Document wKDX6p1ynLKnzKqrg31LvjYBE

CLEENT PRIVATE PROTOCOL TITLE: Metabolism ofT-6292,T-6293, T-6294, and T-6295 by Rat and Human Hepatocytes SRI Study No. B011-95 RESEARCH CLEENT 3M Medical Deparunent ToxicologyServices 3M Center Building220-2E-02 St.Paul,MN 55133-3220 CEi JUL 70 igo "X 3 0 @o LOCI Stud@ Monitor: StevenC. Gordon, Ph.D.,D.A.B.T. TESTING LABORATORY SRI International ToxicologyLaboratory 333 Ravenswood Avenue Menlo Park,CA 94025 Study Director: CarolE. Green,Ph.D.,D.A.B.T. Telephone:(415)859-4083 FAX: (415)859-2889 APPROVALS: Research Client'sAuthorizedRepresentative Date F -- 'S@ Study-Director ZUF@- I I)ate QualityAssurance 1 v. May 3. 1995 'Date CLIENT PRIVATE I. STLTDYOBJECTIVE The objectiovfethistudyistodetermintehemetabolisomfthetesatrticluessing isolatheedpatocytperseparefdromratandhumanliverT.he datageneratebdythisinvitro systemwillbeusedtocomparethemetabolisomfthetesatrticliensthetwospecies. Hepatocyteswillbe isolatefdrom ratand human liverspecimens.The cellswillbe allowedto attachinmonolayer cultureand thenwillbe incubatedwith suitablceoncentrations of thetestchemicals.Aliquotsof theculturemedium containingceuswillbe removed atthe selectedtime-points.The proteincontentand 7-ethoxycoumarin0-deethylaseactivitya, cytochrome P-450-associateadctivityw,illbe determinedinculturesfrom each preparation. U. STATEMEENT OF PURPOSE The purpose ofthisstudyistoprovidedatathatcan be used tosupportapplicationfsor researchormarketingpermitsforproductsregulatedby theFood and Drug Administration and submittedpursuanttosection4s06, 408, 409, 502, 503, 505, 506, 507, 510, 512-516, 518-520,706 or 801, or otherapplicablseectionosftheFederalFood, Drug and Cosmetic Act or Sections351 or 354-36OF ofthePublicHealthServiceAct. This studywillbe conducted accordingtoSRI StandardOperatingProcedures(SOP's)as wellas incompliancewiththe Food and Drug Administratio2n1 CFR Part58 Good LaboratoryPracticeforNonclinical LaboratoryStudies. M. TEST ARTICLE A. TestArticlIedentification Name: T-6292 Lot No.: To be providedby ResearchClient Molecularweight:571.2 2 CLIENT PRIVATE Name: T-6293 Lot No.: To be providedby ResearchClient Molecular weight: 685.28 Name: T-6294 Lot No.: To be providedby ResearchClient Molecular weight: 527.2 Name: T-6295 Lot No.: To be 12rovidebdy Research Client Molecularweight: 538.1 B. Purityand Stability Documentationoftheidentitys,trengthp,uritya,nd stabiliotfythetestarticlewsillbe theresponsibiliotfytheResearchClient.Documentation on the identitaynd purityof the solventcontrol,DMSO, willbe obtainedfrom the supplierofthecompound. C. Handling and Storage On receiptt,hetestarticlewsillbe placedina secondarycontainer(lightproofa)nd storedas recommended by theResearchClient.A MaterialSafetyData Sheet(MSDS) or othercomparable document specif@@gany hazardsand identifyinfgirsatidand clean-up proceduresin caseof an accidentaslpillshallbe providedby the Research Clientand shall accompany thetestarticlesT.he testarticlewsillbe loggedinand storedinBuildingL, Room LB286. 3 CLIENT PRIVATE D. DispositioofntheTestArticle Any remaininugnusedportioonfthetesatrticlweisllbereturnetdotheResearch Clientaftercompletionof thestudy. rV. TEST SYSTEM A. Background and Justificatiofnor Selectionofthe Test System The liveristhemajor siteof metabolismofmost organicchemicals,both endogenous and foreign.Species-relatdeidfferenceisnthe metabolismofxenobioticasrewellknown and have been documented to be correlatetdo thetoxiceffectsof chemicalson certainspecies (Caldwell,1980;Calabrese,1983). The recentavailabiliotfyhuman tissuesforresearch, particularhliygh-qualithyuman liverspecimens,has contributegdreatlytotheknowledge base on human xenobiotimcetabolismcapabilitiesI.n additiont,hesetissuecsan be used forin vitroinvestigatioonns themetabolismoftestchemicalsearlyinthedevelopmentof new productstomore reliablpyredictthemetabolicfateof thechemical inhumans. B. Test Species 1. Rat. AdultSprague-Dawleyrats(3malesand 3 females)willbe used forthe preparatioonf hepatocytes.They willbe purchasedfrom CharlesRiverLaboratoriesand will weigh approximately200-250 g and willbe atleast2 months old atthetime ofreceipt. The ratstobe used forthisstudywillbe housed inBuildingL, where thecellisolation willbe performed. Ratswillbe takenfrom theshippingcontainersw,eighed,examinedfor generalhealth,and placedthreeper cage in 22 x 12'h x 8-inchsuspendedpolycarbomtecages labeledwith theirquarantinneumber assignments.Animals willbe quarantineidnthesame room inwhich they willbe housed. Rats willbe fed (ad libitumP)urinaCertifieRdodent Chow (#5002)and thefeedsupplierwillprovideSRI withan analyticarleportidentifyintghe diettobe freeofcontaminantsforeach lotoffeed received.A copy of each analyticarleport willbe availablaespartofthestudyrecord.Ratswillreceivepurified(deionizeadnd UVtreatedd)rinkingwaterad libitum.Temperaturewillbe maintainedat72 40F and relative humiditymaintainedbetween 35 and 65%. A lightcycleof 12 hours lightand 12 hours dark willbe maintained,withlightstartinagt06:00. The laboratoryspecieswillbe quarantinedforatleast3 days beforeuse in an experiment.The LaboratoryAnimal Medicine DepartmentatSRI willissuedocumentationto 4 CLIENT PRIVATE the Study Directoron the healthofanimalsduringthequarantineperiod.Beforeisolatioonf hepatocytest,he ratswillbe anesthetizewdithsodium pentobarbita(l65 mg/kg). 2. Human. Human liverspecimens willbe acquiredthrough cooperationwith organ procurementorganizationfsrom brain-deadhuman organ donors. Liverfrom two males and two femaleswillbe used. Tissueswillbe processedas fortransplantT.he organs willbe perfusedintheoperatingroom with ice-coldorgan preservatiosnolutionand packed in ice. The tissuewsillthenbe shippedtothe laboratorfyorcellisolatiobny themost expedient method. Hepatocyteswillbe isolateeditherby SRI Internationoarl theywillbe obtainedfrom theHuman CellCultureCenter(FolkstonG,A) . C. Test System Identiflcation Each liverspecimenwillbe identifiebdy a lettedresignatintghespecies(R = rat;H human), followedby a number indicatintgheparticulasrpecimen. D. Specimen Characteristics For themetabolismstudiess,ixratswillbe used (threemales and threefemales)and fourhuman specimens(twomales and two females).For human specimens,informationon age,sex,race,and causeofdeathwillbe providedtotheResearch Client.Other pertinent informationthatison drug exposure,smoking,and medicalhistorymay be available. E. SpecialConsiderationsRelatedto Human Tissues Complete serologytestinwgillbe performedon eachhuman donor specimenand only thosespecimensthattestnegativetohepatitiBsand C virusesh,uman inimunovirus(AIDS), and syphiliwsillbe acceptedforuseon thisproject.Many ofthedonors,however, willhave a positivreeactiotnocytomegaloviru(sCMV). Whatever theresultsof serologytestinga,llhuman tissuewillbe treatedas infectious and technicianwsilltakethenecessaryprecautionisnitsuse forthisproject,includingwearing properattirwehilepreparinghepatocytesor subcellulafrractionasn.d performingincubations. The tissuewsillbe handledina hood toprotecthetechniciafntom aerosoltshatmay form duringexperimentalproceduresand tokeep preparationsterile.Glasswarethatcomes into contactwith human tissuewillbe disinfectewdith sodium perchloratet,henwashed and autoclaved.Disposablematerialwsillbe incinerated. 5 CLIENT PRIVATE. V. EXPERIMENTAL DESIGN A. HepatocyteIsolation Rat hepatocytewsillbe isolatebdy eithewrhole liveror biopsyperfusionas convenient.Human hepatocyteswillbe preparedby theperfusionofbiopsysections(Strom et al.,1981;Green etal.,1986;Allenand Green, 1991). Inbrief,thewhole liveror wedges of theorgan willbe perfusedfirswtitha Ca"-freebuffer,followedby a buffercontaining collagenase.Hepatocyteswillbe combed freefrom thedigestedtissueand purifiedby simple differenticaelntrifugation. Isolatedhepatocyteswillbe platedontocollagen-coatecdulturedishes(35 mm diameter)ina modifiedWaymouth's 752/1culturemedium (CMHla) thatcontains11.2Ag/ml alanine,12.8gg/ml serine,24.0 ilglmlasparagine8,4.0Ag/ml gentamicinsulfate0,.168 tig/mlaminolevuliniaccid,5.0 tLg/mloleicacid,5.0 Ag/ml linoleiaccid,1.0AgIml D,Ltocopherol2,88 ng/ml testosterone2,72 ng/ml estradiol3,93 ng/ml dexamethasone,7.9 Ag/rnl thyroxin,30 ng/ml glucagon,0.02 U/ml insulin0,.1% ITS (CollaborativReesearch,Inc.; finalconcentrations5: Ag/ml transferri5n,Aglml insulina,nd 5 ng/ml selenium),0.2 mM Lascorbicacid2-phosphate,and 0.2% BSA. The initiaplatingmedium willcontainfetal bovineserum. After2 to3 hr of incubatioant370C inan atmosphereof95% air:5% C02, themedium willbe aspiratetdoremove nonviableu,nattachedcellsand replacedwith CMHla containingthetestarticle. B. Metabolism Experiments 1. Preparationand AdministrationofTestArticle.The testarticlewsillbe preparedas stocksolutionsin DMSO, maintainingtheDMSO concentratioant0.1% or less They willbe dilutedwith culturemedium togivea finalconcentratiodneterminedby discussionwsith theStudy Monitor and basedon theresultosf therange-fmdingcytotoxicity experiment.Stock solutionosfthetestarticlewsillbe preparedimmediatelypriortothe experimentand keptintherefrigerator on iceuntiluse. Aliquotsofthesestocksolutions willbe savedforlateranalysibsy Advanced BioanalyticSaelrvices(IthacaN,Y) toverifythe concentrationasnd homogeneity of the stocksolutions. 2. Preparationof ConditionedMedium. Rat hepatocyteswillbe isolatedand culturedon collagen-coatecdulturedishesfor0 and 6 hr. At theappropriatteime point,the hepatocyteswillbe scrapedintotheculturemedium. The cellsand medium willbe aspirated and immediatelymixed with an equalvolume of icecoldmethanol. The conditionedmedium willthen be centrifugeadtapproximately1200 X g for5 minutesand subsequentlyfrozenfor 6 CLIENT PRIVATE shipmenttotheResearchClient.Approximately500 ml ofconditionemdedium ateach time pointwillbe prepared. 3. IncubationofHepatocyteswith Test Article. Cultureswillbe incubatedin duplicatweith thetestchemicalinCMHla. Sampleswillbe takenat2 tiine-poin(t0sand 6 hr). The followingcontrolswillalsobe incubatedand samplestakenat0 and 6 hr: 1) hepatocytesinmedia withouttestarticl(e4setsofquadruplicatiencubations)2;) incubation media containingtestarticlebsut no cells.The attachedcellswillbe scrapedintothe culture medium. Then thecellsand medium willbe aspiratedand immediatelyadded to an equal volume of ice-coldmethanoltostopthe reaction.The sampleswillthenbe centrifugeadthigh speed in a tabletopcentrifugefor5 minutesand subsequentlfyrozenforshipment. 4. ProteinAssay. The proteincontentof thehepatocytecultureswin be determinedby a spectrophotometripcrocedure,Coomassieblueassay(Bradford,1976). 5. Cytochrome P450 Activity.As a controltoindicattehepresenceof cytochromeP450 associateadctivity7,-ethoxycoumarin0-deethylatio(nECOD) activitwyill be determinedineach preparatioonf hepatocytes.The cellswillbe incubatedwith 100,UM 7-ethoxycoumarinfor1 hr. The culturemedium willbe assayedforhydroxycoumarin productionusingthefluorometrimcethod of Greenleeand Poland(1978).ECOD activitwyill be calculateads theamount of hydroxycoumarin produced/hr/mgprotein. 6. Analysisof Metabolism. Samples willbe shippedto Dr. JackHenion at Advanced BioanalyticaSlervices,Inc.for analysisofmetabolism. 7. Data Collection.The proteinassayand ECOD assaywillbe performed using a spectrophotometearnd fluorometer,respectivelyT.he valueswillbe recordedmanually or capturedelectronicalldye,pendingon equipment availabilitDya.ta from both assayswillbe recordedand manipulatedon a MicrosoftExcel spreadsheet.Informationon human liver specimensarekept in a computer database and arealsorecordedin a laboratorynotebook. The resultsofthemetaboliteanalysis(performedby Advanced BioanalyticaSlervices, Inc.)willbe returnedtoSRI and thedatawillbe calculateadsthemnol ofmetabolite/mg protein.These datawillbe includedinthefmalreport.The resultosftheanalysisoftheliver tissueby Mr. James Johnson at3M Environmentaland PollutioCnontrolwillalsobe included inthe fmal report.The raw datageneratedon themetabolitaenalysiwsillbe kept and maintainebdy Advanced BioanalyticSaelrvices.The raw dataon levelosfthetestarticleisn thehuman liversampleswillbe kept and maintainedby 3M. 7 CLEENTPRIVATE D. Sample ShippingAddresses The incubatiosnamples,samplesof frozenhuman and ratlivertissue(approximately 1 g),FBS, BSA, L-ascorbicacid2-phosphate,and media (conditioneadnd fresh)willbe sent to: Dr. JackHenion,Ph.D. Advanced BioanalyticaSlervices,Inc. 15 Catherwood Road Ithaca,NY 14850 A sampleofeachhuman livertissue(approximatel1y g)willbe sentto: James -D.Johnson 3M EnvironmentalEngineeringand PollutioCnontrol 935 Bush Avenue Building2-3E-09 PO Box 33331 St.Paul,MN 55133-3331 The dataon proteincontent,ECOD, reportsa,nd excesstestarticlweillbe sentto: Dr. StevenC. Gordon 3M Medical Department,ToxicologyServices P. 0. Box 33220 St.Paul,MN 55133-3220 VI. CONTROL OF BIAS Human tissueswillbe used as theybecome available. VH. STATISTICAL EVALUATION OF DATA Mean and standarddeviationwsillbe determinedforthereplicatiencubationsforeach testafticle. 8 CLIENT PRIVATE. VM. REPORTS Draftreportswillbe issuedpriortosubmissionof theFinalReport.Two copiesof the FinalReport willbe submittedtotheResearchClient. IX. RECORDS TO BE MAINTAINED The laboratornyotebooks,allraw data,relevantcommunications,and theoriginaclopy ofthe FinalReport willbe transferretdotheSRI Records Center,BuildingB, aftercompletion of thestudy. These materialswillbe maintainedfor 10 years;then theResearchClientwillbe contactedconcerningfuturedispositioonf therecords. X. GOOD LABORATORY PRACTICES (GLP) AND QUALITY ASSURANCE This studywillbe conductedinaccordancewith Good LaboratoryPracticeRegulations. The dutiesof SRIs QualityAssuranceUnit(QAU) willincludeinspectintghecalculations, inspectinlgaboratorwyork, certifyinpgroperidentificatiaonnd notebookentryof samples,and reviewingtheFinalReport. A QualityAssuranceStatementwillaccompany theFinalReport ofthe study. Afterthestudyhasbeen initiatemdo,dificationosftheprotocol-by eithertheTesting L.aboratoroyr theSponsor-willbe submittedinwritingtotheotherparty.AR agreed-upon modificationwsillbe intheform ofProtocolAmendments, thatwillstatethespecific modificationasnd thereasonsforthemodificationasnd willbe signedand datedby the Sponsor'sRepresentativaend theTestingLaboratory'sStudy Director.All procedureswillbe performed in accordancewithSRI StandardOperatingProcedures. The StudyDirectorwillbe made aware ofany GLP noncompliancedirectly.Ifthis areaofnoncompliancesignificantalfyfecttsheresultosfthe study,the Sponsorwillbe notified immediately.This willbe documentedand theagreedupon outcome willbe forwardedtothe QAU. , Authorizeddesignateosf theSponsormay inspecthisstudyduringregularworking hours forqualityassurancepurposesand may copy any study-relateodriginaldata. 9 CLIENT PRIVATE REFERENCES Allen,K. L. and C. E. Green. In: Methods inToxicologyV,olume 1,In VitroBiological Systems,PartA. C. A. Tyson and J.Frazier(eds)Academic PressInc.,pp. 262-270, 1993. Bradford,M. M. Anal.Biochem.72, 248-254,1976. Calabrese,E. J. John Wiley & Sons,New York, pp.203-282,1983. Caldwell,J. InEnzymaticBasisofDetoxicationV,ol.I. WilliamB. Jakoby (Ed.),Academic Press,New York, pp. 85-114,1980. DeLean, A.,P.J.Munson, and D. Rodbard. 1978. Am. J PhysioL235,E97-EI02. Green, C. E., J.E. Dabbs, and C. A. Tyson. Anal.Biochem. 129,269-276,1983. Green,C. E.,S. E. LeVaUey, and C. A. Tyson. J.Pharmacol.Exp. Ther.237,931-936, 986. Greenlee,W. F. and A. Poland. Jour.Pharmacol.Exp. Ther.205,596-605,1978. Strom, S. C.,R. L. JirtleR,. S. Jones,D. L. Novicid,M. R. Rosenberg,A. Novotny, G. Irons,J.R. McLain, and G. Michalopoulos.J.Natl.Cancer Inst.68,771-778,1982. 10