Drug Metabolism
S.P. Markey
Laboratory of Neurotoxicology
NIMH, NIH
Nov. 14, 2002
Evolution of Drug Metabolism As a
Science
Post WWII Pioneers
• R.T. 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
• B.B. Brodie, U.S.
– NYU and Laboratory of Industrial Hygiene, NYC 1949 –
Metabolic fate of acetanilide and phenacetin in man
(with J. 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
(Dick Adamson, Jim Gillette, and Sidney Udenfriend)
– 1950s, Brodie lab developed the
spectrophotofluorimeter (R. 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
Drug
NADP+
CYP
eR-Ase
PC
CYP Fe+3
Drug
Drug OH
NADPH
CO
CYP-Fe+2
Drug
CO
hu
CYP Fe+3
Drug OH
CYP Fe+2
Drug
eO2
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
• Twelve 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
CYP Tables
• Human CYPs - variability and importance in drug
metabolism
• Isoforms in metabolism of clinically important drugs
• Factors that influence CYP activity
• Drugs that inhibit CYPs
• Non-Nitrogenous CYP inhibitors
• Extrahepatic CYPs
Human Liver Drug CYPs
CYP
enzyme
1A2
1B1
2A6
2B6
2C
2D6
2E1
2F1
2J2
3A4
4A, 4B
Level
(%total)
~ 13
<1
~4
<1
~18
Up to 2.5
Up to 7
Extent of
variability
~40-fold
Up to 28
~20-fold
~30 - 100-fold
~50-fold
25-100-fold
>1000-fold
~20-fold
2E
S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997
Factors Influencing Activity and Level of CYP Enzymes
Nutrition
1A1;1A2;2E1; 3A3; 3A4,5
Smoking
1A1;1A2
Alcohol
2E1
1A1,1A2; 2A6; 2B6; 2C;
Drugs
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
S. Rendic & F. J. Di Carlo Drug Metab Rev 29: 413-580, 1997
Participation of the CYP Enzymes in Metabolism of
Some Clinically Important Drugs
CYP
Enzyme
1B1
2F1
4A
1A1
2A6
Participation in
Drug metabolism
(%)
~1.3
2.5
2.5
2B6
3.4
2E1
4.1
1A2
8.2
Examples of Substrates
17-Estradiol
Ipomeanol
Prostaglandins
R-Warfarin
Cyclophosphamide, Halothane
Zidovudine, AZT
Cyclophosphamide,
Testosterone
Acetaminophen,
Chlorzoxazone
Dapsone. Halothane
Acetaminophen, Caffeine
Phenacetin, (R) –Warfarin
S. Rendic & F.J. Di Carlo, Drug Metab Rev 29:413-580, 1997
Participation of the CYP Enzymes in Metabolism of
Some Clinically Important Drugs (cont’d)
CYP
Enzyme
Participation in
Drug
Metabolism(%)
2C8,9
15.8
2C18, 19
8.3
2D6
18.8
3A4,5
34.1
Examples of Substrates
Tolbutamide, Diclofenac
(S) –Warfarin, Phenytoin
Hexobarbital
Diazepam, Omeprazole
(S) –Mephenytoin
Codeine, Debrisoquine
Dextromethorphan
“Ecstasy”, Bufuralol, Sparteine
Carbamazepine, Cortisol
Dapsone, Diazepam
Erythromycin, Midazolam
Nifedipine, Omeprazole
Testosterone
S. Rendic & F.J. Di Carlo, Drug Metab Rev 29:413-580, 1997
Drugs that Inhibit Drug Metabolism by Forming
Complexes with CYPs
Amphetamine
Cimetidine
Dapsone
2,5-Dimethoxy-4methylamphetamine
Diphenylhydramine
Erythromycin
Fenfluramine
Itraconazole
Ketoconazole
Methadone
Methamphetamine
Nortriptyline
SKF 525A
Sulfanilamide
Modified from: A. Alvares and W.B. Pratt, Pathways of Drug
Metabolism in Principles of Drug Metabolism (Eds. W.B. Pratt,
P.Taylor) 3rd Edition, 1990
Non-nitrogenous Substances that Effect Drug
Metabolism by Forming Complexes with CYPs
• Grapefruit juice - CYP 3A4 inhibitor; highly
variable effects; unknown constituents
– D.G. Bailey, et al.; Br J Clin Pharmacol 1998,
46:101-110
• Isosafrole, safrole - CYP1A1, CYP1A2
inhibitor; found in root beer, perfume
• Piperonyl butoxide & alcohol -CYP1A1,
CYP1A2 inducer; insecticide constituent
Overheard Conversation
• At a B&B breakfast table, 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?”
Effect of Grapefruit Juice on Felodipine Plasma Concentration
5mg tablet
with juice
without
Cl
H
CH 3 O 2 C
CH 3
N
H
Cl
CO 2 CH 3
CH 3
Cl
3A4
Cl
CO 2 CH 3
CH 3 O 2 C
CH 3
N
CH 3
Review- D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110
Grapefruit Juice Facts
• GJ or G (not 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 effected (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
CYP
Enzyme
1A1
1B1
2A6
2B6
2C
2D6
Tissue
Lung, kidney, GI tract, skin, placenta, others
Skin, kidney, prostate, mammary,others
Lung, nasal membrane, others
GI tract, lung
GI tract (small intestine mucosa) larynx, lung
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
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
Aliphatic hydroxylation
R CH2CH3
OH
R CHCH3
Examples: ibuprofen, pentobarbital
CO 2H
CO 2H
HO
ibuprofen
O
O
HN
HN
O
N
O
O
N
H
H
pentobarbital
O
OH
Aromatic Hydroxylation
R
nonenzymatic
R
or
OH
OH
R
O
R
O DNA, Pr ote in
toxic
reactions
OH
R
unstable
arene epoxide
intermediate
OH
HYL1
epoxide
hydrolase
R
OH
Examples: acetanilide, phenytoin, propranolol
Endogenous substrates: steroid hormones (not aromatic amino acids)
phenytoin
N
N
N
N
HYL1
N
CYP2C8,9
N
O
O
HO
OH
3,4-dihydrodihydroxyphentoin
H
O
O
H
phenytoin
N
N
N
N
O
HO
para-hydroxyphenytoin
O
OH
meta-hydroxyphenytoin
Arene epoxide intermediate produces multiple products
propranolol
H
H
N
O
N
O
OH
OH
OH
H
N
O
OH
OH
N (or O, S)-Dealkylation
R N
CH3
-1e -
+
R N
CH2
CH2
R
R N
CH2
CH2
CH2
R
O2
CH3
-H+
CH2
CH2
R
R N
OH
CH2
CH2
CH2
R
R N
H
CH2
CH2
R
+ HCHO
N-demethylation generates formaldehyde
ethylmorphine
N
CH3
N
H
+
O
O
OH
ethylmorphine
O
O
HCHO
OH
desmethyl-ethylmorphine
N-demethylation favored over O-deakylation
propranolol
H
H
N
O
N
O
OH
OH
OH
H
N
O
H
N
O
OH
H
OH
OH
6-methylthiopurine
S
CH3
N
N
N
N
SH
N
N
N
N
+
HCHO
N-Oxidation
R NHOH
R NH2
R N+ R
_
R
O
R N R
R
Examples: chlorpheniramine, trimethylamine
S-Oxidation
R
R
S
R2
Examples: chlorpromazine, cimetidine
R2
S O
chlorpheniramine
Cl
Cl
N
N
O
N
N
chlorpromazine
O
S
S
N
Cl
N
N
Cl
N
Deamination
R CHCH 3
NH2
OH
R C CH3
NH2
O
R C CH 3
Examples: amphetamine, diazepam
+
NH 3
amphetamine
NH2
O
+
NH3
Dehalogenation
R1 R2 R3 C X
R1 R2 R3
C.
+
Cl
RH
-
R1 R2 R3 CH
+
.
R
Example: carbon tetrachloride, others include. halothane, methoxyflurane
CCl 4
CHCl 3
+
.
R
(lipid peroxidation)
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
• 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 effect 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
• 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 (D.
Ziegler, 1993, Ann Rev Pharmacol Toxicol 33:179-199)
– Complete structures defined (Review: J. Cashman, 1995,
Chem Res Toxicol 8:165-181)
– 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
FMO Oxidations
H
H
N
CH 3
N
HN
N
H
H
N
N
N C N
cimetidine
+
N
CH 3
N
O
-
nicotine-N-oxide
nicotine
S
FMO3
FMO3
S
HN
N
O
H
H
N
N
N C N
cimeditine S-oxide
Hydrolysis Reactions
Esters
O
O
R1
O
R2
R1
OH
+
R2 OH
Example: aspirin (others include procaine, clofibrate)
CO 2 H
OCOCH 3
CO 2 H
OH
Hydrolysis Reactions
Amides
O
R1
O
NH
R2
+
R1
R2 NH2
OH
Example:lidocaine; others include peptide drugs
O
N
OH
N
H
N
O
+
NH2
Conjugation Reactions
Glucuronidation
CO2H
O
OH
HO O
OH
O P O P O CH2
OH O OH
ON
O
NH
O
UDP- -D-glucuronic acid
+
ROH
or
R 3N
UGT
CO2H
OO R
OH
OH
OH
O-glucuronide
CO2H R
+R
ONR
OH
OH
OH
N+-glucuronide
Liver has several soluble UDP-Gluc-transferases
HO 3
N
O
N CH3
6
O
CH3
N
N
HO
Morphine
Amitriptyline
Cotinine
Glucuronic acid conjugation to
phenols, 3°-amines, aromatic amines
Conjugation Reactions
Sulfation
O
R O S OH
O
R OH
NH2
+
N
N
N
N
H
H
HO
O H
OH O
OH
O P O S
O
O
(PAPS, 3’-phosphoadenosine5’-phosphosulfate)
H
OH
Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin
H2N
N
N
H2N
O
N
O
N
N
N
HO S O
NH 2
Minoxidil
O
NH
Minoxidil-sulfate
Sulfation may produce active metabolite
Conjugation Reactions
Acetylation
O
Ar NH 2
O
CoA S
R NH 2
R OH
R
SH
Ar N
H
+
Acetyl transferase
O
R O
CH3
CH3
O
O
R N
H
CH3
R
S
CH3
Examples: Procainamide, isoniazid, sulfanilimide, histamine
NAT enzyme is found in many tissues, including liver
Procainamide
O
Unchanged
in Urine, 59%
H2 N
N
H
24% Fast
17% Slow
H
N
Unchanged
in Urine, 85%
N
3%
O
N
H
O
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
N
H
trace metabolite
HO
H
N
O
N
N
H
non-enzymatic
O
O N
N
H
N
Lupus?
Drug Conjugation Example:
Isoniazid - N-acetyltransferase
• First line drug in the treatment of TB; normally given
at a does of 5 mg/kg, max. 300 mg/day for period of 9
months
• Rapid and slow acetylators first seen in TB patients;
t1/2 for fast is 70 min; t1/2 for slow is 180 min
• N-acetyltransferase (NAT2 isoform) is in liver, gut
• Peripheral neuropathy (about 2% patients; higher
doses produce effects in 10-20%) seen in slow
acetylators (reversible with pyridoxine)
• Hepatotoxicity also seen, esp. in older patients
N
N
NAT2
O
H
H
N N H
H
O
Isoniazid
CH 3
N
N
H
O
N-Acetylisoniazid
N
minor
O
OH
CH3
CYP1A2
OH
NH
NH 2
N+
NAT2
C O
O
NH
Carcinogenic DNA Adduct
Reactive
Nitrenium ion
N-Acetylation may trigger nitrenium ion formation
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
target genes
CAR
xenobiotics
RXR
xenoprotection
PXR
cytoplasm
nucleus
S.A. Kliewer
CYP3A Inducers Activate
Human, Rabbit, and Rat PXR
rifampicin
PCN
Cell-based
reporter assay
dexamethasone
RU486
clotrimazole
troglitazone
tamoxifen
1
3
5
7
9
11
13
15
17
19
Reporter activity (fold)
S.A. Kliewer
CYP3A Regulation
• Expressed in liver and intestine
• Activated by xenobiotics
• Bind to Xenobiotic Response Elements
xenobiotics
rifampicin
PCN
dexamethasone
RU486
clotrimazole
troglitazone
tamoxifen
?
XRE
xenobiotics
(e.g., drugs)
endobiotics
(e.g., steroids)
CYP3A
liver
intestine
CYP3A
HO-xenobiotics HO-endobiotics
• Protect against xenobiotics
• Cause drug-drug interactions
S.A. Kliewer et al.
Endo Rev 23:687, 2002
Pregnane X Receptor (PXR)
human PXR
DNA
Ligand
rabbit PXR
94%
82%
mouse PXR
96%
77%
rat PXR
96%
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 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)
•
•
•
•
•
CAR
CAR
CAR
DNA
Ligand
PXR
PXR
PXR
66%
41%
Highly expressed in liver and intestine
Binds response elements as RXR heterodimer
High basal transcriptional activity without ligand
Sequestered in cytoplasm
Activated by xenobiotics
– phenobarbital, TCPOBOP (1,4-bis[2-(3,5dichloropyridyloxy)]benzene)
S.A. Kliewer
Plasticity in the PXR Binding Pocket
Volume:
SR12813
hyperforin
1280 Å3
1544 Å3
S.A. Kliewer
PXR Structure
• Large, elliptical hydrophobic cavity
• The cavity changes shape to accommodate different
ligands
• PXR is ideally suited to function as xeno-sensor
xenobiotics
PXR RXR
xenobiotic
metabolism
S.A. Kliewer
PXR and CAR Regulate Overlapping Genes
PCN (PXR)
TCPOBOP (CAR)
• Phase I enzymes
Cyp3a11
Cyp2b10
Aldh1a1
Aldh1a7
(3.5x)
(12x)
(2.1x)
(1.6x)
(3.4x)
(110x)
(1.9x)
(1.9x)
(2.8x)
(16x)
(15x)
• Phase II enzymes
Liver RNA
Ugt1a1
Gst-a1
• Transporters
Mrp2
Mrp3
Oatp2
(3.0x)
(9.2x)
(2.0x)
(1.9x)
S.A. Kliewer
Acetaminophen
• 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; 1946, Lester
reported conjugated para-aminophenol as major
metabolite of acetanilide
• 1948-49 Brodie and Axelrod recognized
acetaminophen as the major active metabolite in
phenacetin
• CAR modulates acetaminophen toxicity [Science
(Oct 11) 298:422, 2002]
Acetaminophen and p-Aminophenols
HN
COCH 3
HN
COCH 3
NH2
Acetanilide, 1886
(accidental discovery of
antipyretic activity; high toxicity)
75-80%
70-90%
NH2
OC 2 H5
HN
OC 2 H5
Phenacetin or
acetophenetidin, 1887
(nephrotoxic,
methemoglobinemia)
COCH 3
Recognized as active metabolite
of acetanilide and phenacetin
in 1948 (Brodie &Axelrod);
popular in US since 1955
OH
Acetaminophen, 1893
Acetominophen Metabolism
HN
COCH 3
~60%
HN
O
COCH 3
O
OH
CO 2 H
OH
HO
OH
~35%
CYP2E1*
CYP1A2
CYP3A4
N
COCH 3
HN
O
COCH 3
SO 3 H
*induced by ethanol, isoniazid
Protein adducts,
O
NAPQI
Oxidative stress
N-acetyl-p-benzoquinone imine
Toxicity
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]
•Addition of N-acetyl cysteine to acetaminophen tablets
proposed to prevent liver toxicity. [British Medical
Journal, Vol. 323, Sept. 15, 2001]
Acetaminophen Protein Adducts
HN
COCH 3
N
COCH 3
CYP2E
HS-Protein
O
OH
H2NProtein
Protein S N
COCH 3
HN
COCH 3
HN
COCH 3
S Protein
O
OH
NH Protein
OH
S.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995)
J.L. Holtzman, Drug Metab. Rev. 27: 277-297 (1995)
NAPQI toxicity linked to CAR activation,
GSH depletion
N
COCH 3
SH
HN
COCH 3
glu-cys-gly
GLY
S CYS
Glutathione S-Transferase (GST Pi)
O
OH
GLU
SH
glu-cys-gly
Phenobarb
TCPOBOP
CAR
androstanol
GST Pi
toxicity
oxidative stress
mechanism ?
Protective effect. Liver cells die (pale areas) when exposed to high doses of acetaminophen (left),
but a CAR inhibitor protects against such damage (right).
Jun Zhang,* Wendong Huang,* Steven S. Chua, Ping Wei, David D. Moore
Science, October 11, 298:422, 2002
Acetaminophen toxicity mechanism
• Mice nulled for glutathione S-transferase are resistant to
acetaminophen toxicity
– equal amounts of acetaminophen protein adducts formed in null and wild
type suggesting protein adducts may not be toxic
– hepatic GSH lowered in wild type (but not in KO) after acetaminophen
• CAR nulled mice are also resistant to acetaminophen toxicity
– hepatic GSH lowered in wild type (but not in KO) after acetaminophen
– CAR-humanized mice demonstrate same toxicity response
• N-acetyl cysteine is an effective agent to block GSH depletion
and rescue from liver damaging toxicity
• NAPQI-protein adduction or NAPQI-GSH depletion-oxidative
stress....to be continued
Terfenadine (Seldane©)
OH
HO
N
Terfenadine in the News
• DHHS/FDA: Terfenadine; Proposal to Withdraw
Approval of Two New Drug Applications
– Federal Register 62, January 14, 1997
• Hoechst Marion Roussel To Promote Switch From
Seldane to Allegra
– Independent News Service, January 14, 1997
• Citing Its Side Effects, F.D.A. Weighs Ban on
Allergy Drug
– The New York Times, January 14, 1997
• FDA Wants Drug Seldane Off Market
– The Washington Post, January 14, 1997
• Hoechst’s First Quarter Results Below Forecasts
– Independent News Service, May 7, 1997
Terfenadine
• Developed in 1980s as a 2nd generation H1antihistamine; from introduction in 1985, prescriptions
> 16 million in 1991
• First generation antihistamines are lipophilic ethylamine
derivatives that readily penetrate the CNS and placenta
- objective of 2nd generation is minimal CNS effects
(non-sedating), not crossing the blood brain barrier;
longer acting
• Cardiac side-effects are serious - inhibition of
potassium channels by unmetabolized parent drug
causes prolongation of QT interval leading to life
threatening arrythmia (torsades de pointes); first
recognized at USUHS in 1989 (Monahan BP et al, JAMA
1990; 264:2788-2790.)
• Drugs or substances inhibiting terfenadine metabolism
(grapefruit juice, ketoconazole, itraconazole,
antimicrobials) or liver dysfunction exacerbate the side
effects
Terfenadine Metabolism
OH
HO
N
Terfenadine
(Seldane)
CYP3A4
OH
HO
N
Fexofenadine
(Allegra)
CO 2 H
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.panvera.com/tech/dmeguide/index.html
– Drug Metabolism Resource Guide - catalog with useful
information and characteristics of natural and recombinant
drug metabolizing enzymes; assay methods
•http://www.netsci.org/Science/Special/feature06.html
– Site contains essay “The emerging role of ADME in optimizing
drug discovery and design” RJ Guttendorf, Parke-Davis
•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.