Biotransformation involves chemical alteration of drugs in the body through two phases. Phase I makes the drug more polar through reactions like oxidation, reduction and hydrolysis. This is mainly done by cytochrome P450 enzymes in the liver. Phase II further increases polarity through conjugation reactions like glucuronidation and sulfation. This makes the drug water-soluble and ready for excretion to remove it from the body. Biotransformation is an important mechanism for maintaining homeostasis after exposure to various xenobiotics.

1. BIOTRANSFORMATION
Biotransformationmeanschemical alterationof the druginthe body.Itis neededtorender
nonpolar(lipid-soluble) compoundspolar(lipid-insoluble)sothattheyare notreabsorbedinthe
renal tubulesandare excreted.Inthe absence of metabolism, bodywill notbe able togetridof
lipophilicsubstances,andtheywill become verylongacting.Mosthydrophilicdrugs,e.g.
streptomycin,neostigmine,pancuronium, etc.are littlebio-transformedandare largelyexcreted
unchanged.Mechanismswhichmetabolize drugs(essentiallyforeignsubstancesorXenobiotic) have
developedtoprotectthe bodyfromingestedtoxinandotherenvironmentalchemicals.The primary
site fordrug metabolismisliver;othersare-kidney,intestine,lungsandplasma.Biotransformationof
drugsmay leadto the following.
(i) Inactivation- Mostdrugsandtheiractive metabolitesare renderedinactiveorlessactive,e.g.
ibuprofen,paracetamol,lidocaine,chloramphenicol,propranololanditsactive metabolite 4-
hydroxypropranolol.Thus,biotransformationprovidesanalternative methodof terminatingdrug
actionto excretion.
(ii) Active metabolite fromanactive drug- Many drugshave beenfoundtobe partiallyconvertedto
one or more active metabolite;the effectsobservedare the sumtotal of that due to the parentdrug
and itsactive metabolite(s)
(iii) Activationof inactivedrug- Fewdrugsare inactive assuchand needconversioninthe bodyto
one or more active metabolites.Suchadrug is calledaprodrug.The prodrugmay offeradvantages
overthe active formin beingmore stable,havingbetterbioavailabilityorotherdesirable
pharmacokineticpropertiesorlessside effectsandtoxicity.Someprodrugsare activatedselectively
at the site of action.
Biotransformationreactionscanbe classifiedinto:
(a) Nonsynthetic/Phase l/Functionalizationreactions:afunctional group(-OH, -COOH, -CHO, -NH2,-
SH) isgeneratedorexposed-metabolite maybe active orinactive.
(b) Synthetic/Conjugation/Phase IIreactions: anendogenousradical isconjugatedtothe drug-
metabolite ismostlyinactive;exceptfew drugs,e.g.glucuronideconjugate of morphine andSulfate
conjugate of minoxidil are active. Certaindrugsalreadyhave functionalgroupsand are directly
conjugated,whileothersundergoaphase Ireactionfirstfollowedbyaphase IIreaction.
NON SYNTHETIC REACTIONS-
1) Oxidation/reductionreactions- Oxidationreactionsinvolve membrane-associatedenzymes
expressedinthe endoplasmicreticulum(ER) of hepatocytesand,toa lesserextent,of cells
inothertissues.The enzymesthatcatalyse these phase Ireactionsare typicallyoxidases; the
majorityof these enzymesare heme proteinmono-oxygenasesof the cytochrome P450
class.Cytochrome P450 enzymes(sometimesabbreviatedCYP) are also known as microsomal
mixed-function oxidases and areinvolved in the metabolismof approximately 75% of all drugs used
today. (The term P450 refers to the 450-nm absorption peak characteristic of these heme proteins
when they bind carbon monoxide.)
The net resultof a cytochrome P450-dependent oxidation reaction is:

2. The reaction proceeds when the drug binds to the oxidized (Fe3+) cytochrome P450 to form a complex, which
is then reduced in two sequential oxidation/reduction steps as outlined in figureA. Nicotinamideadenine
dinucleotidephosphate(NADPH) donates the electrons in both of these steps via a flavoprotein reductase.In
the firststep, the donated electron reduces the cytochrome P450–drug complex. In the second step, the
electron reduces molecular oxygen to form an activated oxygen–cytochrome P450–drugcomplex. Finally,as
the complex becomes more activethrough rearrangement, the reactive oxygen atom is transferred to the
drug, resultingin the formation of the oxidized drugproduct and recyclingoxidized cytochromeP450 in the
process.The mechanismof these reactions is illustrated in Figure B.
Most livercytochrome P450 oxidasesexhibitbroadsubstrate specificity.Thisisdue inpartto the
activatedoxygenof the complex,whichisapowerful oxidizingagentthatcanreact witha varietyof
substrates.The namesof the cytochrome P450 enzymesare sometimesdesignatedby“P450”
followedbythe numberof the P450 enzyme family,capital letterof the subfamily,andanadditional
numberto identifythe specificenzyme (e.g.,P4503A4). Many of the P450 enzymeshave partially
overlappingspecificitiesthattogetherallow the livertorecognize andmetabolize awide arrayof
xenobiotics.

3. Together,P450-mediatedreactionsaccountformore than 95% of oxidative bio-transformations.
Otherpathwaysmayalso oxidize lipophilicmolecules.A pertinentexampleof anon-P450 oxidative
pathwayisthe alcohol dehydrogenasepathwaythatoxidizesalcoholstotheiraldehyde derivatives
as part of the overall processof excretion.These enzymesare alsothe basisforthe toxicityof
methanol.Methanol isoxidizedbyalcohol dehydrogenase toformaldehyde,whichcando
considerable damage tosome tissues.The opticnerve isparticularlysensitive toformaldehyde,and
methanol toxicitycancause blindness.
Anotherimportantnon-P450enzyme ismonoamineoxidase (MAO).Thisenzyme isresponsible for
the oxidationof amine-containingendogenouscompoundssuchascatecholaminesandtyramine
and some xenobiotics,includingdrugs.
Reduction- Thisreactionisthe converse of oxidationandinvolvescytochrome P-450enzymes
workinginthe opposite direction.Alcohols,aldehydes,quinonesare reduced.Drugsprimarily
reducedare chloralhydrate,chloramphenicol,halothane,andwarfarin.
2) Conjugationandhydrolysisreactions provide asecondsetof mechanismsformodifying
compoundsforexcretion.Althoughhydrolysisof ester- andamide-containingdrugsis
sometimesincludedamongthe phase Ireactions(inthe olderterminology),the
biochemistryof hydrolysisismore closelyrelatedtoconjugationthanto
oxidation/reduction. Substratesforthese reactionsinclude bothmetabolitesof oxidation
reactions(e.g.,epoxides)andcompoundsthatalreadycontainchemical groupsappropriate
for conjugation,suchashydroxyl (-OH),amine(-NH2),orcarboxyl (-COOH) moieties.These
substratesare coupledbytransferenzymestoendogenousmetabolites(e.g.,glucuronicacid
and itsderivatives, sulphuricacid,aceticacid,aminoacids,andthe tripeptide glutathione) in
reactionsthatofteninvolve high-energyintermediates.The conjugation andhydrolysis
enzymesare locatedinboththe cytosol andthe endoplasmicreticulumof hepatocytes(and
othertissues).Inmostcases,the conjugationprocessmakesthe drugmore polar.Virtually
all of the conjugatedproductsare pharmacologicallyinactive,withsome important
exceptions(e.g.,morphineglucuronide).
Some conjugationreactionsare importantclinicallyinthe case of neonates,whohave notyet
fullydevelopedthe capacitytocarry out thissetof reactions.UDP-glucuronyl transferases
(UDPGT) isresponsibleforconjugatingbilirubininthe liverandfacilitatingitsexcretion.A
relative deficiencyof thisenzymeatthe time of birthputs infantsatriskfor neonatal jaundice,
whichresultsfromincreasedserumlevelsof unconjugatedbilirubin.Neonataljaundice isa
problembecause neonateshave notonlyunderdevelopedactivityof thisenzyme butalsoan

4. undevelopedblood–brainbarrier.Unconjugatedbilirubiniswater-insoluble andverylipophilic;
it bindsreadilytothe unprotectedneonatal brainandiscapable of causingsignificantdamage to
the central nervoussystem.Thispathologicconditionisknownasbilirubinencephalopathyor
kernicterus.Neonatal hyperbilirubinemia(unconjugated) canbe treatedwithphototherapywith
450-nm light,whichconvertscirculatingbilirubintoanisomerthatis more rapidlyexcreted.
Anothereffective treatmentisthe administrationof small dosesof the barbiturate
phenobarbital,whichpowerfullyup-regulatesthe expressionof the enzymeUDPGTand thereby
reducesserumlevelsof unconjugatedbilirubin.Thisexampleillustratesarecurringtheme:
understandingdrugmetabolismcanhelppredictbothadverseandpotentiallyadvantageous
drug–drug interactions.
It isimportantto note that conjugationand hydrolysisreactionsdonotnecessarilyconstitute
the last stepof biotransformation.Since the conjugationof these highlypolarmoietiesoccurs
intracellularly,theyoftenrequire active transportacrosscellularmembranestobe excreted
(active transportof the parentdrug can also occur). Moreover,some conjugationproductsmay
be subjectedtofurthermetabolism.

5. 3) Hydrolysis- Thisiscleavage of drugmoleculebytakingupa molecule of water.
Similarly,amidesandpolypeptidesare hydrolysedby amidasesandpeptidases.Inaddition,
there are epoxide hydrolaseswhichdetoxify epoxide metabolitesof some drugsgenerated by
CYP oxygenases.Hydrolysisoccursinliver, intestines,plasmaandothertissues.Examples of
hydrolyseddrugsare choline esters,procaine, lidocaine,procainamide,aspirin,indomethacin,
carbamazepine-epoxide,pethidine,andoxytocin.
4) Cyclization- Thisisformationof ring structure from a straightchaincompound,e.g.
cycloguanil fromproguanil.
5) Decyclization- Thisimpliesopeningupof ringstructure of the cyclicdrug molecule,such as
barbiturates, phenytoin.Thisisgenerallya minorpathway.

6. PHASE 2 METABOLISM
These reactionsinvolve conjugationof the drugor itsphase I metabolitewithanendogenous
substrate, usuallyderivedfromcarbohydrate oraminoacid,to forma polar highlyionizedorganic
acid,whichis easilyexcretedinurine orbile.Conjugationreactionshave highenergyrequirement
and are generallyfasterthanphase Ireactions.
(i) Glucuronide conjugation- Thisisthe mostimportantsyntheticreactioncarriedoutbya
groupof UDP-glucuronosyl transferases(UGTs).Compoundswithahydroxylor
carboxylicacidgroupare easilyconjugatedwithglucuronicacidwhichisderivedfrom
glucose.Examplesare— chloramphenicol, aspirin,paracetamol,diazepam, lorazepam,
morphine,andmetronidazole.Notonly drugsbutendogenoussubstrateslikebilirubin,
steroidal hormonesand Thyroxine utilize this pathway.Glucuronidationincreasesthe
molecularweightof the drugwhichfavoursitsexcretioninbile.Drugglucuronides
excretedinbile canbe hydrolysedbybacteriainthe gut—the liberateddrugis
reabsorbedandundergoesthe same fate.Thisenterohepaticcyclingof the drug
prolongsitsaction,e.g. - phenolphthalein,oral contraceptives.
(ii) AcetylationCompoundshavingaminoorhydrazine residuesare conjugatedwiththe
helpof acetyl coenzyme-A,e.g.sulfonamides,isoniazid,PAS,dapsone,hydralazine,
clonazepam,procainamide.Multiplegenescontrol the N-acetyl transferases(NATs),and
rate of acetylationshowsgeneticpolymorphism(slow andfastacetylators).
(iii) Methylation- The aminesandphenolscanbe methylatedbymethyltransferases(MT);
methionineandcysteineactingasmethyl donors,e.g.adrenaline,histamine,nicotinic
acid,methyldopa,captopril,mercaptopurine.

7. (iv) Sulfate conjugation- The phenoliccompoundsandsteroidsare sulphatedby
sulfotransferases(SULTs),e.g.chloramphenicol,methyldopa,adrenal andsex steroids.
(v) Glycination- Glycineandglutamate appeartobe the mostcommon acceptorsof amino
acidsin mammals.Inhumans,more than95% of bile acidsare N–acyl amidateswith
glycine ortaurine.Althoughproductsof aminoacidconjugationare consideredtobe
metabolicallystableandnontoxic, ithasbeensuggestedthatthe firstreactionof amino
acid conjugationleadsinsome casestoformationof potentiallytoxicintermediates.This
toxificationpathwayinvolvesconjugationof N–hydroxyaromaticamineswiththe
carboxylicacidgroupof serine andproline.Aminoacidactivatedbyaminoacyl–tRNA–
synthetase (Fig.9) subsequentlyreactswithanaromatichydroxylamine toformN–ester
that can degrade to produce a reactive nitreniumion.Ingeneral,the toxicityof
nitreniumionsisclinicallyrelevantsince these electrophilespossessingDNA–binding
abilityare responsible forcarcinogenicityof aromaticamines. Salicylates,nicotinicacid
and otherdrugshavingcarboxylicacid groupare conjugatedwithglycine,butthisis not
a major pathwayof metabolism.

8. (vi) Glutathione conjugation- Thisiscarried outbyglutathione-S-transferase (GST) forming a
mercapturate.Itisnormallya minorpathway. However,itservestoinactivate highly
reactive quinone orepoxide intermediatesformedduring metabolismof certaindrugs,
e.g.paracetamol. Whenlarge amountof such intermediatesare formed(inpoisoningor
afterenzyme induction), glutathione supplyfallsshort—toxicadducts are formedwith
tissue constituentsresultingin hepatic,renal andothertissue damage.
(vii) Ribonucleoside/nucleotidesynthesis- Thispathwayisimportantforthe activation of
manypurine andpyrimidine antimetaboliteusedincancerchemotherapy
SUMMARY
PHASEI – make the drug polarby unmasking polarfunctional groups
Microsomal enzymesmeansenzymesthattypicallyfoundinendoplasmicreticulumof
hepatocytes
- By microsomal enzyme CytP450
- Microsomal enzymes
• Flavinmonooxygenase
• Cytochrome P450
• UDP glucoronyl transferase
• Glutathion -S- transferase
• Epoxide hydrolase
• CarboxyEsterase
CytP450 enzymes:
1) CyP2D6 – 20-30% of drugs metabolismoccursbythisenzyme
Ex. Betablockers,Antidepressants,Antiarrhythmic
2) CyP2C9/8- 10% drugmetabolised
Ex.Celecoxib,Warfarin,phenytoin
3) C yP2C19-
4) Cyp1A1 and CyP1A2– Ex. Theophylline,paracetamol
5) CyP2E1 – Ex. Enflurane

9. 6) CyP3A4 – most drug metabolizedbythisenzyme)
Ex. Protease inhibitors,statins,benzodiazepine,Amiodarone,Estrogen,Erythromycin
PHASEII – alsoknownas true detoxification
- Make the drug polarby conjugationoraddinga polar functional group
- Drug acquire polarity
- Excretedoutof the body
- By Non– microsomal enzymes
- Exceptglucuronide conjugation
ENZYMES INVOLVED–
• Sulphonyl transferase –Additionof sulphate groupinxenobiotic
• UDP – glucoronyl transferase - Additionof glucoronicacid
• Glutathion-s-transferase- Additionof glutathione
• N – acetyl transferase – additionof acetyl group
• Methyl transferase –Additionof methyl group
Xenobioticbiotransformationisanimportantmechanismformaintaininghomeostasisfollowing
exposure tonumerousxenobiotics,suchasmedications,industrialchemicals,orfood
procarcinogens.The liveristhe primarylocationof expressionforxenobiotic–metabolizingenzymes
inhumansand othermammals,althoughextra hepaticallylocalisedenzymesappeartobe quite
importantas well.Several drugmetabolisingenzymes,forexample,are thoughttoreduce the
bioavailabilityof orallydeliveredmedicationsorto activate environmental carcinogensinthe colon.
Phase IIreactionsmay or maynot be precededbyPhase Ireactions.Phase IIenzymesprobablyplay
an essential partinthe detoxificationof differentxenobiotics.Furthermore,theyplayanimportant
role inregulatinghomeostasisbybinding,transporting,orinactivatingphysiologicallyactive
chemicalssuchas hormones,bile acids,orothersubstances. Inadditiontotheirpositive effects,
these enzymesare involvedinthe creationof reactive intermediatesof avarietyof chemicals.The
conjugationof N–hydroxyaromaticaminesisthe most–discussedexample of atoxificationreaction.
Numerousprocessesconvertthese moleculestohazardousmetabolites,includingN–
glucuronidationbyUGTs,O–acetylationby NATs,O–sulfonationbySULTs,andconjugationwith
aminoacidsby aminoacyl–tRNA–synthetase.Because of covalentattachmenttoDNA or other
biomolecules,the newlyproducedreactive electrophilicnitreniumandcarboniumionscanact as
carcinogensandmutagens.AnotherimportantconcernisgeneticvariationsinPhase IIenzymes.
Impaireddrugmetabolismdue togeneticallybasedfailure of competentenzymesmayresultin
harmful consequencesof clinicallyutilisedmedicines. Furthermore,geneticvariationsinthese
enzymesare clearlyresponsible forthe developmentof avarietyof neurological diseasesor
malignancies.Tosummarise,Phase IIenzymesare anintriguingresearchtopicbecause theyplayan
importantrole inthe metabolismof hundredsof foreign compounds,aswell asinthe regulationof
metabolismanddispositionof variousendogenousbiologicallyactive substances,thereby
maintaininghomeostasisinthe humanbody.