Drug Metabolism

S.P. Markey Laboratory of Neurotoxicology NIMH, NIH
Nov. 16, 2006

Evolution of Drug Metabolism As a Science Post WWII Pioneers
• Richard Tecwyn Williams – Great Britain
– 1942, worked on the metabolism on TNT with regard to toxicity in munitions workers; due to the war he assembled teams to work on metabolism of sulfonamides, benzene, aniline, acetanilide, phenacetin, and stilbesterol – Developed concept of Phase 1 & Phase 2 Reactions. • Biotransformation involves metabolic oxygenation, reduction, or hydrolysis; result in changes in biological activity (increased or decreased) • Second phase, conjugation, in almost all cases resulted in detoxication.

Evolution of Drug Metabolism As a Science Post WWII Pioneers
• Bernard B. Brodie, U.S.
– NYU and Laboratory of Industrial Hygiene, NYC 1949 – Metabolic fate of acetanilide and phenacetin in man (with Julius Axelrod) – 1950s, NIH – pioneering studies on all aspects of drug metabolism; esp. reserpine, serotonin;hexobarbital tolerance – 1952 – R.T. Williams spent 6 months at NIH; subsequently many students went between both labs (Richard Adamson, James Gillette, and Sidney Udenfriend) – 1950s, Brodie lab developed the spectrophotofluorimeter (Robert Bowman)

Drug Metabolism
Extrahepatic microsomal enzymes
(oxidation, conjugation)

Hepatic microsomal enzymes
(oxidation, conjugation)

Hepatic non-microsomal enzymes
(acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

Liver Microsomal System •Oxidative Reactions: Cytochrome P450 mediated • Examples – Formation of an inactive polar metabolite • Phenobarbital – Formation of an active metabolite • By Design: Purine & pyrimidine chemotherapy prodrugs • Inadvertent: terfenadine – fexofenadine – Formation of a toxic metabolite • Acetaminophen – NAPQI .

NADP+ Drug CYP e R-Ase - CYP Fe+3 PC Drug Drug OH CYP Fe+3 Drug OH e- NADPH CO CYP-Fe+2 Drug CO hυ CYP Fe+2 Drug O2 O2 CYP Fe+2 Drug H2O 2H+ Electron flow in microsomal drug oxidizing system .

kidney. NADPH. G.An Overview • NADPH + H+ + O2 + Drug → NADP+ + H2O + Oxidized Drug • Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name) • CYP monooxygenase enzyme family is major catalyst of drug and endogenous compound oxidations in liver. phosphatidylcholine and molecular oxygen • CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio • The reductase serves as the electron source for the oxidative reaction cycle . the reductase. lungs • Oxidative reactions require the CYP heme protein. skin. tract.Cytochrome P450 Isoforms (CYPs) .I.

and the categories are based upon protein sequence homology • Most of the drug metabolizing enzymes are in CYP 1.CYP Families • Multiple CYP gene families have been identified in humans. two or more enzymes can catalyze the same type of oxidation. indicating redundant and broad substrate specificity. 2. & 3 families . its presence in the GI tract is responsible for poor oral availabilty of many drugs . • CYP3A4 is very common to the metabolism of many drugs. • Frequently. • CYPs have molecular weights of 45-60 kDa.

CYP Nomenclature • Families . eg. CYP1) Subfamily . CYP1A2 Italics indicate gene (CYP1A2).additional arabic numeral when more than 1 subfamily has been identified.edu/human. regular font for enzyme Comprehensive guide to human Cyps http://drnelson. utmem. eg.40-55% homology of amino acid sequence.P450.table. eg. CYP1A Subfamily .CYP plus arabic numeral (>40% homology of amino acid sequence.html • • • • .

CYP Tables • Human CYPs .variability and importance in drug metabolism • Isoforms in metabolism of clinically important drugs • Factors that influence CYP activity • Non-Nitrogenous CYP inhibitors • Extrahepatic CYPs .


DiCarlo. Wojnowski.J. 2004 . 1997 *L.Human Liver Drug CYPs CYP enzyme 1A2 1B1 2A6 2B6 2C 2D6 2E1 2F1 2J2 3A4 4A.5 Up to 7 Extent of variability ~40-fold ~30 . 4B 2E Level (%total) ~ 13 <1 ~4 <1 ~18 Up to 2. Drug Metab Rev 29:413-80. Ther Drug Monit 26: 192-199. Rendic & F.100-fold ~50-fold 25-100-fold >1000-fold ~20-fold Up to 28 30-60* ~20-fold 90-fold* S.

Cyclophosphamide. Phenytoin.9. Zidovudine (2C8. Testosterone Acetaminophen. S. Codeine. Rendic Drug Metab Rev 34: 83-448.Warfarin. Caffeine. Tolbutamide (2C9). Erythromycin. SWarfarin (2C9. Caffeine. Debrisoquine Acetaminophen. R-Warfarin Acetaminophen. Testosterone. Acetaminophen. Testosterone Cyclophosphamide. 2002 . Chlorzoxazone. Carbamazepine.19). Halothane Acetaminophen. Hexobarbital. R-Warfarin 17β -Estradiol. Testosterone. R. Erythromycin. Codeine. Phenytoin. Halothane. Phenacetin.and RWarfarin.Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs CYP Enzyme Examples of substrates 1A1 1A2 2A6 2B6 2C-family Caffeine. Caffeine.19). Zidovudine 2E1 2D6 3A4 Adapted from: S. Testosterone. Cortisol.

1 A2.19 . 2D6 . 2A6.5 1A1.1A2. Rendic Drug Metab Rev 34: 83-448. 2E1 2E1 1A1. 3A4.Factors Influencing Activity and Level of CYP Enzymes Nutrition Sm oking Alcohol Drugs En vironm ent Genetic Pol ym orphism 1A1.5 1A.1A2. 1B1 . 1A2. 2C9 . 2C. 2E1. 2002 . 3A4. 3A3. 2C8. 2B6. 5 1A1.19 . 2A6. 2A6. 2D6. 2A6.9 . 1B . 2B6 . 3A3. 3A4. 2D6 . 2E1 Red indicates enzymes important in drug metabolism Adapted from: S.

Non-nitrogenous Substances that Affect Drug Metabolism • Grapefruit juice . 2006.G and Bailey. fucocoumarins – Bailey. other herbal products – Tirona. Br J Clin Pharmacol 1998. safrole – CYP1A1. 4:281-97.G et al.CYP 3A4 inhibitor. Br J Clin Pharmacol. found in root beer. CYP1A2 inhibitor. D.G. 46:101110 – Bailey. • St John’s wort. Am J Cardiovasc Drugs 2004. R.. D. perfume . et al.. . D.G.61: 677-81 • Isosafrole. highly variable effects.

after grapefruit juice was served. someone remarked “A friend read the package insert with her prescription and the fine print warned against drinking grapefruit juice…is this true? Should it be avoided with all medications? How about grapefruit itself? How about orange juice?” .Overheard Conversation • At a B&B breakfast table.

46:101-110 .Effect of Grapefruit Juice on Felodipine Plasma Concentration 5mg tablet with juice without Cl Cl CO 2 CH3 CH3 Cl CH3 O 2 C CH3 N H H 3A4 CH3 O 2 C CH3 N Cl CO 2 CH3 CH3 Review. Br J Clin Pharmacol 1998.. et al.G.D. Bailey.

Grapefruit Juice Facts • GJ or G. lime. liver not as markedly affected (i.v. require new enzyme synthesis • Effect cumulative (up to 5x Cmx ) and highly a variable among individuals depending upon 3A4 small bowel basal levels . not elimination t1/2 • GJ reduced metabolite/parent drug AUC ratio • GJ caused 62% reduction in small bowel enterocyte 3A4 and 3A5 protein. or Sun Drop Citrus soda. Seville OJ(not most OJ) elevates plasma peak drug concentration. pharmacokinetics unchanged) • GJ effects last ~4 h.

Human Drug Metabolizing CYPs Located in Extrahepatic Tissues S. DiCarlo. Drug Metab Rev 29:413-80. 1997 .J. Rendic & F.

Human Drug Metabolizing CYPs Located in Extrahepatic Tissues (cont’d) CYP Enzyme 2E1 2F1 2J2 3A 4B1 4A11 Tissue Lung. Drug Metab Rev 29:413-80. placenta Heart GI tract. 1997 . Rendic & F. placenta. lung. DiCarlo. others Lung. fetus. uterus. kidney Lung.J. placenta. placenta Kidney S.

CYP Biotransformations • Chemically diverse small molecules are converted. generally to more polar compounds • Reactions include (see text): – – – – – Aliphatic hydroxylation. aromatic hydroxylation Dealkylation (N-.O-. S-) N-oxidation. S-oxidation Deamination Dehalogenation .

Isoniazid – Other Conjugation Reactions: O-Methylation. glutathione) – Many conjugation enzymes exhibit polymorphism . taurine. SMethylation. Amino Acid Conjugation (glycine.Non-CYP Drug Biotransformations • Oxidations • Hydrolyses • Conjugation (Phase 2 Rxs) – Major Conjugation Reactions • Glucuronidation (high capacity) • Sulfation (low capacity) • Acetylation (variable capacity) • Examples:Procainamide.

drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA). ethanol metabolism Xanthine Oxidase . serotonin. and then to uric acid. norepinephrine. Drug substrates include theophylline.Non-CYP drug oxidations (1) • Monoamine Oxidase (MAO). Diamine Oxidase (DAO) . delays metabolism of other substrates. epinephrine). effective for treatment of gout. Alcohol & Aldehyde Dehydrogenase .non-specific enzymes found in soluble fraction of liver. DAO substrates include histamine and polyamines. MPTP converted to toxin MPP+ through MAO-B. 6mercaptopurine.MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine. • • . Allopurinol is substrate and inhibitor of xanthine oxidase.converts hypoxanthine to xanthine.

Pharmacogenomics 2002. et al. drugs. Cashman. lung (S. 3:325-39) – Require molecular oxygen. Chem Res Toxicol 8:165-181. 1995. Drug Metab Rev 2002. sulfur – particularly facile formation of N-oxides – Different FMO isoforms have been isolated from liver. NADPH. flavin adenosine dinucleotide (FAD) – Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent – FMOs are heat labile and metal-free. phosphorus.K.Non-CYP drug oxidations (2) • Flavin Monooxygenases – Family of enzymes that catalyze oxygenation of nitrogen. 34:523-32) – Complete structures defined (Review: J. unlike CYPs – Factors affecting FMOs (diet. Krueger. sex) not as highly studied as CYPs .

Diczfalusy. M.Hydrolysis secondary to Cyp Metabolism QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. M. Alexson. S Swedmark Drug Metab Disp 30: 643-647. H. Involvement of Liver Carboxylesterases in the In Vitro Metabolism of Lidocaine S. 2002 . E. Halldin.

Conjugation Reactions Glucuronidation CO2H O OH HO O OH O P O P O CH 2 OH O OH ON O NH O + ROH or R 3N UGT CO2H OO R OH OH OH O-glucuronide CO2H R + N R O R OH OH OH UDP.α-D-glucuronic acid N+-glucuronide Liver has several soluble UDP-Gluc-transferases .

3°-amines. aromatic amines .HO 3 N O N CH 3 6 O N N CH 3 HO Morphine Amitriptyline Cotinine Glucuronic acid conjugation to phenols.

p-hydroxyacetanilide. 3-hydroxycoumarin . 3’-phosphoadenosine5’-phosphosulfate) H HO H OH Examples: ethanol.Conjugation Reactions Sulfation R OH NH2 N N + N N H O H OH O OH O P O S O O O R O S OH O (PAPS.

H2N N O N N H2N O N HO S O O N N NH 2 NH Minoxidil Minoxidil-sulfate Sulfation may produce active metabolite .

sulfanilimide.Conjugation Reactions Acetylation O Ar NH 2 CoA S O Ar R NH 2 R OH R SH N H R O CH 3 O R N H CH 3 R S O CH 3 O CH 3 + Acetyl transferase Examples: Procainamide. histamine NAT enzyme is found in many tissues. including liver . isoniazid.

59% H2 N N H N 24% Fast 17% Slow Unchanged in Urine. 85% H N O 3% O N H N 1% NAPA 0.Procainamide O Unchanged in Urine.3% H N O O N H H N O H2 N N H H N .

Procainamide O H2 N N H N trace metabolite HO H N O N H N non-enzymatic O O N N H N Lupus? .

Additional Effects on Drug Metabolism • Species Differences – Major differences in different species have been recognized for many years (R. guinea pig. and dog and 3 days in humans. • Phenylbutazone half-life is 3 h in rabbit. – Induction appears to be environmental adaptive response of organism – Orphan Nuclear Receptors (PXR.T. • Induction – Two major categories of CYP inducers • Phenobarbital is prototype of one group . CAR) are regulators of drug metabolizing gene expression .enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells. ~6 h in rat. Williams). • Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene).

PXR and CAR Protect Against Xenobiotics co-activator PBP target genes CAR xenobiotics RXR PXR xenoprotection cytoplasm nucleus S.A. Kliewer .

CYP3A Regulation • Diverse drugs activate through heterodimer complex • Protect against xenobiotics • Cause drug-drug interactions T.M. A. Kliewer 2002:1. 259-266 . S. Wilson.

Rabbit. Kliewer . and Rat PXR rifampicin PCN dexamethasone RU486 clotrimazole troglitazone tamoxifen 1 3 5 7 9 11 13 15 17 19 Cell-based reporter assay Reporter activity (fold) S.A.CYP3A Inducers Activate Human.

Kliewer .A. including pregnenolone 16α -carbonitrile (PCN) • Cloned due to homology with other nuclear receptors • Highly active in liver and intestine • Binds as heterodimer with retinoic acid receptor (RXR) S. • Named on basis of activation by natural and synthetic C21 steroids (pregnanes).Pregnane X Receptor (PXR) human PXR rabbit PXR mouse PXR rat PXR DNA 94% 96% 96% Ligand 82% 77% 76% • PXR is one of Nuclear Receptor (NR) family of ligand-activated transcription factors.

anchored by PPAR-binding protein (PBP) • Binds response elements as RXR heterodimer • High basal transcriptional activity without ligand • Activated by xenobiotics – phenobarbital.Constitutive Androstane Receptor (CAR) CAR CAR CAR PXR PXR PXR DNA 66% Ligand S.4-bis[2-(3. Kliewer 41% • Highly expressed in liver and intestine • Sequestered in cytoplasm • Co-factor complex required for activation.A. TCPOBOP (1.5dichloropyridyloxy)]benzene) .

Kliewer .5x) (12x) (2.6x) (3.PXR and CAR Regulate Overlapping Genes PCN (PXR) • Phase I enzymes Cyp3a11 Cyp2b10 Aldh1a1 Aldh1a7 (3.9x) (1.1x) (1.2x) (2.8x) (16x) (15x) • Transporters Mrp2 Mrp3 Oatp2 (3.4x) (110x) (1.0x) (9.A.9x) TCPOBOP (CAR) • Phase II enzymes Liver RNA Ugt1a1 Gst-a1 (2.9x) S.0x) (1.

and extensively used in analgesic mixtures until implicated in analgesic abuse nephropathy • Acetaminophen recognized as metabolite in 1899 • 1948-49 Brodie and Axelrod recognized methemoglobinemia due to acetanilide and analgesia to acetaminophen • 1955 acetaminophen introduced in US . • Phenacetin introduced in 1887. excessively toxic (methemoglobinemia).Acetaminophen (Paracetamol) • Acetanilide – 1886 – accidentally discovered antipyretic. para-aminophenol and derivatives were tested.

1948) popular in US since 1955 Acetaminophen. (Brodie &Axelrod. high toxicity) 70-90% NH2 OC 2 H5 OC 2 H5 75-80% Phenacetin or acetophenetidin. 1887 (nephrotoxic.Acetaminophen and p-Aminophenols HN COCH 3 NH2 HN COCH 3 Acetanilide. methemoglobinemia) HN COCH 3 Metabolic pathway quantified. 1886 (accidental discovery of antipyretic activity. 1893 OH .

•The American Liver Foundation reports that 35% of cases of severe liver failure are caused by acetaminophen poisoning which may require organ transplantation. especially if administered within 10 h of ingestion [NEJM 319:15571562. 2003 . •N-acetyl cysteine is an effective antidote. 1988] •Management of acetaminophen overdose [Trends Pharm Sci 24:154-157.Acetaminophen Toxicity •Acetaminophen overdose results in more calls to poison control centers in the United States than overdose with any other pharmacologic substance.

isoniazid Protein adducts.Acetominophen Metabolism HN COCH 3 ~60% ~35% OH HN COCH 3 O HO O CO 2 H OH CYP2E1* CYP1A2 CYP3A11 N COCH 3 HN COCH 3 O OH SO 3 H *induced by ethanol. O NAPQI Oxidative stress N-acetyl-p-benzoquinone imine Toxicity .

27: 147-177 (1995) K. Drug Metab.Acetaminophen Protein Adducts HN COCH 3 N COCH 3 CYPs HS-Protein OH O H2N-Protein Protein S N COCH 3 HN COCH 3 HN COCH 3 S Protein O NH Protein OH OH S. Rev.D. Chem Res Toxicol 18:924-33 (2005) .D.. Welch et al. Nelson.

Acetaminophen toxicity mechanism • N-acetyl cysteine is an effective agent to block GSH depletion and rescue from liver damaging toxicity • CAR and PXR modulate acetaminophen toxicity (2002. 2004) • CAR-null mice are resistant to acetaminophen toxicity – hepatic GSH lowered in wild type (but not in KO) after acetaminophen – CAR-humanized mice demonstrate same toxicity response • Activation of PXR induces CYP3A11 and markedly enhances acetaminophen toxicity in wild type mice • CAR transcription co-activator KO blocks toxicity (2005) .

2004.NAPQI toxicity linked to PXR activation G. Toxicol Sci 82(2):374-80 HN COCH 3 N COCH 3 CAR PXR OH CYP2E1* CYP3A11 O toxicity Xenobiotics HN COCH 3 SH glu-cys-gly GLY S CYS OH toxicity oxidative stress mechanism ? GLU . Guo et al.

2C19. 3A4) available for drug metabolism studies . comprehensive web site regarding all aspects of chemical structure (sequence and 3D) of P450 proteins from all species.icgeb. steroid ligands.WWW Information Resources •http://www.html – Six freeze dried human CYPs (1A2.fda. 2E1. FDA Guidance for Industry •http://www.sigmaaldrich.it/p450/ – Directory of P450 Containing Systems.biocatalytics. 2D6.trieste.html – Site has many commercially available drug metabolizing enzymes and useful links to multiple drug metabolism resources •http://www. links to related sites including leading researchers on P450 •http://www.gov/cder/guidance/ – Site contains many useful documents regarding drug metabolism and FDA recommendations including "Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro".Drug Metabolism .com/p450.com/Area_of_Interest/Biochem icals/Enzyme_Explorer. 2C9.