JournalofReceptorsandSignalTransductionDownloadedfrominformahealthca.re.c0mbyKerriSpenniechiaonO3f[l3r'l5
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`J. OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH.
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`l7(l—3). 177-184 (1997)
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`CHARACTERISATION OF [311]-DARIFENACIN AS A NOVEL RADIOLIGAND
`FOR THE STUDY OF MUSCARINIC M3 RECEPTORS
`
`Carolyn M. Smith * and Rob M. Wallis
`Discovery Biology. Pfizer Central Research,
`Sandwich. Kent CTI3 9N1. England.
`
`ABSTRACT
`
`Dar-ifenaein, (S)-2-[1-[2,3-dihydrobenzofuran-5-yl]-3-pyrrolidinylI-2,2-diphenylacetamide, is a
`novel muscarinic M3 antagonist.
`In this study we have compared the binding of {3I-1]-
`clarifenacin to the five cloned human muscarinic receptors (ml
`- mg] expressed in CHO cells.
`[SH]-darifenacin binds with 6 fold higher ztffnity to m; (Kn = 0.33 nmol/1) over mi (KB = 1.6
`nmol/l) receptors. There was no specific binding of [3]-I]-darifenacin to H12 receptors and
`specific binding to m4 and T115 receptors was insufficient to detennine 21 K0. Binding of [3H]-
`daiifenacin to m; and rm was displaced by atropine ("mg pK, = 9.36, T113 pK; = 9.4), 4—DAMP
`(ml pK; = 9.04. 11']; pK.= 9.19), pirenzepine (m, pK. = 8.63, I113 pK. = 6.85), methoctramine (ml
`pK; =7.28, rm pK. = 6.63), and darifenacin (ml NC. = 8.36, l’Tl_1 mi. :9. 14). demonstrating that
`[JH]-darifenaein represents the first selective m3 radioligand.
`
`INTRODU
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`ON
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`Five subtypes of human muscarinic receptor, designated as ml, 5113. mg, no and H15. have
`
`been identified and cloned (I). The pharmacologically defined M1. M2. M3 and N14 receptors
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`correspond to the cloned mi,
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`[Tl].
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`[U3 and im receptors, however,
`
`the pharrnacological M5
`
`receptor corresponding to the ms gene product has yet to be reported. Although a few
`
`rnuscarinic antagonists have proved useful
`
`in characterising muscarinic receptor subtypes,
`
`definitive pharmacological characterisation has been hampered by the lack of subtype-specific
`
`compounds. Most commonly used standard antagonists include pirenzepine (M; selective},
`
`AF~DX-1 16(1 l[[2-Idimethylan1ino)rnethyl]-1-piperidinyl]acetyl]—5.1 1-dihydro—6H-pyrido[2,3—
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`6][1,4] benzodiazepine-6-)onc) and methoctramine (M2 selective). 4-diphenylncetoxy-N
`
`C|'[‘)'llg1lll
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`.\l:II‘t‘e| [)L‘l\kL'l'.
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`lu-..
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`177
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`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0001
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`Illul-ITE Llnr H.-I}
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`178
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`SMITH AND WALLIS
`
`methylpiperadine methobromide (4—DAMP), hexahydrosiladitenidol (HHSiD) and p-fluor0-
`
`HI-lSiD (MIIM3 selective). Darifenacin is a novel muscai-inic antagonist shown to be potent
`
`and selective for the M3 receptor both in isolated tissue studies (2) and in radioligand binding
`
`studies to the cloned human muscarinic subtypes (3). In this study we have compared the
`
`binding of [31-I]-darifenacin to the five cloned human muscarirlic receptors expressed in CHO
`cells.
`
`MATERIALS
`
`[3H]-Darifenacin (Specific Activity 22Ci/mrnoi) was prepared by tritiation of the benzofuran
`
`ring to give 2,3-ditritiobenzofuran by Amersham Intemat onal
`
`(Buckinghamshire. UK).
`
`Atropine, 4-DAMP, methoctrarnine and pircnzepine, were purchased from RBI (Natick, MA).
`
`Darifenacin was synthesised in our laboratories at Pfizer Central Research.
`
`METH DS
`
`Membrane Preggggfign.
`
`The CHO-Kl cells stably expressing the human in, — mg receptors have been described
`
`previously (4) and were obtained from Dr. Tom Bonner (National Institute of Neurological
`
`Disorders and Stroke, Bethesda, MD). Transfected cells were grown to 80% confluency and
`
`washed twice in phosphate buffered saline (PBS) prior to harvc sting by scraping. Scraped cells
`
`were resuspendcd in PBS, pelleted at 1000 xg for 10 min. and stored at —80°C until use.
`
`Thawed membranes were homogenised in ice cold HEPES bufier (20 mM pH 7.4) and washed
`
`twice by centrifugation at 38,000 x g for 20 min.
`
`The protein concentration of the
`
`resuspended membranes was determined using a Sigma protein assay kit. Membranes were
`
`stored in aliquots at -80°C before use.
`
`Radioli
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`n Bintlin
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`u i 5.
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`All membranes, drugs and radioligand solutions were made up in 20 mM HEPES buffer
`
`(pH 7.4 at 25°C). Final protein concentrations were 120 ttg/ml (ml), 60 ]J.g/ml (ITI3) and 400
`
`pg/ml (I112, rm, m5). Assays were perfon-nod in a total volume of 500 _u.l. Saturation analysis
`
`for [3H}—darifenacin binding to IT|.] receptors was performed over the concentration range 0.1 -
`
`10 nmolfl and for m1, m2_ m4 and ms over the range 0.1 - 25 nmol/1. For competition
`
`experiments, pKt s were determined by displacement of [’H]—da'ifenacin (0.4 nmolll for m3 and
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`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0002
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`lllul-ITE LINK-I}
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`JournalofReceptorsandSignalTransductionDownloadedfrominformahealthcareeombyKerriSpennicchiaonl]3.’03fl5
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`[3H]—D/\RlFENACIN
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`179
`
`l nmol/l for mt) using 12 concentrations of antagonist. Time course experiments with the ITI3
`
`receptor were performed using 0.4nmol/l
`
`[3H]—dai-ifenacin at various incubation times. Non
`
`specific binding (NSB) was defined using ll.LlVl atropine.
`
`Incuhations were initiated by the
`
`addition of [3H]—darifenacin and canned out at 25°C for 2 hr. Binding was terminated by rapid
`
`filtration through a Brandell cell harvester onto Whatman GFIB filters followed by 3 washings
`
`with ice-cold HEPES buffer. Each filter was dried and trapped radioactivity was measured by
`
`liquid scintillation counting using Meltilex solid scintillant and a Wallac 1204 Beta counter.
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`Data Analysis.
`
`Results are expressed as mean values 2 the standard deviation of
`
`rt experiments. The
`
`maximal number of binding sites {B.,,..,.) and the equilibrium dissociation constant (Kn) were
`
`determined from direct analysis of the saturation binding data using Grafit. Hill coefficients
`
`and [C50 values were obtained from competition experiments by the method of Hill (5) using an
`
`in—house data fitting programme. K; values were derived from 1C5.) values using the Cheng-
`
`Prussoff IC5ocorrection (6).
`
`RESULT§
`
`Kinetics of [JH]-Darifenacin Binding.
`
`Specific binding of [31-I]-darifenacin to m, and my receptors (which was >80% of total
`
`binding) was timc—dependent and reached equilibrium at 25°C after 2 h. A representative
`
`association time-curve for [3I~i]~d-arifenacin binding to the m3 receptor is shown in Figure 1.
`
`Representative saturation curves for specific binding of [AH]-darifenaein to m] and H13
`
`receptors are shown in Figure 2. The linear representation of the data following Scatchard
`
`transformation indicates the presence of a single homogeneous population of sites in each cell
`
`line. The equilibrium binding parameters Kr, and BM, for m. and mg are shown in Table 1.
`
`It was not possible to determine K1; and Em, for [SH]-darifenacin binding to the rug receptor
`
`as there was no specific binding of [3H]-darifenacin even at concentrations as high as 25
`
`nmolll. Similarly, binding of [’H]-darifenacin to m.. and ms receptors was not sufficient to
`
`allow accurate detennination of K” and Bum When the same membrane preparations of H11,
`
`rm and ms used to investigate [5H]—darifenacin binding were incubated with the non-selective
`
`antagonist
`
`[’H]- N-methyl scopol-amine ([311]-NMS)
`
`(0.lnmol!l)
`
`. high levels of specific
`
`binding ( > 80% of total binding) were observed (data not shown) indicating that muscarinic
`
`receptors were present in these preparations.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0003
`
`Illul-ITE LINH1}
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`

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`ISO
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`SMITH AND WALLIS
`
`
`
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`Time (min)
`
`FIG. 1 Time course of association of [ll-I]-darifenacin binding to the cloned human [H3 receptor.
`Membranes expressing ml receptor (60 pg protein I ml) were incubated at 25 “C with 0.4 nmol/l
`[31-l']—darifenacin for the times indicated. Each data point is the nean of 6 determinations from 1
`representative experiment. NSB = non-specific binding
`
`|’H I-Darifgnagh fljsnlagemgm Exnggimgnts.
`
`Table 2 shows the selectivity of standard muscarinic antagonists at displacing [3H]-
`
`darifenaein from ml and m receptors. None of the Hill coefftc ents were significantly different
`
`from unity (range 0.8 - 1.1), consistent with the presence of a single population of receptors in
`
`each cell line. Representative binding curves to show displacement of [3H]-darifenacin from
`
`the mgl receptor are shown in Figure 3.
`
`Darifenaein was 6-fold selective for I113 over ml. Atropine and 4-DAMP had similar
`
`affinities towards ml and ma. Pirenzepine was 60-fold more seletive for ml over mg and
`
`methoctramine was 4-fold more selective for ml over 1113.
`
`DISCUSSION
`
`The number of ml and mg binding sites labelled by [‘H]—-Jarifenaein was consistent with
`
`those we have obtained previously using the nomselective muscarinic antagonists PH}-
`
`quinuclidinyl benzilate (PH:-QNB) (3) and [3H]-NMS (data not shown). Similarly, the binding
`
`afiinity and receptor seiectivities of standard musearinie antagonists (atropine, 4-DAMP,
`
`methoetramine and pirenzcpine) at ml and m;, receptors were similar to that reported for [3H]-
`
`NMS (4,7). However, the potencies of compounds in this study were consistently 0.5 —
`
`1 log
`
`unit greater than we have previous reported for the same compounds against [3I-I]-QNB. Our
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0004
`
`lllul-ITE LINK-I}
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`JournalofReceptorsandSignalTransductionDownloadedfrominformahealthcarecombyKerriSpennicchiaonl]3.’03fl5
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`FIG. 2 Saturation isotherms of specific [JHJ-danfenacin binding to the cloned human m. and m
`receptors. Membranes expressing rn. (120 pg protein! ml) (upperfigure) and ITI3 receptor (60 ttg
`protein 1' ml)
`(lower figure) were incubated at 25 °C for 2 h over the range of [ H]-darifenacin
`concentrations indicated. Data shown are taken from representative experiments carried out in
`duplicate. Scatchard analysis of the data is shown in the insets. Values for K9 and Bmx are given
`in Table I.
`
`3
`Equilibrium Binding Parameters (Kr, and Bmx.) for [‘H]—Darifenacin Binding to the Cloned
`Human Muscarinie Receptor Subtypes
`
`TABLE 1.
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`
`The estimates of K.) and BW were obtained from saturation experiments as shown in Fig. 2.
`Data are expressed as the mean : SD. from 46 experiments.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0005
`
`lllul-ITE LINN.-I}
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`€
`

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`182
`
`SMITH AND WALLIS
`
`TABLE 2.
`
`(‘nmparison til‘ the pK, values for Muscarinic Antagonists Displueing ["1H|—Duril'emu:in from the
`Cloned Human mi and mi Receptor.
`
`Atropine
`
`pl-it 13H]-Darifenaein
`H1}
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`9.142012
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`6.85 : 0.08
`
`Dztrifenaein
`4vDAMP
`Methoetramine
`Pirenze inc
`
`pK, values were derived from ['iH]-darifenaein displacement experiments and represent the
`mean 1- SD,
`[
`r1 = 4-6) for the negative logarithm of the K.. K, represents the inhibition
`constant obtained using the Cheng—Prussoi'f equation and the K” values given in Table 1.
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`FIG. 3 Displacement of specific ["H]—darifenacin binding to the human cloned mi receptor.
`Membranes expressing ITI3 receptor (60 ttg protein I ml) were incubated at 25 °C with 0.4 nmoUl
`11H]~darifenacin for 2 h. Data shown are from representative experiments where each data point
`is the mean of three values. Variability nfdata between experiments is indicated in Table 2.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0006
`
`Illul-ITE t_Ir'..tr-'.-I}
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`JournalofReceptorsandSignalTransductionDownloadedfrominformahealtheareeombyKerriSpennieehiaonU3.’03fl5
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`personaluseonly
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`JournalofReceptorsandSignalTransductionDownloadedfrominforrnahealthearecombyKerriSpennicchiaon03/03/15For
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`[JHLDARIFENACIN
`
`183
`
`earlier study was conducted using TRIS buffer (50mM) compared to HEPES buffer (20mM) in
`
`the present study. We believe that the higher potency of standard antagonists and darifcnacin
`
`reported in this study is consistent with the use of a buffer of lower ionic strength. Indeed,
`
`previous studies have shown that lowering ionic strength increases the binding affinity of
`
`muscarinic antagonists without altering the binding capacity (8,9). Our unpublished data
`
`indicates that the affinity of PH]-NMS for m3 receptors is greater in HEPES buffer than TRIS
`
`buffer. Clearly, the fact that the binding of muscarinic antagonists is sensitive to incubation
`
`conditions means that careful consideration needs to be made when designing binding assays.
`
`In this study we have characterised the binding of 13H]-darifenacin to the human cloned
`
`muscarinic receptor subtypes (rm - H15) and [31-1]-darifenacin has been shown to bind with high
`
`affinity to m; and m; receptors with 6-fold higher affinity for m over ml. Furthermore, there
`
`was no specific binding of [al-I]-darifenacin to H12 and only low levels of specific binding to rm
`
`and ms receptors even though perfonned in parallel with the non-selective antagonist [SH]-
`
`NMS confirmed the presence of muscarinic receptors in the membrane homogenates.
`
`In
`
`conclusion, these data indicate that [3l~l]-darifenacin has unique selectivity profile and therefore
`
`represents the first my — selective radioligand which should prove to be a useful tool in the
`
`study of muscarinic receptors.
`
`A KNOWLED EMENTS
`
`We would like to acknowledge the work of Dr AR MacKenz.ie and Mr SM Demon who
`designed the chemical synthesis for the preparation of [3HJ-darifenacin
`
`REFEREN ES
`
`Bonner, T.I.; Buckley, N.J.; Young, A.; and Brann, M.R.
`1.
`muscarinic receptor genes. Science 237, 527-532, 1987.
`
`Identification of a family of
`
`2. Wallis. R.M.; Burges, R.A.; Cross, P.E.; and MacKen7.ie A.R. Darifenacin, a selective M3
`antagonist. J. Phan-nacol. Res. 315, S4, 1995.
`
`Nunn, P.A.; Greengrass, P.M.; Ncwgreen, D.T.; Naylor, A.M.; and Wallis, R.M. The
`3.
`binding profile of the novel muscarinic receptor antagonist darifenacin. against the live cloned
`human musearinic receptors expressed in CHO Cells. Brit. J. Pharmacol Proceedings of
`Brighton Meeting, 1996.
`
`Buckley, N.J.; Bonner, T.; Buckley, C.M.; and Brann, M.R. Antagonist binding
`4.
`properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol.
`35, 469-476, 1989.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0007
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`Illul-ITE I_In.tt-'.-I}
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`personaluseonly.
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`184
`
`SMITH AND WALLIS
`
`Hill, A.V. The possible effects of the aggregation of the molecules of haemoglobin on the
`5.
`dissociation curve. J. Physiol. 40, iv-vii, 1910.
`
`Cheng, Y,C.; and Prusoff, W.H.; Relationship between the inhibition constant (K) and
`6.
`the concentration of inhibitor which causes 50 per cent inhibition (IC5o) of an enzymatic
`reaction. Biochem. Pharmacol. 22. 3099-3108. 1973.
`
`(3.; Tacke, R.; Mutschler, E.; and Brann, M.R.
`Dorje, Fr, Wess, J.; Lambrecht,
`7.
`Antagonist binding profiles of five cloned human musearinic receptor subtypes.
`J. Pharmacol.
`Exp. Ther. 256, 727-733, 1991.
`
`Birdsall. N.J.; Burgcn. EC.; Hulrnc, E.C.‘. and Wells. The effects of ions on the binding of
`8.
`agonists and antagonists to muscarinic receptors Br. J. Pharmacol. 67, 371-377, 1979.
`
`Hulmc, E.C.; Birdsall, N.J.M.'. and Buckley, NJ. Musttarinic receptor subtypes. Annu.
`9.
`Rev. Pharmacol. Toxicol. 30, 633-73, 1990.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2041 - 0008
`
`RIGHTS LINH-I}