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1
Drug MetabolismClinical Pharmacology
Spring Course 2006
M. E. Blair Holbein, Ph.D.
Clinical Pharmacologist
Presbyterian Hospital
2
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 drugPhase I product(s)
3
Biotransformations Phase I
OxidationCytochrome P450 monooxygenase systemFlavin-containing monooxygenase systemAlcohol dehydrogenase and aldehyde dehydrogenaseMonoamine oxidase (Co-oxidation by peroxidases)
ReductionNADPH-cytochrome P450 reductaseReduced (ferrous) cytochrome P450
HydroloysisEsterases and amidasesEpoxide 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
OxidationCytochrome P450 monooxygenase systemFlavin-containing monooxygenase systemAlcohol dehydrogenase and aldehyde dehydrogenaseMonoamine oxidase (Co-oxidation by peroxidases)
ReductionNADPH-cytochrome P450 reductaseReduced (ferrous) cytochrome P450
HydroloysisEsterases and amidasesEpoxide hydrolase
Phase II Glutathione S-transferases Mercapturic acid biosynthesis UDP-Glucoron(os)yltranasferases N-Acetyltransferases Amino acid N-acyl transferases Sulfotransferases
5
Biotransformations Phase I
OxidationCytochrome P450 monooxygenase systemFlavin-containing monooxygenase systemAlcohol dehydrogenase and aldehyde dehydrogenaseMonoamine oxidase (Co-oxidation by peroxidases)
ReductionNADPH-cytochrome P450 reductaseReduced (ferrous) cytochrome P450
HydroloysisEsterases and amidasesEpoxide hydrolase
Phase II Glutathione S-transferases Mercapturic acid biosynthesis UDP-Glucoron(os)yltranasferases N-Acetyltransferases Amino acid N-acyl transferases Sulfotransferases
6
Drug Metabolism - Determinants of Activity
Inhibition of enzyme activity Patterns
CompetitiveNoncompetitiveUncompetitive
Effects not mediated by enzyme activity, e.g. free fraction, membrane effects, etc.
Inducibility Rate-limitations
SubstratesFirst-pass metabolism, high-extraction drugs
Co-factors Turnover
Polymorphism Predictability of in vivo effects based on in vitro data is
highly variable
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Kinetic equations for inhibition of metabolizing enzymes
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Drug Metabolism - Determinants of Activity
Inhibition of enzyme activity Patterns
CompetitiveNoncompetitiveUncompetitive
Effects not mediated by enzyme activity, e.g. free fraction, membrane effects, etc.
Inducibility Rate-limitations
SubstratesFirst-pass metabolism, high-extraction drugs
Co-factors Turnover
Polymorphism Predictability of in vivo effects based on in vitro data is
highly variable
9
Biotransformations Phase I
OxidationCytochrome P450 monooxygenase systemFlavin-containing monooxygenase systemAlcohol dehydrogenase and aldehyde dehydrogenaseMonoamine oxidase (Co-oxidation by peroxidases)
ReductionNADPH-cytochrome P450 reductaseReduced (ferrous) cytochrome P450
HydroloysisEsterases and amidasesEpoxide hydrolase
Phase II Glutathione S-transferases Mercapturic acid biosynthesis UDP-Glucoron(os)yltranasferases N-Acetyltransferases Amino acid N-acyl transferases Sulfotransferases
10
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:XenobioticsEndobiotics
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.
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Catalytic reaction cycle CYP450 and the oxidation of xenobiotics
e-
e-
CYP Reductase
NADP+
NADP
Fe2+CYP
Fe3+CYP
DRUGDRUG
DRUGDRUG
O2
Fe2+CYP
2H+
H2O
Fe3+CYP
DRUGDRUGOH
O2
DRUGDRUG DRUGDRUGOH
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Catalytic reaction cycle involving cytochrome P450 in the oxidation of xenobiotics
Oxidized Drug + NADP+ + H2O
Drug + NADPH + H+ + O2
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CYP450 Mediated Chemical Transformation
Hydroxylation Aliphatic Aromatic
N-Dealkylation, O-Dealkylation, S-Dealkylation Oxidative Deamination Dehalogenation N-Oxidation S-Oxidation
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CYP Mediated Oxidation
Aliphatic Hydroxylation
RCH2CH3
OHRCHCH3
Ex: Hydroxylation of ibuprofen
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CYP Mediated Oxidation
Aromatic Hydroxylation
Ex: Hydroxylation of acetanilide to 4-hydroxyacetanilide
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CYP Mediated Oxidation
Aromatic Hydroxylation Directly through asymmetric oxygen transfer Through an unstable arene oxide intermediate
PredictabilityInfluence of environment
Ex:Hydroxylation of aromatic carbon atoms
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CYP Mediated Oxidation
Aromatic Hydroxylation Results in several oxidized metabolites
Ex:Metabolism of phenytoin
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CYP Mediated Oxidation
Dealkation (N-, O-, S-)
Ex: N-demethylation of ethylmorphine
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CYP Mediated Oxidation
N-demethylation generates formaldehyde as a by-product
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CYP Mediated Oxidation
Dealkation (N-, O-, S-)
Ex: N-demethylation and hydroxylation of propranolol
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CYP Mediated Oxidation
Oxidative Deamination
Ex: General mechanism for oxidative deamination
22
CYP Mediated Oxidation
Oxidative Deamination
Ex: Deamination of amphetamine to inactive ketone
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CYP Mediated Oxidation
Dehalogenation
Ex: Dehalogenation generates reactive free radicals.Metabolism of carbon tetrachloride generates oxidized lipids
24
CYP Mediated Oxidation
N-Oxidation may produce toxic by-products
25
CYP Mediated Oxidation
N-Oxidation
Ex: N-oxidation of Dapsone
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CYP Mediated Oxidation
S-OxidationEx: General schemeEx: CYP3A and Flavin monooxygenase produce same metabolite
27
CYP Mediated Oxidation
S-OxidationEx: A-oxidation of tazofelone
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Drug Metabolism CYP450
Cytochrome P450 system responsible for the majority of oxidative reactions
Significant polymorphism in many. CYP2C9, CYP2C19, and CYP2D6—can be even be genetically absent!
Drugs may be metabolized by a single isoenzyme Desipramine/CYP2D6; indinavir/CYP3A4; midazolam/CYP3A;
caffeine/CYP1A2; omeprazole/CYP2C19 Drugs may be metabolized by multiple isoenzymes
Most drugs metabolized by more than one isozymeImipramine: 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
29
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: CYP2D6Root: cytochrome P450 CYPGenetic Family: CYP2 Genetic Subfamily: CYP2DSpecific Gene: CYP2D6
NOTE that this nomenclature is genetically based; it has NO functional implication
Phase I Oxidation: Cytochrome P450 (CYP) Isoenzymes
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Proportion of Drugs Metabolized by CYP450 Enzymes in Humans
CYP2C198%
CYP1A211%
CYP2A63%
CYP2C916%
CYP2E14%
CYP3A438%
CYP2D620%
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Cytochrome P450 3A4,5,7
Largest number of drugs metabolized Present in the largest amount in the liver.
Present in GI tract
Not 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
Wilkinson, G. R. N Engl J Med 2005;352:2211-2221
Mechanism of Induction of CYP3A4-Mediated Metabolism of Drug Substrates (Panel A)
The Resulting Reduced Plasma Drug Concentration (Panel B)
33
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.
Substrates include: many beta-blockers – metoprolol, timolol, amitriptylline, imipramine, paroxetine, haloperidol, risperidone, thioridazine, codeine, dextromethorphan, ondansetron, tamoxifen, tramadol
Inhibited by: amiodarone, chlorpheniramine, cimetidine, fluoxetine, ritonavir
Common Drug Substrates and Clinically Important Inhibitors of CYP2D6
Wilkinson, G. R. N Engl J Med 2005;352:2211-2221
35
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
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Cytochrome P450 1A2
Substrates include: theophylline, caffeine, imipramine, clozapine
Inhibited by: many fluoroquinolone antibiotics, fluvoxamine, cimetidine
Induced by: smoking tobacco
37
38Copyright restrictions may apply.
Cornelis, M. C. et al. JAMA 2006;295:1135-1141.
Coffee Intake and Relative Risk of Myocardial Infarction by CYP1A2 Genotype
39
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
40
Cytochrome P450 2B6
Substrates include: bupropion, cyclophosphamide, efavirenz, methadone
Inhibited by: thiotepa Induced by: phenobarbital, rifampin
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Cytochrome P450 2E1
Substrates include: acetaminophen
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Cytochrome P450 2C8
Substrates; paclitaxel, torsemide, amodiaquine, cerivastatin, repaglinide
Inhibited by: trimethoprim, quercetin, glitazones, gemfibrozil, montelukast
Induced by: rifampin
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Biotransformations Phase I
OxidationCytochrome P450 monooxygenase systemFlavin-containing monooxygenase systemAlcohol dehydrogenase and aldehyde dehydrogenaseMonoamine oxidase (Co-oxidation by peroxidases)
ReductionNADPH-cytochrome P450 reductaseReduced (ferrous) cytochrome P450
HydroloysisEsterases and amidasesEpoxide hydrolase
Phase II Glutathione S-transferases Mercapturic acid biosynthesis UDP-Glucoron(os)yltranasferases N-Acetyltransferases Amino acid N-acyl transferases Sulfotransferases
44
Non-CYP Mediated Chemical Transformation
Hydrolysis Reduction Oxidations
Flavine monooxygenases Monoamine and diamine oxidases Alcohol and aldehyde dehydrogenase
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Non-CYP Mediated Biotransformation
Hydrolysis Esterases, amidases and proteases Non-microsomal (cytosolic) Widely distributed in most tissues
46
Non-CYP Mediated Biotransformation
Reduction
Ex: Reduction of side-chain of digoxin produces inactive metabolite
47
Non-CYP Mediated Chemical Transformation
Hydrolysis Reduction Oxidations
Flavine monooxygenases Monoamine and diamine oxidases Alcohol and aldehyde dehydrogenase
48
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
49
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
50
Flavine Monooxygenases Reactions
S-Oxygenation Spironolactone Cimetidine
Stereoselective: FMO3 forms the (+) enantiomer and FMO1 forms (-) enantiomer
N-Oxygenation Imipramine Nicotine
51
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
52
Flavine Monooxygenase Cycle
FMO Cycle
NADP+
+ H2OEnz | Flox
DRUGDRUGDRUG - ODRUG - O
NADPH
Enz | FlH2 +
NADP+
O2
Enz | FlHOH
Enz | FlOOH
53
Catalytic reaction cycle CYP450 and the oxidation of xenobiotics
e-
e-
CYP Reductase
NADP+
NADP
Fe2+CYP
Fe3+CYP
DRUGDRUG
DRUGDRUG
O2
Fe2+CYP
2H+
H2O
Fe3+CYP
DRUGDRUGOH
O2
DRUGDRUG DRUGDRUGOH
54
Flavine Monooxygenase Cycle
FMO Cycle
NADP+
+ H2OEnz | Flox
DRUGDRUGDRUG - ODRUG - O
NADPH
Enz | FlH2 +
NADP+
O2
Enz | FlHOH
Enz | FlOOH
55
Non-CYP Mediated Oxidation
Oxidation: Flavine Monooxygenases
Ex: N-Oxidation of nicotine, catalyzed by FMO3
56
Non-CYP Mediated Oxidation
Oxidation: Flavine Monooxygenases
Ex: S-Oxidation of cimetidine, catalyzed by FMO3
57
Non-CYP Mediated Chemical Transformation
Hydrolysis Reduction Oxidations
Flavine monooxygenases Monoamine and diamine oxidases Alcohol and aldehyde dehydrogenase
58
Non-CYP Mediated Oxidation
Oxidation: Monoamine Oxidases Mitochondrial enzymes Deaminate endogenous neurotransmitters
DopamineSerotoninNorepinephrineEpinephrine
Same type of products as other oxidizing enzymes Distinguish source enzyme of metabolites by other means
Found in liver, kidney, intestine, brain
59
Non-CYP Mediated Oxidation
Oxidation: Diamine Oxidases Endogenous amines
HistaminePolyamines
Putrescine Cadaverine
Amines converted to aldehydes (in presence of O2)
Contribute to oxidation of some drugs Found in liver, intestine, placenta
60
Non-CYP Mediated Chemical Transformation
Hydrolysis Reduction Oxidations
Flavine monooxygenases Monoamine and diamine oxidases Alcohol and aldehyde dehydrogenase
61
Non-CYP Mediated Oxidation
Alcohol and Aldehyde Dehydrogenases
Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.
62
Non-CYP Mediated Oxidation
Alcohol and Aldehyde Dehydrogenases
Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.
Relative Contribution to Drug Metabolism - Phase I
Evans & Relling Science 1999
64
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)
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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
66
Phase II Biotransformations (Conjugations)
Glutathione Catalyzed by glutathione-S-transferases
Cytosolic and microsomal Detoxification of electrophilic (and potentially carcinogenic)
molecules
67
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
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Phase II Biotransformations (Conjugations)
GlucuronidationEx: N- and O- linked glucuronide formation markedly enhances the polarity and water solubility.
69
Phase II Biotransformations (Conjugations)
Glucuronides can be generated from a variety of substrates
70
Phase II Biotransformations (Conjugations)
Sulfation
General pathway for enzymatic sulfation
71
Phase II Biotransformations (Conjugations)
Sulfation
Ex: Minoxidil
72
Phase II Biotransformations (Conjugations)
Acetylation
Ex: Acetyl transferase donates the acyl group from Coenzyme A to drug substrates
73
Phase II Biotransformations (Conjugations)
Acetylation
Ex: Isoniazid inactivation by acetylation
74
Phase II Biotransformations (Conjugations)
Hydroxylation and acetylationEx: Reactive nitrenium ions may be produced in the metabolism of aromatic amines through hydroxylation and acetylation
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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
76
Questions?
Blair Holbein, Ph.D., BCAPPresbyterian Hospital of Dallas
Email: [email protected] Website: http://phdres.caregate.net Annotated bibliography
77
References
Wright JM. Drug Interactions. In: 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.
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.