Biotransformation of Xenobiotics

Biotransformation of Xenobiotics
Biotransformation of Xenobiotics...
Overview
• Major Phase I and Phase II enzymes
• Reaction mechanisms, substrates
• Enzyme inhibitors and inducers
• Genetic polymorphism
• Detoxification
• Metabolic activation
• FDA guidances related to biotransformation
2
Introduction
• Purpose
– Converts lipophilic to hydrophilic compounds
– Facilitates excretion
• Consequences
– Changes in PK characteristics
– Detoxification
– Metabolic activation
3
Comparing Phase I & Phase II
Enzym e Phase I Phase I I

Types of reactions Hydrolysis Conjugations
Oxidation
Reduction
Increase in Small Large
hydrophilicity
General mechanism Exposes functional Polar compound added
group to functional group
Consquences May result in Facilitates excretion

metabolic activation
4
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
5
Phase I: Hydrolysis
• Carboxyesterases & peptidases
– hydrolysis of esters
– eg: valacyclovir, midodrine
– hydrolysis of peptide bonds
– e.g.: insulin (peptide)
• Epoxide hydrolase
– H2O added to expoxides
– eg: carbamazepine
6
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
7
Reductions
• Carbonyl reduction
– Alcohol dehydrogenase (ADH)
• Chloral hydrate is reduced to trichlorothanol
• Disulfide reduction
– First step in disulfiram metabolism
• Sulfoxide reduction
– NSAID prodrug Sulindac converted to active
sulfide moiety
8
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
9
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
10
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
11
Phase I: Monooxygenases
• Monoamine oxidase
– Primaquine, haloperidol, tryptophan are
substrates
– Activates 1-methyl-4-phenyl-1,2,5,6-
tetrahydropyridine (MPTP) to neurotoxic toxic
metabolite in nerve tissue, resulting in
Parkinsonian-like symptoms
12
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 13
Monooxygenases
• Flavin-containing mono-oxygenases
– Generally results in detoxification
– Microsomal enzymes
– Substrates: nicotine, cimetidine,
chlopromazine, imipramine
– Repressed rather than induced by
phenobarbital, 3-methylcholanthrene
14
Phase I: Cytochrome P450
• Microsomal enzyme ranking first among

Phase I enzymes with respect to catalytic
versatility
• Heme-containing proteins
– Complex formed between Fe2+ and CO absorbs
light maximally at 450 (447-452) nm
• Overall reaction proceeds by catalytic cycle:
RH+O2+H++NADPH ROH+H2O+NADP+
15
Cytochrome P450
catalytic 16
Cytochrome P450 reactions
• Hydroxylation of aliphatic or aromatic

carbon
– (S)-mephenytoin to 4’-hydroxy-(S)-
mephenytoin (CYP2C19)
– Testosterone to 6-hydroxytestosterone
(CYP3A4)
17
• Expoxidation of double bonds

– Carbamazepine to 10,11-epoxide
• Heteroatom oxygenation, N-hydroxylation
– Amines to hydroxylamines
– Omeprazole to sulfone (CYP3A4)
18
• Heteroatom dealkylation
– O-dealkylation (e.g., dextromethorphan to
dextrophan by CYP2D6)
– N-demethylation of caffeine to:
theobromine (CYP2E1)
paraxanthine (CYP1A2)
theophylline (CYP2E1)
19
• Oxidative group transfer

– N, S, X replaced with O
– Parathion to paroxon (S by O)
– Activation of halothane to
trifluoroacetylchloride (immune hepatitis)
20

• Cleavage of esters
– Cleavage of functional group, with O incorporated
into leaving group
– Loratadine to Desacetylated loratadine (CYP3A4,
2D6)
21
• Dehydrogenation
– Abstraction of 2 H’s with formation of C=C
– Activation of Acetaminophen to hepatotoxic
metabolite N-acetylbenzoquinoneimine
22
Cytochrome P450 expression
• Gene family, subfamily names based on

amino acid sequences
• At least 15 P450 enzymes identified in
human liver microsomes
23
Cytochrome P450 expression
• Variation in levels, activity due to:

– Genetic polymorphism
– Environmental factors: inducers, inhibitors,
disease
– Multiple P450’s can catalyze same reaction
(lowest Km is predominant)
– A single P450 can catalyze multiple pathways
24
Major P450 Enzymes in Humans
CYP1A1/ 2
Expressed Substrates Inducers Inhibitors

in:
Liver Caffeine Cigarrette Furafylline

Lung Theophylline smoke; (mechanism-
Skin Cruciferous based);
GI veggies; ∀-naphtho-
Placenta Charcoal- flavone
broiled meat (reversible)
25
CYP2B6

in:
Liver Diazepam ??? Orphenadrine
Phenanthrene (mechanism-
based)
26
CYP2C19
Genetic polymorphism Substrates Inducers Inhibitors
Poor metabolizers have defective Phenytoin Rifampin Sulfafenazole

CYP2C9 Piroxicam
Tolbutamide
Warfarin
27
CYP2C19
Genetic polymorphism Substrates Inducers Inhibitors
 Rapid and slow S-mephenytoin Rifampin Tranylcypromine

metabolizers of S- (4’-hydroxylation
mephenytoin is catalyzed by
 N-demethylation CYP2C19)
pathway of S-
mephenytoin
metabolism
predominates in slow
metabolizers
28
CYP2D6
Genetic polymorphism Substrates I nducers Inhibitors
 Poor metabolizers lack Propafenone None known Fluoxetine

CYP2D6 Desipramine Quinidine
 Debrisoquine causes marked, Propanolol
prolonged hypotension in Codeine
slow metabolizers Dextromethorphan
 No effect on response to Fluoxetine
propanolol in poor Clozapine
metabolizers; alternate Captopril
pathway (CYP2C19) will
predominate Poor metabolizers
 5-10% of Caucasians are identified by
poor metabolizers urinary exrection of
 < 2% of Asians, African Dextrorphan
Americans are poor
metabolizers
29
CYP2E1
Expressed in: Substrates Inducers Inhibitors
Liver Ethanol Ethanol Disulfiram

Lung Acetaminophen Isoniazid
Kidney Dapsone
Lympocytes Caffeine
Theophylline
Benzene
30
CYP3A4
Expressed Substrates Inducers I nhibitors

in:
Liver; Acetaminophen Rifampin Ketoconazole;

Kidney; Carbamazepine Carbamazepine Ritonavir;
Intestine; Cyclosporine Phenobarbital Grapefruit juice;
Most Dapsone Phenytoin Troleandomycin
abundant Digitoxin
P450 Diltiazem
enzyme in Diazepam
liver Erythromycin
Etoposide
Lidocaine
Loratadine
Midazolam
Lovasatin
Nifedipine
Rapamycin
Taxol
Verapamil
31
CYP4A9/ 11

in:
Liver Fatty acids and ??? ???
derivaties;
Catalzyes  - and
 1-hyroxylation
32
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)
33
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
34
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) 35
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
36
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 37
Glucuronidation & -
glucuronidase
• Conjugates excreted in bile or urine (MW)
 -glucuronidase from gut microflora cleaves
glucuronic acid
• Aglycone can be reabsorbed & undergo
enterohepatic recycling
38
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
39
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) 40
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
41
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)
42
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)
43
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 44
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
45
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
46
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 47
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 48
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
49
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
50
• 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
51
• Four classes of soluble glutathione-S-

transferase ( , , ,  )
• Distinct microsomal and cytosolic glutathione-
S-transferases
• Genetic polymorphism
52
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
53
• 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
54
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
55
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
56
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
57
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
58
In vitro guidance:
• 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
59
In vitro guidance:
• 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
60
In vitro guidance:
• 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***
61
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
62
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
63
References
• Casarett and Doull’s Toxicology, The Basic Sciences of
Poisons, 5th Edition, Klassen, Amdur & Doull (eds),
Macmillan Publishing Co.
• CDER Guidance for Industry: Drug Metabolism/Drug
Interaction Studies in the Drug Development Process:
Studies in Vitro, April 1997, CLIN 3
• Davit B, Reynolds K, Yuan R et al. FDA evaluations using
in vitro metabolism to predict and interpret in vivo
metabolic drug-drug interactions: impact on labeling. J
Clin Pharmacol 1999 Sep;39(9):899-910
64

Biotransformation of Xenobiotics

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Biotransformation of Xenobiotics

Comparing Phase I & Phase II

Enzym e Phase I Phase I I

Consquences May result in Facilitates excretion

First Pass Effect

• Peroxidases couple oxidation to reduction of

Phase I: Cytochrome P450

• Microsomal enzyme ranking first among

Cytochrome P450 reactions

• Hydroxylation of aliphatic or aromatic

Cytochrome P450 reactions

• Expoxidation of double bonds

Cytochrome P450 reactions

Cytochrome P450 reactions

• Oxidative group transfer

Cytochrome P450 reactions

Cytochrome P450 reactions

Cytochrome P450 expression

• Gene family, subfamily names based on

Cytochrome P450 expression

• Variation in levels, activity due to:

Major P450 Enzymes in Humans

Expressed Substrates Inducers Inhibitors

Liver Caffeine Cigarrette Furafylline

Major P450 Enzymes in Humans

Expressed Substrates Inducers Inhibitors

Major P450 Enzymes in Humans

Genetic polymorphism Substrates Inducers Inhibitors

Poor metabolizers have defective Phenytoin Rifampin Sulfafenazole

Major P450 Enzymes in Humans

Genetic polymorphism Substrates Inducers Inhibitors

 Rapid and slow S-mephenytoin Rifampin Tranylcypromine

Major P450 Enzymes in Humans

Genetic polymorphism Substrates I nducers Inhibitors

 Poor metabolizers lack Propafenone None known Fluoxetine

Major P450 Enzymes in Humans

Expressed in: Substrates Inducers Inhibitors

Liver Ethanol Ethanol Disulfiram

Major P450 Enzymes in Humans

Expressed Substrates Inducers I nhibitors

Liver; Acetaminophen Rifampin Ketoconazole;

Major P450 Enzymes in Humans

Expressed Substrates Inducers Inhibitors

Metabolic activation by P450

Induction and P450

Phase II: Glucuronidation

• Major Phase II pathway in mammals

• Metabolic activation of 2.6-dinitrotoluene)

Phase II: Sulfation

• Sulfotransferases are widely-distributed

• Sulfation is a high affinity, low capacity

• Four sulfotransferases in human liver cytosol

Phase II: Methylation

• Common, minor pathway which generally

Phase II: Acetylation

• Major route of biotransformation for aromatic

Phase II:Amino Acid Conjugation

Amino Acid Conjugation

• Substrates: bile acids, NSAIDs

Phase II:Glutathione Conjugation

• Enormous array of substrates

• Two types of reactions with glutathione

• Four classes of soluble glutathione-S-