新型抗胆碱能药物盐酸戊乙奎醚及其光学异构体的细胞毒性评价

新型抗胆碱能药物盐酸戊乙奎醚及其光学异构体的细胞毒性评价 新型抗胆碱能药物盐酸戊乙奎醚及其光学 异构体的细胞毒性评价 ?基础研究? ? 1385? 中国临床药理学与治疗学 中国药理学会主办 CNM-一I【206/R.ISSN1009—250J I~-mail;cep196@21cncorn 2007I);12(12):1385—1389 Cytotoxicityevaluationofanovelanticholinergic drugpenehyclidinehydrochlorideanditsopticalisomers WANGYi—mei,PENGShuang—qing,ZHONGBo—hua,LIUKe—liang ,2gInstituteofPharmacologyandToxicology,27TaipingRoad,HaidianDistrict,Beijing100 850,China ABS,】:RACrAIM:Toevaluatethecytotoxicityofa novelanticholinergicdrugpenehyclidinehydrochloride (PHC)anditsfouropticalisomersR-1,2,1,and 2.M匝THoDS:Twonvitroassays,MTYassayand neutralreduptakeassay,wereusedtoevaluatethecyto. toxicityfollowingPHCanditsisomersexposuretoHepG2 cellsatdifferentconcentrations.RESUUrS:PHCandits isomersinduceddecreasesofviabilityofHepG2cellsina concentration.dependentmanlier.Comparisonofthecyto. toxicityofthefiveanticholinergicagentswith50%inhibi. toryconcentration(IC5o)valuesindicatedthattheorder ofpotencywasPHC>R-2>R-1>S-2>S-1forMTF assay.andR-2>PHCR-1>S-2>S-1forneutralred uptakeassay.CoNCLUSION:Withrespecttothecyto. toxicityofthefourisomersonHepG2cells:theRconfig. urationwasmorepotentthantheSconfiguration,andR. 2wasthemostpotentisomerwhereasS.1wastheleast potentisomeramongthefouropticalisomers. KEYWORDSanticholinergicdrug;penehyrclidine hydrochloride;opticalisomers;cytotoxicity CLCNumber:R965.2 Doeumen~:A ArticleII):1009.2501(2007)12.1385—05 Overthepastcentury,anticholinergicdrugshas Received2007-07—20Accepted2007—11一l6 111isworkwassupportedbytheNationalNaturalScienceFoundationofChina (328l3251). WANGYi-mei,male,PhD,engagedinpharmacologyandtoxicologyre— search. Tel:010,66949631E-mail:wangyiraei2006@126.tom PENGShuang-qing,correspondenceauthor,male,PhD,professor,engaged inpharmacologyandtoxicologyre~arch. Tel:010—66949631E-mail:pengsq@hotmail.con beenwidelyusedfortreatmentofcertaindiseasessuchas chronicobstructivepulmonarydisease(COPD)in"and overactivebladder.However,thetherapeuticapplica— bilityofanticholinergicdrugswasrestrictedbytheirside effectsinbothperipheralandcentralnervoussystemdue tothediversityofcholinergicreceptors,andfewhi【ghly subtype—selectivemuscarinicreceptorantagonistshave beendevelopedandusedintherapeutics.Therefore, therearestillsomespacesinsearchingforbetterselective muscarinicreceptorantagonistswithnewmolecularstrue— tures. Penehyclidinehydrochloride(PHC)isanovelanti— cholinergicdrugdevelopedbyourinstitute.Sincethere aretwochiralcarbonatomsinthemolecularstructure, PHCisaracemeconsistingof4opticalisomersR-1. 2,S-1andS一2.Thisracemiccompoundpossessesboth antimuscarinicmadantinieotinieactivities,andreceptor bindingassayindicatesthattheracemehashigherselec— tivityforM3receptorsubtype,whichmakesitbeusedfor thetreatmentofrespiratorydisorderssuchasCOPD. However,thiskindofanticholinergicdrughasalsopre— sentedsideeffectsasclassicalantimuseariniedrugssuch asdrymouth,flushinganddizziness.Originally,PHC wasdevelopedforanantieholinergiedrugasaracemeon accountofthedifficultyinsynthesizingsingleenantiomer, thoughithadshownthatthereweredistinguisheddiffer— encesinthebiologicalactivitiesandphannaeokinetie propertiesamongthefourisomers_一.Recently.afac— ileandefficientproceduretopreparesingleenantiomerof PHCbyemployingdiastereoseleetivesyntheticmethodhas beendeveloped'..whichisofgreatsignificancetofur? therstudythepharmaceuticalpropertiesofeachisomm.. Thepresentstudyattemptstoevaluatetheeytotoxicityof PHCanditsopticalisomers. ? 1386? 1.1DrugsandreagentsPHCanditsfouroptical isomemR-1,2,1andS-2weresynthesizedand providedbyBeijingInstituteofPharmacologyandToxicol— ogy.3一(4,5一dimethyl—thiazol一2一y1)一2,5一diphenyhet- razoliumbromide(肼)andneutralredwaspurchased fromSigmaChemicalCo.(St.Louis,MO,USA).AU otherreagentswereofanalyticalorregentgrade. 1-20e?cultureHepG2cellswereoriginallyfrom ATCC(?HB8065),andwerecryopreservedinliquid nitrogeninourlaboratory.CellswereculturedinDulbec. co'smodifiedEWe'smedium(GIBCOBRL,GrandIs. 1and,NY,USA)containing10%heat.inactivatedfetal bovineselEiln(GIBCOBRL,GrandIsland,NY,USA)at 37inahumidifiedatmosphereof95%airand5% CO2. 1.3Mr兀'assayThecytotoxicityoftheseanticholine. cagentswasevaluatedbyM1TrassayaccordingtoMo- smannlllJwithslightmodifications.Thismethodisbased onthereductionof册intoablueformazancrystalby mitoehondrialdehydrogenasesofviablecells.111eamount offormazanisproportionaltothenumberofviableceils. Forthisassay,cellsfromexponentiallygrowingcultures wereused.Briefly,HepG2cellswereseededin96.well platesatadensityof104cells/mL(10oLperwel1). After24hofpre—incubation,themediumwasreplacedby newmediumcontainingdifferentconcentrationsofPHCor itsopticalisomemdissolvedinmedium(fourwellsper concentration).Following24hofincubation,themedi. ulnwasremovedfromallplatesand100"L0fM1_rsolu. tion(0.5me/mE)wasaddedtoeachwellandtheplates wereincubatedat37oCfor4h.Themediumwasthen gentlyaspirated,followingwhichtheformedformazan crystalsweresolubilizedbyadditionof100"LDMSO. 111eplateswereshakenfor10minandmeasuredonami. croplatereader(WellscansMK3,ThermoLabsystems, Finland)at570nn1.Foreachconcentration,wellscon— tainingallreagentsexceptcellswereservedasreference blank.Thepercentageofviablecellsascomparedwith controlwascalculatedas(absorbanceofdrug.treated wells-absorbanceofblank)/(absorbanceofcontro1.absor. banceofblank)×100./C卯values(concentrationsoftest compoundsrequiredtoreducethecellviabilityby50% ascomparedwiththecontrolcells)wereobtainedfromthe sigmoidalfittingdose—responseCHIVESbyusingOrigin snftware. ChinJClinPharmacolTher2007Dec;12(12) 1.4NeutralRedUptakeassayThecytotoxicityof testcompoundswasverifiedwithneutralreduptakeas. say.Neutralredisavitaldyewhichisactivelytransport. edintothelysosomesofviablecells.Chemicalsthatdam. ageplasmaand/orlysosomalmembraneswilldecFeasethe abilityofcellstotakeupneutralred.Theintracellular dyeuptakeisthemfomproportionaltothenumberofvia— blecells.Thiscytotoxicityassaywasperformedasprevi— ouslydescribed_l2J.Briefly,'theprocedureofcellseeding andtreatmentswassanleasthatofMTYassayasde. scribedabove.Followinganexposureperiodof24h,test compoundswereremovedandreplacedth200"L0f neutralredsolution(50me/mE),andtheplateswerein. cubatedat37oCinacultureincubator.After3h.the cellswereextensivelywashedwithphosphate.bufferedsa. 1ineandneutralredwithinviablecellswasextractedby additionof50%aqueousethanolcontaining1%acetic acid(aceticacid:ethanol:water=1:50:49).Ab. sorbanceofeachwellwasmeasuredonamicroplatereader at540nm.Cellviabilitywasexpressedasapercentageof contro1.Themethodforcalculationof/Cmvalueswas similartothatofMTYassay. 1.5StatisticalanalysisDatawerepresentedasmean 4-SDofatleastthreeseparateexperiments.Thediffer. encesamongdifferentgroupswereanalyzedusingone—way analysisofvariance(ANOVA).APvaluelessthan 0.05wasconsideredstatisticallysignificant. 2REsIJIS 2.1CytotoxicityofPHCanditsopticalisome~on HepG2cellsThecytotoxicityofPHCanditsopticaliso— merswereevaluatedbyMTYassayandneutralreduptake assay.AsshowninFig1AandFig1B,exposureof HeDC2cellstoPHCanditsisomeminaconcentration rangeof0—600tanol/Lfor24hresultedindecreasesof cellviabilityinaconcentration.dependentmanner,re. spectively.Asfortheresultsdeterminedby肼assay. asFig1Ashowed.thedecreasesincellviabilitywerenot significantfollowingtreatmentofHepG2cellswithR一2at concentrationslessthan100tanol/L,whereasasignifi— cantdecreaseofcellviability(about10%)wasobserved at200tanol/L(P<0.05),andthecellviabilitywas dramaticallyreducedtoabout60%ofcontrolwhenthe concentrationwasiReleasedto300tanol/L.Similarly, otherisomemdisplayednomarkedeffectsonthecellvia— bilityatrelativelowconcentrations,whereastheyinduced concentration-dependentreductionsincellviabilitywith 中国临床药理学与治疗学2007Dec;12(12 increasingconcentrations.Asforneutralreduptakeas- say,asshowninFig1B,thetrendsofcytotoxicityin- ? 1387? ducedbytheseanticholinergicagentsaresimilartothose ofMTFassay. 01002003004005006000100200300400500600 Concentration0amol/L)Concentration(tamol/L) Fig1CytotoxicityofPHCanditsfouropticalisomersonHepG2~ells(~?s,n=3) HepG2cellswere.】 q)osedtoarangeofconcentrations(0,25,50,100,200,300,4O0,500and600gmol/L)ofPHCa nditsopticalisomersR-1,R-2,S- 1,andS-2for24h,thecytotoxicitywasevaluatedbyM'ITassay(A)andneutralreduptakeassa y(B). 2.2ComparisonofthecytotoxicityinducedbyPHC anditsopticalisomersThe/C5ovaltles,whichrepre- senttheconcentrationsoftestcompoundsrequiredtore- ducethecellviabilityby50%wereemployedtocompare thecytotoxicityofPHCanditsopticalisomers.The/C5o valuesderivedfromthesigmoidalfittingoftheconcentra- tion-responsecurvesfromthetwoinvitroassayswere showninFig2A(MqTassay)andFig2B(neutralred PHC尺一1尺一212 uptakeassay).Comparisonofthecytotoxicitywith/C5o valuesindicatedthattheordersofpotencywerePHC>R- 2>R-1>S-2>S-1forMTFassay,andR-2>PHC l>S-2>S-1forneutralreduptakeassay,respective- ly.Furthermore,itseemedthat'neutralreduptakeassay wasmoresensitivethanMTI"assayaccordingtothecon- centration.responsecurvesandtheIC5ovalues. 350 , 300 0 兰250 200 150 10O PHC尺.1尺.212 Fig2/Cs0values(pmol/L)ofPHCanditsopticalisomersonHepG2cells(~?s) CytotoxicityofPHCanditsopticalisomersR-1,R-2,S-I,andS-2wasevaluatedbyMTFassay (A)andneutralreduptakeassay(B).1Csovalueswerede' rivedfromthesionoidalfittingoftheConcentration—responseCUlWes. 3DIsCUSSION Singleenantiomerdrugspossessseveraladvantages suchasbetterpharmacokineticprofileandsafetycon. paredwithracemates,itisthereforeessentialtothorough- lyevaluatethepharmacologicalandtoxicologicalproper- tiesoftheisomerstoidentifythemostsuitableenantiomer fordevelopmentintoatherapeuticdrug.Sincetheinter- actionofonedrugwiththereceptorhasbeenknowntobe stereoselective,theremaybesignificantdifferencesin bothpharmacologicalandtoxicologicalpropertiesamong theisomersofaracemicdrug.Ithadbeenshownthatthe R.configurationofsubstitutedalkoxylandalkalinicalco- holgroupsinPHCwasmoresuitabletothestereo-struc- tureofthebindingsiteofmuscarinicreceptorsthantheS- configuration【.whichresultedindirencesintheaf- finiwoftheisomerstobrainmuscarinicreceptors. Therewerealsodistinctdifferencesinthebiologicalactiv- itiesamongtheisomers,andthepotencyorderwasR-2 >PHC>S-1>R-1>S-2. 一一A1一IIqB1>=o0 ??? 554433 一IoE.j^c】 ? 1388? Earlyinvitrotoxicityscreeningmightimprovethe successrateofnewchemicalentitiesinphammceutical development.Toourknowledge,nostudyhasbeenper— formedtorevealthepotentialtoxicitydifferencesamong thefourisomemofPHC.Inthepresentstudy,weem. ployedtwoinvitrocytotoxicityassays,MTrassayand neutralreduptakeassay,toevaluateandcomparethecy- totoxicityoftheisomemofPHC.Bothassayshavebeen extensivelyusedfortheevaluationofcytotoxicityofdiffer- entchemicalsincludingdrugs[一. Inthisstudy.ahu. man-derivedhepatomacelllineHepG2isadoptedasan invitrocytotoxicityscreeningmodel,becausethecell lineretainsmanycharacteristicsandfunctionsofhuman hepatocytessuchasPhaseIandPhaseIImetabolicenz- ymelevelsIl8_,andmanystudieshaveshownthat HepG2celllineisavaluabletoolforcytotoxicityscreen- ingintheearlyphaseofpharmaceuticaldevelopment. Asassessedby/C50values,thegeneralorderofcy- totoxicitypotencywasR-2>R-1>S-2>S-1.andthe RconfigurationseemedmorepotentthantheSconfigura- tion. Asfarastheassaysemployedareconcerned.the twoassaysarehighlyconsistent,anditappearsthatneu- tralreduptakeassayismoresensitivethanMTFassay. Takenphammcokineticpropertiesandbiologicalactivities 0ftheisomersintoconsiderations.itseemsthatthereare somenegativecorrelationsbetweenthebiologicalactivities andthecytotoxicityoftheisomers.Thedifferencesinthe cytotoxicityamongtheisomersmayresultfromthediffer— encesintheconfigurationatchiralcentersand/orthena- tureofthesubstituentgroups,andtheexactmechanisms needfurtherresearches. Takentogether,thepreliminarycytotoxicitystudy indicatesthattherealedifferencesinthecytotoxicityof thefourisomers.ThisfindingmayprovideSomebenefi- cialinformationfordevelopingtheindividualisomersof PHCaspotentialnovelreceptor-selectiveanticholinergic drugs. 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