
`a high affinity and specificity for muscarinic receptors (Nil-
`vebrant et al. 1994 & 1996). In human isolated urinary
`bladder preparations tolterodine potently antagonises con-
`tractions induced by either carbachol or electrical stimula-
`tion (Naerger et al. 1995; Nilvebrant et al. 1995). In vivo, a
`selectivity of tolterodine for the urinary bladder over sali-
`vary glands has been demonstrated in the anaesthetised cat
`(Gillberg et al. 1994; Nilvebrant et al. 1996). At least 5 genes
`encoding for molecularly distinct subtypes of muscarinic re-
`ceptors (ml-m5) have been cloned and expressed in cell
`lines and 3 of these subtypes can be distinguished pharma-
`cologically (M,-M3) (Caulfield 1993; Hulme et al. 1990).
`However, the selectivity profile of tolterodine in vivo cannot
`be explained in terms of selectivity for one of these muscar-
`inic receptor subtypes (Nilvebrant et al. 1996).
`Metabolism studies in humans have revealed that toltero-
`dine undergoes oxidation via cytochrome P450 2D6 to a 5-
`hydroxymethyl derivative, PNU-200577 (labcode DD 01
`[(R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphen-
`
`Author for correspondence: Lisbeth Nilvebrant, Medical Depart-
`ment Urology, Pharmacia & Upjohn AB, s-752 81 Uppsala,
`Sweden (fax +46 18 166464).
`
`yl)-3-phenylpropanamine) (Anderson et al. 1995; Postlind
`et al. 1996). PNU-200577 has been synthesised and found
`to be pharmacologically active in preliminary experiments.
`While serum levels of the metabolite in humans are gener-
`ally comparable to those of tolterodine following oral ad-
`ministration of the parent compound (unpublished obser-
`vations), tolterodine and PNU-200577 differ in terms of the
`extent of protein binding. In human and cat serum, PNU-
`200577 exists as >30% unbound drug while tolterodine is
`>95% protein bound (unpublished observations). Taken to-
`gether, these data suggest that PNU-200577 contributes to
`the therapeutic action of tolterodine. Thus, the aim of this
`study was to determine the pharmacological profile of
`PNU-200577 both in vitro and in vivo. For comparative pur-
`poses, previously published data on tolterodine (Nilvebrant
`et al. 1994 & 1996) are included.
`
`Materials and Methods
`Functional in vitro studies. Male guinea-pigs (Dunkin Hartley
`strain) weighing 300-500 g were used in all experiments. Antimus-
`carinic potency of PNU-200577 was determined in guinea-pig iso-
`lated urinary bladder strips, as previously described (Nilvebrant et
`al. 1996). Following equilibration, the urinary bladder preparations
`were repeatedly exposed to a standard concentration of the muscar-
`inic receptor agonist carbachol (3 pM; ECso). A reproducible con-
`trol response was established before the effects of PNU-200577 were
`determined. Following the generation of a cumulative concen-
`tration-response curve to carbachol (control), PNU-200577 was
`added to the tissue bath for 60 min. (Nilvebrant 1986) and a second
`cumulative concentration-response curve to carbachol generated in
`the presence of PNU-200577. The effect of PNU-200577 was
`studied at 4 5 different concentrations using separate tissue prep-
`arations. Responses were expressed as a percentage of the maximal
`contractile response elicited by carbachol in the control curve.
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1015 - Page 1
`
`

`
`170
`
`LISBETH NILVEBRANT ET AL.
`
`Radioligand binding studies. The affinity of PNU-200577 for mus-
`carinic receptors was determined by radioligand binding studies in
`homogenates of guinea-pig tissues (urinary bladder, parotid gland,
`heart and cerebral cortex) and in Chinese hamster ovary cells ex-
`pressing the human muscarinic receptor subtypes (ml-m5). Briefly,
`tissue homogenates were incubated at 25" with the radioligand 1-
`quinuclidinyl [phenyl-4
`benzilate [( -)3H-QNB] and different
`concentrations of unlabelled antagonist under conditions of equilib-
`rium: urinary bladder, 60 min. (Nilvebrant & Sparf 1983a); parotid
`gland, 210 min. (Nilvebrant & Sparf 1982); and heart and cerebral
`cortex, 80 min. (Nilvebrant & Sparf 1986). Incubations were ter-
`minated by centrifugation. The pellets were washed three times with
`buffer before radioactivity was determined by liquid scintillation
`spectrometry as previously described (Nilvebrant & Sparf 1983a).
`Radioligand binding studies in Chinese hamster ovary cells were
`carried out in culture plates. Briefly, cell homogenates were incu-
`bated with (-)3H-QNB and different concentrations of unlabelled
`antagonist under conditions of equilibrium (37" for 300 rnin.). In-
`cubations were terminated by rapid filtration and the amount of
`radioactivity determined by liquid scintillation spectrometry. Total
`binding of (-)3H-QNB was determined in the absence of any com-
`peting ligand, while non-specific binding was determined in the
`presence of unlabelled atropine (10 pM). Receptor-specific binding
`in each experiment was defined as total non-specific binding.
`
`In vivo studies. The antimuscarinic effects of PNU-200577 in vivo
`were studied in the anaesthetised cat, as previously described (Nil-
`vebrant e/ a/. 1996). Ten female European short-haired cats (2.2-
`3.1 kg, 8-10 months of age) were used in the experiments (PNU-
`200577, n=5; control, n=5). Briefly, bladder contractions were
`elicited by intraarterial administration of a submaximal dose of ace-
`tylcholine (1-2 pg . kg-'), while salivation was induced by supra-
`maximal electrical stimulation (6 V, 2 msec, 5 Hz) of the parasym-
`pathetic chorda-lingual nerve over 2 min. Acetylcholine was ad-
`ministered before and approximately 9 and 16 min. after each dose
`of PNU-200577 (2-203 nmol . kg-' [0.001-0.1 mg . kg-'1) or saline,
`which was administered by intravenous infusion in the right femoral
`vein at a rate of 1 ml . min.-' . kg-'. Electrical stimulation of the
`chorda-lingual nerve was performed before and approximately 7
`min. after each dose of PNU-200577 or saline.
`
`Data analysis. The concentration of carbachol that produced 50%
`of the maximal contractile response (ECSo) in guinea-pig isolated
`urinary bladder strips was determined in the absence and presence
`of different concentrations of PNU-200577, respectively. The affin-
`ity of PNU-200577 was calculated as the dissociation constant KB
`(Schild 1949). In the radioligand binding experiments, the concen-
`tration of PNU-200577 that inhibited (-)3H-QNB binding by 50%
`was determined from the experimental concentration-inhi-
`bition curves. Dissociation constants (Ki) were calculated by cor-
`recting the ICSo values for the radioligand-induced parallel shift and
`the differences in receptor density, using the method described by
`Jacobs et al. (1975); see also Nilvebrant & Sparf (1982 & 1983b) for
`details. Differences between the inhibitory effect exerted by PNU-
`
`200577 on urinary bladder contraction and on salivary secretion in
`the anaesthetised cat were analysed for each dose using a paired
`Student's t-test. Differences between the doses of antagonist causing
`a 50% inhibition of these responses (ID5,,) were also analysed. All
`data are expressed as mean2S.E.M. P values cO.05 were considered
`statistically significant.
`
`Drugs and chemicals. The following drugs and chemicals were used:
`PNU-200577 [(R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethyl-
`phenyl)-3-phenylpropanamine, as the mandelate salt (batch nos. CA
`007 075:l and CA 007 075:3); Pharmacia & Upjohn AB, Sweden];
`atropine sulphate, carbachol (carbamylcholine chloride), acetylcho-
`line chloride and phenyl methyl sulphonyl fluoride (Sigma Chemical
`Company, U.S.A.); (-)3H-QNB (specific radioactivity 38.845.4
`Ci . mmol-'
`(1.43-1.63 Tbq . mmol-'); Du Pont NEN Research
`Products, U.S.A.); Dulbecco's modified Eagle's medium and HAM'S
`F12 medium (National Veterinary Institute, Sweden); foetal bovine
`serum albumin (HyClone Lab, U.S.A.); L-glutamine and penicillin/
`streptomycin (ICN Biomedicals, U.S.A.). Other chemicals used
`(analytical grade) were purchased from general commercial sources.
`(-)3H-QNB was diluted in absolute ethanol. Fresh solutions of
`PNU-200577, carbachol, atropine and acetylcholine were prepared
`for each experiment; for functional in vilro studies dilutions were
`made in double distilled water, while for in vivo studies solutions
`were diluted in saline.
`
`Results
`Functional in vitro studies.
`PNU-200577 produced a concentration-dependent. parallel,
`rightward shift in the concentration-response curve to car-
`bachol in guinea-pig urinary bladder strips. The maximum
`response to carbachol was not depressed. The mean KB
`value for PNU-200577 was 0.84+0.09 nM (n=20). Schild
`plot analysis for PNU-200577 determined a pA2 value of
`9.14 and a slope that was close to unity (0.99). This indi-
`cates that PNU-200577 is a competitive antagonist at the
`muscarinic receptors in the guinea-pig urinary bladder.
`
`Radioligand binding studies.
`PNU-200577 caused a concentration-dependent inhibition
`of ( -)3H-QNB binding in homogenates of guinea-pig uri-
`nary bladder, parotid gland, heart and cerebral cortex
`(table 1). The PNU-200577 concentration-inhibition curves
`of (-)3H-QNB binding were parallel and had Hill coef-
`ficients ( ~ t ~ )
`close to unity (table l), indicating that PNU-
`200577 binds to a single population of muscarinic binding
`sites in each tissue.
`
`Table 1.
`Dissociation constants (K,) and Hill coefficients (nH) for PNU-200577 and tolterodine, as determined by competitive radioligand binding
`studies in homogenates of guinea-pig tissues.
`
`Tissue
`Urinary bladder
`Parotid gland
`Heart
`Cerebral cortex
`a Data from Nilvebrant et al. (1996).
`Results are mean2S.E.M. of n separate experiments.
`
`PNU-200577
`K, (nM)
`2.920.3
`5.220.3
`1.1 20.1
`0.6020.04
`
`nH
`0.9820.07
`1.0620.07
`1.04+0.04
`0.9320.04
`
`Tolterodine"
`K, (nM)
`2.720.2
`4.820.3
`1.620.04
`0.7520.01
`
`nH
`1.02?0.03
`1.0420.03
`1.04?0.06
`1.0520.03
`
`n
`6
`5
`5
`5
`
`n
`8
`7
`6
`7
`
`~
`
`~
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1015 - Page 2
`
`

`
`ANTIMUSCARINIC POTENCY/BLADDER SELECTIVITY O F A TOLTERODINE METABOLITE
`
`171
`
`Table 2.
`Dissociation constants (K,) and Hill coefficients (nH) for PNU-
`200577 and tolterodine at human muscarinic receptors expressed in
`Chinese hamster ovary cells.
`PNU-200577
`Receptor
`K, (nM)
`subtype
`nH
`2.350.2
`1.0220.03
`ml
`2.020.5 0.9220.03
`m2
`2.520.5 0.9320.04
`m3
`2.820.2 0.9720.04
`m4
`2.950.4
`I.Ollr0.04
`m5
`Data from Nilvebrant rt al. (1996).
`Results are expressed as mean2S.E.M. of 4 6 separate experiments.
`
`To1 terodined
`nH
`Ki (nM)
`3.050.2
`1.03?0.04
`3.820.7 1.0020.04
`3.420.8 1.06-tO.03
`5.0?0.8
`1.05+-0.07
`3.420.8 1.00+-0.05
`
`PNU-200577 was a potent inhibitor of ( -)3H-QNB bind-
`ing in homogenates of Chinese hamster ovary cells express-
`ing human muscarinic ml-m5 receptors (table 2). Overall,
`Ki values for PNU-200577 were similar at the respective
`muscarinic receptor subtypes.
`
`In vivo studies.
`intraarterially) produced
`Acetylcholine (0.5-8 (g * kg-'
`dose-dependent contraction of the urinary bladder in the
`anaesthetised cat, with a dose of 1-2 pg . kg-' intraarteri-
`ally producing a reproducible submaximal response (data
`not shown). PNU-200577 produced dose-dependent inhi-
`bition of acetylcholine-induced urinary bladder contraction
`(fig. I), with a mean IDSo of 1554 nmol * kg-' (n=5). The
`threshold dose (ID30) for inhibition of acetylcholine-in-
`duced urinary bladder contraction was 7 2 3 nmol . kg-I.
`Electrical stimulation of the chorda-lingual nerve in-
`duced a mean salivary secretion of 321231 p l . 2 min.-'.
`PNU-200577 produced dose-dependent inhibition of elec-
`trically stimulated salivation (fig. I ) with an ID30 and IDSO
`of 21 t 2 and 4022 nmol * kg-I, respectively (P<0.05 and
`
`U Bladder contraction
`-C Salivation
`
`75
`
`-50 1
`
`r
`1
`
`8
`
`~
`
`"
`
`-
`
`
`
`~ Q I
`10
`
`r ~
`
`~
`
`100
`
`1 ~
`~
`
`~
`
`~
`~
`1000
`
`r
`
`l
`
`-
`
`Dose (nrnol*kg-')
` Effect of PNU-200577 on acetylcholine-induced urinary
`Fig. I ,
`bladder contraction and electrically stimulated salivary secretion in
`the anaesthetised cat. Results are expressed as percentage inhibition
`of the maximum response in each experiment, and are the mean
`(S.E.M. of 5 separate experiments. *=P<0.05 versus inhibition of
`salivary secretion (paired Student's t-test).
`
`P<O.Ol versus inhibition of bladder contraction). This indi-
`cates that PNU-200577 is three times more potent at the
`urinary bladder compared to the salivary gland.
`In control animals the administration of saline had only
`minor effects on the basal response to bladder contraction
`and salivation. Neither PNU-200577 nor saline had a sig-
`nificant effect on heart rate (data not shown).
`
`Discussion
`In the anaesthetised cat, PNU-200577 produced a dose-de-
`pendent inhibition of acetylcholine-induced urinary bladder
`contraction and electrically stimulated salivation, and was
`almost three times more potent for inhibition of urinary
`bladder contractions compared with salivation (ID50 15 and
`40 nmol . kg-', respectively). It may be argued that it would
`have been preferable to induce bladder contractions and
`salivation using an identical means of stimulation, i.e. either
`electrically or chemically with a muscarinic agonist given
`intravenously. However, both methods have practical limi-
`tations. For example, bladder contractions induced by elec-
`trical stimulation of the pelvic nerve are partly resistant to
`blockade by atropine and other muscarinic receptor antag-
`onists; indeed, the cholinergic component constitutes only
`about 30% of the response (unpublished observations),
`while the remainder is non-cholinergic non-adrenergic med-
`iated. This is well known to be the case in animal bladders,
`as shown in numerous in vitro and in vivo studies (Anders-
`son 1988 & 1993; Wein et al. 1994). However, since it is
`generally accepted that contractions of the human bladder
`are mediated mainly by muscarinic receptors (Andersson
`1993; Wein et al. 1994), we found it more relevant to study
`only the cholinergic component of the bladder response in
`the cat. Acetylcholine must be administered close to the tar-
`get organ in order to avoid degradation. We therefore used
`intraarterial injections because it is not possible to simul-
`taneously stimulate both the bladder and the salivary
`glands by intravenous administration of acetylcholine with-
`out using doses high enough to kill the animals. We also
`tried to administer stable analogues of acetylcholine (e.g.
`carbachol, methacholine) by intravenous injection. In gen-
`eral, however, this method resulted in an unstable baseline
`bladder response over time, together with low and variable
`stimulation of salivation. The inhibition exerted by muscar-
`inic antagonists could therefore not be reliably determined
`and reproduced between animals (data not shown). In the
`present study, chemical stimulation of the bladder with i.a.
`acetylcholine and electrical stimulation of the salivary
`glands were therefore the methods of choice in terms of
`reliable and consistent responses. Both responses were
`q
`
`~
`elicited by the natural transmitter, acetylcholine, but the
`concentration of acetylcholine at the muscarinic receptors
`in the bladder and salivary gland is obviously unknown, as
`always in in vivo experiments. Thus, it cannot be excluded
`that a difference in end-organ activation to some extent can
`explain the bladder selectivity of PNU-200577. However, in
`this context, it should be noted that the corresponding in
`
`I
`
`
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1015 - Page 3
`
`

`
`172
`
`LISBETH NILVEBRANT ET AL.
`
`vivo data on atropine and oxybutynin in previous studies
`were clearly in line with the general clinical experience with
`these drugs (Gillberg et al. 1994; Nilvebrant et al. 1996).
`The selectivity profile of PNU-200577 observed in this
`study is identical to that of tolterodine (Nilvebrant et al.
`1996), although PNU-200577 is more potent than the par-
`cnt compound in vivo. ID50 values for tolterodine were 101
`and 257 nmol * kg-I, respectively, for the inhibition of uri-
`nary bladder contraction and salivation (Nilvebrant et ul.
`1996). A likely explanation for the higher potency of PNU-
`200577 in vivo is that a very low percentage (<5%) of tolter-
`odine is unbound in serum, whereas >30% of PNU-200577
`exists as the unbound drug (unpublished observations).
`Since the total serum drug concentrations of tolterodine
`and PNU-200577 are similar following oral administration
`of the parent compound (unpublished observations), the re-
`sponse observed in vivo following oral administration of tol-
`terodine is likely to be, in part, the result of the activity of
`unbound PNU-200577.
`The findings of this study indicate that the pharmaco-
`logical profile of PNU-200577 in vitro is also essentially
`identical to that of tolterodine (Nilvebrant et al. 1996). In-
`deed, PNU-200577 was a potent and competitive antagonist
`at muscarinic receptors in guinea-pig isolated urinary blad-
`der strips and exhibited a high affinity for muscarinic recep-
`tors in the urinary bladder, parotid gland, heart and cer-
`ebral cortex. In addition, PNU-200577 had similar affinity
`for all five muscarinic receptor subtypes expressed in Chi-
`nese hamster ovary cells (K, 2.0-2.9 nM), as previously
`shown for tolterodine (K, 3 .O-5.0 nM) (Nilvebrant et al.
`1996). Thus, as for tolterodine, the selectivity profile of
`PNU-200577 in vivo cannot be attributed to selectivity for
`a single muscarinic receptor subtype (Nilvebrant et al.
`1996). It is interestingly to note that oxybutynin, a muscar-
`inic antagonist that exhibits a ten-fold higher selectivity for
`m3 over m2 receptors in vitro, displays the reverse selectivity
`profile to PNU-200577 and tolterodine in the anaesthetised
`cat, i.e. oxybutynin inhibits salivation by 80% at doses re-
`quired for 50% inhibition of the urinary bladder response
`(Nilvebrant et ul. 1996). Thus, the reverse selectivity pattern
`observed for oxybutynin compared with PNU-200577 and
`tolterodine may be related to the difference in relative affin-
`ity for M3/ni3 and M2/m2 receptors. Other compounds with
`selectivity for M3/m3 over M2/m2 receptors in vitro, such as
`darifenacin and UH-AH 37, exhibit an identical selectivity
`profile to oxybutynin in the anaesthetised cat (Nilvebrant et
`ti/. 1996). In contrast, the M2/m2 selective compound AQ-
`RA 741 was found to exhibit a bladder selectivity in vivo,
`which was similar to that demonstrated for tolterodine (Nil-
`vebrant et al. 1996). The data on AQ-RA 741 may suggest
`a functional role for M2/m2 receptors in bladder contrac-
`tion and, thus, in the selectivity for bladder over salivary
`glands demonstrated for PNU-200577 in vivo.
`In summary, the pharmacological in vitro and in vivo pro-
`files of PNU-200577 are almost identical to those of toltero-
`dine, the parent compound. In view of its high antimuscar-
`
`inic potency, lower degree of protein binding and similar
`serum concentration in humans after oral administration of
`the parent compound, PNU-200577 may contribute to the
`therapeutic action of tolterodine.
`
`References
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`Andersson. S. H. G., A. Lindgren & P-0. Edlund: Metabolism q/’
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`rice and application (2nd ed). Eds.: R. A. Munday, T. P Stephen-
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`43-70.
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1015 - Page 4