Download Characteristics and common properties of inhibitors, inducers, and

DRUG METABOLISM REVIEWS, 34(1&2), 17–35 (2002)
CHARACTERISTICS AND COMMON PROPERTIES
OF INHIBITORS, INDUCERS, AND ACTIVATORS
OF CYP ENZYMES
Paul F. Hollenberg
The University of Michigan, Ann Arbor, Michigan
HISTORICAL BACKGROUND
The modification of the enzymatic activity of the cytochrome P450 (CYP)
enzymes by inhibition, induction, or activation is of great interest to
enzymologists, pharmacologists, and chemists who are interested in mechanisms
relating to these three effectors of enzyme activity. When the activity of a CYP is
modified in vivo in humans, these effects are a major concern for clinicians and
patients due to the potential for these alterations to greatly change the metabolism
of the drug substrate(s) for that enzyme and thereby alter the biological activity of
the drug leading to the potential for harmful drug– drug interactions. The
inhibition and induction of the CYP enzymes are probably the most common
causes for most drug interactions that have been documented in the literature (1).
The inhibition of the metabolism of one drug, as a result of competition
between two different drugs for metabolism by the same CYP, may result in
unexpected elevations in the plasma concentrations of one or both drugs that can
ultimately result in a variety of minor as well as serious adverse effects. These
types of interactions have led to very serious adverse events including fatalities
and ultimately have resulted in several prominent drugs being withdrawn from the
market (2,3). The onset of inhibition is usually rapid following a single
administration of the inhibitory compound. However, as will be discussed later,
some types of inhibition are not manifested rapidly. On the other hand, enzyme
induction, which results in an increase in the total amount of an enzyme with a
concomitant increase in its drug metabolizing ability, may attenuate the
pharmacological effect of a drug as a result of increasing the metabolism and
subsequent elimination leading to marked decreases in plasma concentrations.
17
Copyright q 2002 by Marcel Dekker, Inc.
www.dekker.com
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Unlike inhibition, the onset of induction is generally relatively slow and it may
take days or even weeks for the full effects to be manifested. Although inhibitory
effects generally involve the direct interaction of the inhibitory drug with the CYP
it is effecting, the effects of inducers are generally indirect in that in most cases
they do not interact physically with the enzyme in order to cause induction.
Significant drug interactions leading to limited utility of a given drug entity
or even its withdrawal from the market may result in significant economic losses
for the pharmaceutical companies. Therefore, pharmaceutical companies are
developing and utilizing many new approaches that can be used to predict the
possibility that new drug candidates will cause significant drug interactions
through inhibition, induction, or activation of the CYPs. During the last decade,
our knowledge of the structures and the regulatory mechanisms of the CYP
enzymes have grown remarkably as a result of the many major advances in
biochemical technology and molecular biology. Although the mechanistic aspects
of both CYP inhibition and induction are much better understood than previously,
the accurate prediction of the occurrence and consequences of drug– drug
interactions continues to be an important unsolved challenge.
INHIBITORS OF THE CYTOCHROME P450 ENZYMES
As can be seen from the tabular presentation included as part of this issue of
Drug Metabolism Reviews, many inhibitors of the CYP enzymes have been
identified and although some are inhibitory for a number of different CYPs, a fair
number are very selective for only one enzyme. This property is particularly
important for the identification of the role that a particular enzyme plays in the
metabolism of a given drug substrate and for the development of drugs that may
target a specific enzyme.
The nature of the catalytic cycle of the CYP enzymes presents a number of
potential points at which inhibition of substrate metabolism may occur. The
primary steps in the catalytic cycle for the reactions catalyzed by the CYPs are: 1)
substrate binding; 2) one-electron reduction of the ferric (Fe+3) enzyme to the
ferrous (Fe+2) enzyme; 3) binding of molecular oxygen to the ferrous (Fe+2) iron;
4) transfer of the second electron to the ferrous-oxy-substrate complex leading
to the release of water and the formation of an activated oxygen intermediate; 5)
the catalytic insertion of the activated oxygen into the substrate to form the
oxygenated product; and 6) the release of the oxygenated product resulting in
the release of the native ferric (Fe+3) form of the enzyme that can then undergo
another catalytic cycle (4). Three of these steps: 1) substrate binding; 2) the
binding of molecular oxygen to the ferrous (Fe+2) enzyme; and 3) the catalytic
step in which the activated oxygen is transferred from the heme iron to the
substrate, appear to be particularly susceptible to inhibition (5). An additional
target for inhibitors is the transfer of electrons from the CYP reductases to the
CYP, which occurs following substrate addition and after the binding of molecular
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
19
oxygen to the ferrous CYP. These inhibitors generally interfere with the transfer of
electrons to the CYP by accepting electrons directly from the reductase and
consequently do not cause inhibition by interacting directly with the CYP.
Therefore, they are generally nonspecific with respect to the CYP forms they
inhibit.
The basic types of enzyme inhibitors include: 1) competitive; 2)
noncompetitive; 3) uncompetitive; 4) product inhibition; 5) transition-state
analogs; 6) slow, tight binding inhibitors; and 7) irreversible inhibitors (6).
Inhibitors of the CYPs can be divided into three general categories that differ in
their mechanisms (5). They are: 1) compounds that bind reversibly; 2)
compounds that form quasi-irreversible complexes with the iron of the heme
prosthetic group; and 3) compounds that bind irreversibly to the prosthetic
heme or to the protein, or that cause covalent binding of the heme prosthetic
group or its degradation product to the apoprotein. In general, those inhibitors
that interfere with the catalytic cycle prior to the formation of the activatedoxygen intermediate are reversible inhibitors that act as competitive,
noncompetitive, uncompetitive, product, or transition-state inhibitors. Those
compounds that act during or subsequent to the formation of the activated
oxygen intermediate are generally either quasi-irreversible or irreversible
inhibitors and, in many cases, have been shown to be mechanism-based
inactivators (5). Since the inhibitors of the human CYPs are indicated in the
Table in this issue, they will not be discussed individually here but we will
focus on the types and mechanisms of inhibition.
Generally, reversible inhibition is thought to be the most common cause of
drug– drug interactions. Reversible inhibition of CYPs is transient and the normal
metabolic functions of the enzymes will continue following elimination of the
inhibitor from the body. As opposed to the irreversible and quasi-irreversible
inhibitors that exhibit both dose-dependent and time-dependent inhibition of
substrate metabolism, reversible inhibitors exhibit only dose-dependent inhibition
(7). As already indicated, reversible inhibition be can further classified as
competitive, noncompetitive, and uncompetitive and may involve product
inhibition, or inhibition by transition-state analogs or slow, tight binding
inhibitors.
In competitive inhibition, binding of the inhibitor to the enzyme prevents the
binding of the substrate to the active site of the enzyme. This generally is believed
to be the result of the inhibitor sharing some degree of structural similarity with
the substrate(s) of that CYP. Depending on the substrate specificity of the CYP,
some of which exhibit relatively little apparent specificity, the structural
similarities between the competitive inhibitor and the substrate whose metabolism
is inhibited may or may not be apparent. Although the competitive inhibitor may
be a substrate also for the CYP that it inhibits, that is not necessarily the case. The
competition by the inhibitor for occupancy of the CYP active site may involve
simple competition for binding to a lipophilic domain in the active site or it may
involve hydrogen bonding or ionic bonds with specific amino acid residues in the
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active site (5). This type of inhibition is most commonly observed when two
different substrates of the same CYP enzyme are present. In the case of classic
competitive inhibition where the inhibitor is not a substrate of the enzyme, the Km
apparent for the substrate increases in the presence of the inhibitor; however, there
is no change in the Vmax. The standard approach to characterizing competitive
inhibition involves performing metabolism studies with varying concentrations of
both the substrate, S, and the inhibitor, I, and analyzing the data using a
Lineweaver – Burk plot (1/v vs. 1/S ) or other transformations of the Michaelis –
Menten equation. In the Lineweaver– Burk plot, competitive inhibition is
identified by a common intersection point for all the lines on the ordinate axis.
The Dixon plot in which 1/v is plotted vs. S is an alternative method which is often
used (8). Since all of the linear transforms of kinetic data have some problems
with the weighting of the data points (9), a variety of nonlinear regression
programs have been developed and are readily available for the data from studies
on the effects of inhibitors on metabolism. One approach that has been developed
for the analysis of enzyme inhibitor mechanisms involves the use of virtual
kinetics (10).
When the inhibitor is not metabolized by the CYP being studied, Ki is the
actual binding constant for the inhibitor, unlike the Km, which is usually not equal
to the binding constant of the substrate (8). However, when the inhibitor is also
metabolized by the CYP, then Ki may not be a valid measure of the binding
constant. In this case, the two substrates are exhibiting mutually competitive
inhibition of each other.
Competitive inhibition is encountered relatively frequently in drug
metabolism studies both when using microsomal preparations or purified
reconstituted enzyme systems in vitro as well as in metabolism studies performed
in vivo. Approaches have been developed for characterizing inhibitory
interactions in vivo through analysis of various pharmacokinetic parameters
(11,12). Since many of the CYPs have numerous drugs as substrates, competition
of various drugs for metabolism by a specific CYP is a common occurrence
leading to drug– drug interactions in patients who are simultaneously administered
several different drugs.
In noncompetitive inhibition, the inhibitor binds to the enzyme at a site other
than the active site of the enzyme and has no effect on substrate binding. However,
the enzyme – substrate– inhibitor complex is unable to function catalytically. In
this case, a decrease in the Vmax is observed without a concomitant change in the
Km. Although this type of inhibition is discussed routinely in any presentation of
enzyme kinetics, examples of such inhibition are relatively uncommon and they
are rarely observed, if at all, in studies of metabolism by CYPs. In studies with
microsomes or the expressed enzyme, this type of kinetic behavior would be
expected to be observed also when some of the enzyme involved in metabolism is
inactivated, e.g., by a mechanism-based inactivator or by the formation of an MI
complex (to be discussed later).
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Uncompetitive inhibition, like noncompetitive inhibition, is readily defined
but it also is seldom seen in drug metabolism. In this case, the inhibitor, instead
of binding to the free enzyme, binds to the enzyme– substrate complex resulting
in the formation of a nonproductive enzyme –substrate– inhibitor complex. In
this case, both Vmax and Km are decreased proportionately so that the ratio
Vmax/Km remains constant. In a Lineweaver –Burk plot of the data, parallel lines
would be observed with different concentrations of the inhibitor.
Some reversible inhibitors act by coordinating with the prosthetic heme iron
atom. The coordination of a strong ligand to the heme iron shifts the iron from the
high- to the low-spin form giving rise to the “Type II” difference spectrum (13).
This change in the spin state occurs concomitantly with a change in the redox
potential of the CYP that makes reduction by the CYP reductase more difficult
(14). Thus, the inhibition of CYPs by strong iron ligands is a result not only of the
occupation of the sixth coordination site of the heme iron but also the change in the
reduction potential of the iron.
Inhibitors that bind to both a hydrophobic binding site in the CYP active site
and to the prosthetic heme-iron are generally much more effective inhibitors than
those that utilize only one of those interactions (5). In general, these relatively
potent reversible inhibitors are nitrogen-containing aliphatic and aromatic
compounds. Some of the most widely used compounds in this class are derivatives
of imidazole and pyridine.
A second category of inhibitors includes those compounds that form quasiirreversible complexes with the heme iron-atom. These compounds generally
require catalytic activation by the enzyme to transient intermediates that then
coordinate very tightly to the prosthetic heme in the CYP active site leading to
inhibition. This coordination is generally so tight that the inhibitory complexes can
only be broken down to release the native catalytically active enzyme under
special experimental conditions. The formation of these metabolic intermediates
that can coordinate so effectively with the CYP to convert it to a catalytically
nonfunctional state is associated with several different classes of compounds
including those containing a dioxymethylene function and nitrogen-containing
compounds including 1,1-disubstituted hydrazines, acyl hydrazines, and a variety
of alkylamines that may be converted to nitroso metabolites. Alkyl and aryl
methylenedioxy compounds are oxidized by CYPs to yield species that coordinate
tightly to the prosthetic heme iron leading to metabolite intermediate complexes
(MI complexes) that are extremely stable. A requirement for an initial catalytic
event has been unequivocally demonstrated (15). The stability of the ferrous MI
complex is demonstrated by the fact that it can be isolated intact from rats treated
with isosafrole. The ferric MI complex is much less stable than the ferrous
complex and incubation with lipophilic compounds that can displace the inhibitor
from the active site lead to regeneration of the native catalytically active enzyme
(16). The nature of the side-chain substituent on the methylenedioxy compound is
an important determinant of the formation of the MI complex and its stability.
Both the size and lipophilicity of the alkyl group are important since analogues
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with alkyl chains of one to three carbons give relatively unstable complexes while
those with larger alkyl groups are stable (17).
A second class of agents that forms MI complexes with the CYP heme
iron is the relatively large class composed of alkyl and aromatic amines. This
class includes a number of clinically important antibiotics such as erythromycin
and troleandomycin (18). The oxidation of these amines yields intermediates
that coordinate tightly to the ferrous heme resulting in an optical spectrum with
an absorbance maximum at 445– 455 nm. Primary amines, but not secondary or
tertiary amines, are capable of forming the MI complexes following oxidation.
Secondary and tertiary amines may also form MI complexes following
N-dealkylation to the primary amines. The primary amines appear to be
hydroxylated initially followed by a two-electron oxidation of the resulting
hydroxylamine to give the nitroso group, which currently is thought to be
involved in chelation with the iron to form the MI complex (18).
A final category of inhibitors are those compounds that bind irreversibly to
the prosthetic heme or to the protein, or that cause covalent binding of the heme
prosthetic group, or its degradation product to the apoprotein. These compounds
require metabolic activation by the CYPs and are part of a class of inhibitors
commonly referred to as “catalysis-dependent”, “suicide”, or “mechanism-based”
inactivators (19,20,21).
Mechanism-based inactivation is generally regarded to be a relatively
unusual occurrence with most enzymes. However, it is observed in reactions
catalyzed by CYPs in somewhat higher frequency than would normally be
expected, possibly as a result of the reactivity of the oxygenated intermediates
formed during the course of the oxygenation reactions. The utility of mechanismbased inactivators in the design of new drugs that are highly selective for a given
CYP enzyme has attracted great interest recently since, in principle, these
compounds could be designed so that they would only inhibit the target enzyme
(21). These inactivators also have attracted considerable interest as a result of their
utility in elucidating enzyme mechanisms. Finally, a great deal of effort has been
expended on the development of mechanism-based inactivators as inhibitors of
specific CYP enzymes that can be used as diagnostic tools to identify which
form(s) of the CYPs are involved in catalyzing a particular reaction in microsomal
preparations. These compounds result in the formation of covalent bonds that
cannot be broken to regenerate catalytically active enzyme. Although the onset of
inhibition by these compounds may occur in vivo more slowly than that observed
with reversible or quasi-irreversible inhibitors, the final effect of the mechanismbased inactivators is generally much more profound and inhibition of drug
metabolism can be reversed only by the synthesis of new, catalytically active
CYPs.
A variety of compounds have been shown to be good mechanism-based
inactivators for CYPs. These include: 1) acetylenes and terminal alkyl and aryl
olefins such as 2-ethynylnaphthalene, 9-ethynylphenanthrene, 5-phenyl-1-pentyne, 10-undecynoic acid, and 17a-ethynyl-estradiol; 2) a variety of organosulfur
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
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compounds such as disulfiram, cimetidine, dialkylsulfides, parathion, diethyldithiocarbamate, isothiocyanates, thioureas, and xanthates; 3) halogenated
compounds such as chloramphenicol and N-monosubstituted dichloroacetamides;
4) 1-aminobenzotriazole and its N-aralkylated derivatives; and 5) furanocoumarins such as methoxypsoralins, L -754, 394 (a Merck compound synthesized as a
potential HIV protease inhibitor) and bergamottin (5,21,22).
Reactions that result in the mechanism-based inactivation of CYPs involve
the formation of a complex between the CYP and a reactive intermediate that
can then either react with the CYP to form a covalent adduct resulting in
inactivation or it can dissociate resulting in product formation and the
regeneration of the native catalytically active enzyme. The derivation of the
various kinetic constants for this type of reaction has been described (20). Key
to this type of inactivation is the concept of the partition ratio (23). The
partition ratio can be thought of as the number of latent activator molecules
metabolized and released as product for each molecule metabolized to give the
inactivated form of the enzyme. This can be thought of also as the number of
cycles of metabolism the enzyme can traverse, on the average, before it is
inactivated. The partition ratio is generally considered a measure of the
efficiency of the inactivator. The most efficient mechanism-based inactivator
would have a partition ratio of zero and in a standard assay where metabolite
formation is measured it would not be considered a substrate since product
formation could not be measured. Mechanism-based inactivators having
partition ratios ranging from almost zero to several thousand have been
reported.
As pointed out by Ortiz de Montellano and Correia (5), mechanism-based
inactivators may exhibit better enzyme specificity than reversible inhibitors
since: 1) the initial binding of the inhibitor to the specific CYP enzyme must
satisfy all of the constraints imposed on reversible inhibitors; 2) the inhibitor
must be acceptable as a substrate for that CYP due to the requirement for
catalytic activation to a reactive species; and 3) the formation of the reactive
species during metabolism leads to an irreversible modification of the enzyme
that permanently removes it from the pool of active enzymes.
The apparent flexibility of the active sites of many of the CYPs, as
evidenced by the sizes and numbers of different substrates they can metabolize,
offers the possibility of designing many different mechanism-based inactivators
having a variety of potentially reactive moieties. Due to their broad substrate
specificities, a major problem in designing mechanism-based inactivators for
CYPs lies in designing ones that will be specific for a single CYP.
The development of reversible, quasi-irreversible, and irreversible inhibitors
of CYPs and our understanding of the mechanisms of action of these various types
of inhibitors have increased remarkably over the past few years and have provided
important insights for the development of highly selective isozyme-specific
inhibitors of the CYPs. These inhibitors are of substantial interest not only for
studies probing the structures, mechanisms of action, and biological roles of
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specific P450s, but also because of their potential as modulators of CYP activity
that can be used as therapeutic agents in a manner analogous to the way inhibitors
of the steroidogenic P450s have been used to treat endocrine disorders and as anticancer agents. Since different CYP enzymes play major roles in the metabolic
activation and detoxication of various chemical carcinogens and other toxins, the
development of agents that can be used to selectively inactivate these enzymes in
order to shift the balance between the various metabolic pathways such that
metabolic activation is minimized whereas detoxication is enhanced would be of
great value.
INDUCERS OF THE CYTOCHROME P450 ENZYMES
The induction of the drug metabolizing enzymes was recognized first
because of the profound effects that it had on the pharmacological responses to
drugs and other xenobiotics (24). For example, animals exposed chronically to
barbiturates were shown to exhibit “tolerance” to the hypnotic effect of the
barbiturates as a result of the induction of the CYPs responsible for their
metabolism. Induction of CYPs with specific inducers also was shown to decrease
tumor formation in animals exposed to chemical carcinogens. These two examples
illustrate two important aspects of induction that were understood early on. First,
inducers are substrates for the enzymes they induce and secondly, enzyme
induction oftentimes may enhance detoxication; particularly for exposure to
relatively low concentrations of substrate. Depending on the CYP enzyme(s)
being induced and the compounds that the animal is exposed to, induction may be
extremely deleterious also. For example, if the induced CYP catalyzes the
metabolic activation of a toxin or carcinogen, exposure of the organism to that
compound following induction may greatly enhance its toxicity or carcinogencity
(24).
The effect of an inducer is to increase the activity of one or more enzymes by
causing an increase in the intracellular concentration of the induced enzyme(s).
Although the increase in the level of the induced protein is oftentimes the result of
an increase in the transcription of the gene associated with the induced protein,
this is not always the case. Enzyme induction generally exhibits typical doseresponse relationships. Although a single CYP enzyme can often account for most
of the induction by a given inducer, there may be significant inductive effects on
one or more additional CYPs. Induction of forms that are either absent or at
extremely low levels constitutively may have profound effects on the metabolism
of some substrates.
The term induction in general has been restricted to cases in which protein
synthesis is stimulated. However, it is important to recognize that the steady-state
concentration of a protein is determined by both its rate of synthesis and its rate of
degradation. Therefore, any change in the steady-state levels of a CYP due to
exposure to an inducer could reflect changes in either process or even both
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
25
synthesis and degradation. Although there are a few exceptions, inducers of the
CYP enzymes have generally been demonstrated to stimulate de novo synthesis of
the protein. The rate of CYP synthesis is dependent on the concentration of the
mRNA for that enzyme. As was already indicated to be the case for the steadystate concentrations of the proteins, the mRNA concentrations for a given CYP
also reflect the rate of transcription of the gene as well as the rate of degradation of
the mRNA.
In most cases, CYP induction involves an increase in gene transcription.
The sequence of events for induction due to increases in gene transcription
usually involves a transient increase in the rate of transcription that begins soon
after exposure to the inducer with maximum transcriptional activity observed at
about 10 –12 hr and the activity returns to basal within approximately 18 hr,
depending on the dose of the inducer. The concentration of the specific message
increases in parallel with the increase in transcriptional activity. However, the
mRNA is generally degraded relatively slowly and therefore, it will accumulate
and remain elevated following the return of transcription activity to basal
levels.
The time required for a protein to reach a new steady-state level as a
consequence of an increase in its rate of synthesis is determined by its rate of
degradation. The degradation of proteins generally has been shown to be a firstorder process and, depending on the specific CYP, the half-life is anywhere
from 8 to 30 hr. As a consequence, the increase in the concentration of the CYP
will lag behind the rise in synthesis due to increases in the message.
When induction is due to a decrease in the rate of protein degradation, the
concentration of the CYP may increase more rapidly than when it is due solely
to transcriptional activation. Erythromycin and troleandomycin appear to induce
primarily by decreasing the degradation rate for the 3A protein (25). They form
MI complexes with the CYP that are relatively resistant to degradation and
have been shown to extend the half-life of 3A1 to more than 60 hr.
Induction of the CYPs increases the capability for metabolic detoxication
and elimination, and thus is considered an important part of the defense system
against exposure to xenobiotics. It was noted in the early 1950s that feeding
3-methylcholanthrene to rats reduced the carcinogenic activity of some
aminoazo dyes by increasing their N-demethylation (26) and that phenobarbital
(PB) elicited sleep times were shortened following chronic administration of
barbiturates due to increases in the metabolism of the barbituates. Although
induction of the CYPs may be advantageous in most cases, it can have a variety
of pharmacological consequences including alterations in drug efficacy, drug –
drug interactions, and increases in the metabolic activation of procarcinogens.
Consequently induction of the CYPs can be viewed as a “double-edged sword”
for the organism involved.
Most of the CYP enzymes appear to be inducible, although to various
extents. Human CYP 1A1/2, 2A6, 2C9, 2C19, 2E1, and 3A4 are all known to be
inducible (27). The molecular mechanisms for CYP induction have been
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extensively studied in rodents beginning with the pioneering studies of Poland and
co-workers (28).
The enzymes in the CYP1 family are involved in the metabolism of many
planar aromatic compounds and aromatic amines. The induction of the genes in
this family (CYP1A1, CYP1A2, and CYP1B1) is under the control of the arylhydrocarbon (Ah) receptor. In the absence of ligand, most of this protein exists
in the cytosol in a complex with Hsp90, a heat-shock chaperone protein.
Binding of an inducing agent to the receptor dissociates this complex leading to
translocation of the Ah receptor into the nucleus, where it forms a second
heterodimer with the Ah-receptor nuclear transporter (ARNT), a nuclear basic
helix – loop– helix protein. This heterodimer then binds to a response element,
the xenobiotic response element (XRE), present in multiple copies in the 50 flanking region of the CYP1 genes and functions as a transcriptional enhancer
to stimulate gene transcription (29,30). Although this scheme is widely accepted
as a general scheme for the induction of CYP1 family members, it is clear that
the induction mechanisms may be much more complex under a variety of
conditions (27). In addition to the XRE, human CYP1A promoters have a
number of other regulatory elements including a glucocorticoid response
element (GRE) that mediates glucocorticoid potentiation of CYP1A1 induction
by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic
hydrocarbons (PAHs) (31). There is also a negative regulatory element on
the 50 -flanking region of the human and rat CYP1A1 genes (32,33) that binds a
member of the NF-Y transcription family (33) and the human CYP1A2 gene
has an AP-1 site (34). The induction of CYP1 enzymes in humans has been
well established (27).
The ability of PB to cause marked induction of liver microsomal drug- and
steroid-metabolizing enzymes was one of the fundamental observations leading to
interest in the P450s. Subsequently, numerous compounds which are structurally
unrelated to PB (e.g., allylisopropylacetamide, chlordane, DDT, trans-stilbene
oxide, diortho-substituted polychlorinated biphenyls) were shown to induce
similar patterns of enzyme activities and are considered to induce in a “PB-like”
fashion (35). In rat liver, PB induces a number of different P450s including 2A1,
2B1/2, 2C6/7/11, and 3A1/2. Induction responses range from 50 to 100-fold (2B1/
2) to 2- to 4-fold (2A1 and 2C6).
The CYP2B family of enzymes in rodents has been shown to play
important roles in catalyzing the metabolism of a large number of drugs and
other xenobiotics in laboratory animals. However, the information on the
metabolic capabilities of CYP 2B6, the human orthologue, and its role in
metabolism is relatively limited. Hepatic expression of 2B6 is low, averaging
about 1% of the total P450, and there is no evidence for inducibility in humans.
In contrast to the detailed mechanistic understanding developed for the
CYP1 family over the past 20– 30 years the induction mechanisms responsible for
increases in CYP2B expression in mammalian systems following exposure to
“PB-like” inducers remained largely unknown until recently when several key
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27
discoveries were made regarding the mechanisms by which hepatic CYPs are
induced by xenochemicals (36,37). Three members of the “orphan” nuclear
receptor family of ligand-activated transcription factors (CAR, PXR, and PPAR)
have been shown to play crucial roles in the induction of the hepatic CYP2, CYP3,
and CPA4 families, respectively, following exposure to the prototypical inducers
phenobarbital (CAR), rifampicin and pregnenolone 16a-carbonitrile (PXR), and
clofibric acid (PPAR). The acronym CAR stands for constitutive androstane
receptor, PXR for pregnane X receptor, and PPAR for peroxisome proliferatoractivated receptors. Liver X receptor (LXR) and Farnesol X receptor (FXR), two
other nuclear receptors, are activated by oxysterols and bile acids, respectively,
and are involved in regulating the expression of the cholesterol 7a-hydroxylase, a
key enzyme in bile acid biosynthesis. All five of these receptors involved in the
regulation of CYPs belong to the same family of nuclear receptors (NR1). They
share the same heterodimerization partner , the retinoid X-receptor (RXR) and
they interact with other nuclear receptors as well as with a variety of other
intracellular signaling pathways. Endogenous ligands have been identified
recently for each of the nuclear receptors and information has been obtained on
their physiological receptor functions (36). The CAR receptor is constitutively
active and its endogenous ligands include androstanol and androstenol, which both
are inhibitory. Stimulatory ligands for PXR include corticosterone and
pregnenolone and for PPAR they include linoleic acid and arachidonic acid
(36). To exert their effects the receptors bind to the DNA response elements
indicated: CAR (DR4), PXR (DR3, ER6), PPAR (DR1), LXR (DR4), and FXR
(IR1) (36).
Because of its important role in the metabolic activation of a variety of
drugs, chemical carcinogens, and other toxicants, the regulation of CYP2E1
has been the subject of intensive investigation and these studies have
demonstrated the involvement of multiple induction mechanisms (27).
Induction in rodents has been shown to involve effects on essentially all
regulatory levels including increases in transcription, mRNA stabilization,
translational efficiency increases, and post-translational protein stabilization
(27,38). Starvation, higher levels of ethyl alcohol, and hypophysectomy
appear to be associated with increased transcription of 2E1 in rats. CYP2E1
induction observed in diabetic rats may be due to mRNA stabilization.
Acetone and pyridine have been reported to exert effects at the level of
translational efficiency. This is accompanied by a shift of the polysomal
distribution to a higher density along with an increase in incorporation into
the newly synthesized protein of radiolabeled amino-acids (39,40). Ethyl
alcohol as well as a variety of other small organic molecules appear to
induce by post-transcriptional stabilization of the CYP 2E1 apoprotein as
evidenced by significant changes in the protein half-life (41). Since these
separate induction mechanisms can all occur simultaneously, it has been
suggested that the theoretical maximum induction of 50- to 100-fold could
be observed (38).
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Enzyme induction studies in cells in culture or in experimental animals can
be performed readily since the amounts of the CYPs induced and their catalytic
activities can be measured directly. Direct evidence of CYP induction in humans is
much more difficult to obtain due to practical and ethical considerations. However,
there are some reports that demonstrate large interindividual variability in CYP
protein levels (or mRNA) as well as catalytic activity in response to inducers. Ged
and co-workers (42) reported results from liver biopsies collected before and after
treatment with rifampicin (600 mg/day) for four days. They observed increases
due to induction ranging from 160 to 2900%. Kolars et al. (43) also observed
considerable interindividual variability in rifampicin induction of CYP3A4 in the
small intestine in five healthy volunteers where the increase in message ranged
from 0 to 1200%.
As mentioned previously, reports of direct evidence of the induction of CYPs
in vivo based on measurements of protein levels and catalytic activity are limited.
However, there are numerous studies in which the reduction in plasma areas under
the curve (AUCs) have been determined as indirect measures of enzyme induction.
This approach is based on the belief that induction causes a change in the level of
the enzymes involved in metabolizing a probe substrate but not in the identities or
the properties of the CYPs. Although these beliefs may be true generally, they are
probably not universally true. However, assuming the general applicability of this
hypothesis, then enzyme induction leads to an increase in the intrinsic clearance
(Vmax/Km) due to an increase in the Vmax that is directly proportional to the
increase in the CYP activity due to induction. Thus, the concept of intrinsic
clearance is used to relate enzyme induction to changes in the AUC. Large
individual variations in enzyme induction have been reported based on this
indirect approach. In eight healthy volunteers, induction by rifampicin caused
decreases in the oral AUC for S-verapamil ranging from 5- to 60-fold with a mean
value of 30-fold (44). Rifampicin treatment also was shown to decrease the AUCs
for midazolam by 11.6- to 55-fold in another study of 10 subjects (45). Increases in
the oral clearance of cyclosporin ranging from 2.5- to 6.6-fold were seen following
treatment with rifampicin (46). Major reasons for these large interindividual
variabilities in enzyme induction probably include environmental, dietary, age,
and genetic factors.
Although there is significant variability among individuals with respect to
the magnitude of the inductive effect, there does appear to be a limiting value
above which there is no greater increase in activity. That is to say that among
individuals, the enzyme levels following maximal induction are quantitatively
similar. For example, the maximally induced values of CYP3A in six human
hepatocyte cultures were essentially identical, probably representing the normal
level to which CYP3A could be induced in those cells. Interestingly, the induction
of the activity for the 4-hydroxylation of oxazaphosphorine was inversely related
to the basal activity in human hepatocytes (47), suggesting that those individuals
with lower basal levels of a given CYP might exhibit a greater degree of enzyme
induction.
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
29
ACTIVATORS OF THE CYTOCHROME P450 ENZYMES
A variety of compounds have been identified that enhance the catalytic
activity of the CYPs by some type of activation or stimulation mechanism as
opposed to induction. Although examples of in vivo stimulation of drug
metabolism have been reported, they are rare and the relevance of these types of
interactions resulting in enzyme activation in vivo leading to drug –drug
interactions is not clear. If CYP stimulation occurs in vivo, the outcome would be
likely to be the same as induction. However, one difference that might be expected
would be that the effect of an activator would be extremely rapid as opposed to
induction, which may require days and multiple doses before the effects of the
inducer on drug metabolism are manifested.
Examples of activation or stimulation of drug metabolizing enzyme activity
in microsomes or in the reconstituted system using purified enzymes of the CYPs
are relatively abundant. A variety of compounds are known to stimulate CYP
activity in either of these two systems and they are listed in the accompanying
tabular presentation included as part of this issue of Drug Metabolism Reviews. In
order to stimulate the rate of an enzyme-catalyzed reaction, the stimulatory
compound must cause an increase in the rate-limiting step for the overall catalytic
reaction. Given the multitude of different CYP enzymes and substrates, it is likely
that a variety of rate-limiting steps may be targets for the stimulation of various
reactions catalyzed by the CYPs. These would include the first electron transfer,
substrate binding, oxygen binding, transfer of the second electron, oxygen
activation, insertion of the activated oxygen into the substrate, and product release
(4). Depending on the site of action of the activator and its mechanism of action,
one might observe increases in the Vmax, decreases in the Km, or both. Possible
mechanisms would include allosteric effects on substrate binding, effects on the
redox potential of the heme ion, alterations in the interactions between the
reductase and the CYP, shunting of electrons from one CYP enzyme to another in
microsomes, alterations in the fluidity or other physical or chemical characteristics
of the microsomal membrane, or destabilization of the enzyme – product complex.
Stimulation of microsomal drug-metabolism involving the CYPs was an area
of great interest and experimental activity in the 1970s and some of the key results
have been reviewed in excellent reviews by Anders (48), Cinti (49), and Holtzman
(50). It is interesting to note that some compounds that inhibit the microsomal
metabolism of certain substrates cause marked stimulation of the metabolism
of other substrates. For example, metyrapone, which inhibits the oxidative
metabolism of tyramine, morphine, hexobarbital, and aminopyrine, significantly
increased the microsomal metabolism of acetanilide and trichloroethylene (51).
The enhancement of liver microsomal drug oxidations by acetone was first
reported by Anders (52). Acetone was shown to stimulate the para-hydroxylation
of aniline, acetanilide, and N-butylaniline in microsomal preparations. The
acetone stimulation exhibited increased activity with increasing pH and it
caused increases in both the Km and Vmax with increasing concentrations.
30
HOLLENBERG
Although a number of investigators have attempted to elucidate the mechanism of
the acetone enhancement of aniline hydroxylation, the detailed mechanism is still
not known (49).
Some of the most interesting and enlightening studies involving activation
of microsomal drug metabolism have been associated with 7,8-benzoflavone (anaphthoflavone), a synthetic flavonoid that was used widely in studies of
chemical carcinogenesis in the 1970s and 1980s (49). Studies by Conney and
co-workers showed that the addition of 7,8-benzoflavone to homogenates or
microsomes from human liver increased the rates of metabolism of a variety of
substrates including benzo[a]pyrene, aflatoxin B1, antipyrine, and zoxazolamine,
but had little or no effect on the metabolism of coumarin, 7-ethoxycoumarin, or
hexobarbital. This specificity for certain substrates suggested that the flavonoids
may exert their stimulatory effects on specific CYP enzymes. This was
subsequently demonstrated using purified CYP enzymes in the reconstituted
system (53). The CYP3A4 was shown to be activated by several different
flavonoids during the bioactivation of benzo[a]pyrene and aflatoxin B1, in
studies using the purified recombinant enzyme and specific antibodies (54). A
particularly intriguing aspect of the stimulation of CYP3A4 is the regioselectivity exhibited with substrates that can undergo multiple routes of
metabolism. One example involves the metabolism of aflatoxin B1 by 3A4.
Although 7,8-benzoflavone enhances the 8,9-epoxidation of this substrate, it
inhibits its 3a-hydroxylation (55).
Detailed studies on purified CYPs in the reconstituted system have lead to
the suggestion that the stimulatory influences of xenobiotics on the various CYP
enzymes may be due to either homotropic or heterotropic cooperativity (reviewed
in Refs. 56– 58). Homotropic cooperativity, also referred to as substrate activation,
is an activating effect due to the substrate itself. In this case, a plot of enzyme
activity vs. substrate concentration would exhibit a sigmoidal pattern.
Heterotropic cooperativity refers to the situation in which the activity for the
metabolism of one substrate is increased by the presence of another compound,
often referred to as the effector. By definition, homotropic cooperativity would not
lead to drug– drug interactions whereas heterotropic cooperativity has the
potential for significant drug– drug interactions, the outcome of which would be
very similar to those seen with enzyme induction.
The mechanisms for homotropic and heterotropic cooperativity of the CYPs
are not well understood at this time. However, several models have been proposed
describing possible interactions between the CYP and the substrate(s) and/or
effector (58 – 60). The stimulation of benzo[a]pyrene metabolism by 7,8-benzoflavone was initially suggested to be due to increased efficiency of the NADPHCYP reductase. However, this explanation for 7,8-benzoflavone stimulation lost
credibility following the observation that although 7,8-benzoflavone stimulated
CYP3A4 catalyzed 8,9-epoxidation of aflatoxin B1, it inhibited its 3ahydroxylation. In addition, it has been suggested that the CYP3A4 active site
may have multiple ligand-binding sites such that the substrate molecule and the
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
31
effector (which may be a second substrate molecule) may be present
simultaneously in the CYP 3A4 active site. Based on studies of several sitespecific mutants, a model has been developed to explain the cooperativity of
CYP3A4 that consists of two substrate-binding domains and an effector-binding
site (60– 62). A recent model to explain the cooperativity of CYP3A4 is the
“nested allosterism” model in which it is proposed that there is a complex of two
conformers of the enzyme with each conformer being able to accommodate two
binding domains (63).
When considered in conjunction with the concept of multiple binding
domains, the conformation hypothesis appears to be a reasonable mechanism that
can explain both homotropic and heterotropic cooperativity. However, it must be
recognized that this is a very abstract concept with no physical representation. In
addition, it is based on studies using recombinant enzymes in a reconstituted
system that may not reflect concentrations of drugs, effectors or cofactors,
interactions, etc., experienced by the drug-metabolizing enzyme systems in vivo in
human liver or in extra-hepatic tissues. Thus, the clinical significance of the
stimulatory effects (homotropic or heterotropic cooperativity) of any xenochemicals on CYP3A4 or any other CYP remains to be determined. Because of the
complexities involved in dealing with human subjects and performing drug
metabolism in vivo as well as the large interindividual variability which may
constantly change in response to changes in diet, etc., it may not be possible to
demonstrate stimulation of a specific CYP activity in vivo. However, this does not
negate the importance of understanding the mechanism for the cooperative effects
of various compounds in the CYPs and identifying those compounds that may act
as potent effectors of one or more CYP enzymes.
REFERENCES
1.
2.
3.
4.
5.
6.
Lin, J.H.; Lu, A.Y.H. Inhibition and Induction of Cytochrome P450 and the Clinical
Implications. Clin. Pharmacokinet. 1998, 35, 361– 390.
Ferslew, K.E.; Hagardorn, A.N.; Harlan, G.C.; McCormick, W.F. A Fatal Drug
Interaction between Clozapine and Fluoxetine. J. Forensic Sci. 1998, 43,
1082 – 1085.
Kudo, K.; Imamura, T.; Jitsufuchi, N.; Zhang, X.X.; Tokunaga, H.; Nagata, T. Death
Attributed to the Toxic Interaction of Triazolam, Amitryptyline and Other
Psychotropic Drugs. Forensic Sci. Int. 1997, 86, 35 – 41.
White, R.E.; Coon, M.J. Oxygen Activation by Cytochrome P450. Annu. Rev.
Biochem. 1980, 49, 315 –356.
Ortiz de Montellano, P.R.; Correia, M.A. Inhibition of Cytochrome P450 Enzymes.
In Cytochrome P450: Structure, Mechanism, and Biochemistry; 2nd Ed.; Ortiz de
Montellano, P.R., Ed.; Plenum Press: New York, 1995; 305 –364.
Guengerich, F.P. Inhibition of Drug Metabolizing Enzymes: Molecular and
Biochemical Aspects. In Handbook of Drug Metabolism; Woolf, T.F., Ed.; Marcel
Dekker: New York, 1999; 203– 227.
32
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
HOLLENBERG
Lin, J.H.; Chen, I.-W.; Chiba, M.; Nishime, J.A.; de Luna, F.A. Route-Dependent
Nonlinear Pharmacokinetics of a Novel HIV Protease Inhibitor: Involvement of
Enzyme Inactivation. Drug Metab. Dispos. 2000, 28, 460 – 466.
Segel, I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and
Steady-State Enzyme Systems; Wiley and Sons: New York, 1975.
Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Butterworths: London,
1979.
Bronson, D.D.; Daniels, D.M.; Dixon, J.T.; Redick, C.C.; Haaland, P.D. Virtual
Kinetics: Using Statistical Experimental Design for Rapid Analysis of Enzyme
Inhibitor Mechanisms. Biochem. Pharmacol. 1995, 50, 823– 831.
Renwick, A.G. Toxicokinetics: Pharmacokinetics in Toxicology. In Principles and
Methods of Toxicology; 4th Ed.; Hayes, A.W., Ed.; Raven Press: New York, 2001;
137– 192.
Black, D.J.; Kunze, K.L.; Wienkers, L.C.; Gidal, B.E.; Seaton, T.L.; McDonnell,
N.D.; Evans, J.S.; Bauwens, J.E.; Trager, W.F.; Warfarin-Fluconazole, V. A
Metabolically Based Drug Interaction: In Vivo Studies. Drug. Metab. Dispos. 1996,
24, 422 – 428.
Schenkman, J.B.; Sligar, S.G.; Cinti, D.L. Substrate Interactions with Cytochrome
P-450. Pharmacol. Ther. 1981, 12, 43 –71.
Guengerich, F.P. Oxidation – Reduction Properties of Rat Liver Cytochromes P450
and NADPH-Cytochrome P-450 Reductase Related to Catalysis in Reconstituted
Systems. Biochemistry 1983, 22, 2811– 2820.
Franklin, M.R. The Enzymic Formation of a Methylenedioxyphenyl Derivative
Exhibiting an Isocyanide-like Spectrum with Reduced Cytochrome P-450 in Hepatic
Microsomes. Xenobiotica 1971, 1, 581– 591.
Elcombe, C.R.; Bridges, J.W.; Gray, T.J.B.; Nimmo-Smith, R.H.; Netter, K.J.
Studies on the Interaction of Safrole with Rat Hepatic Microsomes. Biochem.
Pharmacol. 1975, 24, 1427– 1433.
Murray, M.; Hetnarski, K.; Wilkinson, C.F. Selective Inhibitory Interactions of
Alkoxymethylene-Dioxybenzenes Towards Mono-Oxygenase Activity in Rat
Hepatic Microsomes. Xenobiotica 1985, 15, 369– 379.
Mansuy, D.; Gans, P.; Chottard, J.-C.; Bartoli, J.-F. Nitrosoalkanes as Fe(II) Ligands
in the 455-nm-Absorbing Cytochrome P-450 Complexes Formed from Nitroalkanes
in Reducing Conditions. Eur. J. Biochem. 1977, 76, 607 –615.
Ator, M.A.; Ortiz de Montellano, P.R. Mechanism-Based (Suicide) Enzyme
Inactivation. In The Enzymes: Mechanisms of Catalysis; 3rd Ed.; Segman, D.S.,
Boyer, P.D., Eds.; Academic Press: New York, 1990; Vol. 19, 214– 282.
Silverman, R.B. Mechanism-Based Enzyme Inactivation: Chemistry and Biology;
CRC Press: Boca Raton, FL, 1988.
Kent, U.M.; Jushchyshyn, M.I.; Hollenberg, P.F. Mechanism-Based Inactivators as
Probes of Cytochrome P450 Structure and Function. Curr. Drug. Metab. 2001, 2,
215– 244.
He, K.; Iyer, K.; Hayes, R.N.; Sinz, M.W.; Woolf, T.F.; Hollenberg, P.F. Inactivation
of P450 3A4 by Bergamottin, a Component of Grapefruit Juice. Chem. Res. Toxicol.
1998, 11, 252 – 259.
Wang, E.; Walsh, C. Suicide Substrates for the Alanine Racemase of Eschericia coli
B. Biochemistry 1978, 17, 1313– 1321.
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
24.
33
Whitlock, J.P., Jr.; Denison, M.S. Induction of Cytochrome P450 Enzymes that
Metabolize Xenobiotics. In Cytochromes P450: Structure, Mechanism, and
Biochemistry; 2nd Ed.; Ortiz de Montellano, P.R., Ed.; Plenum Press: New York,
1995; 367– 398.
25. Watkins, P.B.; Wrighton, S.A.; Scheutz, E.G.; Maurel, P.; Guzelian, P.S. Macrolide
Antibiotics Inhibit the Degradation of the Glucocorticoid-Responsive Cytochrome
P-450p in Rat Hepatocytes In Vivo and in Primary Monolayer Culture. J. Biol.
Chem. 1986, 261, 6264– 6271.
26. Conney, A.H.; Miller, E.C.; Miller, J.A. The Metabolism of Methylated Aminoazo
Dyes. Evidence for Induction of Enzyme Synthesis in the Rat by 3-Methylcholanthrene. Cancer Res. 1956, 16, 450 –459.
27. Ronis, M.J.J.; Ingelman-Sundberg, M. Induction of Human Drug-Metabolizing
Enzymes: Mechanisms and Implications. In Handbook of Drug Metabolism; Woolf,
T.F., Ed.; Marcel Dekker: New York, 1999; 239– 262.
28. Poland, A.P.; Glover, E.; Kende, A.S. Sterospecific, High Affinity Binding of
2,3,7,8-Tetrachlorodibenzo-p-dioxin by Hepatic Cytosol: Evidence that the Binding
Species Is a Receptor for the Induction of Aryl Hydrocarbon Hydroxylase. J. Biol.
Chem. 1976, 251, 4936– 4946.
29. Perdew, G.H. Association of the Ah receptor with the 90 kDa Heat Shock Protein.
J. Biol. Chem. 1988, 263, 13802– 13805.
30. Hankinson, O. A Genetic Analysis of Processes Regulating Cytochrome P450 1A1
Expression. Adv. Enzyme Regul. 1994, 34, 159 –171.
31. Mathis, J.M.; Houser, W.H.; Bresnick, E.; Cidlowski, J.A.; Hines, R.N.; Prough,
R.A.; Simpson, E.R. Glucocorticoid Regulation of the Rat Cytochrome P450c
(P4501A1) Gene: Receptor Binding within Intron I. Arch. Biochem. Biophys. 1989,
269, 93 –105.
32. Boucher, P.D.; Hines, R.N. In Vitro Binding and Functional Studies Comparing
Human CYP1A1 Regulatory Element with the Orthologous Sequences from Rodent
Genes. Carcinogenesis 1995, 16, 383 –392.
33. Boucher, P.D.; Piechocki, M.P.; Hines, R.N. Partial Characterization of the Human
CYP1A1 Negatively Acting Transcription Factor and Mutational Analysis of Its
Cognate DNA Recognition Sequence. Mol. Cell. Biol. 1995, 15, 5144 – 5155.
34. Quattrochi, L.C.; Vu, T.; Tukey, R.H. The Human CYP1A2 Gene and Induction by
3-Methylcholanthrene. A Region of DNA Which Supports Ah Receptor Binding and
Promotor Specific Induction. J. Biol. Chem. 1994, 269, 6949– 6954.
35. Waxman, D.J.; Azaroff, L. Phenobarbital Induction of Cytochrome P-450 Gene
Expression. Biochem. J. 1992, 281, 577– 592.
36. Waxman, D.J. P450 Gene Induction by Structurally Diverse Xenochemicals: Central
Role of Nuclear Receptors CAR, PXR, and PPAR, Arch. Biochem. Biophys. 1999,
369, 11 –23.
37. Honkakoski, P.; Negishi, M. Regulation of Cytochrome P450 (CYP) Genes by
Nuclear Receptors. Biochem. J. 2000, 347, 321– 337.
38. Ronis, M.J.J.; Lindros, K.O.; Ingelman-Sundberg, M. The CYP2E Family. In
Cytochromes P450: Metabolic and Toxicological Aspects; Ioannides, C., Ed.; CRC
Press: Boca Raton, FL, 1996; 211 –239.
39. Kim, S.G.; Shehin, S.E.; States, C.; Novak, R.F. Evidence for Increased
Translational Efficiency in the Induction of P450 IIE1 by Solvents: Analysis of
34
HOLLENBERG
P450 IIE1 mRNA Polyribosomal Distribution. Biochem. Biophys. Res. Commun.
1990, 172, 767 –774.
40. Kraner, J.C.; Lasker, J.M.; Corcoran, J.B.; Ray, S.D.; Raucy, J.L. Induction of P450
2E1 in Isolated Rabbit Hepatocytes: Role of Increased Protein and mRNA Synthesis.
Biochem. Pharmacol. 1993, 45, 1483– 1488.
41. McGee, R.E., Jr.; Ronis, M.J.J.; Cowherd, R.M.; Ingelman-Sundberg, M.; Badger,
T.M. Characterization of Cytochrome P450 2E1 Induction in a Rat Hepatoma FGC-4
Cell Model by Ethanol. Biochem. Pharmacol. 1994, 48, 1823 – 1833.
42. Ged, C.; Rouillon, J.M.; Pichard, L.; Combalbert, J.; Bressot, N.; Bories, P.; Michel,
H.; Beaune, P.; Maurel, P. The Increase in Urinary Excretion of 6(-Hydroxycortisol)
as a Marker of Human Hepatic Cytochrome P450IIIA Induction. Br. J. Clin.
Pharmacol. 1989, 28, 373– 387.
43. Kolars, J.C.; Schmiedlin-Ren, P.; Scheutz, J.D.; Fang, C.; Watkins, P.B.
Identification of Rifampicin-Inducible P450IIA4 (CYP3A4) in Human Small
Bowel Enterocytes. J. Clin. Invest. 1992, 90, 1871 –1878.
44. Fromm, M.F.; Dilger, K.; Busse, D.; Kroemer, H.; Eichelbaum, M.; Klotz, U. Gut
Wall Metabolism of Verapamil in Older People: Effects of Rifampicin-Mediated
Enzyme Induction. Br. J. Clin. Pharmacol. 1998, 45, 247 –255.
45. Backman, J.T.; Olkkola, K.T.; Neuvonen, P.J. Rifampicin Drastically Reduces
Plasma Concentrations and Effects of Oral Midazolam. Clin. Pharmacol. Ther. 1996,
59, 7 – 13.
46. Hebert, M.F.; Roberts, J.P.; Prueksaritanont, T.; Benet, L.Z. Bioavailability
of Cyclosporin with Concomitant Rifampin Administration is Markedly Less
Than Predicted by Hepatic Enzyme Induction. Clin. Pharmacol. Ther. 1992, 52,
453– 457.
47. Chang, T.K.; Yu, L.; Maurel, P.; Waxman, D.J. Enhanced Cyclophosphamide and
Ifosphamide Activation in Primary Human Hepatocyte Cultures: Response to
Cytochrome P-450 Inducers and Autoinduction by Oxazaphosphorines. Cancer Res.
1997, 57, 1946 – 1954.
48. Anders, M. Enhancement and Inhibition of Microsomal Drug Metabolism. Prog.
Drug. Res. 1973, 17, 11 –32.
49. Cinti, D.L. Agents Activating the Liver Microsomal Mixed Function Oxidase
System. Pharmacol. Ther. 1978, 2, 727 –749.
50. Holtzman, J.L. The Role of the Stimulation of NADPH-Cytochrome P-450
Reductase Activity in Hepatic, Microsomal Mixed Function Oxidase Activity.
Pharmacol. Ther. 1979, 4, 601 –627.
51. Leibman, K. Effects of Metyrapone on Liver Microsomal Drug Oxidations. Mol.
Pharmacol. 1969, 5, 1– 9.
52. Anders, M. Acetone Enhancement of Microsomal Aniline para-Hydroxylase
Activity. Arch. Biochem. Biophys. 1968, 126, 269 –275.
53. Huang, M.-T.; Johnson, E.; Muller-Eberhard, V.; Koop, D.R.; Coon, M.J.; Conney,
A.H. Specificity in the Activation and Inhibition by Flavonoids of Benzo[a]pyrene
Hydroxylation by Cytochrome P-450 Isozymes from Rabbit Liver Microsomes.
J. Biol. Chem. 1981, 256, 10897 – 10901.
54. Bauer, E.; Guo, Z.; Ueng, Y.-F.; Bell, L.C.; Zeldin, D.; Guengerich, F.P. Oxidation
of Benzo[a]pyrene by Recombinant Cytochrome P450 Enzymes. Chem. Res.
Toxicol. 1995, 8, 136 –142.
INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES
55.
35
Ueng, Y.-F.; Kuwabara, T.; Chun, Y.-J.; Guengerich, F.P. Coooperativity in
Oxidations Catalyzed by Cytochrome P450 3A. Biochemistry 1997, 36, 370– 381.
56. Guengerich, F.P. Cytochrome P-450 3A4: Regulation and Role in Drug Metabolism.
Annu. Rev. Pharmacol. Toxicol. 1999, 39, 1– 17.
57. Ekins, S.; Ring, B.J.; Binkley, S.N.; Hall, S.D.; Wrighton, S.A. Autoactivation and
Activation of the Cytochrome P450s. Int. J. Clin. Pharmacol. Ther. 1998, 36,
642 – 651.
58. Tang, W.; Stearns, R.A. Heterotropic Cooperativity of Cytochrome P450 3A4 and
Potential Drug – Drug Interactions. Curr. Drug Metab. 2001, 2, 185 –198.
59. Huang, M.-T.; Chang, R.L.; Fortner, J.G.; Conney, A.H. Studies on the Mechanism
of Activation of Microsomal Benzo[a]pyrene Hydroxylation by Flavonoids. J. Biol.
Chem. 1981, 256, 6829– 6836.
60. Harlow, G.R.; Halpert, J.R. Analysis of Human Cytochrome P450 3A4
Cooperativity. Construction and Characterization of a Site-Directed Mutant That
Displays Hyperbolic Steroid Hydroxylation Kinetics. Proc. Natl Acad. Sci. USA
1998, 95, 6636 –6641.
61. Korzekwa, K.R.; Krishnamachary, N.; Shou, M.; Ogai, A.; Parise, R.A.; Rettie, A.E.;
Gonzalez, F.J.; Tracey, T.S. Evaluation of Atypical Cytochrome P450 Kinetics with
Two-Substrate Models: Evidence That Multiple Substrates Can Simultaneously
Bind to Cytochrome P450 Active Sites. Biochemistry 1998, 37, 4137 –4147.
62. Domanski, T.L.; He, Y.-A.; Harlow, G.R.; Halpert, J.R. Dual Role of Human
Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity.
J. Pharmacol. Exp. Ther. 2000, 293, 585– 591.
63. Atkins, W.M.; Wang, R.W.; Lu, A.Y.H. Allosteric Behavior in Cytochrome
P450-Dependent In Vitro Drug– Drug Interactions: A Prospective Based on
Conformational Dynamics. Chem. Res. Toxicol. 2001, 14, 338 –347.