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BIOTRANSFORMATION OF DRUGS
Contents
PART A: Biotransformation reactions
 Introduction
 Phase I reactions
 Phase II reacions
 The effect of biotransformation on the biological
activity of drugs: How the biological (pharmacological and
toxic) activity of the metabolite compares to that of the parent
compound?
PART B: Alterations in biotransformation
 Biological alterations
- Decreased biotransformation capacity in neonates
- Genetic alterations in biotransformation
Mutation or multiplication of genes coding for drug metabolizing
enzymes – possible consequences
 Chemical alterations
- Induction of CYP – possible consequences
- Inhibition of CYP – possible consequences
INTRODUCTION
ELIMINATION MECHANISMS
Physical mechanism:
Chemical mechanism:
EXCRETION
BIOTRANSFORMATION
CONTRIBUTION OF EXCRETION AND BIOTRANSFORMATION
TO ELIMINATION OF DRUGS - examples
Drugs NOT biotransformed,
excreted unchanged
Drugs excreted both
unchanged and as metabolites
Drugs fully biotransformed,
excreted only as metabolites
Benzylpenicilin
Aminoglycosides
Metformin
Tubocurarine
Amantadine
Salicylates
Acetaminophen
Phenobarbital
TCADs
Phenothiazines
Chloramphenicol
The sites of biotransformation:
 Predominantly, the liver
The liver contributes to both
the presystemic and the systemic elimination of many drugs.
 Often other tissues, as well.
e.g., in intestinal mucosa cells  presystemic elimination of several drugs
in renal tubular cells, etc
 The colon, by bacteria - e.g., azo reduction, hydrolytic reactions
BIOTRANSFORMATION: classification
Phase II reactions
(conjugations)
Phase I reactions
1. Glucuronidation
2. Sulfation
3. Conjugation with glycine (Gly)
4. Conjugation with glutathione (GSH)
1. Oxidation
2. Reduction

5. Acetylation
6. Methylation
3. Hydrolysis
The chemical role of Phase I and Phase II biotransformations:
A functional group is added to the molecule
or explored in the molecule
at which conjugation can take place
An organic acid (or acetyl or methyl group)
is conjugated to the molecule
at a preexisting functional group or
at a functional group acquired
in Phase I biotransformation
General examples
RH
R-HC=O
R-O-glucuronic acid
oxidation
O
reduction
2H
R1-O-(C=O)-R2
R-OH
R-O-sulfonic acid
R-CH2-O-glucuronic acid
R-CH2-OH
hydrolysis
HOH
R-CH2-O-sulfonic acid
R-O-glucuronic acid
R1-OH
R-O-sulfonic acid
The pharmacological role of Phase I and Phase II biotransformations:
How the fate and effect of metabolites
compare to the fate and effect of the parent drug?
Phase I metabolites
Phase II metabolites (conjugates)
Water-solubility:  (slightly faster excretion)
Water-solubility: for 1-4:  (rapid excr.)
for 5-6:  (slower excretion)
Biological activity: in general 
but often 
Biological activity: almost always 
very seldom

Part A. BIOTRANSFORMATION REACTIONS
1. PHASE I BIOTRANSFORMATIONS
1.1. OXIDATION
1.1.1. Microsomal oxidation
1.1.1.1. CYP-catalyzed reactions
1.1.1.1.1. Oxigenation
1.1.1.1.1.1. Insertion of oxygen produces a stable oxigenated metabolite
1.1.1.1.1.1.1. C-hydroxylation: aliphatic hydroxylation, aromatic hydroxylation
1.1.1.1.1.1.2. N-hydroxylation
1.1.1.1.1.1.3. Epoxidation
1.1.1.1.1.2. Insertion of oxygen produces an unstable oxigenated metabolite
which undergoes spontaneous cleavage into two molecules
1.1.1.1.1.2.1. Oxidative dealkylation (N- and O-dealkylation)
1.1.1.1.1.2.2. Oxidative deamination
1.1.1.1.1.2.3. Oxidative dehalogenation
1.1.1.1.1.2.4. Oxidative desulfuration
1.1.1.1.2. Dehydrogenation
1.1.1.1.3. Reduction (reductive dehalogenation)
1.1.1.2. FMO-catalyzed oxidations
1.1.2. Non-microsomal oxidation
1.1.2.1. MAO-catalyzed oxidations
1.1.2.2. Oxidations catalyzed by molydenum-containing oxidases
1.1.2.2.1. Xanthine oxidase-catalyzed oxidations
1.1.2.2.2. Aldehyde oxidase-catalyzed oxidations
1.1.2.3. Alcohol dehydrogenase and aldehyde dehydrogenase-catalyzed oxidations
1.2. REDUCTION
1.2.1. Azo-reduction
1.2.2. Nitro-reduction
1.2.3. Carbonyl-, aldehyde- and aldose-reduction (by aldo-keto reductases; AKR)
1.3. HYDROLYSIS
1.3.1. Hydrolysis by carboxylesterases
1.3.2. Hydrolysis by alkaline phosphatase
1.3.3. Hydrolysis by paraoxonases
1.3.4. Hydrolysis by epoxide hydrolase (illustrated under epoxidation)
1.3.5. Hydrolysis by microbial hydrolases in the colon
2. PHASE II BIOTRANSFORMATIONS (CONJUGATIONS)
2.1. GLUCURONIDATION
2.2. SULFATION
2.3. CONJUGATION WITH GLYCINE
2.4. CONJUGATION WITH GLUTATHIONE (Glu-Cys-Gly)
2.5. ACETYLATION
2.6. METHYLATION
PHASE I BIOTRANSFORMATIONS
OXIDATION - summary
MICROSOMAL
NON-MICROSOMAL
catalyzed by:
catalyzed by:
 Cytochrome P-450 (CYP)
 Monoamino-oxidases (MAO) - mitochondrial
 Mo-containing oxidases (cytosolic):
 Microsomal flavine-containing
monooxigenase (FMO)
Xanthine oxidase (XO) and Aldehyde oxidase (AOX)
 Alcohol- and aldehyde dehydrogenases
(ADH, ALDH) - cytosolic
OXIDATIONS CATALYZED BY CYTOCHROME P-450 (CYP)
CYP is a heme-containing protein embedded in the membranes of the smooth
endoplasmic reticulum (SER), the fragments of which in a tissue homogenate are
sedimented after ultracentrifugation (at 100,000g) in the microsomal fraction.
Origin of the name cytochrome P-450:
 Cytochrome: it is a coloured intracellular protein
 P: it is pink
 450: its absorption spectrum has a maximum at 450 nm
CYP constitutes a superfamily of enzymes that are classified into families
(numbered) and subfamilies (marked with capital letters), the latter of which contain
the individual enzymes (numbered), e.g., CYP1A2, CYP2C9, CYP2D6, CYP3A4.
CYTOCHROME P-450 (CYP)-CATALYZED REACTIONS  Mechanisms
CYP is an extremely versatile enzyme as it can catalyze numerous types of reaction, out of which
three types are presented below.
Oxygenation involves insertion of an O atom (from O2) into a C-H bond, forming a hydroxylated
metabolite, or into a C=C double bond, forming an epoxide. A hydroxylated metabolite may be stable,
or unstable. From an unstable hydroxylated metabolite a group may break off spontaneously: an alkyl
group, ammonia, a halogen atom, or sulfur atom; such reactions are called oxidative dealkylation,
oxidative deamination, oxidative dehalogenation, and oxidative desulfuration, respectively.
CYP can also catalyze dehydrogenation, i.e. removal of 2 H atoms from a drug molecule (this is
how the reactive hepatotoxic paracetamol metabolite is formed).
Surprisingly, CYP may also catalyze reduction, by transferring only 1 electron to a compound (e.g. in
a reductive dehalogenation reaction) or as many as 6 electrons to a nitro group, thus converting it
into an amino group (nitro reduction). As catalyzed by CYP, these reductions are also shown here.
I. OXYGENATION - the most typical CYP-catalyzed reaction
General scheme: RH + O2 + (NADPH + H)+
CYP
NADPH-CYP reductase
R-OH + HOH + NADP+
Note: 1 atom of O2 is inserted into the drug, whereas the 2nd O atom is reduced to HOH by NADPH-CYP red.
Catalytic cycle (simplified):
1
Product (ROH)
Fe
3+
Fe
ROH
7
6
(FeO)3+
RH
HOH
H
+
2+
Fe OOH
RH
Substrate (RH)
3+
In the course of oxygenation
by CYP, O2 is activated by electron
uptake to form reactive O atoms.
The first reactive form is
the hydroperoxy form
(produced in step 5).
From this, 1 O is inserted into a
water molecule (HOH).
The second reactive form
is the oxene (produced in step 6).
From this, the reactive O atom
is inserted into a C-H bond
of the drug, forming a hydroxylated
metabolite in step 7, which is then
released from CYP.
CYP-catalyzed
dehydrogenation and
reduction are explained below.
5
H
Note that Fe represents
the iron in CYP.
e
+
_
3+
Fe
RH
2
e
_
2+
Fe
RH
3
O2
3+
_
Fe O2
RH
4
NADPH-cytochrome
P450 reductase
II. DEHYDROGENATION e.g. acetaminophen (=paracetamol), nifedipine, ethanol
6
(FeO)3+
RH2
Fe
R
NOTE: The 2nd O atom is also
reduced to water, using 2 H atoms
from the drug molecule.
3+
7
HOH
III. REDUCTION e.g. nitro reduction of clonazepam; reductive dehalogenation of CCl4
2+
4
Fe O2
RH
2+
e
_
Fe O
_2
[RH]
5
NOTE: The second electron is not
transferred to the CYP-bound
oxygen, but to the drug molecule.
CYP-CATALYZED REACTIONS  Overview
OXYGENATION (insertion of one O atom) - two variations:
1. INSERTION OF O PRODUCES A STABLE METABOLITE:
a. Hydroxylation
 C-hydroxylation (insertion of O into a C-H bond to form a hydroxyl group):
- Aliphatic hydroxylation:
- Aromatic hydroxylation:
tolbutamide (-), terfenadine (+), cyclophosphamide (+)
warfarin (-), phenytoin (-), propranolol (-)
 N-hydroxylation (insertion of O into an N-H bond): dapsone (†)
b. Epoxidation (insertion of O into a C=C bond to form an epoxide): carbamazepine (-/o)
2. INSERTION OF O PRODUCES AN UNSTABLE METABOLITE
WHICH SPONTANEOUSLY BRAKES INTO TWO MOLECULES:
a. Oxidative dealkylation ( dealkylated metabolite + an aldehyde):
 N-dealkylation:
Diazepam  nordiazepam (+),
Carisoprodol  meprobamate (+),
Amitriptyline  nortriptyline (+)  desmethyl-nortriptyline (-)
Lidocaine  monoethylglycylxylidine (+)  glycylxylidine (-)
Caffeine  paraxanthine (-), or theophylline, or theobromine
 O-dealkylation:
Codeine  morphine (+)
Dextromethorphan  dextrorphan (=D form of levorphanol) (+)
Phenacetin  acetaminophen (=paracetamol) (+)
b. Oxidative deamination ( O-containing metabolite + NH3):
Amphetamine  phenylacetone (-)
c. Oxidative dehalogenation ( O-containing metabolite + HBr):
Halothane  trifluoroacetyl chloride (†)
d. Oxidative desulfuration ( O-containing metabolite + S):
Thiopenthal  pentobarbital (+),
Parathion  paraoxon (+); thio-TEPA  TEPA (+)
DEHYDROGENATION (removal of 2 H atoms):
 Acetaminophen  N-acetylbenzoquinone imine (†)
 Nifedipine (a dihydropyridine parent compund)  ”pyridine” metabolite (-)
REDUCTION (reductive dehalogenation, nitro reduction):
 CCl4  CCl3 (†) + Cl¯; Halothane  debrominated halothane free radical (†) + Br¯
 For reduction of nitro group of clonazepam (or nitrazepam) to amino group by CYP,
see under nitro reduction (6-electron reduction)
+ = active metabolite;  = inactive metabolite; † = toxic metabolite
CYP-CATALYZED REACTIONS:
1. NSERTION OF O PRODUCES A STABLE METABOLITE
HYDROXYLATION = insertion of O into a C-H or N-H bond
a) C-HYDROXYLATION: aliphatic
O
O
S NH C NH
O
CH3
CYP2C9
Tolbutamide (antidiabetic)
Hydroxymethyl-tolbutamide (inactive)
CH3
CH3
HOOC
CH2 CH
CH
CH3
Ibuprofen
(NSAID)
CH3
CH3
HOOC
CH
O
O
S NH C NH
O
HO CH2
CH2 OH
CH3
HOOC
CH2 C OH
CH
CH2 CH
CH3
CH3
3-hydroxy-ibuprofen
CONJUGATES
2-hydroxy-ibuprofen
b) C-HYDROXYLATION: aromatic
Phenytoin
(antieileptic, antiarrhythmic)
O
HN
HN
NH
C6H5
4'-Hydroxyphenytoin
(inactive)
O
CYP2C9
NH
C6H5
O
O
OH
O
O
OH
CYP2C9
HO
O
O
H
H
C
C
CH2 C
Warfarin (anticoagulant)
CH3
OH
CH2 C
7-hydroxy-warfarin
(inactive)
O
CH3
O
c) N-HYDROXYLATION
O
H2 N
S
O
Dapsone (antileprotic)
NH2
CYP2C9
O
H2 N
S
O
Dapsone-hydroxylamine
(hemotoxic and allergenic)
NH OH
EPOXIDATION = insertion of O into a C=C double bond
H
O
H
CYP
N
N
C
O
NH2
Carbamazepine-10,11-epoxide
(less active metabolite)
C
O
NH2
Carbamazepine
(antiepileptic)
Epoxide
hydrolase
CYP
O
CYP
(+)
(+)
(+)
O
(+)
HO
HO
OH
benzo(a)pyrene
benzo(a)pyrene-7,8-oxide benzo(a)pyrene-7,8-dihydro-diol
OH
benzo(a)pyrene7,8-dihydro-diol-9,10-epoxide
"ultimate carcinogen"
NOTE:
Leucotriene A4 (LTA4) is also an epoxide!
LTA4 is converted into either LTB4 (a diol) by epoxide hydrolase, or into LTC4 (a GSH
conjugate) by glutathione S-transferase.
CYP-CATALYZED REACTIONS:
2. INSERTION OF O PRODUCES AN UNSTABLE METABOLITE
WHICH UNDERGOES SPONTANEOUS CLEAVAGE INTO TWO MOLECULES
First, the drug becomes hydroxylated at the C atom of the alkyl group that is linked to the N (or the O)
atom. This hydroxylated metabolite is unstable. It breaks spontaneously into two molecules: the
dealkylated metabolite (e.g., an amine or alcohol/phenol), and an aldehyde (e.g., formaldehyde after
demethylation, acetaldehyde after deethylation, etc.).
N-dealkylation
General scheme:
NOTE: After hydroxylation at the
R NH2 N-bound C atom of the alkyl group,
the alkyl group breaks off (as an aldehyde)
O from the N atom, producing a
dealkylated amine metabolite
H C
H of the drug molecule.
OH
R NH CH2
R NH CH3
Examples:
CH3
H
O
N
O
N
CYP2C19
CYP3A4
Cl
N
HCHO
Diazepam
Cl
N
Other examples:
lidocaine
imipramine
erythromycine
monoethylglycylxylidine
desmethylimipramine
desmethylerythromycine
Nordiazepam
Theophylline may undergo:
- N-demethylation at N1 and N3 by CYP
- C-hydroxylation at C8 by CYP
- N- methylation at N7 by MT
O
N3-demethylation
H3C
N
N
CYP1A2
O
H
N
N
H
1-Methylxanthine
(25 %)
O
H3C
O
CH3
N
N1
7
3
N
N
O
H3C
MT
SAM
O
H
N
N
N
CH3
CH3
Caffeine
Theophylline
N
In neonates more caffeine is
formed from theophylline than
in adults because the CYP
levels in the neonatal liver
are low, however, the
methyltransferase (MT)
levels are relatively high.
O
H3C
Hydroxylation
CYP3A4, CYP2E1
H
O
N
N
OH
O
N
N
CH3
O
N1-demethylation
CYP1A2
O
CH3
N
N
N
CYP1A2
H
N
N
OH
O
N
N
H
1,3-Dimethyluric acid
(50 %)
H
N3-demethylation
H3C
N
CH3
3-Methylxanthine
(15 %)
1-Methyluric acid
(5 %)
O-dealkylation
General scheme:
R O CH3
NOTE: After hydroxylation at the
R OH O-bound C atom of the alkyl group,
the alkyl group breaks off (as an aldehyde)
O from the O atom, producing a
dealkylated hydroxylated metabolite
H C
of the drug molecule.
H
OH
R O CH2
Example:
Other examples:
codeine
phenacetine
HO
CH3 O
CYP2D6
HCHO
N
CH3
Dextromethorphan
(antitussive drug)
N
CH3
Dextrorphan
(active metabolite)
morphine
paracetamol
Oxidative deamination
CYP
CH2 CH CH3
OH
O
CH2 C CH3
CH2 C CH3
NH2
NH2
NH3
Amphetamine
(centrally acting sympathomimetic)
Phenylacetone
(inactive)
Oxidative dehalogenation
F
F
Cl
C
C
F
H
F
Br
C YP2E1
F
Cl
(+)
F
C
C
F
OH
F
Br
C
HBr
Cl
C
O
F
T rifluoro-acetyl-chloride
binds covalently to hepatic proteins,
thus form ing a neoantigene and
causing "halothane hepatitis"
Halothane
(inhalation anesthetic)
Oxidative desulfuration
S
C2H 5 O
P
C 2H 5O
O
NO 2
O
P
CYP
C 2H5 O
P
NO 2
O
C 2H 5O
[S]
Parathion
(an organophosphate insecticide)
S
S
HN
O
H5 C2
NH
N
CYP
O
CH CH3
CH2 CH2 CH3
Thiopental
(an i.v. anesthetic)
H5 C 2
NO 2
O
HN
NH
N
O
Paraoxon
(active insecticide,
an irreversible inhibitor of
acethylcholinesterase)
O
HN
O
O
C 2H 5 O
S
C 2H 5O
O
CH CH 3
CH 2CH 2CH3
[S]
O
NH
N
H5 C 2
O
CH CH3
CH 2 CH 2 CH 3
Pentobarbital
(active as hypnotic)
The arrows point to the bond into which an O atom is inserted, which then promotes
cleavage of the molecule to release NH3, HBr, or sulfur.
CYP-CATALYZED REACTIONS: 3. DEHYDROGENATION
General scheme:
6
(FeO)3+
RH2
Fe
R
3+
7
HOH
Examples:
H
N
O
C
CH3
N
O
C
CH3
CYP1A2
CYP3A4
Acetaminophen
analgetic and
antipyretic
drug
NO2
O
C OCH3
O
H3CO C
CYP2E1
OH
NOTE: While an oxygenation produces 1
molecule hydroxylated metabolite plus 1
molecule HOH, dehydrogenation produces
2 molecules HOH.
plus a dehydrogenated metabolite.
NO2
O
C OCH3
O
H3CO C
CYP3A4
O
H3C
CH3
N
N-Acetyl-p-benzoH
quinoneimine
(NAPBQI)
Nifedipine
hepatotoxic
a dihydropyridine Ca++-channel blocker
H3C
N
CH3
"Pyridine metabolite"
inactive
Other example: Dehydrogenation of nicotine to nicotine 1’(5’) iminium ion (see under aldehyde
oxidase)
CYP-CATALYZED REACTIONS: 4. REDUCTION
In CYP-catalyzed reduction, the second electron is not transferred to the CYP-bound O2, but to the
CYP-bound substrate, e.g. carbon tetrachloride or clonazepam (see also under nitro reduction). With
carbon tetrachloride, its reduced intermedier then undergoes a homolytic cleavage to form a free
radical and a chloride anion. The reactive trichloromethyl free radical formed in the liver, can initiate
lipid peroxidation in the liver cell membranes and can thus induce hepatic necrosis.
General scheme:
2+
4
2+
Fe O2
RH
e
Fe O
_2
[RH]
_
5
Examples:
1. Reductive dehalogenation of carbon tetrachloride (1-electron reduction):
Carbon
tetrachloride Cl
solvent
Cl
C Cl
Cl
CYP2E1
Cl
reduction
_
Cl-
Cl
Cl
C : Cl
Cl
Cl
Cl
C.
Tricloromethyl
free radical
initiates lipid peroxidation
and liver injury
2. Nitro-reduction of clonazepam to 7-amino-clonazepam (6-electron reduction):
H
N
H
N
O
O
CYP3A4
O2N
CYP2C19
N
Clonazepam
anxiolytic and
antiepileptic drug
N
Cl
Cl
2e-
R-NO
HOH
7-aminoclonazepam
inactive
2e-
2e-
R-NO2
2H+
H2N
R-NH2
R-NH-OH
2H+
2H+
HOH
NOTE for those interested: Homolytic cleavage occurs when a covalent bond (formed by two
electrons shown as :) is cleaved in a way so that one electron remains with one of the breakage
products, whereas the other electron will belong to the other breakage product. Thus, homolytic
cleavage yields products with an unpaired electron (called free radicals), which are reactive.
OXIDATIONS CATALYZED BY
MICROSOMAL FLAVIN-CONTAINING MONOOXYGENASE (FMO)
FMO-CATALYZED OXIDATIONS  Overview
Take place on heteroatoms in drugs:
Oxidation of S atom:
Cimetidine → cimetidine-S-oxide
Oxidation of tert. N atom: Nicotine → nicotine-1’-N-oxide
Trimethylamine → trimethylamine N-oxide
(Deficiency: Fish-odor syndrome)
OXIDATIONS CATALYZED BY FMO - Examples:
O
N-CN
CH2 S CH2 CH2 NH C
N
NH CH3
N-CN
CH2 S CH2 CH2 NH C
N
NH CH3
FMO
N
H
CH3
N
H
Cimetidine
(an H2-receptor antagonist)
CH3
Cimetidine S-oxide
(inactive)
FMO
N
N
Nicotine-1'-N-oxide
(inactive)
Nicotine
CH3
CH3 N
CH3
Trimethylamine
endogenous volatile compound;
accumulates in FMO3 deficiency,
causing fish odor syndrome
CH3
N
CH3
N
O
FMO3
CH3
CH3 N
O
CH3
Trimethylamine N-oxide
(water-soluble metabolite
excreted in urine)
OXIDATIONS CATALYZED BY NON-MICROSOMAL ENZYMES
NON-MICROSOMAL OXIDATIONS  Overview
The oxygen incorporated into the substrate is derived from HOH rather than O2.
OXIDATION CATALYZED BY MAO
MAO catalyzes oxidative deamination of amines.
Examples:
- Dopamine → 3,4-dihydroxy-phenylacetaldehyde
- Norepinephrine → 3,4-dihydroxy-mandelic aldehyde (-)
- N-deisopropyl-propranolol → the respective aldehyde (-)
- MPTP → MPP+(†) – kills the DAergic neurons, causing Parkinson’s disease
OXIDATIONS CATALYZED BY
MOLYBDENUM-CONTAINING OXIDASES:
 XANTHINE OXIDASE-CATALYZED OXIDATION:
Examples:
Hypoxanthine → xanthine → uric acid
Allopurinol → alloxanthine (compet. inhibition of uric acid formation)
6-Mercaptopurine → 6-thiouric acid (-)
 ALDEHYDE OXIDASE-CATALYZED OXIDATION:
Examples:
- Nicotine 1’(5’) iminium ion → cotinine
- Benzaldehyde → benzoic acid
- 3-Methoxy 4-hydroxy-mandelic aldehyde → VMA
OXIDATIONS CATALYZED BY
ALCOHOL- AND ALDEHYDE DEHYDROGENASES:
Examples:
- Methanol → formaldehyde → formic acid (†) – causes blindness
- Ethanol → acetaldehyde (†) → acetic acid
- Ethylene glycol →→
glycolic acid (†) → glyoxylic acid → oxalic acid (†) – cause renal injury
- Chloral (= trichloroacetaldehyde) → trichloroethanol (+) – ADH in the
reverse reaction (using NADH) can reduce chloral; ethanol facilitates this by increasing
NADH supply.
MAO-CATALYZED OXIDATIVE DEAMINATION OF AMINES INTO ALDEHYDES
General scheme:
Dopamine
HO
HO
CH2 CH2 NH2
FAD
1
FOUR STEPS - the byproducts are ammonia and HOOH!
1. R-CH2NH2 + FAD > R-CH=NH + FADH2 Dehydrogenation
2. R-CH=NH + H2O > R-CH(OH)-NH2
Hydration
3. R-CH(OH)-NH2
> R-CH=O + NH3
Deamination
4. FADH2 + O2
> FAD + H2O2
FAD
regeneration
HOOH !
4
FADH2
HO
HO
O2
CH2 CH NH
HO
O
HOH
CH2 C
HO
2
HO
NH3
HO
CH2 CH NH2
3
OH
OH
acid (DOPAC)
HO
O
CH2 C
HO
H
Oxidation by ALDH
Reduction by AR
HO
3,4-dihydroxyphenylacetyl aldehyde
(DOPAL)
CH2 CH2OH
HO
alcohol
Example:
Propranolol
OH
N-Desisopropylpropranolol
OH
CH3
O CH2 CH CH2 NH2
O CH2 CH CH2 NH CH
CH3
CYP2D6
N-dealkylation
O2 + H2O
CYP2D6
MAO-A
aromatic hydroxylation
NH3 + H2O2
OH
CH3
O CH2 CH CH2 NH CH
OH
CH3
OH
4-Hydroxypropranolol
O
O CH2 CH C
H
Oxidation
by AOX
Reduction
by AR
Carboxylic acid
Glycol
OXIDATIONS CATALYZED BY Mo-CONTAINING OXIDASES,
XANTHINE OXIDASE (XO) and ALDEHYDE OXIDASE (AOX)
Mechanism of oxygenation by XO and AOX – two steps:
NOTE: The oxygen atom incorporated into the substrate is derived from water rather than O2.
(1) A water molecule is added into the parent compound, forming an intermediary metabolite.
(2) Thereafter, 2 H atoms are removed from the intermediary metabolite by molecular oxygen (O2),
thus forming hydrogen peroxide (HOOH) and the oxygenated metabolite, which carries now a ketogroup or a carboxylic acid group. These 2 steps are shown only in the reactions presented for AOX.
For simplicity, they are not shown in the reactions presented for XO.
Oxygenations catalyzed by XO and/or AOX:
1. Oxygenation of a C atom double-bonded to a N atom in a heterocyclic ring,
such as:  in purines, e.g. hypoxanthine and xanthine (preferred by XO),
 in purine analogues, e.g. 6-mercaptopurine and 6-thioxanthine (preferred by XO),
 in zaleplone (preferred by AOX)
 in the dehydrogenated nicotine metabolite, nicotine-1’(5’)-iminium ion (mainly by AOX)
2. Oxygenation of aromatic (but not aliphatic) aldehydes, such as benzaldehyde
(product of benzyl alcohol oxidation by ADH – see gasping syndrome).
XANTHINE OXIDASE-CATALYZED OXIDATIONS
O
N
HN
N
O
O
Hypoxanthine
HN
XO
N
H
N
O
O
HN
XO
O
O
N
H
N
H
HN
N
H
N
H
Uric acid
Xanthine
N
H
N
N
H
N
Allopurinol
to decrease hyperuricemia
N
HN
N
S
S
S
N
H
6-Mercaptopurine
antitumor drug
XO
HN
N
N
N
H
H
6-Thioxanthine
O
XO
HN
H
N
O
O
N
N
H
H
6-Thiouric acid
inactive
Administration of the XO inhibitor allopurinol to patients treated with 6-mercaptopurine (6MP) would
decrease the elimination of 6-mercaptopurine and in turn could result in 6MP-induced toxicity, i.e.
bone marrow depression. Therefore, allopurinol is contraindicated for the 6-mercaptopurinetreated patient even if hyperuricemia developed. Patients have died because this contraindication
was disregarded by ignorant physicians!
NOTE: 6MP can also be eliminated by methylation at the SH group by thiopurine methyltransferase
(TPMT, see later). TPMT deficiency increases the toxicity of 6MP, just like cotreatment with
allopurinol does.
ALDEHYDE OXIDASE-CATALYZED OXIDATIONS
O
O
C
CH3
C
N
CH3
C
N
CH2 CH3
AOX
N
HO
N
N
H
CN
Zaleplon
short-acting hypnotic
CH3
N
CH2 CH3
HOH
N
N
O
CH2 CH3
AOX
O2
N
HOOH
N
O
CN
N
H
N
CN
5-oxo-zaleplon
5-hydroxy-zaleplon
NOTE: Zaleplon is also N-deethylated by CYP3A4.
CYP2A6
CYP2B6
N
+
N
AOX
AOX
OH
N
N
dehydrogenation
N
CH3
Nicotine
CH3
N
HOH
CH3
N
O2
HOOH
nicotine-1'(5')-iminium ion
O
C
H
Benzaldehyde
AOX
HOH
OH
AOX
OH
C
H
O2
HOOH
CH3
N
cotinine
O
C
OH
Benzoic acid
formed from benzylic alcohol,
(see gasping syndrome)
NOTE:
Cotinine is the main metabolite of nicotine and as such it may be used as a biomarker of nicotine
exposure (i.e. smoking). Cotinine is conveniently analyzed from the saliva of the smoker.
O
ALCOHOL DEHYDROGENASE (ADH)- AND
ALDEHYDE DEHYDROGENASE (ALDH)-CATALYZED OXIDATIONS
+
NADH + H +
NAD
CH3 CH2 OH
+
CH3 OH
CH2 OH
CH2 OH
H
NADH + H
O
HOH
H
3,4-dihydroxy-phenylacetyl
aldehyde (DOPAL)
formed by MAO
in dopaminergic neurons
acetic acid
C
NADH + H
+
O
H C
H
ALDH
OH
formic acid
Formic acid causes acidosis
and retinal injury (blindness)
H2C OH
ALDH
COOH
glycol
aldehyde
Rate
limiting
step
COOH
HC O
ADH
ALDH
COOH
glyoxylic
acid
glycolic
acid
COOH
oxalic
acid
Acidic metabolites cause acidosis and renal injury
HO
ALDH
OH
formaldehyde
HC O
HO
CH2 C
+
OH
H2C OH
O
ALDH
NAD
O
CH3 C
H
OH
H
+
NADH + H
ADH
NADH + H +
DISULFIRAM
to promote
alcohol withdrawal
H
ETHANOL
FOMEPIZOLE
(antidotes)
+
NAD
+
NAD
OH
H C
ADH
Ethylene
glycol
ETHANOL
FOMEPIZOLE
(antidotes)
HO
CH3 C
acetaldehyde
NAD
+
OH
CH3 C
ADH
Ethanol
Methanol
O HOH
H3CO
H3CO
O
O
HO
CH2 C
HO
OH
3,4-dihydroxyphenylacetic acid
(DOPAC)
CH
OH
O
ALDH
C
HO
CH
H
OH
Vanillyl mandelic aldehyde
formed by COMT and MAO
from epinephrine and NE
NOTES: 1. Fomepizole (=4-methylpyrazole; ANTIZOL®) is a potent
competitive inhibitor of ADH and is the most reliable antidote in
poisonings with methanol or ethylene glycol. Fomepizole is also used to
alleviate the acetaldehyde syndrome caused by ethanol consumption in
disulfiram-treated patients.
C
OH
Vanillyl mandelic acid
(VMA)
CH3
N
N
H
fomepizole
4-methylpyrazole
ANTIZOL
2. As shown in Part 6 (Appendix-2), disulfiram is biotransformed into an electrophilic metabolite
which covalently binds to and irreversibly inhibits aldehyde dehydrogenase. For this reason, a
disulfiram-treated individual should not consume alcohol for at least a week after discontinuation of
disulfiram administration.
3. Genetic and gender variations in the biotransformation of ethanol are presented in Part 6,
Appendix-3.
4. Elimination of the potentially toxic DOPAL by ALDH from nigrostriatal dopaminergic neurons is
a protective mechanism. Inhibition of ALDH by some pesticides (e.g. the fungicide benomyl) has
been speculated to be a mechanism of pesticide-induced Parkinson disease.
5. VMA is the final product of the biotransformation of epinephrine (E) and norepinephrine (NE) via
the COMT – MAO – ALDH pathway. Thus, VMA is the biomarker of E and NE production. Urinary
excretion of large amounts of VMA is a diagnostic sign for pheochromocytoma.
REDUCTION  Overview
AZO-REDUCTION (R1-N=N-R2  R1-NH2 + R2-NH2)
catalyzed by microbial enzymes in the colon
 Prontosyl  sulfanilamide + triaminobenzene
 Sulfasalazine (salicylazosulfapyridine)
 5-aminosalycylic acid + sulfapyridine
NITRO-REDUCTION (R-NO2  R-NH2):
catalyzed by CYP
 Clonazepam  7-amino-clonazepam
 Chloramphenicol  an arylamine metabolite (†)
CARBONYL-, ALDEHYDE-, and ALDOSE-REDUCTION
(R-C=O → R-C-OH):
catalyzed by cytosolic enzymes of the aldo-keto reductase (AKR)
superfamily, using NADPH




Haloperidol  reduced haloperidol
Oxcarbazepin  10-hydroxy-carbazepin (+)
Doxorubicin  doxorubicinol (†)
3,4-Dihydroxyphenylacetaldehyde (DOPAL; produced from DA by MAO)
 3,4-dihydroxyphenylethanol (DOPET)
REDUCTION
AZO-REDUCTION R N N R'
R NH2 + R' NH2
O
H2N
N N
O
H2N
S NH2
NH2
colonic
bacteria
O
NH2
Prontosil
S N
H
N N
HO
colonic HOOC
bacteria
N
S NH2
NH2
(Gerhard Domagk, Nobel Prize, 1939)
O
H2N
O
tiraminobenzene
HOOC
+
sulfanilamide
(antibacterial,
the prototype of sulfonamides)
O
NH2
HO
+
S N
H
H2N
O
O
Sulfasalazine (Salicylazosulfapyridine)
(to treat ulcerative colitis)
NITRO-REDUCTION (R-NO2
Clonazepam
(anxiolytic and
antiepileptic effects)
N
R-NO
H
N
sulfapyridine
5-aminosalycylic acid
(antiinflammatory)
R-NHOH
R-NH2)
H
N
O
O
CYP3A4
O2N
CYP2C19
N
H2N
N
R CH2 OH
CARBONYL-REDUCTION R CH O
O
7-aminoclonazepam
(inactive)
Cl
Cl
OH
OH
C CH2 CH2 CH2 N
AKR
OH
CH CH2 CH2 CH2 N
Cl
Cl
Haloperidol
(antipsychotic)
F
Oxcarbazepine
(antiepileptic,
inactive prodrug)
reduced haloperidol
(inactive)
F
O
OH
AKR
N
O
O
10-hydroxycarbazepine
(active)
C
N
O
NH2
OH
C
O
O
C CH2OH
NH2
OH
OH
CH CH2OH
OH
OH
AKR
OCH3 O
Doxorubicin
(antitumor drug)
OH
O
O
OCH3 O
OH
O
CH3
CH3
HO
HO
NH2
AKR = aldo-keto reductase
NH2
O
doxorubicinol
(cardiotoxic)
HYDROLYSIS  Overview
BY CARBOXYLESTERASES (CES, microsomal enzymes) and/or
BUTYRYLCHOLINESTERASES (BChE, soluble enzymes, also in plasma)
 HYDROLYSIS OF CARBOXYLIC ACID ESTERS: rapid
Examples:
- Drugs:
Succinylcholine, mivacurium, procaine, tetracaine, cocaine, esmolol,
meperidine, remifentanil, acetylsalicylic acid, clopidogrel, oseltamivir
– the active drugs are converted into inactive (or less active) metabolites
- Prodrugs: Fibrate esters, e.g. fenofibrate (-)  fenofibric acid (+);
ACE-inhibitor esters, e.g. enalapril (-)  enalaprilat (+)
Antipsychotic esters, e.g. pipothiazine palmitate (-)  pipothiazine (+)
Mycophenolate mofetil (-)  mycophenolic acid (+) – by CES
Bambuterol  terbutaline (+); Also: heroin (+)  morphine (+)
 HYDROLYSIS OF CARBOXYLIC ACID AMIDES: slow
Example:
Procainamide
BY ALKALINE PHOSPHATASES: hydrolyzes phosphoric acid monoesters
Examples (i.v. injectable prodrugs): Fosphenytoin (-)  phenytoin (+)
Fospropofol (-)  propofol (+)
Clindamycin phosphate (-)  clindamycin (+)
BY PARAOXONASES (Lactonases)
 HYDROLYSIS OF PHOSPHORIC ACID TRI-ESTERS
Examples: Paraoxon and other organophosphate insecticides
 HYDROLYSIS OF LACTONES
Examples: Statins, e.g. lovastatin, or simvastatin (lactone)  hydroxy-acid (+),
Spironolactone  hydroxy-acid (-)
BY EPOXIDE HYDROLASE: hydrolyzes the epoxide into dihydrodiols
Examples: Carbamazepine-epoxide (little-reactive epoxide, active metabolite)
Benzpyrene-epoxide (DNA-reactive epoxide, mutagenic and carcinogenic)
NOTE: Leucotriene A4 (LTA4) is also an epoxide! It is converted into either LTB4 (a diol) by epoxide
hydrolase, or into LTC4 (a GSH conjugate) by glutathione S-transferase.
BY PANCREATIC LIPASE IN THE SMALL INTESTINE
Example: castor oil (ricinoleic acid-containing triglyceride)  ricinoleic acid (+)
BY MICROBIAL HYDROLASES IN THE COLON
Examples (laxative prodrugs): Sennoside A, bisacodyl, sodium picosulfate
(-) = inactive metabolite, (+) = active metabolite, ASA = acetylsalicylic acid
NOTE: BChE is protective against organic phosphate ester insecticides and “nerve agents” whose
oxone form phosphorylates and irreversibly inactivates not only acetylcholinesterase (causing the
toxicity) but also BChE, which has no role in the toxic action. Thus BChE represents a “silent binding
site” for OP insecticides and chemical warfare agents by preventing their binding to the target of toxic
action, i.e. the neuronal acetylcholinesterase.
HYDROLYSIS BY CARBOXYLESTERASES and/or BUTYRYLCHOLINESTERASES
esterase
HOH
General scheme: R C O R'
O
R COOH + HO R'
DRUGS inactivated by hydrolysis:
H3C
O
CH NH CH2 CH CH2 O
H3 C
CH2 CH2 C O CH3
OH
+
O
CH3
Esmolol
(short acting beta-antagonist)
O
CH3
CH2 C O CH2 CH2 N CH3
+
CH3
CH2 C O CH2 CH2 N CH3
CH3 O C
O
N CH2 CH2 C O CH3
Succinylcholine
O
CH3 CH2 C N
O
CH3
(short acting muscle relaxant)
Remifentanil
(short acting opioid)
CH2 CH3
O
C
H
H2N
O CH3
Clopidogrel
(inhibitor of
platelet aggregation)
N
Cl
C O CH2 CH2 N
O
CH2 CH3
Procaine
(short acting local anesthetic; rapidly hydrolyzed)
S
O
COOH
O C
CH2 CH3
H2N
CH3
C NH CH2 CH2 N
O
Acetylsalicylic acid
(aspirin)
CH2 CH3
Procainamide
(antiarhytmic drug; slowly hydrolyzed)
PRODRUGS activated by hydrolysis:
O
COOH
C O C2H5
CH3
CH2 CH2 CH NH CH C N
Enalapril
Cl
CH3 O
C
O
CH3
C O CH
CH3
CH3 O
O C
Fenofibrate
(ACE inhibitor)
(triglyceride-lowering drug)
CH2 CH2 CH2 N
CH2 CH2 O
C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
O
O
N
CH3
S N
O
CH3
Pipothiazine palmitate
(PIPORTIL a depot antipsychotic,
injected i.m. as an oily injection once a month)
S
Some drugs (e.g., enalapril) containing carboxyl groups are esterified with an alcohol
to make them more lipophilic for better absorption by diffusion from the GI tract. After
absorption, such a drug is readiliy hydrolyzed by carboxylesterase and the original
organic acid (e.g., enalaprilate from enalapril) is released. Enalapril is an inactiv drug
(i.e., prodrug), whereas enalaprilate is the active ACE inhibitor metabolite. Similarly,
the lipophilic heroin readily diffuses into brain, where its hydrolysis produces morphine.
HYDROLYSIS BY ALKALINE PHOSPHATASE (AP)
NOTE: Fosphenytoin, fospropofol and clindamycin phosphate are phosphate ester
prodrugs that were synthesized in order to convert the poorly water-soluble phenytoin,
propofol and clindamycin, respectively, into water-soluble, i.v.-injectable forms.
As phenytoin does not contain a hydroxyl group at which to esterify it with phosphoric acid, and the
hydroxyl group of propofol is sterically hindered from phosphoric acid by the neighboring isopropyl
groups, first a -CH2-OH group had to be linked to these molecules. This linker group breaks off
spontaneously as formaldehyde after the AP-catalyzed hydrolysis.
HOH
H
N
O
N
O
OH
HO
OH
O
O
O
O CH2 OH
P
HCHO
O
Propofol
OH
N
HOH
Cl
Cl
AP
N
H
N
H
OH
O
O
SCH3
HO
P
OH
O
O
HO
SCH3
OH
OH
Clindamycin
phosphate
OH
OH
Phospropofol
N
O P O
OH
O
spontaneous
cleavage
AP
HO
OH
HCHO
CH2 OH
HOH
OH
HO
O
N
H
Phenytoin
CH2 O P O
O
N
OH
Phosphenytoin
OH
P
H
N
O
OH
CH2 O P O
spontaneous
cleavage
H
N
AP
Clindamycin
OH
HYDROLYSIS BY PARAOXONASES (PON)
Hydrolysis of phosphoric acid tri-esters:
O
H3C CH2 O
P
H3C CH2 O
O
H3C CH2 O
PON
+
P
O
NO2
H3C CH2 O
HO
NO2
OH
Paraoxon
(the active metabolite of parathion, an OP insecticide,
irreversble acethylcholinesterase inhibitor)
Hydrolysis of lactone rings:
O
HO
O
PON
HO
O
OH
OH
C
PON
O
CH3
O
H3C
O
H3C
CH3
O
O
active
lovastatin
H3C
O
H3C
O
C
OH
OH
inactive
spironolacton
S C CH3
Lovastatin
Spironolactone
(HMG-CoA reductase inhibitor, cholesterol lowering drug)
(aldosterone antagonist, K-sparing diuretic)
Notes:
- PON is also called aromatic esterase 1 or serum aryl-dialkylphosphatase 1
- PON1 is synthesized in the liver and transported along with HDL in the plasma. It functions as an
antioxidant; it is an anti-atherosclerotic component of HDL.
HYDROLYSIS BY MICROBIAL HYDROLASES IN THE COLON
Hydrolysis produces stimulatory laxatives in the colon
OH
Sennoside A
HO
OH
O
O
HO
OH
O
OH
O
COOH
COOH
COOH
2 HOH
COOH
COOH
2 glucose
COOH
O
O
OH
HO
O
HO
OH
Bisacodyl
OH
active antraquinone
aglycone
antraquinone glycoside
(prodrug)
OH
O
N
N
2 HOH
O
O
2 acetate
H3C
O
O
HO
CH3
Active diphenol-methane
laxative metabolite
Diacetic acid ester of
diphenol-methane laxative (prodrug)
Sodium
picosulfate
N
N
2 HOH
O
O
S
NaO
2 sulfate
S
O
O
OH
O
ONa
HO
OH
O
Disulfonic acid ester of
diphenol-methane laxative (prodrug)
Active diphenol-methane
laxative metabolite
PHASE II BIOTRANSFORMATIONS (CONJUGATIONS)
GLUCURONIDATION
Enzyme
Cosubstrate
Substrates: drugs, endogenous compounds
UDP-glucuronosyl
transferases
UDP-glucuronic OH-containing compounds: morphine, paracetamol,
acid
chloramphenicol, oxazepam, phenolphthalein,
(in the endoplasmic
propofol, diethylstilbestrol, estradiol, thyroxine
reticulum = microsomes)
OH-contaning metabolites: 6-OH phenytoin, etc.
N-OH-dapsone
COOH-containing compounds: valproic acid,
diclophenac, furosemide, telmisartan, bilirubin
COOH-containing metabolites: fenofibric acid
N-containing drugs: lamotrigine
SULFATION
Enzyme
Cosubstrate
Substrates
Sulfotransferases
PAPS
OH-containing compounds: pravastatin + see above
OH-contaning metabolites: see above
N-oxide-containing compounds: minoxidil
(in cytosol)
CONJUGATION WITH GLYCINE
Enzyme
Cosubstrate
Acyl-CoA synthetase
ATP
+ Glycine N-acyltransf. + CoA-SH
(in mitochondrial matrix) + Glycine
Substrates
COOH-containing compounds: salicylic cid,
benzoic acid,
nicotinic acid
CONJUGATION WITH GLUTATHIONE (Glu-Cys-Gly)
Enzyme
Cosubstrate
Substrates
Glutathione S-transf.
Glutathione
(glu-cys-gly)
Electrophilic C-containing compounds:
ethacrinic acid, epoxides (e.g. LTA4)
Electrophilic C-containing metabolites:
N-acetyl-p-benzoquinone imine, acrolein
(in cytosol)
ACETYLATION
Enzyme
Cosubstrate
Substrates
N-acetyltransferases
(in cytosol)
Acetyl-CoA
NH2-containing compounds:
NAT1 substrates: p-aminobenzoic acid,
sulfamethoxazol
NAT2 substrates: isoniazid, procainamide, dapsone,
hydralazine, sulfamethazine,
METHYLATION
Enzyme
Cosubstrate
Substrates
Methyltransferases
S-adenosylmethionine
OH-containing compounds: L-Dopa (COMT)
N-containing compounds: histamine, nicotine
SH-cont. compounds: 6-mercaptopurine (TPMT)
(in cytosol)
GLUCURONIDATION
Enzymes: UDP-glucuronosyltransferases (UDP-GT, UGT)
Cosubstrate:
O
UDP-glucuronic acid (UDP-GA)
NH
O
O
N
_
COO
O
O
CH2 O P O P O
O
O
_
O
_
HO OH
OH
OH
OH
Examples:
- Ether glucuronide formation (from compounds with a hydroxyl group)
COO
_
O
OH
O2N
OH
UDP-GT
CH CH CH2 OH
NH
UDP-GA
O2N
HO OH
CH CH CH2 O
C CHCl 2
C CHCl2 OH
O
NH
O
Chloramphenicol
(antibiotic)
Chloramphenicol glucuronide
(rapidly excreted in urine)
Others: - Endogenous compounds: Thyroxine, Estradiol
- Drugs: Acetaminophen, Diethylstilbestrol, Ezetimibe, Morphine, Oxazepam, Phenolphthalein
- Hydroxylated metabolites of drugs
- Ester glucuronide formation (from compounds with a carboxylic group)
CH3 CH2 CH2
O
UDP-GT
CH
C
CH3 CH2 CH2
COO
CH3 CH2 CH2
CH
UDP-GA
O
O
C
CH3 CH2 CH2
OH
_
HO OH
O
OH
Valproic acid
(antiepileptic drug)
Valproic acid glucuronide
(rapidly excreted in urine)
Others: - Bilirubin (forms mono- and diglucuronides)
- Drugs: NSAID (e.g., diclofenac), furosemide, telmisartan
- Acidic metabolites (e.g., fenofibric acid) produced from fibric acid esters by ester hydrolysis
- N-glucuronide formation
N
H2N
N
N
UDP-GT
Lamotrigine
(antiepileptic drug)
Other: Carbamazepine
NH2
N
Cl
Cl
_
O
+
HO OH
N
UDP-GA
Cl
Cl
N
H2N
NH2
COO
OH
Lamotrigine N2-glucuronide
(rapidly excreted in urine)
SULFATION
Enzymes: Sulfotransferases (SULT)
Cosubstrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS)
NH2
N
N
O
O
N
N
O S O P O CH2
O
O
O
O
OH
PO32-
Examples:
NH C CH3
O
NH C CH3
O
SULT
PAPS
OH
Acetaminophen = paracetamol
(analgetic and antipyretic)
Others: catecholamines (e.g., dopamine)
minoxidyl (see later)
hydroxylated metabolites of drugs
O
O S O
O
acetaminophen-sulfate = paracetamol-sulfate
(rapidly excreted in urine)
CONJUGATION WITH GLYCINE
Enzymes: (1) acyl-CoA synthetase (ACS)
(2) acyl-CoA: glycine N-acyltransferase (GNT)
Both enzymes are located in the mitochondrial matrix.
Cosubstrates: (1) ATP, (2) Coenzyme A (CoA-SH), (3) Glycine (Gly)
Examples:
C
O
C
OH
OH
ACS
O
S CoA
OH
GNT
Salicyl-CoA
NH CH2 COOH
OH
Salicyl-glycine = salicyluric acid
(rapidly excreted in urine)
carboxylesterase
acetylation
and inactivation of COX
Acetylsalicylic acid
(aspirin)
GNT
ACS
Benzoic acid
O
Gly
ATP, CoA-SH
Salicylic acid
C
ATP, CoA-SH
Benzoyl-CoA
Gly
Benzoyl-glycine
(hippuric acid)
AOX
Benzadehyde
ADH
Benzyl alcohol (an antiseptic;
see under gasping syndrome)
Note: Benzoyl-glycine was the first identified metabolite of a xenobiotic. In 1830, it was
found in the urine of horses given benzoic acid. Then it was named hippuric acid
(hippos means horse in Greek).
CONJUGATION WITH GLUTATHIONE
Enzymes: Glutathione S-transfrerases (GST)
Cosubstrate: Glutathione (GSH)
COO
CH
_
O
O
CH2 CH2 C
C
NH
NH
CH2 COO
_
CH
NH2
CH2
SH
..
-glutamic acid
cysteine
glycine
Notice that the SH group in GSH
is electron-rich (i.e., nucleophilic),
therefore it is reactive with
electron-deficient (i.e., electrophilic)
atoms, thus forming a glutathione conjugate.
As drugs and chemicals with elecrophilic
atoms may also bind to protein-cysteines,
conjugation with glutathione prevents
their covalent binding to proteins. Therefore,
conjugation with GSH is an important
protective mechanism against reactive
electrophiles, such as NAPBQI.
Examples: Compounds with electrophilic (electron deficient, partially positive) carbon atoms
O CH2 COOH
O CH2 COOH
Cl
Cl
GST
GSH
Cl
Cl
O C C CH2 CH3
(+) CH
O C CH CH2 CH3
CH2 SG
2
Ethacrynic acid (EA)
EA-glutathione
conjugate
(loop diuretic)
Note: the carbon with (+) sign is
electron-deficient (contains a partial positive
charge), thus it is electrophilic, therefore
reactive with GSH, whose SH group is
electron-rich (nucleophilic).
O
(+)
Acrolein,
a reactive electrophilic CH2 CH C
H
aldehyde that causes
hemorrhagic cystitis
in CP-treated patients
EA-Cys conjugate
(active diuretic metabolite)
O Acrolein-glutathione
conjugate
GS CH2 CH2 C
(detoxified)
H
GSH
Cyclophosphamide (CP)
(antitumor drug, a nitogen-mustard)
HN
C CH3
O
N
C CH3
O
CYP2E1
toxication
OH
Acetaminophen
= paracetamol
HN
C CH3
O
GSH
(+)
(+)
detoxication
O
N-acetyl-p-benzoquinoneimine
(NAPBQI)
ELECTROPHILIC METABOLITE
MAY CAUSE HEPATIC NECROSIS
SG
OH
Acetaminophen-glutathione
conjugate
(readily excreted)
ACETYLATION
Enzymes: N-acetyltransferases (NAT)
Cosubstrate: Acetyl coenzyme A (Ac-CoA)
NH2
N
N
O
CH3
O
O
N
N
NH C CH C CH2 O P O P O CH2
OH CH3
CH2
O
O
O
CH2
C
O
O
O
NH CH2 CH2 S C CH3
PO3H
OH
-
Examples:
O
O
C
C
NH NH2
O
NH NH C CH3
NAT2
Ac-CoA
N
Isoniazid
(antituberculotic)
N
Acetylisoniazid
(inactive metabolite)
Others: NAT1 substrates: p-Aaminobenzoic acid, p-Aminosalicylic acid, Sulfamethoxazole
NAT2 substrates: Dapsone, Procainamide, Hydralazine, Sulfamethazine
METHYLATION
Enzymes: Methyltransferases (MT)
Cosubstrate: S-adenosylmethionine (SAM)
NH2
N
N
_
CH3
OOC CH CH2 CH2 S
+
NH2
N
N
CH2
O
OH
OH
Examples:
entacapone
O-methylation
CH3 O
HO
CH2 CH COO
HO
_
NH2
COMT
SAM
CH2 CH COO
HO
_
NH2
L-Dopa
(antiparkinson drug, dopamine precursor)
3-O-Methyl-L-Dopa
N-methylation
CH2 CH2 NH2
CH2 CH2 NH2
HMT
HN
N
SAM
CH3 N
N
N-Methylhistamine
Histamine
S-methylation
SH
S CH3
N
N
N
N
H
6-Mercaptopurine
(antitumor drug)
TPMT
N
N
SAM
N
N
H
6-Methylmercaptopurine
Note: In patients with thiopurine methyltransferase (TPMT) deficiency (like in patients
treated with allopurinol – see earlier), 6-mercaptopurine elimination is slow. In such
patients, this drug may not cause apoptosis of leukemic cells only (which is desirable),
but may also induce apoptosis of normal bone marrow cells as well!
EFFECT OF BIOTRANSFORM ATION ON BIOLOGICAL ACTIVITY
1. TYPICALLY: ACTIVE DRUG  INACTIVE METABOLITE
 warfarin
 phenytoin
 theophylline
 morphine
 acetaminophen
 isoniazide






7-hydroxy-warfarin
4-hydroxy-phenytoin
1- or 3-methylxanthine
morphine-3-glucuronide
acetaminophen-glucuronide
acetyl-isoniazide
CYP2C9
CYP2C9
CYP2A1
UDP-GT
UDP-GT
NAT2
2. EXCEPTIONALLY: ACTIVE METABOLITE IS FORMED
a. Inactive parent compound (PRODRUG)  active metabolite
 cyclophosphamide 
 tamoxifen

 parathion

 terfenadine

 chloral hydrate

 sulfasalazine

 oxcarbazepine

 lovastatin (lactone) 
 enalapril (ester)

 fenofibrate (ester) 
 ezetimibe

 minoxidyl

 ethacrynic acid (EA) 
phosphoramide mustard
4-hydroxy-tamoxifen
paraoxon
alcohol  acid
trichloroethanol
5-aminosalicylic acid
10-hydroxy-carbazepine
lovastatin (free acid)
enalaprilate (free acid)
fenofibric acid (free acid)
ezetimibe-glucuronide
minoxidyl-sulfate
EA-cysteine
CYP2B6
CYP2D6
CYP3A4
CYP3A4
ADH (rev.), AR
Azo-reductase
AK-reductase
Paraoxonase
Esterase
Esterase
UDP-GT
SULT
GSTGGT, DP
b. Active parent compound  active metabolite
 phenylbutazone
 carisoprodol
 risperidone
 imipramine
 codeine
 diazepam
 morphine







-OH-phenylbutazone
meprobamate
9-OH-risperidone (paliperidone)
desmethyl-imipramine
morphine
nordiazepam  oxazepam
morphine 6-glucuronide
CYP2D6, 2C9
CYP2C19
CYP2D6
CYP2D6
CYP2D6
CYP  CYP
UDP-GT
c. „Non-toxic” parent compound  toxic metabolite
 acetaminophen

 halothane

 cyclophosphamide 
 methanol

 ethylene glycol

 doxorubicin

N-acetyl-p-benzoquinoneimine
trifluoroacetyl chloride
acrolein
formic acid
glycolic-, glyoxylic- oxalic acid
doxorubicinol
CYP2E1
CYP2E1
CYP3A4, 2B6
ADH  ALDH
ADH  ALDH
AK-reductase
BIOTRANSFORMATION OF DIAZEPAM,
FIRST INTO ACTIVE PHASE-I METABOLITES (NORDIAZEPAM, OXAZEPAM),
AND FINALLY INTO INACTIVE OXAZEPAM-GLUCURONIDE
PHASE-I biotransformation
CH3
O
N
Cl
Examples of drugs
whose active metabolites are also drugs:
Diazepam
(antianxiety and
antiepileptic drug)
active
[VALIUM]
N
T1/2= 40 hrs
diazepam VALIUM > oxazepam SERAX
risperidone RISPERDAL > paliperidon INVEGA
terfenadine TELDANE* > fexofenadine TELFAST
sulfasalazine SALAZOPYRIN > mesalazine SALOFALK
enalapril ENALAPRIL tabl > enalaprilate VASOTEC inj
* Withdrawn - see later
N-demethylation CYP2C19
CYP3A4
H
O
N
Cl
Nordiazepam
active
metabolite
N
PHASE-II biotransformation
(conjugation)
Hydroxylation CYP
H
H
O
N
O
N
UDP-GT
OH
Cl
UDP-GA
N
O -Glucuronic
Cl
acid
N
T1/2= 8 hrs
oxazepam
active metabolite
and also anti anxiety drug [SERAX]
oxazepam-glucronide
inactive
and readily excreted metabolite
HYDROXYLATION OF RISPERIDONE TO AN ACTIVE METABOLITE
NOTE that both risperidone and its metabolite, paliperidone, are sold as drugs.
F
F
OH
H3C
N
H3C
N
CYP2D6
N
N
O
N
N
N
O
O
Risperidone
(antipsychotic; RISPERDAL),T1/2 = 3 hrs
N
O
9-hydroxy-risperidone (active)
paliperidone (INVEGA), T1/2 = 25 hrs
BIOTRANSFORMATION OF CYCLOPHOSPHAMIDE INTO
THERAPEUTICALLY ACTIVE METABOLITE (PHOSPHORAMIDE MUSTARD),
SOME INACTIVE METABOLITES,
AND A TOXIC METABOLITE (ACROLEIN)
Cl
O
cyclophosphamide
M=
P
(antitumor drug)
N
N
H
Cl
O
O
GS
N
H
O
O
P
P
N
M
O
P
M
N
H
OH
4-glutathionylcyclophosphamide
M
CYP3A4
CYP2C9
CYP2B6
1
O
imminocyclophosphamide
O
C
H
M
H2N
M
detoxication
O
O
ALDH1A1
P
O
4-hydroxycyclophosphamide
spontaneous
cleavage
2
O
aldophosphamide
NOTE: Steps 1 and 2
represent N-dealkylation,
starting with hydroxylation on
the C atom linked to the N atom,
then chain breaking at the
arrow, and forming an aldehyde.
Here the aldehyde is
aldophosphamide.
O
C
HO
O
spontaneous
cleavage
H
C
SG
GSH
detoxication H
O
O
C
HO P
H2N
M
Acrolein
reacts covalently with protein-SH
Phosphoramide mustard
reacts covalenly with DNA bases
SPECIFIC TOXIC EFFECTS
ANTITUMOR EFFECT
and
GENERAL TOXIC EFFECT
of antitumor drugs
e.g., bone marrow depression
Liver:
damage to the
sinusoidal endothelial cells,
causing
sinusoidal obstruction sy. (SOS)
Bladder:
damage to the
urothelial cells,
causing
hemorrhagic cystitis
H2N
M
O-carboxyethylphosphoramide mustard
3
O
P
BIOTRANSFORMATION OF TERFENADINE (A PRODRUG ANTIHISTAMINE THAT
IS PRONE TO CAUSE POLYMORPHIC VENTRICULAR TACHYCARDIA)
INTO FEXOFENADINE (AN ACTIVE ANTIHISTAMINE)
Terfenadine is INACTIVE as an
antihistamine (it is a prodrug).
As a K+ channel inhibitor, terfenanidine
iinhibits repolarization of cardiomyocytes
CH3
and is arrhythmogenic. It has caused
C CH3
polymorphic ventricilar extrasystoles
(called
torsades de pointes) especially
CH3
when the patient also received a CYP3A4
inhibitor drug, such as ketoconazole or
erythromycin.
Terfenadine
[TELDANE]
(withdrawn)
OH
HO C
N (CH2)3 CH
Hydroxylation
CH3
OH
HO C
N (CH2)3 CH
C CH2OH
CH3
alcoholic
metabolite
Dehydrogenation
Hydration
Dehydrogenation
OH
HO C
N (CH2)3 CH
CYP
CH3
C COOH
CH3
carboxylic acid
metabolite
Ketoconazole
Erythromycin
CYP3A4
The carboxylic acid metabolite
is ACTIVE as an antihistamine!
Now it is marketed as
fexofenadine [TELFAST]
CYP3A4 inhibitors (e.g., erythromycin, ketoconazole) increased the
arrhythmogenic effect of coadministered terfenadine and have caused several
fatalities by precipitating polymorphic ventricular extrasystole (torsades de
pointes). Now terfenadine is withdrawn and replaced with fexofenadine
(TELFAST).
AN EXCEPTIONAL WAY FOR ACTIVATION OF A DRUG:
THE PRODRUG MINOXIDIL FORMS AN ACTIVE SULFATE CONJUGATE
N
N
NH2
MINOXIDIL
(prodrug,
inactive as a vasodilator)
N
NH2
O
PAPS
Sulfotransferase
N
N
NH2
MINOXIDIL-SULFATE
N
NH2
OH
O
S
O
O
N
+
N
NH3
N
NH2
O
O
MINOXIDIL-SULFATE inner salt form
(active vasodilator metabolite,
excreted slowly)
S
O
O
The vast majority of sulfate conjugates are highly water-soluble (due to their anionic
charge), pharmacologically inactive and readily excreted (e.g., acetaminophen-sulfate).
In contrast, minoxidyl-sulfate, owing to inner salt formation which masks the + and –
charges of this conjugate, is poorly water-soluble, pharmacologically active and slowly
excreted.
On a similar basis, the sulfate conjugate of 5-hydroxy-triamterene (the metabolite
of the diuretic triamterene) is also an active and poorly water-soluble; in fact it may
cause crystalluria by being precipitated out from the urine (see under Diuretics).
AN EXCEPTIONAL WAY FOR ACTIVATION OF MORPHINE:
BY CONJUGATION WITH GLUCURONC ACID AT THE 6-OH GROUP
The superactive nature of Mo-6-glucuronide is explained by inner salt formation
(like for minoxidyl-sulfate, see above)
Morphine-3-glucuronide
INACTIVE
cannot form an inner salt
Morphine
ACTIVE
Morphine-6-glucuronide
SUPERACTIVE
can form an inner salt
COOH
H
O
H
OH
H
H
OH
HO
H
O
HO
3
O
OH
UDP-GT
O
UDP-GA
UDP-GT
O
NH
6
COOH
NH
O
HO
HO
NH
UDP-GA
O
H
H
OH
H
H
OH
OH
H
AN EXCEPTIONAL WAY FOR ACTIVATION OF EZETIMIBE:
BY FORMATION OF A GLUCURONIDE THAT REACHES AND INHIBITS
THE INTESTINAL CHOLESTEROL TRANSPORTER
COOH
O
OH
O
OH
OH
OH
UDP-GT
F
HO
OH
UDP-GA
N
O
Ezetimibe
F
F
N
O
Ezetimibe-glucuronide (EG)
active metabolite
produced in the enterocytes
and the liver
F
Mrp-2
transporter
the absorption of
cholesterol decreases
THE SERUM CHOLESTEROL
LEVEL DECREASES
EG inhibits the
intestinal cholesterol
transporter (NPC1L1)
Bile, intestinal tract
DETOXIFICATION OF ACETAMINOPHEN (PARACETAMOL)
BY GLUCURONIDATION (OR SULFATION) AT ITS HYDROXYL GROUP AND
TOXIFICATION OF ACETAMINOPHEN BY CYP2E1-CATALYZED DEHYDROGENATION
TO A REACTIVE ELECTROPHILIC QUINONEIMINE (NAPBQI).
O
HN
C
Acetaminophen
= Paracetamol
O
O
CH3
toxication
by dehydrogenation
CYP2E1
N
C
(+)
HN
CH3
(+)
protein
OH
O
detoxication
of paracetamol
CH3
S
NAPBQI
OH
C
detoxication
of NAPBQI
glutathione
(glu-cys-gly)
UDP-GA
UDP-GT
O
O
HN
C
HN
CH3
C
CH3
S
O
glutathione
OH
glucuronic acid
EXCRETION
HEPATOCELLULAR
NECROSIS
You must know this!
NAPBQI IS NORMALLY DETOXIFIED BY CONJUGATION WITH GLUTATHIONE.
HOWEVER, WHEN ACETAMINOPHEN IS OVERDOSED, NAPBQI IS PRODUCED IN SO LARGE
QUANTITIES THAT GLUTATHIONE BECOMES CONSUMED AND DEPLETED. THEN NAPBQI
BINDS COVALENTLY TO THIOL GROUPS IN HEPATOCELLULAR PROTEINS, INACTIVATING
THESE PROTEINS (enzymes, transporters, mitochondrial e-transport proteins, etc.)
AND ULTIMATELY CAUSING LIVER NECROSIS.
Alcoholists are hypersensitive to acetaminophen-induced liver injury, because (1) in the
alcoholist’s liver the amount of CYP2E1 is increased, therefore more NAPBQI is formed, and
because (2) in the alcoholist’s liver the amount of glutathione is decreased, therefore
glutathione is more readily depleted, allowing NAPBQI to react with protein-thiols.
NAPBQI = N-acetyl-para-benzoquinoneimine
Part B. ALTERATIONS IN BIOTRANSFORMATION
1. BIOLOGICAL ALTERATIONS
IN BIOTRANSFORMATION - summary
 Age-related alterations -
Neonatal immaturity of biotransformation
 Genetic alterations:
- Mutation of genes encoding drug metabolizing enzymes
 the mutant gene may code for an inactive or unstable enzyme protein
 POOR or SLOW metabolizer phenotype
Such individuals may be hyperreactive to the drug
if the active parent compound is slowly converted into inactive metabolite.
- Multiplication of genes encoding drug metabolizing enzymes
 the gene in multiple copies codes for multiple amounts of enzyme protein
 EXTENSIVE or RAPID metabolizer phenotype
Such individuals may be non-reactive to the drug
if the active parent compound is rapidly converted into inactive metabolite.
NEONATAL IMMATURITY OF BIOTRANSFORMATION
summary
UDP-GT immaturity
 Bilirubin
 Chloramphenicol


Physiological jaundice (see FIG)
Grey baby syndrome (see FIG)
Immature glycine conjugation
 Benzyl alcohol

Gasping syndrome (see FIG)

Floppy infant syndrome (see FIG)
Immature CYP, UDP-GT
 Diazepam
UDP-GT immaturity: physiological jaundice, chloramphenicol-induced grey baby sy.
Bilirubin (not excreted into bile)
H
N
O
CH
H3C
H
N
H3C
CH 2 CH
H
N
CH2
CH 2
CH2
CH2
CH2
C
C
HO
O
O
Bilirubin diglucuronide (excreted into bile)
H
N
CH
CH 3
CH
H
N
O
O
CH
H
N
CH2
UDP-GA
H3C
H3C
CH 2 CH
CH 3
_
OOC
OH
O
CH 2
CH2
CH2
CH2
C
C
O
O
OH
H
N
CH2
UDP-GT
O
H
N
CH
CH 3
CH
O
O
COO
HO
OH
Chloramphenicol glucuronide
O2N
CH CH CH2 OH
O2N
UDP-GA
CH CH CH2 O
C CHCl2
NH
COO
_
O
OH
UDP-GT
_
OH
HO
OH
CH2
CH 3
HO
Chloramphenicol (CL)
O
NH
O
C CHCl2
HO OH
OH
O
At high concentration, CL inhibits mitochondrial protein synthesis, causing:
> cardiomyopathy
> myopathy
= GREY BABY SYNDROME
Immature glycine conjugation: benzyl alcohol-induced gasping syndrome
CH2 OH
CYP
ADH
AOX
BCS
BC:GT
Benzyl-alcohol
BA accumulation
acidosis
gasping
Cytochrome P450
Alcohol-dehydrogenase
Aldehyde-oxydase
Benzoyl-CoA-synthetase
Benzoyl-CoA:glycine-N-acyl-transferase
LOW CAPACITY STEP
IN NEONATES
ADH
O
O
C
C
H
C
OH
benzaldehyde
S-CoA
BCS
BC:GT
CoA-SH
glycine
AOX
O2=
O
O
benzoic acid
(BA)
benzoyl-CoA
C
NH CH2 COOH
URINE
benzoyl-glycine
(hippuric acid)
Note: The use of benzyl alcohol is prohibited in neonatology care units.
Immature CYP, UDP-GT: diazepam-induced floppy infant syndrome (FIS)
CH3
O
N
Cl
diazepam
active
[VALIUM]
N
FIS developes in a baby
whose mother received diazepam
before delivery.
T1/2= 40 hrs
SLOW STEPS
IN NEONATES
CYP2C19
CYP3A4
H
H
O
N
H
O
N
CYP
Cl
OH
Cl
N
N
O
N
UDP-GT
UDP-GA
O glucuronic acid
Cl
N
T1/2= 8 hrs
oxazepam
active
[SERAX]
nordiazepam
active
oxazepam-glucronide
inactive
BLOOD ETHANOL CONC. (mg%)
Immature alcohol dehydrogenase: delayed elimination of ethanol from the baby
monkey whose mother received ethanol before delivery
140
NEONATAL
MATERNAL
120
100
80
60
40
DELIVERY
20
0
0
50
100
150
200
250
300
350
TIME (min)
Interpretation:
Before delivery, ethanol elimination in the mother-fetus “unit” was entirely carried out
by ADH in the maternal tissues. After delivery, the ethanol that remained in the baby
can only be eliminated at a very slow rate because ADH is barely expressed in the
neonatal tissues. This also happens to diazepam in floppy infant sy (see above).
GENETIC ALTERATIONS IN BIOTRANSFORMATION
I. Mutation of genes encoding drug metabolizing enzymes
 the mutant gene may code for an inactive or unstable enzyme protein
 POOR metabolizer (hyperreactive) phenotype
1. CYP deficiencies
 CYP2C9 deficiency
 Warfarin hydroxylation
Adverse effects at regular dose:

 CYP2C19 deficiency
 Diazepam N-demethylation 
 CYP2D6 deficiency
 Debrisoquin hydroxylation 
 N-dealkylation of TCADs

 N-dealkyl. of antipsychotics 
Bleeding
Prolonged sedation
Pronounced hypotension
Pronounced sedation, toxicity
Tardive dyskinesia
(involuntary movements, tics)
2. Carboxylesterase (pseudocholinesterase) deficiency
Succinylcholine

Prolonged paralysis
3. NAT2 deficiency  slow acetylator phenotype
Isoniazide
Procainamide, hydralazine


 Neurotoxicity, hepatotoxicity
 Systemic lupus erythemathodes
4. Thiopurine methyltransferase deficiency
6-Mercaptopurine

 Myelotoxicity
5. Dihydropyrimidine dehydrogenase deficiency
5-Fluorouracyl

 Myelotoxicity
II. Multiplication of genes encoding drug metabolizing enzymes
 the gene in multiple copies codes for multiple amounts of enzyme protein
 EXTENSIVE metabolizer (non-reactive) phenotype
Multiplication of CYP2D6 gene  ultrarapid metabolizer phenotype
Psychotropic drugs, e.g., - TCADs (imipramine, nortriptyline),
(most are CYP2D6 substrates) - SSRIs (fluoxetine, paroxetine) and
- Others (clozapine, mianserine)
are all ineffective at regular doses
2. CHEMICAL ALTERATIONS
IN BIOTRANSFORMATION - summary
 Overexpression of enzymes by inducers
 Inhibition of enzymes by inhibitors
Cytochrome P450 enzymes are often induced or inhibited by certain drugs or
other chemicals. CYP inducers and inhibitors thus may significantly affect the
fate and the effect of other drugs that are biotransformed by CYP enzymes.
CYP superfamily organization:
 CYP families – labeled with numbers; only CYP1, CYP2 and CYP3 families are
involved in biotransformation of drugs. (CYP7A, for example, is cholesterol 7hydroxylase, the rate limiting enzyme of bile acid synthesis.)
 CYP subfamilies – labeled with capital letters, e.g. CYP2B, CYP2C, CYP2E
 CYP family members – labeled with numbers after the letter of the subfamily,
e.g., CYP2C9, CYP2C19, CYP2D6, CYP3A4
Of all the CYPs, CYP3A4 is the most abundant in human liver (35% of total) and
in human small intestinal mucosa cells (80% of total intestinal CYP).
 Some drugs are biotransformed largely or exclusively by one CYP.
For example:
- Phenytoin and warfarin are hydroxylated by CYP2C9
- Halothane is oxidatively debrominated by CYP2E1
 Other drugs may be biotransformed by several CYPs.
For example:
- Acetaminophen is dehydrogenated by CYP2E1 (mostly), CYP1A2 and CYP3A4
- Diazepam is N-demethylated by CYP2C19 and CYP3A4
- Dextromethorphan is O-demethylated by CYP2D6 and CYP3A4
A drug that is biotransformed by multiple CYPs, is typically less susceptible to
pharmacokinetic interactions by CYP inhibitors, which often inhibit one specific
CYP enzyme, and therefore CYPs that remain uninhibited may still eliminate the
drug.
ENZYME INDUCTION:
Induction means increased synthesis of the enzyme protein.
Induction is usually caused by increased gene transcription, which is mediated
by ligand-activated transcription factors, such as aryl hydrocarbon receptor
(AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR).
(see the table on the next page).
The inducer is a drug or other chemical which binds as a ligand to the ligandactivated transcription factor (intracellular receptor) and activates it. The
activated transcription factor (with its dimerizing partner) binds to a cognate
sequence in the regulatory region of one or more CYP genes, thus promoting
the transcription of such genes, ultimately increasing CYP protein synthesis.
Inducers may induce multiple CYP members. For example, phenobarbital
induces CYP2 members (except CYP2D) and CYP3A4, whereas ethanol induces
only CYP2E1 (called ethanol-inducible CYP form; although CYP2E1 is not truly
induced by ethanol but is stabilized by it.)
THE MECHANISM OF CYP3A4 INDUCTION BY DRUGS:
Increased CYPA4 gene transcription by activated pregnane X-receptor (PXR)
Xenobiotic*
(Ligand)
Increased
expression of
CYP3A4
PXR
Increased
transcription of
CYP3A4 mRNA
Plasma
membrane
DNA
ER6
Nucleus
RXR
*Ligands: rifampicin, phenobarbital, hyperforin (St. John's wort), etc.
PXR = Pregnane X receptor
RXR = Retinoid X receptor
CYP3A4 (CYP3A7)
CYP
family
CYP1
Drug substrates
A1: barely expressed, unless induced.
A2: caffeine, theophylline, tizanidine, ondansetron,
acetaminophen
Inducer
chemicals
Inducer
Inhibitors (often
receptors (TFs) competing substrates)
Drugs: omeprazole
AHR:
A2: ciprofloxacine
Others: PAHs (charcoalfluvoxamine
broiled meat, smoking), Aryl
cimetidine
TCDD, indole-3-carbinol hydrocarbon
acyclovir
(in brussels sprouts,
receptor
broccoli, cabbage)
CYP2
CYP3
(3A4)
B6: cyclophosphamide, bupropion, ketamine
C9: phenytoin, warfarin, tolbutamide
C19: omeprazole, diazepam, fluvastatin
D6: Psychotropic drugs: SSRI (citalopram, fluoxetine,
paroxetine), TCAD (amitriptyline, clomipramine,
desipramine, imipramine, nortriptyline), clozapine,
mianserine, mirtazapine, venlafaxine. Cardiovascular
drugs: -blockers (alprenolol, bufuralol, metoprolol,
propranolol, timolol), encainide, flecainide,
propafenone. Miscellaneous drugs: codeine,
debrisoquine, dextromethorphan, phenformin
E1: halothane, acetaminophen, dapsone, theophylline
Benzodiazepines: alprazolam, clonazepam,
clorazepate, diazepam, flurazepam, halazepam,
midazolam, prazepam, triazolam. Calcium channel
blockers: amlodipine, diltiazem, felodipine, isradipine,
lercanidipine, nifedipine, nisoldipine, nitrendipine,
verapamil. HIV antivirals: indinavir, nelfinavir, ritonavir,
saquinavir. Immunesuppressive drugs: cyclosporine,
tacrolimus, sirolimus. Macrolide antibiotics:
clarithromycin, erythromycin. Steroids: estradiol,
hydrocortisone, progesterone, testosterone. Statins:
lovastatin, simvastatin, atorvastatin. Others: buspirone,
codeine, dextromethorphan, methadone, taxol
B6, C9, C19:
phenobarbital
rifampin
B6: phenytoin
E1: ethanol,
isoniazide
CAR:
Constitutive
androstane
receptor
(CYP2D6 is NOT
inducible)
PXR:
rifampin, rifabutin
phenobarbital
Pregnane X
phenytoin
receptor
carbamazepine
nifedipine
dexamethasone
spironolactone
lovastatin
simvastatin
hyperforin (an
antidepressant in St
John's wort)
B6: clopidogrel
ticlopidin
C9: amiodarone
(+D6), fluconazole
C19: moclobemide
D6: cimetidine
quinidine
terbinafine
fluoxetine
paroxetine
E1: disulfiram
A4:
Drugs:
Azole antifungals
Macrolide antibiot.
HIV protease inhib
Gestodene (suicide
inhibitor contrac. ster)
Dietary chemicals:
naringenin: a flvonoid,
bergamottin: a furano
-cumarin (both present
in grapefruit juice)
CONSEQUENCES OF CYP INDUCTION:
1. Enhanced inactivation of a drug after repeated administration
Occurs when the drug is both a substrate and an inducer of a CYP;
thus it accelerates its own biotransformation into inactive metabolites
 diminished effect (self-induced tolerance)
e.g. Barbiturates (both substrates and inducers of CYP2 and CYP3A4)
Meprobamate (both substrate and inducer of CYP)
2. Enhanced inactivation of some coadministered drugs
after repeated administration of the inducer
Occurs when the inducer drug induces a CYP which transforms the
coadministered drug into inactive metabolite;
thus the inducer accelerates the biotransformation of the coadministered
drug into inactive metabolites  diminished effect
e.g. CYP2C9 inducers (phenobarbital, rifampin)
+ warfarin (anticoagulant, CYP2C9 substrate)
 INSUFFICIENT ANTICOAGULANT EFFECT of warfarin!
 THROMBOSIS, despite warfarin therapy!
CYP3A4 inducers (phenobarbital, rifampin, phenytoin, carbamazepine)
+ an oral contraceptive (contains ethinyl estradiol, a CYP3A4 substrate)
 INSUFFICIENT CONTRACEPTIVE EFFECT of the contraceptive
 UNWANTED PREGNANCY, despite taking a contraceptive!
CYP3A4 inducers (phenobarbital, rifampin, phenytoin, carbamazepine)
+ immunophyllin-binding immunosuppressive drug
(e.g., cyclosporine A, tacrolimus, sirolimus; all are CYP3A4 substrates)
 INSUFFICIENT IMMUNOSUPPRESSION
 TRANSPLANT REJECTION, despite immunosuppressive therapy!
3. Enhanced toxification of a coadministered drug
after repeated administration of the inducer
Occurs when the inducer drug induces a CYP which transforms the
coadministered drug into a toxic metabolite;
thus the inducer accelerates the biotransformation of the coadministered
drug into toxic metabolites  increased toxicity
e.g. CYP2E1 inducer (ethanol) + acetaminophen overdose (CYP2E1 substr.)
 increased formation of N-acetyl-p-benzoquinone imine (NAPBQI)
 HEPATIC NECROSIS!
Another cause of increased susceptibility of drinkers to paracetamolinduced liver injury: lower hepatic GSH  detoxication of NAPBQI
CYP2E1 inducer (ethanol) + halothane anesthesia
 increased formation of trifluoroacetyl chloride
 the probability for halothane HEPATITIS may increase!
CONSEQUENCES OF CYP INHIBITION:
1. Decreased inactivation of an active drug  increased therap. effect:
e.g. a CYP2C9 inhibitor (e.g., amiodarone)
+ the oral anticoagulant warfarin  BLEEDING!
a CYP3A4 inhibitor (e.g., itraconazole, clarithromycin)
+ the oral antidiabetic repaglinide (CYP3A4 substr.) HYPOGLYCEMIA!
a CYP3A4 inhibitor (e.g., ketoconazole)
+ cyclosporine A (CsA; an immunosuppressive drug; CYP3A4 substr.)
  oral bioavailability and  elimination of CsA
 Two consequences (Two birds are killed with one stone!):
  effectiveness (dose reduction  saving on the drug)
 the antifungal ketokonazole prevents fungal infection of the
immunosuppressed patient
2. Decreased inactivation of an active drug  increased adverse effect:
e.g. a CYP3A4 inhibitor (e.g., itraconazole, clarithromycin, bergamottin)
+ a CYP3A4 substrate statin (not pravastatin, fluvastatin, rosuvastatin)
 MYOTOXICITY! – see FIG.
a CYP3A4 inhibitor (e.g., ketoconazole, erythromycin)
+ terfenadine (a withdrawn antihistamin; CYP3A4 substrate)
 POLYMORPHIC VENTRICULAR ES!– see FIG.
a CYP1A2 inhibitor (e.g., ciprofloxacin, fluvoxamine)
+ tizanidine (a central muscle relaxant 2-receptor agonist; CYP1A2 substr.)
 EXCESSIVE SEDATION, HYPOTENSION! – see FIG.
3. Decreased activation of a prodrug  decreased therapeutic effect
e.g. a CYP2D6 inhibitior (e.g., fluoxetine or paroxetine)
+ tamoxifen (an antiestrogen prodrug, CYP2D6 substrate)
 decreased conversion into 4-hydroxytamoxifen (active antiestrogen)
– see FIGURE
Thus, CYP2D6 inhibitors can compromise the effectiveness of tamoxifen
against estrogen-dependent breast cancers!
4. Decreased toxification of a drug  decreased toxic effect
e.g. CYP2E1 inhibitior (disulfiram)
+ halothan (an inhalation anesthetic, CYP2E1 substrate)
 decreased conversion into trifluoroacetyl chloride (reactive metab.)
Thus, disulfiram pretreatment before halothane anesthesia could be
used to decrease the likelihood of halothane hepatitis.
GRAPEFRUIT JUICE (contains bergamottin which inhibits CYP3A4 as well as the
OATP transporter-mediated hepatic uptake of statins) INCREASES THE PLASMA
CONCENTRATION OF SIMVASTATIN (SV; A PRODRUG)
AND ITS ACTIVE METABOLITE SIMVASTATIN ACID SEVERAL FOLD.
(Lilja et al., Clin. Pharmacol. Ther. 64: 477-483, 1998.)
15
Simvastatin acid (ng/ml)
Simvastatin (ng/ml)
30
25
grapefruit juice + SV
20
15
10
water + SV
5
0
10
grapefruit juice + SV
5
water + SV
0
0
2
4
6
8 10 12 14 16 18 20 22 24
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (h)
Time (h)
BIOTRANSFRORMATION PATHWAYS FOR THE CHOLESTEROL-LOWERING
DRUG SIMVASTATIN INTO ACTIVE SIMVASTATIN ACID BY PARAOXONASE
AND INACTIVE METABOLITES BY CYP3A4
HO
O
HO
COOH
O
O
OH
O
Paraoxonase
O
O
HOH
Simvastatin
(inactive)
Simvastatin acid
(active)
CYP3A4
HO
naringenin, bergamottin; itraconazole, verapamil
O
HO
O
O
O
HO
O
O
O
O
O
O
O
O
HO
OH
6'-hydroxy SV
3'-hydroxy SV
H2C
6'-exomethylene SV
CYP3A4 inhibitors, such as constiuents of grapefruit juice (naringenin and
bergamottin) as well as drugs (itraconazole, verapamyl, erythromycin, etc.) may
increase the myotoxicity of statins by diminishing their elimination by CYP3A4.
OATP inhibitors, such as the grapefruit juice constituent bergamottin and the fibratetype triglyceride-lowering drugs (e.g., gemfibrozil), may also increase the myotoxicity of
statins by diminishing their hepatic uptake mediated by OATP.
BIOTRANSFORMATION OF TERFENADINE (A PRODRUG ANTIHISTAMINE THAT
IS PRONE TO CAUSE POLYMORPHIC VENTRICULAR TACHYCARDIA)
INTO FEXOFENADINE (AN ACTIVE ANTIHISTAMINE)
Terfenadine is INACTIVE as an
antihistamine (it is a prodrug)
As a K+ channel inhibitor, terfenanidine
iinhibits repolarization of cardiomyocytes
CH3
and is arrhythmogenic. It has caused
C CH3
polymorphic ventricilar extrasystoles
(called torsades de pointes) especially
CH3
when the patient also received a CYP3A4
inhibitor drug, such as ketoconazole or
erythromycin.
Terfenadine
[TELDANE]
(withdrawn)
OH
HO C
N (CH2)3 CH
Hydroxylation
CH3
OH
HO C
N (CH2)3 CH
C CH2OH
CH3
alcoholic
metabolite
Dehydrogenation
Hydration
Dehydrogenation
OH
HO C
N (CH2)3 CH
CYP
CH3
C COOH
CH3
carboxylic acid
metabolite
Ketoconazole
Erythromycin
CYP3A4
The carboxylic acid metabolite
is ACTIVE as an antihistamine!
Now it is marketed as
fexofenadine [TELFAST]
CYP3A4 inhibitors (e.g., erythromycin, ketoconazole) increased the
arrhythmogenic effect of coadministered terfenadine and have caused several
fatalities by precipitating polymorphic ventricular extrasystole (torsades de
pointes). Now terfenadine is withdrawn and replaced with fexofenadine
(TELFAST).
N
N
S
S
N
Cl
H
N
N
Cl
hydroxylation
H
N
CYP1A2
NH
Tizanidine
N
N
NH
S
N
N
O
Tizanidine, a centrally acting muscle relaxant, is rapidly
biotransformed by CYP1A2. This is responsible for both its
hepatic presystemic elimination (F = 0.7) and rapid
elimination (T1/2 = 2.5 hrs) of tizanidine. Therefore, drugs
that inhibit CYP1A2 (e.g., fluvoxamine, ciprofloxacin,
rofecoxib) can increase the AUC of tizanidine several fold.
At high level, tizanidine, which is a 2-receptor agonist like
clonidine, can cause marked hypotension, bradycardia and
sedation.
N
S
N
H
N
NH
FLUVOXAMINE, CIPROFLOXACIN, ROFECOXIB
OH
Cl
H
N
CYP1A2
HO
hydroxylation CYP1A2
N
Cl
dehydrogenation
NH
N
-------------------------------------------------------------------------------------------------------------------
O
CH3
O
N
N
CH3
CYP2D6
tamoxifen
(prodrug)
CH3
SSRI
(e.g. fluoxetine
paroxetine)
CH3
-hydroxytamoxifen
(active antiestrogen;
used against breast cancer)
OH
CYP2D6 inhibitors (e.g., fluoxetine or paroxetine) can compromise the
therapeutic effectiveness of tamoxifen against estrogen-dependent breast
cancer because they inhibit the conversion of tamoxifen into 4hydroxytamoxifen, the active antiestrogen metabolite of tamoxifen.
THERAPEUTIC USE OF ENZYME INHIBITORS
CYP inhibitors:
Ketoconazol – to increase effectiveness of cyclosporine (to save on the drug)
Disulfiram – to decrease the likelihood of
• halothane hepatitis or
• acetaminophen-induced hepatic necrosis
MAO inhibitors:
MAO A inhibitors (e.g., moclobemide) – to treat major depression
MAO B inhibitors (e.g., selegiline) – to treat Parkinson's disease
COMT inhibitors:
Entacapone – to treat Parkinson's disease
XO inhibitors:
allopurinol – to decrease uric acid formation (in gout)
(BUT: it decreases the elimination and thus increases the toxicity of 6-mercaptopurine!)
Alcohol dehydrogenase inhibitors:
Fomepizol, ethanol – to treat methanol or ethylene glycol intoxication
Aldehyde dehydrogenase (ALDH) inhibitors:
Disulfiram – to induce „acetaldehyde syndrome”
and thus promote alcohol withdrawal
Note, disulfiram is not only an irreversible ALDH inhibitor, but a CYP inhibitor as well.
Drugs with unwanted ”disulfiram-like effect”:
- Some cephalosporins: cefamandol, cefoperazone
(Their metabolite, 1-methyltetrazole-5-thiol, inhibits aldehyde dehydrogenase, like
disulfiram does.)
- Chloramphenicol
- Metronidazole
- Procarbazine