Drug Interactions
Content
Drug Interactions
Evolution of Drug Metabolism As a Science
• 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.
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 R-Ase e
-
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
Cytochrome P450 Isoforms (CYPs) - 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, kidney, G.I. tract, skin, lungs • Oxidative reactions require the CYP heme protein, the reductase, NADPH, 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
CYP Families
• Multiple CYP gene families have been identified in humans, and the categories are based upon protein sequence homology • Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families . • CYPs have molecular weights of 45-60 kDa. • Frequently, two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity. • CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availabilty of many drugs
CYP Nomenclature
• • • Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg. CYP1) Subfamily - 40-55% homology of amino acid sequence; eg. CYP1A Subfamily - additional arabic numeral when more than 1 subfamily has been identified; eg. CYP1A2 Italics indicate gene (CYP1A2); regular font for enzyme Comprehensive guide to human http://drnelson.utmem.edu/human.P450.table.html Cyps
• •
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
ROLE OF CYP ENZYMES IN HEPATIC DRUG METABOLISM
CYP1A2 2% CYP2C9 10% Others 3% Others 20% CYP1A 8% CYP3A 31%
CYP2D6 30%
CYP3A4 55%
CYP2C6 12%
CYP2E1 13%
CYP2C11 16%
Proportion of drug metabolized by CYP
Percentage in the human liver microsomes
As of October 2006 there were 6422 P450 enzymes, organized into 708 families, which were identified in species, although only 2279 in 99 families in animals. Only the 50 P450 enzymes described in man are likely to be of any clinical relevance, and even then only the P450s in families 1, 2, and 3 appear to be responsible for the metabolism of drugs and therefore are potential sites for drug interactions.
Human Cytochrome P450 Superfamily
Human Liver Drug CYPs
CYP enzy me 1A2 1B1 2A6 2B6 2C 2D6 2E1 2F1 2J2 3A4 4A, 4B
2E
Level (%total) ~ 13 <1 ~4 <1 ~18 Up to 2.5 Up to 7
Ex tent of vari ability ~40 -fold ~30 - 100 -fold ~50 -fold 25-100 -fold >1000 -fold ~20 -fold
Up to 28 30-60 *
~20 -fold 90-fold*
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997 *L. Wojnowski, Ther Drug Monit 26: 192-199, 2004
Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs
CYP Enzyme Examples of substrates
1A1 1A2 2A6 2B6 2C-family Caffeine, Testosterone, R-Warfarin Acetaminophen, Caffeine, Phenacetin, R-Warfarin 17β-Estradiol, Testosterone Cyclophosphamide, Erythromycin, Testosterone Acetaminophen, Tolbutamide (2C9); Hexobarbital, SWarfarin (2C9,19); Phenytoin, Testosterone, R- Warfarin, Zidovudine (2C8,9,19); Acetaminophen, Caffeine, Chlorzoxazone, Halothane Acetaminophen, Codeine, Debrisoquine Acetaminophen, Caffeine, Carbamazepine, Codeine, Cortisol, Erythromycin, Cyclophosphamide, S- and RWarfarin, Phenytoin, Testosterone, Halothane, Zidovudine
2E1 2D6 3A4
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Factors Influencing Activity and Level of CYP Enzymes
Nutrition Smoking Alcohol Drugs 1A1;1A2; 1B1, 2A6, 2B6, 2C8,9,19; 2D6, 3A4,5 1A1;1A2, 2E1 2E1
1A1,1A2; 2A6; 2B6; 2C; 2D6; 3A3, 3A4,5 1A1,1A2; 2A6; 1B; 2E1; Environment 3A3, 3A4,5 Genetic 1A; 2A6; 2C9,19; 2D6; Polymorphism 2E1
Red indicates enzymes important in drug metabolism
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Non-nitrogenous Substances that Affect Drug Metabolism • Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; fucocoumarins
– Bailey, D.G. et al.; Br J Clin Pharmacol 1998, 46:101-110 – Bailey, D.G et al.; Am J Cardiovasc Drugs 2004, 4:281-97.
• St John’s wort, other herbal products
– Tirona, R.G and Bailey, D.G. ; Br J Clin Pharmacol. 2006,61: 677-81
• Isosafrole, safrole
– CYP1A1, CYP1A2 inhibitor; found in root beer, perfume
Effect of Grapefruit Juice on Felodipine Plasma Concentration
5mg tablet with juice without
Cl Cl CO 2 CH 3 CH 3
Cl
CH 3 O 2 C CH 3 N H
H
3A4
CH 3 O 2 C CH 3 N
Cl CO 2 CH 3 CH 3
Review- D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110
Grapefruit Juice Facts
• GJ or G, lime, or Sun Drop Citrus soda, Seville OJ(not most OJ) elevates plasma peak drug concentration, not elimination t1/2 • GJ reduced metabolite/parent drug AUC ratio • GJ caused 62% reduction in small bowel enterocyte 3A4 and 3A5 protein; liver not as markedly affected (i.v. pharmacokinetics unchanged) • GJ effects last ~4 h, require new enzyme synthesis • Effect cumulative (up to 5x Cmax) and highly variable among individuals depending upon 3A4 small bowel basal levels
Human Drug Metabolizing CYPs Located in Extrahepatic Tissues
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997
Human Drug Metabolizing CYPs Located in Extrahepatic Tissues (cont’d)
CYP Enzyme 2E1 2F1 2J2 3A 4B1 4A11 Tissue Lung, placenta, others Lung, placenta Heart GI tract, lung, placenta, fetus, uterus, kidney Lung, placenta Kidney
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997
CYP Biotransformations
• Chemically diverse small molecules are converted, generally to more polar compounds • Reactions include
– – – – – Aliphatic hydroxylation, aromatic hydroxylation Dealkylation (N-,O-, S-) N-oxidation, S-oxidation Deamination Dehalogenation
Non-CYP Drug Biotransformations
• Oxidations • Hydrolyses • Conjugation (Phase 2 Rxs)
– Major Conjugation Reactions • Glucuronidation (high capacity) • Sulfation (low capacity) • Acetylation (variable capacity)
• Examples:Procainamide, Isoniazid
– Other Conjugation Reactions: O-Methylation, SMethylation, Amino Acid Conjugation (glycine, taurine, glutathione) – Many conjugation enzymes exhibit polymorphism
Non-CYP drug oxidations (1)
• Monoamine Oxidase (MAO), Diamine Oxidase (DAO) - MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA); MPTP converted to toxin MPP+ through MAO-B. DAO substrates include histamine and polyamines. Alcohol & Aldehyde Dehydrogenase - non-specific enzymes found in soluble fraction of liver; ethanol metabolism Xanthine Oxidase - converts hypoxanthine to xanthine, and then to uric acid. Drug substrates include theophylline, 6mercaptopurine. Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout.
•
•
Non-CYP drug oxidations (2)
• Flavin Monooxygenases
– Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides – Different FMO isoforms have been isolated from liver, lung (S.K. Krueger, et al. Drug Metab Rev 2002; 34:523-32) – Complete structures defined (Review: J. Cashman, 1995, Chem Res Toxicol 8:165-181; Pharmacogenomics 2002; 3:325-39) – Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD) – Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent – FMOs are heat labile and metal-free, unlike CYPs – Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs
Conjugation Reactions Glucuronidation
CO 2H O OH HO O OH O P O P O CH 2 OH O OH ON O NH O
+ ROH or R 3N UGT
CO 2H OO R OH OH OH
O-glucuronide
CO 2H R +R N O R OH OH OH
UDP- α-D-glucuronic acid
N+-glucuronide
Liver has several soluble UDP-Gluc-transferases
HO 3 N O N CH 3
6
O
N N
CH 3
HO
Morphine
Amitriptyline
Cotinine
Glucuronic acid conjugation to phenols, 3°-amines, aromatic amines
Conjugation Reactions Sulfation
R OH
NH 2 N N
+
N N H O H OH O OH O P O S O O H OH
O R O S OH O
(PAPS, 3’-phosphoadenosine5’-phosphosulfate)
H HO
Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin
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
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, isoniazid, sulfanilimide, histamine NAT enzyme is found in many tissues, including liver
Procainamide
O
Unchanged in Urine, 59%
H2 N
N H
N
24% Fast 17% Slow Unchanged in Urine, 85%
H N O
3%
O N H N
1%
NAPA 0.3%
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.T. Williams).
• Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat, guinea pig, and dog and 3 days in humans.
•
Induction – Two major categories of CYP inducers
• Phenobarbital is prototype of one group - enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells. • Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene).
– Induction appears to be environmental adaptive response of organism – Orphan Nuclear Receptors (PXR, CAR) are regulators of drug metabolizing gene expression
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. Wilson, S. A. Kliewer 2002:1, 259-266
CYP3A Inducers Activate Human, Rabbit, 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. Kliewer
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. • Named on basis of activation by natural and synthetic C21 steroids (pregnanes), 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.A. Kliewer
Constitutive Androstane Receptor (CAR)
CAR CAR CAR PXR PXR PXR
DNA
66%
Ligand
S.A. Kliewer
41%
• Highly expressed in liver and intestine • Sequestered in cytoplasm • Co-factor complex required for activation; anchored by PPAR-binding protein (PBP) • Binds response elements as RXR heterodimer • High basal transcriptional activity without ligand • Activated by xenobiotics – phenobarbital, TCPOBOP (1,4-bis[2-(3,5dichloropyridyloxy)]benzene)
Acetaminophen (Paracetamol)
• Acetanilide – 1886 – accidentally discovered antipyretic; excessively toxic (methemoglobinemia); para-aminophenol and derivatives were tested. • Phenacetin introduced in 1887, 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
Acetaminophen and p-Aminophenols
HN COCH
3
HN
NH 2
COCH
3
Acetanilide, 1886 (accidental discovery of antipyretic activity; high toxicity) 70-90%
NH 2
OC 2 H5
OC 2 H5
75-80%
Phenacetin or acetophenetidin, 1887 (nephrotoxic, methemoglobinemia)
HN
COCH
3
Metabolic pathway quantified; (Brodie &Axelrod, 1948) popular in US since 1955 Acetaminophen, 1893
OH
Acetaminophen Toxicity
•Acetaminophen overdose results in more calls to poison control centers in the United States than overdose with any other pharmacologic substance. •The American Liver Foundation reports that 35% of cases of severe liver failure are caused by acetaminophen poisoning which may require organ transplantation. •N-acetyl cysteine is an effective antidote, especially if administered within 10 h of ingestion [NEJM 319:15571562, 1988] •Management of acetaminophen overdose [Trends Pharm Sci 24:154-157, 2003
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
Protein adducts, Oxidative stress Toxicity
O
SO 3 H
*induced by ethanol, isoniazid
NAPQI N-acetyl-p-benzoquinone imine
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.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995) K.D. Welch et al., Chem Res Toxicol 18:924-33 (2005)
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)
Drug Metabolism - WWW Information Resources •http://www.icgeb.trieste.it/p450/
– Directory of P450 Containing Systems; comprehensive web site regarding all aspects of chemical structure (sequence and 3D) of P450 proteins from all species; steroid ligands; links to related sites including leading researchers on P450
•http://www.fda.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", FDA Guidance for Industry
•http://www.sigmaaldrich.com/Area_of_Interest/Biochem icals/Enzyme_Explorer.html
– Site has many commercially available drug metabolizing enzymes and useful links to multiple drug metabolism resources
•http://www.biocatalytics.com/p450.html
– Six freeze dried human CYPs (1A2, 2C9, 2C19, 2D6, 2E1, 3A4) available for drug metabolism studies
Evolution of Drug Metabolism As a Science
• 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.
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 R-Ase e
-
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
Cytochrome P450 Isoforms (CYPs) - 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, kidney, G.I. tract, skin, lungs • Oxidative reactions require the CYP heme protein, the reductase, NADPH, 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
CYP Families
• Multiple CYP gene families have been identified in humans, and the categories are based upon protein sequence homology • Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families . • CYPs have molecular weights of 45-60 kDa. • Frequently, two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity. • CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availabilty of many drugs
CYP Nomenclature
• • • Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg. CYP1) Subfamily - 40-55% homology of amino acid sequence; eg. CYP1A Subfamily - additional arabic numeral when more than 1 subfamily has been identified; eg. CYP1A2 Italics indicate gene (CYP1A2); regular font for enzyme Comprehensive guide to human http://drnelson.utmem.edu/human.P450.table.html Cyps
• •
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
ROLE OF CYP ENZYMES IN HEPATIC DRUG METABOLISM
CYP1A2 2% CYP2C9 10% Others 3% Others 20% CYP1A 8% CYP3A 31%
CYP2D6 30%
CYP3A4 55%
CYP2C6 12%
CYP2E1 13%
CYP2C11 16%
Proportion of drug metabolized by CYP
Percentage in the human liver microsomes
As of October 2006 there were 6422 P450 enzymes, organized into 708 families, which were identified in species, although only 2279 in 99 families in animals. Only the 50 P450 enzymes described in man are likely to be of any clinical relevance, and even then only the P450s in families 1, 2, and 3 appear to be responsible for the metabolism of drugs and therefore are potential sites for drug interactions.
Human Cytochrome P450 Superfamily
Human Liver Drug CYPs
CYP enzy me 1A2 1B1 2A6 2B6 2C 2D6 2E1 2F1 2J2 3A4 4A, 4B
2E
Level (%total) ~ 13 <1 ~4 <1 ~18 Up to 2.5 Up to 7
Ex tent of vari ability ~40 -fold ~30 - 100 -fold ~50 -fold 25-100 -fold >1000 -fold ~20 -fold
Up to 28 30-60 *
~20 -fold 90-fold*
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997 *L. Wojnowski, Ther Drug Monit 26: 192-199, 2004
Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs
CYP Enzyme Examples of substrates
1A1 1A2 2A6 2B6 2C-family Caffeine, Testosterone, R-Warfarin Acetaminophen, Caffeine, Phenacetin, R-Warfarin 17β-Estradiol, Testosterone Cyclophosphamide, Erythromycin, Testosterone Acetaminophen, Tolbutamide (2C9); Hexobarbital, SWarfarin (2C9,19); Phenytoin, Testosterone, R- Warfarin, Zidovudine (2C8,9,19); Acetaminophen, Caffeine, Chlorzoxazone, Halothane Acetaminophen, Codeine, Debrisoquine Acetaminophen, Caffeine, Carbamazepine, Codeine, Cortisol, Erythromycin, Cyclophosphamide, S- and RWarfarin, Phenytoin, Testosterone, Halothane, Zidovudine
2E1 2D6 3A4
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Factors Influencing Activity and Level of CYP Enzymes
Nutrition Smoking Alcohol Drugs 1A1;1A2; 1B1, 2A6, 2B6, 2C8,9,19; 2D6, 3A4,5 1A1;1A2, 2E1 2E1
1A1,1A2; 2A6; 2B6; 2C; 2D6; 3A3, 3A4,5 1A1,1A2; 2A6; 1B; 2E1; Environment 3A3, 3A4,5 Genetic 1A; 2A6; 2C9,19; 2D6; Polymorphism 2E1
Red indicates enzymes important in drug metabolism
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Non-nitrogenous Substances that Affect Drug Metabolism • Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; fucocoumarins
– Bailey, D.G. et al.; Br J Clin Pharmacol 1998, 46:101-110 – Bailey, D.G et al.; Am J Cardiovasc Drugs 2004, 4:281-97.
• St John’s wort, other herbal products
– Tirona, R.G and Bailey, D.G. ; Br J Clin Pharmacol. 2006,61: 677-81
• Isosafrole, safrole
– CYP1A1, CYP1A2 inhibitor; found in root beer, perfume
Effect of Grapefruit Juice on Felodipine Plasma Concentration
5mg tablet with juice without
Cl Cl CO 2 CH 3 CH 3
Cl
CH 3 O 2 C CH 3 N H
H
3A4
CH 3 O 2 C CH 3 N
Cl CO 2 CH 3 CH 3
Review- D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110
Grapefruit Juice Facts
• GJ or G, lime, or Sun Drop Citrus soda, Seville OJ(not most OJ) elevates plasma peak drug concentration, not elimination t1/2 • GJ reduced metabolite/parent drug AUC ratio • GJ caused 62% reduction in small bowel enterocyte 3A4 and 3A5 protein; liver not as markedly affected (i.v. pharmacokinetics unchanged) • GJ effects last ~4 h, require new enzyme synthesis • Effect cumulative (up to 5x Cmax) and highly variable among individuals depending upon 3A4 small bowel basal levels
Human Drug Metabolizing CYPs Located in Extrahepatic Tissues
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997
Human Drug Metabolizing CYPs Located in Extrahepatic Tissues (cont’d)
CYP Enzyme 2E1 2F1 2J2 3A 4B1 4A11 Tissue Lung, placenta, others Lung, placenta Heart GI tract, lung, placenta, fetus, uterus, kidney Lung, placenta Kidney
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997
CYP Biotransformations
• Chemically diverse small molecules are converted, generally to more polar compounds • Reactions include
– – – – – Aliphatic hydroxylation, aromatic hydroxylation Dealkylation (N-,O-, S-) N-oxidation, S-oxidation Deamination Dehalogenation
Non-CYP Drug Biotransformations
• Oxidations • Hydrolyses • Conjugation (Phase 2 Rxs)
– Major Conjugation Reactions • Glucuronidation (high capacity) • Sulfation (low capacity) • Acetylation (variable capacity)
• Examples:Procainamide, Isoniazid
– Other Conjugation Reactions: O-Methylation, SMethylation, Amino Acid Conjugation (glycine, taurine, glutathione) – Many conjugation enzymes exhibit polymorphism
Non-CYP drug oxidations (1)
• Monoamine Oxidase (MAO), Diamine Oxidase (DAO) - MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA); MPTP converted to toxin MPP+ through MAO-B. DAO substrates include histamine and polyamines. Alcohol & Aldehyde Dehydrogenase - non-specific enzymes found in soluble fraction of liver; ethanol metabolism Xanthine Oxidase - converts hypoxanthine to xanthine, and then to uric acid. Drug substrates include theophylline, 6mercaptopurine. Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout.
•
•
Non-CYP drug oxidations (2)
• Flavin Monooxygenases
– Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides – Different FMO isoforms have been isolated from liver, lung (S.K. Krueger, et al. Drug Metab Rev 2002; 34:523-32) – Complete structures defined (Review: J. Cashman, 1995, Chem Res Toxicol 8:165-181; Pharmacogenomics 2002; 3:325-39) – Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD) – Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent – FMOs are heat labile and metal-free, unlike CYPs – Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs
Conjugation Reactions Glucuronidation
CO 2H O OH HO O OH O P O P O CH 2 OH O OH ON O NH O
+ ROH or R 3N UGT
CO 2H OO R OH OH OH
O-glucuronide
CO 2H R +R N O R OH OH OH
UDP- α-D-glucuronic acid
N+-glucuronide
Liver has several soluble UDP-Gluc-transferases
HO 3 N O N CH 3
6
O
N N
CH 3
HO
Morphine
Amitriptyline
Cotinine
Glucuronic acid conjugation to phenols, 3°-amines, aromatic amines
Conjugation Reactions Sulfation
R OH
NH 2 N N
+
N N H O H OH O OH O P O S O O H OH
O R O S OH O
(PAPS, 3’-phosphoadenosine5’-phosphosulfate)
H HO
Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin
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
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, isoniazid, sulfanilimide, histamine NAT enzyme is found in many tissues, including liver
Procainamide
O
Unchanged in Urine, 59%
H2 N
N H
N
24% Fast 17% Slow Unchanged in Urine, 85%
H N O
3%
O N H N
1%
NAPA 0.3%
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.T. Williams).
• Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat, guinea pig, and dog and 3 days in humans.
•
Induction – Two major categories of CYP inducers
• Phenobarbital is prototype of one group - enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells. • Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene).
– Induction appears to be environmental adaptive response of organism – Orphan Nuclear Receptors (PXR, CAR) are regulators of drug metabolizing gene expression
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. Wilson, S. A. Kliewer 2002:1, 259-266
CYP3A Inducers Activate Human, Rabbit, 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. Kliewer
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. • Named on basis of activation by natural and synthetic C21 steroids (pregnanes), 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.A. Kliewer
Constitutive Androstane Receptor (CAR)
CAR CAR CAR PXR PXR PXR
DNA
66%
Ligand
S.A. Kliewer
41%
• Highly expressed in liver and intestine • Sequestered in cytoplasm • Co-factor complex required for activation; anchored by PPAR-binding protein (PBP) • Binds response elements as RXR heterodimer • High basal transcriptional activity without ligand • Activated by xenobiotics – phenobarbital, TCPOBOP (1,4-bis[2-(3,5dichloropyridyloxy)]benzene)
Acetaminophen (Paracetamol)
• Acetanilide – 1886 – accidentally discovered antipyretic; excessively toxic (methemoglobinemia); para-aminophenol and derivatives were tested. • Phenacetin introduced in 1887, 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
Acetaminophen and p-Aminophenols
HN COCH
3
HN
NH 2
COCH
3
Acetanilide, 1886 (accidental discovery of antipyretic activity; high toxicity) 70-90%
NH 2
OC 2 H5
OC 2 H5
75-80%
Phenacetin or acetophenetidin, 1887 (nephrotoxic, methemoglobinemia)
HN
COCH
3
Metabolic pathway quantified; (Brodie &Axelrod, 1948) popular in US since 1955 Acetaminophen, 1893
OH
Acetaminophen Toxicity
•Acetaminophen overdose results in more calls to poison control centers in the United States than overdose with any other pharmacologic substance. •The American Liver Foundation reports that 35% of cases of severe liver failure are caused by acetaminophen poisoning which may require organ transplantation. •N-acetyl cysteine is an effective antidote, especially if administered within 10 h of ingestion [NEJM 319:15571562, 1988] •Management of acetaminophen overdose [Trends Pharm Sci 24:154-157, 2003
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
Protein adducts, Oxidative stress Toxicity
O
SO 3 H
*induced by ethanol, isoniazid
NAPQI N-acetyl-p-benzoquinone imine
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.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995) K.D. Welch et al., Chem Res Toxicol 18:924-33 (2005)
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)
Drug Metabolism - WWW Information Resources •http://www.icgeb.trieste.it/p450/
– Directory of P450 Containing Systems; comprehensive web site regarding all aspects of chemical structure (sequence and 3D) of P450 proteins from all species; steroid ligands; links to related sites including leading researchers on P450
•http://www.fda.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", FDA Guidance for Industry
•http://www.sigmaaldrich.com/Area_of_Interest/Biochem icals/Enzyme_Explorer.html
– Site has many commercially available drug metabolizing enzymes and useful links to multiple drug metabolism resources
•http://www.biocatalytics.com/p450.html
– Six freeze dried human CYPs (1A2, 2C9, 2C19, 2D6, 2E1, 3A4) available for drug metabolism studies
