Transcript Biotransformation of Xenobiotics - Lectures For UG-5

BIOTRANSFORMATION OF
XENOBIOTICS
Overview
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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
Increase in
Small
hydrophilicity
General
Exposes functional
mechanism
group
Conjugations
Consquences
Facilitates excretion
May result in
metabolic activation
Large
Polar compound added
to functional group
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 & peptidases
Hydrolysis of esters
eg: valacyclovir, midodrine
Hydrolysis of peptide bonds
e.g.: insulin (peptide)
Epoxide hydrolase
H2O added to epoxides
eg: carbamazepine
Phase I: Reductions
Azo Reduction
N=N to 2 -NH2 groups
eg: prontosil to sulfanilamide
Nitro Reduction
N=O to one -NH2 group
eg: 2,6-dinitrotoluene activation
N-glucuronide conjugate hydrolyzed by gut microflora
Hepatotoxic compound reabsorbed
Reductions
Carbonyl reduction
Chloral hydrate is reduced to trichlorothanol
Disulfide reduction
First step in disulfiram metabolism
Reductions
Quinone reduction
Cytosolic flavoprotein NAD(P)H quinone
oxidoreductase
two-electron reduction, no oxidative stress
high in tumor cells; activates diaziquone to more
potent form
Flavoprotein P450-reductase
one-electron reduction, produces superoxide ions
metabolic activation of paraquat, doxorubicin
Reductions
Dehalogenation
Reductive (H replaces X)
Enhances CCl4 toxicity by forming free radicals
Oxidative (X and H replaced with =O)
Causes halothane hepatitis via reactive acylhalide
intermediates
Dehydrodechlorination (2 X’s removed, form C=C)
DDT to DDE
Phase I: Oxidation-Reduction
Alcohol dehydrogenase
Alcohols to aldehydes
Genetic polymorphism; Asians metabolize alcohol
rapidly
Inhibited by ranitidine, cimetidine, aspirin
Aldehyde dehydrogenase
Aldehydes to carboxylic acids
Inhibited by disulfiram
Phase I: Monooxygenases
Monoamine Oxidase
Primaquine, haloperidol, tryptophan are substrates
Activates 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine (MPTP) to neurotoxic toxic
metabolite in nerve tissue, resulting in
Parkinsonian-like symptoms
MonoOxygenases
Peroxidases couple oxidation to reduction of H2O2
& lipid hydroperoxidase
Prostaglandin 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-oxygenases
Generally results in detoxification
Microsomal enzymes
Substrates: Nicotine, Cimetidine, Chlopromazine,
Imipramine
Phase I: Cytochrome P450
Microsomal enzyme ranking first among Phase I
enzymes
Heme-containing proteins
Complex 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 BONDS
Carbamazepine to 10,11-epoxide
HETEROATOM OXYGENATION
Omeprazole to sulfone (CYP3A4)
Cytochrome P450 reactions
HETEROATOM DEALKYLATION
O-dealkylation (e.g., dextromethorphan to dextrophan
by CYP2D6)
N-demethylation of caffeine to:
theobromine (CYP2E1)
paraxanthine (CYP1A2)
theophylline (CYP2E1)
Cytochrome P450 reactions
Oxidative Group Transfer
N, S, X replaced with O
Parathion to paroxon (S by O)
Activation of halothane to
trifluoroacetylchloride (immune hepatitis)
Cytochrome P450 reactions
Cleavage of Esters
Cleavage of functional group, with O incorporated into
leaving group
Loratadine to Desacetylated loratadine (CYP3A4, 2D6)
Cytochrome P450 reactions
Dehydrogenation
Abstraction of 2 H’s with formation of C=C
Activation 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 expression
VARIATION IN LEVELS activity due to
Genetic Polymorphism
Environmental Factors: inducers, inhibitors, disease
Multiple 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;
Charcoalbroiled meat
Furafylline
(mechanismbased);
-naphthoflavone
(reversible)
Major P450 Enzymes in Humans
CYP2B6
Expressed
in:
Substrates
Inducers Inhibitors
Liver
Diazepam
???
Phenanthrene
Orphenadrine
(mechanismbased)
Major P450 Enzymes in Humans
CYP2C19
Genetic polymorphism
Substrates Inducers Inhibitors
Poor metabolizers have Phenytoin Rifampin Sulfafenaz
defective CYP2C9
Piroxicam
ole
Tolbutami
de
Warfarin
Major P450 Enzymes in Humans
CYP2C19
Genetic polymorphism
Substrates
Inducers
 Rapid and slow
metabolizers of Smephenytoin
 N-demethylation
pathway of Smephenytoin
metabolism
predominates in slow
metabolizers
S-mephenytoin
Rifampin
(4’-hydroxylation
is catalyzed by
CYP2C19)
Inhibitors
Tranylcypromine
Major P450 Enzymes in Humans
CYP2D6
Genetic polymorphism
Substrates
 Poor metabolizers lack
CYP2D6
 Debrisoquine causes marked,
prolonged hypotension in
slow metabolizers
 No effect on response to
propanolol in poor
metabolizers; alternate
pathway (CYP2C19) will
predominate
 5-10% of Caucasians are
poor metabolizers
 < 2% of Asians, African
Americans are poor
metabolizers
Propafenone
None known
Desipramine
Propanolol
Codeine
Dextromethorphan
Fluoxetine
Clozapine
Captopril
Poor metabolizers
identified by
urinary exrection of
Dextrorphan
Inducers
Inhibitors
Fluoxetine
Quinidine
Major P450 Enzymes in Humans
CYP2E1
Expressed in:
Substrates
Inducers
Inhibitors
Liver
Lung
Kidney
Lympocytes
Ethanol
Acetaminophen
Dapsone
Caffeine
Theophylline
Benzene
Ethanol
Isoniazid
Disulfiram
Major P450 Enzymes in Humans
CYP3A4
Expressed
in:
Substrates
Inducers
Inhibitors
Liver;
Kidney;
Intestine;
Most
abundant
P450
enzyme in
liver
Acetaminophen
Carbamazepine
Cyclosporine
Dapsone
Digitoxin
Diltiazem
Diazepam
Erythromycin
Etoposide
Lidocaine
Loratadine
Midazolam
Lovasatin
Nifedipine
Rapamycin
Taxol
Verapamil
Rifampin
Carbamazepine
Phenobarbital
Phenytoin
Ketoconazole;
Ritonavir;
Grapefruit juice;
Troleandomycin
Major P450 Enzymes in Humans
CYP4A9/11
Expressed Substrates
in:
Liver
Inducers Inhibitors
Fatty acids and ???
derivaties;
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 acetylto 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, Smethyltransferase
 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
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

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-Stransferases
 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 wellcharacterized
 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