1 Drug Metabolism Clinical Pharmacology Spring Course 2006 M. E. Blair Holbein, Ph.D. Clinical...

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Drug MetabolismClinical Pharmacology

Spring Course 2006

M. E. Blair Holbein, Ph.D.

Clinical Pharmacologist

Presbyterian Hospital

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)

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

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

Kinetic equations for inhibition of metabolizing enzymes

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

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.

Catalytic reaction cycle CYP450 and the oxidation of xenobiotics

CYP Reductase

Fe2+CYP

Fe3+CYP

DRUGDRUG

Fe2+CYP

Fe3+CYP

DRUGDRUGOH

DRUGDRUG DRUGDRUGOH

Catalytic reaction cycle involving cytochrome P450 in the oxidation of xenobiotics

Oxidized Drug + NADP+ + H2O

Drug + NADPH + H+ + O2

CYP450 Mediated Chemical Transformation

Hydroxylation Aliphatic Aromatic

N-Dealkylation, O-Dealkylation, S-Dealkylation Oxidative Deamination Dehalogenation N-Oxidation S-Oxidation

CYP Mediated Oxidation

Aliphatic Hydroxylation

RCH2CH3

OHRCHCH3

Ex: Hydroxylation of ibuprofen

Aromatic Hydroxylation

Ex: Hydroxylation of acetanilide to 4-hydroxyacetanilide

Aromatic Hydroxylation Directly through asymmetric oxygen transfer Through an unstable arene oxide intermediate

PredictabilityInfluence of environment

Ex:Hydroxylation of aromatic carbon atoms

Aromatic Hydroxylation Results in several oxidized metabolites

Ex:Metabolism of phenytoin

Dealkation (N-, O-, S-)

Ex: N-demethylation of ethylmorphine

N-demethylation generates formaldehyde as a by-product

Dealkation (N-, O-, S-)

Ex: N-demethylation and hydroxylation of propranolol

Oxidative Deamination

Ex: General mechanism for oxidative deamination

Oxidative Deamination

Ex: Deamination of amphetamine to inactive ketone

Dehalogenation

Ex: Dehalogenation generates reactive free radicals.Metabolism of carbon tetrachloride generates oxidized lipids

N-Oxidation may produce toxic by-products

N-Oxidation

Ex: N-oxidation of Dapsone

S-OxidationEx: General schemeEx: CYP3A and Flavin monooxygenase produce same metabolite

S-OxidationEx: A-oxidation of tazofelone

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

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

Proportion of Drugs Metabolized by CYP450 Enzymes in Humans

CYP2C198%

CYP1A211%

CYP2A63%

CYP2C916%

CYP2E14%

CYP3A438%

CYP2D620%

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)

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

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

Cytochrome P450 1A2

Substrates include: theophylline, caffeine, imipramine, clozapine

Inhibited by: many fluoroquinolone antibiotics, fluvoxamine, cimetidine

Induced by: smoking tobacco

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

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

Cytochrome P450 2B6

Substrates include: bupropion, cyclophosphamide, efavirenz, methadone

Inhibited by: thiotepa Induced by: phenobarbital, rifampin

Cytochrome P450 2E1

Substrates include: acetaminophen

Cytochrome P450 2C8

Substrates; paclitaxel, torsemide, amodiaquine, cerivastatin, repaglinide

Inhibited by: trimethoprim, quercetin, glitazones, gemfibrozil, montelukast

Induced by: rifampin

Non-CYP Mediated Chemical Transformation

Hydrolysis Reduction Oxidations

Flavine monooxygenases Monoamine and diamine oxidases Alcohol and aldehyde dehydrogenase

Non-CYP Mediated Biotransformation

Hydrolysis Esterases, amidases and proteases Non-microsomal (cytosolic) Widely distributed in most tissues

Non-CYP Mediated Biotransformation

Reduction

Ex: Reduction of side-chain of digoxin produces inactive metabolite

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

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

Flavine Monooxygenases Reactions

S-Oxygenation Spironolactone Cimetidine

Stereoselective: FMO3 forms the (+) enantiomer and FMO1 forms (-) enantiomer

N-Oxygenation Imipramine Nicotine

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

Flavine Monooxygenase Cycle

FMO Cycle

+ H2OEnz | Flox

DRUGDRUGDRUG - ODRUG - O

Enz | FlH2 +

Enz | FlHOH

Enz | FlOOH

Catalytic reaction cycle CYP450 and the oxidation of xenobiotics

CYP Reductase

Fe2+CYP

Fe3+CYP

DRUGDRUG

Fe2+CYP

Fe3+CYP

DRUGDRUGOH

DRUGDRUG DRUGDRUGOH

Flavine Monooxygenase Cycle

FMO Cycle

+ H2OEnz | Flox

DRUGDRUGDRUG - ODRUG - O

Enz | FlH2 +

Enz | FlHOH

Enz | FlOOH

Non-CYP Mediated Oxidation

Oxidation: Flavine Monooxygenases

Ex: N-Oxidation of nicotine, catalyzed by FMO3

Oxidation: Flavine Monooxygenases

Ex: S-Oxidation of cimetidine, catalyzed by FMO3

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

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

Alcohol and Aldehyde Dehydrogenases

Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.

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

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)

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

Phase II Biotransformations (Conjugations)

Glutathione Catalyzed by glutathione-S-transferases

Cytosolic and microsomal Detoxification of electrophilic (and potentially carcinogenic)

molecules

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

GlucuronidationEx: N- and O- linked glucuronide formation markedly enhances the polarity and water solubility.

Glucuronides can be generated from a variety of substrates

Sulfation

General pathway for enzymatic sulfation

Sulfation

Ex: Minoxidil

Acetylation

Ex: Acetyl transferase donates the acyl group from Coenzyme A to drug substrates

Acetylation

Ex: Isoniazid inactivation by acetylation

Hydroxylation and acetylationEx: Reactive nitrenium ions may be produced in the metabolism of aromatic amines through hydroxylation and acetylation

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

Questions?

Blair Holbein, Ph.D., BCAPPresbyterian Hospital of Dallas

Email: bholbein@hcin.net Website: http://phdres.caregate.net Annotated bibliography

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.

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