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432
INTRODUCTION
edge and understanding rather than encyclopedic information. Readers wanting to go further
in the study of xenobiotic metabolism may consult various classic or recent books (1-10).
1.1
1 introduction
Drws
Food constituents devoid of physiological roles
Food additives (preservatives, coloring and
flavoring agents, antioxidants, etc.)
Chemicals of leisure, pleasure, and abuse (ethanol,
coffee and tobacco constituents, hallucinogens,
doping agents, etc.)
Agrochemicals (fertilizers, insecticides, herbicides,
etc.)
Industrial and technical chemicals (solvents, dyes,
monomers, polymers, etc.)
Pollutants of natural origin (radon, sulfur dioxide,
hydrocarbons, etc.)
Pollutants produced by microbial contamination
(e.g., aflatoxins)
Pollutants produced by physical or chemical
transformation of natural compounds (polycyclic
aromatic hydrocarbons by burning, Maillard
reaction products by heating, etc.)
sum of the processes affectingthe fate of a chemical substance in the body) and with biotransformation as understood in this chapter (12).
In pharmacology, one speaks of pharmacodynamic effects to indicate what a drug does to
the body and pharmacokinetic effects to indicate what the body does to a drug; two aspects
of the behavior of xenobiotics that are strongly
interdependent. Pharmacokinetic effects will
obviously have a decisive influence on the intensity and duration of pharmacodynamic effects, whereas metabolism will generate new
chemical entities (metabolites) that may have
distinct pharmacodynamic properties of their
own. Conversely, by its own pharmacodynamic effects, a compound may affect the state
of the organism (e.g., hemodynamic changes,
enzyme activities) and therefore the organism's capacity to handle xenobiotics. Only a
systemic approach can help one appreciate the
global nature of this interdependence (13).
1.2 Types of Metabolic Reactions Affecting
Xenobiotics
1 Introduction
GLUC
GLUC
/ \
GLUC
HO
SULF
GLUC
SULF
GLUC
Figure 13.1. The metabolism of propranolol(1)in humans, accounting for more than 90%of the
dose. GLUC, glucuronide(s);SULF, sulfate(s) (22).
SULF
2 Functionalization Reactions
437
Enzymes
Number of
Substratesb
Overall
Contribution
to Drug
Metabolism
and the overall contribution to drug metabolism does not need to be perfect, because some
enzymes show a limited capacity (e.g., sulfotransferases), whereas others make a significant contribution to the biotransformation of
their substrates (e.g., hydrolases).
reactions are of major significance in drug metabolism and are mediated by various enzymes
that differ markedly in their structure and
1. The enzyme in its ferric (oxidized)form exists in equilibrium between two spin states,
2 FUNCTIONALIZATION REACTIONS
2.1 Introduction
Table 13.5 The Human CYP Gene Superfamily: A Table of the Families and Subfamilies of
Gene Products (7,24-28)
Families
Subfamilies
--
P450 1A Subfamily
P450 1B Subfamily
P450 2A Subfamily
P450 2B Subfamily
(Includes
phenobarbitalinducible forms)
P450 2C Subfamily
(Constitutive
forms; includes
sex-specific
forms)
P450 2D Subfamily
P450 2E Subfamily
(Ethanolinducible)
P450 2F Subfamily
P450 25 Subfamily
P450 3A Subfamily
P450 4A Subfamily
P450 4B Subfamily
P450 4F Subfamily
P450 5A Subfamily
P450 7A Subfamily
P450 7B Subfamily
P450 8A Subfamily
P450 8B Subfamily
P450 11A
Subfamily
P450 11B
Subfamily
(Steroid
hydroxylases)
CYP2F1
cYP2J2
CYP3A4, CYP3A5, CYP3A7 fital CYP
enzyme), CYP3A43
CYP4A11 (Fatty acid w and ( ~ 1 ) hydroxylases)
CYP4B1
CYP4F2, CYP4F3, CYP4F8, CYP4F12
CYP5A1 (TXAsynthase)
CYP7A1
CYP7B1
CYP8Al (Prostacyclin synthase)
CYP8B1
CYPllAl (Cholesterol side-chain '
cleavage)
CYPllBl, CYP11B2
P450 26A
Subfamily
P450 27A
Subfamily
P450 27B
Subfamily
CYP39
CYP46
CYP51 (Lanosterol 14a-demethylase)
This list reports all human CYPs with known substrate(s) andlor inhibitor(s1. At the time of writing, human CYPs of
unknown function were 2Al,2R1,251,2Ul, 2W1,4A20, 4A22,4Fll, 4F22,4V2,4Xl, 20,26B1,26Cl, and 27C1 (28).
2 Functionalization Reactions
439
Table 13.6 Levels and Variability of Human CYP Enzymes Involved in Drug
Metabolism (27,28)
CYP
Level of
Enzyme in
Liver
(% of Total)
As
Substrates
As.
Inhibitors
As Inducers1
Activators
6 (33)
3 (25)
1 (9)
2 (1)
13 (no data)
21 (6)
2 (2 activators)
7 (7)
45 (17)
2.
3.
4.
5.
6.
7.
8.
XOH
Hz0
Figure 13.2. Catalytic cycle of cytochrome P450 associated with monooxygenase reactions. (Fe3+),
ferricytochrome P450; hs, high spin; Is, low spin; (Fez+), ferrocytochrome P450; F,,, flavoprotein
1-NADPH-cytochrome P450 reductase; F,,, NADH-cytochrome b, reductase; cyt b,, cytochrome
b,; XH, substrate (modified from Ref. 6).
of lesser relevance (31). Aldehyde reductases are widely distributed in nature and occur in a considerable number of mammalian
tissues. Their subcellular location is primarily
cytosolic, and in some instances is also mitochondrial. The so-called ketone reductases include a- and P-hydroxysteroid dehydrogenases (e.g., EC 1.1.1.50 and EC 1.1.1.51),
various prostaglandin ketoreductases (e.g.,
prostaglandin-F synthase, EC 1.1.1.188; prostaglandin-E, 9-reductase, EC 1.1.1.189), and
many others that are comparable with aldehyde reductases. One group of particular
importance are the carbonyl reductases
(NADPH; EC 1.1.1.184). Furthermore, the
many similarities (including some marked
overlap in substrate specificity) between monomeric, NADPH-dependent aldehyde reductase (AKRl),aldose reductase (AKR2),and carbony1 reductase ( A m ) have led to their
designation as aldo-ketoredudases (AKRs) (32).
Other reductases that have a role to play
in drug metabolism include glutathione reduc-
I
i
2 Functionalization Reactions
1
f
8.
I
:
nient from a mechanistic viewpoint to distinguish between sp3-, sp2-, and sp-carbon atoms.
2.3.1 sp3-Carbon Atoms. Reactions of oxi-
Figure 13.4. Halothane (2) and two of its metabolites, namely trifluoroacetic acid (3) produced by oxidation and a reactive radical (4) produced by reduction.
pathways, catalytic mechanisms, and products (Fig. 13.5). Thus, the oxidation of aromatic rings generates a variety of (usually stable) metabolites. Their common precursor is
often a reactive epoxide (reaction 1-A), which
can either be hydrolyzed by epoxide hydrolase
(reaction 1-B) to a dihydrodiol or rearranged
under proton catalysis to a phenol (reaction
1-C).The production of a phenol is a very com- '
mon metabolic reaction for drugs containing
one or more aromatic rings. Thepara-position
is the preferred position of hydroxylation for
unsubstituted phenyl rings, but the regioselectivity of the reaction becomes more complex with substituted phenyl or with other aromatic rings.
Dihydrodiols are seldom observed, as are
catechol metabolites produced by their dehydrogenation catalyzed by dihydrodiol dehydrogenase (reaction 1-D). It is interesting to
note that this reaction restores the aromaticity that had been lost on epoxide formation.
The further oxidation of phenols and phenolic
metabolites is also possible, the rate of reaction and the nature of products depending on
the ring and on the nature and position of its
substituents. Catechols are thus formed by reaction 1-E, whereas hydroquinones are sometimes also produced (reaction 1-F).
In a few cases, catechols and hydroquinones have been found to undergo further oxidation to quinones (reactions 1-G and 1-11.
Such reactions occur by two single-electron
steps and can be either enzymatic or nonenzymatic (i.e., resulting from autoxidation and
yielding as by-product the superoxide anionradical 0,'-). The intermediate in this reaction is a semiquinone. Both quinones and
semiquinones are reactive, particularly to-
R'
(2) R'-CH-CH-R"
R"
/"="\R"' - w.-&R
OH R"
R"PfR
R
OH
Figure 13.5. Major functionalization reactions involving an sp2- or spcarbon in substrate molecules. These reactions are oxidations (oxygenationsand dehydrogenations), reductions (hydrogenations), and hydrations, plus some postenzymatic rearrangements.
2 Functionalization Reactions
R-NH2
R-NHOH
G R-N=O
d
R-NH-NH-R'
R-N=N-R'
R-NO2
R-N=N-R'
Figure 13.7. Major functionalization reactions involving nitrogen atoms in substrate molecules.
The reactions shown here are mainly oxidations (oxygenations and dehydrogenations) and reductions (deoxygenations and hydrogenations).
to oximes (reaction 5-C), which are in equilibrium with their nitroso tautomer (reactions
5-F and 5-G).
1,4-Dihydropyridines,and particularly calcium channel blockers such as nivaldipide (7)
(Fig. 13.8), are effectively oxidized by cytochrome P450. The reaction is one of aromatization (reaction 6 in Fig. 13.7), yielding the
corresponding pyridine.
Dinitrogen moieties are also targets of oxidoreductases. Depending on their substituents, hydrazines are oxidized to azo compounds (reaction 7-A), some of which can be
oxygenated to azoxy compounds (reaction
1-D). Another important pathway of hydrazines is their reductive cleavage to primary
amines (reaction 7-C). Reactions 7-A and 7-D
are reversible and the corresponding reduc-
2 Functionalization Reactions
(7)
dration between a thiol and a sulfenic acid (reaction 1-B). However, our understanding of sulfur biochemistry is largely incomplete, and
much remains to be learned. This is particularly
true for reductive reactions. Whereas it is well
known that reaction 1-C is reversible (i.e., reaction 1-D),the reversibility of reaction 1-A is unclear and reduction of sulfinic and sulfonic acids
seems unlikely.
The metabolism of sulfides (thioethers) is
rather straightforward. Besides the S-dealkylation reactions discussed earlier, these
compounds can also be oxygenated by monooxygenases to sulfoxides (reaction 2-A) and
then to sulfones (reaction 2-C). Here, it is
known with confidence that reaction 2-A is indeed reversible, as documented by many examples of reduction of sulfoxides (reaction
2-B), whereas the reduction of sulfones has
never been found to occur.
Thiocarbonyl compounds are also substrates
of monooxygenases, forming S-monoxides
(sulfines, reaction 3-A) and then S-dioxides
(sulfenes, reaction 3-C). As a rule, these metabolites cannot be identified as such because of
their reactivity. Thus, S-monoxides rearrange
to the correspondingcarbonyl by expelling a sulfur atom (reaction 3-D). This reaction is known
as oxidative desulfuration and occurs in thioarnides and thioureas (e.g., thiopental). As for the
S-dioxides, they react very rapidly with nucleophiles and particularly with nucleophilic sites in
biological macromolecules. This covalent binding results in the formation of adducts of toxico-
\-+
R-SH
R-SOH
R-S02H
R-S03H
R- S- S- R'
S
(3)
II
R- C- R'
Y R- CO- R'
NHCOCH3
Q-
NCOCH3
l$
OH
(8)
(9)
Figure 13.10. Paracetamol (8) and its toxic quinoneimine metabolite (9).
(4)
R-AsyAs-R
(5)
R-SeH
R-As=O
c--C
R-As03H2
R-SeOH
---+
R-Se02H
2 Functionalization Reactions
(3)
(4)
R-COO-R'
R-ON02
R-0S03H
R-CONHR'
R-COOH
R-OH
'-OH
R-COOH
R'-OH
HN03
H2S04
R'-NH2
CONJUGATION REACTIONS
3.1
lntroduction
Methylation
3.2.1 lntroduction. Reactions of methyl-
3 Conjugation Reactions
(5)
..
R-SH
R-S-CH3
Sulfation
3 Conjugation Reactions
(11
R'\
CH- OH
R'
(3)
R'\
N- OH
R"
R'\
R'\
CH-OS03
R/
N-OS03
R'\
+
R"
R'\
/CH+
R
Figure 13.18. Major sulfation reactions involving primary and secondary alcohols, phenols, hydroxylamines and hydroxylamides, and
amines.
3.4
major and very frequent reaction of conjugation. It involves the transfer to the substrate
of a molecule of glucuronic acid from the
cofactor uridine-5'-diphospho-a-D-glucuronic
acid (23) (UDPGA; Fig. 13.20). The enzyme
catalyzing this reaction is known as UDP-glucuronyltransferase (UDP-glucuronosyltransferase; EC 2.4.1.17, UDPGT) and consists of a
number of proteins coded by genes of the UGT
superfamily. The human UDPGT known to
metabolize xenobiotics is the product of two
gene families, UGTl and UGT2. These enzymes include UGTlAl (bilirubin UDPGTs)
and several UGTlA, as well as numerous phenobarbital-inducible or constitutively expressed UGT2B (57-61).
In addition to glucuronidation, this section
briefly mentions glucosidation, a minor metabolic reaction seen for a few drugs. Candidate
enzymes catalyzing this reaction could be
COOH
An important pathway of O-glucuronidation is the formation of acyl-glucuronides (reaction 3). Substrates are arylacetic acids (e.g.,
diclofenac) (15) (Fig. 13.16) and aliphatic acids (e.g., valproic acid). Aromatic acids are seldom substrates; a noteworthy exception is diflunisal(20) (Fig. 13.191, which yields both the
acyl and phenolic glucuronides. The significance of acyl glucuronides has long been underestimated, perhaps because of analytical
difficulties. Indeed, these metabolites are quite
reactive, rearranging to positional isomers and
binding covalently to plasma and seemingly also
tissue proteins (63). Thus, acyl glucuronide formation cannot be viewed solely as a reaction of
inactivation and detoxification.
A special class of acyl glucuronides are the
carbamoyl glucuronides (reaction 4 in Fig.
13.21). A number of primary and secondary
amines have been found to yield this type of
conjugate, whereas, as expected, the intermediate carbamic acids are not stable enough to
be characterized. Carvedilol (24) (Fig. 13.22)
is one drug exemplifying the reaction, in addition to forming an 0-glucuronide on its alcohol
group and a carbazole-N-linkedglucuronide (see
below) (64). Much remains to be understood
concerning the chemical and biochemical reactivity of carbamoyl glucuronides.
Hydroxylamines and hydroxylamides may
also form 0-glucuronides (reaction 5, Fig.
13.21). Thus, a few drugs and a number of
aromatic amines are known to be N-hydroxylated and then 0-glucuronidated. The glucuronidation of N-OH groups competes with
0-sulfation, but the reactivity ofN-0-glucuronides to undergo heterolytic cleavage and
form nitrenium ions does not seem to be well
characterized.
R- COOH
R'\
/
R'\
N-COOH
"
R'\
N-CO-0-GLU
R'
N-OH
GLU
R-CO-N-R'
R-CO-N-R'
R-SH
R- CSSH
GLU
R-SO2-N-R'
R-S-GLU
R-CS-S-GLU
GLU
(14) R-CO-CH2-CO-R'-
R-CO-CH-CO-R'
COOH
GLU =
Figure 13.21. Major glucuronidation reactions involving phenols, alcohols, carboxylic acids, carbamic acids, hydroxylamines and hydroxylamides, carboxamides, sulfonamides, various amines, thiols, dithiocarboxylic acids, and 1,3-dicarbonyl compounds.
More and more drugs of this type (e.g., antihistamines and neuroleptics), are found to undergo this reaction to a marked extent in humans, e.g., cyproheptadine (27) in Fig. 13.23).
Third in importance are the S-glucuronides formed from aliphatic and aromatic
thiols (reaction 12 in Fig. 13.21) and from dithiocarboxylic acids (reaction 13) such as diethyldithiocarbamic acid, a metabolite of disulfiram. As for C-glucuronidation (reaction
14),this reaction has been seen in humans for
l,3-dicarbonyl drugs such as phenylbutazone
and sulfinpyrazone (28) (Fig. 13.23).
3.4.3 Clucosidation Reactions. A few drugs