Download Inhibition and induction of human cytochrome P450 (CYP) enzymes

x e n o b io t ic a
, 1998, v o l . 28, n o . 12, 1203± 1253
Inhibition and induction of hum an cytochrom e P450
(C YP) enzym es
O. PELKONEN ‹ *, J. M AÈ ENPAÈ AÈ Œ , P. TAA VITSAINEN ‹ ,
A. RAUTIO ‹ and H. RAU NIO ‹
‹ Department of Pharmacology and Toxicology, University of Oulu, FIN-90220 Oulu,
Finland
ΠClinical Research, Leiras OY, PO Box 325, FIN-00101, Helsinki, Finland
Received January 1998
In trodu ction
Detailed knowledge of metabolism of drugs is crucial for two main reasons.
First, metabolism determines to a large extent pharmacokinetic behaviour, interindividual variability and interactions of a drug, all matters of great importance
in drug treatment. Second, diå erences in metabolism are also often behind the
diæ culties in the extrapolation from animals to man, which is a serious obstacle in
drug testing and development.
There is a large number of factors aå ecting drug metabolism and they are usually
classi® ed into genetic and non-genetic host and environm ental factors. In the last
category, chemical exposures, including drug treatment, occupational exposure to
chemicals or environmental pollution can lead either to induction or inhibition of
drug metabolism.
Induction is de® ned as the increase in the amount and activity of a drugmetabolizing enzyme, which is a long-term (hours and days) consequence of a
chemical exposure. Inhibition of drug metabolism in general may mean either an
acute decrease of metabolism of a particular substrate by another simultaneously
present chemical or a time-dependent decrease in the amount of a drug-metabolizing
enzyme by several factors, such as a chemical injury or a disease process. In this
review, we will deal only with interactions at the level of enzymes.
Previously, the study of induction and inhibition of drug metabolism was largely
empirical and phenomenological, and prediction beyond the compounds under
study was very diæ cult, if at all possible. During the past decade, how ever, and
particularly as a consequence of the detailed know ledge obtained about cytochrome
P450 (CY P) enzymes, both induction and inhibition can be understood on a detailed
mechanistic basis and the predictability of pharmacological and toxicological
consequences has become possible.
As to clinical consequences of induction and inhibition, the nature of the
products determine the outcome. If the reaction to be studied leads to inactive
product(s), induction results in attenuation and inhibition results in exaggeration of
the eå ects of a drug. If the product is active, either pharmacologically or
toxicologically, the reverse outcome is observed.
This review covers the phenomena of induction and inhibition of human CYPs
and concentrates upon quantitative aspects of in vitro and in vivo studies. This
* Author for correspondence.
0049± 8254} 98 $12 . 00 ’
1998 Taylor & Francis Ltd
1204
O. Pelkonen et al.
approach is hoped to provide a background for quantitative extrapolation of results
obtained from in vitro experimental systems to the in vivo situation, both for
induction and inhibition.
C haracteriza tion of hu m an C YP s in th e liver
Hepatic patterns of CYP enzymes
Since the 1980s our know ledge on speci® c forms of the P450 system in human
tissues has increased enormously. As a result of protein puri® cation, antibody
production, immunoinhibition, use of panels of substrates and inhibitors, and the
cloning, sequencing and heterologous expression of CY P cDNAs, a detailed
know ledge on speci® c properties of enzymes has been achieved. For further
information see the recent extensive reviews of Nebert (1989, 1991), Gonzalez
(1990, 1992), Guengerich (1992, 1994) and W righton and Stevens (1992).
A schematic presentation of some pertinent characteristics of the major human
hepatic CYP enzymes is given in ® gure 1. This qualitative ® gure serves as a
background and a synopsis for the sections dealing with quantitative aspects of
inhibitors and inducers with special emphasis on CYP speci® city and on semiquantitative extrapolation.
From the pharmacological and toxicological point of view, each enzyme can be
characterized on the basis of more or less selective substrates, inhibitors and
inducers. The relative amounts of various enzymes are naturally of importance, but
it should be kept in mind that the kinetic characteristics of enzymes towards
particular substrates and inhibitors are actually of importance for metabolism and
clearance of drugs and for metabolic interactions.
Interindividual variability of CYP enzymes
A phenom enon that cannot be overemphasized in the ® eld of xenobiotic
metabolism is interindividual variability, which results in very individualized
patterns of enzyme composition and hence metabolic activities. Permanent
determinants causing variability are genetic factors, which result in pharmacokinetically distinct subpopulations, for example extensive and poor metabolizers
due to polym orphisms in CYP 2D6 (Meyer 1994) and CYP2C1 9 (Goldstein and De
M orais 1994). It seems probable that there is at least some element of genetic
component in the variability of every CYP-associated activity (Pelkonen and Raunio
1997). On the other hand, numerous environmental factors add further variation,
which are not usually permanent, but transient. Induction and inhibition are
typically transient environmental factors, although it seems clear that the extent
(and m aybe also the pattern) of induction may be determined by genetic factors.
Figure 1 depicts schematically the situation in the liver. There are some
enzymes, such as CY P2F1 and CYP4B 1, that are expressed almost exclusively in
certain extrahepatic tissues (Raunio et al. 1995). Some enzymes, CYP 1A1 (Raunio
et al. 1995) and CYP1B1 (Sutter et al. 1994, Hakkola et al. 1997) foremost, seem to
be present and } or induced mainly (if not solely) in extrahepatic tissues and are
therefore unlikely to be quantitatively of great importance in pharmacokinetics. The
rest of the CYP forms display a substantial variability, which has to be taken into
consideration in in vitro± in vivo extrapolation, but this is seldom currently done.
Figure 1. Schematic representation of human hepatic P450 enzymes with model substrates, inhibitors and inducers (modi® ed from Pelkonen and
Breimer 1994). The size of the circles is roughly proportional to the relative amounts in human liver. Broken circles indicate that the presence
of enzymes is uncertain, or very low, or may appear only after induction.
CYP3A4/5/7
Dextromethorphan
P450 inhibition and induction in man
1205
1206
Table 1.
O. Pelkonen et al.
Compounds and reactions claimed to demonstrate a high degree of human CYP speci® city.
CYP
Preferred substrate and reaction
1A2
phenacetin O-deethylation
ethoxyresoru® n O-deethylation
coumarin 7-hydroxylation
2A6
2B6
2C8
4-tri¯ uoro-7-etho xycoumarin
O-deethylase
taxol hydroxylation
2C9
tolbutamide methylhydroxylation
2C19
diclofenac hydroxylation
S-warfarin 7-hydroxylation
S-mephenytoin 4-hydroxylation
2D6
omeprazole oxidation
debrisoquine 4-hydroxylation
3A4
30
0.2
0.4
800
3.6
50
7
20
18
50
400
15
45
0.5
5
10
165
6
2
5
5
bufuralol 1 ´-hydroxylation
40
12
chlorzoxazone 6-hydroxylation
aniline 4-hydroxylation
testosterone (steroid) 6bhydroxylation
midazolam 1-hydroxylation
40
15
47
90
90
25
4
50
nifedipine dehydrogenation
15
900
K and V
m
V
max
(nmol}
mg 3 h)
4
4
60
dextromethorpan O-deethylation
2E1
K
m
(l m )
max
Reference
Bourrie et al. (1996)
Bourrie et al. (1996)
Pearce et al. (1992)
Bourrie et al. (1996)
Buters et al. (1993)
Harris et al. (1994)
Sonnichsen et al. (1995)
Knodell et al. (1987)
Bourrie et al. (1996)
Transon et al. (1996)
table 6
Kato et al. (1992)
Chiba et al. (1993)
Relling et al. (1989)
Andersson et al. (1993)
Boobis and Davies (1984)
Narimatsu et al. (1993)
Fischer et al. (1992)
Transon et al. (1996)
Rodrigues and Roberts (1997)
Bourrie et al. (1996)
Kerry et al. (1994)
Ching et al. (1995)
Le Guellec et al. (1993)
Boobis and Davies (1984)
Halliday et al. (1995)
Yamazaki et al. (1994)
Peter et al. (1990)
Bourrie et al. (1996)
Waxman et al. (1983)
Kronbach et al. (1989)
Schmider et al. (1995)
Ghosal et al. (1996)
Transon et al. (1996)
Bourrie et al. (1996)
are approximate and some are con® rmed and modi® ed by our own unpublished studies.
Two subfamilies, namely CYP 2C and CY P3A, are somewhat problematic
because they contain several closely related enzymes and there is still some
uncertainty about the assignment of speci® c activities with speci® c forms.
Substrate and inhibitor selectivity
From the point of view of this review, the most interesting characteristics of CY P
enzymes are substrate speci® city and inhibitor selectivity and in tables 1 and 2
several ` speci® c ’ or ` diagnostic ’ substrates and inhibitors have been listed, as they
are currently used. It must be stressed here that speci® city has in most cases only
a relative meaning, as will be later shown for some of these compounds. The term
` selectivity ’ should, in principle, be more appropriate. For example, substrates
which earlier were often used as ` isoform-speci® c ’ (at the time when the dichotomy
was principally between cytochromes P448 and P450), such as benzo(a)pyrene or
P450 inhibition and induction in man
Table 2.
CYP
Enzyme-speci® c ` diagnostic ’ inhibitory probes for human P450 enzymes.
Inhibitor
Target CYP
Inhibition
(K , l m )
furafylline
0.7
2A6
¯ uvoxamine
methoxsalen
0.2
0.3
2D6
2E1
3A4
pilocarpine
sulfaphenazole
teniposide
¯ uconazole
quinidine
diethyldithiocarbamate
troleandomycin
ketoconazole
gestodene
Next sensitive
CYP (K , l m )
i
1A2
2C9
2C19
1207
i
"
10
4
0.3
12
2
0.06
"
8 .2 (CYP2D6)
2 (CYP2E1)
10 (CYP2C9)
25
not known
8 (2C9)
10 (3A4)
2
"
18
0.1
7
"
7 (CYP2A6)
ND*
10
ND*
References
Bourrie et al. (1996)
Clarke et al. (1994)
Nemeroå et al. (1996)
Ma$ enpa$ a$ et al. (1994)
Yamazaki et al. (1992)
Bourrie et al. (1996)
Bourrie et al. (1996)
Relling et al. (1989)
Kunze et al. (1996)
Bourrie et al. (1996)
Guengerich et al. (1986)
Yamazaki et al. (1992)
Zhou et al. (1993)
Bourrie et al. (1996)
Schmider et al. (1995)
Guengerich et al. (1992)
ND, not determined.
benzphetamine, are in fact not very speci® c (Levin 1990, Soucek and Gut 1992).
Later on, research has been directed towards ® nding truly enzyme-speci® c
substances, or compounds which are metabolized at speci® c positions by speci® c
enzymes, e.g. testosterone (Waxman et al. 1983, 1991) or warfarin (Kaminsky 1989,
Rettie et al. 1989). Obviously ` enzyme-speci® city ’ is not a suæ cient prerequisite
enough for a substance that is intended to be used also in vivo, but it is an important
starting point.
W ith respect to inhibitors of enzyme activity, many substances are relatively
non-speci® c and even those claimed to be enzyme-speci® c usually have aæ nity to
other enzymes, although this occurs only at higher concentrations (table 2). One
good example is cimetidine, a well-known inhibitor of P450-linked reactions
(Puurunen et al. 1980). It has been shown that cimetidine interacts with at least
human hepatic CYP 1A, 2C, 2D, 2E and 3A forms, but with widely variable aæ nities
(K nodell et al. 1991).
Further information on P450 substrates and inhibitors can be found in reviews
by Testa and Jenner (1981), Gonzalez (1992), M urray (1992), Vesell (1993),
Rodriguez (1994) and Guengerich (1995).
CYP speci® city of metabolism of a particular drug
A prerequisite for rational study and prediction of metabolic interactions is the
know ledge of CYP speci® city of metabolism or aæ nity of the compound under
study. Currently there is a number of approaches available to study of the role of
know n CYP s in the metabolism and aæ nity of any xenobiotic. M ore extensive
coverage of these approaches can be found in recent reviews (Rodrigues 1994).
A simple approach is to study the inhibitory eå ect of a compound on model
reactions (table 1) catalysed by human liver microsomes or recombinant enzymes. If
a compound inhibits a particular activity, it has a certain aæ nity towards the
enzyme, although it is not possible to tell whether it is metabolized. If the primary
1208
O. Pelkonen et al.
metabolic routes of a compound have been elucidated and a method is available for
their quantitation in in vitro incubations, it is possible to employ ` diagnostic ’
inhibitors (table 2) and to look which of them, and at which concentrations, inhibit
metabolic routes. It is also possible to use enzyme-speci® c antibodies and to test
which metabolic routes are inhibited and to what extent by a particular anti-CYP
antibody. In a panel of human liver microsomes it is possible to correlate the
metabolism of a compound under study with the activities of CYP-speci® c model
reactions and thus get an idea about enzyme(s) catalysing the reaction. Practically all
major CYP enzymes have been expressed in various host cells, such as bacteria, yeast
and mammalian cells, and it is relatively straightforward to study either the
metabolism of, or inhibition by, a compound under study in a cell system expressing
a particular CYP enzyme.
It is possible to make a number of predictions on the basis of the known
characteristics of each CYP enzyme and on the basis of the known CY P-speci® city
of the metabolism of a com pound. For example, if it is known that the CYP 3A4
enzyme participates in the metabolism or interactions of a particular substance, it is
possible to identify some matters of concern on the basis of what is generally known
about CYP 3A4. The following list of predictions is from the review articles of
W atkins (1994) and Wilkinson (1996) and some of these phenomena will be dealt
with more thoroughly in later sections.
CYP 3A4 is induced by rifampicin, antiepileptics, dexamethasone etc and
consequently, the elimination of the compound might be enhanced in situations
involving administration of these drugs.
CYP 3A4 levels are inhibited by ketoconazole, itraconazole and a large number of
other compounds, as well as by grapefruit juice. The metabolism of the
compound under study might be inhibited by these substances.
CYP 3A4 is activated by several ¯ avones and endogenous steroids. The ¯ avones,
which are constituents of food, may enhance the metabolism of substrates of
CYP 3A4.
CYP 3A4 is very variable between individuals. Also, the elimination of the
studied compound may be variable.
CYP 3A4 is present in intestinal epithelium. This fact may lead to a ® rst-pass
eå ect with respect to the compound under study.
CYP 3A4 displays an age-related reduction in activity. The elimination of the
compound of interest may show the same phenomenon.
CYP 3A4 activity is decreased in liver cirrhosis. The elimination of the studied
compound is expected to be decreased in severe liver disease.
E
E
E
E
E
E
E
Probe drugs
The term ` probe drug ’ , also called ` marker drug ’ , was introduced into clinical
pharmacology during the 1970s when considerable interest arose on the in¯ uence of
environm ental factors on drug-metabolizing enzyme activity. A probe drug is
devised to provide information, which allows for an extrapolation to other important
issues (enzyme activity, rate of m etabolism of other compounds). There have been
attempts to envisage an ` ideal ’ probe drug, but obviously some of the more desirable
characteristics are that the probe drug is CYP -speci® c, safe to be used in vivo in man
and widely available, easily and reliably assayed in suitable body ¯ uids (including
P450 inhibition and induction in man
Table 3.
1209
Probe or model drugs } substances claimed to be useful in vivo CYP identi® cation in man.
Probe substrate
Methods available a
Enzymes*
Aminopyrine
Antipyrine
Caå eine
Chlorzoxazone
Coumarin
Dapsone
Debrisoquine
Dextromethorphan
Diazepam
Diclophenac
Erythromycin
Hexobarbital
Lidocaine
Lorazepam
Mephenytoin
metronidazole
Midazolam
Nifedipine
Omeprazole
Paracetamol
p } b, m(r) } ex, pm } u
p } b, p } s, pm } u
pm } u
pm } b, pm } u
pm } u, (pm } b)
pm } u
pm } u
pm } u
pm } b, pm } u, m(r) } ex
pm } b
m(r) } ex
p } b, pm } u
adm iv, pm } b
pm } u
pm } u
p } b, pm } u
pm } b, pm } u
pm } b, pm } u
pm } u
pm } u
Pentobarbital
Phenacetin
Phenytoin
Propranolol
Sparteine
Sulfamethazine
Theophylline
Tolbutamide
Trimethadione
Warfarin
p} b
p } b, m(r) } ex
pm } b, pm } u
p } b, pm } u
pm } u
pm } u
p } b, pm } u
p } b, pm } u
p } b, pm } u
p } b, pm } u
1A, 3A, NAT2
1A, 3A, (others)
1A2, NAT2
2E1,(1A2)
2A6
NAT2
2D6
2D6, (3A4)
2C19, 2D6
2C9
3A4
2C19, (others)
3A4, (1A2)
UGT
2C19
nk
3A4
3A4
2C19
2E1, 1A2, GST, GT,
ST
nk
1A2, 2E1
2C8} 9
2C19, 2D6
2D6
NAT2
1A2
2C9
1A2, 3A
2C9, 1A2, 3A4, 2C19
6b -hydroxycortisol
d -Glucaric acid
endogenous
endogenous
3A4
nk
Modi® ed from Pelkonen and Breimer (1994), where original references can
be found.
a p, Parent drug ; m, metabolite(s) ; b, blood (plasma, serum) ; u, urine ; s,
saliva ; (r), radioactive label ; ex, exhaled air ; adm iv, administered intravenously ; nk, not known.
* NAT, N-acetyltransferase ; UGT, UDP-glucuronosyltransferase ; GST,
glutathione S-transferase ; ST, sulphotransfe rase.
metabolites), and its pharmacokinetics is predominantly determined by metabolism
and not by liver blood ¯ ow or protein binding. In addition, the system should be
predictable, i.e. a limited number of samples should yield quantitative information
on the rate of metabolism and } or the rate of metabolite formation. Further
discussion on these aspects is found in a recent review by Kivisto$ and Kroemer
(1997).
A list of drugs (and some endogenous substances) which are claimed to be useful
as in vivo probe drugs for various purposes is given in table 3. Here some
information on CYP selectivity has been indicated, although we do not try to give
more detailed and quantitative information about this important characteristic of
any probe drug. Later on, we provide some detailed examples, including antipyrine,
a classical ` general ’ probe and warfarin. It would be of considerable importance to
analyse in a detailed and quantitative manner the applicability and usefulness of the
various proposed probe drugs.
1210
O. Pelkonen et al.
In hibition : m echan ism s and quan titation
The in-depth treatment and formal derivation of equations to characterise
various m odes of inhibition can be found in appropriate textbooks and handbooks.
A good introduction to the basic phenomena of inhibition of drug m etabolism is by
Boobis (1995). Here we will deal with only those aspects of inhibition that are
needed to understand the quantitative information given in subsequent tables.
Inhibitory potency in vitro
The most important single measure for inhibitory potency of a given compound
is the K i , or inhibition constant, which expressed an aæ nity of a compound to an
enzyme. It should be stressed here that K i is characteristic for each particular
inhibitor and enzyme, and it is not dependent on any particular substrate used for
the quantitation of an enzyme. W ith respect to human hepatic P450 enzymes, this
value can be easily measured with standard in vitro approaches, in which various
concentrations of a substance are incubated with human liver microsomes and an
inhibition of a CYP -speci® c model reaction is quantitated. A substance may have
aæ nity for an enzyme without being metabolized by the same enzyme or it may be
an alternative substrate of the enzyme and serve as an inhibitor on this basis. In both
cases the K i is derived from an in vitro experiment, but for an alternative substrate,
a K i should be the same as its K m .
It may be worth stressing that assay conditions such as protein concentration,
buå er, ions, pH, and so on, may critically aå ect the inhibitory potency of the
compound (Ekins et al. 1998, M a$ enpa$ a$ et al. 1998) and should be thoroughly
investigated.
Inhibition of clearance
For any substrate, the ratio Vmax } K m is a measure of intrinsic clearance, which
relates to the eæ cacy of an enzyme to metabolize a substrate. Usually, in clinical
usage, drug concentrations are far below their K m , and in this situation it can be
demonstrated that the intrinsic clearance is decreased dependent on the ratio
between the concentration of an inhibitor to its K i , [I ] } K i . This statement is true for
whatever the mechanism of inhibition may be. In tables 4 and 5, and in some
subsequent tables, calculations based on this simple model have been performed :
assuming competitive inhibition and the substrate concentration far below its K m
(i.e. [S] ’ K m ), the percentage inhibition can be simply calculated according to the
equation I(I 1 K i )3 100. It has to be stressed that the number achieved is a very
crude ` ® rst guess ’ and depends on a number of other factors which will be discussed
below in some detail.
However, when substrate concentrations approach and exceed the K m , the
mechanism of inhibition becomes important. In a competitive mode of inhibition,
increasing substrate concentration abolishes inhibition because the inhibitor is
increasingly removed from the active site of an enzyme. In this case, the denominator
of the above mentioned simple equation should contain the term (1 –[S]} K m ) ; the
higher the substrate concentration [S], the lower the percentage inhibition.
However, in a non-competitive mode of inhibition, a certain proportion of an
enzyme, which is determined by the ratio [I ] } K i, is ` inactivated ’ for a more
420
455
550
N-deisopropylation
l -demethylation
3-demethylation
8-hydroxylation
Propranolol
Theophylline
100 (60)
100 (60)
100 (60)
1.2 (90)
1.2 (90)
?
10 (?)
1 (77)
0.1 (70)
33 (20)
0.2 (?)
0.2 (?)
10 (?)
50 (35)
[C]vivo"
(l m )
18
15
19
11
0.6
?
99
83
0.5
0.8
99
25
I#
–
–
1
1
1
–
–
?
(1 )
(1
1
1
Interaction
potential
in vivo $
#
Maximal concentration of the drug in vivo after clinically relevant doses.
I = inhibition percentage : assuming competitive inhibition and the substrate concentration ’
equation I} (I 1 K )3 100.
i
$ Qualitative evidence for in vivo interactions caused by the drug.
% Inhibitor of the reaction above.
"
10 (1A1)
200 (1A2)
O-deethylation
Phenacetin
Inh : furafylline
Inh : ¯ uvoxamine
39
0.07
0.2
7,8-hydroxylation
oxidation
Ondansetron
Paracetamol
38
24
0.1
200
N-demethylation
7-hydroxylation
3-demethylation
Reaction
or K
i
(l m )
m
References
m
K , the percentage inhibition was calculated according to the
Robson et al. (1987), Kunze et al. (1993), Gu et al. (1992),
Rasmussen et al. (1994), Tjia et al. (1996)
Marathe et al. (1994)
Marathe et al. (1994)
Sesardic et al. (1988), Brosen et al. (1993)
Sesardic et al. (1990)
Brosen et al. (1993)
Berthou et al. (1993)
Patten et al. (1993)
Ring et al. (1996)
Ring et al. (1996)
Grant et al. (1988), Butler et al. (1989), Tassaneeyakul
et al. (1993)
Substrates and inhibitors of the human CYP1A2 enzyme.
Olanzapine
Inh % : furafylline
Caå eine
Drug
K
Table 4.
P450 inhibition and induction in man
1211
40
40
6
R-4´-hydroxylation
S-4´-hydroxylation
5´-hydroxylation
5´-hydroxylation
5´-hydroxylation
7-hydroxylation
competitive inhibition
Phenytoin
Piroxicam
Tenoxicam
Tienilic acid
S-warfarin
Inh : sulfaphenazole
4
0.15
120
1.6
27
23
128
(99)
(65)
(99)
(99)
10 (99)
80 (65)
33 (99)
20 (99)
20 (99)
80 (92)
80 (92)
10
80
10
20
370
10 (99)
80 (65)
10
80 (65)
10 (99)
[C]vivo"
(l m )
71
100
85
33
33
62
89
8
100
27
47
74
25
100
40
100
71
I#
#
(1997)
(1997)
(1997)
(1997)
K
for a substrate, the percentage inhibition was calculated
Rettie et al. (1992)
Tracy et al. (1997)
Lopez Garcia et al. (1993)
1
Leemann et al. (1993)
1
1
Leemann et al. (1993)
1
1
m
al.
al.
al.
al.
Veronese et al. (1991), Bajpai et al. (1996)
1
et
et
et
et
Rodrigues et al. (1996), Tracy et al. (1997)
Tracy et al. (1997)
Tracy et al. (1997)
Tracy et al. (1997)
Tracy et al. (1997)
Hamman
Hamman
Hamman
Hamman
1
?
?
?
1
?
1
1
1
Leemann et al. (1993), Transon et al. (1996)
1
1
References
Interaction
potential
in vivo $
Maximal concentration of the drug in vivo after clinically relevant doses.
I = inhibition percentage : assuming competitive inhibition and the substrate concentration is ’
according to the equation I} (I 1 K )3 100.
i
$ Qualitative evidence for in vivo interactions caused by the drug.
% Inhibitor of the reaction above.
"
50
10
O-demethylation
competitive inhibition
competitive inhibition
competitive inhibition
competitive inhibition
Naproxen
Inh : sulfaphenazole
Inh : warfarin
Inh : piroxicam
Inh : tolbutamide
4
2-hydroxylation
competitive inhibition
3-hydroxylation
competitive inhibition
38± 47
0.11± 0 .12
21± 29
0.06± 0 .07
4-hydroxylation
or K
m
i
(l m )
S,R-Ibuprofen
Inh % : sulfaphenazole
S,R-ibuprofen
Inh : sulfaphenazole
Reaction
K
Substrates and inhibitors (Inh) of the human CYP2C9 enzyme as assessed in human liver microsomes.
Diclofenac
Drug} group
Table 5.
1212
O. Pelkonen et al.
P450 inhibition and induction in man
1213
prolonged period of tim e, being unavailable for catalysis, and the inhibition cannot
be abolished by increasing the substrate concentration.
M echanism-based inhibition
For the P450 enzymes, the inhibitory species may not be the substrate but a
metabolite, which is then complexed or covalently bound to a metabolising enzyme
itself (` suicide inhibition ’ ) or to other enzymes nearby. The consequence is a
removal of a variable proportion of an enzyme from active catalysis, i.e. a noncompetitive mode of inhibition. However, the detection of mechanism-based
inhibition requires speci® c incubation conditions. A preincubation of liver microsomes in the presence of an inhibitor under the metabolising conditions is necessary,
because the presence of a substrate might competitively inhibit the metabolism of a
mechanism-based inhibitor.
A speci® c case of mechanism-based inhibition is the situation in which an
enzyme is inactivated very slowly during in vivo conditions. In this case it is diæ cult
to reveal inhibition in in vitro experiments.
Concentration of the inhibitor
W hatever the exact K i is, it does not directly tell us inhibition will be observed
during the in vivo use of a compound. The critical factor in the term [I ]} K i is the
concentration of an inhibitor, which ideally means the concentration at the active
site or a modulatory site. Obviously, this particular concentration is not known and
surrogate values are usually used, such as total or free concentration in the plasma.
M ost authors think that the unbound (i.e. free concentration) is the most appropriate
to use, because it is only free drug that is able to transfer to hepatocytes and to the
vicinity of P450 enzymes. H owever, it is conceivableÐ and for some drugs even
shownÐ that many lipid-soluble drugs are concentrated in hepatocytes and
consequently the actual concentration in the liver far exceeds that in plasma. Even
the measurement of the partition between liver and plasma does not necessarily
indicate the available portion of a drug to an enzyme, because a drug may be very
tightly bound inside hepatocytes and may not be available to the active site of the
enzyme. A detailed and extensive treatment of modelling and predicting interactions
of drug metabolism, including factors aå ecting partition between liver and plasma,
can be found in Leemann and Dayer (1995). In the current review, we have used
plasma concentrations as such, taken mostly from general sources (Dollery et al.
1991, Hardman et al. 1996), but we have also tabulated plasma protein binding of the
drugs, so that the interested reader could calculate the theoretical inhibition
percentages by the ` free ’ drug. Diå erent sources give slightly diå erent plasma
concentrations, but we have usually selected the highest therapeutic concentration,
if known.
Clinical signi® cance of an interaction
Aæ nity and CYP speci® city can be studied in vitro and thus a potential of a drug
to cause interactions can be revealed. However, this does not yet mean that the
compound would cause clinically signi® cant interactions. For such interactions to
occur, two prerequisites have to be ful® lled :
1214
O. Pelkonen et al.
The concentration of the drug in clinical situation should be high enough, so that
inhibition would be manifested in vivo.
The therapeutic index of the drug should be narrow, such that a change caused
by an interacting drug would cause side eå ects.
E
E
The clinical signi® cance of a drug interaction involves also a judgmental
component, which in most cases is rather large. The judgmental components
involve the severity of potential harm to the patient, assessment of decreased
therapeutic outcome and so on. This makes it diæ cult to say unequivocally whether
an interaction is ` clinically signi® cant ’ . Semiquantitative classi® cations have been
constructed, such as that of Preskorn (1993) using the terms ` substantial ’ ,
` moderate ’ , ` mild ’ , ` unlikely ’ , ` not clinically signi® cant ’ . However, in the end
clinical assessment and judgment is the ® nal arbiter as to the clinical and therapeutic
signi® cance of an interaction and this assessment m ay be diæ cult to put into exact
numbers and may cause disagreement even between experts.
E xa m ples of substrates and in hibitors w ith aæ n ity pred om inan tly to a
single C YP enzym e
In the following sections we make an attempt towards semiquantitative
assessment of the inhibitory potential of some substrates and inhibitors with
variable speci® cities towards CY P forms. The K m and K i are taken from the
appropriate in vitro studies. Evidently there is some variation in the exact numbers
taken from studies performed in various laboratories. In this treatment, we do not
usually present Vmax and clearances, although they would allow for calculation of the
extent to which the metabolism of a compound is aå ected by various inhibitors.
Clearances for individual CYPs are especially important for substrates which are
metabolized signi® cantly via several more or less equally important enzymes.
However, usually it is rather diæ cult to decide the approximate proportion of the
total clearance that is due to a particular CYP enzyme. In terms of potential
signi® cant interactions, the often cited view is that the inhibition of the clearance has
to be " 50 % for the interaction to be ` clinically signi® cant ’ . However, any exact
lim it is debatable, because ` clinically signi® cant interaction ’ is strongly dependent
on the narrowness of the therapeutic to toxic dose levels and on the generality or
speci® city of the target site of toxicity.
Calculations for inhibitory potencies are based on the simple equation presented
above. These calculations can certainly be re® ned by taking into consideration some
additional factors in the models, such as plasma protein binding, absorptive phase
concentrations in the portal blood, partition of a drug between liver and plasma,
organelle accumulation in the hepatocyte and so on. The problem is that we do not
usually know many of those factors. W e have also collected some data on
metabolism-related interactions of the compounds tabulated. These data are taken
mainly from textbooks, handbooks or desk reference sources (Dollery et al. 1991,
Hardman et al. 1996) and are presented in a simplistic way. Nevertheless, we hope
that some conclusions can be made from these data.
CYP1A2
In m an, the CY P1A1 protein is expressed at a very low level in the liver (Wrighton
et al. 1986), whereas CY P1A1 and its associated activities can be detected and are
inducible by cigarette smoke and PAHs in extrahepatic tissues like the lung and
P450 inhibition and induction in man
1215
placenta (Pasanen and Pelkonen 1994, Raunio et al. 1995). Although CY P1A1 is able
to oxidize a number of drug substrates (as has been demonstrated with, e.g.,
heterologously expressed CYP 1A1), we will not deal with this enzyme further in this
review.
The CYP1A2 gene product is clearly the predominant hepatic enzyme of
CY P1A subfamily in man, although it is quite variably expressed in human liver
(Shimada et al. 1994). There is no evidence of signi® cant expression of CYP 1A2 in
extrahepatic tissues.
Substrates and inhibitors. Some examples of substrates and inhibitors for CYP 1A2
are shown in table 4. The puri® ed human CY P1A2 protein was originally shown to
catalyse phenacetin O-deethylation (Distlerath et al. 1985). Caå eine has been used
as an in vivo metabolic probe for CYP 1A2 (Butler et al. 1989). Fast and slow
metabolisers of caå eine 3-demethylation have been identi® ed, although the genetic
basis for this distinction is not clear (Butler et al. 1992). Also theophylline has been
reported to be a speci® c substrate for this enzyme in man (Robson et al. 1987) and
concurrent treatment with theofylline and inhibitors of CYP1A2 may lead to
harmful drug interactions (Stockley 1996). Both xanthines are rather interesting in
that their K m for CYP 1A2 are very high (hundreds of l m ), but they have also very
high plasma concentrations, making it probable that they might cause interactions
with other drugs metabolized via CYP 1A2 (table 4).
a -Naphtho¯ avone (7,8-benzo¯ avone) has been shown to be a potent and
relatively speci® c inhibitor of both CY P1A isoforms (Burke et al. 1977). However,
it has not been used in vivo in m an. Furafylline, a methylxanthine analogue, is a
potent inhibitor of several CYP 1A2-associated metabolic reactions (tables 2 and 4),
whereas it has only a weak eå ect on CYP 1A1 (Sesardic et al. 1990). However,
furafylline is not available for in vivo use because it causes severe interactions with
caå eine (Tarrus et al. 1987). A selective serotonin reuptake inhibitor, ¯ uvoxamine
has also been reported to be a potent inhibitor of CYP 1A2, as exempli® ed by the
inhibition of phenacetin O-deethylation and theofylline metabolism (Brosen et al.
1993, Rasmussen et al. 1995). However, it seems not to be as speci® c as furafylline.
M ore information on ¯ uvoxamine will be presented in a later section.
CYP2C9
The human genome has been shown to contain several genes belonging to the
CY P2C subfamily (Goldstein and de M orais 1994) and they have been shown to be
expressed at signi® cant levels only in the liver. The metabolic roles of the diå erent
hepatic enzymes in this subfamily are still rather poorly de® ned and here we deal
only with CYP 2C9 and CYP 2C19 in some detail. Nevertheless, CYP2C8 has been
puri® ed from human liver in diå erent laboratories (Wrighton et al. 1987, Ged et al.
1988, Leo et al. 1989). It has a role in the metabolism of endogenous substances like
retinol and retinoic acid and drugs such as benzphetamine (Wrighton et al. 1987,
Leo et al. 1989). Tolbutamide is also metabolized by CY P2C8, although the aæ nity
of tolbutam ide for this isoform is clearly lower than for CYP 2C9 (Relling et al. 1990,
Veronese et al. 1993).
Substrates and inhibitors of CYP2C9. A number of important drugs are substrates
of CYP 2C9 (table 5). CYP2C9 participates in the hydroxylation of tolbutamide and
1216
O. Pelkonen et al.
hexobarbital (Shimada et al. 1986, Brain et al. 1989) as well as phenytoin and
warfarin (Veronese et al. 1991, Rettie et al. 1992). Currently it seems that diclofenac
4-hydroxylation is becoming a useful probe drug for both in vitro and in vivo studies
(table 2). A lot of in vitro information has been published on ibuprofen and naproxen
(table 5). Both substrates are stereoselectively metabolized by CYP2C9, and this has
been demonstrated also with a recombinant enzyme (Hamman et al. 1997, Tracy et
al. 1997). The K m for ibuprofen is about 20± 50 l m and that for naproxen about
120 l m (a high-aæ nity ® gure), but when compared with their in vivo concentrations
they are similar enough to expect signi® cant interactions. However, when one takes
into consideration an extensive plasma protein binding, the calculated in vivo
inhibition percentages remain rather small. This same phenomenon seems to be true
with respect to most anti-in¯ ammatory (and other) drugs listed in table 5.
Nevertheless, at least some interactions based on metabolism have been reported in
monographs dealing with these compounds. Obviously we need m uch more
information about the eå ect of plasma protein binding on hepatic uptake and
accumulation.
Sulphaphenazole is a potent and speci® c inhibitor of the CYP 2C9 enzyme and it
appears to inhibit the metabolism of various NSAIDs as well as tolbutamide with
a roughly similar potency (table 5) (Brian et al. 1989, Veronese et al. 1993).
Sulphaphenazole is also an eå ective in vivo inhibitor (Birkett et al. 1993).
CYP2C9 and other CYPs in warfarin metabolism. W arfarin, a coumarin-type
anticoagulant, is extensively oxidized in human and rodent liver microsomes,
principally by P450-mediated reactions (Kaminsky 1989, Rettie et al. 1989). The Rand S-enantiomers of warfarin are metabolized by diå erent metabolic pathways.
S-warfarin is mainly metabolized to 6- and 7-hydroxyw arfarin. Small amounts of
other hydroxy metabolites and warfarin alcohol are also formed. R-warfarin is
oxidized presumably by P450 enzymes to 6-, 7-, 8-, and 10-hydroxywarfarin, but
the main metabolic pathway is reduction by soluble enzymes to warfarin alcohol
(K aminsky and Zhang 1997).
The warfarin alcohols and hydroxy metabolites are excreted in the urine and in
bile, and also enterohepatic circulation occurs. Of the dose, 85 % may be recovered
as metabolites in urine with ! 1 % as the unchanged drug. The mean plasma halflife of warfarin is about 36 h, with a relatively wide variation from 10 to 45 h.
However, the S-enantiomer has a shorter half-life of 18± 35 compared with 20± 60 h
for the R-enantiomer. Total plasma warfarin clearance ranges from 2 .5 to
6 .4 ml3 h Õ " 3 kg Õ " . Therapeutic plasma concentrations at steady state range from
300 l g } l to 3 mg } l with a wide interindividual variation. W arfarin is highly albumin
bound with values ranging from 97 to 99 .5 % (D ollery et al. 1991).
In vitro studies with human liver m icrosomes have demonstrated the predictive
value of a simple inhibition screening with warfarin as an inhibitor. The inhibitory
eå ect of racemic warfarin on CYP model activities have indicated that warfarin
inhibited CY P2C9-catalysed tolbutamide methylhydroxylation with a K i of about
6± 12 l m . Values for other CYPs were at least 30± 40 times higher (unpublished data).
This simple experiment demonstrates the predominant aæ nity of warfarin towards
CY P2C9, suggesting a need for more thorough studies. The K i for the inhibition of
tolbutamide methylhydroxylation (6± 12 l m ), indicates a relatively high aæ nity and
if this aæ nity is associated also with metabolism of warfarin it might indicate an
enzyme that is metabolizing warfarin at therapeutic concentrations of 2 mg } l
P450 inhibition and induction in man
Table 6.
1217
Kinetics of oxidative metabolism of warfarin by human liver microsomes and recombinant
expressed CYP enzymes.
CYP"
S-warfarin
R-warfarin
Metabolite
K
m
(l m )
M
M
7OH
6OH
4
3
300
r2C9
r2C9
r3A4
7OH
60H
6OH
4
4
300
M
M
M
M
6OH
7OH
8OH
10OH
265, 1412
159, 1580
162, 1500
400
r1A2
6OH, 7OH,
8OH
8OH, 6OH,
7OH
10OH
r2C19
r3A4
C
V
max
(nmol } mg 3
h)
0.5
0.1
1.1
Formation
clearance
in vivo#
1847
400
(40) $
(7)
(90)
0.9, 11 .6
0.3, 2.7
0.9, 2.2
2.4
1600
not available
200
not available
400
(200)
462
227
338
342
Data derived from Kunze and Trager (1996) and Kunze et al. (1996).
" M, human liver microsomes ; r, recombinant.
# ml3 h Õ " 3
kg Õ " 10Õ $ , from Black et al. (1996).
$ Figures in parentheses mean activity in pmol} mg protein 3 min.
(10 l m ). Quantitative prediction is possible only when the kinetic parameters for
warfarin metabolism have been determined. Inhibition screening does not give this
information. However, by using an in vitro inhibition screening study it is possible
to pinpoint a high-aæ nity CYP form for warfarin. Even if CYP 2C9 is not a
metabolizing enzyme, the high aæ nity would indicate a possibility for interactions.
W arfarin metabolism in hum an liver microsomes has been studied with
diagnostic inhibitors and antibodies and correlation analysis, as well as with
recombinant enzymes. W ith all of these approaches, the identity of enzyme(s)
catalysing various oxidative pathways of warfarin metabolism as well as the kinetic
parameters have convincingly been demonstrated (table 6 ; Kunze et al. 1996). On
the basis of comparison of K m for the formation of various warfarin metabolites it
can be anticipated that (S)-7- (and 6-) hydroxymetabolite is (are) predominantly
formed at clinically achievable warfarin concentrations and the predominant
catalysing enzyme is CY P2C9. W ith respect to R-warfarin clearance, at least three
CY Ps participate, but the K m are almost two orders of magnitude higher than that
for CY P2C9 (table 6).
The formation clearances for each of the metabolites formed from the (R)- and
(S)-warfarin in human subjects have been recently determined (Black et al. 1996).
Com parison of the above in vitro data with metabolite formation clearances in vivo
seem to show a relatively direct correspondence (table 6). The formation clearances
of (S)-6- and (S)-7-hydroxyw arfarin represent up to 90 % of the total metabolite
clearance of S-warfarin, a ® gure that is in an excellent correlation with the role of
CY P2C9 in the in vitro metabolism of S-warfarin.
Because CYP 2C9 is such a predominant catalyst of S-warfarin clearance,
clinically signi® cant interactions (inhibition and induction) could have been
predicted on this basis (see above, and also a later section on induction). Several
P450 enzymes, including at least CYP1A2, CYP2C19 and CYP 3A4, catalyse the
1218
O. Pelkonen et al.
formation of (R)-hydroxywarfarins (table 6). Also on the basis of in vivo ® ndings
with inducers and inhibitors of the P450 system, the participation of the above
mentioned CY Ps can be at least tentatively identi® ed.
On the basis of the above ® ndings it can be concluded that the most important
warfarin-oxidising enzyme, CYP 2C9, has been identi® ed by in vitro approaches.
Because the K m for other P450 forms are at least 40± 50 times larger, it can be
concluded that their contribution to the overall metabolism of warfarin must be
small, if substantial CYP2C9 activity is present. The knowledge of general
properties of CYP 2C9 would also have enabled at least qualitative, if not
quantitative, predictions to be made about the pharmacokinetic behaviour and
potentially signi® cant inhibition and induction interactions of warfarin.
It has been repeatedly suggested on the basis of in vitro studies, that warfarin
would seem to be a promising probe compound for in vivo studies. However, it has
been used only to a very limited extent. One of the reasons for this is that as an
anticoagulant warfarin has potentially hazardous side eå ects, although the use of a
single, smaller-than-therapeutic dose may not manifest prolongation of bleeding
time. Another potential problem in the use of warfarin as a probe drug is its high
degree of protein binding. Furthermore, a complicating factor with warfarin is the
stereochemical selectivity in its m etabolism which requires stereoselective analysis
of parent enantiomers and metabolites (Lam 1988). Currently it can only be said that
the formation rate of the 7-hydroxymetabolite of S-warfarin could be used as an
index of the CY P2C9 activity in vivo, but the usefulness of other metabolites as
indices for other CY Ps remains to be dem onstrated.
CYP2C19
CYP 2C19-mediated 4 ´-hydroxylation of S-mephenytoin is polymorphically
expressed in humans and recent studies have demonstrated that the polymorphism
is due to at least two major and several minor variant alleles of CY P2C19 (Goldstein
and de M orais 1994). The PM phenotype based on two major variant alleles is rather
infrequent among Caucasians (2± 4 % ), but is much more common in Orientals
(around 20 % ) (Wedlund et al. 1984, Alvan et al. 1990).
Substrates and inhibitors. There are a number of substrates for the CYP 2C19
enzyme, but very few even remotely speci® c inhibitors (Guengerich 1995b).
Proguanil, omeprazole and imipramine are metabolized by CYP2C19, but also
other CYP s are im portant catalysts of the metabolism of these drugs (Andersson et
al. 1993, Birkett et al. 1994). Omeprazole may be the most promising probe for in
vivo studies and the search for speci® c inhibitors continues. Recently, ¯ uconazole
and ¯ uvoxamine have been shown as potent inhibitors of CYP2C19-mediated Rwarfarin 8-hydroxylation in vitro (Kunze et al. 1996) and CYP2C19-mediated
proguanil bioactivation in vivo (Jeppesen et al. 1997), respectively, but both
compounds seem rather unspeci® c.
CYP2D6
Individuals can be classi® ed into extensive (EM ) and poor metabolizers (PM )
according to their genetically determined ability (phenotype) to oxidize a number of
drugs, such as debrisoquine, sparteine, bufuralol and dextromethorphan (Mahgoub
P450 inhibition and induction in man
1219
et al. 1977, Eichelbaum et al. 1979). The molecular basis of this polymorphism
(called CYP2D6 polym orphism) has been elucidated in great detail (Meyer et al.
1990). About 7 % of the Caucasian population are PM s (Alvan et al. 1990), because
mutations in CYP2D6 gene have led to an absence of a functional CY P2D6 protein
(G onzalez 1990, M eyer 1994). Also individuals carrying multiple copies (i.e. the
ampli® cation) of the active CYP2D6 gene have been detected (Johansson et al.
1993). It is remarkable that the CY P2D6 enzyme seems to be resistant to xenobiotic
induction, which aå ects the activities of other P450 enzymes. The only clear
example of an exogenous in¯ uence is the competitive inhibition of the enzyme by a
number of drugs, including quinidine and some neuroleptics (Brosen and Gram
1989). Thus, the study of environmental in¯ uences on CYP 2D6 is of interest, but
mainly because of the possible interference upon the phenotyping of the trait and
clinically important drug interactions.
Substrates and inhibitors of CYP2D6. The im portance of CYP2D6 polymorphism
is substantial, since numerous drugs, including cardiovascular drugs, b -adrenergic
blocking agents (bufuralol, metoprolol and propranolol), tricyclic antidepressants
(amitriptyline, nortriptyline and imipramine), neuroleptics (perphenazine, thioridazine, haloperidol and clozapine) and miscellaneous other drugs like codeine,
dextromethorphan and phenformin are substrates for CYP 2D6 (Cholerton et al.
1992). It is important to know which substances interact with CYP2D6, since many
of the therapeutic drugs listed above have a narrow therapeutic window. consequently, dangerous drug interactions may occur when using drugs that are oxidized
by CYP 2D6.
The inhibitor spectrum of CYP 2D6 has been thoroughly studied. Quinidine
is a highly selective and potent inhibitor, although it is not a substrate of the
CY P2D6 enzyme (Guengerich et al. 1986) (table 2). In a survey of diå erent
chemicals on their eå ects on bufuralol 1-hydroxylase, an activity speci® c for
CY P2D6, several alkaloids and neuroleptics were found to be potent inhibitors
(Fonne-P® ster and M eyer 1988). The K i of the alkaloid ajmalicine was as low as
3 .3 n m . M any of a new class of antidepressant drugs, selective serotonin reuptake
inhibitors or SSRIs are substrates for CYP2D6 and } or inhibit it (Brosen 1993) as we
describe in a later section.
CYP2E1
Only one gene belonging to this subfamily has been identi® ed in the human
genome, namely CYP2E1 (Ronis et al. 1996). The activity of CYP2E1 is aå ected by
numerous factors, including alcohol drinking, several drugs such as isoniazid and
some pathophysiological conditions such as diabetes, ketonemia and obesity (Koop
1992, Ronis et al. 1996). The inducing eå ect of ethanol on CY P2E1 is discussed in
a later section. It seems probable that CY P2E1 is expressed and induced also in some
extrahepatic tissues, but the signi® cance of extrahepatic activity in the kinetics of
drugs in vivo is not clear (Shimizu et al. 1990). Since the rodent and human CY P2E1
enzymes catalyze similar reactions, rat and mouse are good models when screening
for substrates of this enzyme.
Substrates and inhibitors of CYP2E1. Over 60 substrates have been shown to be
metabolized by this enzyme (Koop 1992). M ost substrates are carcinogens or other
1220
O. Pelkonen et al.
toxicants and there are only a few drug substrates. Because of the proposed relatively
small substrate pocket of the enzyme, CYP 2E1 accepts various volatile anaesthetic
agents as substrates (Koop 1992). Chlorzoxazone has become a widely used substrate
for CYP 2E1 in vitro (table 2). The advantage of using this compound is that the
chlorzoxazone 6-hydroxylase assay is very sensitive compared with the former
CY P2E1-speci® c assays used. Chlorzoxazone might also be an appropriate probe to
study CYP 2E1 function in vivo in man and its role in pathogenesis of diå erent
diseases like alcoholism and diabetes (Kim et al. 1995). However, recent studies
indicate that CY P1A1 is also able to m etabolize chlorzoxazone (Ono et al. 1995).
Because CYP1A1 may be a prom inent enzyme in extrahepatic tissues especially after
PAH-type induction, chlorzoxazone may not be used as a speci® c probe for CY P2E1
in extrahepatic tissues.
There are several more or less speci® c inhibitors of CY P2E1. Disul® ram inhibits
CY P2E1-associated activities in man (Guengerich et al. 1991). Disul® ram is
reduced to diethyldithiocarbamate which inhibits CYP 2E1 relatively potently, but
it is also an almost equally potent inhibitor of CY P2A6 (Brady et al. 1991,
Guengerich et al. 1991). Also 3-amino-1,2,4-triazole, phenethyl isothiocyanate and
dihydrocapsaicin are speci® c mechanism-based inhibitors of CYP2E1 in rodents
(K oop 1992).
It should be stressed that ethanol and acetone, as well as several volatile
anaesthetics, all substrates for CYP2E 1, can attain relatively high levels in the
body and might thus interfere with CYP 2E1-catalysed reactions. In experimental
conditions, many organic solvents that are widely used as vehicles of compounds to
be studied in in vitro incubations with tissue preparations, are relatively potent
inhibitors of CYP2E 1 and could give completely erroneous results if not properly
used.
Human CYP3A subfamily
The members of the CYP 3A subfamily are CYP 3A4, CYP 3A5 and CYP3A7.
These enzymes have a central role in drug metabolism since they are the most
abundant forms of P450 (20± 60 % ) in human liver (Guengerich 1995b). In addition,
CY P3A4 is expressed in the human intestine and it catalyses drug metabolism there
as well (Kolars et al. 1992b, Guengerich 1995b). CYP 3A4 is expressed in all human
livers and about 50 % of drugs currently in the m arket are substrates for it. The
CY P3A5 protein is expressed at detectable levels in the human liver in about 25 %
of individuals. The third member of the CYP 3A subfamily is CYP 3A7 that is
particularly expressed in human foetal liver (Wrighton and Stevens 1992). A
number of structurally diå erent compounds are substrates for these isoforms
including steroids, macrolide antibiotics, benzodiazepines and other miscellanous
substances (Wrighton and Stevens 1992).
Substrates and inhibitors. It seems that all the members of CYP 3A subfamily have
similar substrate preferences (Gonzalez 1992b, Guengerich 1995b). However,
CY P3A5 may have some diå erences in its aæ nity to bind substrates when compared
with CYP 3A4 (Wrighton et al. 1989, 1990). cDNAs expressing CYP3A4 and
CY P3A4 eå ectively catalyse the oxidation of testosterone, progesterone and
androstenedione, which may be physiologically important reactions (Waxman et al.
1991). CYP3A enzymes metabolise many drugs including cortisol, quinidine,
nifedipine, diltiazem, lidocaine, lovastatin, erythromycin, troleandom ycin, cyclo-
P450 inhibition and induction in man
1221
sporin, warfarin, triazolam and midazolam (Guengerich and Shimada 1991,
W righton and Stevens 1992). many procarcinogens like AFB1 are also activated by
CY P3A enzym es (Aoyama et al. 1990, Guengerich 1993). In conclusion, the CYP3A
subfamily is very important in catalysing the metabolism of diå erent drugs,
carcinogens and endogenous substances.
In recent years diå erent diagnostic in vivo probes measuring CYP3A activity
have been developed. The ® rst described in vivo system was the non-invasive
method of Saenger et al. (1981) to measure the amount of 6 b -hydroxycortisol in
urine. Erythromycin N-demethylase activity can be measured by the 14[C]erythromycin breath test (Watkins et al. 1989). Other in vivo probes of CYP 3A4
tested include midazolam, nifedipine, dapsone and lidocaine (Watkins 1994).
M idazolam is a well characterised probe for CYP3A4 (see below). However,
correlations between diå erent in vivo CYP 3A probes in man are not always very
good and may arise from the heterogeneity of CYP3A isoforms. It is not always
clear which CYP 3A isoform is responsible for the metabolism of a drug in question.
There is a num ber of isoform-speci® c inhibitors of the members of CYP3A
subfamily. Troleandomycin (TAO ) has been shown to form a metabolic-intermediate complex with CY P3A isoforms (Pessayre et al. 1983) and seems to be
relatively selective. G estodene is also a selective mechanism-based inhibitor of
CY P3A4 and CYP3A5 (Guengerich 1990, W righton et al. 1990). These inhibitors
have to be initially oxidized before they form complexes with speci® c P450s. Also,
many substrates listed above inhibit CYP 3A mediated reactions.
Grapefruit juice has been shown to inhibit the metabolism of a number of
CY P3A substrates (Bailey et al. 1991, Soons et al. 1991). The com ponents of
grapefruit juice, like ¯ avonoids and furanocoumarins have been claimed to inhibit
CY P3A enzymes, and further the metabolism of CY P3a substrates like felodipine,
cyclosporine, terfenadine and midazolam just a few to mention (Ameer and
W eintraub 1997). However, it was recently shown by Lown et al. (1997) that the
inhibition of the metabolism of CYP3A substrates by grapefruit juice may be due to
reduction of the CY P3A4 protein in small intestine and not to the inhibitory role on
CY P3A4 of ¯ avones found in grapefruit juice (Guengerich 1995b).
An interesting feature of CY P3A4 is that it has been shown to be stimulated by
various substances like ¯ avones (Guengerich 1995b). Further, autostimulation by
the substrate itself has been shown to occur with several substrates (Ekins et al.
1998). The stimulators have to be keep apart from inducers, which increase the
protein expression in the cell. The mechanism may vary depending on the stimulator
in question. The stimulation of the enzyme may occur when the substrate or
stimulator binds to an allosteric site of the enzyme leading to a conformational
change of the enzyme (Ekins et al. 1998). It has also been suggested that the
stimulation may occur by enhancing the interaction of NADPH-P450 reductase
with CYP 3A4 or the stimulator and the substrate bind simultaneously to diå erent
sites in the active centre of CYP3A4 (Guengerich 1995, Ekins et al. 1998). Recently,
Koley et al. (1997) suggested that the stimulator may activate an inactive
subpopulation of CY P3A4. The most potent stimulator of CYP 3A4 catalytic
activity known is a -naphtho¯ avone, although many other ¯ avones also stimulate
this activity (Shou et al. 1994). Flavonoids are widespread in natural foods (Yang
et al. 1992) and therefore the stimulation of CYP 3A4 activity may have clinical
signi® cance. Further, endogenous substances like progesterone and testosterone
have also been shown to stimulate CYP 3A-mediated reactions (Johnson et al. 1988,
1222
O. Pelkonen et al.
Kerr et al. 1994, M a$ enpa$ a$ et al. 1998). However, the clinical signi® cance of these
® ndings is unclear, but potentially the stimulation of CYP 3A may result in low
plasma levels of CYP3A substrates or the stimulators may enhance the activation of
carcinogens by CYP 3A. The stimulation of midazolam metabolism is discussed
below.
CYP 3A enzymes are induced by several antiepileptics, rifampicin and corticosteroids which may lead to many clinically signi® cant drug interactions as discussed
in detail later.
CYP3A4 and inhibition of cyclosporin oxidation. Pichard et al. (1990) have
published a very extensive paper where they studied the inhibition of cyclosporin
metabolism by a large number of potential CYP3A4 substrates and inhibitors in
isolated hum an hepatocytes. The com pounds studied, as well as some additional
information, are listed in table 7. Several important conclusions can be made on the
basis of this information.
It seems that apparently there is very little correlation between the percentage
inhibition, calculated on the basis of a K i and in vivo plasma concentration, and
the potential of a compound to cause interactions that are regarded as ` clinically
signi® cant ’ .
If plasma protein binding is taken into consideration in the calculations (i.e. free
concentrations are used), even smaller percentage inhibition would be obtained
and the discrepancy between the calculated inhibition and the expectation of
` clinically signi® cant ’ interactions becomes even more noticeable.
Some substances, especially cimetidine and erythromycin, are clearly more prone
to cause in vivo interactions than would be predicted on the basis of in vitro
studies (K i ) and in vivo achievable concentrations. For these com pounds the
obvious reason is their conversion to reactive products which cause mechanism
based inhibition. How much ` suicide inhibition ’ would explain other discrepancies (e.g. see bromocriptine) remains to be evaluated. Another possibility
is that the drug is converted into a metabolite or metabolites, which is (are) the
predominant species in the body and which cause potential interactions.
E
E
E
At the present moment, the reasons for poor correlations are unclear. However, the
secondary sources from where we extracted the information on potential interactions, may list some interactions on the basis of what is expected from the
know ledge that two compounds are metabolized by the same enzymes, and not on
the basis of actual positive studies. It remains to be seen whether a detailed, more
quantitative analysis would yield a better correlation between in vitro predictions
and actual in vivo changes (table 7).
Drug interactions with midazolam, a probe drug for CYP3A enzymes. M idazolam is
a short-acting benzodiazepine derivative that has been used as a hypnotic agent
(D undee et al. 1984). The metabolic pathways of midazolam have been identi® ed
both in vitro and in vivo (Guengerich 1995b). Further, interactions between
midazolam and many other commonly used drugs have been thoroughly studied
both in vitro and in vivo. Therefore we chose midazolam as an example to discuss the
advantages and problems found when analysing in vitro studies to predict drug
interactions in vivo. It is also evident that CYP3A4 is a unique P450 enzyme because
of its complex properties that make the in vitro± in vivo correlations diæ cult to judge.
P450 inhibition and induction in man
1223
Table 7. Inhibition aæ nity of drugs for CYP3A4, as measured by inhibition of the oxidative CYP3A4mediated metabolism of cyclosporin in human cultured hepatocytes, and comparison with in vivo
observed interactions (inhibition potency data taken from Pichard et al. 1990).
Inhibitor
Clotrimazole
Ketoconazole
Miconazole
Itraconazole
Nicardipine
Bromocriptine
Troleandomycin
Nifedipine
Terfenadine
Ergotamine
Isradipine
Josamycin
Midecamycin
Dihydroergotamine
Verapamil
Midazolam
Progesterone
Fluconazole
Diltiazem
Erythromycin
Glibenclamide
Cortisol
Ethinylestradiol
Prednisone
Me-predniso ne
Prednisolone
K (l m )
C (l m )#
in vivo
Calculated
inhibition
(% )"
0 .1
0 .7
0 .9
1 .2
8
8
10
10
10
12
12
19
22
23
24
40
45
60
63
75
78
125
172
190
190
210
2 .5
10 (98)
2 .5 (92)
0 .4 (99)
0 .3 (95)
0 .001 (96)
3
0 .3 (90)
0 .01
?
0 .15 (96)
3
3
?
1 .5 (90)
0 .25 (96)
0 .04 (97)
70
0 .3 (85)
3 (83)
0 .1 (99)
0 .6 (95)
0 .5 (95)
0 .7 (80)
0 .7 (80)
0 .7 (80)
96
93.5
73.5
25
3.6
0.01
23
3.0
0.1
?
1.2
14
12
?
6
0.6
0.1
54
0.5
4
0.1
0.5
0.3
0.4
0.4
0.3
i
Interaction
potential#
?
1
1
1
1
1
1
1
?
?
1
1
1
?
1
–
?
1
1
1
?
1
?
1
1
1
" Assuming competitive inhibition and the substrate concentration ’
K
m
for cyclosporin metabolism, the percentage inhibition was calculated according
to the equation I(I 1 K ) 3 100.
i
# Data on in vivo maximal concentrations, extent of plasma protein binding
(in parentheses) and interaction potential have been collected mainly from
monographs and handbooks (Dollery et al. 1991, Hardman et al. 1996). Plussign means that clinical studies have indicated interactions between the
inhibitor and the CYP3A4-mediated elimination and } or metabolite formation
of cyclosporin or other CYP3A4-associated drugs.
In vitro metabolism. M idazolam is metabolized to 1 ´-hydroxy (1 ´-hydroxymidazolam) and 4-hydroxy midazolam (4-hydroxymidazolam ) in vitro by human
liver microsomes (Kronbach et al. 1989, Gorski et al. 1994). The in vitro metabolism
of midazolam is catalysed solely by CYP3A enzymes. Human CYP 3A4 and
CY P3A5 enzymes have been shown to have similar substrate preferences (see above)
and 1 ´-hydroxymidazolam and 4-hydroxymidazolam formation are catalyzed by
both CYP3A4 and CYP 3A5 isoforms (Kronbach et al. 1989, Gorski et al. 1994).
However, it has been reported that microsomal samples containing high levels of
CY P3A5 had a higher 1 ´-hydroxymidazolam } 4-hydroxymidazolam ratio than the
samples containing only CYP3A4 (Ma$ enpa$ a$ et al. 1998). In addition, CYP 3A7 is
responsible for 1 ´-hydroxymidazolam and 4-hydroxymidazolam formation in
human foetal liver microsomes (Gorski et al. 1994 ; M a$ enpa$ a$ et al. 1998).
In vivo metabolism. M idazolam is also metabolised to 1 ´-hydroxymidazolam and 4hydroxymidazolam in vivo. Both metabolites are pharmacologically active and both
1224
O. Pelkonen et al.
Table 8. Eå ect of several inhibitors and inducers of CYP3A4 on 1 ´-hydroxymidazolam formation in
vitro and on midazolam AUC ± ¢ in vivo in human volunteers.
(!
IC
Inhibitor or inducer*
Erythromycin
Azithromycin
Verapamil
Fluconazole
Itraconazole
Ketoconazole
Rifampicin*
or K
i
(l m )
&!
194**
170
100
" 80**
1
0 .1*
inducer
AUC, % of control
(placebo)
442
87
292
373
1080
1590
4
Data are derived from the following : Gascon and Dayer (1991), Olkkola et
al. (1993, 1994) Backman et al. (1994, 1995, 1996), Wrighton and Ring (1994),
Ahonen et al. (1997).
metabolites are rapidly conjugated by glucuronic acid to form an inactive product
(D undee et al. 1984). However, only very low levels of 4-hydroxymidazolam are
detected in plasma after taking midazolam (Mandema et al. 1992). The main
metabolite of midazolam, 1 ´-hydroxymidazolam, has also been shown to be
produced by CYP 3A4 in vivo (Thummel et al. 1994a, b). M any diagnostic inhibitors
of CY P3A reduce the clearance of midazolam as discussed further below. Additional
indication of the involvement of CY P3A isoforms in the in vivo metabolism of
midazolam has been obtained from a study showing a signi® cant correlation between
midazolam clearance and the erythromycin breath test (Lown et al. 1995).
CYP 3A4 is expressed in relatively large amounts in the luminal epithelium of the
small intestine (Kolars et al. 1994). Recently, it was shown that midazolam is
signi® cantly metabolised in the human small intestine (Paine et al. 1996). Therefore,
many clinically signi® cant drug interactions discussed below may occur in the small
intestine.
Inhibitors, activators and inducers of midazolam metabolism. The role of CYP3A
enzymes in midazolam metabolism has been further indicated by CYP 3A speci® c
inhibitors. 1 ´-Hydroxymidazolam formation is inhibited by substrates and } or
inhibitors of CY P3A like cyclosporine, erythromycin, itraconazole, ketoconazole
and terfenadine (Gascon and Dayer 1991, W righton and Ring 1994, G oldberg et al.
1996). Further, midazolam has been shown to inhibit the metabolism of terfenadine
and quinine, which both are substrates of CYP 3A (Jurima-Romet et al. 1994, Zhang
et al. 1997). As already discussed above, grapefruit juice inhibits the metabolism of
CY P3A4 substrates and it also inhibits midazolam metabolism (Kupferschmidt et
al. 1995, Ameer and W eintraub 1997). Large diå erences have been observed in the
ability of CYP 3A inhibitors to inhibit m idazolam metabolism in vitro and the results
are not always proportional to the in vivo situation. Relatively weak inhibitors of
midazolam metabolism, like erythromycin and verapamil, have been shown to be
potent inhibitors of midazolam metabolism in vivo (table 8). Further, azithromycin
which is as potent an inhibitor of midazolam metabolism as erythromycin in vitro,
did not inhibit midazolam m etabolism in vivo at all (table 8). Therefore, it is not
always straightforward to make predictions of the in vivo situation based on in vitro
data. In the case of erythromycin, its inability to produce a signi® cant inhibitory
eå ect on CYP3A4 in vitro may be due to the fact that the mechanism of inhibition of
macrolide antibiotics occurs via metabolic-intermediate complexes with CYP3A
P450 inhibition and induction in man
1225
(Wrighton and Stevens 1992). Trolendomycin, another m acrolide antibiotic,
produces a metabolic-intermediate complex rapidly (Murray 1987), whereas
erythromycin does it at a much slower rate (Wrighton and Ring 1994). Indeed, in the
in vivo situation where erythromycin was given for 5 days to the volunteers prior to
taking midazolam, a signi® cant interaction was observed between erythromycin and
midazolam (Olkkola et al. 1993). H owever, in a similar clinical study design
azithromycin was not able to inhibit midazolam metabolism (Backman et al. 1996).
Antimycotics, including ketoconazole, itraconazole and ¯ uconazole are potent
inhibitors of midazolam metabolism both in vitro and in vivo (table 8). M oreover,
their ability to inhibit midazolam metabolism is proportional to their in vitro
potency to inhibit 1 ´-hydroxymidazolam formation.
The stimulation of CYP3A isoforms has been shown also by using midazolam as
a substrate. Recently, a -naphtholavone was shown to be a potent stimulator of 1 ´hydroxymidazolam formation (Ghosal et al. 1996, M a$ enpa$ a$ et al. 1998). However,
a -naphtholavone had no eå ect on the CYP3A mediated 4-hydroxymidazolam
formation, although the inhibitors of midazolam m etabolism have been shown to
inhibit both 1 ´-hydroxymidazolam and 4-hydroxymidazolam formation (Gascon
and Dayer 1991). Two other CYP3A substrates, terfenadine and testosterone,
regioselectively stimulated 1 ´-hydroxymidazolam formation and 4-hydroxymidazolam formation, respectively (Ma$ enpa$ a$ et al. 1998). The regioselective
stimulation of midazolam is another indication of the complexity of the regulation of
CY P3A enzymes. Further, the stimulatory potency of terfenadine was highly
dependent on the assay conditions used. Terfenadine was a potent inhibitor of
midazolam metabolism in certain assay conditions (buå er, ionic strength) whereas
it was a potent stimulator of midazolam metabolism in other assay conditions. Again
these factors further complicate the ability to make conclusions of drug interactions
in vivo based on in vitro data. 1 ´-Hydroxymidazolam formation was stimulated by a nephtho¯ avone in isolated human hepatocytes providing further evidence that the
stimulation of CY P3A may occur in vivo as well (Ma$ enpa$ a$ et al. 1998).
The eå ect of various CYP3A4 inducers like rifampicin, phenytoin and
carbamazepine have also been shown to dramatically decrease the C max and AUC of
midazolam in man (Backman et al. 1996 (table 8). Further, the hypnotic eå ects of
midazolam were minimal in volunteers and patients after receiving inducing agents
(Backman et al. 1996). Therefore, when midazolam is given orally, inducers of
CY P3A4 should be avoided.
In vitro studies are a valuable tool to predict drug interactions in vivo in most
instances. However, caution should be exercised when extrapolating possible drug
interactions in vivo by using in vitro data, especially in the case of CYP 3A substrates.
E xa m ples of substrates and in hibitors w ith aæ n ity for several C YP s
To illustrate induction and inhibition phenomena in connection with diå erent
chemicals, we present here in more detail some well-known drugs and groups of
drugs, which are extensively metabolized by several P450 enzymes. Adm ittedly,
warfarin is also oxidized by several CY Ps, at least in vitro, but as described earlier,
by far the most important enzyme in vivo for warfarin metabolism is CYP2C9.
These examples have been selected so that possibilities of in vitro± in vivo
extrapolations are analysed in a more thorough fashion and that the clinical
relevance of induction and inhibition phenomena will be illuminated through some
examples.
1226
Table 9.
O. Pelkonen et al.
Kinetics and CYP-associated catalysis of pathways of the oxidative metabolism of antipyrine.
Reaction
K
m
(mm )"
V
max
(nmol } mg*
min)"
4-hydroxylation
5.2± 23.1
0 .57± 1.40
N-demethylation
5.9± 26.3
0 .34± 2.23
3-Methylhydroxylation
9.0± 21.1
0 .59± 1.41
CYPs participating in the
reaction #
3A4(5) up to 65 %
1A2 about 30 %
2A6, 2B6
2C(9 } 19) 75± 80 %
1A2 20± 25 %
2A6, 2C8, 2C18, 2D6, 2E1, 3A
1A2 50 %
2C(9) 50 %
2C8, 2C9, 2E1
" Ranges for the K and V
have been taken from Boobis et al. (1981), Engel et al. (1996) and Sharer
m
max
and Wrighton (1996).
# Contributions of the major CYP(s) catalysing the reaction has been estimated on the basis of
diagnostic inhibitors, antibodies, and recombinant expressed enzymes (Engel et al. 1996, Sharer and
Wrighton 1996).
Antipyrine
Antipyrine as a measure of in vivo oxidative drug metabolism has been very
extensively studied (almost 3000 references in a M edline search between 1961 and
1990, Poulsen and Loft, personal communication) and has been dealt with in a large
number of reviews (for references, see Poulsen and Loft 1988, Pelkonen and
Breimer 1994). The elimination rate of antipyrine is sensitive to induction by
antiepileptic and other drugs, by cigarette smoking and it is inhibited by various
liver diseases and several concomitantly administered drugs. The measurement of
urinary metabolites of antipyrine and thereby the rates of formation of metabolites
adds further information on the diå erential eå ects of inducing or inhibiting
substances with respect to diå erent isoforms, but this issue has only been
investigated to a lim ited extent (Poulsen and Loft 1988). Antipyrine seems to be a
quite useful and universal probe to detect the in¯ uence of common environmental
factors (including drug treatment) and disease processes on overall P450 activity.
Until very recently, there was not much inform ation on isoforms involved in
antipyrine metabolism, except the classical inducers of the M C-type and the PBtype aå ect the metabolic pathways diå erentially. However, on the basis of studies
with some diagnostic inhibitors it seemed probable that CYP2C (sulphaphenazole),
CY P2D (debrisoquine, quinidine) and CYP 3A (nifedipine) or at least certain
enzymes belonging to these subfamilies do not participate in antipyrine metabolism
(Pelkonen and Breimer 1994).
The recent studies of Sharer and W righton (1996) and Engel et al. (1996) have
changed the situation completely. Through their work it is know n that practically all
know n hepatic P450 enzymes participate in the oxidative metabolism of antipyrine,
at least to a minor extent (table 9). Although the clearance of antipyrine via three
major metabolic pathways is roughly equal, all these individual pathways are
catalysed by several P450 enzymes with variable K m and Vmax characteristics. In this
light it becomes understandable why antipyrine has been characterized as ` a
general ’ probe and why almost any chemical exposure aå ects its clearance. On this
basis, antipyrine may be quite suitable for initial screening purposes, but does not
detect eå ects on speci® c CYP enzymes. Assessment of metabolite formation is only
of limited value in this respect.
P450 inhibition and induction in man
1227
However, three enzymes seem to be of major importance for antipyrine
clearance, namely CYP 1A2, CYP 2C(9) and CY P3A(4). Consequently, considering
the properties of these enzymes (see above) it becomes apparent why antipyrine
elimination is increased by cigarette smoking (CYP1A2 is induced) and antiepileptic
drugs (CYP 3A4 and CYP 2C9 are induced) and why a large number of drugs retard
its clearance (those three enzymes are responsible for the clearance of a majority of
pharmaceuticals, as far as is currently known). Typically, the eå ect of inducers or
inhibitors on antipyrine clearance is only about 10± 50 % (Poulsen and Loft 1988). It
is clear that these modest and clinically insigni® cant changes are due to multiple
CY P enzymes participating in antipyrine metabolism. Consequently, a general
probe such as antipyrine is not very eæ cient in revealing increases or decreases of
speci® c CYP enzymes. Furthermore, the impact of an environmental factor on the
elimination of a drug metabolized by a single CYP enzym e may be an order of
magnitude larger than what may erroneously be anticipated on the basis of
information obtained from antipyrine.
An early claim that the production of the main primary metabolites of antipyrine
is catalysed by polymorphically regulated P450 enzymes (Penno and Vesell 1983)
did not receive, even then, a complete acceptance. W hether correct or not, it was
thought that in most cases environmental and host factors in¯ uence the overall
antipyrine metabolism, which may therefore mask any polymorphic pattern in
metabolite formation. It is known that at least CYP2D6 participates in the
metabolism of antipyrine, but its contribution to the overall clearance is so small that
it is unlikely to have anything but an extremely minor eå ect. It is possible that there
is still an unrecognized polymorphism behind the ® ndings of Penno and Vesell
(1983), but this remains to be demonstrated.
Citalopram metabolism
Citalopram is a widely used antidepressant and is considered to be the most
selective of the serotonin selective reuptake inhibitors (SSRI). The terminal
elimination half-life of citalopram is 1 .5 days. It is metabolized by successive Ndemethylations to N-desmethylcitalopram and N-didesmethylcitalopram, both of
which are detected in plasma, although the levels are roughly one-third and onetenth of the parent compound, respectively. Citalopram N-oxide and the deaminated
propionic acid derivative are minor urinary metabolites. About 10± 20 % of the drug
is excreted unchanged (Baumann and Larsen 1995).
Recent investigations on citalopram nicely illustrate the two major goals of in
vitro studies : (1) to identify CY P enzymes metabolizing a compound under study or
to which a compound has aæ nity without being metabolized, and (2) to analyse
whether it would have been possible to predict in vivo metabolism and potential
drug± drug interactions on the basis of in vitro data.
In vitro studies. The eå ect of citalopram on various CYP -speci® c model reactions
in hum an liver microsomes are presented in table 10. Aæ nities for most enzymes
studied are relatively low, with very little inhibition at concentrations ! 100 l m .
One exception is CYP2D6-catalysed reactions, for which K i vary from 5 to 19 l m
(Brosen 1994, 1996). Thus it seems that, CYP 2D6 excluded, citalopram has a
relatively low aæ nity towards most human hepatic CYPs. M ainly due to the
1228
Table 10.
O. Pelkonen et al.
Inhibitory eå ects of citalopram on CYP-speci® c model reactions in human liver microsomes.
CYP
Reaction studied
% of control at
100 l m
citalopram
K (l m )
i
1A1
ethoxyresoru® n O-deethylation
82
"
1A2
ethoxyresoru® n O-deethylation
theophylline N-demethylations
96
92± 95
"
"
100
100
2A6
coumarin 7-hydroxylation
92
"
100
2C9
tolbutamide methylhydroxylation
88
"
100
2C19
S-mephenytoin 4-hydroxylation
78
"
100
2D6
dextromethorpan O-deethylation
sparteine oxidation
imipramine 2-hydroxylation
2E1
chlorzoxazone 6-hydroxylation
92
"
100
3A4
testosterone 6b -hydroxylation
cortisol 6b -hydroxylation
98
71
"
100
100
100
7
5.1
19
"
Data derived from Rasmussen et al. (1995).
Table 11.
Eå ects of diagnostic inhibitors on citalopram N-demethylation in human liver microsomes.
Inhibitor
Inhibitor
concentration
(l m )
Inhibition
(%)
"
10
5
! 5
1A2 1
1A2 –
1A2 –
20
!
5
2A6 –
10
!
5
2C9 –
10
10
2C19 1
2C19 1
10
10
2D6 1
2D6 1
5
5
2E1 –
2E1 –
10
10
3A4 } 5 1
3A4 } 5 1
Fluvoxamine
Furafylline
Phenacetin
12 .5
10
10
Coumarin
Sulfaphenazole
"
Quinidine
Paroxetine
5
20
"
Methylpyrazole
DEDC
20
20
Ketoconazole
Troleandomycin
2 .5
50
!
100
500
Omeprazole
Mephenytoin
Prediction "
"
"
!
"
"
!
Data derived from Rochat et al. (1997) and Kobayashi et al. (1997).
"
1 , Participation of the respective enzyme is predicted ; – , the contrary
to the plus sign.
relatively narrow range and low concentrations of citalopram used in those studies,
it is diæ cult to pinpoint low-aæ nity enzymes. In retrospect, it would have been
better to start with m uch higher (i.e. 1± 5 m m ) citalopram concentrations, which may
have allowed for the detection of low-aæ nity enzymes (see below).
Studies on citalopram N-demethylation in human liver microsomes in vitro have
revealed biphasic kinetics (Rochat et al. 1997). Consequently, there are at least two
major enzymes catalysing citalopram N-demethylation in vitro. High-aæ nity and
low-aæ nity components have roughly similar intrinsic clearances. Inhibition by
chemical inhibitors of citalopram N-demethylation has been studied by screening
experiments (table 11 ; Rochat et al. 1997). Studies with these ` diagnostic ’ inhibitors
P450 inhibition and induction in man
Table 12.
1229
N-demethylation of citalopram enantiomers by cDNA-expressed human liver micrososal
cytochrome P450 enzymes.
(pmol} h
max
3 pmol CYP)
V
CYP
1A2
3.0
2A6
not detectable
2B6
not detectable
2C9
not detectable
2C19
S-CIT 78 .1
R-CIT 53 .1
2D6
S-CIT 5.0
R-CIT 8. 5
2E1
not detectable
3A4
S-CIT 62 .1
R-CIT 43 .6
K
m
(l m )
ND (high)
198
211
Intrinsic
clearance
(CL )
i
ND
0.39
0.25
18.2
22.1
0.27
0.38
169.0
163.0
0.37
0.27
Data derived from Rochat et al. (1997) and
Kobayashi et al. (1997).
S-CIT and R-CIT refer to the S and R isomers
of citalopram, respectively.
(table 2) suggest that at least CYP3A4 } 5, CY P2C19 and CYP 2D6 participate in
citalopram N-demethylation. The role of CYP1A2 remains unclear, because the
inhibition results with ¯ uvoxamine could be explained on the basis of inhibition of
CY Ps other than CY P1A2. Furthermore, furafylline and phenacetin, which are
probably more selective towards CY P1A2, do not inhibit citalopram N-demethylation at the concentrations used.
Citalopram N-demethylation by cDNA-expressed CYPs. Table 12 presents the
results obtained from two laboratories for the N-demethylation of citalopram by
cDNA-expressed human CYP s. Expressed enzymes with relatively high turnover
numbers were CYP 2C19, CYP 2D6, and CY P3A4. Also CYP 1A2 showed little
activity. CYP 2D6 seems to be a high-aæ nity enzyme, but intrinsic clearance
calculations demonstrated that all three enzymes were roughly equally active. W hen
compared with results obtained with human liver microsomes, CYP 2D 6 seems to
represent the high-aæ nity (but low capacity) component, and CYP 2C19 and
CY P3A4 the low-aæ nity component.
Although there is substantial interindividual variability in the content of the
individual CYP enzymes, it can be assumed on the basis of studies using human liver
microsomes in vitro that CYP3A, CY P2C19 and CY P2D6 represent about 30, 4 and
2 % of total P450 content, respectively (Shimada et al. 1994). Because the intrinsic
clearances of drugs by these CYP s are rather similar (see above), their contributions
to the overall metabolism of citalopram should be in the order of their abundance.
Studies on the diagnostic inhibitors point to the same conclusion : ketoconazole
inhibited approximately 60 % of the microsomal N-demethylation of citalopram,
whereas the percentages for omeprazole (CYP 2C19) and quinidine (CYP 2D6) were
maximally 30 and 15 of total N-demethylation, at their CYP-speci® c concentrations.
In conclusion, the major P450 enzymes catalysing the principal pathway of
citalopram metabolism, N-demethylation, have been shown to be CYP3A4,
CY P2C19 and CY P2D6. The aæ nity of CY P2D6 is roughly one order of magnitude
1230
Table 13.
O. Pelkonen et al.
Inhibition of CYP2D6-mediated desipramine 2-hydroxylation by SSRI-compounds in
human liver microsomes in vitro and calculated inhibition in vivo.
SSRI
K
i
(l m ) "
Fluoxetine
Nor¯ uoxetine
Fluvoxamine
Paroxetine
Sertraline
Norsertralin e
Citalopram
Norcitalopram
Quinidine
3, 0 .6
2, 0 .43
20, 8 .2
2, 0 .15
20, 0 .7
15, NK
80, 5 .1
100, NK
0 .05
"
C
max
(l m )#
Inhibition
in vivo
(%)$
1 (94)
1 (94)
1 (77)
0.2 (95)
0.1 (99)
0.1
0.4 (70)
0.4
10
25
33
5
10
0 .5
0 .7
0 .5
! 0 .5
100
Eå ect in vivo %
"
"
"
350 %
350 %
14 %
300 %
26± 72 %
26± 72 %
46 %
46 %
potent
" First K values are taken from Moltke et al. (1994) and are based on
i
inhibition of desipramine 2-hydroxylation activity, except for citalopram and
norcitalopram, for which the values are calculated on the basis of relative
inhibition of imipramine 2-hydroxylation (Skjelbo and Brosen 1992). The
second values are for sparteine oxidation in vitro (Brosen 1993). NK, not
known.
# C
denotes the (peak) plasma concentration of a SSRI drug in vivo
max
(l m ). Plasma protein binding (in parentheses), which aå ect the free concentration, has not been taken into consideration. Liver} plasma partition ratio
has been assumed to be 1, although it may actually be considerably higher for
some SSRIs. It should be stressed that ¯ uoxetine and nor¯ uoxetine both
together produce plasma concentration of about 1 l m .
$ Assuming competitive inhibition and the substrate concentration ’
K
m
for desipramine metabolism, the percentage inhibition was calculated according to the equation I } (I 1 K ) 3 100.
i
% Percent increase in the area under the plasma concentration-time curve of
desipramine (AUC) (Brosen 1996). In vivo data on sparteine elimination
(Jeppesen et al. 1996) is in a good agreement with desipramine data.
greater than that of CYP 3A4 or CY P2C19, but the intrinsic clearances of these
enzymes are roughly equal. Consequently, because of the relative abundances of
these enzymes, none of them is overwhelmingly important for the clearance of
citalopram and one would not expect any major consequences for induction or
inhibition of P450 enzymes. This speci® c point is further elaborated in the next
section.
SSRI-antidepressants and quantitative prediction of drug± drug interactions
There are some quantitative in vitro inhibition and aæ nity data available for
all ® ve SSRI-compounds for CYP2D6 and CY P3A4 interactions which make it
possible to calculate potential in vivo inhibition for representative CYP 2D6-,
CY P3A4-, and CYP 1A2-catalysed metabolic reactions (desipramine, midazolam
and phenacetin, respectively).
W ith respect to CYP 2D 6 (on the basis of the data in table 13) clinically relevant
concentrations of nor¯ uoxetine and ¯ uoxetine seem to lead to a signi® cant in vivo
inhibition of the CYP 2D6-mediated 2-hydroxylation of desipramine. Also
paroxetine and ¯ uvoxamine are calculated to cause some inhibition. Comparison of
the inhibitory potencies of ¯ uoxetine (plus nor¯ uoxetine) and paroxetine observed
in in vivo studies are in line with in vitro inhibition results when sparteine oxidation
was used as a model reaction for CY P2D6, whereas the potency of paroxetine would
P450 inhibition and induction in man
1231
Table 14. Aæ nity of SSRI-compounds for CYP3A4 in human liver microsomes in vitro, and calculated
inhibition of in vivo midazolam metabolism (according to von Moltke et al. 1994, 1996).
SSRI
Aæ nity in
vitro (l m )"
Fluoxetine
Nor¯ uoxetine
Fluvoxamine
Paroxetine
Sertraline
Norsertralin e
Citalopram
Ketoconazole
7.1, 44 .3
2.7, 8.0
5.6, 20 .2
3.8, 14 .3
3.5, 20 .3
3.5, 10 .7
165
0.02
C
max
(l m )#
1 (94)
1 (94)
1 (77)
0.2 (95)
0.1 (99)
0.1
0.4 (70)
10
I in vivo (%)$
12 .3, 2 .2
27 .0, 11.1
15 .2, 4 .7
5 .0, 1 .3
2 .8, 0 .5
2 .8, 0 .9
0 .2
100
Eå ect in vivo%
detectable ?
detectable ?
detectable ?
absent
absent
absent
strong
" Aæ nity values are K of inhibition of two midazolam CYP3A4-mediated reactions (von Moltke et
i
al. 1996), except for citalopram where the value is the K for citalopram N-demethylation (Rochat et al.
m
1997).
# C
denotes the (peak) plasma concentration of a SSRI drug in vivo (l m ). Plasma protein binding
max
(in parentheses), which aå ect the free concentration, has not been taken into consideration. Liver} plasma
partition ratio has been assumed to be 1, although it may actually be considerably higher for some SSRIs.
It should be stressed that ¯ uoxetine and nor¯ uoxetine both together produce plasma concentration of
about 1 l m .
$ Assuming competitive inhibition and the substrate concentration ’ K for midazolam metabolism,
m
the percentage inhibition was calculated according to the equation I } (I 1 K ) 3 100.
i
% Assessment is based on Nemeroå et al. (1996). Eå ect in vivo means whether interaction with other
CYP3A4-catalysed reactions have been observed in vivo.
have been underestimated if the 2-hydroxylation of desipramine had been used as a
model reaction. It seems that various model reactions may lead to both underestimation or overestimation of the inhibitory potency of a particular SSRI.
However, for example, plasma protein binding and liver to plasma concentration
ratios have not been taken into consideration and may be of importance in such
calculations (von Moltke et al. 1994).
W ith respect to CYP3A4 (table 14), calculations indicate that nor¯ uoxetine
(which is the predominant plasma constituent of long-term ¯ uoxetine treatment)
and ¯ uvoxamine potentially cause in vivo inhibition " 15 % of midazolam
metabolism. There is some evidence that ¯ uoxetine treatment actually leads to
increased plasma concentrations and } or retarded elimination of alprazolam, carbamazepine, terfenadine and diazepam whereas ¯ uvoxamine treatment inhibits the
elimination of alprazolam and terfenadine (Nemeroå et al. 1996). However, the data
of Stevens and W righton (1993) do not support a signi® cant inhibition of midazolam
hydroxylation by ¯ uoxetine. W ith respect to citalopram, the only values for aæ nities
for CY P3A4 are available from Rochat et al. (1997) and Rasmussen et al. (1995) and
are 165 and " 100 l m , respectively, indicating a relatively low aæ nity and making
it unlikely that citalopram would cause drug± drug interactions via CY P3A4.
W ith respect to CYP 1A2, only ¯ uvoxamine seems to have a high enough aæ nity
for the enzyme to cause clinically signi® cant interactions (table 15). Actually these
comparative studies previously led to suggestions that ¯ uvoxamine might be the
inhibitor of choice for CYP 1A2. However, recent results suggest that ¯ uvoxamine
has a relatively high aæ nity towards some other CYP enzymes (Rochat et al. 1997).
1232
O. Pelkonen et al.
Table 15.
Ability of SSRI-antidepressants and their metabolites to inhibit CYP1A2-mediated reactions
vitro and in vivo.
Drug
K (l m ) "
Fluoxetine
"
100
1 (94)
!
Nor¯ uoxetine
"
100
1 (94)
!
i
0 .2
Fluvoxamine
C
max
(l m ) #
I in vivo (%) $
1 (77)
Eå ect in vivo %
1
caå eine ( –)
clozapine ( 1 ?)
1
?
83
caå eine ( 1 1 1 )
theophylline ( 1 1 1
clozapine ( 1 1 1 )
imipramine
amitriptyline
clomipramine
Paroxetine
45
0 .2 (95)
0.4
caå eine ( –)
Sertraline
70
0 .1 (99)
0.1
not known
100
0 .4 (70)
0.4
caå eine ( –)
Citalopram
"
!
)
"
K in vitro for phenacetin O-deetylation (Brosen et al. 1993).
i
C
denotes the (peak) plasma concentration of the SSRI drug in vivo (l m ). Plasma protein binding
max
(in parentheses), which aå ect the free concentration, has not been taken into consideration. Liver} plasma
partition ratio has been assumed to be 1, although it may actually be considerably higher for some SSRIs.
It should be stressed that ¯ uoxetine and nor¯ uoxetine both together produce plasma concentration of
about 1 l m .
$ Assuming competitive inhibition and the substrate concentration ’
K for desipramine metabm
olism, the percentage inhibition was calculated according to the equation I} (I 1 K ) 3 100.
i
% Caå eine results from Jeppesen et al. (1996) : ( 1 1 1 ) strong, ( 1 1 ) moderate and ( 1 ) slight eå ect
on caå eine elimination in vivo, ( –) very small or absent eå ect.
#
In duction
Induction in general
Classically, the de® nition of induction is the de novo synthesis of new enzyme
molecules as a result of an increased transcription of the respective gene after an
appropriate stimulus. However, in drug metabolism research the term induction has
been used as a generic term, describing an increase in the amount and } or activity of
a drug metabolising enzyme as a result of an exposure to an ` inducing chemical ’ ,
whatever the underlying mechanism. However, in the usual sense of induction,
there is a certain lag phase before an increase in enzyme activity can be observed.
This lag phase is due to the fact that, whatever the underlying mechanism, it takes
time to increase the amount of enzym e molecules, either as a result of increased
transcription and translation or as result of the stabilisation of an enzyme by a
substrate, which leads to a new steady-state level between synthesis and degradation.
An increase in enzyme activity, due to activation, is not usually included under
the term induction. Some examples include the eå ect of dexamethasone on the
elimination of some drugs and a rapid enhancement of antipyrine elimination by
heme arginate in porphyric patients (Mustajoki et al. 1992), probably is due to the
restoration of holoenzyme by heme in the presence of intact apoenzyme.
Based on mostly animal experiments, inducers have been categorised into several
classes (table 16), which can be characterized mainly on the basis of the spectrum of
enzymes induced and the potency of induction. This table gives only a qualitative
view of the spectrum and mechanisms of induction and in the following section more
background is given on mechanistic details and quantitative aspects of induction in
man or human-derived systems. It has to be stressed that in many cases we have to
rely on what we know from animal experiments.
P450 inhibition and induction in man
Table 16.
Class
1233
Classi® cation of inducers of drug-metabolizing enzymes.
Prototype inducer
Principal enzymes aå ected
PAH-type
2,3,7,8Tetrachlorodibenzo-pdioxin
CYP1A, UDPglucuronosyltransferase
Ethanol-type
Ethanol
CYP2E1
Phenobarbital-type
Phenobarbital
CYP1A, CYP2A, CYP2B, CYP3A
Glucocorticoid-type
Dexamethasone
CYP3A
Peroxisome proliferator-type
Clo® brate
CYP4
This classi® cation is based mainly on animal studies, and the types of induction are not as clear-cut
in man.
Quantitation of induction
The basic tenet is that induction leads to an increased amount of an existing
enzyme (or enzymes) and not to a qualitatively diå erent enzyme. This means that
in the quantitative analysis the only changing measure is Vmax . Obviously, when
more than one enzyme is induced, calculations will become more complicated, but
still there are no ` new ’ players present. The overall eå ect in vivo will still depend on
the aæ nities and rates of m etabolism of various enzymes participating in the
metabolism of a compound under study.
Spectrum and mechanisms of induction
Several individual agents that induce CY P enzymes have been identi® ed in man,
and the list of drugs whose pharmacokinetics and pharmacodynamics are aå ected by
induction is rather long. For comprehensive updates on such drugs the reader is
referred to relevant monographs (Wrighton and Stevens 1992, Goldstein and de
M orais 1994, Guengerich 1995, W ilkinson 1996). Only the basic classes of induction
as well as the mechanisms involved will be dealt with here.
Cigarette smoking and PAH-like inducers. Decreased half-life and} or increased
clearance of several drugs have been demonstrated in smokers (Sotaniemi and
Pelkonen 1987). The common denominator for these drugs is that they are
metabolised by CYP 1A forms. Examples include theophylline, caå eine, antipyrine,
imipramine, paracetamol (acetam inophen), and phenacetin (table 5). The metabolism of these drugs is mediated predominantly by CY P1A2, which represents
approxim ately 10 % of the total hepatic P450 content (Shimada et al. 1994). Not
only CYP1A-mediated reactions, but also glucuronide conjugation of, for example,
mexiletine is increased due to cigarette smoking (Sotaniemi and Pelkonen 1987).
The inducing eå ects of cigarette smoking are attributed to the polycyclic aromatic
hydrocarbon (PAH) class of compounds. Consistent with this, CY P1A2 activity is
increased in human prim ary hepatocytes by the prototype PAH inducer 3methylcholanthrene (Morel et al. 1990).
CYP 1A1 is mainly an extrahepatic enzyme. It is highly induced in the lung,
mammary gland, lymphocytes, and placenta by PAHs and cigarette smoke (Raunio
et al. 1995). The regulatory mechanisms of CY P1A induction have been thoroughly
elucidated (Hankinson 1995). CY P1A inducers interact with the so-called Ah (Aryl
1234
O. Pelkonen et al.
hydrocarbon) receptor, which upon ligand binding is activated and translocated to
the nucleus as a complex which includes also the AR NT (aryl hydrocarbon nuclear
translocator) protein. The complex binds to speci® c regions in the regulatory areas
of the CYP1A genes, the Ah-receptor regulatory elements (AhRE ), also known as
xenobiotic- or drug-responsive elements. This interaction leads to increased
transcription of the CYP1A genes and the de novo production of CYP1A protein.
Increased amounts of CYP 1A enzymes may have two diå erent types of consequences : increased toxicity due to more eæ cient activation of protoxins and
procarcinogens that are substrates of these enzymes (toxic response), or decreased
toxicity as a result of enhanced inactivation reactions (adaptive response) (Schmidt
and Brad® eld 1996).
The CYP1A1 gene is distributed in a polymorphic pattern in the human
population. The two main variant alleles CYP1A1 are an MspI RFLP in the 3 ´noncoding region of the gene, and a second one is the closely linked point mutation
in exon 7, creating a substitution of valine % ’ # for isoleucine % ’ # (Kawajiri et al. 1993).
Several attempts have been made to correlate these polymorphisms to the
inducibility and function of the CY P1A1 enzyme. The initial reports (Petersen et al.
1991, Landi et al. 1994) on the higher inducibility of the MspI allele compared with
the wild-type allele have been questioned in other studies in which no diå erences in
the induction properties between these two alleles were found (Crofts et al. 1994,
W edlund et al. 1994, Jacquet et al. 1996). Recent studies with heterologously
expressed CYP 1A1Val % ’ # alleles clearly show that the catalytic and kinetic properties
of this enzyme do not diå er from those of the wild-type (CY P1A1Ile % ’ # ) enzyme
(Zhang et al. 1996, Persson et al. 1997). It may be that the high-inducibility
CY P1A1 phenotype will be explained by variations in the regulatory genes rather
than the structural gene. Despite convincing evidence that mutations in the Ahreceptor gene confer high and low inducibility in inbred strains of mouse, attempts
to correlate CYP 1A1 inducibility with known polymorphisms in the human AHreceptor gene have yielded negative results (Micka et al. 1997).
The regulation of CYP 1A2 is not as well characterized as that of CYP 1A1. It is
inducible by smoking, charbroiled food, cruciferous vegetables, omeprazole and
even vigorous exercise (Wrighton and Stevens 1992a, Guengerich 1995a). Induction
of CYP 1A2 by PAHs is mainly transcriptional and involves the Ah-receptor, but
also other, currently unknown factors (Quattrochi et al. 1994). Two CYP1A2 knock
out mouse strains have been constructed (Pineau et al. 1995, Liang et al. 1996).
These m ice develop normally apart from de® cient metabolism of some xenobiotics
metabolised by CY P1A2. Thus CY P1A2 appears not to have any crucial endogenous
function.
CYP 1B1, a novel member in the CYP 1 family, has catalytic properties similar
but not identical to CYP 1A members (Shim ada et al. 1996). In rodents, CY P1B1
is highly inducible by PAHs in several extrahepatic tissues, but the inducibility in
human tissues appears to diå er from that of CYP 1A1 (Hakkola et al. 1997).
Omeprazole and congeners. The CY P1A-inducing capacity of omeprazole in the
human liver and primary hepatocytes was ® rst reported in 1990 by Diaz et al. (1990).
Shortly afterwards, omeprazole was shown to induce CYP1A also in the human
alimentary tract (McDonnell et al. 1992). Both of these ® ndings have been con® rmed
by diå erent methodological approaches (Nousbaum et al. 1994, Buchthal et al. 1995,
Kash® et al. 1995), but also negative ® ndings have been reported, especially using
P450 inhibition and induction in man
1235
the standard therapeutic doses of omeprazole (Andersson et al. 1991, Galbraith and
M ichnovicz 1993, Rizzo et al. 1996). In human primary hepatocytes, omeprazole
and lanzoprazole also appear modestly to induce CYP 3A members (Curi-Pedrosa et
al. 1994), and both agents stimulate CY P1A1 in the human colon adenocarcinoma
derived cell line Caco-2 (Daujat et al. 1996).
Omeprazole is not a direct ligand for the Ah receptor (Daujat et al. 1992, CuriPedrosa et al. 1994, Lesca et al. 1995). However, the induction of CYP1A by
omeprazole is mediated by enhanced translocation of the Ah receptor to the nuclei
and binding to the regulatory elements upstream of the CYP 1A coding genes
(Quattrochi and Tukey 1993). Recent evidence suggests that omeprazole is
metabolised to a sulfenam ide intermediate that interacts with the ligand binding
domain of the Ah-receptor (Dzeletovic et al. 1997). The inducing eå ect is strictly
species speci® c, since the CYP1A1 gene is activated in man but not in mouse
hepatocytes, possibly due to a repressor mechanism in mouse cells (Kikuchi et al.
1995, Dzeletovic et al. 1997). Thus, omeprazole is an addition to the growing list of
agents that induce CYP1A by activating the Ah-receptor without binding directly to
it, possibly involving ligand binding of a metabolite or inducer-elicited changes in
the phosphorylation of proteins regulating the Ah-receptor (H ankinson 1995).
The overall omeprazole-dependent increases in CYP1A activities in the liver and
gut in vivo and rather low (usually ! 2-fold) and high doses and } or prolonged
treatments are needed to produce the inducing eå ect. In addition, the inducibility of
CY P1A2 by omeprazole is aå ected by the CYP 2C19 status, since omeprazole is
metabolized by CYP 2C19. For example, a dose of 120 mg } day omeprazole for
7 days causes ! 30 % increase in the N-3-demethylation of caå eine in vivo in
CY P2C19 extensive metabolizers, whereas a 40 % increase is elicited in CYP 2C19
poor metabolizers (Rost and Roots 1994). The inducing eå ect using the standard
dose of 40 mg } day is pronounced only in individuals having a defective CYP 2C19
enzyme (Rost et al. 1992, 1994, Sarich et al. 1997). Taken together, the evidence
suggests that the induction caused by omeprazole is unlikely to have practical
consequences. Concerns that elevated CYP1A levels due to omeprazole could result
in increased procarcinogen activation or acetaminophen toxicity do not appear to be
substantiated, since the magnitude of induction is so small compared with cigarette
smoking, and no such adverse eå ects have been associated with omeprazole
treatment (Petersen 1995). The clinical use of om eprazole and related proton pump
inhibitors is currently extensive all over the world and major drug interactions due
to induction have not been reported. In line with this notion, a recent study (Sarich
et al. 1997) reported that the omeprazole-elicited 75 % increase in plasma clearance
of caå eine, as a marker of induced CYP 1A2 activity, is not accompanied by changes
in the metabolic activation of paracetamol.
Ethanol. Ethanol induces liver drug metabolism in man as measured by both in
vivo and in vitro parameters (Sotaniemi and Pelkonen 1987). The presence of an
inducible microsomal ethanol-oxidizing enzyme system, clearly distinct from
alcohol dehydrogenases and catalases, was reported in the late 1960s (Lieber and
DeCarli 1968). This system has been characterized in great detail, and it has become
evident that CYP2E1 is the mediator of the inducible oxidation of ethanol and it may
metabolize up to 10 % of the ingested alcohol (Fraser 1997). CYP 2E1 also
metabolizes a wide variety of drugs and toxic chemicals, including several
procarcinogens, making its inducibility of great practical importance (Lieber 1997).
1236
O. Pelkonen et al.
Tsutsumi et al. (1989) reported that the amount of immunodetectable CY P2E1
apoprotein in the liver was 4-fold higher in alcoholics than in non-drinkers or
alcoholics who had abstained from drinking. Ethanol intake causes up to a 3-fold
elevation in the amounts of both CY P2E1 protein and mRNA in the human liver
(Perrot et al. 1989, Takahashi et al. 1993). The plasma clearance of chlorzoxazone,
a drug metabolized by CYP 2E1, is increased almost 2-fold in individuals consuming
excessive amounts (" 300 g } day) of alcohol (Girre et al. 1994).
Of the numerous other agents capable of CY P2E1 induction in the rat (Ronis et
al. 1996), isoniazid also appears to be an inducer in man since it increases the in vivo
metabolism of en¯ urane (Mazze et al. 1982) and chlorzoxazone (Zand et al. 1993).
Isoniazid is also an inhibitor of the CYP2E1 enzyme and therefore a washout period
of 48 h after the last dose of a prolonged regimen of isoniazid administration is
needed for a manifest inducing eå ect to occur (O’ Shea et al. 1997). The inducing
eå ect is dependent on the N-acetylation status, either slow or extensive acetylators
being more prone to CY P2E1 induction depending on the length of the washout
period applied (Chien et al. 1997, O’ Shea et al. 1997).
Like most other CY P forms, CY P2E1 is expressed at highest levels in the
perivenous hepatocytes (zone 3) with a diminishing gradient towards the periportal
area (zone 1) (Lindros 1997). In rat and man, ethanol-dependent increases in
CY P2E1 expression occur in both the perivenous and midzonal areas (Takahashi et
al. 1993). In primary human hepatocytes, ethanol treatment increases the activity of
p-nitrophenol hydroxylase (Donato et al. 1995) and elevates the amounts of CY P2E1
and CYP 3A apoproteins (Kostrubsky et al. 1995). In HepG2 cells transfected with
the coding sequence of CYP 2E1 cDNA, ethanol increased CYP 2E1 protein but not
mRNA levels, indicating that the elevation is due to protein stabilization (Carroccio
et al. 1994).
The mechanism of CYP2E 1 induction by ethanol has been extensively studied in
the rat, and due to the conserved nature of the CYP2E1 gene and protein, the
regulation of induction may be similar in man. During chronic ethanol intake,
CY P2E1 induction occurs in two phases : at blood levels ! 300 mg } dl the CY P2E1
protein levels are increased without changes in mRNA, and higher blood ethanol
levels also cause increases in the amount CYP 2E1 mRNA (Ronis et al. 1996). The
mechanisms of increases in CYP 2E1 protein levels include enhanced translation and
protein stabilization. One mechanism for stabilization of CY P2E1 is protection of
the protein from cAM P-mediated degradation by the enzyme-bound substrate
(Eliasson et al. 1992). Likewise, CYP2E1 mRNA levels are elevated by increased
transcription or stabilization of the message, depending on the stimulus causing
induction (Ronis et al. 1996). DNA footprinting analysis of the ® rst kilobase of the
CYP2E1 5 ´-¯ anking sequence revealed 13 protected regions, but none appeared to
participate in enhanced transcription of the CYP2E1 gene, indicating that regions
further upstream of the gene may be involved in ethanol-mediated increase of
transcription (McGehee et al. 1997).
Several polym orphisms in the CYP2E1 gene have been detected. Of these
polymorphisms, the one generating a PstI site and the lack of a RsaI site in the 5 ´¯ anking region of the CYP2E1 gene (so-called c2 allele) has been reported to confer
higher transcriptional activity and elevated enzymatic activity than the wild-type
allele among Japanese population (Watanabe et al. 1994). In an in vivo study using
chlorzoxazone metabolism as a marker, Lucas et al. (1995) did not detect any
diå erences in basal CYP 2E1 activities in Caucasian individuals carrying the c2 allele
P450 inhibition and induction in man
1237
versus wild-type homozygotes, and the inducing eå ect of ethanol appeared to be
weaker in individuals with the mutated CY P2E1 alleles. Thus, it is likely that
additional factors, perhaps other mutations in the CYP2E1 gene (Hu et al. 1997) will
explain the discordant results concerning high CY P2E1 inducibility.
Phenobarbital and other antiepileptic drugs. Phenobarbital is the archetypical
inducer of drug metabolism (Waxman and Azaroå 1992). Phenobarbital is still
being used in the therapy of epilepsy, and it has long been known to be a strong and
broad-spectrum in vivo inducer of drug metabolism. As an example of the potency
of induction, the dose of warfarin required for the anticoagulant eå ect can be
increased up to ten-fold during phenobarbital treatment (Patsalos and Duncan
1993). Also other antiepileptic drugs, especially phenytoin and carbamazepine, have
been shown to induce drug metabolism in man (Perucca 1978, Park and
Breckenridge 1981, Brodie 1992). For example, phenytoin therapy strongly reduces
the C max and AUC of cyclosporin A in vivo (Freeman et al. 1984), and studies in
human primary hepatocytes have shown that phenytoin elevates the activity of
cyclosporin A oxidase (Pichard et al. 1990). Carbamazepine is a broad-spectrum
inducer, enhancing the metabolism of numerous drugs, including warfarin,
theophylline, oral contraceptives and carbamazepine itself (autoinduction) (Brodie
and Dichter 1996).
In rodents, phenobarbital induces CY P forms in several subfamilies, including
CY P1A, CYP2A, CY P2B and CYP 3A, the members in the CY P2B subfamily
reacting most sensitively (Waxm an and Azaroå 1992). Several lines of evidence
suggest that in man the CYP 3A forms are the ones most aå ected by phenobarbital,
carbamazepine and other antiepileptic drugs (Roots et al. 1979, Ohnhaus et al. 1989,
Bertilsson et al. 1997). Recent data obtained with primary human hepatocytes
suggest that CYP 2B6 is also inducible by phenobarbital as well as by rifampicin and
dexamethasone (Chang et al. 1997). In addition, members of the CY P2C subfamily
(CYP2C8 and CY P2C9) are inducible by these agents (Morel et al. 1990, Chang et
al. 1997), and there is also evidence for the in vivo induction of CY P2A6 in response
to antiepileptic drug treatment (Rautio et al. 1994). The inducing eå ect of
antiepileptic drugs on several CY P forms explains the clinical observations that
several of the antiepileptics aå ect a number of structurally unrelated pharmaceuticals by reducing their bioavailability.
The new antiepileptic drugs gabapentin (Goa and Sorkin 1993), lamotrigine
(G oa et al. 1993), and vigabatrin (Connelly 1993) appear to be devoid of clinically
signi® cant inducing properties. Oxcarbazepine, the 10-keto-derivative of carbamazepine, lacks autoinduction properties and does not aå ect the pharmacokinetics
of warfarin (Ka$ lvia$ inen et al. 1993). In a prospective study, Isoja$ rvi et al. (1994)
showed that replacing carbamazepine with oxcarbazepine resulted in an increase in
the half-life and a decrease in the clearance of antipyrine, re¯ ecting a normalization
of liver CYP function. Although clearly being a less potent CYP inducer than
carbamazepine, oxcarbazepine reduces the bioavailability of ethinylestradiol and
levonorgestrel, thus diminishing the action of oral contraceptives containing these
hormones (Jensen et al. 1992, Ka$ lvia$ inen et al. 1993).
The mechanisms mediating phenobarbital induction in rodents have not yet
been thoroughly characterised. It appears that there are no cellular receptors
binding phenobarbital. Rather, the induction is a consequence of complex
rearrangements in putative positive and negative regulatory proteins acting at the 5 ´-
1238
O. Pelkonen et al.
regulatory region of the responsive CYP genes (Waxman and Azaroå 1992). Despite
a greatly increased knowledge on the regulatory factors mediating phenobarbital
induction in experimental animals, virtually nothing is known abut the mechanisms
of induction of the human CYP forms by phenobarbital and other antiepileptic
drugs.
Rifampicin and corticosteroids. Rifampicin is a widely used antibiotic for the
treatment of tuberculosis. The inducing eå ects of rifampicin on drug metabolism in
vivo were noticed soon after its introduction to clinical practice (Baciewicz and Self
1984, Baciewicz et al. 1987). For example, rifampicin accelerates the elimination of
quinidine, 17a-ethinylestradiol, cyclosporine and a number of other drugs
(Venkatesan 1992). Consistent with the fact that most drugs aå ected by rifampicin
are substrates of CYP 3A4, rifampicin has been shown to induce mainly CYP3A
enzymes in the liver in vivo (Watkins et al. 1985, Ged et al. 1989). Slight inducing
eå ects on metabolic pathways mediated by other CYP forms have also been reported
(K ostrubsky et al. 1995).
Human primary hepatocytes have proved to be very sensitive to the inducing
eå ect of rifampicin. Treatment of primary hepatocytes with rifampicin produces
increases in several CYP 3A-mediated catalytic activities, including oxidation of
cyclosporine (Pichard et al. 1990), lidocaine (Li et al. 1995), and the oxazaphosphorine cancer drugs cyclophosphamide and ifosfamide (Chang et al. 1997).
These eå ects are caused by rifampicin concentrations that are equal to the 2± 30 m m
serum concentrations achieved after standard therapeutic doses (Acocella 1978).
Rifampicin increases the amounts of CYP3A4 mRNA and apoprotein, but does not
aå ect the amount of CYP3A5 in primary hepatocytes (Schuetz et al. 1993, Chang et
al. 1997). A more pronounced eå ect on CYP 3A4 was noticed in HepG2 cells, and
CY P3A7 was also elevated in this cell line (Schuetz et al. 1993). An interesting
® nding is that the mRNA encoding CYP 3A7, a form present almost exclusively in
the foetal liver, is inducible by rifampicin in primary hepatocytes derived from adult
liver (Greuet et al. 1996). CYP3A5 appears to be induced by rifampicin in human
colon carcinoma-derived cell lines (Schuetz et al. 1996). In addition to its inducing
eå ects on CYP3A, rifampicin elevates also CYP 2A (Dalet-Beluche et al. 1992) and
CY P2C (Morel et al. 1990) apoprotein levels in human primary hepatocytes,
resembling phenobarbital in this respect.
CYP 3A enzymes are also present at high levels in the human alimentary tract
(K aminsky and Fasco 1992). Induction of CYP 3A4 has been shown to occur in small
bowel enterocytes in response to rifampicin treatment (Kolars et al. 1992). Using the
CY P3A4 substrate cyclosporine as a marker, Hebert et al. (1992) reported that
rifampicin treatment decreases cyclosporine bioavailability more than would be
predicted from by increased hepatic m etabolism. This phenomenon was ascribed to
an elevation of intestinal CYP 3A4-mediated metabolism of cyclosporine (Hebert et
al. 1992). This is important, since combination of cyclosporine with CYP inducers
leads to decreased cyclosporine concentrations in blood and the risk of organ
rejection, and, upon termination of CY P-inducing drug therapy, cyclosporine
concentrations rise to levels which may cause adverse eå ects (Christians and Sewing
1993).
The induction of drug metabolism has been claimed to be also the primary cause
of drug interactions observed with corticosteroids. The analysis of inducing
properties of corticosteroids is complicated by the fact that they are often also
P450 inhibition and induction in man
1239
Figure 2. Role of metabolism in the detoxi® cation and activation of paracetamol. Paracetamol is
normally eliminated as glucuronide and sulphate conjugates. If ingested in high doses ( "
4 g} day), these pathways can be saturated, and more of the parent compound is available for CYP
enzymes to convert to the reactive intermediate NAPQI. This metabolite is scavenged by
glutathione S-transferase, but if the hepatocyte glutathione stores are depleted, formation of
macromolecule adducts with NAPQI occurs in the liver. Conditions which enhance the
toxi® cation process (CYP induction) or decrease the detoxi® cation functions (malnourishment,
diseases) augment paracetamol toxicity. Adapted from Zimmerman and Maddrey (1995), Park
et al. (1996). SG, glutathione adduct.
substrates and hence inhibitors of the reactions under study. For example,
methylprednisolone, prednisolone, and prednisone either increase or decrease
cyclosporin A clearance, depending on the experimental set-up (Christians and
Sewing 1993). However, CY P3A4 expression is increased due to dexamethasone
treatment in vivo (Molowa et al. 1986), and dexamethasone increases the catalytic
activities mediated by CYP 3A4 in human primary hepatocytes (Pichard et al. 1990,
Donato et al. 1995). Prednisone, but not prednisolone or methylprednisolone,
elevates the amounts of CYP3A mRNA, protein, and catalytic activity in human
primary hepatocytes (Pichard et al. 1992).
CYP 3A4 is inducible not only by rifampicin and glucocorticoids but also by
phenobarbital, phenytoin, clotrimazole, spironolactone, and sulfadimidine (Watkins
et al. 1985, Pichard et al. 1990, M orel et al. 1990, Kocarek et al. 1995). The 5 ´
1240
O. Pelkonen et al.
regulatory region of the CYP3A4 gene contains putative binding sites for the
glucocorticoid receptor and an element designated NFSE (P450NF-speci® c
element), which may participate in the induction process (Hashimoto et al. 1993).
Functional analysis proving this is still lacking. Recently, the induction of CYP 3A5
by glucocorticoids was shown to be mediated by a 219-bp enhancer, which
contained two glucocorticoid-responsive element half-sites (Schuetz et al. 1996).
This sequence is unique to CY P3A5, since a similar sequence is lacking from
CY P3A4 and the rat CYP 3A1 (Schuetz et al. 1996). In addition, other consensus
sequences possibly mediating induction in the promoter regions of CYP 3A genes
have been described, and it has become apparent that the host cell environment also
strongly in¯ uences the inducibility of CYP 3A genes (Barwick et al. 1996).
In individuals who are extensive metabolisers of debrisoquine (normal CYP 2D 6
function), 3-week rifampicin pretreatment caused a reduction in m orphine plasma
concentrations and a signi® cant attenuation of codeine’ s respiratory and psychomotor eå ects after a single dose of codeine (Caraco et al. 1997). This may be
explained by an induction of hepatic CYP3A4, which is the major enzyme mediating
codeine N-demethylation, an inactivating pathway competing with the morphineproducing O-demethylation (Caraco et al. 1997).
Peroxisome proliferators. It is well established that several agents that cause
peroxisome proliferation in the liver, such as clo® brate and nafenopin, are potent
hepatocarcinogens and inducers of the CYP 4A subfamily forms in rodents (Johnson
et al. 1996). However, humans are resistant to the peroxisome proliferating eå ects
produced by this class of compounds, and they are not considered to pose a
hepatocarcinogenic hazard (Bentley et al. 1993, Lake 1995). Since members in the
CY P4A subfamily participate in the maintenance of tissue homeostasis, including
regulation of blood ¯ ow in the kidney and brain (Simpson 1997), any changes in the
activities of CYP 4A enzymes might theoretically aå ect these vital functions.
Evidence for this, how ever, is lacking in humans. Due to the very low abundance of
CY P4A protein in the human liver and paucity of relevant drug substrates, its role
in the overall pharmacokinetics of commonly used drugs m ust be considered as
negligible.
Consequences of enzyme induction
For drugs that are active in their parent form, induction may increase the drug’ s
elimination and decrease its pharmacological eå ect. For prodrugs, compounds that
require metabolic activation and whose eå ects are produced by the active
metabolites, enhanced pharmacodynamic eå ects may be expected. The toxicological
implications of enzyme induction have been discussed by Park et al. (1996).
A good example of an adverse consequence due to enzyme induction is the
increased toxicity of paracetamol (acetaminophen). Long-term consumption of
alcohol is associated with liver damage from therapeutic doses (! 4 g } day) of
paracetamol (Nelson 1990). As illustrated in ® gure 2, CYP2E1 is the major enzyme
in converting paracetamol to the reactive intermediate, N-acetyl-p-aminobenzoquinone (NAPQI). Thus, conditions which increase the activity of CY P2E1
may sensitise an individual to the toxic eå ects of paracetamol. This has been shown
to occur in man and experimental animals, particularly associated with chronic
ethanol exposure (Zimmerman and M addrey 1995). In accordance with this, a
P450 inhibition and induction in man
1241
recently developed CYP 2E1 knock-out mouse strain (Cyp2e1 Õ /Õ ) was shown to be
considerably more resistant to the hepatotoxic eå ects of paracetamol than the
corresponding wild-type mice (Lee et al. 1996). These ® ndings would implicate a
dominant role for CYP 2E1 in ethanol-caused paracetamol toxicity, but recent
evidence that ethanol also induces CYP3A forms (Kostrubsky et al. 1995) and the
ability of CYP3A inhibitors to prevent ethanol-induced liver damage in rat
(K ostrubsky et al. 1997) suggest that CYP 3 enzymes may also mediate paracetamol
activation to toxic intermediates.
R esearch needs an d future tren ds
This review is just one attempt to treat (semi)quantitatively in vitro± in vivo
extrapolation of drug metabolism and interactions, and future studies are described
below.
For obvious reasons, human liver microsomes are the gold standard for in vitro
studies. However, because of practical and ethical reasons, their availability is
lim ited and we need a renewable source of human enzymes, such as recombinant
expressed enzymes in suitable host cells. For them to be useful and reliable, we need
more comparative studies in which human liver m icrosomal and recombinant
enzymes are being characterized at the same time and under the same experimental
conditions.
The large variability in human CYP -associated activities needs to be dealt with
in a meaningful way. The ® rst obvious task would be to evaluate to what extent
sometimes extreme variations seen in original studies are due to technical reasons
and } or to ` genuine ’ biological reasons. This type of evaluation has not been
performed to any considerable extent. After this analysis, calculations should be
performed with diå erent, even extreme, scenarios in mind to get some information
about rare deviant possibilities.
For in vitro± in vivo extrapolation, we need more information about factors that
determine the concentration of a drug at the site of an enzyme. Currently we have to
resort in most cases to plasma concentrations, or free concentrations after allowing
for plasma protein binding, but more research is required to de® ne hepatic uptake
and persistence, and non-metabolic processes in the liver and extrahepatic tissues
aå ecting the concentrations of com pounds under study.
A thorough analysis and identi® cation of what are the suæ cient parameters for
drug metabolism and elimination in vivo remains to be performed. Formation
clearances of important metabolites together with knowledge of non-metabolic
absorption characteristics and clearance(s) might be appropriate and suæ cient
know ledge for attempts to perform in vitro± in vivo extrapolations. Interindividual
variability should also be taken into account.
However, identifying relevant parameters to describe in vivo changes in drug
clearance as a consequence of interaction is only a beginning. From the clinical
standpoint, it is of importance to judge whether the change is actually clinically
signi® cant. This is not an easy task, because at ® rst glance, every drug is diå erent in
terms of frequency, severity and dose-dependency of side eå ects, which determine
the clinical signi® cance. It is diæ cult with our current state of knowledge to identify
quantitative rules or classi® cations to con® dently predict whether or not a given
interaction would lead to a clinically signi® cant outcome.
1242
O. Pelkonen et al.
A ckn ow ledgem ents
This review was written to contribute to the goals of the COST Action B1. The
work in the authors’ laboratory has been supported by The Academy of Finland
M edical Research Council (Contract Nos 1051029 and 34555), by the Biomed1
project and by the Biomed2 project EUROCYP.
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