Muscarinic Antagonists：Preclinical Evaluation, BI的,把几个在研药物比较了一番√

Preclinical Evaluation of Long-Acting Muscarinic Antagonists:Comparison of Tiotropium and Investigational DrugsPaola Casarosa,Thierry Bouyssou,Sabine Germeyer,Andreas Schnapp,Florian Gantner,and Michael PieperDepartment of Pulmonary Diseases Research,Boehringer Ingelheim Pharma GmbH &Co.KG,Biberach an der Riss,GermanyReceived February 16,2009;accepted May 26,2009ABSTRACTChronic obstructive pulmonary disease (COPD)is character-ized by progressive airflow limitation caused by persistent in-flammatory processes in the airways.An increased cholinergic tone mediates different pathophysiological features of COPD,such as bronchoconstriction and mucus hypersecretion,mostly through activation of the human muscarinic M 3receptor (hM 3)subtype.Tiotropium bromide (Spiriva)is a well estab-lished muscarinic antagonist in the pharmacological manage-ment of COPD with a once-daily posology.The rationale behind the sustained bronchodilation obtained with tiotropium con-sists in its slow dissociation from hM 3receptors.In this study,we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs)currently in clinical development,namely aclidinium bromide and glycopyrrolate.The different muscarinic antago-nists were characterized for their 1)affinity toward the different human muscarinic receptor subtypes expressed in Chinesehamster ovary cells and kinetics of receptor dissociation,2)potency in inhibiting the agonist-induced activation of musca-rinic receptors through measurement of second messengers,and 3)efficacy and duration of bronchoprotection,as tested in a model of acetylcholine-induced bronchoconstriction in anes-thetized dogs over a period of 24h.All of the tested LAMAs showed high affinity and potency toward the hM 3receptor (tiotropium,p A 2ϭ10.4;aclidinium,p A 2ϭ9.6;and glycopyr-rolate,p A 2ϭ9.7).However,dissociation half-lives of the LA-MAs from the hM 3receptor differed significantly (tiotropium,t 1⁄2ϭ27h;aclidinium,t 1⁄2ϭ10.7h;and glycopyrrolate,t 1⁄2ϭ6.1h).In line with their kinetic properties at the hM 3,the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24h after administration (tiotropium ϭ35%,acli-dinium ϭ21%,and glycopyrrolate ϭ0%at 24h)when applied at equieffective doses.The term COPD describes a clinical condition character-ized by chronic airway obstruction caused by an abnormal inflammatory response of the respiratory system.The main causal factor in the pathogenesis of COPD is sustained inha-lation of cigarette smoke (Rabe et al.,2007).Occasionally,other factors are involved,including a genetically induced deficiency of the enzyme ␣1-antitrypsin or exposure to nox-ious agents at work and in the environment.COPD is char-acterized by a predominantly neutrophilic inflammation of the airway walls,and airflow limitation is mainly a result of hyperplasia of mucosal glands,hypertrophy,and,in partic-ular,constriction of the bronchial smooth muscle in the small airways (Barnes,2000).COPD is associated with a signifi-cant level of morbidity and mortality (Rabe et al.,2007).The Global Burden of Disease Study has projected that COPD,which ranked sixth as the cause of death in 1990,will become the third leading cause of death worldwide by 2020(Murray and Lopez,1997).National and international guidelines recommend that pa-tients with COPD should receive bronchodilators as first-line maintenance treatment,because they increase expiratory flow by decreasing airway smooth muscle tone,thus leading to reduced lung hyperinflation (Rabe et al.,2007).The most frequently prescribed bronchodilators for COPD treatment are muscarinic antagonists and ␤2-agonists.In COPD,bron-choconstriction and mucus secretion are increased,and the airways become hyper-responsive to contractile agents.These changes are caused mostly by increased parasympa-thetic nerve activity (Barnes,2004).Acetylcholine (ACh),released from parasympathetic nerve endings,activatesArticle,publication date,and citation information can be found at .doi:10.1124/jpet.109.152470.ABBREVIATIONS:COPD,chronic obstructive pulmonary disease;[3H]NMS,[N -methyl -3H]scopolamine methyl chloride;hM,human muscarinic receptor;ACh,acetylcholine;LAMA,long-acting muscarinic antagonist;CHO,Chinese hamster ovary;PTX,pertussis toxin;ipratropium bromide,(1R ,3r ,5S ,8r )-8-isopropyl-3-[(Ϯ)-tropoyloxy]tropanium bromide monohydrate;tiotropium bromide,6␤,7␤-epoxy-3␤-hydroxy-8-methyl-1␣H ,5␣H -tropanium bromide di-2-thienylglycolate;LAS34273,aclidinium bromide,(3R )-3-{[hydroxydi(thiophen-2-yl)acetyl]oxy}-1-(3-phenoxypropyl)-1␭5-azabicyclo[2.2.2]octan-1-ylium bromide;NVA-237,glycopyrrolate bromide,3-(␣-cyclopentylmandeloyloxy)-1,1-dimethylpyrrolidinium bromide.0022-3565/09/3302-660–668$20.00T HE J OURNAL OF P HARMACOLOGY AND E XPERIMENTAL T HERAPEUTICSVol.330,No.2Copyright ©2009by The American Society for Pharmacology and Experimental Therapeutics 152470/3498433JPET 330:660–668,2009Printed in U.S.A.660by guest on April 22, 2011 Downloaded frompostjunctional muscarinic M 3receptors present on airway smooth muscle and submucosal glands to induce bronchocon-striction and mucus secretion,respectively.Thus,anticholin-ergic bronchodilators have a particular value in the treat-ment of COPD because they block the effects of an increased vagal cholinergic tone.In clinical trials,muscarinic antago-nists have been shown to produce an improvement in lung function,exercise endurance,and health-related quality of life and significant reductions of exacerbations,and—in sum-mary—to improve the clinical course of COPD (Vincken et al.,2002;Tashkin et al.,2008).Importantly,muscarinic an-tagonists show a favorable side-effect profile.Thanks to the quaternary structure of modern anticholinergics,which pre-vents a substantial absorption from mucosal surfaces and penetration of the blood-brain barrier,and the inhalative administration route,which further limits systemic expo-sure,these drugs are virtually free of clinically relevant side effects,besides occasional reports of dry mouth (Gross,2006).Tiotropium (Spiriva),introduced recently for the treatment of COPD,is the first example of a long-acting muscarinic antagonist (LAMA):following a single dose of tiotropium,clinically relevant improvement in lung function lasts for more than 24h,allowing once-daily dosing (Vincken et al.,2002).The mechanism behind its long duration of action relates to the slow rate of dissociation from its target,the human M 3muscarinic receptor (Disse et al.,1999).Long duration of action (preferably 24h)is an important feature of drugs intended to treat chronic diseases,enabling both pro-longed efficacy (Tashkin,2005)and a simple,once-daily dos-age regime that improves patient compliance (Tamura and Ohta,2007).Other drugs are currently being evaluated in clinical trials for their ability to function as LAMAs with a potential for once-daily administration,i.e.,aclidinium [also known as LAS34273from Almirall Prodesfarma (Barcelona,Spain),currently in phase IIb trials]and glycopyrrolate [NVA-237,from Novartis (Basel,Switzerland),in phase III trials].Currently,however,limited preclinical information is published concerning the pharmacological properties of these investigational drugs.Thus,in the present study,we have directly compared the pharmacology of the different LAMAs,namely tiotropium,aclidinium,and glycopyrrolate,in in vitro and in vivo models.To understand the antagonists’behavior at the molecular level,interaction with the different muscarinic receptor subtypes was analyzed in binding and functional assays.Given the rationale behind tiotropium’s long duration of action,particular attention was given to the kinetics of dissociation from the muscarinic receptors.Effi-cacy and duration of bronchoprotection were tested in a phar-macological model of acetylcholine-induced bronchoconstric-tion in anesthetized dogs over a period of 24h.Materials and MethodsChemicals and Reagents.[N -methyl -3H]Scopolamine methyl chloride ([3H]NMS,specific activity,82Ci/mmol)was obtained from PerkinElmer Life and Analytical Sciences (Waltham,MA).MgCl 2,carbachol,muscarine,atropine sulfate,pirenzepine,N -methyl sco-polamine bromide,EDTA,3-isobutyl-1-methylxanthine,NaCl,and HEPES were obtained from Sigma-Aldrich (St.Louis,MO).Ipratro-pium bromide,tiotropium bromide,aclidinium bromide,and glyco-pyrrolate bromide were synthesized in the chemical laboratories of Boehringer Ingelheim (Biberach an der Riss,Germany).The tritia-tion of tiotropium was performed by RC Tritec AG (Teufen,Switzer-land).[3H]Tiotropium was purified by high-performance liquid chro-matography on a XBridge C-8column (Waters GmbH,Eschborn,Germany)resulting in a radiochemical purity of Ն98%,and a specific activity of 65Ci/mmol.All cell culture reagents were purchased from Invitrogen (Carlsbad,CA).Cell Culture Techniques.Chinese hamster ovary (CHO)cells transfected with the cDNAs encoding the human M 1to M 5musca-rinic acetylcholine receptors were purchased from PerkinElmer Life and Analytical Sciences.CHO cells were grown in Ham’s F-12me-dium supplemented with 10%fetal calf serum in the presence of the selection agent G418(400␮g/ml).Cells were maintained at 37°C in humidified air containing 5%CO 2.Equilibrium Binding Experiments.Membrane isolation and purification from CHO cells stably expressing the human M 1to M 5receptors was performed as described previously (Casarosa et al.,2005).In brief,cells were suspended in buffer A (15mM Tris-HCl,pH 7.5,2mM MgCl 2,0.3mM EDTA,1mM EGTA),homogenized,and spun down for 30min at 48,000g .The pellet was resuspended in buffer B (7.5mM Tris-HCl,pH 7.5,12.5mM MgCl 2,0.3mM EDTA,1mM EGTA,250mM sucrose),aliquoted,and stored at Ϫ80°C until use.Protein content was measured with the BCA kit (Thermo Fischer Scientific,Rockford,IL).In all radioligand experiments,the binding buffer consisted of 10mM HEPES,1mM MgCl 2,pH 7.4.After the indicated incubation period,bound and free [3H]NMS were separated by rapid vacuum filtration using a Brandel Harvester (Gaithersburg,MD)on GF/B filters presoaked in 0.5%polyethylenimine and rapidly washed three times with ice-cold binding buffer.Filter disks were added to 3ml of scintillation fluid (Ultima Gold from PerkinElmer Life and Analyti-cal Sciences)in pony-vials,and radioactivity was quantified by use of liquid scintillation spectrometry on a Tri-Carb 2900TR Liquid Scin-tillation Analyzer (PerkinElmer Life and Analytical Sciences).In all experiments,total binding never reached 10%of that added,limiting complications associated with depletion of the free-radioligand con-centration.Saturation binding experiments were performed by incubating membranes (usually 5–10␮g/sample,adjusted according to the B max of the individual cell line),with a range of concentrations of [3H]NMS (4pM to 8nM)in a total volume of 4ml,to avoid significant ligand depletion at the lower concentrations.Samples were incubated at room temperature for 2h under gentle agitation before filtration.Nonspecific binding was determined for each radioligand concentra-tion by coincubating in parallel a set of membranes with an excess of unlabeled atropine (10␮M).To obtain affinity estimates of unlabeled antagonists,heterolo-gous competition experiments against [3H]NMS were performed at equilibrium.Membranes were incubated in the presence of [3H]NMS (final concentration,approximately 0.1nM)and different concentra-tions of unlabeled antagonist at room temperature with gentle agi-tation for 18to 20h before petition displacement binding data were fitted to the equation described by Hill (1909),and IC 50values obtained from the inhibition curves were converted to K i values by use of the method of Cheng and Prusoff (1973).Kinetic Studies.To determine dissociation kinetic parameters according to the classical method,membranes expressing the differ-ent muscarinic receptor subtypes were first allowed to equilibrate with 0.45nM [3H]NMS or 90pM [3H]tiotropium at room tempera-ture for at least 2h (300␮l/sample).Subsequently,samples were added to test tubes already containing 3ml of binding buffer with atropine 10Ϫ5M (time 0)to start dissociation.At the different time points,samples were filtered by use of a Brandel harvester,as described above.To monitor the potential membrane degradation occurring at later time points,some samples were added at time 0to tubes containing 3ml of binding buffer with the same concentration of radioligand present in the preincubation step (i.e.,0.45nM [3H]NMS or 90pM [3H]tiotropium),so that dissociation was not started and decrease in radioligand binding would reflect receptor breakdown.Dissociation data were fitted to a single-phase exponen-Preclinical Pharmacology of Long-Acting Anticholinergics661by guest on April 22, 2011 Downloaded fromtial decay function,and the K off rate obtained was transformed into a t 1⁄2value (dissociation half-life)using the following equation:t 1⁄2ϭln 2/K off .The dissociation kinetic parameters of unlabeled ligands were assessed with the use of the method of Motulsky and Mahan (1984).With this approach,the kinetics of an unlabeled ligand are deter-mined via competition with a radioligand ([3H]NMS),whose kinetic parameters have already been determined for a given receptor.This method involves the simultaneous addition of both radioligand and unlabeled competitor to the membranes,so that at time 0all recep-tors are unoccupied.Membranes were added at time 0to tubes containing 0.3nM [3H]NMS in the presence or absence of three different concentrations of unlabeled competitor (approximately 30-,100-,and 300-fold K i ).At the different time points (up to 8h),the degree of [3H]NMS bound to receptor was assessed by filtration harvesting,as described above.Dissociation rates for unlabeled an-tagonists were calculated with Prism (GraphPad Software Inc.,San Diego,CA)using the equation for kinetics of competitive binding by fitting (least-squares)the data from the competition kinetic experi-ments to a two-component exponential curve,with all parameters fixed apart from k 3and k 4.The kinetics of [3H]NMS,determined independently (average of three independent experiments)and used in these equations,are as follows:for hM 1receptor:k on ϭ3.22ϫ108M Ϫ1min Ϫ1and K off ϭ0.019min Ϫ1;for hM 2receptor:k on ϭ6.96ϫ108M Ϫ1min Ϫ1and K off ϭ0.035min Ϫ1;for hM 3receptor:k on ϭ5.96ϫ108M Ϫ1min Ϫ1and K off ϭ0.013min Ϫ1.cAMP Assay.To determine the functional antagonistic potency of the different muscarinic antagonists at the hM 2receptor,changes in intracellular cAMP levels were determined with CHO-hM 2cells in suspension (5000cells/well)with use of Lance technology (PerkinElmer Life and Analytical Sciences)and the 384-well plate format (Optiplate;PerkinElmer Life and Analytical Sciences),ac-cording to the manufacturer’s protocol.In brief,cells were stimulated with a range of agonists’concentrations (either carbachol or musca-rine,from 10Ϫ3to 10Ϫ12M)in Hanks’buffered saline solution sup-plemented with 5mM HEPES,0.1%bovine serum albumin,500mM 3-isobutyl-1-methylxanthine,and 1␮M forskolin in the absence or presence of at least six different concentrations of antagonists (usu-ally from 10Ϫ5to 10Ϫ10M)for 30min at room temperature.A 15-min preincubation with antagonists was allowed to proceed before the addition of the agonist.Cells were lysed with use of Lance reagents,and after an additional 2h,plates were read on an Envision plate reader (PerkinElmer Life and Analytical Sciences).The concentra-tion of cAMP in the samples was calculated from a standard curve.The Gaddum equation was used to fit the data,and shifts in the dose ratio were plotted according to Schild.Inositol Phosphate Accumulation Assay.Changes in intracel-lular levels of inositol phosphates were monitored in CHO-hM 1and CHO-hM 3cells by use of the IPone kit (Cisbio Bioassays,Bagnols-sur-Ce `ze Cedex,France)according to the manufacturer’s instruc-tions.In brief,cells were plated 1day in advance in 384-well white plates (Greiner Bio-One GmbH,Frickenhausen,Germany),with a density of 20,000cells per well.On the day of the assay,cells were stimulated with a range of agonists’concentrations (either carbachol or muscarine,from 10Ϫ3to 10Ϫ12M)in a buffer consisting of 10mM HEPES,1mM CaCl 2,0.5mM MgCl 2,4.2mM KCl,146mM NaCl,5.5mM glucose,and 50mM LiCl (final pH 7.4)in the absence or presence of at least six different concentrations of antagonists (usu-ally from 10Ϫ5to 10Ϫ10M).A 15-min preincubation with antagonists was allowed to proceed before addition of the agonist.After 1-h stimulation at 37°C,cells were lysed using reagents provided by the kit,and after an additional hour,plates were read on an Envision plate reader.The concentration of inositol phosphates in the samples was calculated from a standard curve.The Gaddum equation was used to fit the data,and shifts in the dose ratio were plotted accord-ing to Schild.In Vivo Experiments.Bronchoprotective effects of the musca-rinic antagonists were investigated in a model of acetylcholine(ACh)-induced bronchoconstriction in anesthetized,ventilated bea-gle dogs.All animal experiments were conducted in accordance with the German Animal Welfare Law and received ethics committee approval.Male dogs weighing between 10and 12kg were randomly assigned to four groups,each consisting of four animals.The dogs were anesthetized with a bolus injection of propofol (10mg/kg i.v.)followed by an infusion of 30mg/kg i.v.propofol per hour (B.Braun Melsungen,Melsungen Germany)into the cephalic vein.The dogs were intubated and ventilated with volume-controlled pressure with a mixture of room air and oxygen (3:1)with a Siemens respirator at a rate of 15strokes per minute.Optimal ventilation was ensured by regular measurement of acid-base status and oxygen saturation in the blood.Muscarinic antagonists were dissolved in water/ethanol (40:60,v/v)and administered by inhalation by use of the Respimat Soft Mist Inhaler (Boehringer Ingelheim Pharma GmbH,Ingelheim,Germany)connected to the tracheal tube of the ventilated dogs in a volume of 30␮l.Increases in airway resistance were induced by repeated intravenous injections of 10␮g/kg acetylcholine (Sigma-Aldrich).Experiments were performed as dose range-finding studies for 3h before studies for 24h.For the 3-h study setup,intravenous acetylcholine challenges were injected at:Ϫ45,Ϫ30,Ϫ15,5,10,30,60,90,120,150,and 180min after inhalation of the test compound.For the 24-h study setup,ACh was administered 30and 15min before and 5,10,and 30min and 6,12,and 24h after inhalation of the test compounds.Bronchoprotection was expressed as percentage of inhibition of the ACh-induced increase in airway resistance before the administration of the compound (predrug values).Data Analysis.All experiments were analyzed by either linear or nonlinear regression analysis with the equations mentioned under the different assay methodology using Prism version 5.1(GraphPad Software,San Diego,CA).Data are expressed as mean ϮS.E.M.ResultsDetermination of Ligand Affinity for the Human Muscarinic M 1to M 5Receptors.To analyze the pharma-cological behavior of the different muscarinic antagonists in vitro,CHO cell lines selectively and stably expressing either of the human muscarinic M 1,M 2,M 3,M 4,and M 5receptors were used to ensure that measurements were made at a single receptor subtype.For each cell line,the B max values and affinities toward [3H]NMS,as determined in saturation experiments,are reported in Table 1.The antagonists’affinities for the different receptor subtypes were determined in heterologous competitive binding experi-ments against [3H]NMS.Because the time needed to reach equilibrium can change significantly,depending on the kinetic parameters of the unlabeled compound (Motulsky and Mahan,1984),initial experiments were performed to decide how long incubation should proceed.This aspect is of particular impor-tance for compounds with longer kinetics of receptor interac-TABLE 1Pharmacological characterization of the different CHO cell lines stably expressing human muscarinic receptor subtypesDissociation constants for ͓3H ͔NMS (expressed in picomolars)and levels of receptor expression (expressed in picomoles per milligram of protein content of the membrane preparations)were determined in saturation binding assays,as described under Materials and Methods .The average of at least two independent experiments per-formed in triplicate is shown.Receptor SubtypeK d B max pMpmol/mghM 160Ϯ1 1.91Ϯ0.09hM 266Ϯ49.10Ϯ0.10hM 378Ϯ7 2.97Ϯ0.03hM 454Ϯ14 2.39Ϯ0.03hM 5201Ϯ168.99Ϯ0.20662Casarosa et al.by guest on April 22, 2011 Downloaded fromtion,as shown for tiotropium (Disse et al.,1993),where too short incubation would result in underestimation of receptor affinity.As a model system,membranes expressing the hM 3receptor were used,and the different muscarinic antagonists,i.e.,tiotropium,ipratropium (with fast dissociation from the hM 3receptor,see next paragraph),aclidinium and glycopyrro-late,were allowed to equilibrate in the presence of [3H]NMS for different times (Fig.1).Ipratropium reached equilibrium after 2h,as indicated by the absence of any further change in its IC 50over time.In contrast,tiotropium needed at least 20h of incu-bation time to reach pseudo-equilibrium,as indicated by the shift in pIC 50values at shorter time points (pIC 50values after 2,5,and 10h were statistically different from that obtained at 20h,P Ͻ0.05).An intermediate behavior was observed for aclidinium and glycopyrrolate,which needed at least 5h to reach pseudo-equilibrium in this experimental setting.Because the time needed to reach equilibrium is related to the receptor dissociation kinetics for each cold test compound (Motulsky and Mahan,1984),these data suggest that,among the substances tested,tiotropium has the longest half-life at the hM 3receptor.Based on these results,an assay incubation time of approxi-mately 20h was used in all subsequent experiments,because this provided a satisfactory compromise between attaining equilibrium while avoiding membrane degradation.Displacement curves were analyzed according to a model of orthosteric competition between [3H]NMS and the unlabeled competitors,so that IC 50values were transformed into K i val-ues according to the Cheng-Prusoff equation.To prove that this is the correct model for interpreting the data,the shifts in IC 50values in relation to the increase in amount of radioligand used in a given experiment were monitored for different ligands:the IC 50values shifted linearly in correlation with the increase of radioactive probe ([3H]NMS),as assumed in the model for com-petitive interaction (Cheng and Prusoff,1973)(data not shown).With this method,the different muscarinic antagonists were tested,and the p K i values obtained under these conditions are summarized in Table 2.Kinetics of Dissociation from Human Muscarinic Re-ceptors.Another important parameter in describing the in-teraction taking place between ligand and receptor is the dissociation rate constant,K off ,which refers to the rate at which the drug receptor complex dissociates.It has previ-ously been demonstrated for tiotropium that its slow disso-ciation from the target receptor hM 3is key to its long dura-tion,which allows for once-daily administration of tiotropium (Disse et al.,1999).The dissociation rates of compounds are traditionally assessed directly by monitoring the specific binding of a radiolabeled form of the ligand of interest over time.As an alternative,Motulsky and Mahan (1984)intro-duced a method by which a kinetically characterized radioli-gand is added simultaneously with the unlabeled ligand to the receptor preparation of interest.Because the pattern of radioligand binding over time depends on both the concen-tration and kinetic parameters of the competitor,the disso-ciation rate of the unlabeled compound can be derived exper-imentally in this setting.This methodology has been used previously with success to assess the dissociation of ligands from the human M 3receptor (Dowling and Charlton,2006).The dissociation kinetic parameters for tiotropium were determined with the classical method (i.e.,monitoring disso-ciation of [3H]tiotropium from hM 3receptors in time;Fig.2A)and with the “Motulsky and Mahan”method (i.e.,via com-petition kinetic experiments between unlabeled tiotropium and [3H]NMS,as explained under Materials and Methods ;Fig.2B).The dissociation half-life values of tiotropium ob-tained with the two different methods were in close agree-ment (K off 0.028Ϯ0.003and 0.026Ϯ0.005h Ϫ1derived with the classical and alternative method,respectively);therefore,the kinetic values of the other compounds were determined with the competition kinetic experiments,which do not re-quire the preparation of a specific radioligand for each com-pound to be tested.The K off and dissociation half-life values of the different muscarinic antagonists at the hM 1,hM 2,and hM 3receptors are reported in Table 3.The half-life ratioofFig.1.Shift of pIC 50values obtained in heterologous binding assays for different muscarinic antagonists over time.CHO-hM 3cell membranes were incubated with [3H]NMS and a range of competitor concentrations (ipratropium,E ;tiotropium,F ;aclidinium,‚;glycopyrrolate,Ⅺ)at room temperature for different times (i.e.,2,5,10,20,and 24h)before har-vesting through filtration.IC 50values were obtained by fitting a sigmoi-dal function.Data are shown as means ϮS.E.M.for three experiments,each performed in triplicate.TABLE 2Binding affinities of different muscarinic antagonists against the five human muscarinic receptor subtypesThe equilibrium dissociation constants of the different muscarinic antagonists were determined in heterologous competition experiments against ͓3H ͔NMS.The p K i values shown are the average of at least three independent experiments performed in triplicate,and the means ϮS.E.associated with the measurements were 0.1or less.hM 1hM 2hM 3hM 4hM 5Atropine 9.779.479.689.979.50NMS10.3110.2510.3210.429.59Ipratropium 9.409.539.589.659.07Pirenzepine 8.63Ͻ7.07.037.917.32Tiotropium 10.8010.6911.0211.029.96Aclidinium 10.7810.6810.7410.8410.26Glycopyrrolate 10.099.6710.0410.269.74Preclinical Pharmacology of Long-Acting Anticholinergics663by guest on April 22, 2011 Downloaded fromeach antagonist for dissociation from the hM 3and hM 2re-ceptors is included in Table 3as well.Quantification of Functional Competitive Antago-nism:Analysis of the G q Pathway.The M 1to M 5receptors can be subdivided into two major functional classes according to their G-protein-coupling preference.The “odd-numbered”M 1,M 3,and M 5receptors selectively couple to G-proteins of the G q /G 11-family,whereas the “even-numbered”M 2and M 4receptors preferentially activate G i /G o -type G-proteins (Wess et al.,2007).Given that the hM 4and hM 5receptors are expressed almost exclusively in the central nervous system and are basically absent in the lungs where predominantly hM 3and hM 2are expressed (Lee et al.,2001),the functional analysis of muscarinic antagonists was restricted to the hM 1to hM 3receptor subtypes.The assays of choice to monitor receptor activation con-sisted in measurement of second messengers (i.e.,inositol phosphates for G q -coupled receptors andcAMP for the G i -coupled receptors,respectively).Two different agonists be-longing to different chemical classes,i.e.,carbachol and mus-carine,were tested.Both behaved as full agonists (␣ϭ1)on the hM 1and hM 3receptors,with muscarine being slightly more potent (p D 2values of muscarine and carbachol are:6.7Ϯ0.1and 6.0Ϯ0.1at the hM 1receptor;7.3Ϯ0.1and 6.7Ϯ0.1at the hM 3receptor,respectively;n Ն11).To measure the antagonism of the different anticholin-ergics,the shift in the agonist response curves induced by the presence of different antagonist concentrations were plotted according to Schild (e.g.,in Fig.3),and p A 2values were determined.All muscarinic antagonists tested showed a com-petitive and surmountable antagonistic behavior (exempli-fied for ipratropium in Fig.3A).Most of the tested anticho-linergic drugs (i.e.,ipratropium,atropine,NMS,and pirenzepine)induced a parallel shift of the agonist concen-tration-response curve that could be perfectly fitted with the Schild equation,with a slope not significantly different from 1(e.g.,ipratropium,Fig.3,A and B).A different profile was observed for tiotropium,aclidinium,and glycopyrrolate,where fitting the dose ratios over a large range of antagonist concentrations (10Ϫ10-10Ϫ5M)resulted in a regression with a slope different from 1.Slopes are 1.29Ϯ0.05,1.27Ϯ0.07,and 1.27Ϯ0.05for tiotropium,aclidinium,and glycopyrrolate,respectively (see dashed lines in Fig.3,B–D).A slope that is significantly greater than 1could indicate a lack of antagonist equilibrium,as can be expected when compounds with slow receptor dissociation kinetics are tested.Because in conditions of nonequilibrium the speed of ligand association to the receptor is proportional to its concentration,the fractional antagonist receptor occu-pancy will be lower (compared with that at equilibrium)for the lower concentrations (Kenakin,1984),and this causes the slope of the Schild regression to be more than 1.To solve this problem,these antagonists were tested at higher con-centrations (i.e.,from 10Ϫ8to 10Ϫ5M)with half-logarithmic concentration steps.With this adjustment,Schild regression turned out to be linear with unit slope (Fig.3,B–D),allowingFig.2.Determination of the K off parameter of tiotropium for the hM 3receptor by two independent methods.A,according to the traditional method,CHO-hM 3cell membranes were preincubated with [3H]tiotro-pium,and then at time 0dissociation was started (F )by adding an excess of atropine,and the amount of receptor-bound [3H]tiotropium was mon-itored by filtering samples at the indicated time points.Data were best-fitted using a one-phase exponential decay.In some samples (E ),disso-ciation was not started,and total binding was monitored at the different time points to rule out membrane degradation.The average of three independent experiments,each performed in triplicate,is shown.B,to determine the K off value according to the method described by Motulski and Mahan (1984),competition kinetics curves were performed with CHO-hM 3membranes in the presence of [3H]NMS (approximately 0.45nM)and tiotropium (F ,0;f ,0.3nM;E ,1nM;Ⅺ,3nM).Samples were incubated at room temperature for the indicated time points before fil-tration occurred.Data were fitted with Prism equations as described under Materials and Methods to calculate K off values.Because the total binding somewhat varied from experiment to experiment,data are pre-sented as mean ϮS.D.from one representative of three independent experiments performed in triplicate.DR,drug receptor.D.P.M.,desinte-grations per minute.TABLE 3Koff values (h Ϫ1)and dissociation half-lives (h)for the different muscarinic antagonists against the human M 1,M 2,and M 3receptor subtypesThe dissociation constants were determined according to the method described by Motulski and Mahan (1984),by analyzing competition kinetics curves in the presence of ͓3H ͔NMS and different concentrations of unlabeled antagonist,as shown in Fig.2B for tiotropium.Data shown are the mean K off values ϮS.E.M.of three independent experiments,each performed in triplicate,and the corresponding dissociation half-life (in hours).hM 1hM 2hM 3t 1/2Ratio M 3/M 2K off t 1/2K off t 1/2K off t 1/2h Ϫ1hh Ϫ1hh Ϫ1hIpratropium 6.75Ϯ0.150.117.7Ϯ0.30.03 3.09Ϯ0.090.227.3Tiotropium 0.066Ϯ0.0110.50.26Ϯ0.05 2.60.026Ϯ0.0052710.4Aclidinium 0.11Ϯ0.007 6.40.39Ϯ0.03 1.80.071Ϯ0.0110.7 5.9Glycopyrrolate 0.35Ϯ0.072 1.84Ϯ0.10.370.11Ϯ0.02 6.116.5664Casarosa et al.by guest on April 22, 2011 Downloaded from。