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`J P. HL'lD()BR()-T(')R().San1i::go: EC".
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`'l'nLy0: J.N. PE.\-‘.\IEiE‘A'I'l1EiR. Pxukuilincz A. PERTOVAARA. Turku; R
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`QUIRIUK’. Verdun; U. RAVI
`‘S, E.\.~cu‘. M.[3.;\. RF.|'I‘H.
`('hia.':1gu: H. S(.'HOE.V[/XKER. Bugncux; J.l".M. S.'VH’l'S. Msliislrichtz K.
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`
`|5aitent Uviher, UCB Pharma GmbH — Exhibit 2032 - 0001
`
`

`
` European Journal of Pharinacology 327 (l99'l} 195 --20?
`
`Tolterodine — a new bladder—selectivc antimuscarinic agent
`
`Lisbeth Nilvebrant “", Karl—Ei'ik Andersson ", Per-Goran Gillberg °, Matthias Stahl “,
`Bengt Sparf ‘
`.9wi1r.i'i.=n
`.‘lvlrdi'<.‘ui' Department Ural'ii_t.;y. Pharmuria & t'.."pjohrr AB. Sa7.§.’r’iI. Upp.€ai'rr.
`!' Department ofCi'i'm'cui’ PfrarmaL'u!ugv_ Ufllil.‘t'.’rSli")‘ Hospital, Limd, Sir:-o'er:
`' Degummem ufPharmuc'oI'ogy. Pharmrariarli U}'J,I'oi1rr iii‘). !..i'pp.m.'a. Sweden
`Received I? October 1996; revised [3 March l99'i". accepted lll March I997
`
`I
`
`Abstract
`
`Toltcrodine is a new muscarinic receptor antagonist intended for the treatment of urinary urge incontinence and other symptoms
`related to an overactive bladder. The aim of the present study was to compare the antiitiuscarinie properties of tollerodine with those of
`oxybutynin.
`in vitro and in vivo. Tolterodine effectively inhibited earbachol-indiiced C0l'tl|’itCllOnS of isolated strips of urinary bladder
`from guinea pigs (Kn 3,0 nM; p.‘\2 8,6; Schild slope 0.97) and humans (KM 4.U nM; PA: 8,4; Sehild slope
`l.(l-1)
`in ii
`C0ncentratiorrdepcndeitt. competitive manner. The affinity of tolterodine was similar to that derived for oxybutynin (KB 4.4 nM; pA2
`8.5; Schild slope 0.89)
`in the guinea-pig bladder. Tolterodine (Ill -2103 rimol/kg (0.01-l mg/kg);
`intravenous infusion) was
`significantly more potent
`in inliibiiing zicetylclioline-induced urinary bladder contraction than electrically-induced salivation in the
`anaesthetised eat.
`in contrast, oxybutynin displayed the opposite tissue selectivity. Radioligand binding data showed that tolterodirie
`bound with high affinity to museziririie receptors in iiririiiry bladder (K, 2.7 nM), heart (K,
`l.6 nM). cerebral cortex (K. 0.75 nM) and
`parotid gland (K; 4.8 nMl from guinea pig: and in urinary bladder from humans (K, 3.3 :iM).
`'l'olti:rodine and oxybuiynin were
`equipotent. except in the parotid gland. where uxybutynin bound with 8-times higher affinity (K: 0.62 nM). Binding data on human
`niiiniczirinic ml—m5 receptors expressed in Chinese hamster ovary cells showed that oxybiitynin,
`in contrast
`to tolterudiniz. exhibits
`selectivity (10-fold) for muscarinic m3 over m2 receptors. The KB value determined for Oxybutynin (4_4 nM) in functional Studies on
`guinea-pig bladder correlated better with the binding affinity at rnuscarinic M2/m2 receptors (K, 2.3 and 6.7 nM) than at inuscarinic
`M_,/iri3 receptors (K, 0.62 and 0.67 nM). The tissue selectivity demonstrated for tolterodinc in vivo cannot be attributed to selectivity for
`a single muscarinic receptor subtype. However, the combined in vitro and in vivo data on tollerodine and oxybutynin may indicate either
`that miiscartnie M,/m3 receptors in glands are more sensitive to blockade than those in bladder smooth muscle. or that musearinic
`M 1/m2 receptors contribute to bladder contraction.
`
`KL‘_t=\t'0rt:t’.i" Urinary bladder contraction: Saliiatiort; Muscarinic receptor subtype; Muscariitie receptor antagonist; (Guinea pig); (Cat): (Human)
`
`I. Introduction
`
`Tolterodine (Fig. I) is a new musearinie receptor antag-
`onist
`intended for the treatment of urinary urge inconti-
`nence and other symptoms of an overactive bladder. The
`pharmacological treatment of urge incontinence has for a
`long time been based on musearinie receptor antagonists,
`e.g.. proparitlieline, emeproniurn and oxybiitynin (Anders-
`son, I988; Wcin et al..
`l994}. and it
`is generally agreed
`that contractions of the human urinary bladder are medi-
`ated mainly through stimulation of acetylcholine mus-
`
`’ Corresponding author ‘tel: (46-lit] lfi-4-120;
`
`l-‘ax
`
`(46-18) If:-6464.
`
`I993). Oi-tybulynin is cur-
`earinie receptors (Aitdcrsson,
`rently considered to be the drug of choice in the treatment
`of bladder overactivity (Yarker et al., I995}. The effective-
`ness of oxybutynin has been demonstrated in several con-
`trolled clinical studies, but
`the incidence of classical an-
`timuscarinie side effects often leads to discontinuation of
`treatment. Dryncss ofthe mouth is the most prevalent side
`effect, experienced by at least 50% of patients on oxybii-
`tynin therapy (Yarlccr ct al., 1995).
`The existence of molecularly distinct muscarinic recep-
`tor subtypes has been well established (I-lulme et al., I990;
`Caulfield. l993). Thus. live genes encoding for musearinic
`receptors (ml—m5) have been cloned and expressed in cell
`lines, while three mitscarinie receptor subtypes (M,—M3)
`
`0014-2999/97/S17 tit) Copyright 1‘) I997 tilqei-ier Science B.V. All rights reserved.
`PU soot 4-2999t97)0006't.tt
`
`N01 iCE:
`THIS MATEPAAL MAY BE
`PROTECTED BY COPYRIGHT
`LAW (TITLE 7. us. CODE)
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2032 - 0002
`
`

`
`I96
`
`I.
`
`.‘Vi.lt-I.-brunt (4 m’ / European .Im4rnai'oj Pharmncnfogt‘ 3.7? H99 71 I95 -207’
`
`Fig.
`
`I. Chemical structure uftnlterntline.
`
`can be distinguished by pharmacological means. A fourth
`pharmacological subtype (M4) has also been defined, al-
`though muscarinie My-selective antagonists are not yet
`available. Roughly, muscarinie M,
`receptors prevail
`in
`neuronal
`tissues while the heart appears to contain a
`homogenous population of muscarinie M2 receptors and
`exocrine glands are considered to contain mainly mus-
`carinie M3 receptors. The muscarinie M,
`receptors are
`found in striatum, cortex and peripheral rabbit lung. How-
`ever,
`it
`is commonly found that more than one receptor
`subtype coexist within a tissue or cell (I-lulme et al., 1990;
`Caulfield, I993).
`Many smooth muscles contain both muscarinie M3
`(‘cardiac’) and muscarinie M, (‘glandular’) receptors (for
`review. see Eglen et al., I994). The presence of mRNA for
`muscarinie m2 and m3 receptors has been demonstrated in
`the urinary bladder of rat, pig (Macda et al.,
`I988) and
`humans (Yamaguchi ct al..
`I994). Radioligand binding
`data indicate the presence of muscarinie M2 and N13, as
`well as muscarinie M,, receptors in human bladder tissue
`and that muscarinie M3 receptors may predominate (Kondo
`et al., 1995; Wammack et aI., I995). On the other hand,
`irnmunoprecipitation data indicate that
`the proportion of
`muscarinic m2 anti m3 receptors is 3:1 in the bladders
`from humans, guinea pigs and rabbits. and 9:I
`in the rat
`bladder {Wang ct al.. 1995). Radioligand binding data on
`selective antagonists in the guinea-pig bladder (Nilvebrant
`and Sparf, 1986, I988) and other smooth muscles (Girtddo
`et al.. I987; Michel and Whiting, 1988: Eglcn et al., 1994)
`most often reflect the pharmacology of the muscarinic M:
`receptors. This probably rclleets a predominance of mus-
`carinie M3/m2 receptors in smooth muscles and the rather
`limited subtype selectivity of available antagonists. llow-
`ever.
`functional
`in vitro data on a number of selective
`antagonists indicate that
`the contraction of most smooth
`muscles.
`including the urinary bladder,
`is mediated by
`muscarinie M,
`receptors (Caulfield,
`I993; Eglen et al.,
`I994; Tobin and Sjijgreri,
`I995; Wang et al.,
`I995). The
`functional role of muscarinic M3 receptors is still mysteri-
`ous, but emerging evidence suggests that the importance of
`the muscarinic M,/m2 receptors in various smooth mus-
`cles might have been underestimated (Zhang and Buxton,
`I991; Griffin and Ehlert, I992; Caulficld, I993; Thomas ct
`al., I993; Eglcn ct aI., I994; Chen et al., (995; Roddy et
`al., 1995).
`
`to compare the
`The aim of the present study was
`antimuscarinie properties of tolterodine and oxybutynin.
`Oxybutynin has been characterised as
`a muscarinie
`M,/M3-selective antagonist (Nilvebrttnt and Sparf, 1982,
`l983b,
`I986; Lazareno et al.,
`I990; Noronha-Blob and
`Kachur,
`l99l). For purposes of comparison,
`the classical
`non-selective muscarinie receptor antagonist atropine was
`also included. The inhibitory effects oftolterodine, oxybLI—
`tynin and atropine on urinary bladder contraction and
`salivary secretion in vivo were studied in the anaesthetised
`cat. The in vitro studies comprised ratlioligand binding and
`functional studies on bladders from guinea pigs and hu-
`mans. Radioligand binding studies were also carried ottt on
`parotid gland, heart and cerebral cortex from guinea pigs,
`as well as on human muscarinie ml-—m5 receptors ex-
`pressed in Chinese hamster ovary (CHO) cells.
`
`2. Materials and methods
`
`2.1. In vitro studies
`
`i
`2.1.}. Guinea—pig ri.s‘srres
`Male guinea pigs (Dunkin Hartley. weighing 300-500
`g) were killed by a blow on the neck and exsanguinated.
`The cerebral cortex was dissected on ice and the other
`
`tissues in Krebs—l-lenseleit solution, as previously described
`(Nilvebraut and Sparf,
`I982.
`I9R3ti. 1986). Urinary blad-
`ders for
`functional
`in vitro studies were cut
`into strip
`preparations (3><3 X I2 mm). Tissues
`for
`radioligand
`binding studies were homogenised, as described below.
`
`2.} 2. Human urimzry bladder
`Detrusor specimens from the dome of the bladder were
`obtained front 23 patients (mean age 63 ;t 2 years; range
`4-4—7S years) undergoing surgery for bladder malignancy.
`Some of the patients had received preoperative radiation
`therapy (20 Gy). However, the muscarinie receptor density
`in the human bladder and the affinity for muscarinie
`receptor antagonists does not appear to be influenced by
`age, sex and radiotherapy (Nilvebrartt et al., 1985). All
`specimens were taken from a macroscopically normal pan
`of the detrusor. No mueosal
`tissue was
`included. For
`
`in vitro studies, dissected bladder specimens
`functional
`into strip preparations (1 X I X3 mm; 6-I2
`were cut
`strips were obtained from each patient). Tissue for radioli-
`gand binding studies was frozen at -70°C‘ and stored at
`this temperature until used in the assays.
`
`2.1.3. Cht'ne.se hamster ovary (CHO) cells
`CHO cell
`lines transfeeted with genes encoding the
`human muscarinic receptor subtypes ml. m2, m3 and m4,
`respectively, were obtained from Dr T. Bonner, National
`Institutes of Health. USA, while cells expressing the m5
`subtype were obtained from Dr M. Brann. University of
`Vermont, USA (Buckley et al., 1989). The cells were
`
`'l5'atent 0Tv'ner", UC'B'"Pharma GmbH — Exhibit 2032 - 0003
`
`

`
`I. Niles-‘Iran! 9: or’ /‘ European Jmrrrra! of Pharmacology" 327 {I997} I95-207
`
`I97
`
`cultured in a medium consisting of equal parts of Dul-
`becco’s modified Eagle’s medium (DMEM) and HATWS
`Fl2,
`respectively. The medium was supplemented with
`foetal bovine serum (l()%), I.-glutamine (4 mM) and peni-
`cillin/strcptomycin (I00 U/ml), at 37°C in a humidified
`atmosphere (7% CO2, 93% air) in tissue culture flasks or
`roller bottles. Cultures were continuously harvested every
`second or third day and stored at
`-7U°("'unti| usctl
`itl
`radioligand binding studies.
`
`2.1.4. Firnctirmal in citm .s'rudics
`
`Guinea-pig and human bladder strips were transferred
`to jacketed tissue baths (5 ml) and mounted between two
`hooks. One of the hooks was connected to a force trans-
`ducer (FT 03. Grass instruments). The baths contained a
`temperature-controlled (37°C) Krebs-Henseleit solution,
`which was continuously aerated with carbogen (93.5% or
`95% O2/6.5% or 5% CO3) to maintain pH at 7.4. Isomet-
`ric tension was recorded by a Polygraph (Grass. model 79
`D). A resting tension of 5 mN was initially applied to each
`preparation. During stabilisation (45-60 min) the strips
`were repeatedly washed and the resting tension was ad-
`justed. The Krebs-Henselcit solution used for guinea-pig
`bladder contained (mM): NaCl ([19), KCl (4.6), CaCl_,
`(L5), MgCl2 (1.2),
`l\"aHCOJ (20), NaH,FO, (L4) and
`glucose (I 1). The Krebs-llenseleit buffer used for human
`preparations had a slightly different composition (mM):
`NaCl (118). KCl(4.6), CaCl3 (1.5), MgSO4 (L2), Natl-{CO3
`(24.9). KH_.PO, (1.4) and glucose (l 1). When responses
`of human strips to K‘ were studied, the NaCl in the Krebs
`solution was replaced by KC] to give a K ' concentration
`of 124 mM.
`
`Guinea-pig preparations were exposed to a standard
`concentration of the muscarinic receptor agonist carbachol
`(3 p.M; ECXD)
`to establish reproducible responses (three
`subsequent contractions with s 10% variation in ampli-
`tude) bclbre a concentration t response curve to car-bachol
`(control) was generated using cumulative addition. After
`exposure of the bladder strips to a fixed concentration of
`antagonist for 60 min (Nilvebrant, 1986),
`the concentra-
`tion—rcsponsc curve to carbachol was repeated in the
`presence oi‘ antagonist. Responses were expressed as a
`percentage of the maximal contractile response elicited by
`earbachol in the control curve.
`The viability of human bladder preparations was con-
`trolled by exposure to K* (I24 mM) before cumulative
`concentration—rcsponsc curves to carbachol were estab-
`lished (control). Two consecutive control curves were
`generated before incubation with tolterodine. but all
`rc-
`sponscs were expressed as a percentage of the maximal
`contractile response in the control curve immediately pre-
`ceding incubation with toltcrodinc. The strips were ex-
`posed to tolterodine for 20 min before the concentration-
`response curve to carbachol was repeated.
`When human bladder strips were stimulated electrically,
`two electrodes were placed on each side of the preparation
`
`to it. Stimulation was delivered by a Grass
`and parallel
`S88 electrical stimulator as single square-wave pulses with
`a duration of 0.8 ms. The voltage was suprarnaximal and
`the stimulation was given in 5 s trains with an interval of2
`min. The frequency interval used was
`I-45 Hz. The
`frequency-response relations were established for each
`Strip, and a frequency producing 70—80% of maximum
`response was selected. The strips were stimulated at this
`frequency at 2-min intervals. and tolterodine was then
`added cumulatively.
`EC50 values for carbachol were determined in the ab-
`sence and presence of antagonist, respectively. The affinity,
`of the antagonist was calculated as the dissociation con-
`stant Kn (Schild, 1949). The mean: S.E.M. of the KB
`values determined using different antagonist concentra-
`tions in (ii) preparations was calculated. The ICSO value
`for tolterodine in electrically stimulated preparations was
`calculated by linear interpolation.
`
`2.1.5.
`studies
`
`il/frmbrane preparations for
`
`radirfligand binding
`
`All preparation steps were carried out on ice. lluman
`bladder specimens obtained from different patients were
`never pooled. Thawed human bladder samples, dissected
`guinea-pig bladders, parotid glands and hearts were ho-
`mogenised in ten volumes of ice-cold sodium-potassium
`phosphate buffer (50 mM; pH 7.4) containing 1 mM
`phenylmethylsulphonyl tluoride, using a Polytron PT-3000
`(guinea-pig urinary bladder and parotid gland: 26 000 rpm,
`2X 15 s; guinea-pig heart:
`i300 rpm, 2 X 15 s; human
`urinary bladder: 26000 rpm, 3 X 15 s; with a 30 s cooling
`interval between bursts). Guinea-pig cerebral cortex was
`homogenised using a Potter-Elvehjem Tcllon/glass ho-
`mogeniser. Before use in the assays, tissue homogenates
`were
`diluted with
`ice-cold phosphatc/phcnylmethyl-
`sulphonyl fluoride buffer to a final protein concentration of
`50.3 mg/ml.
`llomogcnatcs of CHO cells containing
`muscarinie ml—mS receptors were prepared in 1-2 ml of
`ice-cold sodium phosphate buffer (25 mM, pH 7.4) con-
`taining 5 mM MgCl1 (Buckley ct al., 1989) and I mM
`phenylmethylsulphonyl
`fluoride. using a glass—glass ho-
`mogeniscr. The cell homogenates were diluted with ice-
`eold phosphate/phenylmethylsulphonyl fluoride buffer to
`l0O(l—|000O times the original wet weight (protein con-
`centrations of 4-190 pg/ml) before use in the assays.
`Protein concentration in the final tissue homogenates and
`in cell homogenates containing > 25 pg/ml were deter-
`mined by the method of‘ Lowry et al. (1951). The BioRad
`Protein Assay (Bradford, 1976) was used for cell ho-
`mogenates with protein concentrations below 25 pg/ml.
`Bovine serum albumin was used as standard for both
`methods.
`
`2.I.6. Radiofigarid binding as'.s'u)'.s
`The affinities of the unlabelled drugs were determined
`by competition experiments, using [3l-l](—)-l—quir1uC-
`
`ZPateTnt Otmei: UCB Pharma GmbH — Exhibit 2032 - 0004
`
`

`
`|9R
`
`I. Niirehrarrr ct tzl / Ertrapprm Journal rifPharmr7rnlngy 327H99 7) !9.5—2t?."
`
`lidinyl benzilate as tracer. Non-specific binding was deter-
`mined in the presence of IO u.M atropine. Total and
`non—s.pecific binding of [Jl-l](—)-l-quinuelidinyl benzilate
`was detemtined in six samples each, while samples con-
`taining unlabelled antagonist were run in triplicate.
`Tissue homogenates (1.0 ml) were incubated with
`[3H](—)—1—qu‘muclidinyl benzilate (2 nM) and different
`concentrations of unlabelled antagonist under conditions of
`equilibrium: guinea-pig urinary bladder, 25°C for 60 min
`(Nilvebrant and Sparf, 19833.); parotid gland, 25"C for 2l0
`min (Nilvebrant and Sparf. 1982); heart and cerebral cor-
`tex, 25°C for 80 min (Nilvcbrant and Span". 1985); and
`human bladder, 25°C for I80 min (Nilvcbrant ct al., 1985).
`lncubations were tenninated by centrifugation (Nilvebrant
`and Sparf, 1983a). The pellets were washed and the tubes
`allowed to dry before the tips containing the pellets were
`cut off and transferred to scintillation vials. Soluene-I00
`(Packard) was added (200 ttl/tube) and the samples were
`left overnight for solubilisation ofthe pellet. A scintillation
`cocktail (lnstafluor or Ultimn Gold. Packard) was added
`and radioactivity detemtined by liquid scintillation spec-
`trometry (Packard TriCarb model 2200 CA or 2500 TR). A
`quench correction curve was used for the conversion of
`cpm to dpm.
`lines containing human
`Binding studies on CHO cell
`muscarinic ml—m5 receptors were run in 24-well cultures
`plates. Cell homogenates (l.0 ml) were incubated with
`["ll](~)~l-quinuclidinyl bertzilate (1 nM) and different
`concentrations of unlabelled drug under conditions of equi-
`librium (37°C for 300 min). The optimal protein concentra-
`tions and the time required to reach equilibrium for each
`cell
`line was detcnnined in a separate series of experi-
`ments (data not shown).
`lncubations were tenninated by
`filtration through a cell harvester (Packard) onto GF/C
`filters (Packard). The filter plates were dried, 200 pl of
`Microscint (Packard) added and the radioactivity was de-
`termined in a Packard Top Count scintillation counter
`(et'l'tcicncy of 44-46%).
`Data are presented as mean: S.E.M. of (n) experi-
`ments.
`In the competition binding experiments,
`[C50 val-
`ues for the antagonists were determined from the experi-
`mental concentration—inhibition curves. Dissociation cott-
`
`stants (K) were calculated by correcting the ICE) values
`for the radioligand-induced parallel shift and the differ-
`ences in receptor concentration, using the method de-
`scribed by Jacobs et al. (I975); see also Nilvebrant and
`Spat‘f(l9S2, 1983b) for details.
`ln order to verify that the density of muscarinic recep-
`tors
`in the human bladder and the affinity (Kd)
`for
`[3Hl(-)-l-quinuclidinyl benzilate were within the ranges
`previously reported (Nilvebrant cl al.,
`I985), saturation
`experiments were performed in specimens from six indi-
`viduals. Samples containing different concentrations of
`[3H]( — )-l-quinuclidinyl benzilate (0.05—5 nM) were incu-
`bated under Conditions of equilibrium (25°C for [80 min),
`using atropine for determination of non-specific binding.
`
`Receptor density and Kd were determined by Scatchard
`analysis of the saturation data. These parameters (receptor
`density 122:9 fmol/mg protein (range 93-146); Kd
`0.12 i 0.02 nM) were within the range of those previously
`detemiined in a series of I4 patients (receptor density
`1641* 15 fmol/mg protein (range 77-231); Kd 0.14-_t-_
`0.02 n:Vl) (Nilvcbrant et al., I985).
`
`22. In cico studies
`
`2.2. I. /lm'mal'.r and anaesthesia
`
`Twenty-five female European short-haired cats (2.2—3.4
`kg; 6-10 months old) were used. The animals were de-
`privcd of food overnight before the experiment. They were
`initially anaesthetised with an intrapcritoncal
`injection of
`pentobarbital sodium (30-39 mg/kg), followed by intra-
`venous (i.v.) administration of or-ehloralusc (25 mg/ml in
`‘Va sodium tetraborate; 25 mg/kg).
`The animal was placed on an electrically heated pad
`and the hotly temperature was controlled by a feedback
`loop from a thermometer in the anus. Additional doses of
`or-chloralosc were given (l76—3l2 ul/kg per h) when
`required to maintain anaesthesia.
`In order to maintain
`homeostasis, a Ringer solution was administered (2.2-7.8
`tnl/kg per h).
`
`'
`22.2 Surgery
`The animal was tracheotomised and a glass respiratory
`cannula was inserted and connected to a volumetric low-
`
`pressure transducer (Grass PTSA) for recording of respira-
`tory volumc. Both femoral arteries were cathctcriscd with
`polyethylene tubing (PE90). One of these was Connected to
`a blood pressure transducer (Statham P23DC). The other
`was used for intra-arterial (t.a.) administration of acetyl-
`cholinc and collection of blood samples for determination
`of haematocrit and blood gases. The right femoral vein
`was calheterised (PE90) and cotmcetcd to a 3-way stop-
`cock for i.v. infusion of test compounds and Ringer solu-
`tion.
`
`The hypogastric and pelvic nerves were identified and
`transected. The ureters were cut. and cathctcrised (PE10).
`A catheter (PE240) inserted into the urinary bladder through
`an incision in the proximal urethra. At the beginning of the
`experiment, this catheter was connected to an open vessel
`filled with temperature-controlled (37°C) saline that was
`placed approximately 15 cm above the animal. During
`stabilisation (about I h), the bladder relaxed and gradually
`became filled with saline under constant hydrostatic pres-
`sure. The bladder catheter was later connected to a pres-
`sure transducer (Statham p23A) for recording of intravcsi-
`cal pressure. The duct of the submandibular gland was
`exposed in the neck and catheteriscd (PEll)). The parasym-
`pathetic ehorda-lingual nerve was exposed and cut as
`proximally as possible. The peripheral nerve stump was
`placed on a bipolar electrode. lntravesical pressure, blood
`pressure, heart rate (derived from the blood pressure sig-
`
`'Patent"0wner, UCB Pharma GmbH — Exhibit 2032 - 0005
`
`

`
`L.
`
`."l«'i'It'ehran! at at’ /' European Jourrio! afPFmrrnocoi'ogy 327 (I99?) I95—.?fl7
`
`I99
`
`ride, plttznylmelhylsiilphonyl fluoride, oxybutynin hydro-
`chloride and atropine sulphate (Sigma, USA}; []Hl( —)-1-
`quinuclidinyl benzilate. 32-52 Ci/mmol (equivalent
`to
`l.l8--1.94 TBq/Inrnol} (fiiniersham, UK or Du Pont NEN,
`USA); pentobarbital sodium (Apotel-tsbolaget, Sweden);
`oi-chloralosc (Merck. Germany); sodium boratc (Mallinl<-
`rodt, USA); DMEM and I-lAM's I-"I2 (National Veterinary
`Institute, Sweden); foetal bovine serum albumin (HyClorie
`Lab, USA); L-gltitamine and penicillin/streptomycin (ICN
`Biomedicals. USA). Other chemicals used (analytical
`grade) were purchased from general commercial sources.
`The radioligand [JH]( - )-l-quinuelidinyl benzilate was
`diluted in absolute ethanol. Stock solutions of tolterodine.
`oxybutyniri, atropine and carbzichol for functional in vitro
`studies were prepared and diluted in redistilled water.
`Stock solutions of tolterodine, oxybutynin. atropine and
`acctylcholine for in vivo studies were prepared and diluted
`in saline. Fresh solutions were prepared for each experi-
`merit.
`
`3. Results
`
`3. I. In uilro .rt‘udie.s
`
`3.1.}. Funcrionai .S!udt€.t‘ on isolated urinmfv bladder
`Tolterodine effectively inhibited earbaehol-induced con-
`tractions of guinea-pig isolated urinary bladder strips in a
`concentration-dependent manner (Fig. 2). The Schild plot
`(not shown) was linear (slope 0.97; pA2 8.6), indicating a
`simple competitive blockade oi‘
`the bladder muscarinic
`receptors. Similar data were obtained for oxybutynin
`{Schild slope 0.89; pA2 8.5). Tolterodine was thus equipo-
`tent to oxybutynin, but less potent than atropine (Table l).
`
`100
`
`8°
`
`60
`
`40
`
`20
`
`0
`
`B
`
`7
`
`4
`5
`5
`—l_og lcarbacnoll (M)
`
`3
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`(1;
`
`rial) and respiration rate were recorded on a Polygraph
`(Grass mode! 7D). Blood pressure, heart rate, respiration,
`blood gases and haernatocrit were measured In order to
`monitor the general condition of the animal during the
`course of the experiment.
`
`2.2.3 Expe'r'i'nierm7I' procedure
`receiving
`The
`cats were divided into four groups
`tolterodine (n = 5), oxybutynin (ii = 5). atropine (rt 7 5),
`or saline (H = 10; control group), respectively. Tolterodine
`(21-2103 nmol/kg (0.0l—l mg/kgl), oxybutynin (25-
`2538 nmol/kg (0.0l—l mg/kg)),
`atropine
`(0.864-lo
`nmol/kg (0.0(}03—0.03 mg/kg)) or saline were adminis-
`tered by i.v.
`infusion at a rate of l ml/kg per min. The
`animals were left to recover For at
`least 24 min between
`
`doses. Urinary bladder contractions were evoked by La.
`injection of a standard (submaximal) dose ol‘ acetylcholine
`(l-4 ug/kg), which produced a short-lasting (< I niin),
`large (> 9 cmll3O) and reproducible (< l5% variation}
`increase in intravesical pressure. This standard dose was
`selected from a dose-response curve to acetylcholine (0.5 -8
`pg/kg) generated at
`the beginning of each experiment.
`Aeetyleholine was administered before and approximately
`9 and H3 min after each dose ofantagonist. Salivation was
`induced by suprarnaitinial electrical stimulation (6 V, 2 ms.
`5 Hz) ofthe churda-lingual nerve. The frequency-response
`relationship was established at the beginning of each ex-
`periment using frequencies of 0.5,
`l, 2 and 5 H7. over 2
`min. The frequency used in the experiments (5 Hz) pro-
`duced a mean salivary secretion of 372 1 24 ul during 2
`min. Electrical stimulation of the ehorcla-liiiguzil nerve was
`perfonned before and approximately 7 min after each dose
`of antagonist. Arterial blood samples for determination of
`haematocrit and blood gases were drawn before the admin-
`istration of each dose of test compound.
`
`2.3. Cmculattoos and .YIt1'l.i.tflL’.§‘
`
`Effects of drugs on urinary bladder contractions and
`salivary secretion are expressed as percentage inhibition of
`the control responses. Data are presented as mean i S.E.M.
`of (ii) animals. Differences between the inhibitory effect
`exerted by the antagonist on urinary bladder contractions
`and salivary secretion were analysed for each dose level
`using a paired Student's :-test. Differences between the
`doses of antagonist causing a 50% inhibition of" these
`responses (lC_.,,) were also analysed. P values < 0.05
`were considered significant.
`
`2.4. Dmgs and L‘I1emiccii'.t'
`
`The following drugs and chemicals were used: to1tero-
`dine tartrate {tR)-N,N—diisopropyl-3-(2-hydroxy-5-rnetbyl-
`pheny1)-3-phenylpropanaminc hydrogen L-(+)-tartrate].
`saline and Ringer solution (Pharmaeia&Upjohn, Sweden);
`czirbamyleholine chloride (carbachol), acetylcholine chlo-
`
`2. Effect of Iolterodinc on the concentration-response curve to
`Fig.
`cart-mchol
`in guinea-pig isolated iiniiary bladder. The contractile rc-
`spnnses elicited by increasing concentrations of carbachol (cumulatn-e
`udditinns) were recorded in the absence (control) and presence, respec-
`tively, oftolterodine at concentrations of 10 nM (Y ). 30 nM ( 6) and 500
`nM (A). The time of incubation with toltcrodine was 60 min. Control
`data (0) represent the mean (iS.l’..M.) of I-1 individual experiments
`The I:IC5,, value for crirbachol was 2 4 :0 3 uM.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2032 - 0006
`
`

`
`I
`Table:
`Aflinilics, expressed as the dissociation constants (R5,). for inuscurinic receptors in guzncmpig cerebral uorlcx. heart unil parnlid gland
`(.‘ompound
`Unnary bladder
`Cerebral cortex
`Hcari
`
`Tnl1cmdii1e
`Oxyburynin
`Almpini:
`
`K" EnM]
`3.0 i (1.2 (15)
`4.4-:0.6(l-4}
`0.7 i0.1 (10)
`
`Ki (nM)
`2.7-:02 ((-5
`4.0104(6)
`10:13.] (7)
`
`n"
`I .02 $0.03 (6)
`0.99iU.01{6}
`1.00 10.02 (7)
`
`KI (nM)
`0,75 1 ().(!l (S)
`0.4-E 1005(5)
`0.32:0.02 (4)
`
`:1”
`J 05 ;|;0.U3 {5}
`09'-'-:00!
`{5}
`1,0o:0,oo3 (4)
`
`K‘ (nMJ
`l.6 i 0.04 (5)
`2.3tU.3 (5)
`0.891-0.06 (3)
`
`H"
`1.04 _-1 0.06 (5)
`I 05 1:01)} {5}
`1.05 10.00 (3)
`
`l’ar(md gland
`
`K; (nM)
`(5)
`4.8 -_I_ 0.3
`0.62:0.05(6)
`0.35 30.05 (9)
`
`:1”
`L04 ; 0.03 (5)
`l.U| :0.03 (6)
`0.95 10.01 (9)
`
`Daia on urinary bladder muscarinic recepmrs were derived from functional in vitm studies (K,,) on carhachol-induced contractions of bladder strips and from competition binding studies (K_) in bladder
`homogenates. rcspcciivcly. K. values were derived from competition binding experiments with [‘H]( - l-I-quinuclidinyl henzilaic, as described in Section 2.