Drug Metabolism
Clinical Pharmacology
Spring Course 2006
M. E. Blair Holbein, Ph.D.
Clinical Pharmacologist
Presbyterian Hospital
1
Drug Metabolism - History
 “Xenobiotic
metabolism” established by Richard Tecwyn
Williams
 First
paper with an identified “metabolite” in Nature 1931
 Wrote first book on the Detoxification Mechanisms” 1959
 Focus on elimination of foreign compounds
 Proposed
a delineation of:
Phase I (oxidation, reduction, hydrolysis) biotransformations as
primary covalent chemical modifications to administered
compound
 Phase II (conjugation) with an endogenous polar species

To either parent drug
Phase I product(s)
2
Biotransformations

Phase I

Oxidation
Cytochrome P450 monooxygenase system
Flavin-containing monooxygenase system
Alcohol dehydrogenase and aldehyde dehydrogenase
Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
NADPH-cytochrome P450 reductase
Reduced (ferrous) cytochrome P450

Hydroloysis
Esterases and amidases
Epoxide hydrolase

Phase II
Glutathione S-transferases
 Mercapturic acid biosynthesis
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

3
Biotransformations

Phase I

Oxidation
Cytochrome P450 monooxygenase system
Flavin-containing monooxygenase system
Alcohol dehydrogenase and aldehyde dehydrogenase
Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
NADPH-cytochrome P450 reductase
Reduced (ferrous) cytochrome P450

Hydroloysis
Esterases and amidases
Epoxide hydrolase

Phase II
Glutathione S-transferases
 Mercapturic acid biosynthesis
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

4
Biotransformations

Phase I

Oxidation
Cytochrome P450 monooxygenase system
Flavin-containing monooxygenase system
Alcohol dehydrogenase and aldehyde dehydrogenase
Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
NADPH-cytochrome P450 reductase
Reduced (ferrous) cytochrome P450

Hydroloysis
Esterases and amidases
Epoxide hydrolase

Phase II
Glutathione S-transferases
 Mercapturic acid biosynthesis
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

5
Drug Metabolism - Determinants of Activity
 Inhibition
of enzyme activity
 Patterns
Competitive
Noncompetitive
Uncompetitive
 Effects
not mediated by enzyme activity, e.g. free fraction,
membrane effects, etc.
 Inducibility
 Rate-limitations
 Substrates
First-pass metabolism, high-extraction drugs
 Co-factors
 Turnover
 Polymorphism
 Predictability
of in vivo effects based on in vitro data is
highly variable
6
Kinetic equations for inhibition of metabolizing enzymes
7
Drug Metabolism - Determinants of Activity
 Inhibition
of enzyme activity
 Patterns
Competitive
Noncompetitive
Uncompetitive
 Effects
not mediated by enzyme activity, e.g. free fraction,
membrane effects, etc.
 Inducibility
 Rate-limitations
 Substrates
First-pass metabolism, high-extraction drugs
 Co-factors
 Turnover
 Polymorphism
 Predictability
of in vivo effects based on in vitro data is
highly variable
8
Biotransformations

Phase I

Oxidation
Cytochrome P450 monooxygenase system
Flavin-containing monooxygenase system
Alcohol dehydrogenase and aldehyde dehydrogenase
Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
NADPH-cytochrome P450 reductase
Reduced (ferrous) cytochrome P450

Hydroloysis
Esterases and amidases
Epoxide hydrolase

Phase II
Glutathione S-transferases
 Mercapturic acid biosynthesis
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

9
Phase I Oxidation: Cytochrome P450 Isoenzymes
Background:
 Huge
superfamily of highly versatile enzymes (over 3800
sequences identified)
 Found in the genomes of virtually all organisms
 Heme-containing proteins named for the absorption band at 450
nm when combined with carbon monoxide
 NADP(H) used with molecular oxygen to produce oxidation of a
variety of compounds:
Xenobiotics
Endobiotics
 In
prokaryotes, P450s are soluble proteins.
 In eukaryotes, they are usually bound to the endoplasmic
reticulum or inner mitochondrial membranes.
Human drug metabolism primarily in the endoplasmic reticulum of
hepatocytes. Also in the small intestine, kidney, lung and brain.
More than thirty (30) CYP human isoenzymes have been identified.
10
Catalytic reaction cycle CYP450 and the oxidation of xenobiotics
OH
e-
NADP+
NADP
DRUG
CYP
Reductase
Fe3+
CYP
DRUG
CYP
Fe3+
CYP
Fe2+
OH
DRUG
DRUG
e-
O2
O2
CYP
Fe2+
DRUG
H2O
2H+
11
Catalytic reaction cycle involving cytochrome
P450 in the oxidation of xenobiotics
Drug + NADPH + H+ + O2
Oxidized Drug + NADP+ + H2O
12
CYP450 Mediated Chemical Transformation
 Hydroxylation
 Aliphatic
 Aromatic
 N-Dealkylation,
O-Dealkylation, S-Dealkylation
 Oxidative Deamination
 Dehalogenation
 N-Oxidation
 S-Oxidation
13
CYP Mediated Oxidation
 Aliphatic
Hydroxylation
RCH2CH3
OH
RCHCH3
Ex: Hydroxylation of ibuprofen
14
CYP Mediated Oxidation
 Aromatic
Hydroxylation
Ex: Hydroxylation of acetanilide to 4-hydroxyacetanilide
15
CYP Mediated Oxidation
 Aromatic
Hydroxylation
 Directly
through asymmetric oxygen transfer
 Through an unstable arene oxide intermediate
Predictability
Influence of environment
Ex:Hydroxylation of aromatic carbon atoms
16
CYP Mediated Oxidation
 Aromatic
 Results
Hydroxylation
in several oxidized metabolites
Ex:Metabolism of phenytoin
17
CYP Mediated Oxidation
 Dealkation
(N-, O-, S-)
Ex: N-demethylation of ethylmorphine
18
CYP Mediated Oxidation

N-demethylation generates formaldehyde as a by-product
19
CYP Mediated Oxidation
 Dealkation
(N-, O-, S-)
Ex: N-demethylation and hydroxylation of propranolol
20
CYP Mediated Oxidation
 Oxidative
Deamination
Ex: General mechanism for oxidative deamination
21
CYP Mediated Oxidation
 Oxidative
Deamination
Ex: Deamination of amphetamine to inactive ketone
22
CYP Mediated Oxidation
 Dehalogenation
Ex: Dehalogenation generates reactive free radicals.
Metabolism of carbon tetrachloride generates oxidized lipids
23
CYP Mediated Oxidation
 N-Oxidation
may produce toxic by-products
24
CYP Mediated Oxidation
 N-Oxidation
Ex: N-oxidation of Dapsone
25
CYP Mediated Oxidation
 S-Oxidation
Ex: General scheme
Ex: CYP3A and Flavin monooxygenase produce same metabolite
26
CYP Mediated Oxidation
 S-Oxidation
Ex: A-oxidation of tazofelone
27
Drug Metabolism CYP450


Cytochrome P450 system responsible for the majority of oxidative
reactions
Significant polymorphism in many.


Drugs may be metabolized by a single isoenzyme


CYP2C9, CYP2C19, and CYP2D6—can be even be genetically absent!
Desipramine/CYP2D6; indinavir/CYP3A4; midazolam/CYP3A;
caffeine/CYP1A2; omeprazole/CYP2C19
Drugs may be metabolized by multiple isoenzymes

Most drugs metabolized by more than one isozyme
Imipramine: CYP2D6, CYP1A2, CYP3A4, CYP2C19



If co-administered with CYP450 inhibitor, some isozymes may “pick up
slack” for inhibited isozyme.
Drugs may be metabolized by several different enzyme systems;
e.g. CYP450 and MFO.
This enzyme system notably susceptible to induction.

Inherent turnover; highly variable response
28
Cytochrome P450 (CYP) Isoenzymes
 All
CYP isoenzymes in the same family have at least
40% structural similarity, and those in the same
subfamily have at least 60% structural similarity.
 Nomenclature
ex: CYP2D6
Root: cytochrome P450 CYP
Genetic Family: CYP2
Genetic Subfamily: CYP2D
Specific Gene: CYP2D6
NOTE that this nomenclature is genetically based; it has NO
functional implication
Phase I Oxidation: Cytochrome P450 (CYP) Isoenzymes
29
Proportion of Drugs Metabolized by CYP450
Enzymes in Humans
CYP2D6
20%
CYP3A4
38%
CYP2C19
8%
CYP1A2
11%
CYP2C9
16%
CYP2E1
4%
CYP2A6
3%
30
Cytochrome P450 3A4,5,7
 Largest
number of drugs metabolized
 Present in the largest amount in the liver.
 Present
 Not
in GI tract
polymorphic
 Inherent
activity varies widely
 Activity has been shown to predominate in the gut.
 Substrates:
 Most calcium channel blockers: nifedipine, amlodipine; HMG Co A
 Most benzodiazepines: diazepam, midazolam
 Most HIV protease inhibitors: indinavir, ritonavir
 Most HMG-CoA-reductase inhibitors: atorvastatin, lovastatin
 Cyclosporine, tacrolimus
 Most non-sedating antihistamines
 Cisapride
 Macrolide antibiotics: clarithromycin, erythromycin
 Chlorpheniramine;
 Also: haloperidol, buspirone; sildenafil, tamoxifen, trazodone, vincristine
31
Mechanism of Induction of
CYP3A4-Mediated Metabolism of
Drug Substrates (Panel A)
Wilkinson, G. R. N Engl J Med 2005;352:2211-2221
The Resulting Reduced
Plasma Drug
Concentration (Panel B)
Cytochrome P450 2D6
 Second
largest number of substrates.
 Polymorphic distribution
 Majority
of the population is characterized as an extensive or
even ultra-extensive metabolizer.
 Approximately 7% of the U.S. Caucasian population and 1-2% of
African or Asian inheritance have a genetic defect in CYP2D6
that results in a poor metabolizer phenotype.
include: many beta-blockers – metoprolol,
timolol, amitriptylline, imipramine, paroxetine,
haloperidol, risperidone, thioridazine, codeine,
dextromethorphan, ondansetron, tamoxifen, tramadol
 Inhibited by: amiodarone, chlorpheniramine, cimetidine,
fluoxetine, ritonavir
 Substrates
33
Common Drug Substrates and Clinically Important Inhibitors of CYP2D6
Wilkinson, G. R. N Engl J Med 2005;352:2211-2221
Cytochrome P450 2C9
 Note: Absent
in 1% of Caucasian and African-
Americans.
 Substrates include: many NSAIDs – ibuprofen,
tolbutamide, glipizide, irbesartan, losartan, celecoxib,
fluvastatin, phenytoin, sulfamethoxazole, tamoxifen,
tolbutamide, warfarin
 Inhibited by: fluconazole, isoniazid, ticlopidine
 Induced by: rifampin
35
Cytochrome P450 1A2
 Substrates
include: theophylline, caffeine, imipramine,
clozapine
 Inhibited by: many fluoroquinolone antibiotics,
fluvoxamine, cimetidine
 Induced by: smoking tobacco
36
37
Coffee Intake and Relative Risk of Myocardial Infarction by CYP1A2 Genotype
Cornelis, M. C. et al. JAMA 2006;295:1135-1141.
Copyright restrictions may apply.
38
Cytochrome P450 2C19
 Note: Absent
in 20-30% of Asians, 3-5% of Caucasians
 Substrates include: omeprazole, diazepam, phenytoin,
phenobarbitone, amitriptylline, clomipramine,
cyclophosphamide, progesterone
 Inhibited by: fluoxetine, fluvoxamine, ketoconazole,
lansoprazole, omeprazole, ticlopidine
39
Cytochrome P450 2B6
 Substrates
include: bupropion, cyclophosphamide,
efavirenz, methadone
 Inhibited by: thiotepa
 Induced by: phenobarbital, rifampin
40
Cytochrome P450 2E1
 Substrates
include: acetaminophen
41
Cytochrome P450 2C8
Substrates; paclitaxel, torsemide, amodiaquine,
cerivastatin, repaglinide
 Inhibited by: trimethoprim, quercetin, glitazones,
gemfibrozil, montelukast
 Induced by: rifampin

42
Biotransformations

Phase I

Oxidation
Cytochrome P450 monooxygenase system
Flavin-containing monooxygenase system
Alcohol dehydrogenase and aldehyde dehydrogenase
Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
NADPH-cytochrome P450 reductase
Reduced (ferrous) cytochrome P450

Hydroloysis
Esterases and amidases
Epoxide hydrolase

Phase II
Glutathione S-transferases
 Mercapturic acid biosynthesis
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

43
Non-CYP Mediated Chemical Transformation
 Hydrolysis
 Reduction
 Oxidations
 Flavine
monooxygenases
 Monoamine and diamine oxidases
 Alcohol and aldehyde dehydrogenase
44
Non-CYP Mediated Biotransformation
 Hydrolysis
 Esterases,
amidases and proteases
 Non-microsomal (cytosolic)
 Widely distributed in most tissues
45
Non-CYP Mediated Biotransformation
 Reduction
Ex: Reduction of side-chain of digoxin produces inactive metabolite
46
Non-CYP Mediated Chemical Transformation
 Hydrolysis
 Reduction
 Oxidations
Flavine monooxygenases
 Monoamine and diamine oxidases
 Alcohol and aldehyde dehydrogenase
47
Flavine Monooxygenases
 Wide
variety of substrates
 First isolated from pig liver
 Originally termed N-oxidase (or Ziegler’s enzyme)
 Products are generally polar, non-toxic compounds
 Some
generation of reactive intermediates, esp. S-oxides
48
Flavine Monooxygenases Isoenzymes
 Six
genes in mammals
 Nomenclature based on sequence homology
 FMO1:
major human fetal liver; adult kidney
 FMO2: lung (most species)
 FMO3: major adult liver form; major form in brain
Interindividual variability
 FMO4:
atypical
 FMO5: trace
 FMO6: reported
 Question
of inducibility (vs. P450)
 Genetic polymorphism
49
Flavine Monooxygenases Reactions
 S-Oxygenation
 Spironolactone
 Cimetidine
Stereoselective: FMO3 forms the (+) enantiomer and FMO1 forms () enantiomer
 N-Oxygenation
 Imipramine
 Nicotine
50
Flavine Monooxygenases Mechanism
 Requires
O2 and NADPH
 Unlike P450 which forms oxidizing intermediate AFTER
binding substrate, FMO exists in “preloaded” state and
will oxygenate any lipophilic substrate that binds with it.
 Individual FMOs have broader substrate range than
individual CYP
51
Flavine Monooxygenase Cycle
NADP+
+ H 2O
Enz | Flox
NADPH
FMO Cycle
Enz | FlH2 + NADP+
Enz | FlHOH
O2
Enz | FlOOH
DRUG - O
DRUG
52
Catalytic reaction cycle CYP450 and the oxidation of xenobiotics
OH
e-
NADP+
NADP
DRUG
CYP
Reductase
Fe3+
CYP
DRUG
CYP
Fe3+
CYP
Fe2+
OH
DRUG
DRUG
e-
O2
O2
CYP
Fe2+
DRUG
H2O
2H+
53
Flavine Monooxygenase Cycle
NADP+
+ H 2O
Enz | Flox
NADPH
FMO Cycle
Enz | FlH2 + NADP+
Enz | FlHOH
O2
Enz | FlOOH
DRUG - O
DRUG
54
Non-CYP Mediated Oxidation
 Oxidation:
Flavine Monooxygenases
Ex: N-Oxidation of nicotine, catalyzed by FMO3
55
Non-CYP Mediated Oxidation
 Oxidation:
Flavine Monooxygenases
Ex: S-Oxidation of cimetidine, catalyzed by FMO3
56
Non-CYP Mediated Chemical Transformation
 Hydrolysis
 Reduction
 Oxidations
 Flavine
monooxygenases
 Monoamine and diamine oxidases
 Alcohol and aldehyde dehydrogenase
57
Non-CYP Mediated Oxidation
 Oxidation:
Monoamine Oxidases
 Mitochondrial
enzymes
 Deaminate endogenous neurotransmitters
Dopamine
Serotonin
Norepinephrine
Epinephrine
 Same
type of products as other oxidizing enzymes
 Distinguish
 Found
source enzyme of metabolites by other means
in liver, kidney, intestine, brain
58
Non-CYP Mediated Oxidation
 Oxidation:
Diamine Oxidases
 Endogenous
amines
Histamine
Polyamines
 Putrescine
 Cadaverine
 Amines
converted to aldehydes (in presence of O2)
 Contribute to oxidation of some drugs
 Found in liver, intestine, placenta
59
Non-CYP Mediated Chemical Transformation
 Hydrolysis
 Reduction
 Oxidations
 Flavine
monooxygenases
 Monoamine and diamine oxidases
 Alcohol and aldehyde dehydrogenase
60
Non-CYP Mediated Oxidation
 Alcohol
and Aldehyde Dehydrogenases
Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.
61
Non-CYP Mediated Oxidation
 Alcohol
and Aldehyde Dehydrogenases
Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.
62
Relative Contribution to Drug Metabolism - Phase I
Evans & Relling Science 1999
Phase II Biotransformation: Conjugation:
Glucuronidation, Sulfation, Acetylation
 Addition
of hydrophilic groups (glucuronic acid, sulfate,
glycine, or acetyl) onto the drug or drug metabolite
 Catalyzed by a group of enzymes called transferases.
 Located
in cytosol
 Microsomal enzyme: Uridine diphosphate
glucuronosyltransferase (UGTs)
64
Biotransformations

Phase I

Oxidation
 Cytochrome P450 monooxygenase system
 Flavin-containing monooxygenase system
 Alcohol dehydrogenase and aldehyde dehydrogenase
 Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
 NADPH-cytochrome P450 reductase
 Reduced (ferrous) cytochrome P450

Hydroloysis
 Esterases and amidases
 Epoxide hydrolase

Phase II
Glutathione S-transferases
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

65
Phase II Biotransformations (Conjugations)
 Glutathione
 Catalyzed
by glutathione-S-transferases
Cytosolic and microsomal
 Detoxification
of electrophilic (and potentially carcinogenic)
molecules
66
UDP-Glucuronosyltransferase (UGT)



Catalyses conjugation of glucuronic acid with a substrate with a
suitable functional group
The most important (quantitatively) conjugation step
Substrates
Xenobiotics (drugs, dietary chemicals, carcinogens, environmental
pollutants)
 Endobiotics (steroid hormones, bilirubin, bile acids, fatty acids)



Altered activity important (toxicology, pharmacologically)
Microsomal location in endoplasmic reticulum on opposite side of
membrane from CYP



Transporter functions for cofactors
Polymorphic (at least two families)
Rare disorders associated with genetic abnormalities

Criglar-Najjar Syndromes (types1,2)
Absence of bilirubin conjugation enzyme and marked unconjugated
hyperbilirubinemia & jaundice

Gilbert Syndrome
Partial block in bilirubin conjugation; benign elevation in total and
unconjugated bilirubin
67
Phase II Biotransformations (Conjugations)
 Glucuronidation
Ex: N- and O- linked glucuronide formation markedly enhances the polarity and
water solubility.
68
Phase II Biotransformations (Conjugations)
 Glucuronides
can be generated from a variety of
substrates
69
Phase II Biotransformations (Conjugations)
 Sulfation
General pathway for enzymatic sulfation
70
Phase II Biotransformations (Conjugations)
 Sulfation
Ex: Minoxidil
71
Phase II Biotransformations (Conjugations)
 Acetylation
Ex: Acetyl transferase donates the acyl group from Coenzyme A to drug substrates
72
Phase II Biotransformations (Conjugations)
 Acetylation
Ex: Isoniazid inactivation by acetylation
73
Phase II Biotransformations (Conjugations)
 Hydroxylation
and acetylation
Ex: Reactive nitrenium ions may be produced in the metabolism of aromatic amines
through hydroxylation and acetylation
74
Biotransformations

Phase I

Oxidation
 Cytochrome P450 monooxygenase system
 Flavin-containing monooxygenase system
 Alcohol dehydrogenase and aldehyde dehydrogenase
 Monoamine oxidase (Co-oxidation by peroxidases)

Reduction
 NADPH-cytochrome P450 reductase
 Reduced (ferrous) cytochrome P450

Hydroloysis
 Esterases and amidases
 Epoxide hydrolase

Phase II
Glutathione S-transferases
 UDP-Glucoron(os)yltranasferases
 N-Acetyltransferases
 Amino acid N-acyl transferases
 Sulfotransferases

75
Questions?
 Blair
Holbein, Ph.D., BCAP
Presbyterian Hospital of Dallas
 Email:
bholbein@hcin.net
 Website: http://phdres.caregate.net
 Annotated bibliography
76
References
 Wright
JM. Drug Interactions.
Carruthers SG, Hoffman BB, et al.s, ed. Melmon and Morrelli’s
Clinical Pharmacology: Basic Principles in Therapeutics, 4th ed.
New York 2000 :McGraw-Hill.
 In:
 Markey
SM.Pathways of Drug Metabolism
 In: Atkinson AJ,
Daniels CE, Dedrick RL, et al., ed. Principles of
Clinical Pharmacology, New York 2001: Academic Press.
77