From the Department of Drug Metabolism, Merck
Research Laboratories, West Point, Pennsylvania 19486 and
Laboratory of Molecular Carcinogenesis, NCI, National Institutes
of Health, Bethesda, Maryland 20892, and ** Camitro Corp.,
Menlo Park, California 94025
Received for publication, September 26, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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In many cases, CYP3A4 exhibits unusual kinetic
characteristics that result from the metabolism of multiple substrates
that coexist at the active site. In the present study, we observed that
Multiple drug therapy is practiced widely either to treat a single
medical disorder or to treat more than one simultaneous illness in the
same patient (1). Thus, drug-drug interactions have become an important
clinical issue due to the effects of one drug on the efficacy,
toxicity, or disposition of another drug. The estimated incidence of
clinically significant drug-drug interactions is as high as 20% in
patients receiving multiple drugs (2). The consequences of drug-drug
interactions often are detrimental to clinical therapeutics and
frequently result in an alteration of blood drug levels, leading in
severe cases to life-threatening adverse reactions. For example,
terfenadine cardiotoxicity has been observed when the drug is
coadministered with certain agents such as ketoconazole (3) and
erythromycin (4). Furthermore, mortality has occurred in patients who
were dosed concurrently with sorivudine and fluoropyrimidines (5). Oral
contraceptive failures have been noted in several populations of women
taking rifampin, griseofulvin, and anticonvulsants (6, 7). However,
coadministration of multiple drugs also can be clinically useful. For
instance, phenobarbital, which induces enzyme expression, has been
beneficial in treating patients with unconjugated and neonatal
hyperbilirubinemia (8). The majority of cases of drug-drug interactions
are a result of pharmacokinetic (metabolic clearance of one or more
drugs) or pharmacodynamic (antagonistic or additive drug effects)
alterations. Although interacting agents can affect all aspects of drug
disposition, including absorption, distribution, metabolism, and
excretion through a variety of mechanisms (9), the most common drug
interactions can be understood in terms of alterations in metabolism,
which are associated primarily with changes in the activities of
cytochromes P450, a gene superfamily consisting of at least 40 human
isoenzymes (10). Currently, only families CYP1, -2, and -3 are thought to be important in drug metabolism (11, 12). Of the individual isozymes, CYP3A4, CYP2D6, and CYP2C9 appear to be responsible for the metabolism of the greatest number of drugs. Metabolism-based drug interactions are primarily mediated by the enhancement or inhibition of enzyme systems that govern the biotransformation of xenobiotics.
The kinetic parameters that describe enzyme-based drug interactions,
e.g. Ki, KA,
Km, and Vmax, can be
estimated by in vitro techniques and appropriate kinetic
models. These estimates then may be used to predict the in
vivo pharmacokinetic and pharmacodynamic consequences associated
with exposure to multiple drugs. Although the kinetic expressions for
many types of drug interaction, such as competitive, noncompetitive,
uncompetitive inhibition, and direct activation, have been described in
detail (13), no kinetic model has yet been reported for a P450 enzyme
that catalyzes the metabolism of two substrates coexisting at the
active site and that differentiates the fate of one another through
mutual inhibition or activation. Due to the complexity of such
interactions, the resulting kinetics are not straightforward and hamper
mechanistic interpretation of the interaction in question. Thus,
kinetic models that adequately describe the interaction between
multiple drugs that bind simultaneously to a single enzyme active site
are needed to aid in the understanding of clinically observed drug-drug
interactions and in an assessment of their clinical significance. To
better understand multiple substrate/effector enzyme kinetics, the
present study investigated the metabolic interaction between losartan and Materials--
CYP3A4 Expression in Baculovirus--
The full-length cDNAs
encoding human CYP3A4 and CYP OR were provided by Dr. Frank J. Gonzalez
(National Cancer Institute, Bethesda, MD). The entire coding region of
each cDNA was inserted into baculovirus shuttle vectors, pBlueBac
(XbaI/KpnI for CYP3A4 and EcoRI for
OR) and then recombined separately into baculoviruses according to the
manufacturer's procedure (Invitrogen, Carlsbad, CA). CYP3A4 and OR
proteins were expressed in Sf21 insect cells coinfected with the
two separate recombinant viruses at a ratio (CYP3A4:OR = 5) of
multiplicity of infection of virus in the presence of hemin (1 µg/ml)
(14). Cells were harvested after 3 days, and microsomes were prepared
by two different centrifugations (15). The CYP3A4 content was
determined by the CO difference spectrum, and ~150 nmol of CYP3A4 in
500 ml cell culture was obtained. The specific activity of the CYP3A4
was measured by the testosterone 6 Metabolism of Losartan and HPLC--
HPLC analyses were performed on a Hewlett-Packard
model 1100 liquid chromatograph equipped with both a diode array
detector and an HP 1046 programmable fluorescence detector. Losartan
and its metabolites were separated on a 20/20 ODS column (4.6 mm x 20 cm, 5 µm; TLC, Springfield, VA) and eluted with a 22-min
linear gradient from 30 to 45% acetonitrile in water containing 0.1% acetic acid at a flow rate of 1 ml/min. The retention times of P1, P2, and losartan were 9.1, 17.2, and 18.9 min, respectively. Fluorescence of metabolites was detected at
wavelengths of 250 nm for excitation and 370 nm for emission. The
quantities of metabolites formed were determined from the appropriate
standard curves. Liquid Chromatography-Tandem Mass Spectrometry
(LC-MS/MS)--
For liquid chromatography-MS/MS studies,
chromatography was conducted on a Hewlett-Packard HP1050 gradient
system. Separation was carried out on a Phenomenex LunaTM
C18 column (2.0 mm x 25 cm, 5 µm) using a mobile phase consisting of
0.1% formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B) and a flow rate of 0.2 ml/min. The HPLC system
was interfaced to a Finnigan TSQ 7000 tandem mass spectrometer,
which was operated using electrospray ionization in the positive ion
mode. The capillary temperature was 200 °C, and the electrospray
ionization ionizing voltage was maintained at 5.0 kV. MS/MS was based
on collision-induced dissociation of ions entering the rf-only octapole
region, where argon was used as the collision gas at a pressure of 1.7 millitorr. A combination of collision offset voltages ranging from Hypothesis for the Model of Two Substrate Enzyme
Kinetics--
Based on the experimental findings of this study (see
below), we observed that (i) Data Analysis--
Values for all kinetic parameters were
calculated by an iterative procedure based on appropriate initial
estimates that best fitted the data using the Marquardt-Levenberg
nonlinear least squares algorithm (17). The resulting equations were
simplified by the program Mathematica 4 (Wolfram Research, Inc.,
Champain, IL). The experimental data were fitted with the equations to
generate the surface plots. Statistics of the data was performed using both the residual sum of squares (RSS) and R2. All kinetic
constant statistics were determined by either SigmaPlot 2000 (SPSS
Inc., Chicago, IL) or Mathmatica 4.
Effect of Identification of Effect of Losartan on the Metabolism of Kinetic Analysis--
Full kinetic analyses were performed on the
effect of
These observed kinetic changes associated with the interaction of two
substrates with the enzyme cannot be explained by simple Michaelis-Menton kinetics, in which all equations are based on the
assumption of the one binding domain in the catalytic portion of the
enzyme. Hence, introduction of a second binding domain in the active
site is needed to explain the observed kinetics. Of the two proposed
binding sites in CYP3A4, the model hypothesizes that the first site can
be occupied by either losartan (S1) or
As seen in Table III, all kinetic
parameters in the model were determined by the equations.
KS1, KS2, and
KF are the estimates of dissociation constants
for three single-substrate-bound species, S1E,
ES2, and FE, respectively, whereas
VmaxP1, VmaxP2, and
VmaxQ are the maximum velocities for individual
products formed at their specific sites. In contrast, the two binding
sites had different K and Vmax for
losartan, namely KS1 and
VmaxP1 for S1E, and
KS2 and VmaxP2 for
ES2. Thus, KS1 (67-107
µM), KS2 (25-41
µM), and KF (158-203
µM) were calculated by the three individual equations (Table III), and VmaxP1,
VmaxP2, and VmaxQ were
determined to be 0.16, 0.007, and 4.1 min The clinical importance of drug-drug interactions is well
recognized. Recent advances in this field have included the development of a better understanding of the mechanism of such interactions and the
application of mechanistic information to the construction of
experimental approaches for evaluating new and existing drugs for their
potential to interact with therapeutic agents. Information on in
vitro drug-drug interactions at the level of cytochrome P450
enzymes can be extremely useful in the evaluation of the potential of a
new agent to cause drug interactions in the clinic. Such data derive
from well defined kinetic models that describe precisely the
consequences of a drug interaction at the enzyme level. However, not
all drug interactions can be described adequately by such approaches,
and there is a need to develop kinetic treatments that address unusual
drug interactions of this type.
Cytochrome P450 enzymes are believed to be monomers that bear a single
catalytic site with a common heme prosthetic group but with distinct
apoprotein structures that determine their broad and overlapping
substrate specificity. CYP3A4 is the major P450 enzyme present mainly
in human liver, the content of which can vary 40-fold among individuals
(12, 21, 22). The enzyme appears to be responsible for the oxidative
metabolism of more than 50% of clinically used drugs, and drug
interactions involving CYP3A4 substrates, inhibitors, and activators
and/or inducers are more prevalent and complex than those of other
members of the P450 family. Substrates for CYP3A4 substrates vary
greatly in their physiochemical properties such as structure, molecular size and shape, lipophilicity, electronic characteristics, and kinetic
interaction with enzyme protein. The fact that CYP3A4 can accommodate
substrates of a relatively large size, e.g. cyclosporin (Mr = 1201), suggests that multiple small or
intermediate-sized molecules might be able to coexist in the active
site of this enzyme. Indeed, some evidence has been obtained to support
this hypothesis based on kinetic studies and NMR data (14, 19, 23, 24).
If an active site is capable of accommodating two substrates
simultaneously, the resulting kinetic properties, e.g. binding affinity and catalytic ability, are likely to be affected differently from those observed with simple Michaelis-Menten inhibition and activation, both of which are derived from the one binding region
in the active site. The unusual kinetics associated with two or more
substrate interactions have been documented in a number of reports (14,
19, 23, 25, 26, 45-49). Thus, the two-site model described in the
present study was developed in an attempt to address the questions
related to the kinetics of complex of drug-drug interactions at the
active site of CYP3A4.
Losartan, an angiotensin II receptor antagonist used for the treatment
of hypertension (27), undergoes metabolism by CYP3A4 and CYP2C9 to two
major products, namely a Enzyme activation refers to the process by which direct addition of one
compound (the activator) to an enzyme enhances turnover of the
substrate. The mechanism by which activation of cytochrome P450 takes
place has not been explored extensively. However, it has been proposed
that activation by agents such as Three velocity equations for P1, P2, and Q,
respectively, assume rapid equilibria between free and bound enzyme
species in the model (Equations 1-3). The terms of
k1, k2, and
kq are the respective catalytic rate constants when
the enzyme is occupied by a single substrate, whereas Table III lists all calculated kinetic parameters. Since the model
results in three product velocity equations, a set of three values each
of KS1, KS2,
and KF has been obtained and were
shown to be consistent, suggesting the three rate equations are valid for assessment of kinetic constants. In consideration of the losartan oxidation to P2 in the presence of The model also indicates that an interaction occurred when the two
substrates (losartan) were identical. As seen in Table III,
KB for S1ES2 In summary, a model has been developed that accounts satisfactorily for
the interaction of two substrates (losartan and -naphthoflavone (
-NF) exhibited a differential effect on CYP3A4-mediated product formation as shown by an increase and decrease,
respectively, of the carboxylic acid (P2) and
-3-hydroxylated (P1) metabolites of losartan, while
losartan was found to be an inhibitor of the formation of the
5,6-epoxide of
-NF. Thus, to address this problem, a kinetic model
was developed on the assumption that CYP3A4 can accommodate two
distinct and independent binding domains for the substrates within the
active site, and the resulting velocity equations were employed to
predict the kinetic parameters for all possible enzyme-substrate
species. Our results indicate that the predicted values had a good fit
with the experimental observations. Therefore, the kinetic constants
can be used to adequately describe the nature of the metabolic
interaction between the two substrates. Applications of the model
provide some new insights into the mechanism of drug-drug interactions
at the level of CYP3A4.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthoflavone
(
-NF)1 modulated by
CYP3A4. These results of investigations, in turn, were employed to
conduct a kinetic model that was used to define the nature of the
losartan-
-NF interaction and that may be applied to other metabolic
interactions occurring at the active site of CYP3A4.
EXPERMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NF was purchased from Sigma. Losartan and its
metabolites, namely the
-3-hydroxy (P1) and carboxylic
acid derivatives (P2), were obtained from Merck. The
purified human P450 oxidoreductase (OR) was kindly provided from Dr.
James P. Hardwick (Northeastern Ohio University College of Medicine).
-hydroxylase assay and found to be
42 nmol of product formed/min/nmol of CYP3A4. The activity and content
of OR present in the microsomal preparations were determined by
assaying cytochrome c reduction and normalized according to
the standard curve measured with purified human OR proteins (16). The
molar ratio of CYP3A4 to OR was 1:2.8.
-NF by CYP3A4--
The incubation
mixture (usually 1-ml final volume) consisted of 50-100 pmol of
microsomal P450 3A4 protein, 0.1 M potassium phosphate
buffer (pH = 7.45), and substrate in the presence or absence of
the second substrate/effector (losartan or
-NF). The concentrations
of losartan and
-NF when employed as substrates were varied from
11.1 to 150 µM and from 6.25 to 200 µM,
respectively, and when used as effectors, ranged from 0 to 150 µM. The incubation mixture was pre-incubated for 2 min at
37 °C in a shaking water bath, and the reaction was initiated with
the addition of 1 mM NADPH and further incubated for 20 min. The reactions were terminated by the addition of 8 volumes of
methylene chloride. The metabolites were extracted, and the organic
phase was dried under a stream of nitrogen gas. The residue was
dissolved in 50% acetonitrile in water and analyzed immediately by
HPLC. Standard curves for the metabolites of interest were prepared
using the corresponding authentic reference materials. The consumption
of each substrate at all concentrations studied was limited to less
than 10%, and the rates of product formation from losartan (20 µM) or
-NF (20 µM) were established to
be linear over at least 30 min of incubation at 37 °C.
-NF and its metabolites were separated on a 20/20
ODS column eluted with a 40-min linear gradient from 50 to 100%
methanol in water and were detected by UV absorbance at 260 nm. The
flow rate was 1 ml/min and the retention times of the 5,6-epoxide
metabolite and
-NF itself were 21 and 30 min, respectively.
35
eV to
55 eV were used for all MS/MS analyses.
-NF modulated CYP3A4 activity by
increasing the formation of the carboxylic acid derivative
(P2) and decreasing the formation of
-3 hydroxylated
derivative (P1), (ii) losartan was an inhibitor of
CYP3A4-mediated formation of the 5,6-epoxide (Q) metabolite of
-NF
(Fig. 1), and (iii) the apparent kinetic constants, e.g.
Km and Vmax, describing the
metabolism of the coexisting substrates as altered significantly by
drug interaction in a manner that could not be explained simply by any
of the Michaelis-Menten kinetic models (inhibition or activation) by an
assumption of the one-binding domain at the active site. Thus, a
kinetic model was proposed to address the complexity of metabolism
exhibited in this study. The model postulates that CYP3A4 contains two
distinct and independent binding sites at the active site that can be
occupied simultaneously by two substrate molecules (either the same or
different molecular species). The binding orientations of substrate
with enzyme are defined on the basis of the metabolic fates of each
substrate as presented in Scheme I. The
two occupied binding sites interact kinetically such that occupancy of
the two sites results in changes in the rates of turnover of the two
substrates and in the apparent Km and
Vmax values. Thus, velocity equations for the
formation of each major product are derived according to the
expressions shown below, and kinetic estimates that can truly reflect
the relationship between substrate(s) and enzyme in any of
substrate/effector-enzyme combinations are resolved. The model is based
on the assumption of rapid equilibrium. Thus, all species in the
reaction described by Scheme I equilibrate rapidly, meaning that the
dissociation of each enzyme-substrate complex is much faster than its
breakdown to product(s).
(Eq. 1)
(Eq. 2)
where [S] and [F] are the concentrations of losartan and
(Eq. 3)
-NF, and VmaxP1,
VmaxP2, and VmaxQ are
expressed by [Et]k1, [Et]k2, and
[Et]kq, respectively, referring to the
maximum velocity of the formation of each metabolite at a singly
occupied site. K values are dissociation constants for individual enzyme-substrate complexes, whereas
,
,
, and
represent a factor that determines the change of the
Vmax at one site when the second site is
occupied by the substrate molecule. Since the enzyme reaction forms two
closed circles as indicated in Scheme I, the terms of
KS1KA and
KS2KSF in the equations
can be substituted by KS2KB and
KFKFS, respectively, and vise
versa. Thus, the velocity equation for each product formation may
be expressed in different ways.
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Scheme I.
Proposed kinetic model for metabolic
interaction of simultaneous-existing dual substrates in the active site
of CYP3A4. S, losartan; F, -NF;
P1,
-3-hydroxyl losartan; P2, losartan
carboxylic acid; Q,
-NF 5,6-epoxide; k, rate
constant; and K, dissociation constant. S1 and
S2 are losartan molecules that bind to the two separate
sites, leading to P1 and P2 formation,
respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NF on the Metabolism of Losartan--
Incubation of
losartan with cDNA-expressed CYP3A4 in the presence of NADPH led to
the formation of two major metabolites, namely P1
(
-3-hydroxy losartan) and P2 (the carboxylic acid
metabolite), which were identified by comparing the HPLC retention
times and MS/MS spectra with authentic standards (Fig.
1). Time-dependent formation
of products in the metabolism of losartan (20 µM) was shown to be linear up to at least 30 min of incubation at 37 °C, whereas less than 10% of the substrate was consumed during the experiment. The ratio of the concentration of substrate to that of
CYP3A4 in the incubation usually was between 250 and 2,000. Thus, the
enzyme-mediated reaction was considered to meet the requirements of
both steady state and rapid equilibrium. The addition of
-NF
(11-100 µM) enhanced significantly the rate of formation of P2 in the metabolism of losartan by 6-11-fold (Fig. 3).
However,
-NF proved to be a potent inhibitor of the
-3
hydroxylation of losartan (P1). When the
-NF
concentration was increased to
25 µM, the
P1 metabolite was no longer detected (Fig.
2). Surface plots that indicate the
effect of
-NF on the concomitant inhibition and activation of
losartan metabolism are shown in Figs. 2 and 3, respectively.
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Fig. 1.
Structures of losartan and
-NF. CYP3A4-mediated product formations of
both substrate molecules are shown in the arrows.
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Fig. 2.
Effect of -NF on the
CYP3A4-catalyzed formation of
-3 hydroxylated
losartan (P1). A surface plot is a predicted result
with Equation 1 (RSS = 0.0213, R2 = 0.992).
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Fig. 3.
Effect of -NF on the
CYP3A4-catalyzed formation of carboxylic acid derivative of losartan
(P2). Surface plot is a predicted result with Equation 2 (RSS = 0.101, R2 = 0.988).
-NF 5,6-Epoxide--
A major metabolite
(retention time = 17 min) of the CYP3A4-catalyzed metabolism of
-NF that accounted for >90% of the HPLC peak area of total
metabolites exhibited upon liquid chromatography-MS analysis an
MH+ ion at m/z 289, suggestive of the
addition of an oxygen atom to
-NF. Upon collision-induced
dissociation, major fragment ions were observed at
m/z 103, 131, 143, and 187, respectively (Fig. 4). Although ion at
m/z 103 indicated that the isolated phenyl ring
of the molecule was intact. The ions at m/z 143, 187, and 131 suggested that the oxygen atom had been incorporated into the naphthalene moiety of the molecule. By comparing the HPLC profile
and mass spectral information with previous reports (18-20), the
metabolite was identified tentatively as
-NF 5,6-epoxide.
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Fig. 4.
Spectrum of product ions obtained by
collision-induced dissociation of the MH+ ion
(m/z 289) of the major
CYP3A4-mediated metabolite of -NF.
-NF--
In the
metabolism of
-NF by CYP3A4, the inclusion of losartan (6.25-150
µM) was found to inhibit the formation of
-NF
5,6-epoxide by up to 64%. In these experiments, inhibition of
-NF
metabolism increased in parallel with increases in losartan
concentration, as shown in Fig. 5.
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Fig. 5.
Effect of losartan on the CYP3A4-catalyzed
formation of the 5,6-epoxide of -NF. A
surface plot is a predicted result with Equation 3 (RSS = 0.0153, R2 = 0.990).
-NF on losartan metabolism. The apparent kinetic
parameters and statistics are given in Table
I. In the absence of
-NF, two apparent
Km values were determined by monitoring the
formation of P1 (KmP1(app)) and
P2 (KmP2(app)); these were 82 and
40.3 µM, respectively. The effect of
-NF on losartan
metabolism indicated that
-NF at 1-150 µM stimulated
dramatically the formation of P2 with a marked increase in
VmaxP2(app) (up to 4.6-fold) and a rapid
decrease in KmP2(app) (up to 4-fold). However,
when the formation of metabolite P1 was quantified,
-NF
exhibited the opposite effect, showing an increase in
KmP1(app) (4.2-fold) but a decrease in
VmaxP1(app). At
-NF concentrations >25
µM, formation of P1 was inhibited completely (
P1
0). In the metabolism of
-NF by
CYP3A4, incorporation of losartan resulted in a considerable decrease
in VmaxQ(app) (35.7% of
VmaxQ(app) in the absence of losartan) and a
slight increase in KmQ(app) (Table
II).
Effect of -NF on the kinetics of losartan metabolism by
cDNA-expressed CYP3A4
-NF.
Effect of losartan on the kinetics of -NF metabolism by
cDNA-expressed CYP3A4
-NF-5,6-epoxide formation in the absence
or presence of losartan.
-NF (F). The
binding of losartan forms S1E and
S1ES2 complexes for the breakdown to
P1, whereas the binding of
-NF forms FE and
FES2 for the production of Q (Scheme I). Two substrates for the binding site are competitive, resulting in an inhibition, as
reflected in part by an increase in KmP1(app))
or KmQ(app) and decrease in
VmaxP1(app) or
VmaxQ(app). Meanwhile, the second site is
occupied by losartan (S2) to form ES2,
FES2, and S1ES2 species for
generating P2 exclusively. Thus, the activation of losartan
oxidation to P2 by
-NF clearly is attributed to
FES2, one of the three P2-forming species,
suggesting that the binding of
-NF to this site changes the kinetic
nature for the other site for losartan (S2), allowing
losartan to be metabolized readily to P2 (probably an
allosteric effect). Since the two binding sites are involved in the
enzyme reaction and numerous substrate-enzyme complexes are formed,
each complex possesses its own dissociation constant(s) and
product-forming rate(s). In fact, the observed rate of formation of a
particular metabolite represents the sum of the rates of all associated
enzyme-substrate complexes generating that metabolite. For example, the
net rate of P2 production is accounted for by the rates of
product formation from ES2, S1ES2, and FES2. Therefore, the resulting apparent
Km and Vmax determined by the
total rate of product formation (Table I and II) do not truly represent
the kinetic characteristics of each individual substrate-enzyme
complex. The model that describes the observed metabolic interaction
leads to the solution of kinetic parameters in equilibria and provides
a prediction of the potential in vitro drug-drug interaction
via the enzyme-mediated reaction.
1,
respectively. When both sites were occupied with losartan,
Vmax values were changed by factors
(0.38)
for S1ES2
P1 and
(0.43) for
S1ES2
P2, leading to an
inhibition by
VmaxP1 < VmaxP1 and
VmaxP2 < VmaxP2. In addition, Km
values were shown to increase for both KB = 435 µM (S1ES2
ES2)
and KA = 150-211 µM (S1ES2
S1E), respectively,
implying that the kinetic interaction of the two substrates at the
active sites occurs. On the other hand, when ES2 and
S1ES2 were occupied/displaced with
-NF,
VmaxP2 for FES2
P2
increased substantially by a factor
(
= 8.3,
VmaxP2 > VmaxP2) with
a little decrease in Km for FES2
FE
(KFS = 15.1 µM < KS2 = 25-40 µM). In contrast,
losartan decreased the Vmax for FES2
Q by a factor
(
= 0.29,
VmaxQ < VmaxQ) but did not alter
Km for FES2
ES2
(KSF = 182-203 µM
KF = 158-203 µM). These results
suggest that the presence of the two substrates (either the same or
different molecular species) elicits changes in the metabolic profile
of the enzyme as a result of several mechanisms of substrate
interactions, e.g. competitive, cooperative, conformational,
steric, and/or electronic effects that cause kinetic and metabolic
multiplicity.
Kinetic parameters calculated from the equations in the text
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-3 hydroxy losartan (P1) and a
carboxylic acid derivative (P2), the latter originating via
an aldehyde intermediate (28, 29). The carboxylic acid metabolite has
been shown to be pharmacologically more active than the parent compound
(30) and is believed to be largely responsible for the long duration of
action of losartan (31).
-NF, like other flavonoids, is a widely
distributed phytochemical that is consumed regularly in the human diet
(32). There is increasing interest in the ability of
-NF to modulate
cytochrome P450-mediated reactions (33, 34). Thus,
-NF has been
shown to increase the total oxidation of losartan by human liver
microsomes and by CYP3A4 expressed in Escherichia coli (28).
As demonstrated in the present study, a major product of this increased
metabolism was the carboxylic acid, P2.
-NF entails allosteric binding of
an activator to the enzyme (20, 35, 36). There are many reports in the
literature of direct activation of CYP3A4 in the in vitro
oxidation of foreign compounds, e.g.
-NF (19, 20,
37-39), carbamazepine (40), acetaminophen (39), steroids (35, 41), and
benzodiazepines (14). In the oxidation of the carcinogen aflatoxin
B1 by CYP3A4 (25, 42),
-NF modulates catalysis in a
regioselective fashion. Thus,
-NF inhibits the 3
-hydroxylation of
aflatoxin B1 but stimulates the 8,9-epoxidation reaction
(22, 25). Since
-NF itself is known to be a substrate of CYP3A4, it
must have access to the active site for metabolism. Interaction of two
substrates according to the one-site model usually results in simple
competitive, noncompetitive inhibition or activation. In the present
study, however, the differential effect of
-NF on the values
Km and Vmax associated with metabolism of losartan to the different products (P1 and
P2), i.e. increase in
KmP1(app) with a decrease in
VmaxP1(app) and decrease in
KmP2(app) with an increase in
VmaxP2(app) cannot be explained by the one-site
model. Hence, the presence of a second binding region in the active
site must be considered to explain these unusual kinetics. According to
such a model,
-NF can bind to one site that is associated with its
metabolism and modulation of enzyme activity while simultaneously
activating the second site for oxidation of losartan to P2.
If
-NF were to bind to an allosteric site independent of the active
site, the kinetic parameters, e.g.
KmP1(app) and KmP2(app)
for losartan, should change to the same extent and in the same
direction. The fact that this was not what was observed (Table I) is
critical to the development of the model. In addition, the inhibitory
effect of either of the two substrates on P1 or Q
production that gives rise to increased Km and
decreased Vmax also is suggestive of
displacement for one site by the two different substrates. In light of
these considerations, an appropriate model that describes the observed
kinetic changes was developed according to the following criteria:
(a)
-NF can only bind to one defined site that is also occupied by losartan in the position required for
-3 hydroxylation (P1), and both substrates can displace each other;
(b) the binding of
-NF to the one site causes an
allosteric effect on the other substrate-bound site required for the
formation of losartan carboxylic acid (P2), thereby
altering active site geometry and oxidation efficiency reflected by a
considerable decrease in KmP2(app) and increase
in VmaxP2(app); and (c) kinetic
properties, e.g. binding affinity and reaction
velocity, for the two-bound sites can be influenced by each other.
Theses changes probably are due to the combined effects of allosteric
modulation, steric effects, and/or electronic characteristics. Hence,
the entire model was built to generate five possible enzyme-substrate
complexes that are used to interpret all metabolic outcomes of drug
interactions with the CYP3A4 enzyme that were observed in the present study.
,
,
,
and
are the limiting factors that indicate the changes in the
velocity of product formation at the one site when the other site is
occupied. K values represent the dissociation constants of
individual enzyme-substrate species during the rapid equilibrium or
steady-state period in which the K value would be equivalent
to Km, reflecting the binding affinity of substrate
to enzyme.
-NF, losartan was
found to have more affinity for the enzyme (KFS = 15.1 µM) than that observed in the absence of
-NF
(KS2 = 25-40 µM), whereas
Vmax for FES2
FE + P2 (
VmaxP2 = 0.058 min
1,
= 8.3) was increased
substantially with respect to VmaxP2 (0.007 min
1), implying that the addition of
-NF
increases the amount of FES2 and accelerates the
P2 formation by 8.3-fold. This suggests that
-NF binding
to the one site changes the kinetic properties, exhibited by
KFS and factor
, of the vacant site for
losartan, which allows losartan to be metabolized readily. However,
-NF inhibited the conversion of losartan to P1, leading
to a significant increase in KmP1(app) and
decrease in VmaxP1(app). The differential effect
of
-NF on the kinetic constants for losartan oxidation is due
apparently to both competition between the two substrates for the one
site and the allosteric effect of
-NF on the other site. Conversely,
losartan has similar but opposite effects on the kinetic properties of
-NF, i.e. the KSF is similar to
KF but
VmaxQ is only
29% of VmaxQ (
= 0.29, Table III).
Additionally, the binding affinity for
-NF
(KmQ(app)) is decreased as the losartan concentration is increased (Table II). These results suggest that (i)
-NF competes with losartan for the one site (low affinity), which
results in the decrease in turnover of both two substrates, and (ii)
the changes in K and Vmax values in
the presence of both substrates are due to kinetic interactions within
the active site, such as competition of the two substrates for the one
site, and/or effects of one occupied site on the other occupied site,
e.g. rigid and allosteric factors. However, since the
Km for S1E
E
(KS1 = 67-107 µM) and
S1ES2
ES2
(KB = 435 µM) are much greater
than the Cmax (< 1 µM) in human
(43), losartan as an inhibitor is unlikely to be relevant to drug-drug
interaction in clinical pharmacokinetics (44).
ES2 (435 µM) was 4-6.5-fold greater than
KS1 for S1E
E (67-107
µM), but
VmaxP1 was 38% of
VmaxP1 (
= 0.38). Similarly,
KA for S1ES2
S1E (150-211 µM) was 3.7-8.4-fold higher than KS2 for ES2
E (25-40.5
µM), whereas
VmaxP2 was 43% of VmaxP2 (
= 0.43). These findings
illustrate that the two different sites are capable of interacting
whenever each is occupied. The appreciable changes in binding affinity
and reaction rate for the formation of P1 and
P2 from losartan are due most likely to the rigidity of the
two molecules with a relatively large size (Mr = 422), which coexist at the active site.
-NF) at the active
site of CYP3A4 and for the differential effect of
-NF on losartan
oxidation. A key feature of this model is that it allows for the
binding of both substrates simultaneously at the active site of the
enzyme and describes in kinetic terms the nature of the interaction
between these molecular species. It is hoped that application of this
model will further aid in our understanding of the complex kinetics
exhibited by CYP3A4 and provide an explanation for drug-drug
interactions at the level of CYP3A4, which hitherto have not been
available to kinetic evaluation.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Drs. A. D. Rodrigues, James Yergey, J. H. Lin, and P. G. Pearson for their valuable discussion.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed to this work equally.
Present address: Metabolism and pharmacokinetics, D13-06,
Bristol-Myers Squibb, P. O. Box 4000, Princeton, NJ 08543.
¶ To whom correspondence should be addressed: Dept. of Drug Metabolism, WP75A-203, Merck Research Laboratories, West Point, PA 19486. E-mail: magang_shou@merck.com. Tel.: 215-652-1899; Fax: 215-652-2410.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008799200
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ABBREVIATIONS |
---|
The abbreviations used are:
-NF,
-naphthoflavone;
OR, oxidoreductase;
HPLC, high performance liquid
chromatography;
MS/MS, tandem mass spectrometry;
RSS, residual sum of
squares.
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