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Contributors
PamelaBachour-ElAzzi
BiopredicInternationalSARL,SaintGregoire,France
EricChunYongChan
DepartmentofPharmacy,FacultyofScience,NationalUniversityofSingapore,Singapore, Singapore
ChristopheChesne
BiopredicInternationalSARL,SaintGregoire,France
StephlinaA.D’Cunha
SchoolofChemistry&MolecularBiosciences,TheUniversityofQueensland,Brisbane, QLD,Australia
AnnK.Daly
TranslationalandClinicalResearchInstitute,NewcastleUniversity,NewcastleUponTyne, UnitedKingdom
M.DeniseDearing
SchoolofBiologicalSciences,UniversityofUtah,SaltLakeCity,UT,UnitedStates
XinxinDing
DepartmentofPharmacologyandToxicology,KenR.CoitCollegeofPharmacy,The UniversityofArizona,Tucson,AZ,UnitedStates
ChieEmoto
LaboratoryofDrugMetabolismandPharmacokinetics,ShowaPharmaceuticalUniversity; TranslationalResearchDivision,ChugaiPharmaceuticalCo.,Ltd.,Tokyo,Japan
ElizabethM.J.Gillam
SchoolofChemistry&MolecularBiosciences,TheUniversityofQueensland,Brisbane, QLD,Australia
F.PeterGuengerich
DepartmentofBiochemistry,VanderbiltUniversitySchoolofMedicine,Nashville,TN, UnitedStates
JamesR.Halpert
DepartmentofPharmacologyandToxicology,CollegeofPharmacy,UniversityofArizona, Tucson,AZ,UnitedStates
SarrahL.Hannon
DepartmentofPharmacologyandToxicology,KenR.CoitCollegeofPharmacy,The UniversityofArizona,Tucson,AZ,UnitedStates
MartinA.Hayes
CompoundSynthesisandManagement,DiscoverySciences,BioPharmaceuticalsR&D AstraZeneca,M€ olndal,Sweden
W.GriffithHumphreys
AranmorePharmaConsulting,Lawrenceville,NJ,UnitedStates
MagnusIngelman-Sundberg
DepartmentofPhysiologyandPharmacology,SectionofPharmacogenetics,Karolinska Institute,Stockholm,Sweden
TrevorN.Johnson
CertaraUKLimited,Sheffield,UnitedKingdom
JacquelineWenHuiLeow
DepartmentofPharmacy,FacultyofScience,NationalUniversityofSingapore,Singapore, Singapore
MicheleM.Skopec
DepartmentofZoology,WeberStateUniversity,Ogden,UT,UnitedStates
MarlainaR.Stocco
DepartmentofPsychologicalandBrainSciences,UniversityofCalifornia,SantaBarbara, SantaBarbara,CA,UnitedStates
HiroshiSuemizu
CentralInstituteforExperimentalAnimals,Kawasaki,Kanagawa,Japan
LloydWeiTatTang DepartmentofPharmacy,FacultyofScience,NationalUniversityofSingapore,Singapore, Singapore
RaineE.S.Thomson
SchoolofChemistry&MolecularBiosciences,TheUniversityofQueensland,Brisbane, QLD,Australia
RachelF.Tyndale
DepartmentofPharmacologyandToxicology;CampbellFamilyMentalHealthResearch Institute,CAMH;DepartmentofPsychiatry,UniversityofToronto,Toronto,ON,Canada
ShotaroUehara
CentralInstituteforExperimentalAnimals,Kawasaki,Kanagawa;ShowaPharmaceutical University,Machida,Tokyo,Japan
YasuhiroUno
JointFacultyofVeterinaryMedicine,KagoshimaUniversity,Kagoshima,Japan
HiroshiYamazaki ShowaPharmaceuticalUniversity,Machida,Tokyo,Japan
Preface
Theyear2022representsthe60thanniversarysincethefirstarticleon cytochromeP450(P450)waspublishedby OmuraandSato(1962).Itis apleasure tocelebrate60yearsofmeaningfulresearchonmanyformsof P450thatexistinmicroorganisms,plants,animals,andhumans.P450shave beenafocusofattentioninmanyindustrialbioengineeringapplicationsand thesynthesesofuniquehumandrugmetabolitesinpharmaceuticaldevelopment.ResearchonP450sinanimalmodelswithintroducedhuman P450 (CYP) genesortransplantedhumanizedliverandwithnonhumanprimates hasextendedintodifferentfields,frommoleculestoinvivosituations,by attractingpharmacologists,toxicologists,andbiochemists.
P450electrontransportismediatedbyamulticomponentmonooxygenasesystemwithreducednicotinamideadeninedinucleotide phosphate(NADPH).MicrosomalP450sreceiveelectronsfromNADPHcytochromeP450reductase.ThecatalyticcycleofP450involvesthe activationofmolecularoxygentoareactiveform(seeFig.3in Chapter1). Inadditiontothebasicscience,researchondrugmetabolism in liverandextrahepaticorgans(e.g.,brain)inanimalmodelsandhumans hasexpandedtooneofimportantareasinclinicalpharmacologyand toxicology.P450researchhasdevelopedfromthestudieswithratliver inthe1960stopersonalizedmedicine,mediatedbystudiesofpolymorphic P450sinindividualpatientsincludingpediatricsandtheelderlyinthe 21stcentury,asdiscussedinthisbookseries.
Theextensivecontributionsofscientiststhroughouttheworldtothe P450 research fieldoverpastsixdecadesshouldbenoted.Thesuccessof P450researchhashadimplicationsinfieldssuchasherbalmedicine,drug interactions,pharmacogenetics,pharmacogenomics,andphysiologically basedpharmacokineticmodeling.Thebasicprincipleforthisbookseries comprisesthreeparts:(i)collectionofacomprehensivecoverageofmajor progressin60yearsinpharmacologyandtoxicology,(ii)discussionfor futuredirectionsoftheresearchonP450s(especiallyforbetterpharmacotherapyinhumans),and(iii)theinvitationtoyoungscientiststojointhis importantandexcitingbasicandadvancedworldofP450.
Thevolumesin AdvancesinPharmacology arepartofaseries.Ihopethat thisbookserieswillstimulateandencouragemanyyoungscientistsinP450 researchtotrynewmethodsandapproaches.Historicalachievementson
P450researchinformertimescannotbedismissedinnewstudies.Forexample,humandrug-metabolizingP450s(inphospholipidmembranes)require anotherprotein,NADPH-cytochromeP450reductase,inappropriateionic strengthenvironmentstoexerttheircatalyticfunctionsofaerobicmetabolisminthepresenceofNADPH.Asthevolumeeditor,Iwillbedelightedif ourbookseriescanextensivelyfacilitatenewresearch.
HIROSHI YAMAZAKI ShowaPharmaceuticalUniversity,Tokyo,Japan
Reference
Omura,T.,&Sato,R.(1962).Anewcytochromeinlivermicrosomes. JournalofBiological Chemistry, 237,1375–1376.
InMemoriam—TsuneoOmura
(byF.PeterGuengerich,BettieSueS.Masters,Ken-Ichirou Morohashi,MasahikoNegishi,andHiroshiYamazaki)
Thebiochemicalcommunity,especiallyhiscolleaguesinthefieldof cytochromeP450,lostoneofitstruepioneerswiththedeathof Prof.TsuneoOmuraonJanuary29,2022.Hediscoveredcytochrome P450inhisworkwiththelateProf.RyoSatoatOsakaUniversity, andaClarivatesearchindicatesthata JBC paper(J.Biol.Chem. 239, 2370–2378,1964)describingtheworkhasbeencitedatleast12,700times. TsuneoOmurawasHonoraryMemberoftheASBMB,adistincthonor.
TsuneoOmurawasbornonJuly29,1930,inShizuokaPrefecture, Japan.HegraduatedfromtheUniversityofTokyowithaBSinchemistry. HethenworkedasaninstructorandlecturerinchemistryatShizuoka University.Thecourseofhisdoctoralworkandadvancementwasrather uniquecomparedtoourcurrentsystems,butin1960hejoinedProf. RyoSato’slaboratoryattheOsakaUniversityInstituteforProtein ResearchasAssociateProfessor.In1961,hewasawardedaDScinbiochemistryfromtheUniversityofTokyo,basedontheworkhehadperformedat ShizuokaUniversity.Itwasduringtheearly1960sinOsakathatOmura
andSatopublishedthreemajorpapersaboutthediscoveryofP450(includingthemosthighlycitedoneinthe JBC),plussevenothersinrelated areas.From1964to1966,OmurawasavisitingscientistattheJohnson FoundationoftheUniversityofPennsylvania(withRonaldW.Estabrook) andthenRockefellerUniversity(withPhilipSiekevitz).Hereturnedto theOsakaInstituteforProteinResearchandthenmovedin1970tothe positionofProfessorofBiologyandMolecularBiologyatKyushu University,apositionheheldthroughouthiscareeruntilheassumed Emeritusstatusin1994.From1995to1997,hewasVisitingProfessorof BiochemistryatVanderbiltUniversity(withMichaelR.Watermanand others).
Prof.OmuramademanycontributionstothefieldofP450research throughouthiscareer.TheseincludestudiesontheregulationofP450s and,inparticular,traffickingofP450sinboththeendoplasmicreticulum andmitochondria.HisstudieswithmitochondrialP450s,specificallythe cholesterolsidechaincleavageenzyme,ledtoanenhancedunderstandingoftheregulationoftheseP450sbyproteinssuchasAd4BP/SF-1,a steroidogenictranscriptionfactor.
Notsurprisingly,Prof.Omurawasaleadingfigureinbiochemistryin Japan,andmanyofhisstudentswentontoveryproductivecareers. AlongwithHonoraryASBMBMembership,Omurareceivedthefirst R.T.WilliamsAwardfromtheInternationalSocietyfortheStudyof Xenobioticsin2001,andhewasalsohonoredatthe1994International MicrosomesandDrugOxidations(MDO)meeting.Omuracontinuedto attendandactivelyparticipateinmeetingsmanyyearsafterhisretirement. Hepresentedaplenarylectureatthe2018MDOmeetinginKanazawa. Tributeswerealsomadetohimataspecial2012meetinginFukuoka, commemorating50yearssincehisdiscoveryofcytochromeP450.
TsuneoOmurawillberememberedasahumbleandverythoughtful man.Hewasveryfriendly,communicative,andalwaysveryanxiousto helpyoungscientistsandlendhisadvice.Hislaboratorywasalwaysopen tovisitorsfromabroad,andhewasveryhappytohelppeoplethroughout the91-plusyearsofhislife.Manyvisitorsrecallhisjoyindrivinghisguests allaroundKyushuwithmanystopsatpottery-makingartisansandnotable sites,includingtheactivevolcano,Mt.Aso.Duetohiswarmpersonality anderuditeknowledge,manystudentswereattractedtohim.During 24yearsofhistenureinKyushuUniversity,112undergraduatestudents and42graduatestudentsjoinedhislaboratory,and33ofthemtookPhD degreesunderhisthoughtfulandpersistentguidance.Allthestudents
spentmeaningfulandvaluabletimeinProf.Omura’slaboratory,andhe createdanatmosphereofcamaraderieandmutualrespect.Hewasatrue sensei ineverysenseofthisJapanesetitleofhonor.
Prof.Omurawasprecededindeathbyhiswife,Yone(December9, 2000),andissurvivedbytheirthreechildren.Obviously,hewasloved bymanyscientistsinthefield,andhewillbemissed.
F.PeterGuengerich∗
DepartmentofBiochemistry,VanderbiltUniversitySchoolofMedicine,Nashville,TN,UnitedStates
∗Correspondingauthor:e-mailaddress:f.guengerich@vanderbilt.edu
Contents
1. Introduction3
2. WhereistheP450fieldtodayandwhatdoweknow?4
2.1 RolesofindividualhumanP450s4
2.2 AbundanceofP450s5
2.3 Regulation7
2.4 Catalyticmechanism9
2.5 StructuresofP450sandbindingofligands11
3. P450sanddrugmetabolism13
3.1 P450sandpharmacokineticissues13
3.2 Drug-druginteractions16
3.3 Toxicityissues26
4. P450sasdrugtargets29
4.1 CurrentP450inhibitorsinuse29
4.2 FutureprospectsforP450inhibition32
4.3 Pestcontrol32
4.4 Targetingaccessoryenzymes34
5. ThefutureofP450research34
5.1 Recentdevelopments34
5.2 Questionsregardingbasicresearch35
5.3 Practicalquestionstobeaddressed35
6. Conclusion37 Acknowledgments37 Conflictofintereststatement38 References38 AdvancesinPharmacology,Volume95Copyright # 2022ElsevierInc. ISSN1054-3589Allrightsreserved. https://doi.org/10.1016/bs.apha.2021.12.001
Abstract
ThedevelopmentofthecytochromeP450(P450)fieldhasbeenremarkableintheareas ofpharmacologyandtoxicology,particularlyindrugdevelopment.Todayitispossible tousetheknowledgebaseandrelativelystraightforwardassaystomakeintelligent predictionsaboutdrugdispositionpriortohumandosing.Muchisknownaboutthe structures,regulation,chemistryofcatalysis,andthesubstrateandinhibitorspecificity ofhumanP450s.Manyaspectsofdrug-druginteractionsandsideeffectscanbeunderstoodintermsofP450s.Thisknowledgehasalsobeenusefulinpharmacypractice,as wellasinthepharmaceuticalindustryandmedicalpractice.However,therearestill basicandpracticalquestionstoaddressregardingP450sandtheirrolesinpharmacologyandtoxicology.AnotheraspectisthediscoveryofdrugsthatinhibitP450totreat diseases.
Abbreviations
Adx adrenodoxin
AhR arylhydrocarbonreceptor
AO aldehydeoxidase
ARNT arylhydrocarbonreceptornucleartransferase
AUC area-under-the-curve
b5 cytochrome b5
CAR constitutivelyactivereceptor
COMT catechol O-methyltransferase
DDI drug-druginteractions
EGFR epidermalgrowthfactorreceptor
FDA (UnitedStates)FoodandDrugAdministration
FMO flavin-containingmonooxygenase
HNF hepaticnuclearfactor
IND InvestigationalNewDrug(application)
Kd dissociationconstant
Ki inhibitionconstant
Km Michaelisconstant
LC-MS combinedliquidchromatography-massspectrometry
MIST metabolitesinsafetytesting
NME newmolecularentity
NMR nuclearmagneticresonance(spectroscopy)
NC non-classical(congenitaladrenalhyperplasia)
P450orCYP cytochromeP450
PDB ProteinDataBank
Pgp P-glycoprotein
POR NADPH-cytochromeP450oxidoreductase
PPAR peroxisomeproliferator-activatedreceptor
PXR pregnaneXreceptor
RAR retinoicacidreceptor
RXR retinoidXreceptor
SNV singlenucleotidevariant
SV simplevirile(congenitaladrenalhyperplasia)
SW salt-wasting(congenitaladrenalhyperplasia)
TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene
UGT uridinediphosphateglucuronosyltransferase
1.Introduction
ThefieldofcytochromeP450(P450orCYP)researchhaditsorigin instudiesonthemetabolismofdrugs,steroids,andcarcinogensinthemiddleofthe20thCentury(Axelrod,1955; Mueller&Miller,1948; Ryan, 1959).However,thediscoveryofP450assuchdidnotoccuruntilafew yearslater(Klingenberg,1958; Omura&Sato,1962,1964).Theevidence foraroleastheterminaloxidaseinahydroxylationdevelopedwiththe17αhydroxylationofasteroid(Cooper,Levine,Narasimhulu,Rosenthal,& Estabrook,1965).StudiesonabacterialP450byGunsalusdeveloped independently(Hedegaard&Gunsalus,1965; Katagiri,Ganguli,& Gunsalus,1968),andthatsystem(P450cam,orCYP101A1)servedasauseful modelformanyyears(Mueller,Loida,&Sligar,1995).Twopapersin1968 and1969byLuandCoonestablishedtheidentityofthelivermicrosomal P450systeminvolvedinoxidations,consistingofthreecomponents:aP450, NADPH-P450reductase(POR),andphospholipid(Lu&Coon,1968; Lu, Junk,&Coon,1969).
Moreaboutthehistoricaldevelopmentofthefieldwasdescribedina recentreview(Guengerich,2019a).Withconsiderableeffort,manyliver P450s(andsomeextrahepaticones)werepurifiedbyconventionalchromatographymethodsandcharacterized.Progresswasalsomadeintermsof mechanismsofcatalysisandgeneregulation.TheintroductionofrecombinantDNAtechnologyledtocloningofcDNAs,expressionofP450sinheterologoussystems,andultimatelyabetterunderstandingofthecomplexity oftheP450SuperfamilywiththecompletionoftheHumanGenome Project.TodaythefieldofP450researchmustbeconsideredasmature, butthatisnottosaythatallimportantquestionshavebeenanswered.As afieldmatures,thebackgroundknowledgeandtheresearchtoolsimprove andmoreimportantquestionscanbeaddressed.
ThefocusofthisbookisonpharmacologyandtherolesofP450enzymes inthemetabolismofdrugs.However,P450salsohaveimportantrolesin themetabolismofsteroids(someofwhichareusedasdrugs),fat-soluble vitamins,fattyacids,chemicalcarcinogens,pesticides,industrialchemicals, foodadditives,andotherchemicals.Collectively >90%ofalloxidationsand reductionsofchemicalsknowntodayarecatalyzedbyP450s(Rendic& Guengerich,2015).ThishighpercentageisalsoinpartduetothepreponderanceofP450reactionsinthebiosynthesisofnaturalproducts (Guengerich,2022b),aswellasdrugsandindustrialchemicals.P450sare foundthroughoutnature,withtheonlycurrentexceptionsbeingsome entericbacteria(e.g., Escherichiacoli, Salmonellatyphimurium).Thenumber of CYP genesinbacteriaandplantsprobablyexceedsthenumberinmammals,inlargepartbecausemostplantshavehundredsandsometimes >1,000 CYP genes(e.g.,wheathas1,285).
2.WhereistheP450fieldtodayandwhatdoweknow?
Thisisanintroductorychapter,andseveralotherchapterswillfocus onsomedetailedaspectsofP450science.Thefocusherewillbeonhuman P450s,althoughtheP450sinexperimentalanimalsarealsostillofgreat interestinthedrugdevelopmentprocess.
2.1RolesofindividualhumanP450s
Oneofthewaysofgroupingthe57humanP450sbyfunctionispresentedin Table1.Ofthese,itisnotclearthat CYP2A7 isexpressedbut CYP4F3 yieldstwoproteins,sothenumberisstill57.Theclassificationbysubstrates isnotwithoutcaveats.SomeP450scanbeclassifiedundermultipleheadings (e.g.,1B1forsteroidsandxenobiotics,27A1forsteroidsandvitamins). SomeoftheP450shavemovedfrom“orphan”status(Unknownin Table1)butitisnotclearhowimportantthesereactionsare(e.g.,2U1, 2S1).P4504X1canslowlyoxidizeanandamide(Stark,Dostalek,& Guengerich,2008)buthasbeenleftintheUnknowncolumn.Itisnotclear howimportantmostofthereactionsofXenobioticsandFattyacidsareto mammalianphysiology.ThepointcanbemadethattheP450sinthe Xenobioticscolumnhaveageneralfunctionofclearingawidevarietyof ingestednaturalproductspresentinourfood(e.g.,terpenes,alkaloids)as ageneralprotectivemechanism,inthesamewaythatexporttransporters do.Studieswithtransgenicmicehaveshownthattheorthologsofmany oftheP450slistedundertheheadingsofXenobioticsandFattyacidsare
Table1 ClassificationofhumanP450sbasedonmajorsubstrateclass. Eicosanoids
SteroidsXenobioticsFattyacidsEicosanoidsVitaminsUnknown
1B1* 1A1* 2J22U12R1* 2A7
7A1* 1A2* 2S14F224A1** 4X1
7B12A6* 2U14F326A120A1
8B12A13* 4A114F826B1
11A1* 2B6* 4A225A127A1
11B1* 2C8* 4B1** 8A1* 27B1
11B2* 2C9* 4F11 27C1
17A1* 2C184F12
19A1* 2C19* 4F22
21A2* 2D6* 4V2
27A12E1* 4Z1
39A12F1
46A1* 2W1
51A1* 3A4* 3A5* 3A7* 3A43
Thisclassificationissomewhatarbitraryinsomecases,e.g.,P450s1B1and27A1could begroupedineitheroftwodifferentcategories. *Crystalstructureavailable. **Crystal structureofanimalorthologueavailable.
notessential(Bissigetal.,2018; Gonzalez&Kimura,2003).However,those involvedinthemetabolismofsteroids,eicosanoids,andvitaminsgenerally areessential.
2.2AbundanceofP450s
Ofthe57P450s(Table1),50areexpressedmainlyintheendoplasmicreticulumandsevenareexpressedbynucleargenesbuttransported(following proteolysis)tothemitochondria(11A1,11B1,11B2,24A1,27A1,27B1, 27C1).Fractionsofsomeoftheendoplasmicreticulum(microsomal) P450sarecleavedandalsoenterthemitochondria(e.g.,1B1,2D6,2E1, 2C8)(Avadhani,Sangar,Bansal,&Bajpai,2011).ThemicrosomalP450s
receiveelectrons(fromNADPH)viathediflavinproteinPORandsometimescytochrome b5 (b5).Thoseinthemitochondriauseasysteminvolving theflavoproteinNADPH-adrenodoxin(Adx)reductaseandAdx.Although themitochondrialP450sclearlyhaveimportantrolesinthemetabolismof steroidsandvitamins(Table1)(Guengerich,2015),insomecasestheycan alsobeinvolvedinthemetabolismofdrugs(Zhangetal.,2012)andother chemicals.
InmammalianlivertheratiooftotalP450toPORhaslongbeenknown tobe10–20:1(Estabrook,Franklin,Cohen,Shigamatzu,&Hildebrandt, 1971).TheconcentrationsofseveralP450sinhumanliverhavebeenestimatedusingimmunochemical(Shimada,Yamazaki,Mimura,Inui,& Guengerich,1994)and,morerecently,massspectrometryproteomic approaches(Achour,AlFeteisi,Lanucara,Rostami-Hodjegan,&Barber, 2017).Theresultsfromseveralstudiesaresummarizedin Fig.1.Whileit isclearthatP4503A4andtwoP450Subfamily2Cenzymes(2C8,2C9) arethemostabundant,thereisalargeamountofvariability,evenincases
Fig.1 PercentagesoftotalP450inhumanliversamplesaccountedforbyeachP450. Thedatapointswerecompiled(Guengerich,2022a)fromfoursetswithmultipleliver samples(Achour,Russell,Barber,&Rostami-Hodjegan,2014; Kawakamietal.,2011; Shimadaetal.,1994)andonewithasingleliversamplehighinP4501A1(Lang, Radtke,&Bairlein,2019).Theestimatesweremadeimmunochemicallyinonecase (Shimadaetal.,1994)andbyLC-MSproteomicmethodsintheothers(Achouretal., 2014; Kawakamietal.,2011; Langetal.,2019).ThevalueforP4501A1isameanofmeasurementsof30samples(Langetal.,2019).Theindividualcolorshavenomeaningbut areaddedtofacilitatevisualization.
wherethesameliversetswereanalyzed(Guengerich,2015).Forinstance,it isnotclearwhetherP450s2A6and2B6shouldbeconsideredminoror abundantenzymes(Fig.1)(Guengerich,2015).
ThecompositionofindividualP450sinhumansmallintestinehasalso beenanalyzed(Paineetal.,2006).Inthisorgan,thedominanceofP450 3A4isevenmorestrikingandthishasrelevanceinconsideringthedispositionofonlyadministereddrugsandinhibitionofdrugmetabolism.The totalamountofP450inthesmallintestineisonlyafewpercentofthat inliver,however,andthispointneedstobeconsideredinthecontextof first-passclearance.
2.3Regulation
ManyoftheP450saresubjecttoenzymeinduction,aswellaslocalizationin differenttissuesduetotheinfluenceoftissue-specificpromoters.Ageneral schemeforinduction(Fig.2)involvesbindingofaligandtoareceptor,formationofaheterodimericpair,nucleartransport,andbindingtospecific sitesofthegenetocause(chromosomerearrangementand)enhanced
Fig.2 GeneralschemefortranscriptionalregulationofP450s.L:ligand,R:receptor, R´-heterodimericpartner,Coactiv:co-activatorprotein(e.g.,hepaticnuclearfactor (HNF) α inthecaseofP4503A4),RNApol:RNApolymerase(Guengerich,2018a,2022a).
transcriptionbyRNApolymerase(Fig.2).Thisisthegeneralpatternseen fortheAhR,PXR,PPARα,andRARsystemsofgeneregulation.With AhRtheheterodimerpartnerisARNT.Withthebulkofthesystems, whichusereceptorsfromthesteroidnuclearreceptorsuperfamily(PXR, PPARα, …),thepartnerisRXR,whichisboundtoretinoicacidor possiblyanotherligand.CAR,involvedinregulationofP450s2B6and 3A4,isdifferentinthatwhileitcanbindsomeligands(e.g.,1,4-bis[2(3,5-dichloropyridyloxy)]benzene,(TCPOBOP)(Maglichetal.,2003), inmostcasesthereceptorisconstitutivelyactiveandnuclearimportis regulatedbyaphosphorylationcascadeinvolvingEGFR(Mutohetal.,2013).
Inductionbydrugsisimportantforseveralreasons:(1)Itleadstochanges inpharmacokineticswhenthedrugofinterestisalsoaninducer. (2)Drug-druginteractionscanbeimportantclinically,asseenintheclassic exampleofenhancedmetabolismof17α-ethnylestradiol(inoralcontraceptives)byP4503A4inducers(Bolt,Kappus,&Bolt,1975).(3)Insomeanimalmodels,enzymeinductioniscorrelatedwithdevelopmentofcertain cancers,particularlyinrodentliver(e.g.,barbiturates,PPARα inducers) (Lubet,Nims,Ward,Rice,&Diwan,1989; Rao&Reddy,1987). Althoughthisismuchlessofaregulatoryconcernthaninthepast,thedevelopmentofrodenttumorsinthedrugdevelopmentscenariomustbe explainedandregulatoryagenciesneedassurancethatthiswillnotbean issueinhumans.
ThemostthoroughlystudiedmodelofP450inductionistranscriptional control.However,regulationcanalsobeatthepost-translationallevel,includingmRNAandproteinstabilization,andepigeneticcontrol.Examplesofroles ofgenemethylation(i.e.,5-methyldeoxycytidine),histonemodification(e.g., acetylation),andmicroRNAinvolvementarenowknownforP450s (Guengerich,2015; Ingelman-Sundbergetal.,2013),althoughthesignificanceinhumansisstillnotestablished.
P450genescanalsoberegulatedbycytokines.InterferonscandownregulateP450s,andthesuppressionofdrugmetabolismbyinterferonshas longbeenknowntobeassociatedwithcolds,flushots,etc.(Mannering, Renton,elAzhary,&Deloria,1980; Renton,1981).Anotherphenomenon observedinratsisthedown-regulationofsomeP450sbysomeofthecommoninducers,e.g.barbituratesandparticularlyFamily1inducers,asseen particularlywithP450s2C11and2E1(Dannan,Guengerich,Kaminsky,& Aust,1983; Guengerich,Dannan,Wright,Martin,&Kaminsky,1982; Thomas,Bandiera,Maines,Ryan,&Levin,1987).Thissuppressionhasbeen showntooccuratthetranscriptionallevel(Sawaya&Riddick,2008)butits relevanceinhumansisunknown.
RodentsdisplaydramaticsexeffectswithregardtoP450regulation (Waxman,Dannan,&Guengerich,1985; Waxman&Holloway,2009). Thebasisofthisiscomplexandinvolvesnotonlyandrogensandestrogens butalsopulsatilepatternsofgrowthhormoneandJAK/STATregulation (Waxman&Holloway,2009; Wiwi&Waxman,2005).Althoughthere aresomereportsofsexdifferencesinsomeP450sinhumans(Wolbold etal.,2003; Zhangetal.,2011),thedifferenceshavenotbeenseenbyothers (Yangetal.,2010)and,atthepharmacokineticlevel,maybeattributableto bodyfat.However,knowledgeofsexdifferencesinrodentP450smaybe importantinunderstandingtheresultsofpre-clinicaltestingindrug development.
2.4Catalyticmechanism
MuchhasbeenwrittenaboutchemicalmechanismsofcatalysisbyP450s elsewhere(Guengerich,2018b; Guengerich&Yoshimoto,2018; Ortiz deMontellano,2015).
Thecatalyticcycleisshownin Fig.3,wheretheP450bindssubstrate (Step1),theironisreduced(Step2),oxygenbinds(Step3),andthesecond electronisdonatedtotheiron(Step4).Atthispointtheintermediatesare unstable,andinformationaboutthemhastakensometimetoaccumulate. TheFe3+-O2 – form(calledCompound0)becomesprotonated(Step5)and thenH2OisreleasedtoleaveCompoundI(afterStep6).InStep7theformalFeO3+ complexabstractsahydrogenatom(oranelectronfroma heteroatomiftheredoxpotentialislowenough)toleavea“caged”radical (Step7),whichundergoesrecombinationwithCompoundII(FeOH3+)to generatetheproductinStep8.Finally,theproduct(ROH)isreleasedin Step9.
TheP450canbereducedwithouthavingsubstratespresent,atleastwith someP450s(Guengerich&Johnson,1997; Johnstonetal.,2011).Insome casesthereisevidencethat b5 providesthesecondelectron(inStep4)butin othercases b5 canstimulateP450reactionswithoutelectrontransfer (Yamazakietal.,2002).Anotherpointisthatthecyclein Fig.3 relatesonly totheelectronicchangesthatoccur,butnumerouschangesinproteinstructureoccuraswell,andevenbindingofasubstratecaninvolveacomplex seriesofsteps(Guengerich,Wilkey,Glass,&Reddish,2019; Guengerich, Wilkey,&Phan,2019; Isin&Guengerich,2006).
AnappreciationofthecatalyticmechanismofP450isimportantin understandingthekindsofreactionsthatP450scando.Inareassuchas drugmetabolismandnaturalproductbiosynthesis,productsmustbe
NADPH-P450 reductasered
NADPH-P450 reductaseox
Compound I
Compound 0
Fig.3 P450catalyticcycle.Theninelabeledstepsshowsequential(1)substratebinding,(2)1-electronreduction,(3)oxygenbinding,(4)second1-electronreduction,(5)protonationof “Compound0,” (6)lossofwatertoform “CompoundI,” (7)hydrogenatom abstractionbyCompoundI,(8)oxygenreboundtoformproduct,and(9)productdissociation.Asindicated,ferrousP450canalsobindsubstrate(Yun,Kim,Calcutt,& Guengerich,2005).Insomecases, b5 canprovidetheelectroninstep2or4.Insome sequentialreactions,step9doesnotoccurandasecondoxidationoftheinitialproduct isobserved(Gonzalez&Guengerich,2017; Reddish&Guengerich,2019).
characterizedandaknowledgeofpossiblemechanismsisneededtodiscern possiblepathways(Guengerich&Yoshimoto,2018; Isin&Guengerich, 2007a).
Althoughpossibilitieshavebeenraisedofvariousotheroxidantforms ofP450invariousoxidations,almostallreactionscanbeexplainedby involvingCompoundIreactions.SomeproposalsforCompound0orother specieshavebeenre-valuatedoranalyzedfurtherandre-interpretedinterms ofCompoundI(Groves,McClusky,White,&Coon,1978; Guengerich& Yoshimoto,2018; Huang&Groves,2017; Krestetal.,2013; Rittle& Green,2010; Yoshimoto&Guengerich,2014).Onlyinafewcaseshas P450CompoundIbeenprepareddirectlyandrigorouslycharacterized (byreactionwithaperacid)(Krestetal.,2013; Rittle&Green,2010). SomebonafideBaeyer-Villiger-typeoxidationsmaystillprovetoinvolve Compound0(Guengerich,2022b).
2.5StructuresofP450sandbindingofligands
AlthoughX-raycrystallographyofP450swaslimitedtosolublebacterial P450sbefore2000,theworkofJohnson(Williams,Cosme,Sridhar, Johnson,&MeRee,2000)andthenothershasledtoaplethoraofP450structures.Asof2021therewereatleast260structuresofmammalianP450sinthe ProteinDataBank,and25ofthe57humanP450shavecrystalstructures available(plusapparentanimalorthologuesofP4504B1and24A1).
AllP450structurestodatehavesimilaroverallfolds(Fig.4).TheinteractionandmovementsamongtheI,F´,andG´helicesareimportantin modulatingligandspecificity.
SomeofthehumanP450shavebeencrystallizedinopen,closed,and intermediateforms(Guengerich,Waterman,&Egli,2016; Poulos& Johnson,2015).AsinglestructureofaP450providesusefulinformation aboutthebondingofaP450withasubstratebutitmaynotpresentapicture ofhowtheP450boundthatsubstrate,i.e.thecourseofeventsleadingto (productive)binding.SomeoftheP450shavebeenfoundtobindsubstrates inmultiplewaysandalsotohavemultipleconformationsintheabsenceofa substrateorotherligand(Ekroos&Sj € ogren,2006; Hsu&Johnson,2019; Porubsky,Battaile,&Scott,2010).
OnehypothesisabouthowenzymessuchasP450sareabletobindso manysubstratesisthatofinducedfit(Fig.5)(Koshland,Nemethy,& Filmer,1966);i.e.bindingofasubstratetoanenzymeinducestheenzyme
Fig.4 AstructureofP4503A4(ProteinDataBank(PDB)1TQN),withmajorhelices labeled(Yanoetal.,2004).Thehemeprostheticgroupisshowningray.
Induced fit hypothesis:
E + S ES E'S EP E + P
Conformational selection hypothesis:
E + S E' + S
ES E'S EP E + P
Fig.5 Hypothesestoexplaincomplexsubstraterecognitiondata(Gianni,Dogan,& Jemth,2014; Vogt&DiCera,2012).
toadoptanewconformationthatismorefavorableforproductivecatalysis. Analternativemechanisminvolvesconformationalselection(Fig.5),where theenzymeexistsinmultipleconformationsintheabsenceofligand,one (ormore)ofwhichbindsthesubstratetoyieldaproductivecomplex (Fig.5).Thesearenotnecessarilycompletelydistinctphenomenaandmay occurtogether.Discerningwhichcourse(Fig.5)isdominantisusuallydifficult,inthatthefreeenergyinvolvedintheroutefromEtoaproductiveE´S complexisidenticalregardlessoftheroute(Chakraborty&DiCera,2017; Vogt,Pozzi,Chen,&DiCera,2014).Onehallmarkofthepresenceof complexbindingpathwaysisslowkinetics,i.e.atlessthandiffusion-limited rates( Johnson,2019).Thetworoutes(Fig.5)canbedistinguishedbymeasuringthekineticsofbindingasafunctionofvaryingtheconcentrationof ligand,enzyme,orboth(Giannietal.,2014; Vogt&DiCera,2012).Such kineticstudieshavebeendonewithseveralhumanP450sandindicatethe dominanceofconformationalselectionpathways(Guengerich,Wilkey, Glass,&Reddish,2019; Guengerich,Wilkey,&Phan,2019).Thebinding ofthepreferredsubstratecamphortobacterialP450cam (P450101A1)appears tobeanexception(Guengerich,Child,Barckhausen,&Goldfarb,2021),but theconformationalselectionmechanismappearedtobemoredominantwith alternatesubstratesofP450cam
ThebindingofinhibitorstoP4503A4hasbeenshowntobeacomplex process,withmultiplestepsandspectrallydetectableintermediates(Fig.6) (Guengerichetal.,2020; Isin&Guengerich,2007b).AchievingfullinhibitionrequirescompletionofthestepsforP4503A4(i.e.,theE*Icomplex in Fig.6).WithP45017A1,multiplespectralintermediatesareseenupon mixingbutinhibitionoccursimmediately,beforethespectralchangesare
Fig.6 SchemesummarizinginteractionofP4503A4withinhibitors.Thetimesof appearanceofindividualspeciesareindicatedinblue(Guengerich,McCarty,& Chapman,2020).
completed(Fig.6)(Child&Guengerich,2020; Guengerich,McCarty, Chapman,&Tateishi,2021).Thedifferencemaybeduetothelargesize oftheactivesiteofP4503A4( 1400A ˚ 3 (Yanoetal.,2004)),whichisable toaccommodatetwomoleculesoftheinhibitorketoconazole(Ekroos& Sj € ogren,2006).NocrystalstructureofaP450containing both asubstrate andinhibitorhasbeenpublishedbutiscertainlyfeasibleforP4503A4 andprobablysomeotherP450s.
3.P450sanddrugmetabolism
IntheearlyhistoryofP450research,littleinformationwasavailable abouthowmanyP450sexisted,howmanyhadmajorrolesindrugmetabolism,andwhichoftheseP450smetabolizedindividualdrugs.Todaythe humanP450sareallknown(Table1),withthecompletionofthehuman genomeandrecognitionoftheP450signaturesequence:
Phe X X Gly X Arg Xb Cys X Gly
wheretheCysisligandedtothehemeironatomandXb isabasicresidue. P450sareinvolvedinthemetabolismof ¾ of(smallmolecule)drugs (Fig.7),andaboutfiveP450sareinvolvedwith90%ofthedrugs (Guengerich,2015; Rendic&Guengerich,2015).Thosefractionshave remainedsimilarfornewdrugs,withP4503A4playinganevenmoredominantrole(Fig.7).Thistrendmaybedue,atleastinpart,to(i)atendency towardslargermolecules,ineffortstoachieveselectivityandpotency,and (ii)effortstoavoiddependenceontheP450sshowingmoregeneticpolymorphism(e.g.,2C19and2D6).
3.1P450sandpharmacokineticissues
Oneissueindrugdevelopmentispredictionofsitesofmetabolism.Over theyearstherehasbeensomeprogressinthe insilico predictionofsites
Fig.7 FractionsofsmallmoleculedrugsapprovedbyUSFDAin2015–2020metabolizedbyindividualenzymes(Bhutanietal.,2021).UGT,uridinediphosphate glucuronosyltransferase;FMO,flavin-containingmonooxygenase;AO,aldehydeoxidase. ReprintedfromBhutani,P.,Joshi,G.,Raja,N.,Bachhav,N.,Rajanna,P.K., Bhutani,H.,etal.(2021).USFDAapproveddrugsfrom2015-June2020:Aperspective. JournalofMedicalChemistry,64(5),2339–2381,Copyright(2021),withpermissionfrom theAmericanChemicalSociety.
(Afzeliusetal.,2007; Boyeretal.,2007; deBruynKops,Sicho,Mazzolari,& Kirchmair,2021; Ekinsetal.,2005; Kirchmairetal.,2015; Martiny& Miteva,2013; Wilson,White,&Mueller,2003),especiallyifthe“top three”sitesareallpredicted.Muchofthesuccesshasbeenachievedwith algorithmsbasedonpriorexamples,asopposedtodockingintoX-raystructures.Nevertheless,therewillprobablyalwaysbesomesurprisesregarding insilicopredictions,e.g.testosteroneishydroxylatedbyP4503A4mainlyat the6β (aswellas2β,1β,and15β)carbonbut4,5-dihydrotestosteroneis hydroxylatedatthe(chemicallymoreinert)18-and19-methylcarbons (Cheng,Sohl,Yoshimoto,&Guengerich,2012).
Asmoleculesprogressinthediscovery/developmentprocess,theydo requiretheuseofanalyticalchemistrytodefinestructuresofmetabolites. ProgressinthepastthreedecadesinLC-MSandNMRhasgreatlyimproved theprocess,andtherearenoveltechniqueswithpossibilities,suchascrystallizationandX-raydiffractionoftrappedcompounds(Rosenberger etal.,2020).
WhatismoredifficultisthepredictionofratesofmetabolismbyP450s, althoughthereareclaimstobeabletodothiswithartificialintelligence (Xiongetal.,2021).Thisisprobablyonlyrealisticinsituationswhere, forinstance,ratesareknownforcloseanalogsandtheeffectsofaddingsubstituentsaresubjecttoHammettanalysisorotherlinearfreeenergyrelationships(Burka,Guengerich,Willard,&Macdonald,1985).Ratesof(total) oxidativemetabolismcanbemeasuredinrelativelyhighthroughputassays withlivermicrosomesandLC-MS,however.Suchassayscanbedonewith hepatocytesbutnotasrapidlyorlarge-scale.Themicrosomalassaysarea rapidmeansofstratifyingfordrugstability.However,ifpharmacologically activeproductsareformed,theresultswillbemisleadingregardingthevalue ofadrugcandidate.
3.1.1Changingmoleculestoattenuatemetabolism
Whenaleaddrugismetabolizedtoorapidly,theremaybepossibilitiesfor slowingthemetabolism.Todothiseffectively,thesiteofoxidationshould beknown.IftheP450involvedinoxidationisknown,itispossibletodock themoleculetosuggestchangesthatmightpreventmetabolismorbioactivationwhilemaintainingpharmacologicalactivity(Brodneyetal.,2015). Strategiesmayinvolve(i)addingamoiety(atthesite)thatwillresistoxidation orpreventbindingtotheP450,(ii)substitutingdeuteriumforprotium(Gant, 2014; Pirali,Serafini,Cargnin,&Genazzani,2019; Stringeretal.,2014),or (iii)addinga“soft”siteelsewhereinthemoleculethat“steer”oxidationthere. Ofthese,thefirstoptionhasbeenthemostuseful.
3.1.2Variationsinpharmacokinetics
Inanidealworld,anewdrugwouldhavethesamemetabolites,half-life,and clearanceinallindividuals,andprescriptionswouldbeeasytodevelop. However,thereareseveralreasonsforvariablepharmacokinetics.
Oneissueisgeneticinter-individualvariability,i.e.geneticdifferences intheP450enzymes.Thisissueisdiscussedindetailinthechapter “PharmacogeneticsofthecytochromesP450:Selectedpharmacological andtoxicologicalaspects”byDaly.
Otherissuesinvolvechangesduetoenzymeinductionandinhibition. Thesecanbeduetothedrugitselfortootherdrugs,orevenchemicals foundinfoods(e.g.,grapefruit)orsocietalhabits(smoking,alcohol). Wheninductionandinhibitionareassociatedwiththedrugitself,thepharmacokineticsofthedrugcanbeexpectedtochangewithtime,eveninthe absenceofotherdrugs.
3.2Drug-druginteractions
Drug-druginteractionsareanimportantissueandaccountforbothasizeablefractionofhospitalizationsandhospitaldeaths(Montane,Arellano, Sanz,Roca,&Farre,2018).Theseproblemsareseenwithmanydiseases andtherapeuticareas(Fig.8A)(Yuetal.,2018).Alargefractionofthepharmacokineticdrug-druginteractionsareseenwithP4503A(4)andsomeof thedrugtransporters(Fig.8B).
Thecomplexityofdrugmetabolismmakesithardtototallyavoid drug-druginteractionsandsomeothertoxicityproblems,asexemplified inthemetabolismofphenacetinandacetaminophen(Fig.9).
Theanalgesicphenacetinisnolongerinusebecauseitwasassociated withratkidneycancers.ItundergoesoxidationinseveralP450-dependent reactions,someofwhichcanleadtothegenerationofreactiveproductsthat cancovalentlybindtoproteinsandDNA.Theproductof O-deethylationis acetaminophen,adrugusedextensivelyforfeverandpain.Acetaminophen isusedtherapeuticallyatleastonceperweekby 23%oftheUSpopulation (Larsonetal.,2005),withbenefit.However,itisalsoinvolvedin ½ ofthe casesofdrug-inducedliverfailure.
CancertreatmentsAntiviralsCardiovasculardrugsCNSagents Gastrointestinalagents Metabolismdisorder/endocrinologytreatmentsRespiratoryagentsAntifungals
Fig.8 Frequencyofnewmolecularentities(NMEs,i.e.newdrugcandidates)in inhibition-baseddrug-druginteractions(DDIs)withdrugsapprovedbytheFoodand DrugAdministration(FDA)intheUnitedStatesbetween2013and2016(Yu,Zhou, Tay-Sontheimer,Levy,&Ragueneau-Majlessi,2018).(A)Groupingbytherapeuticclass. (B)Groupingbyenzymesinvolved.PgpandOAT1B1aretransporters.COMT,catechol O-methyltransferase.
Protein adducts Methemoglobinemia?
Fig.9 RolesofP450sinthebioactivationanddetoxicationofchemicals:thecomplex exampleofphenacetin(Guengerich,2019a).Acetaminophen(paracetamol,Tylenol ®)is widelyusedasananalgesic,safeatlowdosesandhepatotoxicathighlevels(Lee, Buters,Pineau,Fernandez-Salguero,&Gonzalez,1996).Phenacetinhasbeenclassified asacarcinogenandwithdrawnfromuse.Themetabolismofacetaminophenhasbeen investigatedindetail(Dahlin,Miwa,Lu,&Nelson,1984; Dahlin&Nelson,1982; Guengerich,2022a).OnlyinafewcasesarethestructuresoftheproteinandDNA adductsknown.SomeoftheindicatedP450shavebeenidentifiedindifferentspecies, includinghumans(Distlerathetal.,1985; Leeetal.,1996).
Themetabolismofphenacetinisinduced(atleastP4501A2,the O-deethylase)bypolycyclichydrocarbonsandotherP450Family1inducers (AhRagonists)(Conneyetal.,1976; Pantucketal.,1974).Inhumans,the oxidationofacetaminophentoapotentiallytoxiciminoquinoneiscatalyzed mainlybythreeP450s—2E1,1A2,and3A4(Pattenetal.,1993).P4502E1
appearstodominate,andinductionofP4502E1byethanolisgenerally consideredtobethebasisofenhancedhepatotoxicityofacetaminophen inalcoholics(Lee&Kaplowitz,2021).
Drug-druginteractionscaneitherrenderadrugineffectiveorexaggerate thepharmacologyandmakeittoxic.
3.2.1Induction
Themostcommonproblemwithinductionisthelossofdrugefficacydueto enhancedmetabolismofadrug.Aclassicalexampleinvolvestheinduction ofP4503A4byrifampicinorbarbituratesandtheineffectivenessoforalcontraceptivesduetoenhancedclearanceof17α-ethynylestradiol(Bolt,Bolt,& Kappus,1977; Guengerich,1988).Thisphenomenoncontinuestooccur withotherbarbiturates(Wilbur&Ensom,2000)anditisalsoseenwithsome herbalmedicines(Halletal.,2003),inthatSt.John’swortcontainsapotential PXRinducer,hyperforin(Mooreetal.,2000).
P450inducersnotonlyposeproblemsintheclinicbutarealsoissuesin experimentalanimalsintheprocessofsafetyassessment.Somechemicals induceanimalP450sandcanconfoundpre-clinicalpharmacokineticstudies orleadtotoxicityproblems.Eventhoughtheissuesmaynotberelevantto humans,thoseissuesneedtobeexplainedtoregulatoryagencies,andthetestingmaywastevaluableresources.Moreover,someanimaltumorsareseen withcertainmodesofinduction(e.g.,PPARα),eveniftheyarenotrecognizedasbeingrelevanttohumanmedicalsituations.Overall,itisgenerally desirabletoadvancealeaddrugthatisnotaninducer,itthereisachoice andotherfactorsareequal.
3.2.2Inhibition
3.2.2.1Modesofinhibition
Inhibitionisaveryimportantfactorinpharmacokineticdrug-druginteractions.Thesubjecthasbeentreatedextensivelyelsewhere,andonlyabrief treatisewillbeprovidedhere.
AsimplewayofdividingP450inhibitorsisamong(i)reversibleinhibitors,(ii)quasi-reversibleinhibitors,and(iii)irreversibleinhibitors. Reversibleinhibitionisthemoststraight-forwardsituation.Itfollowsthe basicschemesgenerallytaughtinintroductorybiochemistry,i.e.competitive,non-competitive,uncompetitive,andmixedinhibition.Inthesimplest cases,twodrugsareboundtotheenzymeateitherthesameoratdifferent sites.Reversibleinhibitioncanbedetectedquicklywithhighthroughput screening.Themechanismsmaybemorecomplicatedthanjustsimplecompetitionforanactivesite,inthatmultipleligandoccupancyispossiblefor
