Pre clinical studies

Deals with pre-clinical research study aspects in the level of undergraduate students.

1. PRE-CLINICAL SCREENING
KIRSHA K S
ASST. PROFESSOR
DEPT. OF PHARMACY PRACTICE
KMCH COLLEGE OF PHARMACY
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2. PRE-CLINICAL TESTING
Researchusinganimalstofindoutifadrug,procedure,or
treatmentislikelytobeuseful.
Preclinicalstudiestakeplacebeforeanytestinginhumans
isdone.
Aimedtoobtainbasicinformationondrug’seffectthat
maybeusedtopredictsafeandeffectiveuseinhumans.
Objectiveistogeneratealldatathatsatisfyall
requirementsbeforeanewcompoundisdeemedfittobe
testedforthefirsttimeinhumans.
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3. PRECLINICAL TESTING
Pharmacological
screening
Toxicological testing
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4. PHARMACOLOGICAL STUDIES
Pharmacologicalstudiesgaugebiologicaleffect,efficacious
doserangeandoverallpotencyoftheoptimizedlead.
Pharmacodynamic(PD)studies:mechanismofactionofthe
leadandanindepthunderstandingofthedrugaction
Pharmacokinetic(PK)studiesgivesadetailedinsightondrug
distributionindifferentorgansofstudyanimalspostdrug
treatment
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5. PHARMACOLOGICAL SCREENING
Prospectivedrugsubstancesaretestedforbiologicalactivity
toassesstheirpotentialfordevelopingasdrugs.
❖Thesestudiesshouldbedesignedtoidentifyundesirable
pharmacodynamicpropertiesofasubstancethatmayhave
relevancetoitshumansafety
❖Toevaluateadversepharmacodynamicand/or
pathophysiologicaleffectsobservedintoxicologyand/or
clinicalstudies
❖‹Toinvestigatethemechanismoftheadverse
pharmacodynamiceffectsobservedand/orsuspected.
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6. Safetypharmacologystudiesarestudiesthatinvestigate
potentialundesirablepharmacodynamiceffectsofa
substanceonphysiologicalfunctionsinrelationtoexposure
withinthetherapeuticrangeorabove
Essentialsafetypharmacologystudiesaretostudythe
effectsofthetestdrugonvitalfunctions.Vitalorgan
systemssuchascardiovascular,respiratoryandcentral
nervoussystemsshouldbestudied.
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7. Pharmacological
screening
Molecular
level study
Cellular
level study
Whole
animal
study
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8. MOLECULAR LEVEL STUDY
Prospectivesubstanceisstudiedforitsselectivityforvarious
receptorsanditsactivityagainstselectiveenzymesystems
Eg:cellmembranefractionsfromorgans,culturedcells,
clonedreceptors.Adrenalglands,liveretcareused.
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9. CELLULAR LEVEL STUDY
Simulatinghumanandanimalsystemsusingcellandtissue
culture,computerprogrammes.
Useofisolatedtissueslikebloodvessels,heart,lung,ileum
ofratandguineapig
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10. WHOLE ANIMAL STUDY
Todeterminetheeffectofprospectivesubstanceonorgan
systemsanddiseasemodels.
Reservedforsubstanceshowinggoodresultsonin-vitro
testing.
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11. TOXICOLOGY STUDIES
Toxicologyisabranchofsciencethatdealswithtoxins
andpoisonsandtheireffectsandtreatment.
Toxicologicalscreeningisveryimportantforthedevelopment
ofnewdrugsandfortheextensionofthetherapeutic
potentialofexistingmolecules.
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12. Thetoxicityofsubstancescanbeobservedby
(a)studyingtheaccidentalexposurestoasubstance
(b)invitrostudiesusingcells/celllines
(c)invivoexposureonexperimentalanimals.
Thepre-clinicaltoxicitytestinghelpstocalculate“NoObserved
AdverseEffectLevel(NOAEL)”whichisneededtoinitiatethe
clinicalevaluationofinvestigationalproducts.
NOAEListhehighestdoselevel/concentrationofasubstancethatunderdefined
conditionsofexposurecausesnoobservable/detectableadverseeffectson
morphology,functionalcapacity,growth,developmentorlifespanofthetest
animals.
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13. HISTORY OF TOXICITY STUDIES
Paracelsus (1493–1541)
Father of toxicology
Determinedspecificchemicals
responsiblefortheobservedtoxicityof
plantsandanimals.
Demonstratedtheharmlessand
beneficialeffectsoftoxinsandproved
dose-responserelationshipsforthe
effectsofdrugs.
“All substances are poisons; there is
none which is not a poison. The right
dose differentiates a poison and a
remedy.”
Mathieu Orfila (1787–1853)
Father of modern toxicology
Determinedtherelationshipbetween
poisonsandtheirbiologicalproperties
Demonstratedspecificorgandamage
causedbytoxins
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14. mid-1900s
Toxicological
screeningmethods
and toxicological
researchonindividual
substances
1920
useofanimalsin
toxicitystudies
J. W. Trevan
proposed the use of
the 50% lethal dose
(LD50)
1980s
The Organisationfor
Economic Co-
operation and
Development (OECD)
and the International
Conference on
Harmonization (ICH)
brought out the
guidelines for
toxicity testing of
pharmaceutical
substances
mid-20thcentury
environmental
toxicologicalstudies
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15. APPROVAL FORSTUDY
InstituteAnimalEthicsCommittee(IAEC)
InIndia,theCommitteeforthePurposeofControland
SupervisionofExperimentsonAnimals(CPCSEA)guidelines
shouldbefollowedforthemaintenanceofexperimental
animals.
Anapprovedfluidwithdrawalmethodshouldbeused,and
scheduleY(India)shouldbecompliedwithtofulfilthe
regulatoryrequirements
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16. ACUTE TOXICITY STUDIES
Todeterminetheshorttermadverseeffectsofadrugwhen
administeredinsingledoseorinmultipledoseduringaperiod
of24hours.
Acutetoxicitytestingbecarriedoutwithtwodifferentanimal
species(onerodentandonenonrodent).
Inacutetoxicologicaltesting,theinvestigationalproductis
administeredatdifferentdoselevels,andtheeffectisobserved
for14days.
Allmortalitiescausedbytheinvestigationalproductduringthe
experimentalperiodarerecordedandmorphological,
biochemical,pathological,andhistologicalchangesinthedead
animalsareinvestigated.
Acutetoxicitytestingpermitsthe50%lethaldose(LD50)ofthe
investigationalproducttobedetermined. 16

17. SINGLE DOSE ACUTE TOXICITY TESTING
❖In1996,theCenterforDrugEvaluationandResearch(CDER)suggested
asingledoseacutetoxicitytestingprocedureforpharmaceutical
substancesthatusesafixedsafedosethatshouldnotcauseadverse
eventsorthreatenthelifeofananimal.
❖Theexperimentmustbecarriedoutwithaminimumoftwomammalian
species,includinganonrodentspecies,andtheanimalsmustbe
observedfor14days.
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18. REPEATED DOSE TOXICITY TESTING
❖Repeateddosetoxicitytestingiscarriedoutforaminimumof28days.
❖Thetestsubstanceisadministeredregularlyataspecifictime.
❖Usually,arodentofanygenderandage5–6weeksisusedforrepeated
dosetoxicitytesting.
❖Asatellitegroupmaybeincludedinthestudyprotocol.Thisgrouphasboth
acontrolgroupandahigh-dosegroup.
❖Baselineparameterssuchasthebehavioralandbiochemicalparametersof
theanimalsshouldberecorded.
❖Attheendofthestudy,tissuesfrommostoftheorgansareremoved,and
histologicalchangesarerecorded.Ifpossible,immunotoxicity(adverse
effectsontheimmunesystem)studiesareperformedonthesameanimals.
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19. METHODS OF
ACUTE ORAL TOXICITY STUDIES
The fixed dose
procedure (FDP)
The acute toxic
category (ATC)
method
The up-and-down
(UDP) method.
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20. FDP
Assessthenonlethal
toxicityratherthanthe
lethaldose
Theinvestigationalproduct
isadministeredatfixed
doselevelsof5,50,500,
and2000mg/kgandthe
experimentalanimalis
observedforaspecified
period.
ATC
method
Sequentialprocedurein
whichthreeanimalsof
thesamesexareused
ineachstep
Checksfordosecausing
mortality
Four preidentified
startingdosesmaybe
used,andthetestdose
shouldbeselected
basedontheGlobally
Harmonized
Classificationsystem.
UDP testing
approach
Staircasedesign
Reducesthenumberof
vertebrateanimalsin
research
Dosingsingleanimals
sequentiallyat48h
intervals
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21. UDP SCREENING METHOD
❖Thisisthetoxicologicaltestingapproachmostrecommendedby
variousregulatoryagenciesbecausethismethod.
❖TheUDPscreeningmethodinvolvesdosingsingleanimalssequentially
at48hintervals.
❖FemalerodentsarepreferableforUDPtesting.
❖Adoselessthanthebest-estimateLD50doseisselectedand
administeredtoananimal,andtheanimalisobservedfor48h.
❖Ifitsurvives,thestudyiscontinuedwithahigherdose(twicethe
originaldose);iftheanimaldies,testingisconductedwithalower
dosewithanotheranimalofthesamesexastheoriginalanimal.
❖UDPtestingislimitedtodosesupto2000mg/kg.
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22. SUB CHRONIC TOXICITY STUDIES
Sub-chronictoxicitystudieswithrodentsaregenerallyconductedfor90
days(3months)
❖Helppredictappropriatedosesofthetestsubstanceforfuturechronic
toxicitystudies,
❖UsedtodetermineNOELsforsometoxicologyendpoints,and
❖Allowfuturelong-termtoxicitystudiesinrodentsandnon-rodentstobe
designedwithspecialemphasisonidentifiedtargetorgans
Themajordifferencebetweenrepeateddoseandsub-chronictoxicitystudiesis
theduration:repeateddosetoxicitystudiesareconductedoveradurationof
28days,andsub-chronictoxicitystudiesarecarriedoutover90days.
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23. CHRONIC TOXICITY STUDIES
❖Conductedwithaminimumofonerodentandonenonrodentspecies.
❖Thetestcompoundisadministeredovermorethan90days,andthe
animalsareobservedperiodically.
❖Achronictoxicologystudyprovidesinferencesaboutthelong-termeffect
ofatestsubstanceinanimals,anditmaybeextrapolatedtothehuman
safetyofthetestsubstance.
❖Thereportonchronicoraltoxicityisessentialfornewdrugentities.
❖Asatellitegroupmaybeincludedinthestudyprotocol.Thisgrouphasboth
acontrolgroupandhigh-dosegroup.
❖Duringthestudyperiod,theanimalsareobservedfornormalphysiological
functions,behavioralvariationsandalterationsinbiochemicalparameters.
❖Attheendofthestudy,tissuesarecollectedfromallpartsoftheanimal
andsubjectedtohistologicalanalyses
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24. CARCINOGENICITY TESTING
❖Bothrodentsandnonrodentanimalspeciesmaybeusedin
carcinogenicitytesting.
❖Duringandafterexposuretotestsubstances,theexperimentalanimals
areobservedforsignsoftoxicityanddevelopmentoftumors.Ifthese
arenotfound,atestmaybeterminatedafter18monthsinthecaseof
miceandhamstersandafter24monthswithrats.
❖Iftheanimalsarehealthy,hematologicalanalysisisperformedafter
the12monthsandthe18months,respectively,andthestudyis
terminated.
❖Theanimalsaresacrificed,andgrosspathologicalchangesarenoted
andhistopathologicalstudiesarecarriedoutonallthetissues.
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25. REPRODUCTION TOXICITY
TESTING
Thetestcompoundisadministeredtobothmaleandfemale
animals(rodents).
Administrationisforthedurationofonecompletespermatogeniccycle
inmaleanimalsandfortwocompleteestrouscyclesforfemale
animals.
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26. ONE-
GENERATION
REPRODUCTION
TOXICITY TESTING
•Afterthecompletionofthespecified
durationofdrugadministration,the
animalsareallowedtomate.
•Thetestcompoundisadministeredtothe
femaleanimalsduringtheperiodof
pregnancyandnursing.
•Thespermsofmaleanimalsare
collected,andthespermmorphology
andmotilityareanalyzed.
•Duringthestudyperiod,theanimalsare
observedforsignsoftoxicity.Parturition,
thenumberofoffspringandtheirsexes
arerecorded.
•Thenumberofdeadandlivepupsare
noted,andlivepupsareweighedinthe
morningandeveningeachdayduring
thefirst4days.
•Aftertheterminationofthestudy,the
animalsandpupsaresacrificedand
subjectedtoahistopathological
examination
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27. TWO-
GENERATION
REPRODUCTION
TOXICITY
STUDIES
•Aftertheadministrationperiod,theanimalsareintertwined
(parentalmating),afterwhichthefemaleanimalsare
separated.
•Spermsarecollectedfrommaleanimals,andthesperm
morphologyandmotilityareanalyzed.
•Thetestsubstanceisadministeredcontinuouslytopregnant
femaleanimals,whicharemonitoredregularlyformortality
andsignsoftoxicity.
•Afterparturition,nursingratsareadministeredthetestdrug,
andthemortalityofthepups(F1generation)isobserved.
•FromtheF1generation,onemaleandonefemaleanimalare
selected.ThesameprocedureisrepeatedtogettheF2
generationoffspring.
•F1offspring'sarenotallowedtomateuntiltheyhaveattained
fullsexualmaturity,andpairswithoutapregnancyare
evaluatedforinfertility.
•Necropsiesandhistologicalexaminationsarecarriedout.
•Attheendofthestudy,theanimalsaresacrificedandgross
pathologicalandhistologicalexaminationsarecarriedouton
alltheanimals.
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28. TOXICOKINETICS
❖Toxicokineticswhichisanextensionofpharmacokineticsdealswiththe
kineticpatternsofhigherdosesofchemicals/toxins/xenobiotics.
❖Toxicokineticshelpsstudythemetabolismandexcretionpatternof
xenobiotics.
❖Animaltoxicokineticdatahelpextrapolatephysiologicallybased
pharmacokineticsinhumans.
❖Intoxicologicaltesting,pharmacokineticstudiesareusuallycarriedoutin
rodents,rabbits,dogs,nonhumanprimatesandswineusingmanyroutesof
administration.
❖Bloodsamplesarecollectedatvarioustimepointstoanalyze
pharmacokineticdatasuchastheareaunderthecurve,drugdistribution
ratio,Cmax,tmax,andotherpharmacokineticparameters.
❖Toxicokineticstudiesmaybeperformedusinginvitrocelllinesalso
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29. NEUROTOXICITY STUDIES
❖Theeffectsofatestsubstanceonthecentralnervoussystemcanbe
studied
❖Neurotoxicstudiesmaybeemployedtoevaluatethespecific
histopathologicalandbehavioralneurotoxicityofachemicalandare
usedtocharacterizeneurotoxicresponsessuchasneuropathological
lesionsandneurologicaldysfunctions(lossofmemory,sensorydefects,
andlearningandmemorydysfunctions).
❖Usuallyneurotoxicologicalstudiesarecarriedoutinadultrodents.
❖Thetestsubstancemaybeadministeredfor28daysorevenmore
than90days,andneurologicalchangesareevaluated
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30. DEVELOPMENTAL TOXICITY/
EMBRYOTOXICITY STUDIES
•Thecompoundisadministeredbetweenthe8
th
and
14
th
dayofpregnancy,andembryolethaleffects
arestudied.
•Attheendofthestudyoronthe21stdayofthe
study,acaesareansectionisperformedand
parameterssuchasfetuseswithhemorrhagic
bullae,limbmalformations,exencephaly,cleft
palates,openeyelids,andtaildeformitiesaswell
asthemortalityandthenumbersofdeadandlive
pupsarenoted
In Vivo
•Embryonicstemcelltest(EST)forembryotoxicity,
micromassembryotoxicityassay,andwholerat
embryoembryotoxicityassay.
In Vitro
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31. GENETIC TOXICITY TESTING
❖Genetictoxicitytestsareusedtoidentifygenemutations,
chromosomechanges,andalterationsintheDNAsequencing.
❖Thesetestsareusuallyconductedinvariousspeciesincludingwhole
animals,plants,micro-organisms,andmammaliancells.
❖Inthewholeanimalmodel,rodentsarepreferred.
❖Genetictoxicityisassessedusingtherodentchromosomeassay,
dominantlethalassay,mouse-specificlocustest,micronucleustest,
heritabletranslocationassay,andsisterchromatidexchangeassay
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32. REGULATORY REQUIREMENTS
❖Beforeconductinganyclinicalstudy,thesafetyofthetestsubstanceshouldbe
assessedusinganimals.
❖Thetargetorgantoxicity,relationshipbetweenthedoseandresponse,relevant
humaneffects,andanycomplicationsarisingduringtreatment(adversedrug
reactions)shouldbeestablishedthroughpreclinicalevaluations.
❖Thetoxicitystudyshouldbecarriedoutwithaminimumofthreedosesviz.low,
medium,andhighdosesintheexperimentalanimalsandthetoxiceffectcompared
withdatafromacontrolgroupofanimals.
❖TheCommitteeforProprietaryMedicinalProducts(CPMP)hassetguidelinesonthe
toxicologicalexperimentonvariousanimalspecies.Theguidelineinstructsthatthe
maximumselecteddoseshouldbesufficienttoidentifythetargetorgantoxicity.
❖Fromthetoxicologicalevaluation,thenoobservedeffectlevel(NOEL)orNOAEL,
whichmaybeusefulforhumanstudies,maybeestablished.
❖Thelowdose,intermediatedose,andhighdoseusedinthetoxicitytestprovidethe
NOEL,dose–responserelationship,andtargetorgantoxicityinanimals,respectively.
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33. MUTAGENICITY TESTING
UsedtoassesssubmicroscopicchangesinthebasesequenceofDNA,
chromosomalaberrations,andstructuralaberrationsinDNAincluding
duplications,insertions,inversions,andtranslocations.
Mutagenicitystudieswithtransgenicanimalsaremoreappropriateassay
techniquestodeterminethetoxicityofatestsubstance
In vitro testing
Carriedoutintwoorthreedifferentbacteria
andmammaliancellstocovertheendpoints
ofgenemutations,clastogenicity,and
aneuploidy.Thetestgenerallyincludesa
bacterialreversemutationassay
In vivo
mutagenicity
Dosedependentisusedtodeterminethe
case-by-casebasisriskassessmentofthetest
substances
Mutagenicity
testing
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34. 34

35. REFERENCE
S. Parasuraman,Toxicological screening.J Pharmacol Pharmacother.
2011 Apr-Jun; 2(2): 74–79.
doi: 10.4103/0976-500X.81895
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