1. Biotransformation of Xenobiotics
Barbara M. Davit, PhD, DABT
Division of Bioequivalence, Office of Generic Drugs, CDER, FDA
Introduction to the Theory and
Methods in Toxicology
Sept. 17, 2001

2. Overview Major Phase I and Phase II enzymes
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

3. Introduction Purpose Consequences
Introduction
Purpose
Converts lipophilic to hydrophilic compounds
Facilitates excretion
Consequences
Changes in PK characteristics
Detoxification
Metabolic activation

4. Comparing Phase I & Phase II
Comparing Phase I & Phase II

5.
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

6. Phase I: Hydrolysis Carboxyesterases & peptidases Epoxide hydrolase
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

7. Phase I: Reductions Azo reduction Nitro reduction N=N to 2 -NH2 groups
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

8. Reductions Carbonyl reduction Disulfide reduction Sulfoxide reduction
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

9. Reductions Quinone reduction
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

10. Reductions Dehalogenation Reductive (H replaces X)
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

11. Phase I: Oxidation-Reduction
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

12. Phase I: Monooxygenases
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

13.
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

14. Monooxygenases Flavin-containing mono-oxygenases
Monooxygenases
Flavin-containing mono-oxygenases
Generally results in detoxification
Microsomal enzymes
Substrates: nicotine, cimetidine, chlopromazine, imipramine
Repressed rather than induced by phenobarbital, 3-methylcholanthrene

15.
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 ( ) nm
Overall reaction proceeds by catalytic cycle:
RH+O2+H++NADPH ROH+H2O+NADP+

16.
Cytochrome P450
catalytic cycle

17. Cytochrome P450 reactions
Cytochrome P450 reactions
Hydroxylation of aliphatic or aromatic carbon
(S)-mephenytoin to 4’-hydroxy-(S)-mephenytoin (CYP2C19)
Testosterone to 6-hydroxytestosterone (CYP3A4)

18. Cytochrome P450 reactions
Cytochrome P450 reactions
Expoxidation of double bonds
Carbamazepine to 10,11-epoxide
Heteroatom oxygenation, N-hydroxylation
Amines to hydroxylamines
Omeprazole to sulfone (CYP3A4)

19. Cytochrome P450 reactions
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)

20. Cytochrome P450 reactions
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)

21. Cytochrome P450 reactions
Cytochrome P450 reactions
Cleavage of esters
Cleavage of functional group, with O incorporated into leaving group
Loratadine to Desacetylated loratadine (CYP3A4, 2D6)

22. Cytochrome P450 reactions
Cytochrome P450 reactions
Dehydrogenation
Abstraction of 2 H’s with formation of C=C
Activation of Acetaminophen to hepatotoxic metabolite N-acetylbenzoquinoneimine

23. Cytochrome P450 expression
Cytochrome P450 expression
Gene family, subfamily names based on amino acid sequences
At least 15 P450 enzymes identified in human liver microsomes

24. Cytochrome P450 expression
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

25. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

26. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

27. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

28. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

29. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

30. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

31. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

32. Major P450 Enzymes in Humans
Major P450 Enzymes in Humans

33. Metabolic activation by P450
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)

34.
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

35.
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)

36. Phase II: Glucuronidation
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

37. Glucuronidation & genetic polymorphism
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

38. Glucuronidation & -glucuronidase
Glucuronidation & -glucuronidase
Conjugates excreted in bile or urine (MW)
-glucuronidase from gut microflora cleaves glucuronic acid
Aglycone can be reabsorbed & undergo enterohepatic recycling

39. Glucuronidation and -glucuronidase
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

40. Phase II: Sulfation Sulfotransferases are widely-distributed enzymes
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)

41. Sulfation Sulfation is a high affinity, low capacity pathway
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

42. Sulfation Four sulfotransferases in human liver cytosol
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)

43.
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)

44. Methylation & genetic polymorphism
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

45.
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

46. Acetylation & genetic polymorphism
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

47. Phase II:Amino Acid Conjugation
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

48. Amino Acid Conjugation
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

49. Phase II:Glutathione Conjugation
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

50. Glutathione Conjugation
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

51. Glutathione Conjugation
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

52. Glutathione Conjugation
Glutathione Conjugation
Four classes of soluble glutathione-S-transferase ( , , ,  )
Distinct microsomal and cytosolic glutathione-S-transferases
Genetic polymorphism

53. Glutathione-S-transferase
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

54. Glutathione Conjugation
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

55. FDA-CDER Guidances for Industry
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

56.
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:

57. In vitro guidance: assumptions
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

58. In vitro guidance: techniques/approaches
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

59. In vitro guidance: techniques/approaches
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

60. In vitro guidance: techniques/approaches
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

61. In vitro guidance: techniques/approaches
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***

62. In vitro guidance: timing of studies
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

63. In vitro guidance: labeling
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

64.
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):