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, Drugs excreted both Drugs fully biotransformed,
excreted unchanged unchanged and as metabolites excreted only as metabolites

Benzylpenicilin
Aminoglycosides Salicylates TCADs
Metformin Acetaminophen Phenothiazines
Tubocurarine Phenobarbital Chloramphenicol
Amantadine

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
Phase I reactions
(conjugations)
1. Glucuronidation
1. Oxidation 2. Sulfation
3. Conjugation with glycine (Gly)
2. Reduction 4. Conjugation with glutathione (GSH)

3. Hydrolysis 5. Acetylation
6. Methylation

The chemical role of Phase I and Phase II biotransformations:

An organic acid (or acetyl or methyl group)
A functional group is added to the molecule is conjugated to the molecule
or explored in the molecule at a preexisting functional group or
at which conjugation can take place at a functional group acquired
in Phase I biotransformation

General examples
R-O-glucuronic acid
oxidation
RH O
R-OH
R-O-sulfonic acid
R-CH2-O-glucuronic acid
reduction
R-HC=O 2H
R-CH2-OH
R-CH2-O-sulfonic acid

hydrolysis
R-O-glucuronic acid
R1-O-(C=O)-R2 HOH
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 Biological activity: almost always
but often 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 Xanthine oxidase (XO) and Aldehyde oxidase (AOX)
monooxigenase (FMO) 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

CYP
General scheme: RH + O2 + (NADPH + H)+ R-OH + HOH + NADP+
NADPH-CYP reductase
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 Substrate (RH)
Product (ROH)
3+
Fe

3+ 3+
7 Fe In the course of oxygenation Fe 2
ROH by CYP, O2 is activated by electron RH
uptake to form reactive O atoms. _
The first reactive form is e
the hydroperoxy form
(produced in step 5).
From this, 1 O is inserted into a
water molecule (HOH).
2+
(FeO)3+ The second reactive form Fe
6 is the oxene (produced in step 6). 3
RH From this, the reactive O atom RH
is inserted into a C-H bond
of the drug, forming a hydroxylated O2
HOH metabolite in step 7, which is then
+ released from CYP.
H CYP-catalyzed
2+ 3+ _
Fe OOH dehydrogenation and Fe O2
reduction are explained below.
RH RH

5 H
+ 4 NADPH-cytochrome
Note that Fe represents P450 reductase
the iron in CYP. _
e
II. DEHYDROGENATION e.g. acetaminophen (=paracetamol), nifedipine, ethanol
3+ NOTE: The 2nd O atom is also
(FeO)3+ Fe
6 7 reduced to water, using 2 H atoms
RH2 R from the drug molecule.
HOH

III. REDUCTION e.g. nitro reduction of clonazepam; reductive dehalogenation of CCl4

2+ 2+ NOTE: The second electron is not
Fe O2 Fe O 5
4 _
_2 transferred to the CYP-bound
RH e [RH] 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: tolbutamide (-), terfenadine (+), cyclophosphamide (+)
- Aromatic hydroxylation: 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 O O
CYP2C9
CH3 S NH C NH HO CH2 S NH C NH
O O
Tolbutamide (antidiabetic) Hydroxymethyl-tolbutamide (inactive)

CH3 CH3

HOOC CH CH2 CH

CH3
Ibuprofen
(NSAID)

CH3 CH3 CH3 CH2 OH

HOOC CH CH2 C OH HOOC CH CH2 CH

CH3 CH3
2-hydroxy-ibuprofen CONJUGATES 3-hydroxy-ibuprofen

b) C-HYDROXYLATION: aromatic
O Phenytoin O 4'-Hydroxyphenytoin
(antieileptic, antiarrhythmic) (inactive)
HN NH HN NH
CYP2C9
C6H5 C6H5
O O

OH

O O HO O O
CYP2C9
H H
C C
OH CH2 C CH3 OH CH2 C CH3
Warfarin (anticoagulant) O 7-hydroxy-warfarin O
(inactive)
c) N-HYDROXYLATION

O O
CYP2C9
H2 N S NH2 H2 N S NH OH
O O

Dapsone (antileprotic) Dapsone-hydroxylamine

(hemotoxic and allergenic)
EPOXIDATION = insertion of O into a C=C double bond
O
H H

CYP

N N
C C
O NH2 O NH2
Carbamazepine Carbamazepine-10,11-epoxide
(antiepileptic) (less active metabolite)

Epoxide O (+)

CYP hydrolase CYP (+)

(+)

(+)
HO HO
O
OH OH

benzo(a)pyrene benzo(a)pyrene-7,8-oxide benzo(a)pyrene-7,8-dihydro-diol benzo(a)pyrene-

7,8-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
OH R NH2 N-bound C atom of the alkyl group,
the alkyl group breaks off (as an aldehyde)
R NH CH3 R NH CH2
O from the N atom, producing a
H C dealkylated amine metabolite
H of the drug molecule.
Examples:
CH3 H
O O Other examples:
N N
CYP2C19 lidocaine monoethylglycylxylidine
CYP3A4 imipramine desmethylimipramine
Cl N Cl N erythromycine desmethylerythromycine
HCHO

Diazepam Nordiazepam

Theophylline may undergo: O H

- N-demethylation at N1 and N3 by CYP H3C N
N3-demethylation N
- C-hydroxylation at C8 by CYP
CYP1A2
- N- methylation at N7 by MT O N N
H
1-Methylxanthine
(25 %)

O CH3 O H O H O H
H3C N H3C N H3C N H3C N
N1 7 MT N Hydroxylation N N3-demethylation N
OH OH
3 SAM CYP3A4, CYP2E1 CYP1A2
O N N O N N O N N O N N
CH3 CH3 CH3 H
Caffeine Theophylline 1,3-Dimethyluric acid 1-Methyluric acid
(50 %) (5 %)

In neonates more caffeine is

formed from theophylline than H
O CH3
N
in adults because the CYP N1-demethylation N

levels in the neonatal liver CYP1A2

O N
N
are low, however, the CH3
methyltransferase (MT) 3-Methylxanthine
(15 %)
levels are relatively high.
O-dealkylation
General scheme: NOTE: After hydroxylation at the
OH R OH O-bound C atom of the alkyl group,
the alkyl group breaks off (as an aldehyde)
R O CH3 R O CH2
O from the O atom, producing a
H C dealkylated hydroxylated metabolite
of the drug molecule.
H
Example:

CH3 O HO Other examples:

codeine morphine
phenacetine paracetamol
CYP2D6

HCHO
N CH3 N CH3

Dextromethorphan Dextrorphan
(antitussive drug) (active metabolite)
Oxidative deamination
OH O
CYP
CH2 CH CH3 CH2 C CH3 CH2 C CH3
NH2 NH2 NH3
Amphetamine Phenylacetone
(centrally acting sympathomimetic) (inactive)

Oxidative dehalogenation
F Cl F Cl F
(+)
Cl
C YP2E1
F C C Br F C C Br F C C
O
F H F OH HBr F

Halothane T rifluoro-acetyl-chloride
(inhalation anesthetic) binds covalently to hepatic proteins,
thus form ing a neoantigene and
causing "halothane hepatitis"

Oxidative desulfuration

C2H 5 O S C 2H 5O S C 2H 5 O O
P P O P
CYP
C 2H 5O O NO 2 C 2H5 O O NO 2 C 2H 5O O NO 2

[S]
Paraoxon
Parathion (active insecticide,
(an organophosphate insecticide) an irreversible inhibitor of
acethylcholinesterase)

S S O O

HN NH CYP HN NH HN NH

O N O O N O O N O
[S]
H5 C2 CH CH3 H5 C 2 CH CH 3 H5 C 2 CH CH3
CH2 CH2 CH3 CH 2CH 2CH3 CH 2 CH 2 CH 3

Thiopental Pentobarbital
(an i.v. anesthetic) (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:
3+ NOTE: While an oxygenation produces 1
(FeO)3+ Fe molecule hydroxylated metabolite plus 1
6 7
RH2 R molecule HOH, dehydrogenation produces
HOH 2 molecules HOH.
Examples: plus a dehydrogenated metabolite.
O O
H C C
N CH3 N CH3
NO2 NO2
CYP2E1 O O O O
CYP3A4
CYP1A2 H3CO C C OCH3 H3CO C C OCH3
CYP3A4
OH O H3C CH3
H3C N CH3 N
Acetaminophen N-Acetyl-p-benzo- H
analgetic and quinoneimine
antipyretic (NAPBQI) Nifedipine "Pyridine metabolite"
drug hepatotoxic a dihydropyridine Ca++-channel blocker 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+ 2+
4 Fe O2 Fe O
_
_2 5
RH e [RH]
Examples:
1. Reductive dehalogenation of carbon tetrachloride (1-electron reduction):
_
Cl Cl Cl- Cl Tricloromethyl
Carbon CYP2E1
C.
free radical
tetrachloride Cl C Cl Cl C : Cl Cl
reduction initiates lipid peroxidation
solvent
Cl Cl Cl and liver injury

2. Nitro-reduction of clonazepam to 7-amino-clonazepam (6-electron reduction):

H O H O
N N
CYP3A4
O2N N CYP2C19 H2N N

Clonazepam Cl Cl 7-amino-
anxiolytic and clonazepam
antiepileptic drug inactive
2e- 2e- 2e-
R-NO2 R-NO R-NH-OH R-NH2
2H+ HOH 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:

N-CN O N-CN
CH2 S CH2 CH2 NH C NH CH3 CH2 S CH2 CH2 NH C NH CH3
N N
FMO
CH3 CH3
N N
H Cimetidine H Cimetidine S-oxide
(an H2-receptor antagonist) (inactive)

FMO
N N O
N CH3 N CH3

Nicotine Nicotine-1'-N-oxide
(inactive)

CH3 CH3
FMO3
CH3 N CH3 N O
CH3 CH3

Trimethylamine Trimethylamine N-oxide

endogenous volatile compound; (water-soluble metabolite
accumulates in FMO3 deficiency, excreted in urine)
causing fish odor syndrome
 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 Parkinsons 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: FOUR STEPS - the byproducts are ammonia and HOOH!
1. R-CH2NH2 + FAD > R-CH=NH + FADH2 Dehydrogenation
HO Dopamine
2. R-CH=NH + H2O > R-CH(OH)-NH2 Hydration
3. R-CH(OH)-NH2 > R-CH=O + NH3 Deamination
HO CH2 CH2 NH2 4. FADH2 + O2 > FAD + H2O2 FAD
regeneration
FAD HOOH !
1 4
HO FADH2 O2

HO CH2 CH NH
HO
HOH O
2 HO CH2 C
OH
HO HO acid (DOPAC)
NH3
O
Oxidation by ALDH
HO CH2 CH NH2 HO CH2 C
3 Reduction by AR
OH H
HO
3,4-dihydroxy-
phenylacetyl aldehyde
(DOPAL) HO CH2 CH2OH
alcohol

Example:
Propranolol N-Desisopropylpropranolol
OH CH3 OH
O CH2 CH CH2 NH CH O CH2 CH CH2 NH2
CH3
CYP2D6
N-dealkylation

O2 + H2O
CYP2D6
MAO-A
aromatic hydroxylation
NH3 + H2O2

OH CH3 OH
O
O CH2 CH CH2 NH CH O CH2 CH C
H
CH3

OH 4-Hydroxypropranolol Oxidation Reduction

by AOX 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 keto-
group 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 O O
H
N N N
HN XO HN XO HN
O

N N O N N O N N
H O H H
H H
HN
Hypoxanthine N Xanthine Uric acid

N N
H
Allopurinol
to decrease hyperuricemia

S S S
H
N N N
HN XO HN XO HN
O
N O N N O N N
N H H H
H H
6-Mercaptopurine 6-Thioxanthine 6-Thiouric acid
antitumor drug 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-mercaptopurine-
treated 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 O
C CH3 C CH3 C CH3
N N N
CH2 CH3 CH2 CH3 CH2 CH3
AOX AOX

HOH O2 HOOH
N N N
N N N

N HO N O N
CN H CN H CN

Zaleplon 5-hydroxy-zaleplon 5-oxo-zaleplon

short-acting hypnotic
NOTE: Zaleplon is also N-deethylated by CYP3A4.

CYP2A6 +
CYP2B6 AOX OH AOX O
N N N N
dehydrogenation
CH3 CH3 HOH CH3 O2 CH3
N N N N
HOOH
Nicotine nicotine-1'(5')-iminium ion cotinine

O OH O
AOX AOX
C C OH C
H HOH H O2 HOOH OH
Benzaldehyde 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.
 ALCOHOL DEHYDROGENASE (ADH)- AND
ALDEHYDE DEHYDROGENASE (ALDH)-CATALYZED OXIDATIONS
+ +
NAD NADH + H + O HOH OH NAD NADH + H + O
CH3 CH2 OH CH3 C CH3 C H CH3 C
ADH H OH
ALDH OH
Ethanol acetaldehyde DISULFIRAM acetic acid
to promote
alcohol withdrawal
+ + + +
NAD NADH + H HOH OH NAD NADH + H O
O
CH3 OH H C H C H H C
ADH H ALDH OH
OH
Methanol formaldehyde formic acid
ETHANOL
FOMEPIZOLE Formic acid causes acidosis
(antidotes) and retinal injury (blindness)
Rate
+ +
NAD NADH + H limiting
CH2 OH H2C OH H2C OH step HC O COOH
CH2 OH ADH HC O ALDH COOH ADH COOH ALDH COOH
Ethylene glycol glycolic glyoxylic oxalic
glycol ETHANOL aldehyde acid acid acid
FOMEPIZOLE
(antidotes) Acidic metabolites cause acidosis and renal injury

HO HO H3CO H3CO
O O O O
ALDH ALDH
HO CH2 C HO CH2 C HO CH C HO CH C
H OH OH H OH OH
3,4-dihydroxy-phenylacetyl
aldehyde (DOPAL) 3,4-dihydroxy- Vanillyl mandelic aldehyde Vanillyl mandelic acid
formed by MAO phenylacetic acid formed by COMT and MAO (VMA)
in dopaminergic neurons (DOPAC) from epinephrine and NE

NOTES: 1. Fomepizole (=4-methylpyrazole; ANTIZOL) is a potent CH3

competitive inhibitor of ADH and is the most reliable antidote in
N fomepizole
poisonings with methanol or ethylene glycol. Fomepizole is also used to N 4-methylpyrazole
alleviate the acetaldehyde syndrome caused by ethanol consumption in ANTIZOL
H
disulfiram-treated patients.
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 O
H2N N N S NH2 H2N NH2 + H2N S NH2
O colonic O
NH2 bacteria NH2
Prontosil tiraminobenzene sulfanilamide
(Gerhard Domagk, Nobel Prize, 1939) (antibacterial,
the prototype of sulfonamides)
HOOC colonic HOOC
O N O N
bacteria
HO N N S N
H
HO NH2 + H2N S N
H
O O

Sulfasalazine (Salicylazosulfapyridine) 5-aminosalycylic acid sulfapyridine

(to treat ulcerative colitis) (antiinflammatory)

NITRO-REDUCTION (R-NO2 R-NO R-NHOH R-NH2)

Clonazepam H O H O
(anxiolytic and N N
antiepileptic effects) CYP3A4
O2N N CYP2C19 H2N N

Cl Cl 7-amino-
clonazepam
(inactive)

CARBONYL-REDUCTION R CH O R CH2 OH
O OH OH OH
C CH2 CH2 CH2 N AKR CH CH2 CH2 CH2 N
Cl Cl

Haloperidol reduced haloperidol

F
(antipsychotic) F
(inactive)

Oxcarbazepine O OH 10-hydroxycarbazepine
(antiepileptic, (active)
inactive prodrug)
AKR
N N
C C
O NH2 O NH2

O OH O O OH OH
C CH2OH CH CH2OH
OH OH

AKR
OCH3 O OH OCH3 O OH
O O
O O
Doxorubicin CH3 CH3
doxorubicinol
(antitumor drug) (cardiotoxic)
HO HO
NH2 NH2

AKR = aldo-keto reductase

 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

General scheme: R C O R' esterase

R COOH + HO R'
HOH
O
DRUGS inactivated by hydrolysis:

H3C O CH3
+
CH NH CH2 CH CH2 O CH2 CH2 C O CH3 CH2 C O CH2 CH2 N CH3
H3 C OH O CH3
Esmolol
(short acting beta-antagonist) CH3
O +
CH3 O C CH2 C O CH2 CH2 N CH3
O CH3
N CH2 CH2 C O CH3
CH3 CH2 C N O Succinylcholine
O (short acting muscle relaxant)
Remifentanil
(short acting opioid)

CH2 CH3
O
H2N C O CH2 CH2 N
H C O CH3 O CH2 CH3
Clopidogrel
N (inhibitor of
platelet aggregation) Procaine
Cl (short acting local anesthetic; rapidly hydrolyzed)
S

COOH O CH2 CH3

O C CH3 H2N C NH CH2 CH2 N
O CH2 CH3
Acetylsalicylic acid
(aspirin)
Procainamide
(antiarhytmic drug; slowly hydrolyzed)

PRODRUGS activated by hydrolysis:

O COOH
C O C2H5 CH3 CH3
CH2 CH2 CH NH CH C N Cl C O C C O CH
CH3 O O CH3 O CH3
Enalapril
(ACE inhibitor) Fenofibrate
(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 CH3 Pipothiazine palmitate
N S N (PIPORTIL a depot antipsychotic,
O CH3 injected i.m. as an oily injection once a month)

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 spontaneous
H AP H cleavage H
N N N
O O O
OH OH
N N N
O CH2 O P O O CH2 OH HCHO O H
HO P O
OH
Phosphenytoin OH Phenytoin

HOH spontaneous
OH
AP cleavage
O CH2 O P O O CH2 OH OH
OH OH
HCHO
HO P O
Phospropofol OH Propofol

N N
Cl HOH
Cl
AP
N N
H H
O OH O
HO O HO O
HO P O
OH SCH3 OH SCH3
OH
OH
Clindamycin O P O Clindamycin OH
phosphate
OH
HYDROLYSIS BY PARAOXONASES (PON)
Hydrolysis of phosphoric acid tri-esters:

H3C CH2 O O H3C CH2 O O

PON
P P + HO NO2
H3C CH2 O O NO2 H3C CH2 O OH

Paraoxon
(the active metabolite of parathion, an OP insecticide,
irreversble acethylcholinesterase inhibitor)

Hydrolysis of lactone rings:

HO O
PON HO
C
O
O
O
OH
O C
OH PON OH
O O OH
H3C
H3C O
active
CH3 H3C
CH3 lovastatin inactive
spirono-
O lacton
H3C O 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
HO
Sennoside A
OH O O OH HO O OH
O
COOH
COOH COOH
2 HOH

COOH COOH
2 glucose
COOH
O

OH O O OH HO O OH
HO
OH
antraquinone glycoside active antraquinone
(prodrug) aglycone

N N
Bisacodyl
2 HOH
O O
2 acetate
H3C O O CH3 HO OH

Diacetic acid ester of Active diphenol-methane

diphenol-methane laxative (prodrug) laxative metabolite

Sodium N N
picosulfate 2 HOH
O O
S S 2 sulfate
NaO O O ONa HO OH
O O
Disulfonic acid ester of Active diphenol-methane
diphenol-methane laxative (prodrug) laxative metabolite
 PHASE II BIOTRANSFORMATIONS (CONJUGATIONS)
GLUCURONIDATION
Enzyme Cosubstrate Substrates: drugs, endogenous compounds
UDP-glucuronosyl UDP-glucuronic OH-containing compounds: morphine, paracetamol,
transferases 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
(in cytosol) OH-contaning metabolites: see above
N-oxide-containing compounds: minoxidil

CONJUGATION WITH GLYCINE

Enzyme Cosubstrate Substrates
Acyl-CoA synthetase ATP COOH-containing compounds: salicylic cid,
+ Glycine N-acyltransf. + CoA-SH benzoic acid,
(in mitochondrial matrix) + Glycine nicotinic acid

CONJUGATION WITH GLUTATHIONE (Glu-Cys-Gly)

Enzyme Cosubstrate Substrates
Glutathione S-transf. Glutathione Electrophilic C-containing compounds:
(in cytosol) (glu-cys-gly) ethacrinic acid, epoxides (e.g. LTA4)
Electrophilic C-containing metabolites:
N-acetyl-p-benzoquinone imine, acrolein

ACETYLATION
Enzyme Cosubstrate Substrates
N-acetyltransferases Acetyl-CoA NH2-containing compounds:
(in cytosol) NAT1 substrates: p-aminobenzoic acid,
sulfamethoxazol
NAT2 substrates: isoniazid, procainamide, dapsone,
hydralazine, sulfamethazine,

METHYLATION
Enzyme Cosubstrate Substrates
Methyltransferases S-adenosyl- OH-containing compounds: L-Dopa (COMT)
(in cytosol) methionine N-containing compounds: histamine, nicotine
SH-cont. compounds: 6-mercaptopurine (TPMT)
GLUCURONIDATION
Enzymes: UDP-glucuronosyltransferases (UDP-GT, UGT)
Cosubstrate: O
UDP-glucuronic acid (UDP-GA) _
NH COO
O
O O
O CH2 O P O P O
N
O _ _ HO OH
O O

OH

OH OH

Examples:
- Ether glucuronide formation (from compounds with a hydroxyl group) _
COO
O

OH OH
UDP-GT
O2N CH CH CH2 OH O2N CH CH CH2 O HO OH
UDP-GA
NH C CHCl 2 NH C CHCl2 OH
O O
Chloramphenicol Chloramphenicol glucuronide
(antibiotic) (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) _

COO
CH3 CH2 CH2 O CH3 CH2 CH2 O O
UDP-GT
CH C CH C
UDP-GA HO OH
CH3 CH2 CH2 OH CH3 CH2 CH2 O
OH
Valproic acid Valproic acid glucuronide
(antiepileptic drug) (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 _
COO
H2N N NH2 H2N N NH2 O
+
N UDP-GT N
N UDP-GA
N HO OH

Cl Cl OH
Cl Cl
Lamotrigine Lamotrigine N2-glucuronide
(antiepileptic drug) (rapidly excreted in urine)

Other: Carbamazepine
SULFATION
Enzymes: Sulfotransferases (SULT)
Cosubstrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS)
NH2
N

N
O O N
O S O P O CH2 N
O
O O

O OH
PO32-

Examples:
NH C CH3 NH C CH3
O O
SULT
PAPS
O
OH O S O
O
Acetaminophen = paracetamol acetaminophen-sulfate = paracetamol-sulfate
(analgetic and antipyretic) (rapidly excreted in urine)

Others: catecholamines (e.g., dopamine)

minoxidyl (see later)
hydroxylated metabolites of drugs
 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:
O O O
C C C
OH S CoA NH CH2 COOH
OH OH OH
ACS GNT
ATP, CoA-SH Gly

Salicylic acid Salicyl-CoA Salicyl-glycine = salicyluric acid

carboxyl- (rapidly excreted in urine)
esterase
Acetylsalicylic acid acetylation
(aspirin) and inactivation of COX

ACS GNT
Benzoic acid Benzoyl-CoA Benzoyl-glycine
ATP, CoA-SH Gly (hippuric acid)
AOX
ADH
Benzadehyde 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)
Notice that the SH group in GSH
Cosubstrate: Glutathione (GSH) is electron-rich (i.e., nucleophilic),
_ therefore it is reactive with
COO O O electron-deficient (i.e., electrophilic)
_ atoms, thus forming a glutathione conjugate.
CH CH2 CH2 C NH C NH CH2 COO
As drugs and chemicals with elecrophilic
CH atoms may also bind to protein-cysteines,
NH2
CH2 conjugation with glutathione prevents
their covalent binding to proteins. Therefore,
SH conjugation with GSH is an important
.. protective mechanism against reactive
-glutamic acid cysteine glycine 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 O C CH CH2 CH3
(+) CH CH2 SG
2
Ethacrynic acid (EA) EA-glutathione
(loop diuretic) conjugate

Note: the carbon with (+) sign is

electron-deficient (contains a partial positive
charge), thus it is electrophilic, therefore EA-Cys conjugate
reactive with GSH, whose SH group is (active diuretic metabolite)
electron-rich (nucleophilic).

(+) O O Acrolein-glutathione
Acrolein, GSH conjugate
a reactive electrophilic CH2 CH C GS CH2 CH2 C
(detoxified)
aldehyde that causes H H
hemorrhagic cystitis
in CP-treated patients
Cyclophosphamide (CP)
(antitumor drug, a nitogen-mustard)

HN C CH3 N C CH3 HN C CH3

O O O
CYP2E1 GSH
toxication (+) (+) detoxication
SG
OH O OH
Acetaminophen N-acetyl-p-benzo- Acetaminophen-glutathione
= paracetamol quinoneimine conjugate
(NAPBQI) (readily excreted)
ELECTROPHILIC METABOLITE
MAY CAUSE HEPATIC NECROSIS
ACETYLATION
Enzymes: N-acetyltransferases (NAT)

Cosubstrate: Acetyl coenzyme A (Ac-CoA)

NH2
N

N
O CH3 O O N
NH C CH C CH2 O P O P O CH2 N
OH CH3 O O O
CH2
CH2
C O O
O OH
NH CH2 CH2 S C CH3 -
PO3H

Examples:
O O O
C C
NH NH2 NH NH C CH3
NAT2
Ac-CoA
N N
Isoniazid Acetylisoniazid
(antituberculotic) (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 N
_
OOC CH CH2 CH2 S CH2 N
NH2
+ O

OH OH

Examples:
O-methylation entacapone

HO CH3 O

_ COMT _
HO CH2 CH COO HO CH2 CH COO
SAM
NH2 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

Histamine N-Methylhistamine

S-methylation
SH S CH3
N N
N TPMT N
SAM
N N N N
H H
6-Mercaptopurine
(antitumor drug) 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 7-hydroxy-warfarin CYP2C9
phenytoin 4-hydroxy-phenytoin CYP2C9
theophylline 1- or 3-methylxanthine CYP2A1
morphine morphine-3-glucuronide UDP-GT
acetaminophen acetaminophen-glucuronide UDP-GT
isoniazide acetyl-isoniazide NAT2

2. EXCEPTIONALLY: ACTIVE METABOLITE IS FORMED

a. Inactive parent compound (PRODRUG) active metabolite
cyclophosphamide phosphoramide mustard CYP2B6
tamoxifen 4-hydroxy-tamoxifen CYP2D6
parathion paraoxon CYP3A4
terfenadine alcohol acid CYP3A4
chloral hydrate trichloroethanol ADH (rev.), AR
sulfasalazine 5-aminosalicylic acid Azo-reductase
oxcarbazepine 10-hydroxy-carbazepine AK-reductase
lovastatin (lactone) lovastatin (free acid) Paraoxonase
enalapril (ester) enalaprilate (free acid) Esterase
fenofibrate (ester) fenofibric acid (free acid) Esterase
ezetimibe ezetimibe-glucuronide UDP-GT
minoxidyl minoxidyl-sulfate SULT
ethacrynic acid (EA) EA-cysteine GSTGGT, DP

b. Active parent compound active metabolite

phenylbutazone -OH-phenylbutazone CYP2D6, 2C9
carisoprodol meprobamate CYP2C19
risperidone 9-OH-risperidone (paliperidone) CYP2D6
imipramine desmethyl-imipramine CYP2D6
codeine morphine CYP2D6
diazepam nordiazepam oxazepam CYP CYP
morphine morphine 6-glucuronide UDP-GT

c. Non-toxic parent compound toxic metabolite

acetaminophen N-acetyl-p-benzoquinoneimine CYP2E1
halothane trifluoroacetyl chloride CYP2E1
cyclophosphamide acrolein CYP3A4, 2B6
methanol formic acid ADH ALDH
ethylene glycol glycolic-, glyoxylic- oxalic acid ADH ALDH
doxorubicin doxorubicinol AK-reductase
 BIOTRANSFORMATION OF DIAZEPAM,
FIRST INTO ACTIVE PHASE-I METABOLITES (NORDIAZEPAM, OXAZEPAM),
AND FINALLY INTO INACTIVE OXAZEPAM-GLUCURONIDE

PHASE-I biotransformation Examples of drugs

CH3 whose active metabolites are also drugs:
O
N
diazepam VALIUM > oxazepam SERAX
Diazepam risperidone RISPERDAL > paliperidon INVEGA
(antianxiety and terfenadine TELDANE* > fexofenadine TELFAST
Cl N antiepileptic drug) sulfasalazine SALAZOPYRIN > mesalazine SALOFALK
active enalapril ENALAPRIL tabl > enalaprilate VASOTEC inj
T1/2= 40 hrs [VALIUM]
* Withdrawn - see later

N-demethylation CYP2C19
CYP3A4
H
O
N

Nordiazepam
Cl N
active
metabolite

PHASE-II biotransformation
Hydroxylation CYP (conjugation)

H H
O O
N N
UDP-GT
OH O -Glucuronic acid
UDP-GA
Cl N Cl N

T1/2= 8 hrs

oxazepam oxazepam-glucronide
active metabolite inactive
and also anti anxiety drug [SERAX] 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 O N
N O N O
Risperidone 9-hydroxy-risperidone (active)
(antipsychotic; RISPERDAL),T1/2 = 3 hrs paliperidone (INVEGA), T1/2 = 25 hrs
 BIOTRANSFORMATION OF CYCLOPHOSPHAMIDE INTO
THERAPEUTICALLY ACTIVE METABOLITE (PHOSPHORAMIDE MUSTARD),
SOME INACTIVE METABOLITES,
AND A TOXIC METABOLITE (ACROLEIN)

NOTE: Steps 1 and 2

Cl O O
cyclophosphamide represent N-dealkylation,
M= N (antitumor drug) P starting with hydroxylation on
N M the C atom linked to the N atom,
Cl H then chain breaking at the
CYP3A4 arrow, and forming an aldehyde.
1 CYP2C9 Here the aldehyde is
CYP2B6 aldophosphamide.
O O O O O O
4-hydroxy-
P P P cyclo-
phosphamide
N M N M N M
H H
GS OH
4-glutathionyl- immino-
spontaneous
cyclophosphamide cyclophosphamide 2 cleavage

O O O O
ALDH1A1
aldophosphamide C P C P
O detoxication O
H M HO M
H2N H2N
O-carboxyethyl-
3 phosphoramide mustard

spontaneous
cleavage
O O O
SG GSH
C C HO P
H detoxication H M
H2N
Acrolein Phosphoramide mustard
reacts covalently with protein-SH reacts covalenly with DNA bases

SPECIFIC TOXIC EFFECTS ANTITUMOR EFFECT

and
Liver: GENERAL TOXIC EFFECT
damage to the of antitumor drugs
sinusoidal endothelial cells, e.g., bone marrow depression
causing
sinusoidal obstruction sy. (SOS)

Bladder:
damage to the
urothelial cells,
causing
hemorrhagic cystitis
BIOTRANSFORMATION OF TERFENADINE (A PRODRUG ANTIHISTAMINE THAT
IS PRONE TO CAUSE POLYMORPHIC VENTRICULAR TACHYCARDIA)
INTO FEXOFENADINE (AN ACTIVE ANTIHISTAMINE)

Terfenadine Terfenadine is INACTIVE as an

[TELDANE] antihistamine (it is a prodrug).
(withdrawn) As a K+ channel inhibitor, terfenanidine
CH3 iinhibits repolarization of cardiomyocytes
OH
and is arrhythmogenic. It has caused
HO C N (CH2)3 CH C CH3 polymorphic ventricilar extrasystoles
CH3 (called torsades de pointes) especially
when the patient also received a CYP3A4
inhibitor drug, such as ketoconazole or
erythromycin.

Hydroxylation CYP3A4
Ketoconazole
Erythromycin

OH CH3
HO C N (CH2)3 CH C CH2OH
CH3
alcoholic
metabolite

Dehydrogenation
Hydration CYP
Dehydrogenation

OH CH3
HO C N (CH2)3 CH C COOH
The carboxylic acid metabolite
CH3
is ACTIVE as an antihistamine!
carboxylic acid Now it is marketed as
metabolite 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,
N inactive as a vasodilator)
O
NH2

PAPS
Sulfotransferase

N N NH2

MINOXIDIL-SULFATE
N
O OH
NH2
S
O O

+
N N NH3
MINOXIDIL-SULFATE inner salt form
N (active vasodilator metabolite,
O O excreted slowly)
NH2
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 Morphine Morphine-6-glucuronide
INACTIVE ACTIVE SUPERACTIVE
cannot form an inner salt can form an inner salt
COOH
HO
O
H H
H
O HO 3
OH H
O
OH
H OH UDP-GT UDP-GT
O O NH
UDP-GA UDP-GA

NH NH COOH
6
O O
HO HO H
H
H OH
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 HO
OH OH OH
OH
UDP-GT

F N UDP-GA N
O F O

Ezetimibe
the absorption of
cholesterol decreases
Ezetimibe-glucuronide (EG)
F F
active metabolite
produced in the enterocytes
and the liver
Mrp-2
transporter
EG inhibits the
intestinal cholesterol Bile, intestinal tract
transporter (NPC1L1)

THE SERUM CHOLESTEROL

LEVEL DECREASES
 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 O O
HN C CH3 N C CH3 HN C CH3
Acetaminophen toxication
= Paracetamol by dehydrogenation
CYP2E1 (+) (+)
S protein
NAPBQI
OH O OH
detoxication detoxication
of paracetamol of NAPBQI
UDP-GA glutathione
UDP-GT (glu-cys-gly)

O O

HN C CH3 HN C CH3

S glutathione
O glucuronic acid OH

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
alcoholists liver the amount of CYP2E1 is increased, therefore more NAPBQI is formed, and
because (2) in the alcoholists 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 Physiological jaundice (see FIG)
Chloramphenicol Grey baby syndrome (see FIG)

Immature glycine conjugation

Benzyl alcohol Gasping syndrome (see FIG)

Immature CYP, UDP-GT

Diazepam Floppy infant syndrome (see FIG)
UDP-GT immaturity: physiological jaundice, chloramphenicol-induced grey baby sy.
Bilirubin (not excreted into bile) Bilirubin diglucuronide (excreted into bile)
H H H H H H H H
O N CH N CH2 N CH N O O N CH N CH2 N CH N O
UDP-GT

H3C H3C
UDP-GA H3C H3C
CH 3 CH CH2 CH 3 CH CH2
CH 2 CH CH 2 CH2 CH 3 CH 2 CH CH 2 CH2 CH 3
CH2 CH2 _ CH2 CH2 _
OOC O O COO
C C C C
HO O O OH O O O O
OH HO
HO OH
HO OH

Chloramphenicol (CL) Chloramphenicol glucuronide _

COO
OH OH O
UDP-GT
O2N CH CH CH2 OH O2N CH CH CH2 O
UDP-GA HO OH
NH C CHCl2 NH C CHCl2
OH
O 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 Cytochrome P450
ADH Alcohol-dehydrogenase
AOX Aldehyde-oxydase
BCS Benzoyl-CoA-synthetase
BC:GT Benzoyl-CoA:glycine-N-acyl-transferase
Benzyl-alcohol BA accumulation
acidosis LOW CAPACITY STEP
gasping IN NEONATES
ADH

O O O O
C C C C
H OH S-CoA NH CH2 COOH
AOX BCS BC:GT
URINE
CoA-SH glycine
O2=
benzaldehyde benzoic acid benzoyl-CoA benzoyl-glycine
(BA) (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
diazepam FIS developes in a baby
active whose mother received diazepam
Cl N [VALIUM] before delivery.
T1/2= 40 hrs

CYP2C19 SLOW STEPS

CYP3A4 IN NEONATES

H H H
O O O
N N N
CYP UDP-GT
OH O glucuronic acid
UDP-GA
Cl N Cl N Cl N

T1/2= 8 hrs

nordiazepam oxazepam oxazepam-glucronide

active active inactive
[SERAX]

Immature alcohol dehydrogenase: delayed elimination of ethanol from the baby

monkey whose mother received ethanol before delivery
BLOOD ETHANOL CONC. (mg%)

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 Adverse effects at regular dose:
CYP2C9 deficiency
Warfarin hydroxylation Bleeding
CYP2C19 deficiency
Diazepam N-demethylation Prolonged sedation
CYP2D6 deficiency
Debrisoquin hydroxylation Pronounced hypotension
N-dealkylation of TCADs Pronounced sedation, toxicity
N-dealkyl. of antipsychotics Tardive dyskinesia
(involuntary movements, tics)

2. Carboxylesterase (pseudocholinesterase) deficiency

Succinylcholine Prolonged paralysis

3. NAT2 deficiency slow acetylator phenotype

Isoniazide Neurotoxicity, hepatotoxicity
Procainamide, hydralazine 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 ligand-
activated 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 CYP3A4 (CYP3A7)

Nucleus RXR

*Ligands: rifampicin, phenobarbital, hyperforin (St. John's wort), etc.

PXR = Pregnane X receptor
RXR = Retinoid X receptor
CYP Inducer Inducer Inhibitors (often
Drug substrates
family chemicals receptors (TFs) competing substrates)
A1: barely expressed, unless induced. Drugs: omeprazole AHR: A2: ciprofloxacine
CYP1 Others: PAHs (charcoal-
A2: caffeine, theophylline, tizanidine, ondansetron, fluvoxamine
broiled meat, smoking), Aryl
acetaminophen TCDD, indole-3-carbinol hydrocarbon
cimetidine
(in brussels sprouts, acyclovir
receptor
broccoli, cabbage)
CYP2 B6: cyclophosphamide, bupropion, ketamine B6, C9, C19: CAR: B6: clopidogrel
C9: phenytoin, warfarin, tolbutamide phenobarbital ticlopidin
C19: omeprazole, diazepam, fluvastatin rifampin Constitutive C9: amiodarone
D6: Psychotropic drugs: SSRI (citalopram, fluoxetine, B6: phenytoin androstane (+D6), fluconazole
paroxetine), TCAD (amitriptyline, clomipramine, E1: ethanol, receptor C19: moclobemide
desipramine, imipramine, nortriptyline), clozapine, isoniazide D6: cimetidine
mianserine, mirtazapine, venlafaxine. Cardiovascular quinidine
drugs: -blockers (alprenolol, bufuralol, metoprolol, (CYP2D6 is NOT terbinafine
propranolol, timolol), encainide, flecainide, inducible) fluoxetine
propafenone. Miscellaneous drugs: codeine, paroxetine
debrisoquine, dextromethorphan, phenformin E1: disulfiram
E1: halothane, acetaminophen, dapsone, theophylline
CYP3 Benzodiazepines: alprazolam, clonazepam, rifampin, rifabutin PXR: A4:
clorazepate, diazepam, flurazepam, halazepam, phenobarbital Drugs:
(3A4) midazolam, prazepam, triazolam. Calcium channel phenytoin
Pregnane X
Azole antifungals
blockers: amlodipine, diltiazem, felodipine, isradipine, carbamazepine receptor Macrolide antibiot.
lercanidipine, nifedipine, nisoldipine, nitrendipine, nifedipine HIV protease inhib
verapamil. HIV antivirals: indinavir, nelfinavir, ritonavir, dexamethasone Gestodene (suicide
saquinavir. Immunesuppressive drugs: cyclosporine, spironolactone inhibitor contrac. ster)
tacrolimus, sirolimus. Macrolide antibiotics: lovastatin
clarithromycin, erythromycin. Steroids: estradiol, simvastatin Dietary chemicals:
hydrocortisone, progesterone, testosterone. Statins: hyperforin (an naringenin: a flvonoid,
bergamottin: a furano
lovastatin, simvastatin, atorvastatin. Others: buspirone, antidepressant in St -cumarin (both present
codeine, dextromethorphan, methadone, taxol John's wort) 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 paracetamol-
induced 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.)

30 15

Simvastatin acid (ng/ml)

25
Simvastatin (ng/ml)

20 grapefruit juice + SV 10
grapefruit juice + SV
15

10 5
water + SV water + SV
5

0 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 OH
O O
Paraoxonase
O O
HOH

Simvastatin Simvastatin acid

(inactive) (active)
CYP3A4 naringenin, bergamottin; itraconazole, verapamil

HO O HO O HO O

O O O
O O O

O O O

HO

OH H2C

6'-hydroxy SV 3'-hydroxy SV 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 fibrate-
type 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 Terfenadine is INACTIVE as an

[TELDANE] antihistamine (it is a prodrug)
(withdrawn) As a K+ channel inhibitor, terfenanidine
CH3 iinhibits repolarization of cardiomyocytes
OH
and is arrhythmogenic. It has caused
HO C N (CH2)3 CH C CH3 polymorphic ventricilar extrasystoles
CH3 (called torsades de pointes) especially
when the patient also received a CYP3A4
inhibitor drug, such as ketoconazole or
erythromycin.

Hydroxylation CYP3A4
Ketoconazole
Erythromycin

OH CH3
HO C N (CH2)3 CH C CH2OH
CH3
alcoholic
metabolite

Dehydrogenation
Hydration CYP
Dehydrogenation

OH CH3
HO C N (CH2)3 CH C COOH
The carboxylic acid metabolite
CH3
is ACTIVE as an antihistamine!
carboxylic acid Now it is marketed as
metabolite 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 N
S S S
Cl N Cl N Cl N
hydroxylation dehydrogenation
H H H
N NH CYP1A2 N CYP1A2 N
NH NH

N Tizanidine N N
HO O

hydroxylation CYP1A2 FLUVOXAMINE, CIPROFLOXACIN, ROFECOXIB

OH Tizanidine, a centrally acting muscle relaxant, is rapidly

N biotransformed by CYP1A2. This is responsible for both its
S hepatic presystemic elimination (F = 0.7) and rapid
Cl N elimination (T1/2 = 2.5 hrs) of tizanidine. Therefore, drugs
that inhibit CYP1A2 (e.g., fluvoxamine, ciprofloxacin,
H
N NH rofecoxib) can increase the AUC of tizanidine several fold.
At high level, tizanidine, which is a 2-receptor agonist like
N clonidine, can cause marked hypotension, bradycardia and
sedation.

-------------------------------------------------------------------------------------------------------------------

O CH3 O CH3
N N
CH3 CH3

-hydroxytamoxifen
(active antiestrogen;
CYP2D6 used against breast cancer)

tamoxifen
(prodrug) SSRI
(e.g. fluoxetine OH
paroxetine)

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 4-
hydroxytamoxifen, 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