Drug Interactions

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.
Hepatic microsomal enzymes
(oxidation, conjugation)
Extrahepatic microsomal enzymes
(oxidation, conjugation)
Hepatic non-microsomal enzymes
(acetylation, sulfation,GSH,
alcohol/aldehyde dehydrogenase,
hydrolysis, ox/red)
Drug Metabolism
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

Electron flow in microsomal drug oxidizing system
CO
hu
CYP-Fe
+2

Drug
CO
O
2

e
-

e
-

2H
+

H
2
O
Drug
CYP
R-Ase
NADPH
NADP
+

OH
Drug
CYP Fe
+3
PC
Drug
CYP Fe
+2
Drug
CYP Fe
+2
Drug
O
2

CYP Fe
+3
OH
Drug
Cytochrome P450 Isoforms (CYPs) - An Overview
• NADPH + H
+
+ O
2
+ Drug NADP
+
+ H
2
O + 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 Cyps
http://drnelson.utmem.edu/human.P450.table.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
ROLE OF CYP ENZYMES IN HEPATIC
DRUG METABOLISM
Proportion of drug metabolized by CYP
Percentage in the human liver
microsomes
CYP3A4
55%
CYP2D6
30%
CYP2C9
10%
Others
3%
CYP1A2
2%
CYP3A
31%
CYP2C11
16%
CYP2E1
13%
CYP2C6
12%
CYP1A
8%
Others
20%
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
enzyme
Level
(%total)
Extent of
variability
1A2 ~ 13 ~40-fold
1B1 <1
2A6 ~4 ~30 - 100-fold
2B6 <1 ~50-fold
2C ~18 25-100-fold
2D6 Up to 2.5 >1000-fold
2E1 Up to 7 ~20-fold
2F1
2J2
3A4 Up to 28
30-60*
~20-fold
90-fold*
4A, 4B
2 E

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 Caffeine, Testosterone, R-Warfarin
1A2 Acetaminophen, Caffeine, Phenacetin, R-Warfarin
2A6 17-Estradiol, Testosterone
2B6 Cyclophosphamide, Erythromycin, Testosterone
2C-family Acetaminophen, Tolbutamide (2C9); Hexobarbital, S-
Warfarin (2C9,19); Phenytoin, Testosterone, R- Warfarin,
Zidovudine (2C8,9,19);
2E1 Acetaminophen, Caffeine, Chlorzoxazone, Halothane
2D6 Acetaminophen, Codeine, Debrisoquine
3A4 Acetaminophen, Caffeine, Carbamazepine, Codeine,
Cortisol, Erythromycin, Cyclophosphamide, S- and R-
Warfarin, Phenytoin, Testosterone, Halothane, Zidovudine
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002
Red indicates enzymes important in drug metabolism
Factors Influencing Activity and Level of CYP Enzymes
Nutrition
1A1;1A2; 1B1, 2A6, 2B6,
2C8,9,19; 2D6, 3A4,5
Smoking 1A1;1A2, 2E1
Alcohol 2E1
Drugs
1A1,1A2; 2A6; 2B6; 2C;
2D6; 3A3, 3A4,5
Environment
1A1,1A2; 2A6; 1B; 2E1;
3A3, 3A4,5
Genetic
Polymorphism
1A; 2A6; 2C9,19; 2D6;
2E1

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
5mg tablet
with juice
without
Review- D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110

N
CO
2
CH
3
CH
3
O
2
C
CH
3
CH
3
H
H
Cl
Cl
3A4
N
CO
2
CH
3
CH
3
O
2
C
CH
3
CH
3
Cl
Cl
Effect of Grapefruit Juice on
Felodipine Plasma Concentration
Grapefruit Juice Facts
• GJ or G, lime, or Sun Drop Citrus soda, Seville
OJ(not most OJ) elevates plasma peak drug
concentration, not elimination t
1/2

• 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 C
max
) and highly
variable among individuals depending upon 3A4
small bowel basal levels
Human Drug Metabolizing CYPs Located
in Extrahepatic Tissues
CYP
Enzyme
Tissue
1A1 Lung, kidney, GI tract, skin, placenta, others
1B1 Skin, kidney, prostate, mammary,others
2A6 Lung, nasal membrane, others
2B6 GI tract, lung
2C GI tract (small intestine mucosa) larynx, lung
2D6 GI tract


S. Rendic & F.J . DiCarlo, Drug Metab Rev 29:413-80, 1997
Human Drug Metabolizing CYPs Located
in Extrahepatic Tissues (cont’d)
CYP
Enzyme
Tissue
2E1 Lung, placenta, others
2F1 Lung, placenta
2J2 Heart
3A
GI tract, lung, placenta, fetus, uterus,
kidney
4B1 Lung, placenta
4A11 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, S-
Methylation, 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, 6-
mercaptopurine. Allopurinol is substrate and inhibitor of
xanthine oxidase; delays metabolism of other substrates;
effective for treatment of gout.
• 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
Non-CYP drug oxidations (2)
Conjugation Reactions
Glucuronidation

O
OH
OH
O OH
CO
2
H
P O P O
O
HO
OH
O
CH
2
O
N
NH
O
O
O
OH
OH
OH
CO
2
H
O R
+
ROH
or
R
3
N
UGT
UDP- -D-glucuronic acid
O
OH
OH
OH
CO
2
H
N
+
R
R
R
O-glucuronide
N
+
-glucuronide
Liver has several soluble UDP-Gluc-transferases
Glucuronic acid conjugation to
phenols, 3°-amines, aromatic amines

Morphine
O
HO
HO
N CH
3
6
3
Amitriptyline
N
N
N
CH
3
O
Cotinine
Conjugation Reactions
Sulfation
Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin
(PAPS, 3’-phosphoadenosine-
5’-phosphosulfate)

R OH
R O S OH
O
O

H H
NH
2
N
N
N
N
OH
O
H
H
HO
O
P
OH
O
O
S
OH
O
O
+
Sulfation may produce active metabolite

N
N
NH
2
O
H
2
N N
N
N
NH
O
H
2
N N
S
O
HO
O
Minoxidil Minoxidil-sulfate
Conjugation Reactions
Acetylation
Examples: Procainamide, isoniazid, sulfanilimide, histamine

NAT enzyme is found in many tissues, including liver

Ar NH
2
R SH
R OH
R NH
2
+
Ar N
CH
3
O
H
Acetyl transferase
CoA S
O
R N
O
CH
3
H
R O
O
CH
3
R S
O
CH
3
Procainamide
Unchanged
in Urine, 59%
3%
24% Fast
17% Slow
Unchanged
in Urine, 85%
NAPA
0.3%
1%

H
2
N
O
N
H
N

N
O
N
H
N
O
H

H
2
N
O
N
H
N
H

N
O
N
H
N
O
H H
Procainamide
trace metabolite
non-enzymatic
Lupus?

H
2
N
O
N
H
N

N
O
N
H
N
HO
H

N
O
N
H
N O
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
xenobiotics
PXR
RXR
CAR
cytoplasm
nucleus
xenoprotection
target genes
S.A. Kliewer
co-activator
PBP
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
Reporter activity (fold)
troglitazone
1 3 5 7 9 11 13 15 17 19
tamoxifen
Cell-based
reporter assay
S.A. Kliewer
Pregnane X Receptor (PXR)
human PXR
Ligand DNA
mouse PXR
77% 96%
rat PXR
76% 96%
82% 94%
rabbit PXR
• 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 16-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)
• 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-(3,5-
dichloropyridyloxy)]benzene)
CAR
Ligand Ligand
DNA DNA
PXR
66% 66% 41% 41%
CAR
PXR
CAR
PXR
S.A. Kliewer
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
Acetanilide, 1886
(accidental discovery of
antipyretic activity; high toxicity)
Phenacetin or
acetophenetidin, 1887
(nephrotoxic,
methemoglobinemia)
Acetaminophen, 1893
Metabolic pathway quantified;
(Brodie &Axelrod, 1948)
popular in US since 1955
70-90%
75-80%

HN
COCH
3
OH

HN
COCH
3
OC
2
H
5

NH
2
OC
2
H
5

HN
COCH
3

NH
2
•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 319:1557-
1562, 1988]
•Management of acetaminophen overdose [Trends
Pharm Sci 24:154-157, 2003
Acetaminophen Toxicity
Acetominophen Metabolism
~60%
~35%
CYP2E1*
CYP1A2
CYP3A11
NAPQI
N-acetyl-p-benzoquinone imine
*induced by ethanol, isoniazid
Protein adducts,
Oxidative stress
Toxicity

HN
COCH
3
OH

HN
COCH
3
O
SO
3
H

HN
COCH
3
O
O CO
2
H
OH
OH
HO

N
O
COCH
3
Acetaminophen Protein Adducts
CYPs
HS-Protein
H
2
N-
Protein
S.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995)
K.D. Welch et al., Chem Res Toxicol 18:924-33 (2005)

HN
COCH
3
OH

N
O
COCH
3

HN
COCH
3
OH
S Protein

HN
COCH
3
OH
NH Protein

O
COCH
3
N
S Protein
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