overview phase i and phase ii enzymes reaction mechanisms, substrates enzyme inhibitors and...
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BIOTRANSFORMATION OF XENOBIOTICS
Overview Phase I and Phase II enzymes Reaction mechanisms, substrates Enzyme inhibitors and inducers Genetic polymorphism Detoxification Metabolic activation
Introduction
Purpose Converts lipophilic to hydrophilic
compounds Facilitates excretion
Consequences Changes in PK characteristics Detoxification Metabolic activation
Comparing Phase I & Phase II
Enzyme Phase I Phase II
Types of reactions Hydrolysis Oxidation Reduction
Conjugations
Increase in hydrophilicity
Small Large
General mechanism
Exposes functional group
Polar compound added to functional group
Consquences May result in metabolic activation
Facilitates excretion
First Pass Effect
Biotransformation by liver or gut enzymes before compound reaches systemic circulation
Results in lower systemic bioavailbility of parent compound
Examples: Propafenone, Isoniazid, Propanolol
Phase I reactions
Hydrolysis in plasma by esterases (suxamethonium by cholinesterase)
Alcohol and aldehyde dehydrogenase in liver cytosol (ethanol)
Monoamine oxidase in mitochondria (tyramine, noradrenaline, dopamine, amines)
Xanthine oxidase (6-mercaptopurine, uric acid production)
Enzymes for particular substrates (tyrosine hydroxylase, dopa-decarboxylase etc.)
Phase I: Hydrolysis
Carboxyesterases & peptidasesHydrolysis of esters
eg: valacyclovir, midodrine Hydrolysis of peptide bonds
e.g.: insulin (peptide)
Epoxide hydrolaseH2O added to epoxides
eg: carbamazepine
Phase I: Reductions
Azo ReductionN=N to 2 -NH2 groups
eg: prontosil to sulfanilamide
Nitro ReductionN=O to one -NH2 group
eg: 2,6-dinitrotoluene activationN-glucuronide conjugate hydrolyzed by gut
microfloraHepatotoxic compound reabsorbed
Reductions
Carbonyl reductionChloral hydrate is reduced to trichlorothanol
Disulfide reductionFirst step in disulfiram metabolism
Reductions
Quinone reductionCytosolic flavoprotein NAD(P)H quinone
oxidoreductasetwo-electron reduction, no oxidative stresshigh in tumor cells; activates diaziquone to
more potent form
Flavoprotein P450-reductaseone-electron reduction, produces
superoxide ionsmetabolic activation of paraquat,
doxorubicin
Reductions
DehalogenationReductive (H replaces X)
Enhances CCl4 toxicity by forming free radicals
Oxidative (X and H replaced with =O)Causes halothane hepatitis via reactive
acylhalide intermediatesDehydrodechlorination (2 X’s removed, form
C=C)DDT to DDE
Phase I: Oxidation-ReductionAlcohol dehydrogenase
Alcohols to aldehydesGenetic polymorphism; Asians metabolize
alcohol rapidlyInhibited by ranitidine, cimetidine, aspirin
Aldehyde dehydrogenaseAldehydes to carboxylic acidsInhibited by disulfiram
Phase I: Monooxygenases
Monoamine OxidasePrimaquine, haloperidol, tryptophan are
substratesActivates 1-methyl-4-phenyl-1,2,5,6-
tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms
MonoOxygenases
Peroxidases couple oxidation to reduction of H2O2 & lipid hydroperoxidaseProstaglandin H synthetase (prostaglandin
metabolism)Causes nephrotoxicity by activating aflatoxin
B1, acetaminophen to DNA-binding compounds
Lactoperoxidase (mammary gland)Myleoperoxidase (bone marrow)
Causes bone marrow suppression by activating benzene to DNA-reactive compound
Monooxygenases
Flavin-containing Mono-oxygenasesGenerally results in detoxificationMicrosomal enzymesSubstrates: Nicotine, Cimetidine,
Chlopromazine, Imipramine
Phase I: Cytochrome P450
Microsomal enzyme ranking first among Phase I enzymes
Heme-containing proteinsComplex formed between Fe2+ and CO
absorbs light maximally at 450 (447-452) nm
Cytochrome P450 reactions
Hydroxylation
Testosterone to 6-hydroxytestosterone (CYP3A4)
Cytochrome P450 reactions
EPOXIDATION OF DOUBLE BONDSCarbamazepine to 10,11-epoxide
HETEROATOM OXYGENATIONOmeprazole to sulfone (CYP3A4)
Cytochrome P450 reactions
HETEROATOM DEALKYLATIONO-dealkylation (e.g., dextromethorphan to
dextrophan by CYP2D6)N-demethylation of caffeine to:
theobromine (CYP2E1)paraxanthine (CYP1A2)theophylline (CYP2E1)
Cytochrome P450 reactions
Oxidative Group TransferN, S, X replaced with OParathion to paroxon (S by O)Activation of halothane to
trifluoroacetylchloride (immune hepatitis)
Cytochrome P450 reactions
Cleavage of EstersCleavage of functional group, with O
incorporated into leaving groupLoratadine to Desacetylated loratadine
(CYP3A4, 2D6)
Cytochrome P450 reactions
DehydrogenationAbstraction of 2 H’s with formation of C=CActivation of Acetaminophen to
hepatotoxic metabolite N-acetylbenzoquinoneimine
Cytochrome P450 expression
Gene family, subfamily names based on amino acid sequences
At least 15 P450 enzymes identified in human Liver Microsomes
Cytochrome P450 expressionVARIATION IN LEVELS activity due to
Genetic PolymorphismEnvironmental Factors: inducers,
inhibitors, diseaseMultiple P450’s can catalyze same
reaction
A single P450 can catalyze multiple pathways
Major P450 Enzymes in Humans
CYP1A1/ 2
Expressed in:
Substrates Inducers Inhibitors
Liver Lung Skin GI Placenta
Caffeine Theophylline
Cigarrette smoke; Cruciferous veggies; Charcoal-broiled meat
Furafylline (mechanism-based); -naphtho-flavone (reversible)
Major P450 Enzymes in Humans
CYP2B6
Expressedin:
Substrates Inducers Inhibitors
Liver DiazepamPhenanthrene
??? Orphenadrine(mechanism-based)
Major P450 Enzymes in Humans
CYP2C19
Genetic polymorphism Substrates Inducers Inhibitors
Poor metabolizers have defective CYP2C9
Phenytoin Piroxicam Tolbutamide Warfarin
Rifampin
Sulfafenazole
Major P450 Enzymes in Humans
CYP2C19
Genetic polymorphism Substrates Inducers Inhibitors
Rapid and slowmetabolizers of S-mephenytoin
N-demethylationpathway of S-mephenytoinmetabolismpredominates in slowmetabolizers
S-mephenytoin(4’-hydroxylationis catalyzed byCYP2C19)
Rifampin Tranylcypromine
Major P450 Enzymes in HumansCYP2D6
Genetic polymorphism Substrates Inducers Inhibitors
Poor metabolizers lackCYP2D6
Debrisoquine causes marked,prolonged hypotension inslow metabolizers
No effect on response topropanolol in poormetabolizers; alternatepathway (CYP2C19) willpredominate
5-10% of Caucasians arepoor metabolizers
< 2% of Asians, AfricanAmericans are poormetabolizers
PropafenoneDesipraminePropanololCodeineDextromethorphanFluoxetineClozapineCaptopril
Poor metabolizersidentified byurinary exrection ofDextrorphan
None known FluoxetineQuinidine
Major P450 Enzymes in Humans
CYP2E1
Expressed in: Substrates Inducers Inhibitors
LiverLungKidneyLympocytes
EthanolAcetaminophenDapsoneCaffeineTheophyllineBenzene
EthanolIsoniazid
Disulfiram
Major P450 Enzymes in HumansCYP3A4
Expressedin:
Substrates Inducers Inhibitors
Liver;Kidney;Intestine;MostabundantP450enzyme inliver
AcetaminophenCarbamazepineCyclosporineDapsoneDigitoxinDiltiazemDiazepamErythromycinEtoposideLidocaineLoratadineMidazolamLovasatinNifedipineRapamycinTaxolVerapamil
RifampinCarbamazepinePhenobarbitalPhenytoin
Ketoconazole;Ritonavir;Grapefruit juice;Troleandomycin
Major P450 Enzymes in Humans
CYP4A9/ 11
Expressed in:
Substrates Inducers Inhibitors
Liver
Fatty acids and
??? ???
Metabolic activation by P450
Formation of toxic species De-chlorination of chloroform to phosgene De-hydrogenation and subsequent
epoxidation of urethane (CYP2E1) Formation of pharmacologically active
species Cyclophosphamide to electrophilic
aziridinum species (CYP3A4, CYP2B6)
Inhibition of P450
Drug-drug interactions due to reduced rate of biotransformation
Competitive S and I compete for active site e.g., Rifabutin & Ritonavir;
Dextromethorphan & Quinidine
Mechanism-based Irreversible; covalent binding to active
site
Induction and P450
Increased rate of biotransformation due to new protein synthesisMust give inducers for several days for effect
Drug-drug interactionsPossible sub-therapeutic plasma
concentrationseg, co-administration of Rifampin and oral
contraceptives is contraindicated
Some drugs induce, inhibit same enzyme (Isoniazid, Ethanol (2E1), Ritonavir (3A4)
PHASE 2 Reactions
CONJUGATIONS -OH, -SH, -COOH, -CONH with glucuronic acid to give
glucuronides -OH with sulphate to give sulphates -NH2, -CONH2, amino acids, sulpha drugs with acetyl-
to give acetylated derivatives -halo, -nitrate, epoxide, sulphate with glutathione to
give glutathione conjugates
all tend to be less lipid soluble and therefore better excreted (less well reabsorbed)
Phase II: Glucuronidation
Major Phase II pathway in mammalsUDP-glucuronyltransferase forms O-, N-,
S-, C- glucuronides; six forms in human liverCofactor is UDP-glucuronic acidInducers: phenobarbital, indoles, 3-
methylcholanthrene, cigarette smokingSubstrates include dextrophan, methadone,
morphine, p-nitrophenol, valproic acid, NSAIDS, bilirubin, steroid hormones
Glucuronidation & genetic polymorphism
Crigler-Nijar syndrome (severe): inactive enzyme; severe hyperbilirubinemia; inducers have no effect
Gilbert’s syndrome (mild): reduced enzyme activity; mild hyperbilirubinemia; phenobarbital increases rate of bilirubin glucuronidation to normal
Patients can glucuronidate morphine, chloroamphenicol
Glucuronidation &
-glucuronidase
Conjugates excreted in bile or urine (MW)
-glucuronidase from gut microflora cleaves glucuronic acid
Aglycone can be reabsorbed & undergo enterohepatic recycling
Glucuronidation and -glucuronidase
Metabolic activation of 2.6-dinitrotoluene) by -glucuronidase
-glucuronidase removes glucuronic acid from N-
glucuronide nitro group reduced by microbial N-reductase
resulting hepatocarcinogen is reabsorbed
Phase II: Sulfation
Sulfo-transferases are widely-distributed enzymes
Cofactor is 3’-phosphoadenosine-5’-phosphosulfate (PAPS)
Produce highly water-soluble sulfate esters, eliminated in urine, bile
Xenobiotics & endogenous compounds are sulfated (phenols, catechols, amines, hydroxylamines)
Sulfation
Sulfation is a high affinity, low capacity pathwayGlucuronidation is low affinity, high capacity
Capacity limited by low PAPS levels
ACETAMINOPHEN undergoes both sulfation and glucuronidation
At low doses sulfation predominatesAt high doses glucuronidation predominates
Sulfation
Four sulfotransferases in human liver cytosol
Aryl sulfatases in gut microflora remove sulfate groups; enterohepatic recycling
Usually decreases pharmacologic, toxic activity
Activation to carcinogen if conjugate is chemically unstableSulfates of hydroxylamines are unstable (2-AAF)
Phase II: Methylation
Common, minor pathway which generally decreases water solubility
MethyltransferasesCofactor: S-adenosylmethionine (SAM)-CH3 transfer to O, N, S, C
Substrates include phenols, catechols, amines, heavy metals (Hg, As, Se)
Methylation & genetic polymorphism
Several types of methyltransferases in human tissuesPhenol O-methyltransferase, Catechol O-
methyltransferase, N-methyltransferase, S-methyltransferase
Genetic polymorphism in Thiopurine metabolism
high activity allele, increased toxicitylow activity allele, decreased efficacy
Phase II: Acetylation
Major route of biotransformation for aromatic amines, hydrazines
Generally Decreases Water SolubilityN-acetyltransferase (NAT)
Cofactor is AcetylCoenzyme A
Substrates include Sulfanilamide, Isoniazid, Dapsone
Acetylation & genetic polymorphism
Rapid and slow acetylatorsVarious mutations result in decreased enzyme
activity or stabilityIncidence of slow acetylators
70% in Middle Eastern populations; 50% in Caucasians; 25% in Asians
Drug toxicities in slow acetylatorsnerve damage from dapsone; bladder cancer in
cigarette smokers due to increased levels of hydroxylamines
Phase IIAmino Acid Conjugation
Alternative to GlucuronidationTwo principle pathways-COOH group of substrate conjugated with -
NH2 (amine) of glycine, serine, glutamine, requiring CoA activatione.g: conjugation of Benzoic acid with Glycine
to form hippuric acid
Aromatic -NH2 or NHOH conjugated with -COOH of serine, proline, requiring ATP activation
Amino Acid Conjugation
Substrates: Bile Acids, NSAIDsMetabolic activation
Serine or proline N-esters of hydroxyl-amines are unstable & degrade to reactive electrophiles.
Phase IIGlutathione Conjugation
Glutathione-S-transferase catalyzes conjugation with glutathione
Glutathione is tripeptide of glycine, cysteine, glutamic acidFormed by -glutamyl-cysteine synthetase,
glutathione synthetase
Buthione-S-sulfoxine is inhibitor
Glutathione ConjugationTwo types of reactions with glutathione
1.Displacement of halogen, sulfate, sulfonate, phospho, nitro group
2.Glutathione added to activated double bond
Glutathione substratesHydrophobic, Containing electrophilic atomCan react with glutathione non-enzymatically
Glutathione Conjugation
Conjugation of N-acetylbenzoquinoneimine (activated metabolite of acetaminophen)
O-demethylation of organophosphatesActivation of trinitroglycerin
Products are oxidized glutathione (GSSG), dinitroglycerin, NO (vasodilator)
Reduction of hydroperoxidesProstaglandin metabolism
Glutathione Conjugation
Four classes of soluble glutathione-S-transferase
microsomal and cytosolic glutathione-S-transferases
Genetic polymorphism
Glutathione-S-transferase
Inducers (include phenobarbital, corticosteroids, anti-oxidants)
Over expression of enzyme leads to resistance (e.g., insects to DDT, corn to atrazine, cancer cells to chemotherapy)
Species SpecificityAflatoxin B1 not carcinogenic in mice which can conjugate
with glutathione very rapidly
Glutathione Conjugation
Excretion of Glutathione ConjugatesExcreted in bileConverted to Mercapturic Acids in kidney,
excreted in urineEnzymes involved are -glutamyl-trans-
peptidase, aminopeptidase M