WhatisPharmacology?
Iwouldinparticulardrawtheattentiontophysiologiststothistypeofphysiologicalanalysisoforganic systemswhichcanbedonewiththeaidoftoxicagents ....
—ClaudeBernard(1813 78).
1.1AboutThisBook1
1.2WhatisPharmacology?1
1.3TheReceptorConcept3
1.4PharmacologicalTestSystems4
1.5TheNatureofDrugReceptors7
1.6PharmacologicalInterventionand theTherapeuticLandscape7
1.1ABOUTTHISBOOK
1.7System-IndependentDrug Parameters:Affinityand Efficacy10
1.8WhatisAffinity?13
1.9TheLangmuirAdsorption Isotherm13
1.10WhatisEfficacy?15
Essentiallythisisabookaboutthemethodsandtools usedinpharmacologytoquantifydrugactivity.Receptor pharmacologyisbasedonthecomparisonofexperimental dataandsimplemathematicalmodels,witharesulting inferenceofdrugbehaviortothemolecularpropertiesof drugs.Fromthisstandpoint,acertainlevelofunderstandingofthemathematicsinvolvedinthemodelsisuseful butnotimperative.Thisbookisstructuredsuchthateach chapterbeginswiththebasicconceptsandthenmoveson tothetechniquesusedtoestimatedrugparameters,and, finally,forthosesoinclined,themathematicalderivations ofthemodelsused.Understandingthederivationisnota prerequisiteforunderstandingtheapplicationofthemethodsortheresultingconclusion;theseareincludedfor completenessandareforreaderswhowishtopursue explorationofthemodels.Ingeneral,facilitywithmathematicalequationsisdefinitelynotrequiredforpharmacology;thederivationscanbeignoredwithoutanydetriment totheuseofthisbook.
Second,thesymbolsusedinthemodelsandderivations,onoccasion,duplicateeachother(i.e., α isan extremelypopularsymbol).However,theuseofthese multiplesymbolshasbeenretained,sincethispreserves thecontextofwherethesemodelswerefirstdescribedand utilized.Also,changingthesetomakethemuniquewould
1.11Dose ResponseCurves16
1.12ChapterSummaryand Conclusions19
1.13Derivations:Conformational SelectionasaMechanismof Efficacy20 References21
causeconfusionifthesemethodsweretobeusedbeyond theframeworkofthisbook.Therefore,careshouldbe takentoconsidertheactualnomenclatureofeachchapter.
Third,anefforthasbeenmadetominimizetheneed tocross-referencedifferentpartsofthebook(i.e.,whena particularmodelisdescribed,thebasicsarereiterated somewhattominimizetheneedtoreadtherelevantbut differentpartofthebookinwhichthemodelisinitially described).Whilethisleadstoasmallamountofrepeated description,itisfeltthatthiswillallowforamoreuninterruptedflowofreadinganduseofthebook.
1.2WHATISPHARMACOLOGY?
Pharmacology (anamalgamoftheGreek pharmakos, medicineordrug,and logos,study)isabroaddiscipline describingtheuseofchemicalstotreatandcurediseases. TheLatinterm pharmacologia wasusedinthelate 1600s,buttheterm pharmacum wasusedasearlyasthe fourthcenturytodenotetheterm drug or medicine.There aresubdisciplineswithinpharmacologyrepresentingspecialtyareas. Pharmacokinetics dealswiththedisposition ofdrugsinthehumanbody.Tobeuseful,drugsmustbe absorbedandtransportedtotheirsiteoftherapeutic action.Drugswillbeineffectiveintherapyiftheydonot reachtheorgans(s)toexerttheiractivity;thiswillbe
discussedspecificallyinChapter9,Pharmacokinetics,of thisbook. Pharmaceutics isthestudyofthechemicalformulationofdrugstooptimizeabsorptionanddistribution withinthebody. Pharmacognosy isthestudyofplantnaturalproductsandtheiruseinthetreatmentofdisease.A veryimportantdisciplineinthedrug-discoveryprocessis medicinalchemistry,thestudyoftheproductionofmoleculesfortherapeuticuse.Thiscouplessyntheticorganic chemistrywithanunderstandingofhowbiologicalinformationcanbequantifiedandusedtoguidethe syntheticchemistrytoenhancetherapeuticactivity. Pharmacodynamics isthestudyoftheinteractionofthe drugmoleculewiththebiologicaltarget(referredto genericallyasthe“receptor,” videinfra).Thisdiscipline laysthefoundationofpharmacologysincealltherapeutic applicationofdrugshasacommonrootinpharmacodynamics(i.e.,asaprerequisitetoexertinganeffect,all drugmoleculesmustbindtoandinteractwithreceptors).
Thehistoryofpharmacologyistiedtothehistoryof drugdiscovery—seeChapter8,TheOptimalDesignof PharmacologicalExperiments.Asputbythegreat CanadianphysicianSirWilliamOsler(1849 1919;the “fatherofmodernmedicine”),“ thedesiretotake medicineisperhapsthegreatestfeaturewhichdistinguishesmanfromanimals .”Pharmacologyasaseparatescienceisapproximately120 140yearsold.The relationshipbetweenchemicalstructureandbiological activitybegantobestudiedsystematicallyinthe1860s [1].Itbeganwhenphysiologists,usingchemicalstoprobe physiologicalsystems,becamemoreinterestedinthe chemicalprobesthanthesystemstheywereprobing.By theearly1800s,physiologistswereperformingphysiologicalstudieswithchemicalsthatbecamepharmacological studiesmoreaimedatthedefinitionofthebiological activityofchemicals.Thefirstformalizedchairofpharmacology,indicatingaformaluniversitydepartment,was foundedinEstoniabyRudolfBucchiemin1847.In NorthAmerica,thefirstchairwasfoundedbyJohnJacob AbelatJohnsHopkinsUniversityin1890.Adifferentiationofphysiologyandpharmacologywasgivenbythe pharmacologistSirWilliamPaton [2]:
Ifphysiologyisconcernedwiththefunction,anatomywiththe structure,andbiochemistrywiththechemistryoftheliving body,thenpharmacologyisconcernedwiththechangesinfunction,structure,andchemicalpropertiesofthebodybrought aboutbychemicalsubstances
—W.D.M.Paton(1986)
Manyworksaboutpharmacologyessentiallydealin therapeuticsassociatedwithdifferentorgansystemsin thebody.Thus,inmanypharmacologytexts,chaptersare entitleddrugsinthecardiovascularsystem,theeffectof drugsonthegastrointestinal(GI)system,thecentralnervoussystem(CNS),andsoon.However,theunderlying
principlesforalloftheseisthesame,namely,the pharmacodynamicinteractionbetweenthedrugandthe biologicalrecognitionsystemforthatdrug.Therefore,a prerequisitetoallofpharmacologyisanunderstandingof thebasicconceptsofdose responseandhowlivingcells processpharmacologicalinformation.Thisgenerallyis giventheterm pharmacodynamics or receptorpharmacology,where receptor isatermreferringtoanybiologicalrecognitionunitfordrugs(membranereceptors, enzymes,DNA,andsoon).Withsuchknowledgein hand,readerswillbeabletoapplytheseprinciplestoany branchoftherapeuticseffectively.Thisbooktreats dose responsedatagenericallyanddemonstratesmethodsbywhichdrugactivitycanbequantifiedacrossall biologicalsystemsirrespectiveofthenatureofthebiologicaltarget.
Agreatstrengthofpharmacologyasadisciplineisthat itcontainsthetoolsandmethodstoconvert“descriptive data,”i.e.,datathatservestocharacterizetheactivityofa givendruginaparticularsystem,to“predictivedata.” Thislatterinformationcanbeusedtopredictthatdrug’s activityinallorgansystems,includingthetherapeuticone. Thisdefinesthedrug-discoveryprocesswhichisthetestingofnewpotentialdrugmoleculesinsurrogatesystems (whereapotentiallytoxicchemicalcandonolasting harm)beforeprogressiontothenextstep,namely,testing inhumantherapeuticsystems.Themodelsandtoolscontainedinpharmacologytoconvertdrugbehaviorsinparticularorganstomolecularproperties(seeChapter2:How DifferentTissuesProcessDrugResponse)arethemain subjectofthisbook,andthestep-by-stepdesignofpharmacologicexperimentstodothisaredescribedindetailin Chapter8,TheOptimalDesignofPharmacological Experiments(afterthemeaningoftheparticularparametersandtermsisdescribedinpreviouschapters).
Thehuman genome isnowwidelyavailablefordrugdiscoveryresearch.Farfrombeingasimpleblueprintof howdrugsshouldbetargeted,ithasshownbiologiststhat receptor genotypes (i.e.,propertiesofproteinsresulting fromgenetictranscriptiontotheiraminoacidsequence) aresecondarytoreceptor phenotypes (howtheprotein interactswiththemyriadofcellularcomponentsandhow cellstailorthemakeupandfunctionsoftheseproteinsto theirindividualneeds).Sincethearrivalofthehuman genome,receptorpharmacologyasascienceismorerelevantthaneverindrugdiscovery.Currentdrugtherapyis basedonlessthan500moleculartargets,yetestimates utilizingthenumberofgenesinvolvedinmultifactorial diseasessuggestthatthenumberofpotentialdrugtargets rangesfrom2000to5000 [3].Thus,currenttherapyis usingonly5% 10%ofthepotentialtroveoftargets availableinthehumangenome.
Ameaningfuldialogbetweenchemistsandpharmacologistsisthesinglemostimportantelementofthe drug-discoveryprocess.Thenecessarylinkbetween
medicinalchemistryandpharmacologyhasbeenelucidatedbyPaton [2]:
Forpharmacologythereresultsaparticularlycloserelationship withchemistry,andtheworkmayleadquitenaturally,withno specialstressonpracticality,totherapeuticapplication,or(in thecaseofadversereactions)totoxicology.
—W.D.M.Paton(1986)
Chemistsandbiologistsresideindifferentworldsfrom thestandpointofthetypeofdatatheydealwith.Chemistry isanexactsciencewithphysicalscalesthatarenotsubject tosystemvariance.Thus,thescalesofmeasurementare transferable.Biologydealswiththevagariesofcomplex systemsthatarenotcompletelyunderstood.Withinthis scenario,scalesofmeasurementaremuchlessconstantand muchmoresubjecttosystemconditions.Giventhis,agap canexistbetweenchemistsandbiologistsintermsof understandingandalsointermsofthebestmethodtoprogressforward.Intheworstcircumstance,itisagapofcredibilityemanatingfromafailureofthebiologisttomakethe chemistunderstandthelimitsofthedata.Usually,however, credibilityisnottheissue,andthegapexistsduetoalack ofcommonexperience.Thisbookwaswritteninan attempttolimitor,hopefully,eliminatethisgap.
1.3THERECEPTORCONCEPT
Oneofthemostimportantconceptsemergingfromearly pharmacologicalstudiesistheconceptofthe receptor
Pharmacologistsknewthatminuteamountsofcertain chemicalshadprofoundeffectsonphysiologicalsystems.Theyalsoknewthatverysmallchangesinthe chemicalcompositionofthesesubstancescouldleadto hugedifferencesinactivity. Thisledtothenotionthat somethingonorinthecellmustspecificallyreadthe chemicalinformationconta inedinthesesubstancesand translateitintoaphysiologicaleffect.Thissomething wasconceptuallyreferredto asthe“receptor”forthat substance.PioneerssuchasPaulEhrlich(1854 1915, Fig.1.1A)proposedtheexistenceof“chemoreceptors” (actuallyheproposedacollectionofamboreceptors,triceptors,andpolyceptors)oncellsfordyes.Healsopostulatedthatthechemoreceptorsonparasites,cancer cells,andmicroorganismsweredifferentfromhealthy hostandthuscouldbeexploitedtherapeutically.The physiologistturnedpharmacologistJohnNewport Langley(1852 1926, Fig.1.1 B),duringhisstudieswith thedrugsjaborandi(whichcontainsthealkaloidpilocarpine)andatropine,introducedtheconceptthatreceptors wereswitchesthatreceivedandgeneratedsignalsand thattheseswitchescouldbeactivatedorblockedbyspecificmolecules.Theoriginatorofquantitativereceptor theory,theEdinburghpharmacologistAlfredJoseph Clark(1885 1941, Fig.1.1C),wasthefirsttosuggest thatthedata,compiledfromhisstudiesoftheinteractionsofacetylcholineandatropine,resultedfromthe unimolecularinteractionofthedrugandasubstanceon thecellsurface.Hearticulatedtheseideasintheclassic work TheModeofActionofDrugsonCells [4],later
FIGURE1.1 Pioneersofpharmacology.(A)PaulEhrlich(1854 1915).BorninSilesia,Ehrlichgraduated fromLeipzigUniversitytogoontoadistinguishedcareerasheadofinstitutesinBerlinandFrankfurt.Hisstudieswithdyesandbacteriaformedthebasisofearlyideasregardingrecognitionofbiologicalsubstancesbychemicals.(B)JohnNewportLangley(1852 1926).Thoughhebeganreadingmathematicsandhistoryin Cambridgein1871,Langleysoontooktophysiology.HesucceededthegreatphysiologistM.Fostertothechair ofphysiologyinCambridgein1903andbranchedoutintopharmacologicalstudiesoftheautonomicnervous system.Thesepursuitsledtogerminaltheoriesofreceptors.(C)AlfredJ.Clark(1885 1941).Beginningasa demonstratorinpharmacologyinKing’sCollege(London),ClarkwentontobecomeProfessorofpharmacology atUniversityCollegeLondon.FromtherehetookthechairofpharmacologyinEdinburgh.Knownastheoriginatorofmodernreceptortheory,Clarkappliedchemicallawstobiologicalphenomena.Hisbooksonreceptor theoryformedthebasisofmodernpharmacology.
(A)
(B)
(C)
revisedasthe HandbookofExperimentalPharmacology [5] .AsputbyClark
Itappearstothewriterthatthemostimportantfactshownbya studyofdrugantagonismsisthatitisimpossibletoexplainthe remarkableeffectsobservedexceptbyassumingthatdrugsunite withreceptorsofahighlyspecificpattern .Nootherexplanationwill,however,explainatitheofthefactsobserved.
—A.J.Clark(1937).
Clark’snextstepformedthebasisofreceptortheory byapplyingchemicallawstosystemsof“infinitely greatercomplexity” [4].ItisinterestingtonotethescientificatmosphereinwhichClarkpublishedtheseideas. Thedominantideasbetween1895and1930werebased ontheoriessuchasthelawofphasicvariationessentially statingthat“certainphenomenaoccurfrequently.”
HomeopathictheoriesliketheArndt Schulzlawand Weber Fechnerlawwerebasedonlooseideasaround surfacetensionofthecellmembrane,buttherewaslittle physicochemicalbasisfortheseideas [6].Inthisvein, prominentpharmacologistsoftheday,suchasWalter Straub(1874 1944),suggestedthatageneraltheoryof chemicalbindingbetweendrugsandcellsutilizingreceptorswas“ goingtoofar and notadmissible” [6].TheimpactofClark’sthinkingagainsttheseconcepts cannotbeoveremphasizedtomodernpharmacology.
Itispossibletounderestimatetheenormoussignificanceofthereceptorconceptinpharmacologyuntilitis realizedhowrelativelychaoticthestudyofdrugeffect wasbeforeitwasintroduced.Specifically,considerthe myriadofphysiologicalandpharmacologicaleffectsof thehormoneepinephrineinthebody.Asshownin Fig.1.2,ahostofresponsesareobtainedfromtheCNS, cardiovascularsystem,smoothmuscle,andotherorgans. Itisimpossibletoseeathreadwhichrelatesthesevery differentresponsesuntilitisrealizedthatalloftheseare mediatedbytheactivationofasingleproteinreceptor, namely,inthiscase,the β-adrenoceptor.Whenthisis
Bronchial muscle relaxation
Cardiac inotropy
Uterine muscle relaxation
Melatonin synthesis
Pancreatic secretion
Lacrimal gland secretion
Decreased stomach motility
understood,amuchbetterideacanbegainedastohow tomanipulatetheseheterogeneousresponsesfortherapeuticbenefit;thereceptorconceptintroducedorderinto physiologyandpharmacology.
Drugreceptorscanexistinmanyforms,includingcell surfaceproteins,enzymes,ionchannels,membranetransporters,DNA,andcytosolicproteins(see Fig.1.3).There areexamplesofimportantdrugsforallofthese.This bookdealswithgeneralconceptswhichcanbeappliedto arangeofreceptortypes,butmostoftheprinciplesare illustratedwiththemosttractablereceptorclassknownin thehumangenome,namely, seventransmembrane(7TM) receptors.Thesereceptorsarenamedfortheircharacteristicstructurethatconsistsofasingleproteinchainthat traversesthecellmembraneseventimestoproduceextracellularandintracellularloops.Thesereceptorsactivate G-proteinstoelicitresponse,thustheyarealsocommonly referredtoas G-protein-coupledreceptors (GPCRs);this shouldnowbeconsideredalimitingmonikerasthese proteinssignaltoawidevarietyofsignalingmoleculesin thecellandarenotconfinedtoG-proteineffects.There arebetween800and1000 [7] oftheseinthegenome[the genomesequencepredicts650GPCRgenes,ofwhich approximately190(ontheorderof1%ofthegenomeof superiororganisms)arecategorizedasknown7TMRs [8] activatedbysome70ligands].IntheUnitedStates,in 2000,nearlyhalfofallprescriptiondrugsweretargeted toward7TMreceptors [3].Thesereceptors,comprising between1%and5%ofthetotalcellprotein,controla myriadofphysiologicalactivities.Theyaretractablefor drugdiscoverybecausetheyareonthecellsurface,and thereforedrugsdonotneedtopenetratethecelltoproduceeffect.Inthestudyofbiologicaltargetssuchas 7TMRsandotherreceptors,a“system”mustbe employedthatacceptschemicalinputandreturnsbiologicaloutput.Itisworthdiscussingsuchreceptorsystemsin generaltermsbeforetheirspecificusesareconsidered.
1.4PHARMACOLOGICALTESTSYSTEMS
Vascular relaxation
Salivary gland secretion
Cardiac lusitropy
Cardiac chronotropy
Skeletal muscle tremor
Urinary bladder muscle relaxation
FIGURE1.2 Asamplingoftheheterogeneousphysiologicalandpharmacologicalresponsetothehormoneepinephrine.Theconceptofreceptorslinksthesediverseeffectstoasinglecontrolpoint,namely,the β-adrenoceptor.
Molecularbiologyhastransformedpharmacologyandthe drug-discoveryprocess.Aslittleas20yearsago,screening fornewdrugentitieswascarriedoutinsurrogateanimal tissues.Thisnecessitatedaratherlargeextrapolationto spanthedifferencesingenotypeandphenotype.Thebelief thatthegapcouldbebridgedcamefromthenotionthatthe chemicalsrecognizedbythesereceptorsinbothhumans andanimalswerethesame(videinfra).Receptorsare uniqueproteinswithcharacteristicaminoacidsequences. While polymorphisms (spontaneousalterationsinamino acidsequence, videinfra)ofreceptorsexistinthesame species,ingeneraltheaminoacidsequenceofanatural ligand-bindingdomainforagivenreceptortypelargely maybeconserved.Thereareobviouspitfallsofusingsurrogatespeciesreceptorsforpredictinghumandrugactivity,
FIGURE1.3 Schematicdiagramofpotentialdrugtargets.Moleculescanaffectthefunctionof numerouscellularcomponentsbothinthecytosolandonthemembranesurface.Therearemany familiesofreceptorsthattraversethecellularmembraneandallowchemicalstocommunicate withtheinteriorofthecell.
anditnevercanbeknownforcertainwhetheragreement forestimatesofactivityforagivensetofdrugsensures accuratepredictionforalldrugs.Theagreementisvery muchdrugandreceptordependent.Forexample,the humanandmouse α2-adrenoceptorsare89%homologous andthusconsideredverysimilarfromthestandpointof aminoacidsequence.Furthermore,theaffinitiesofthe α2adrenoceptorantagonistsatipamezoleandyohimbineare nearlyindistinguishable(atipamezolehuman α2C10Ki 5 2.9 6 0.4nM,mouse α2-4HKi 5 1.6 6 0.2nM; yohimbinehuman α2-C10Ki 5 3.4 6 0.1nM,mouse α2-4H Ki 5 3.8 6 0.8nM).However,thereisa20.9-folddifferencefortheantagonistprazosin(human α2-C10Ki 5 2034 6 350nM,mouse α2-4HKi 5 97.3 6 0.7nM) [9].Such datahighlightageneralthemeinpharmacologicalresearch, namely,thatahypothesis,suchasoneproposingthattwo receptorswhichareidenticalwithrespecttotheirsensitivitytodrugsarethesame,cannotbeproven,onlydisproven. Whileaconsiderablenumberofdrugscouldbetestedon thetworeceptors(thussupportingthehypothesisthattheir sensitivitytoalldrugsisthesame),thishypothesisis immediatelydisprovenbythefirstdrugthatshowsdifferentialpotencyonthetworeceptors.Thefactthataseries ofdrugstestedshowidenticalpotenciesmaymeanonly thatthewrongsampleofdrugshasbeenchosentounveil thedifference.Thus,nogeneralstatementscanbemade thatanyonesurrogatesystemiscompletelypredictiveof activityonthetargethumanreceptor.Thiswillalwaysbea drug-specificphenomenon.
Thelinkbetweenanimalandhumanreceptorsisthe factthatbothproteinsrecognizetheendogenoustransmitter(e.g.,acetylcholine,norepinephrine),andthereforethe hopeisthatthislinkwillcarryoverintootherdrugsthat recognizetheanimalreceptor.Thisimperfectsystem formedthebasisofdrugdiscoveryuntilhuman cDNA for humanreceptorscouldbeusedtomakecellsexpress
humanreceptors.Theseengineered(recombinant)systemsarenowusedassurrogatehuman-receptorsystems, andtheleapoffaithfromanimalreceptorsequencesto human-receptorsequencesisnotrequired(i.e.,theproblemofdifferencesingenotypehasbeenovercome). However,cellularsignalingisanextremelycomplexprocessandcellstailortheirreceiptofchemicalsignalsin numerousways.Therefore,thewayagivenreceptorgene behavesinaparticularcellcandifferinresponsetothe surroundingsinwhichthatreceptorfindsitself.Thesedifferencesinphenotype(i.e.,propertiesofareceptorproducedbyinteractionwithitsenvironment)canresultin differencesinboththequantityandqualityofasignal producedbyaconcentrationofagivendrugindifferent cells.Therefore,thereisstillacertain,althoughsomewhatlesser,leapoffaithtakeninpredictingtherapeutic effectsinhumantissuesunderpathologicalcontrolfrom surrogaterecombinantorevensurrogatenaturalhumanreceptorsystems.Forthisreason,itisaprimaryrequisite ofpharmacologytoderivesystem-independentestimates ofdrugactivitythatcanbeusedtopredicttherapeutic effectinothersystems.
Aschematicdiagramofthevarioussystemsusedin drugdiscovery,inorderofhowappropriatetheyareto therapeuticdrugtreatment,isshownin Fig.1.4.Asdiscussedpreviously,earlyfunctionalexperimentsinanimal tissuehavenowlargelygivenwaytotestinginrecombinantcellsystemsengineeredwithhuman-receptormaterial.Thishugetechnologicalstepgreatlyimprovedthe predictabilityofdrugactivityinhumans,butitshouldbe notedthattherestillaremanyfactorsthatintervene betweenthegeneticallyengineereddrug-testingsystem andthepathologyofhumandisease.
Afrequentlyusedstrategyindrugdiscoveryisto expresshumanreceptors(through transfection with humancDNA)inconvenientsurrogatehostcells(referred
Pharmacological test systems
Animal receptors Animal tissues
Human receptors
Human receptors
Human target cells
Therapeutic effect in humans
Human receptors
Human target cells under influence of pathology
FIGURE1.4 Ahistoryofthedrug-discoveryprocess.Originally,theonlybiological materialavailablefordrugresearchwasanimaltissue.Withtheadventofmolecularbiologicaltechniquestocloneandexpresshumanreceptorsincells,recombinantsystemssupplantedanimal-isolatedtissuework.Itshouldbenotedthattheserecombinantsystemsstill fallshortofyieldingdrugresponseinthetargethumantissueundertheinfluenceofpathologicalprocesses.
toas“target-based”drugdiscovery;seeChapter10: SafetyPharmacologyforfurtherdiscussion).Thesehost cellsarechosenmainlyfortheirtechnicalproperties(i.e., robustness,growthrate,stability)andnotwithany knowledgeofverisimilitudetothetherapeuticallytargetedhumancelltype.Therearevariousfactorsrelevant tothechoiceofsurrogatehostcell,suchasaverylowbackgroundactivity(i.e.,acellcannotbeusedthat alreadycontainsarelatedanimalreceptorforfearof cross-reactivitytomoleculestargetedforthehuman receptor).Humanreceptorsareoftenexpressedinanimal surrogatecells.Themainideahereisthatthecellisa receptacleforthereceptor,allowingittoproducephysiologicalresponses,andthatactivitycanbemonitoredin pharmacologicalexperiments.Inthissense,humanreceptorsexpressedinanimalcellsarestillatheoreticalstep distancedfromthehumanreceptorinahumancelltype. However,evenifahumansurrogateisused(andthere aresuchcellsavailable),thereisnodefinitiveevidence thatasurrogatehumancellisanymorepredictiveofa naturalreceptoractivitythanananimalcellwhencomparedtothecomplexreceptorbehaviorinitsnaturalhost celltypeexpressedunderpathologicalconditions. Receptorphenotypedominatesintheendorgan,andthe exactdifferencesbetweenthegenotypicbehaviorofthe receptor(resultingfromthegeneticmakeupofthereceptor)andthephenotypicbehaviorofthereceptor(dueto theinteractionofthegeneticproductwiththerestofthe cell)maybecellspecific.Therefore,thereisstillapossiblegapbetweenthesurrogatesystemsusedinthedrugdiscoveryprocessandthetherapeuticapplication. Moreover,mostdrug-discoverysystemsutilizereceptors asswitchingmechanismsandquantifywhetherdrugsturn onorturnofftheswitch.Thepathologicalprocessesthat
westrivetomodifymaybemoresubtle.AsputbypharmacologistSirJamesBlack [10]:
angiogenesis,apoptosis,inflammation,commitmentofmarrowstemcells,andimmuneresponses.Thecellularreactions subsumedintheseprocessesareswitchlikeintheirbehavior biochemicallywearelearningthatinalltheseprocessesmany chemicalregulatorsseemtobeinvolved.Fromtheliteratureon synergisticinteractions,acontrolmodelcanbebuiltinwhich nosingleagentiseffective.Ifanumberofchemicalmessengers eachbringinformationfromadifferentsourceandeachdeliver onlyasubthresholdstimulusbuttogethermutuallypotentiate eachother,thenthedesiredinformation-richswitchingcanbe achievedwithminimumriskofmiscuing.
—J.W.Black(1986).
Suchcomplexendpointsaredifficulttopredictfrom anyoneofthecomponentprocessesleadingtoyet anotherleapoffaithinthedrug-discoveryprocess.For thesereasons,anemergingstrategyfordrugdiscoveryis theuseofnaturalcellularsystems.ThisapproachisdiscussedinsomedetailinChapter11,TheDrugDiscovery Process.
Evenwhenanactivedrugmoleculeisfoundand activityisverifiedinthetherapeuticarena,therearefactorsthatcanleadtogapsinitstherapeuticprofile.When drugsareexposedtohugepopulations,geneticvariations inthispopulationcanleadtodiscoveryof alleles that codeformutationsofthetarget(isogenes),andthesecan leadtovariationindrugresponse.Suchpolymorphisms canleadtoresistantpopulations(i.e.,resistanceofsome asthmaticstothe β-adrenoceptorbronchodilators [11]).In theabsenceofgeneticknowledge,thesetherapeuticfailuresforadrugcouldnoteasilybeavertedsincetheyin
Current state-of-the-art
essenceoccurredbecauseofthepresenceofnewbiologicaltargetsnotoriginallyconsideredinthedrug-discovery process.However,asnewepidemiologicalinformation becomesavailable,thesepolymorphismscannowbe incorporatedintothedrug-discoveryprocess.
Therearetwotheoreticalandpracticalscalesthatcan beusedtomakesystem-independentmeasuresofdrug activityonbiologicalsystems.Thefirstisameasureof theattractionofadrugforabiologicaltarget,namely,its affinity forareceptor.Drugsmustinteractwithreceptors toproduceaneffect,andtheaffinityisachemicalterm usedtoquantifythestrengthofthatinteraction.Thesecondismuchlessstraightforwardandisusedtoquantify thedegreeofeffectimpartedtothebiologicalsystem afterthedrugbindstothereceptor.Thisistermed efficacy.ThispropertywasnamedbyStephenson [12] within classicalreceptortheoryasaproportionalityfactorforthe tissueresponseproducedbyadrug.Thereisnoabsolute scaleforefficacy,butratheritisdealtwithinrelative terms(i.e.,theratiooftheefficacyoftwodifferentdrugs onaparticularbiologicalsystemcanbeestimatedand, underidealcircumstances,willtranscendthesystemand beapplicabletoothersystemsaswell).Itistheforemost taskofpharmacologytousethetranslationsofdrugeffect obtainedfromcellstoprovidesystem-independentestimatesofaffinityandefficacy.Beforespecificdiscussion ofaffinityandefficacy,itisworthconsideringthemolecularnatureofbiologicaltargets.
1.5THENATUREOFDRUGRECEPTORS
WhilesomebiologicaltargetssuchasDNAarenotproteininnature,mostreceptorsare.Itisusefultoconsider thepropertiesofreceptorproteinstoprovideacontextfor theinteractionofsmallmoleculedrugswiththem.An importantpropertyofreceptorsisthattheyhavea3-D structure.Proteinsareusuallycomposedofoneormore peptidechains;thecompositionofthesechainsmakesup theprimaryandsecondarystructureoftheprotein. Proteinsalsoaredescribedintermsofatertiarystructure, whichdefinestheirshapein3-Dspace,andaquaternary structure,whichdefinesthemolecularinteractions betweenthevariouscomponentsoftheproteinchains (Fig.1.5).Itisthis3-Dstructurewhichallowstheprotein tofunctionasarecognitionsiteandeffectorfordrugs andothercomponentsofthecell;inessence,theability oftheproteintofunctionasamessenger,shuttlinginformationfromtheoutsideworldtothecytosolofthecell. For7TMRs,the3-Dnatureofthereceptorformsbinding domainsforotherproteinssuchasG-proteins(theseare activatedbythereceptorandthengoontoactivate enzymesandionchannelswithinthecell;seeChapter2: HowDifferentTissuesProcessDrugResponse)and endogenouschemicalssuchasneurotransmitters,
hormones,andautacoidsthatcarryphysiologicalmessages.Thisimportantclassofdrugtargetisnamedfora characteristicstructureconsistingof7TMdomainsloopingintotheextracellularandintracellularspace—see Fig.1.6.Thesemoleculesarethemaintransferpointsof informationfromtheoutsidetotheinsideofthecell,and suchtransfersoccurthroughchangesintheconformation ofthereceptorprotein(videinfra).Forotherreceptors, suchasionchannelsandsingletransmembraneenzyme receptors,theconformationalchangeperseleadstoa response;eitherthroughanopeningofachanneltoallow theflowofioniccurrentortheinitiationofenzymatic activity.Therapeuticadvantagecanbetakenbydesigning smallmoleculestoutilizethesebindingdomainsorother 3-Dbindingdomainsonthereceptorproteininorderto modifyphysiologicalandpathologicalprocesses.
1.6PHARMACOLOGICALINTERVENTION ANDTHETHERAPEUTICLANDSCAPE
Itisusefultoconsiderthetherapeuticlandscapewith respecttotheaimsofpharmacology.AsstatedbySir WilliamOssler(1849 1919)“... theprimedistinction betweenmanandothercreaturesisman’syearningto takemedicine.”Thenotionthatdrugscanbeusedtocure diseaseisasoldashistory.Oneofthefirstwritten recordsofactual“prescriptions”canbefoundinthe EbersPapyrus(circa1550BC):“ fornightblindness intheeyes liverofox,roastedandcrushedout reallyexcellent!”—see Fig.1.7.Nowitisknownthat liverisanexcellentsourceofvitaminA,aprimetreatmentfornightblindness,butthatchemicaldetailwasnot knowntotheancientEgyptians.Diseasecanbeconsideredundertwobroadcategories:thosecausedbyinvaders suchaspathogensandthosecausedbyintrinsicbreakdownofnormalphysiologicalfunction.Thefirstgenerallyisapproachedthroughtheinvader(i.e.,thepathogen isdestroyed,neutralized,orremovedfromthebody).The oneexceptionofwherethehostistreatedwhenan invaderispresentisthetreatmentofHIV-1infection leadingtoAIDS.Inthiscase,whiletherearetreatments toneutralizethepathogen,suchasantiretroviralstoblock viralreplication,amajornewapproachistheblockadeof theinteractionoftheviruswiththeproteinthatmediates viralentryintohealthycells,thechemokinereceptor CCR5.Inthiscase,CCR5antagonistsareusedtoprevent HIVfusionandsubsequentinfection.Thesecond approachtodiseaserequiresanunderstandingofthepathologicalprocessandrepairofthedamagetoreturntonormalfunction.
Thetherapeuticlandscapeontowhichdrugdiscovery andpharmacologyingeneralcombatdiseasecangenerallybedescribedintermsofthemajororgansystemsof thebodyandhowtheymaygoawry.Ahealthy
Levels of protein (receptor) structure
Primary structure
Sequence of amino acid residues
Secondary structure
Repeating 3-D units such as α-helices and β-sheets (buried main chain H bonds)
Quaternary structure
Arrangement of separate chains
Tertiary structure
Single folded and arranged polypeptide chain, the structure of which is determined by the amino acids
FIGURE1.5 Increasinglevelsofproteinstructure.Aproteinhasagivenaminoacidsequenceto makepeptidechains.Theseadopta3-Dstructureaccordingtothefreeenergyofthesystem. Receptorfunctioncanchangewithchangesintertiaryorquaternarystructure.
FIGURE1.6 Depictionofthestructureofseventransmembrane domainreceptors,oneofthemostifnotthemostimportanttherapeutic targetsavailableinthehumangenome.Chemicalsaccessthereceptor throughtheextracellularspacebybindingtotheextracellulardomains oftheprotein.Thiscausesaconformationalchangeintheproteinthat alterstheinteractionofsignalingproteinsinthecellcytosol.Thislatter processresultsintheinitiationofcellularsignaling.
cardiovascularsystemconsistsofaheartabletopump deoxygenatedbloodthroughthelungsandtopumpoxygenatedbloodthroughoutacirculatorysystemthatdoes notundulyresistbloodflow.Sincetheheartrequiresa highdegreeofoxygenitselftofunction,myocardial ischemiacanbedevastatingtoitsfunction.Similarly,an inabilitytomaintainrhythm(arrhythmia)orlossin strengthwithconcomitantinabilitytoempty(congestive heartfailure)canbefatal.Thelatterdiseaseisexacerbatedbyelevatedarterialresistance(hypertension).A widerangeofdrugsareusedtotreatthecardiovascular system,includingcoronaryvasodilators(nitrates),diuretics,renin-angiotensininhibitors,vasodilators,cardiac glycosides,calciumantagonists,betaandalphablockers, antiarrhythmics,anddrugsfordyslipidemia.Thelungs mustextractoxygenfromtheair,deliverittotheblood, andreleasecarbondioxidefromthebloodintoexhaled air.Asthma,chronicobstructivepulmonarydisease
FIGURE1.7 TheEbersPapyrusisa110-pagescroll(20mlong)thoughttohavebeenwrittenin1550BCbutcontaining informationdatingfrom3400BC.ItisarecordofEgyptianmedicineandcontainsnumerous“prescriptions”someofwhich, thoughempirical,arevalidtherapeuticapproachestodiseases.
(COPD),andemphysemaareseriousdisordersofthe lungsandairways.Bronchodilators(betaagonists),antiinflammatorydrugs,inhaledglucocorticoids,anticholinergics,andtheophyllineanalogsareusedfortreatmentof thesediseases.TheCNScontrolsallconsciousthought andmanyunconsciousbodyfunctions.Numerousdiseasesofthebraincanoccur,includingdepression,anxiety,epilepsy,mania,degeneration,obsessivedisorders, andschizophrenia.Brainfunctionssuchasthosecontrollingsedationandpainalsomayrequiretreatment.Awide rangeofdrugsisusedforCNSdisorders,includingserotoninpartialagonistsanduptakeinhibitors,dopamine agonists,benzodiazepines,barbiturates,opioids,tricyclics,neuroleptics,andhydantoins.TheGItractreceives andprocessesfoodtoextractnutrientsandremoveswaste fromthebody.Diseasessuchasstomachulcers,colitis, diarrhea,nausea,andirritablebowelsyndromecanaffect thissystem.Histamineantagonists,protonpumpblockers, opioidagonists,antacids,andserotoninuptakeblockers areusedtotreatdiseasesoftheGItract.
Theinflammatorysystemisdesignedtorecognizeself fromnonself,andtodestroynonselftoprotectthebody. Indiseasesoftheinflammatorysystem,theselfrecognitioncanbreakdown,leadingtoconditionsin whichthebodydestroyshealthytissueinamisguided attemptatprotection.Thiscanleadtorheumatoidarthritis,allergies,pain,COPD,asthma,fever,gout,graftrejection,andproblemswithchemotherapy.Nonsteroidal antiinflammatorydrugs,aspirinandsalicylates,leukotrieneantagonists,andhistaminereceptorantagonistsare usedtotreatinflammatorydisorders.Theendocrinesystemproducesandsecreteshormonescrucialtothebody forgrowthandfunction.Diseasesofthisclassoforgans canleadtogrowthandpituitarydefects—diabetes;
abnormalityinthyroid,pituitary,adrenalcortex,and androgenfunction;osteoporosis;andalterationsinestrogen progesteronebalance.Thegeneralapproachtotreatmentisthroughreplacementoraugmentationof secretion.Drugsusedarereplacementhormones,insulin, sulfonylureas,adrenocorticalsteroids,andoxytocin.In additiontothemajororganandphysiologicalsystems, diseasesinvolvingneurotransmissionandneuromuscular function,ophthalmology,hemopoiesisandhematology, dermatology,immunosuppression,anddrugaddictionand abuseareamenabletopharmacologicalintervention.
Cancerisaseriousmalfunctionofnormalcellgrowth. Intheyearsfrom1950to1970,themajorapproachto treatingthisdiseasewastotargetDNAandDNAprecursorsaccordingtothehypothesisthatrapidlydividingcells (cancercells)aremoresusceptibletoDNAtoxicitythan normalcells.Sincethattime,awiderangeofnewtherapies basedonmanipulationoftheimmunesystem,inductionof differentiation,inhibitionofangiogenesis,andincreased killerT-lymphocytestodecreasecellproliferationhas greatlyaugmentedthearmamentariumagainstneoplastic disease.Previously,lethalmalignanciessuchastesticular cancer,somelymphomas,andleukemiaarenowcurable.
Threegeneraltreatmentsofdiseasearesurgery,genetic engineering(stillanemergingdiscipline),andpharmacologicalintervention.Whileearlymedicinewassubjectto thetheoriesofHippocrates(460 357BC),whosawhealth anddiseaseasabalanceoffourhumors(i.e.,blackand yellowbile,phlegm,andblood),bythe16thcenturypharmacologicalconceptswerebeingformulated.Thesecould bestatedconciselyasthefollowing [13]:
● Everydiseasehasacauseforwhichthereisaspecificremedy.
TABLE1.1 SomeEndogenousChemicalsControllingNormalPhysiologicalFunction
Neurotransmitters
Acetylcholine2-ArachidonylglycerolAnandamide ATPCorticotropin-releasinghormoneDopamine EpinephrineAspartateGamma-aminobutyricacid GalaninGlutamateGlycine HistamineNorepinephrineSerotonin
Hormones
Thyroid-stimulatinghormoneFollicle-stimulatinghormoneLuteinizinghormone ProlactinAdrenocorticotropinAntidiuretichormone Thyrotropin-releasinghormoneOxytocinGonadotropin-releasinghormone Growth-hormone-releasinghormoneCorticotropin-releasinghormoneSomatostatin MelatoninThyroxinCalcitonin ParathyroidhormoneGlucocorticoid(s)Mineralocorticoid(s) Estrogen(s)ProgesteroneChorionicgonadotropin AndrogensInsulinGlucagon AmylinErythropoietinCalcitriol CalciferolAtrial-natriureticpeptideGastrin SecretinCholecystokininNeuropeptideY Insulin-likegrowthfactorAngiotensinogenGhrelin Leptin
ATP,adenosinetriphosphate.
● Eachremedyhasauniqueessencethatcanbe obtainedfromnaturebyextraction(“doctrineof signatures”).
● Theadministrationoftheremedyissubjecttoa dose responserelationship.
Thebasisforbelievingthatpharmacologicalinterventioncanbeamajorapproachtothetreatmentofdiseaseis thefactthatthebodygenerallyfunctionsinresponsetochemicals. Table1.1 showspartiallistsofhormonesandneurotransmittersinthebody.Manymoreendogenouschemicals areinvolvedinnormalphysiologicalfunction.Thefactthat somanyphysiologicalprocessesarecontrolledbychemicalsprovidestheopportunityforchemicalintervention. Thus,physiologicalsignalsmediatedbychemicalscanbe initiated,negated,augmented,ormodulated.Thenatureof thismodificationcantaketheformofchangesinthetype, strength,duration,orlocationofsignal.
1.7SYSTEM-INDEPENDENTDRUG PARAMETERS:AFFINITYANDEFFICACY
Theprocessofdrugdiscoveryreliesonthetestingof moleculesinsystemstoyieldestimatesofbiologicalactivityinaniterativeprocessofchangingthestructureofthe moleculeuntiloptimalactivityisachieved.Itwillbeseen inthisbookthattherearenumeroussystemsavailableto dothis,andthateachsystemmayinterprettheactivityof moleculesindifferentways.Someoftheseinterpretations
canappeartobeinconflictwitheachother,leadingto apparentcapriciouspatterns.Forthisreason,thewayforwardinthedrugdevelopmentprocessistouseonly system-independentinformation.Ideally,scalesofbiologicalactivityshouldbeusedthattranscendtheactualbiologicalsysteminwhichthedrugistested.Thisisessential toavoidconfusionandalsobecauseitisquiteraretohave accesstotheexacthumansystemunderthecontrolofthe appropriatepathologyavailableforinvitrotesting. Therefore,thedrug-discoveryprocessnecessarilyrelieson thetestingofmoleculesinsurrogatesystemsandthe extrapolationoftheobservedactivitytoallsystems.The onlymeanstodothisistoobtainsystem-independent measuresofdrugactivity,namely,affinityandefficacy.
Ifamoleculeinsolutionassociatescloselywithareceptorprotein,ithasaffinityforthatprotein.Theareawhereit isboundisthebinding domain or locus.Ifthesamemoleculeinterfereswiththebindingofaphysiologicallyactive moleculesuchasahormoneoraneurotransmitter(i.e.,if thebindingofthemoleculeprecludesactivityofthephysiologicallyactivehormoneorneurotransmitter),themolecule isreferredtoasan antagonist.Therefore,apharmacologicallyactivemoleculethatblocksphysiologicaleffectisan antagonist.Similarly,ifamoleculebindstoareceptorand producesitsowneffect,itistermedan agonist.Italsois assumedtohavethepropertyofefficacy.Efficacyis detectedbyobservationofpharmacologicalresponse. Therefore,agonistshavebothaffinityandefficacy.
Classically,agonistresponseisdescribedintwostages, thefirstbeingtheinitialsignalimpartedtotheimmediate
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TWO-LEAVED SOLOMON’S
SEAL: Smilacina trifolia.
Solomon Zigzag. Smilacinaracemosa.
False Spikenard.
Found in June, in moist woods and brookside copses.
The leafy stalk (from 1 to 2 feet high) is oblique and zigzag in gesture; it has a strong fibre, and a smooth surface, and is light green.
The leaf is oval—long in proportion to its width,—tapering to a slender tip, with an entire and much ruffled margin, and 3 noticeable ribs; the surface is finely downy; the color a strong, vigorous green. The leaves have almost no stem at all, and are placed alternately along the stalk.
The flower is small, with 6 petal-like parts and 6 stamens, all greenish white. Many flowers are gathered in a branching cluster upon the end of the leafy curving stalk.
The berries are pale red, speckled with dark. Gray’s Manual in speaking of this genus says its name is a diminution of Smilax, “to which, however, these plants bear little resemblance.” For a similar reason, perhaps, this plant is called “false” after the true Spikenard!
SOLOMON ZIGZAG: S. racemosa.
ARUM FAMILY. ARACEÆ.
Skunk Cabbage. Symplocarpusfœtidus.
Found in March or early April, in damp meadows, and moist or swampy woodlands.
The leaves and hooded flower-clusters rise from the ground.
The large and conspicuous leaves, which do not unfold until the flowering season is past, vary from 1 to 2 feet in length; they are oval in shape with a blunt tip and heart-shaped base, have entire margins, firm texture, and smooth surfaces, and resemble the garden Day-Lily because of their many parallel ribs. In color a light clear green.
The unnoticeable 4-parted greenish-yellow flowers are gathered closely on a fleshy round club (that is about an inch in diameter) and enveloped by a protecting hood. This hood is large and sharppointed, of a very thick and leathery texture, with a smooth and dull glossy surface; it is a dull brown or mahogany color, mottled or streaked with darker purple or red. From 1 to 3 or 4 of these hoodprotected flower-heads are crowded close together, along with the rolled up leaf, in the hold of several dull greenish or slightly purple leaf-like parts which serve as weather blankets wrapped about the whole plant.
After the flowers mature the hood shrivels and falls away, the blankets disappear, and the pointed leaf-bud then unfolds, the leaves pushing forth with fine springing curves. The strong odor of the plant prevents close observation, and denies to it the praise its growth deserves. In habit it is highly gregarious, and favorable meadows are thickly sprinkled with these rich-hued hoods of our earliest spring flower.
SKUNK CABBAGE: Symplocarpus fœtidus.
Golden Club. Orontiumaquaticum.
Found in ponds, or standing water, in early May.
The flower-spike and leaves lie upon the water, or hold themselves just above its surface, springing on long stems from the submerged root.
The oblong, narrow leaf is thick and juicy, with an entire margin, that is conspicuously folded together and joined at the base, and a smooth surface. In color it is a rich-toned green with a bloom above, the under surface being shining and pale and much colored by fine speckles of bronze. The leaf-stem is round, porous, smooth, and bronzy.
The small, flat, indefinite flowers are inset closely upon a round club, in a smooth, curiously patterned mosaic-work of a bright golden-yellow color. This club terminates a smooth porous stem, which becomes large and is flattened on one side beneath the flowers; it is streaked with bronze beneath the water, but clear white above its surface.
“I don’t feel as if I should search for it!” said the farmer when he saw this plant for the first time.
GOLDEN CLUB: Orontium aquaticum.
Green Dragon. ArisæmaDracontium.
Dragon-root.
Found in moist shade, in May.
The root produces usually one leaf-stem, 1 to 2 feet high, which bears the blossom-stem.
The leaflets of the compound leaf are 5 to 13 in number, in shape oblong, tapering at both ends, and entire; the surface is smooth, and a rich dark green color. The stem is smooth, round, and light green.
The little flowers are borne on the base of a long wand, which projects one or two inches beyond the ensheathing wrapper,—both wand and envelope are green; the stem is round, smooth, and green, and grows from the side of the leaf-stem.
This well-named plant has many marks of the dragon upon it; its solitary leaf spreads, particularly when unfolding, a green claw-like hand above the flowers, and all about the club-shaped root-bulb grow numerous flesh-colored bulblets highly suggestive of his dragon-ship’s toes!
GREEN DRAGON: Arisæma Dracontium.
Jack-in-the-Pulpit. Arisæmatriphyllum.
Indian Turnip.
Found in damp shady nooks, blossoming in May.
The root sends up two or three leaf-bearing stems which vary from 8 to 20 inches in height.
The leaf is compound, and often grows to a considerable size; the 3 leaflets are a broad oval shape, tapering at the tip, the ribs much marked, the fibre fine, and surface smooth; a fall, juicy green. The stem is long, round and smooth, and sheathed at the foot.
The inconspicuous flowers are borne at the base of a green wand, which is wrapped around by a leaf-like sheath, its tip curving over the head of Jack and making the sounding board of his airy green pulpit. This sheath is tougher in texture and more shining than the leaves, and varies in color; on the stamen-bearing plant it is green, striped with greenish-white, while that of the pistil-bearing plant is green, striped with blackish violet. The Jack-in-the-Pulpit is borne on a stout, round, shining stem, which springs from between the sheaths of the leaf-stem.
In late summer the ripened seeds are found, a thick short club of bright red berries; the leaves of the seed-bearing plants often grow very large and are a rich, dark green color. It will be observed that Jack-in-the-Pulpit is “brother to dragons”!
