x e n o b i o t i c a , 1998, v o l . 28, n o . 12, 1203± 1253

Inhibition and induction of hum an cytochrom e P450

(CYP) enzym es

O. PELKONEN ‹ *, J. MAÈ ENPAÈ AÈ Œ , P. TAAVITSAINEN ‹ ,

A. RAUTIO ‹ and H. RAUNIO ‹

‹ Department of Pharmacology and Toxicology, University of Oulu, FIN-90220 Oulu,

FinlandΠClinical Research, Leiras OY, PO Box 325, FIN-00101, Helsinki, Finland

Received January 1998

Introduction

Detailed knowledge of metabolism of drugs is crucial for two main reasons.

First, metabolism determines to a large extent pharmacokinetic behaviour, inter-

individual 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 environmental 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 drug-

metabolizing 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, however, and

particularly as a consequence of the detailed knowledge obtained about cytochrome

P450 (CYP) 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.

Characterization of human CYPs in the liver

Hepatic patterns of CYP enzymes

Since the 1980s our knowledge 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 CYP cDNAs, a detailed

knowledge 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 Wrighton 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 semi-

quantitative 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 phenomenon 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 pharmaco-

kinetically distinct subpopulations, for example extensive and poor metabolizers

due to polymorphisms in CYP2D6 (Meyer 1994) and CYP2C19 (Goldstein and De

Morais 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 maybe 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 CYP2F1 and CYP4B1, that are expressed almost exclusively in

certain extrahepatic tissues (Raunio et al. 1995). Some enzymes, CYP1A1 (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.

P450 inhibition and induction in man 1205

CY

P3A

4/5/

7

Dex

trom

etho

rpha

n

Fig

ure

1.

Sch

emat

icre

pre

sen

tati

on

of

hu

man

hep

atic

P4

50

enzy

mes

wit

hm

od

elsu

bst

rate

s,in

hib

itors

and

ind

ucer

s(m

od

i®ed

from

Pel

ko

nen

and

Bre

imer

19

94).

Th

esi

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the

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cles

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ugh

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rop

ort

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ou

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um

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icat

eth

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enzy

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tain

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rvery

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,or

may

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ear

on

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ter

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ucti

on

.

1206 O. Pelkonen et al.

Table 1. Compounds and reactions claimed to demonstrate a high degree of human CYP speci® city.

CYP Preferred substrate and reactionK

m( l m )

Vmax

(nmol }mg 3 h) Reference

1A2 phenacetin O-deethylation 30 800 Bourrie et al. (1996)

ethoxyresoru® n O-deethylation 0.2 3.6 Bourrie et al. (1996)2A6 coumarin 7-hydroxylation 0.4 50 Pearce et al. (1992)

Bourrie et al. (1996)2B6 4-tri¯ uoro-7-ethoxycoumarin

O-deethylase

7 20 Buters et al. (1993)

2C8 taxol hydroxylation 18 50 Harris et al. (1994)

Sonnichsen et al. (1995)2C9 tolbutamide methylhydroxylation 400 15 Knodell et al. (1987)

Bourrie et al. (1996)diclofenac hydroxylation 4 45 Transon et al. (1996)

S-warfarin 7-hydroxylation 4 0.5 table 62C19 S-mephenytoin 4-hydroxylation 60 5 Kato et al. (1992)

Chiba et al. (1993)Relling et al. (1989)

omeprazole oxidation 10 6 Andersson et al. (1993)2D6 debrisoquine 4-hydroxylation 165 2 Boobis and Davies (1984)

Narimatsu et al. (1993)dextromethorpan O-deethylation 5 5 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)

bufuralol 1´-hydroxylation 40 12 Boobis and Davies (1984)Halliday et al. (1995)

Yamazaki et al. (1994)2E1 chlorzoxazone 6-hydroxylation 40 90 Peter et al. (1990)

aniline 4-hydroxylation 15 90 Bourrie et al. (1996)3A4 testosterone (steroid) 6b-

hydroxylation

47 25 Waxman et al. (1983)

midazolam 1-hydroxylation 4 50 Kronbach et al. (1989)

Schmider et al. (1995)Ghosal et al. (1996)

Transon et al. (1996)nifedipine dehydrogenation 15 900 Bourrie et al. (1996)

Km

and Vmax

are approximate and some are con® rmed and modi® ed by our own unpublished studies.

Two subfamilies, namely CYP2C and CYP3A, 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 CYP

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 1207

Table 2. Enzyme-speci® c ` diagnostic ’ inhibitory probes for human P450 enzymes.

CYP Inhibitor

Target CYP

Inhibition(K

i, l m )

Next sensitiveCYP (K

i, l m ) References

1A2 furafylline 0.7 " 10 Bourrie et al. (1996)

Clarke et al. (1994)¯ uvoxamine 0.2 8.2 (CYP2D6) Nemeroå et al. (1996)

2A6 methoxsalen 0.3 2 (CYP2E1) Ma$ enpa$ a$ et al. (1994)Yamazaki et al. (1992)

pilocarpine 4 " 10 (CYP2C9) Bourrie et al. (1996)2C9 sulfaphenazole 0.3 " 25 Bourrie et al. (1996)

2C19 teniposide 12 not known Relling et al. (1989)¯ uconazole 2 8 (2C9) Kunze et al. (1996)

2D6 quinidine 0.06 10 (3A4) Bourrie et al. (1996)Guengerich et al. (1986)

2E1 diethyldithio-carbamate

2 7 (CYP2A6) Yamazaki et al. (1992)

3A4 troleandomycin 18 ND* Zhou et al. (1993)ketoconazole 0.1 " 10 Bourrie et al. (1996)

Schmider et al. (1995)gestodene 7 ND* 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.

With 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 CYP1A, 2C, 2D, 2E and 3A forms, but with widely variable aæ nities

(Knodell et al. 1991).

Further information on P450 substrates and inhibitors can be found in reviews

by Testa and Jenner (1981), Gonzalez (1992), Murray (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

knowledge 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

known CYPs in the metabolism and aæ nity of any xenobiotic. More 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 CYP-speci® city

of the metabolism of a compound. For example, if it is known that the CYP3A4

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 CYP3A4. The following list of predictions is from the review articles of

Watkins (1994) and Wilkinson (1996) and some of these phenomena will be dealt

with more thoroughly in later sections.

E CYP3A4 is induced by rifampicin, antiepileptics, dexamethasone etc and

consequently, the elimination of the compound might be enhanced in situations

involving administration of these drugs.

E CYP3A4 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.

E CYP3A4 is activated by several ¯ avones and endogenous steroids. The ¯ avones,

which are constituents of food, may enhance the metabolism of substrates of

CYP3A4.

E CYP3A4 is very variable between individuals. Also, the elimination of the

studied compound may be variable.

E CYP3A4 is present in intestinal epithelium. This fact may lead to a ® rst-pass

eå ect with respect to the compound under study.

E CYP3A4 displays an age-related reduction in activity. The elimination of the

compound of interest may show the same phenomenon.

E CYP3A4 activity is decreased in liver cirrhosis. The elimination of the studied

compound is expected to be decreased in severe liver disease.

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

environmental 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 metabolism 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 1209

Table 3. Probe or model drugs } substances claimed to be useful in vivo CYP identi® cation in man.

Probe substrate Methods availablea Enzymes*

Aminopyrine p } b, m(r) } ex, pm } u 1A, 3A, NAT2

Antipyrine p } b, p } s, pm } u 1A, 3A, (others)Caå eine pm } u 1A2, NAT2

Chlorzoxazone pm } b, pm } u 2E1,(1A2)Coumarin pm } u, (pm } b) 2A6

Dapsone pm } u NAT2Debrisoquine pm } u 2D6

Dextromethorphan pm } u 2D6, (3A4)Diazepam pm } b, pm } u, m(r) } ex 2C19, 2D6

Diclophenac pm } b 2C9Erythromycin m(r) } ex 3A4

Hexobarbital p } b, pm } u 2C19, (others)Lidocaine adm iv, pm } b 3A4, (1A2)

Lorazepam pm } u UGTMephenytoin pm } u 2C19

metronidazole p } b, pm } u nkMidazolam pm } b, pm } u 3A4

Nifedipine pm } b, pm } u 3A4Omeprazole pm } u 2C19

Paracetamol pm } u 2E1, 1A2, GST, GT,ST

Pentobarbital p } b nkPhenacetin p } b, m(r) } ex 1A2, 2E1

Phenytoin pm } b, pm } u 2C8 } 9Propranolol p } b, pm } u 2C19, 2D6

Sparteine pm } u 2D6Sulfamethazine pm } u NAT2

Theophylline p } b, pm } u 1A2Tolbutamide p } b, pm } u 2C9

Trimethadione p } b, pm } u 1A2, 3AWarfarin p } b, pm } u 2C9, 1A2, 3A4, 2C19

6 b -hydroxycortisol endogenous 3A4d -Glucaric acid endogenous 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 intra-venously ; nk, not known.

* NAT, N-acetyltransferase ; UGT, UDP-glucuronosyltransferase ; GST,glutathione S-transferase ; ST, sulphotransferase.

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.

Inhibition : m echanism s and quantitation

The in-depth treatment and formal derivation of equations to characterise

various modes of inhibition can be found in appropriate textbooks and handbooks.

A good introduction to the basic phenomena of inhibition of drug metabolism 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 Ki, or inhibition constant, which expressed an aæ nity of a compound to an

enzyme. It should be stressed here that Ki

is characteristic for each particular

inhibitor and enzyme, and it is not dependent on any particular substrate used for

the quantitation of an enzyme. With 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 Kiis derived from an in vitro experiment, but for an alternative substrate,

a Ki

should be the same as its Km

.

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, Ma$ enpa$ a$ et al. 1998) and should be thoroughly

investigated.

Inhibition of clearance

For any substrate, the ratio Vmax

} Km

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 Km

, 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 Ki, [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 Km

(i.e. [S] ’ Km

), the percentage inhibition can be simply calculated according to the

equation I(I 1 Ki) 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 Km

, 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] } Km

) ; 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 ] } Ki, is ` inactivated ’ for a more

P450 inhibition and induction in man 1211

Tab

le4

.S

ub

stra

tes

and

inh

ibit

ors

of

the

hu

man

CY

P1A

2en

zym

e.

Dru

gR

eact

ion

Km

or

Ki

(lm

)

[C]v

ivo"

(lm

)I

#

Inte

ract

ion

pote

nti

al

inviv

o$

Ref

eren

ces

Caå

ein

e3-d

emet

hyla

tio

n20

05

0(3

5)

25

1G

ran

tet

al.

(198

8),

Bu

tler

etal.

(19

89),

Tas

san

eey

aku

let

al.

(1993

)

In

h%:

fura

fyll

ine

0.1

10

(?)

99

1

Ola

nzap

ine

N-d

emeth

yla

tion

38

0.2

(?)

0.5

–R

ing

etal.

(199

6)

7-h

yd

roxy

lati

on

24

0.2

(?)

0.8

–R

ing

etal.

(199

6)

On

dan

setr

on

7,8

-hyd

roxy

lati

on

0.1

(70)

Ber

thou

etal.

(1993

)P

arace

tam

ol

oxid

ati

on

33

(20)

Pat

ten

etal.

(1993

)

Ph

enac

etin

O-d

eeth

yla

tion

39

??

?S

esar

dic

eta

l.(1

988

),B

rose

net

al.

(1993

)

In

h:

fura

fyll

ine

0.0

71

0(?

)99

(1

)S

esar

dic

eta

l.(1

990

)In

h:

¯u

voxam

ine

0.2

1(7

7)

83

(1

Bro

sen

etal.

(19

93

)

Pro

pra

no

lol

N-d

eiso

pro

py

lati

on

10

(1A

1)

1.2

(90)

11

–M

arat

he

eta

l.(1

99

4)

20

0(1

A2)

1.2

(90)

0.6

–M

arat

he

eta

l.(1

99

4)

Th

eop

hyll

ine

l-d

emet

hyla

tio

n42

010

0(6

0)

19

1R

ob

son

etal.

(19

87),

Ku

nze

eta

l.(1

99

3),

Gu

eta

l.(1

99

2),

Rasm

uss

enet

al.

(1994

),T

jia

etal.

(19

96)

3-d

emet

hyla

tio

n45

510

0(6

0)

18

18-h

yd

roxy

lati

on

55

010

0(6

0)

15

1

"M

axim

alco

ncen

trat

ion

of

the

dru

gin

viv

oaft

ercl

inic

ally

rele

van

td

ose

s.#

I=

inh

ibit

ion

perc

en

tage

:as

sum

ing

com

pet

itiv

ein

hib

itio

nan

dth

esu

bst

rate

con

cen

trat

ion

’K

m,

the

per

cen

tage

inh

ibit

ion

was

calc

ula

ted

acc

ord

ing

toth

e

eq

uat

ion

I }(I

1K

i)31

00

.$

Qu

alit

ativ

eev

iden

cefo

rin

viv

oin

tera

ctio

ns

cau

sed

by

the

dru

g.

%In

hib

itor

of

the

react

ion

ab

ove.

1212 O. Pelkonen et al.

Tab

le5

.S

ub

stra

tes

and

inh

ibit

ors

(In

h)

of

the

hu

man

CY

P2

C9

enzy

me

asas

sess

edin

hu

man

liv

erm

icro

som

es.

Dru

g}g

rou

pR

eact

ion

Km

or

Ki

(lm

)

[C]v

ivo"

(lm

)I#

Inte

ract

ion

po

ten

tial

inviv

o$

Ref

ere

nce

s

Dic

lofe

nac

4-h

yd

roxy

lati

on

41

0(9

9)

71

1L

eem

an

net

al.

(199

3),

Tra

nso

net

al.

(1996

)

S,R

-Ib

up

rofe

n2-h

yd

roxy

lati

on

38±47

10

(99)

25

1H

amm

anet

al.

(199

7)

In

h%:

sulf

aph

en

azo

leco

mp

etit

ive

inh

ibit

ion

0.1

1±0.1

28

0(6

5)

100

1H

amm

anet

al.

(199

7)

S,R

-ib

up

rofe

n3-h

yd

roxy

lati

on

21±29

10

40

1H

amm

anet

al.

(199

7)

In

h:

sulf

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P450 inhibition and induction in man 1213

prolonged period of time, being unavailable for catalysis, and the inhibition cannot

be abolished by increasing the substrate concentration.

Mechanism-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 non-

competitive mode of inhibition. However, the detection of mechanism-based

inhibition requires speci® c incubation conditions. A preincubation of liver micro-

somes 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

Whatever the exact Kiis, 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 ] } Ki

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.

Most 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. However, 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.

E The concentration of the drug in clinical situation should be high enough, so that

inhibition would be manifested in vivo.

E The therapeutic index of the drug should be narrow, such that a change caused

by an interacting drug would cause side eå ects.

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 may be diæ cult to put into exact

numbers and may cause disagreement even between experts.

Exam ples of substrates and inhibitors with aæ nity predom inantly to a

single CYP 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 CYP forms. The Km

and Ki

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

limit 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. We 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 man, the CYP1A1 protein is expressed at a very low level in the liver (Wrighton

et al. 1986), whereas CYP1A1 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 CYP1A1 is able

to oxidize a number of drug substrates (as has been demonstrated with, e.g.,

heterologously expressed CYP1A1), we will not deal with this enzyme further in this

review.

The CYP1A2 gene product is clearly the predominant hepatic enzyme of

CYP1A 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 CYP1A2 in

extrahepatic tissues.

Substrates and inhibitors. Some examples of substrates and inhibitors for CYP1A2

are shown in table 4. The puri® ed human CYP1A2 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 CYP1A2 (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 Km

for CYP1A2 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 CYP1A2 (table 4).

a -Naphtho¯ avone (7,8-benzo¯ avone) has been shown to be a potent and

relatively speci® c inhibitor of both CYP1A isoforms (Burke et al. 1977). However,

it has not been used in vivo in man. Furafylline, a methylxanthine analogue, is a

potent inhibitor of several CYP1A2-associated metabolic reactions (tables 2 and 4),

whereas it has only a weak eå ect on CYP1A1 (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 CYP1A2, 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.

More information on ¯ uvoxamine will be presented in a later section.

CYP2C9

The human genome has been shown to contain several genes belonging to the

CYP2C subfamily (Goldstein and de Morais 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 CYP2C9 and CYP2C19 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 CYP2C8, although the aæ nity

of tolbutamide for this isoform is clearly lower than for CYP2C9 (Relling et al. 1990,

Veronese et al. 1993).

Substrates and inhibitors of CYP2C9. A number of important drugs are substrates

of CYP2C9 (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 Km

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 much 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 CYP2C9 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. Warfarin, 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 R-

and S-enantiomers of warfarin are metabolized by diå erent metabolic pathways.

S-warfarin is mainly metabolized to 6- and 7-hydroxywarfarin. 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

(Kaminsky 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 half-

life 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 ml 3 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. Warfarin is highly albumin

bound with values ranging from 97 to 99.5 % (Dollery et al. 1991).

In vitro studies with human liver microsomes 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 CYP2C9-catalysed tolbutamide methylhydroxylation with a Ki

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 a æ nity of warfarin towards

CYP2C9, suggesting a need for more thorough studies. The Kifor 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 1217

Table 6. Kinetics of oxidative metabolism of warfarin by human liver microsomes and recombinant

expressed CYP enzymes.

CYP " Metabolite Km

( l m )

Vmax

(nmol } mg 3 h)

Formationclearance

in vivo #

S-warfarin M 7OH 4 0.5 1847M 6OH 3 0.1 400

300 1.1

r2C9 7OH 4 (40) $

r2C9 60H 4 (7)

r3A4 6OH 300 (90)

R-warfarin M 6OH 265, 1412 0.9, 11.6 462M 7OH 159, 1580 0.3, 2.7 227

M 8OH 162, 1500 0.9, 2.2 338M 10OH 400 2.4 342

r1A2 6OH, 7OH, 1600 not available

8OHr2C19 8OH, 6OH, C 200 not available

7OHr3A4 10OH 400 (200)

Data derived from Kunze and Trager (1996) and Kunze et al. (1996)." M, human liver microsomes ; r, recombinant.# ml 3 h Õ " 3 kg Õ " 10 Õ $ , 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 CYP2C9 is not a

metabolizing enzyme, the high aæ nity would indicate a possibility for interactions.

Warfarin metabolism in human liver microsomes has been studied with

diagnostic inhibitors and antibodies and correlation analysis, as well as with

recombinant enzymes. With 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 Km

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 CYP2C9. With respect to R-warfarin clearance, at least three

CYPs participate, but the Km

are almost two orders of magnitude higher than that

for CYP2C9 (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).

Comparison 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-hydroxywarfarin represent up to 90 % of the total metabolite

clearance of S-warfarin, a ® gure that is in an excellent correlation with the role of

CYP2C9 in the in vitro metabolism of S-warfarin.

Because CYP2C9 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 CYP3A4, 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 CYPs 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, CYP2C9, has been identi® ed by in vitro approaches.

Because the Km

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 CYP2C9 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 metabolism 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 CYP2C9 activity in vivo, but the usefulness of other metabolites as

indices for other CYPs remains to be demonstrated.

CYP2C19

CYP2C19-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 CYP2C19 (Goldstein

and de Morais 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 CYP2C19

enzyme, but very few even remotely speci® c inhibitors (Guengerich 1995b).

Proguanil, omeprazole and imipramine are metabolized by CYP2C19, but also

other CYPs are important 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 R-

warfarin 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 polymorphism) has been elucidated in great detail (Meyer et al.

1990). About 7 % of the Caucasian population are PMs (Alvan et al. 1990), because

mutations in CYP2D6 gene have led to an absence of a functional CYP2D6 protein

(Gonzalez 1990, Meyer 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 CYP2D6 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 CYP2D6 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 importance 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, thio-

ridazine, haloperidol and clozapine) and miscellaneous other drugs like codeine,

dextromethorphan and phenformin are substrates for CYP2D6 (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. conse-

quently, dangerous drug interactions may occur when using drugs that are oxidized

by CYP2D6.

The inhibitor spectrum of CYP2D6 has been thoroughly studied. Quinidine

is a highly selective and potent inhibitor, although it is not a substrate of the

CYP2D6 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

CYP2D6, several alkaloids and neuroleptics were found to be potent inhibitors

(Fonne-P® ster and Meyer 1988). The Ki

of the alkaloid ajmalicine was as low as

3.3 n m . Many 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 CYP2E1 is discussed in

a later section. It seems probable that CYP2E1 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 CYP2E1

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). Most 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, CYP2E1 accepts various volatile anaesthetic

agents as substrates (Koop 1992). Chlorzoxazone has become a widely used substrate

for CYP2E1 in vitro (table 2). The advantage of using this compound is that the

chlorzoxazone 6-hydroxylase assay is very sensitive compared with the former

CYP2E1-speci® c assays used. Chlorzoxazone might also be an appropriate probe to

study CYP2E1 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 CYP1A1 is also able to metabolize chlorzoxazone (Ono et al. 1995).

Because CYP1A1 may be a prominent enzyme in extrahepatic tissues especially after

PAH-type induction, chlorzoxazone may not be used as a speci® c probe for CYP2E1

in extrahepatic tissues.

There are several more or less speci® c inhibitors of CYP2E1. Disul® ram inhibits

CYP2E1-associated activities in man (Guengerich et al. 1991). Disul® ram is

reduced to diethyldithiocarbamate which inhibits CYP2E1 relatively potently, but

it is also an almost equally potent inhibitor of CYP2A6 (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

(Koop 1992).

It should be stressed that ethanol and acetone, as well as several volatile

anaesthetics, all substrates for CYP2E1, can attain relatively high levels in the

body and might thus interfere with CYP2E1-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 CYP2E1 and could give completely erroneous results if not properly

used.

Human CYP3A subfamily

The members of the CYP3A subfamily are CYP3A4, CYP3A5 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,

CYP3A4 is expressed in the human intestine and it catalyses drug metabolism there

as well (Kolars et al. 1992b, Guengerich 1995b). CYP3A4 is expressed in all human

livers and about 50 % of drugs currently in the market are substrates for it. The

CYP3A5 protein is expressed at detectable levels in the human liver in about 25 %

of individuals. The third member of the CYP3A subfamily is CYP3A7 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 CYP3A subfamily have

similar substrate preferences (Gonzalez 1992b, Guengerich 1995b). However,

CYP3A5 may have some diå erences in its aæ nity to bind substrates when compared

with CYP3A4 (Wrighton et al. 1989, 1990). cDNAs expressing CYP3A4 and

CYP3A4 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, troleandomycin, cyclo-

P450 inhibition and induction in man 1221

sporin, warfarin, triazolam and midazolam (Guengerich and Shimada 1991,

Wrighton and Stevens 1992). many procarcinogens like AFB1 are also activated by

CYP3A enzymes (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 CYP3A4

tested include midazolam, nifedipine, dapsone and lidocaine (Watkins 1994).

Midazolam is a well characterised probe for CYP3A4 (see below). However,

correlations between diå erent in vivo CYP3A probes in man are not always very

good and may arise from the heterogeneity of CYP3A isoforms. It is not always

clear which CYP3A isoform is responsible for the metabolism of a drug in question.

There is a number of isoform-speci® c inhibitors of the members of CYP3A

subfamily. Troleandomycin (TAO) has been shown to form a metabolic-inter-

mediate complex with CYP3A isoforms (Pessayre et al. 1983) and seems to be

relatively selective. Gestodene is also a selective mechanism-based inhibitor of

CYP3A4 and CYP3A5 (Guengerich 1990, Wrighton et al. 1990). These inhibitors

have to be initially oxidized before they form complexes with speci® c P450s. Also,

many substrates listed above inhibit CYP3A mediated reactions.

Grapefruit juice has been shown to inhibit the metabolism of a number of

CYP3A substrates (Bailey et al. 1991, Soons et al. 1991). The components of

grapefruit juice, like ¯ avonoids and furanocoumarins have been claimed to inhibit

CYP3A enzymes, and further the metabolism of CYP3a substrates like felodipine,

cyclosporine, terfenadine and midazolam just a few to mention (Ameer and

Weintraub 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 CYP3A4 protein in small intestine and not to the inhibitory role on

CYP3A4 of ¯ avones found in grapefruit juice (Guengerich 1995b).

An interesting feature of CYP3A4 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 CYP3A4 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 CYP3A4. The most potent stimulator of CYP3A4 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 CYP3A4 activity may have clinical

signi® cance. Further, endogenous substances like progesterone and testosterone

have also been shown to stimulate CYP3A-mediated reactions (Johnson et al. 1988,

1222 O. Pelkonen et al.

Kerr et al. 1994, Ma$ enpa$ a$ et al. 1998). However, the clinical signi® cance of these

® ndings is unclear, but potentially the stimulation of CYP3A may result in low

plasma levels of CYP3A substrates or the stimulators may enhance the activation of

carcinogens by CYP3A. The stimulation of midazolam metabolism is discussed

below.

CYP3A enzymes are induced by several antiepileptics, rifampicin and cortico-

steroids 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 human hepatocytes. The compounds studied, as well as some additional

information, are listed in table 7. Several important conclusions can be made on the

basis of this information.

E It seems that apparently there is very little correlation between the percentage

inhibition, calculated on the basis of a Ki

and in vivo plasma concentration, and

the potential of a compound to cause interactions that are regarded as ` clinically

signi® cant ’ .

E 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.

E 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 (Ki) and in vivo achievable concentrations. For these compounds the

obvious reason is their conversion to reactive products which cause mechanism

based inhibition. How much ` suicide inhibition ’ would explain other dis-

crepancies (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.

At the present moment, the reasons for poor correlations are unclear. However, the

secondary sources from where we extracted the information on potential inter-

actions, may list some interactions on the basis of what is expected from the

knowledge 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. Midazolam is

a short-acting benzodiazepine derivative that has been used as a hypnotic agent

(Dundee 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 CYP3A4-

mediated metabolism of cyclosporin in human cultured hepatocytes, and comparison with in vivoobserved interactions (inhibition potency data taken from Pichard et al. 1990).

Inhibitor Ki

( l m )C ( l m ) #

in vivo

Calculated

inhibition(%) "

Interactionpotential #

Clotrimazole 0.1 2.5 96 ?

Ketoconazole 0.7 10 (98) 93.5 1Miconazole 0.9 2.5 (92) 73.5 1Itraconazole 1.2 0.4 (99) 25 1Nicardipine 8 0.3 (95) 3.6 1Bromocriptine 8 0.001 (96) 0.01 1Troleandomycin 10 3 23 1Nifedipine 10 0.3 (90) 3.0 1Terfenadine 10 0.01 0.1 ?

Ergotamine 12 ? ? ?Isradipine 12 0.15 (96) 1.2 1Josamycin 19 3 14 1Midecamycin 22 3 12 1Dihydroergotamine 23 ? ? ?Verapamil 24 1.5 (90) 6 1Midazolam 40 0.25 (96) 0.6 –Progesterone 45 0.04 (97) 0.1 ?

Fluconazole 60 70 54 1Diltiazem 63 0.3 (85) 0.5 1Erythromycin 75 3 (83) 4 1Glibenclamide 78 0.1 (99) 0.1 ?

Cortisol 125 0.6 (95) 0.5 1Ethinylestradiol 172 0.5 (95) 0.3 ?

Prednisone 190 0.7 (80) 0.4 1Me-prednisone 190 0.7 (80) 0.4 1Prednisolone 210 0.7 (80) 0.3 1

" Assuming competitive inhibition and the substrate concentration ’ Km

for cyclosporin metabolism, the percentage inhibition was calculated accordingto the equation I(I 1 K

i) 3 100.

# 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). Plus-sign means that clinical studies have indicated interactions between the

inhibitor and the CYP3A4-mediated elimination and } or metabolite formationof cyclosporin or other CYP3A4-associated drugs.

In vitro metabolism. Midazolam is metabolized to 1´-hydroxy (1´-hydroxy-

midazolam) 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 CYP3A4 and

CYP3A5 enzymes have been shown to have similar substrate preferences (see above)

and 1´-hydroxymidazolam and 4-hydroxymidazolam formation are catalyzed by

both CYP3A4 and CYP3A5 isoforms (Kronbach et al. 1989, Gorski et al. 1994).

However, it has been reported that microsomal samples containing high levels of

CYP3A5 had a higher 1´-hydroxymidazolam } 4-hydroxymidazolam ratio than the

samples containing only CYP3A4 (Ma$ enpa$ a$ et al. 1998). In addition, CYP3A7 is

responsible for 1´-hydroxymidazolam and 4-hydroxymidazolam formation in

human foetal liver microsomes (Gorski et al. 1994 ; Ma$ enpa$ a$ et al. 1998).

In vivo metabolism . Midazolam is also metabolised to 1´-hydroxymidazolam and 4-

hydroxymidazolam 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.

Inhibitor or inducer*IC

& !or K

i( l m )

AUC, % of control(placebo)

Erythromycin 194** 442

Azithromycin 170 87Verapamil 100 292

Fluconazole " 80** 373Itraconazole 1 1080

Ketoconazole 0.1* 1590Rifampicin* inducer 4

Data are derived from the following : Gascon and Dayer (1991), Olkkola etal. (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

(Dundee 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 CYP3A4 in vivo (Thummel et al. 1994a, b). Many diagnostic inhibitors

of CYP3A reduce the clearance of midazolam as discussed further below. Additional

indication of the involvement of CYP3A 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).

CYP3A4 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 CYP3A speci® c

inhibitors. 1´-Hydroxymidazolam formation is inhibited by substrates and } or

inhibitors of CYP3A like cyclosporine, erythromycin, itraconazole, ketoconazole

and terfenadine (Gascon and Dayer 1991, Wrighton and Ring 1994, Goldberg et al.

1996). Further, midazolam has been shown to inhibit the metabolism of terfenadine

and quinine, which both are substrates of CYP3A (Jurima-Romet et al. 1994, Zhang

et al. 1997). As already discussed above, grapefruit juice inhibits the metabolism of

CYP3A4 substrates and it also inhibits midazolam metabolism (Kupferschmidt et

al. 1995, Ameer and Weintraub 1997). Large diå erences have been observed in the

ability of CYP3A inhibitors to inhibit midazolam 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 metabolism 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 macrolide 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). However, 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). Moreover,

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, Ma$ enpa$ a$ et al. 1998). However,

a -naphtholavone had no eå ect on the CYP3A mediated 4-hydroxymidazolam

formation, although the inhibitors of midazolam metabolism 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-hydroxy-

midazolam formation, respectively (Ma$ enpa$ a$ et al. 1998). The regioselective

stimulation of midazolam is another indication of the complexity of the regulation of

CYP3A 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 CYP3A 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 Cmax

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

CYP3A4 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 CYP3A substrates.

Exam ples of substrates and inhibitors with aæ nity for several CYPs

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. Admittedly,

warfarin is also oxidized by several CYPs, 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 O. Pelkonen et al.

Table 9. Kinetics and CYP-associated catalysis of pathways of the oxidative metabolism of antipyrine.

Reaction Km

(m m ) "

Vmax

(nmol } mg*min) "

CYPs participating in thereaction #

4-hydroxylation 5.2± 23.1 0.57± 1.40 3A4(5) up to 65 %

1A2 about 30 %2A6, 2B6

N-demethylation 5.9± 26.3 0.34± 2.23 2C(9 } 19) 75± 80 %1A2 20± 25%

2A6, 2C8, 2C18, 2D6, 2E1, 3A3-Methylhydroxylation 9.0± 21.1 0.59± 1.41 1A2 50 %

2C(9) 50 %2C8, 2C9, 2E1

" Ranges for the Km

and Vmax

have been taken from Boobis et al. (1981), Engel et al. (1996) and Sharerand Wrighton (1996).

# Contributions of the major CYP(s) catalysing the reaction has been estimated on the basis ofdiagnostic 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 Medline 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 limited 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 information on isoforms involved in

antipyrine metabolism, except the classical inducers of the MC-type and the PB-

type aå ect the metabolic pathways diå erentially. However, on the basis of studies

with some diagnostic inhibitors it seemed probable that CYP2C (sulphaphenazole),

CYP2D (debrisoquine, quinidine) and CYP3A (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 Wrighton (1996) and Engel et al. (1996) have

changed the situation completely. Through their work it is known that practically all

known 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 Km

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 CYP1A2, CYP2C(9) and CYP3A(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 (CYP3A4 and CYP2C9 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

CYP 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 enzyme 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. Whether 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 N-

demethylations to N-desmethylcitalopram and N-didesmethylcitalopram, both of

which are detected in plasma, although the levels are roughly one-third and one-

tenth 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 CYP 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 human 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 Ki

vary from 5 to 19 l m

(Brosen 1994, 1996). Thus it seems that, CYP2D6 excluded, citalopram has a

relatively low aæ nity towards most human hepatic CYPs. Mainly due to the

1228 O. Pelkonen et al.

Table 10. 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 Ki

( l m )

1A1 ethoxyresoru® n O-deethylation 82 " 100

1A2 ethoxyresoru® n O-deethylation 96 " 100

theophylline N-demethylations 92± 95 " 100

2A6 coumarin 7-hydroxylation 92 " 100

2C9 tolbutamide methylhydroxylation 88 " 100

2C19 S-mephenytoin 4-hydroxylation 78 " 100

2D6 dextromethorpan O-deethylation 7sparteine oxidation 5.1imipramine 2-hydroxylation 19

2E1 chlorzoxazone 6-hydroxylation 92 " 100

3A4 testosterone 6 b -hydroxylation 98 " 100cortisol 6 b -hydroxylation 71 " 100

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(%) Prediction "

Fluvoxamine 12.5 " 10 1A2 1Furafylline 10 ! 5 1A2 –Phenacetin 10 ! 5 1A2 –

Coumarin 20 ! 5 2A6 –

Sulfaphenazole 10 ! 5 2C9 –

Omeprazole 100 " 10 2C19 1Mephenytoin 500 " 10 2C19 1

Quinidine 5 " 10 2D6 1Paroxetine 20 " 10 2D6 1

Methylpyrazole 20 ! 5 2E1 –DEDC 20 ! 5 2E1 –

Ketoconazole 2.5 " 10 3A4 } 5 1Troleandomycin 50 " 10 3A4 } 5 1

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 much 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 1229

Table 12. N-demethylation of citalopram enantiomers by cDNA-expressed human liver micrososal

cytochrome P450 enzymes.

CYP

Vmax

(pmol } h

3 pmol CYP) Km

( l m )

Intrinsicclearance

(CLi)

1A2 3.0 ND (high) ND

2A6 not detectable

2B6 not detectable

2C9 not detectable

2C19 S-CIT 78.1 198 0.39R-CIT 53.1 211 0.25

2D6 S-CIT 5.0 18.2 0.27

R-CIT 8.5 22.1 0.38

2E1 not detectable

3A4 S-CIT 62.1 169.0 0.37R-CIT 43.6 163.0 0.27

Data derived from Rochat et al. (1997) andKobayashi et al. (1997).

S-CIT and R-CIT refer to the S and R isomersof citalopram, respectively.

(table 2) suggest that at least CYP3A4 } 5, CYP2C19 and CYP2D6 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

CYPs other than CYP1A2. Furthermore, furafylline and phenacetin, which are

probably more selective towards CYP1A2, do not inhibit citalopram N-demethyl-

ation 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 CYPs. Expressed enzymes with relatively high turnover

numbers were CYP2C19, CYP2D6, and CYP3A4. Also CYP1A2 showed little

activity. CYP2D6 seems to be a high-a æ nity enzyme, but intrinsic clearance

calculations demonstrated that all three enzymes were roughly equally active. When

compared with results obtained with human liver microsomes, CYP2D6 seems to

represent the high-aæ nity (but low capacity) component, and CYP2C19 and

CYP3A4 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, CYP2C19 and CYP2D6 represent about 30, 4 and

2 % of total P450 content, respectively (Shimada et al. 1994). Because the intrinsic

clearances of drugs by these CYPs 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 (CYP2C19) and quinidine (CYP2D6) 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,

CYP2C19 and CYP2D6. The aæ nity of CYP2D6 is roughly one order of magnitude

1230 O. Pelkonen et al.

Table 13. Inhibition of CYP2D6-mediated desipramine 2-hydroxylation by SSRI-compounds in

human liver microsomes in vitro and calculated inhibition in vivo.

SSRI

Ki

( l m ) "

Cmax

( l m ) #

Inhibitionin vivo

(%) $ Eå ect in vivo %

Fluoxetine 3, 0.6 1 (94) 25 " 350 %Nor¯ uoxetine 2, 0.43 1 (94) 33 " 350 %

Fluvoxamine 20, 8.2 1 (77) 5 14 %Paroxetine 2, 0.15 0.2 (95) 10 " 300 %

Sertraline 20, 0.7 0.1 (99) 0.5 26± 72 %Norsertraline 15, NK 0.1 0.7 26± 72 %

Citalopram 80, 5.1 0.4 (70) 0.5 46 %Norcitalopram " 100, NK 0.4 ! 0.5 46 %

Quinidine 0.05 10 100 potent

" First Ki

values are taken from Moltke et al. (1994) and are based on

inhibition of desipramine 2-hydroxylation activity, except for citalopram andnorcitalopram, for which the values are calculated on the basis of relative

inhibition of imipramine 2-hydroxylation (Skjelbo and Brosen 1992). Thesecond values are for sparteine oxidation in vitro (Brosen 1993). NK, not

known.# C

maxdenotes the (peak) plasma concentration of a SSRI drug in vivo

( l m ). Plasma protein binding (in parentheses), which aå ect the free con-centration, has not been taken into consideration. Liver } plasma partition ratio

has been assumed to be 1, although it may actually be considerably higher forsome 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

mfor desipramine metabolism, the percentage inhibition was calculated ac-cording to the equation I } (I 1 K

i) 3 100.

% Percent increase in the area under the plasma concentration-time curve ofdesipramine (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 CYP3A4 or CYP2C19, 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 CYP3A4 interactions which make it

possible to calculate potential in vivo inhibition for representative CYP2D6-,

CYP3A4-, and CYP1A2-catalysed metabolic reactions (desipramine, midazolam

and phenacetin, respectively).

With respect to CYP2D6 (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 CYP2D6-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 CYP2D6, 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).

SSRIAæ nity invitro ( l m ) " C

max( l m ) # I in vivo (%) $ Eå ect in vivo %

Fluoxetine 7.1, 44.3 1 (94) 12.3, 2.2 detectable ?

Nor¯ uoxetine 2.7, 8.0 1 (94) 27.0, 11.1 detectable ?Fluvoxamine 5.6, 20.2 1 (77) 15.2, 4.7 detectable ?

Paroxetine 3.8, 14.3 0.2 (95) 5.0, 1.3 absentSertraline 3.5, 20.3 0.1 (99) 2.8, 0.5 absent

Norsertraline 3.5, 10.7 0.1 2.8, 0.9Citalopram 165 0.4 (70) 0.2 absent

Ketoconazole 0.02 10 100 strong

" Aæ nity values are Kiof inhibition of two midazolam CYP3A4-mediated reactions (von Moltke et

al. 1996), except for citalopram where the value is the Km

for citalopram N-demethylation (Rochat et al.1997).

# Cmax

denotes the (peak) plasma concentration of a SSRI drug in vivo ( 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

mfor midazolam metabolism,

the percentage inhibition was calculated according to the equation I } (I 1 Ki) 3 100.

% 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 under-

estimation 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).

With 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, carba-

mazepine, terfenadine and diazepam whereas ¯ uvoxamine treatment inhibits the

elimination of alprazolam and terfenadine (Nemeroå et al. 1996). However, the data

of Stevens and Wrighton (1993) do not support a signi® cant inhibition of midazolam

hydroxylation by ¯ uoxetine. With respect to citalopram, the only values for aæ nities

for CYP3A4 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 CYP3A4.

With respect to CYP1A2, 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 CYP1A2. 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 Ki

( l m ) " Cmax

( l m ) # I in vivo (%) $ Eå ect in vivo %

Fluoxetine " 100 1 (94) ! 1 caå eine ( –)clozapine ( 1 ?)

Nor¯ uoxetine " 100 1 (94) ! 1 ?

Fluvoxamine 0.2 1 (77) 83 caå eine ( 1 1 1 )

theophylline ( 1 1 1 )clozapine ( 1 1 1 )

imipramineamitriptyline

clomipramine

Paroxetine 45 0.2 (95) 0.4 caå eine ( –)

Sertraline 70 0.1 (99) 0.1 not known

Citalopram " 100 0.4 (70) ! 0.4 caå eine ( –)

" Ki

in vitro for phenacetin O-deetylation (Brosen et al. 1993).# C

maxdenotes the (peak) plasma concentration of the SSRI drug in vivo ( 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

mfor desipramine metab-

olism, the percentage inhibition was calculated according to the equation I } (I 1 Ki) 3 100.

% 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.

Induction

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 enzyme 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 1233

Table 16. Classi® cation of inducers of drug-metabolizing enzymes.

Class Prototype inducer Principal enzymes aå ected

PAH-type 2,3,7,8-

Tetrachlorodibenzo-p-dioxin

CYP1A, UDP-

glucuronosyltransferase

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-cutin 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 metabolism of various enzymes participating in the

metabolism of a compound under study.

Spectrum and mechanisms of induction

Several individual agents that induce CYP 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

Morais 1994, Guengerich 1995, Wilkinson 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 CYP1A forms. Examples include theophylline, caå eine, antipyrine,

imipramine, paracetamol (acetaminophen), and phenacetin (table 5). The metab-

olism of these drugs is mediated predominantly by CYP1A2, which represents

approximately 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, CYP1A2 activity is

increased in human primary hepatocytes by the prototype PAH inducer 3-

methylcholanthrene (Morel et al. 1990).

CYP1A1 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 CYP1A induction have been thoroughly

elucidated (Hankinson 1995). CYP1A 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 ARNT (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 CYP1A enzymes may have two diå erent types of conse-

quences : 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 CYP1A1 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,

Wedlund et al. 1994, Jacquet et al. 1996). Recent studies with heterologously

expressed CYP1A1Val % ’ # alleles clearly show that the catalytic and kinetic properties

of this enzyme do not diå er from those of the wild-type (CYP1A1Ile % ’ # ) enzyme

(Zhang et al. 1996, Persson et al. 1997). It may be that the high-inducibility

CYP1A1 phenotype will be explained by variations in the regulatory genes rather

than the structural gene. Despite convincing evidence that mutations in the Ah-

receptor gene confer high and low inducibility in inbred strains of mouse, attempts

to correlate CYP1A1 inducibility with known polymorphisms in the human AH-

receptor gene have yielded negative results (Micka et al. 1997).

The regulation of CYP1A2 is not as well characterized as that of CYP1A1. It is

inducible by smoking, charbroiled food, cruciferous vegetables, omeprazole and

even vigorous exercise (Wrighton and Stevens 1992a, Guengerich 1995a). Induction

of CYP1A2 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 mice develop normally apart from de® cient metabolism of some xenobiotics

metabolised by CYP1A2. Thus CYP1A2 appears not to have any crucial endogenous

function.

CYP1B1, a novel member in the CYP1 family, has catalytic properties similar

but not identical to CYP1A members (Shimada et al. 1996). In rodents, CYP1B1

is highly inducible by PAHs in several extrahepatic tissues, but the inducibility in

human tissues appears to diå er from that of CYP1A1 (Hakkola et al. 1997).

Omeprazole and congeners. The CYP1A-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

Michnovicz 1993, Rizzo et al. 1996). In human primary hepatocytes, omeprazole

and lanzoprazole also appear modestly to induce CYP3A members (Curi-Pedrosa et

al. 1994), and both agents stimulate CYP1A1 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, Curi-

Pedrosa 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 CYP1A coding genes

(Quattrochi and Tukey 1993). Recent evidence suggests that omeprazole is

metabolised to a sulfenamide 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 (Hankinson 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

CYP1A2 by omeprazole is aå ected by the CYP2C19 status, since omeprazole is

metabolized by CYP2C19. 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

CYP2C19 extensive metabolizers, whereas a 40 % increase is elicited in CYP2C19

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 CYP2C19

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 omeprazole 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 CYP1A2 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). CYP2E1 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 CYP2E1

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 CYP2E1 protein and mRNA in the human liver

(Perrot et al. 1989, Takahashi et al. 1993). The plasma clearance of chlorzoxazone,

a drug metabolized by CYP2E1, 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 CYP2E1 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 CYP2E1 induction depending on the length of the washout

period applied (Chien et al. 1997, O ’ Shea et al. 1997).

Like most other CYP forms, CYP2E1 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

CYP2E1 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 CYP2E1

and CYP3A apoproteins (Kostrubsky et al. 1995). In HepG2 cells transfected with

the coding sequence of CYP2E1 cDNA, ethanol increased CYP2E1 protein but not

mRNA levels, indicating that the elevation is due to protein stabilization (Carroccio

et al. 1994).

The mechanism of CYP2E1 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,

CYP2E1 induction occurs in two phases : at blood levels ! 300 mg } dl the CYP2E1

protein levels are increased without changes in mRNA, and higher blood ethanol

levels also cause increases in the amount CYP2E1 mRNA (Ronis et al. 1996). The

mechanisms of increases in CYP2E1 protein levels include enhanced translation and

protein stabilization. One mechanism for stabilization of CYP2E1 is protection of

the protein from cAMP-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 polymorphisms 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 CYP2E1 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 CYP2E1 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 CYP2E1 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 Cmax

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 CYP forms in several subfamilies, including

CYP1A, CYP2A, CYP2B and CYP3A, the members in the CYP2B subfamily

reacting most sensitively (Waxman and Azaroå 1992). Several lines of evidence

suggest that in man the CYP3A 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 CYP2B6 is also inducible by phenobarbital as well as by rifampicin and

dexamethasone (Chang et al. 1997). In addition, members of the CYP2C subfamily

(CYP2C8 and CYP2C9) are inducible by these agents (Morel et al. 1990, Chang et

al. 1997), and there is also evidence for the in vivo induction of CYP2A6 in response

to antiepileptic drug treatment (Rautio et al. 1994). The inducing eå ect of

antiepileptic drugs on several CYP forms explains the clinical observations that

several of the antiepileptics aå ect a number of structurally unrelated pharma-

ceuticals by reducing their bioavailability.

The new antiepileptic drugs gabapentin (Goa and Sorkin 1993), lamotrigine

(Goa et al. 1993), and vigabatrin (Connelly 1993) appear to be devoid of clinically

signi® cant inducing properties. Oxcarbazepine, the 10-keto-derivative of carba-

mazepine, lacks autoinduction properties and does not a å 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 CYP3A4, 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

(Kostrubsky 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 CYP3A-mediated catalytic activities, including oxidation of

cyclosporine (Pichard et al. 1990), lidocaine (Li et al. 1995), and the oxaza-

phosphorine 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 CYP3A4 was noticed in HepG2 cells, and

CYP3A7 was also elevated in this cell line (Schuetz et al. 1993). An interesting

® nding is that the mRNA encoding CYP3A7, 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 CYP2A (Dalet-Beluche et al. 1992) and

CYP2C (Morel et al. 1990) apoprotein levels in human primary hepatocytes,

resembling phenobarbital in this respect.

CYP3A enzymes are also present at high levels in the human alimentary tract

(Kaminsky and Fasco 1992). Induction of CYP3A4 has been shown to occur in small

bowel enterocytes in response to rifampicin treatment (Kolars et al. 1992). Using the

CYP3A4 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 metabolism. This phenomenon was ascribed to

an elevation of intestinal CYP3A4-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 CYP-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 byglutathione S-transferase, but if the hepatocyte glutathione stores are depleted, formation of

macromolecule adducts with NAPQI occurs in the liver. Conditions which enhance thetoxi® cation process (CYP induction) or decrease the detoxi® cation functions (malnourishment,

diseases) augment paracetamol toxicity. Adapted from Zimmerman and Maddrey (1995), Parket 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, CYP3A4 expression is increased due to dexamethasone

treatment in vivo (Molowa et al. 1986), and dexamethasone increases the catalytic

activities mediated by CYP3A4 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).

CYP3A4 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, Morel 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 CYP3A5

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 CYP3A5, since a similar sequence is lacking from

CYP3A4 and the rat CYP3A1 (Schuetz et al. 1996). In addition, other consensus

sequences possibly mediating induction in the promoter regions of CYP3A genes

have been described, and it has become apparent that the host cell environment also

strongly in¯ uences the inducibility of CYP3A genes (Barwick et al. 1996).

In individuals who are extensive metabolisers of debrisoquine (normal CYP2D6

function), 3-week rifampicin pretreatment caused a reduction in morphine plasma

concentrations and a signi® cant attenuation of codeine’ s respiratory and psycho-

motor 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 morphine-

producing 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 CYP4A 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

CYP4A 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 CYP4A enzymes might theoretically aå ect these vital functions.

Evidence for this, however, is lacking in humans. Due to the very low abundance of

CYP4A protein in the human liver and paucity of relevant drug substrates, its role

in the overall pharmacokinetics of commonly used drugs must 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-amino-

benzoquinone (NAPQI). Thus, conditions which increase the activity of CYP2E1

may sensitise an individual to the toxic e å ects of paracetamol. This has been shown

to occur in man and experimental animals, particularly associated with chronic

ethanol exposure (Zimmerman and Maddrey 1995). In accordance with this, a

P450 inhibition and induction in man 1241

recently developed CYP2E1 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 CYP2E1 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

(Kostrubsky et al. 1997) suggest that CYP3 enzymes may also mediate paracetamol

activation to toxic intermediates.

Research needs and future trends

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

limited 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 microsomal 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 compounds 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

knowledge 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.

Acknow 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

Medical Research Council (Contract Nos 1051029 and 34555), by the Biomed1

project and by the Biomed2 project EUROCYP.

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