biotransformation of xenobiotics
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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
Expressedin:
Substrates Inducers Inhibitors
LiverLungSkinGIPlacenta
CaffeineTheophylline
Cigarrettesmoke;Cruciferousveggies;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 Humans
CYP2D6
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 Humans
CYP3A4
Expressedin:
Substrates Inducers Inhibitors
Liver;Kidney;Intestine;MostabundantP450enzyme inliver
AcetaminophenCarbamazepineCyclosporineDapsoneDigitoxinDiltiazemDiazepamErythromycinEtoposideLidocaineLoratadineMidazolamLovasatinNifedipineRapamycinTaxolVerapamil
RifampinCarbamazepinePhenobarbitalPhenytoin
Ketoconazole;Ritonavir;Grapefruit juice;Troleandomycin
Major P450 Enzymes in Humans
CYP4A9/ 11
Expressedin:
Substrates Inducers Inhibitors
Liver Fatty acids andderivaties;Catalzyes - and 1-hyroxylation
??? ???
Metabolic activation by P450
Formation of toxic species Dechlorination of chloroform to phosgene Dehydrogenation 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 synthesis Must give inducers for several days for effect
Drug-drug interactions Possible subtherapeutic plasma concentrations eg, co-administration of rifampin and oral
contraceptives is contraindicated Some drugs induce, inhibit same enzyme
(isoniazid, ethanol (2E1), ritonavir (3A4)
Phase II: Glucuronidation
Major Phase II pathway in mammals UDP-glucuronyltransferase forms O-, N-,
S-, C- glucuronides; six forms in human liver Cofactor is UDP-glucuronic acid Inducers: phenobarbital, indoles, 3-
methylcholanthrene, cigarette smoking Substrates 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 p-nitrophenol, 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 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: Sulfation
Sulfotransferases 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 pathway Glucuronidation is low affinity, high capacity
Capacity limited by low PAPS levels Acetaminophen undergoes both sulfation
and glucuronidation At low doses sulfation predominates At 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 unstable Sulfates of hydroxylamines are unstable (2-AAF)
Phase II: Methylation
Common, minor pathway which generally decreases water solubility
Methyltransferases Cofactor: 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 tissues Phenol O-methyltransferase, Catechol
O-methyltransferase, N-methyltransferase, S-methyltransferase
Genetic polymorphism in thiopurine metabolism high activity allele, increased toxicity low activity allele, decreased efficacy
Phase II: Acetylation
Major route of biotransformation for aromatic amines, hydrazines
Generally decreases water solubility N-acetyltransferase (NAT)
Cofactor is AcetylCoenzyme A Humans express two forms Substrates include sulfanilamide,
isoniazid, dapsone
Acetylation & genetic polymorphism
Rapid and slow acetylators Various mutations result in decreased
enzyme activity or stability Incidence of slow acetylators
70% in Middle Eastern populations; 50% in Caucasians; 25% in Asians
Drug toxicities in slow acetylators nerve damage from dapsone; bladder
cancer in cigarette smokers due to increased levels of hydroxylamines
Phase II:Amino Acid Conjugation
Alternative to glucuronidation Two principle pathways
-COOH group of substrate conjugated with -NH2 of glycine, serine, glutamine, requiring CoA activation e.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, NSAIDs Species specificity in amino acid acceptors
mammals: glycine (benzoic acid) birds: ornithine (benzoic acid) dogs, cats, taurine (bile acids) nonhuman primates: glutamine
Metabolic activation Serine or proline N-esters of hydroxylamines are
unstable & degrade to reactive electrophiles
Phase II:Glutathione Conjugation
Enormous array of substrates Glutathione-S-transferase catalyzes
conjugation with glutathione Glutathione is tripeptide of glycine,
cysteine, glutamic acid Formed by -glutamylcysteine
synthetase, glutathione synthetase Buthione-S-sulfoxine is inhibitor
Glutathione Conjugation
Two types of reactions with glutathione Displacement of halogen, sulfate, sulfonate,
phospho, nitro group Glutathione added to activated double bond or
strained ring system Glutathione substrates
Hydrophobic, containing electrophilic atom Can react with glutathione nonenzymatically
Glutathione Conjugation
Conjugation of N-acetylbenzoquinoneimine (activated metabolite of acetaminophen)
O-demethylation of organophosphates Activation of trinitroglycerin
Products are oxidized glutathione (GSSG), dinitroglycerin, NO (vasodilator)
Reduction of hydroperoxides Prostaglandin metabolism
Glutathione Conjugation
Four classes of soluble glutathione-S-transferase ( , , , )
Distinct microsomal and cytosolic glutathione-S-transferases
Genetic polymorphism
Glutathione-S-transferase
Inducers (include 3-methylcholanthrene, phenobarbital, corticosteroids, anti-oxidants)
Overexpression of enzyme leads to resistance (e.g., insects to DDT, corn to atrazine, cancer cells to chemotherapy)
Species specificity Aflatoxin B1 not carcinogenic in mice which
can conjugate with glutathione very rapidly
Glutathione Conjugation
Excretion of glutathione conjugates Excreted intact in bile Converted to mercapturic acids in
kidney, excreted in urine Enzymes involved are -
glutamyltranspeptidase, aminopeptidase M
Activation of xenobiotics following GSH conjugation Four mechanisms identified
FDA-CDER Guidances for Industry
Recommendations, not regulations Discuss aspects of drug
development Used in context of planning drug
development to achieve marketing approval
Among guidances are those dealing with in vitro and in vivo drug interaction studies
In vitro guidance
CDER Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro, April 1997, CLIN 3
Availability: www.fda.gov/cder/guidance/index.htm
In vitro guidance: assumptions Circulating concentrations of parent
drug and/or active metabolites are effectors of drug actions
Clearance is principle regulator of drug concentration
Large differences in blood levels can occur because of individual differences
Assay development critical
In vitro guidance: techniques/approaches
Identify a drug’s major metabolic pathways
Anticipate drug interactions Recommended methods
Human liver microsomes rCYP450s expressed in various cell lines Intact liver systems Effects of specific inhibitors Effects of antibodies on metabolism
In vitro guidance: techniques/approaches
Guidance focuses on P450 enzymes Other hepatic enzymes not as well-
characterized Gastrointestinal drug metabolism is
discussed Metabolism studies in animals
(preclinical phase) should be conducted early in drug development
In vitro guidance: techniques/approaches
Correlation between in vitro and in vivo studies
Should use in vitro concentrations that approximate in vivo plasma concentrations
Should be used in combination with in vivo studies; e.g., a mass balance study may show that metabolism makes small contribution to elimination pathways
In vitro guidance: techniques/approaches
Can rule out a particular pathway If in vitro studies suggest a
potential interaction, should consider investigation in vivo
***When a difference arises between in vivo and in vitro findings, in vivo
should take precedence***
In vitro guidance: timing of studies
Early understanding of metabolism can help in designing clinical regimens
Best to complete in vitro studies prior to start of Phase III
In vitro guidance: labeling
In vivo findings should take precedence in drug product labeling
If it is necessary to include in vitro information, should explicitly state conditions of extrapolation to in vivo
Assumption: if a drug is a substrate for a particular enzyme, then certain interactions may be anticipated