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Vol. 30, Issue 12, 1512-1522, December 2002
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom (A.G., J.B.H.); and Department of Mechanism and Extrapolation Technologies, GlaxoSmithKline, Welwyn, Hertfordshire, United Kingdom (S.E.C.)
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Abstract |
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The selection of appropriate substrates for investigating the potential inhibition of CYP3A4 is critical as the magnitude of effect is often substrate-dependent, and a weak correlation is often observed among different CYP3A4 substrates. This feature has been attributed to the existence of multiple binding sites and, therefore, relatively complex in vitro data modeling is required to avoid erroneous evaluation and to allow prediction of drug-drug interactions. This study, performed in lymphoblast-expressed CYP3A4 with oxidoreductase, provides a systematic comparison of the effects of quinidine (QUI) and haloperidol (HAL) as modifiers of CYP3A4 activity using a selection of CYP3A4 substrates: testosterone (TST), midazolam (MDZ), nifedipine (NIF), felodipine (FEL), and simvastatin (SV). The effect of QUI and HAL on CYP3A4-mediated pathways was substrate-dependent, ranging from potent inhibition of NIF (Ki = 0.25 and 5.3 µM for HAL and QUI, respectively), weak inhibition (TST), minimal effect (HAL on MDZ/SV) to QUI activation of FEL and SV metabolism. Inhibition of TST metabolite formation occurred but its autoactivation properties were maintained, indicating binding of a QUI/HAL molecule to a distinct effector site. Various multisite kinetic models have been applied to elucidate the mechanism of the drug-drug interactions observed. Kinetic models with two substrate-binding sites have been found to be appropriate to a number of interactions, provided the substrates show hyperbolic (MDZ, FEL, and SV) or substrate inhibition kinetic properties (NIF). In contrast, a three-site model approach is required for TST, a substrate showing positive cooperativity in its binding to CYP3A4.
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Introduction |
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To assess the in vivo significance of drug-drug interactions
involving
P4501
inhibition from in vitro data, it is necessary to identify the particular P450 enzymes involved, estimate their contribution to
the overall elimination of the drug, and characterize the inhibition effects (Ito et al., 1998
; Rodrigues et al., 2001
; Tucker et al., 2001
). The latter is the most problematic factor, because it is dependent on the appropriate selection of both an inhibition model to
derive a Ki value and an inhibitor
concentration at the enzyme active site.
The selection of an appropriate inhibition model for CYP3A4 is
particularly difficult because this enzyme frequently does not obey
Michaelis-Menten kinetics, shows substrate-dependent effects (Kenworthy
et al., 1999
; Stresser et al., 2000
; Wang et al., 2000
; Lu et al.,
2001
), and is prone to activation (Shou et al., 1994
; Tang et al.,
1999
; Kenworthy et al., 2001
) in addition to inhibition effects in
drug-drug interaction studies. Substrate auto- and heteroactivation
(Shou et al., 1999
; Domanski et al., 2000
; Ngui et al., 2000
; Kenworthy
et al., 2001
), partial inhibition (Wang et al., 1997
, 2000
), substrate
inhibition (Lin et al., 2001
; Schrag and Wienkers, 2001
), and pathway
differential kinetics (Shou et al., 2001a
) observed for CYP3A4 are
attributed to the different binding domains for the substrate and
modifier within the enzyme active site. Involvement of multiple binding
sites may result in an inhibition effect at only one site or a
differential effect at each site, confounding a straightforward
prediction of a potential in vivo interaction. A description of the
molecular events, incorporating the binding of multiple
substrate/inhibitor molecules, requires relatively complex modeling.
To provide a mechanistic insight for atypical enzyme properties shown
by CYP3A4, various approaches have been reported in recent years (Hosea
et al., 2000
; Tang and Stearns, 2001
), involving either the
simultaneous binding of two molecules (Korzekwa et al., 1998
; Shou et
al., 2001b
) or the existence of a separate effector-binding site (Ueng
et al., 1997
; Kenworthy et al., 2001
). Additional evidence for the
existence of multiple binding sites is provided by site-directed
mutagenesis studies (Harlow and Halpert, 1998
; Domanski et al., 2001
),
indicating that CYP3A4 substrate and effector-binding sites are
separate, but closely linked, and the residues involved in the binding
of either substrate and/or effector depend on the molecule present.
The clinical significance of an observed in vitro heteroactivation of
CYP3A4 is still uncertain, because to date few confirmations in vivo
have been reported. A decrease in diclofenac steady-state plasma
concentrations, observed upon the coadministration of quinidine in
rhesus monkeys, is consistent with an in vitro activation interaction (Tang et al., 1999
; Ngui et al., 2000
). Quinidine also shows the ability to stimulate meloxicam metabolism by CYP3A4 and increases the
contribution of CYP3A4 over CYP2C9 to the overall metabolism by
heteroactivation (Ludwig et al., 1999
).
To explore these confounding factors of CYP3A4 drug-drug interactions
we have selected quinidine (QUI) and haloperidol (HAL) as modifiers.
Selection of QUI, a well known inhibitor of CYP2D6, was based on
previous reports suggesting differential effects on various CYP3A4
substrates, ranging from inhibition (von Moltke et al., 1994
; Kenworthy
et al., 1999
) to activation (Ludwig et al., 1999
; Tang et al., 1999
,
Sai et al., 2000
; Ngui et al., 2001
). Similarly, the effects of HAL on
various subclasses of CYP3A4 substrates (Kenworthy et al., 1999
) were
found to be inconsistent. Midazolam (MDZ), testosterone (TST),
nifedipine (NIF), felodipine (FEL), and simvastatin (SV) were selected
as representatives of a range of CYP3A4 substrates. The choice of TST,
MDZ, and NIF was based on their in vitro kinetic properties, which
include homotropic cooperativity, hyperbolic kinetics, and substrate
inhibition, respectively. FEL was included to provide a comparison with
NIF, based on the similarities in their metabolic pathways and the high
correlation reported for their in vivo clearance (Soons et al., 1993
).
Selection of SV was based on its widespread clinical use and the
significance of its drug-drug interactions.
To describe the experimental data, multisite kinetic models and the
corresponding equations that assume the existence of either two or
three distinct binding domains within the active site of CYP3A4 have
been derived, based on a steady-state, rapid equilibrium approach
(Segel, 1975
). The substrate and effector kinetic properties, and the
alterations in their binding affinities and catalytic efficiency when
simultaneously present at the active site, are characterized by
interaction factors. Based on the findings with HAL and QUI, certain
criteria for a generic two-site model for drug-drug interactions
involving CYP3A4 are defined.
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Materials and Methods |
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Chemicals.
TST, 6
-hydroxytestosterone (6
-HTS), NIF, QUI, MDZ, HAL,
NADP, and isocitric dehydrogenase were purchased from Sigma Chemical (Poole, Dorset, UK). Oxidized NIF (OX NIF), (3S)-3-OH QUI,
and MDZ metabolites were obtained from Ultrafine Chemicals (Manchester, UK). FEL and pyridine metabolite (FEL PYR) were gifts from Astra (Hässle, Mölndal, Sweden). SV was obtained from Merck
(Darmstadt, Germany) and UK-58,790 was from GlaxoSmithKline
(Frythe, UK). All other reagents and solvents were of high analytical
grade. Microsomes from human B-lymphoblastoid cells with coexpressed CYP3A4 and NADPH-cytochrome P450 reductase (CYP3A4/OR) were obtained from Gentest (Woburn, MA).
Incubation Conditions.
Interaction studies were performed at incubation times and protein
concentrations within the linear range for the each substrate. Microsomes from cells containing recombinant human CYP3A4/OR were suspended in 0.1 M phosphate buffer, pH 7.4. The final incubation volume was 0.2 ml, containing 47 to 111 pmol of P450/ml. Samples were
preincubated for 5 min in a shaking water bath at 37°C, and each
reaction was initiated with an NADPH-regenerating system (1 mM
NADP+, 7.5 mM isocitric acid, 15 mM magnesium
chloride, and 0.2 units of isocitric dehydrogenase). The substrates
(concentrations defined below) were added to each incubation in either
methanol or phosphate buffer, depending on the solubility. Neither of
the substrates showed significant microsomal binding (<10%). The
final concentration of methanol in incubation media was
0.5% (v/v).
The range of HAL and QUI concentrations applied was from 0.5 to 100 µM in most studies. The reaction was terminated by 0.1 ml of ice-cold
methanol. Samples were then centrifuged at 13,400g for 5 min
and analyzed by high-performance liquid
chromatography/ultraviolet or high-performance liquid chromatography
with tandem mass spectrometry (Table 1).
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Data Analysis.
The kinetic parameters were calculated from untransformed data by
nonlinear least-squares regression using GraFit 4 (Erithacus Software,
Horley, Surrey, UK). In the case of FEL, MDZ, and SV, the
Michaelis-Menten equation with the weighting factor of 1/y was used to
analyze the data. Analysis of NIF kinetic data was carried out assuming
single site Michaelis-Menten kinetics with substrate inhibition
(Houston and Kenworthy, 2000
). In the case of TST, kinetic parameters
Vmax,
S50, and Hill coefficient
(n) were calculated from untransformed data using Hill
equation. CLmax was calculated in case of TST as
an alternative to CLint (due to autoactivation
phenomenon), providing an estimate for the maximum clearance when the
enzyme is fully activated before the saturation occurs (Houston and
Kenworthy, 2000
). The changes in kinetic parameters observed in the
presence of various modifiers were significance tested using analysis
of variance.
2 and Akaike
information criterion values) between the models and a reduction in the
standard errors of the parameter estimates. Velocity curves for
metabolite formation were simulated using the kinetic parameter
estimates obtained from different multisite kinetic models.
Multisite Kinetic Equilibria Models.
The kinetic models used were adopted from Segel (1975)
and were
based on steady-state and rapid equilibrium approach, allowing simultaneous fit of multiple sets of data to a single equation. The
assumptions of this approach are outlined in this textbook.
Two-Site Models.
The simplest model accommodating atypical kinetic properties when two
molecules of the same substrate bind to the active site is presented in
the Fig. 1A. Two binding sites are
identical, because no orientation differences in binding of S to E were
defined. Alterations in either binding affinity or catalytic efficiency upon binding of a second substrate molecule to a vacant site can describe data both from substrates showing positive cooperativity and
substrate inhibition. The interaction of the substrate molecules is
quantified by the velocity equation shown below:
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(1) |
< 1), or changes in the effective catalytic rate constant (Kp) by the factor
in the
two-substrate-bound complex (
> 1). Changes in
or
in
the opposite direction can yield negative cooperativity (
> 1, resulting in biphasic kinetic profile,
< 1, resulting in
substrate inhibition) (Houston and Kenworthy, 2000
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and
, respectively).
There is the possibility that the enzyme-product complex reduces the
enzyme availability for the S interaction, causing a decreased rate of
the reaction (Narasimhulu et al., 1998Inhibition/Activation of a Substrate with Hyperbolic Kinetics.
Equation 2 is applied for the substrates showing hyperbolic type of
kinetic properties (e.g., FEL). No interaction is observed between the
substrate molecules (autoactivation); therefore, the kinetic model is
simplified eliminating the interaction factor
. This two-site model
can accommodate cases of partial inhibition and changes to
Ks and
Vmax when the formation of a complex
containing two different substrate molecules is more/less favorable,
depending on the
value. Interaction factor
describes the
changes in the binding affinities of the substrate and the modifier in
the presence of each other. The equivalence of two-substrate-binding sites is assumed; therefore,
describing product formation from SES
complex is 2, as Vmax is equivalent to
2Kp[E]t, where
[E]t is the total enzyme concentration. Alterations in
the product formation in the presence of a modifier molecule are
defined by the interaction factor
< 1 for inhibition and
> 1 for activation.
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(2) |
Inhibition of a Substrate Showing Substrate Inhibition Kinetic
Properties.
Recent studies have indicated the utility of the kinetic models with
two substrate-binding sites for the cases of substrate inhibition
kinetics (Houston and Kenworthy, 2000
; Lin et al., 2001
; Schrag and
Wienkers, 2001
). The two-site model applied herein incorporates
sequential binding of substrate molecules, i.e., the substrate
inhibition site cannot be occupied until the active site is filled. The
second site may be independent from the active site. Because the enzyme
has only one catalytically active site, Vmax is equivalent to
Kp[E]t, where [E]t is the total
enzyme concentration. The presence of the effector molecule may
increase the complexity of the model, depending on the effector binding
affinities and its effects on catalytic activities associated with the
substrate-binding sites. Binding of a second substrate or inhibitor
molecule causes a reduction in product formation, characterized by the
interaction factor
(0 <
< 1) (eq. 3), because
SES/IES/SEI are less productive.
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(3) |
Three-Site Models.
These are more complex kinetic models where both substrate and effector
bind to two sites, and one site is unique to either molecule (Kenworthy
et al., 2001
). The principal characteristics of three-site models is
the existence of a distinct effector binding site, with the possibility
of conformational changes upon the binding of the effector molecule,
based on the model suggested by Ueng et al. (1997)
.
Heterotropic Inhibition of a Substrate Showing Sigmoidal
Kinetics.
Equation 4 is derived for the cases where a substrate binds
cooperatively in the presence or absence of the inhibitor. This kinetic
model was previously described for the effects of diazepam on TST
(Kenworthy et al., 2001
) and was applied here for QUI-TST interaction
study. Similar to two-site models, the catalytic sites are assumed to
be equivalent, therefore
would equal 2 in the corresponding
equation and cancels out because Vmax
is equal to 2Kp[E]t,
where [E]t is the total enzyme concentration. The
interaction between two substrate molecules, and the sigmoidal
properties of the substrate, are unaffected by increasing inhibitor
concentration, suggesting that the inhibitor acts at a distinct
effector site. Inhibition is not consistent with a competitive type, as
the modifier causes changes in Vmax,
rather than changes in the substrate-binding constant
Ks.
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(4) |
Partial Inhibition of a Substrate Showing Sigmoidal Kinetics.
Equation 5 defines another type of three-site models with two
catalytically active substrate-binding sites and a distinct effector
site. Similar to previous three-site model, cooperativity in substrate
binding is maintained in the presence of an inhibitor. Binding of an
inhibitor molecule to the separate effector site causes an alteration
in Ki by the factor
(Fig. 1C). In
the cases where
is >1, the affinity of the second inhibitor
molecule is decreased in the presence of the first, consistent with
negative cooperative effect, causing the partial inhibition with the
increasing inhibitor concentration. The concentration and contribution
of I(SEI), I(SE), and I(SES) complexes (Fig. 1C) to [E]t
at higher inhibitor concentration is increased, but these enzyme
species are not productive.
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(5) |
Modifier Concentration-Dependent and Pathway-Differential
Effects.
The three-site kinetic model approach, required to describe
interactions for substrates with sigmoidal kinetic properties, can also
be used to describe the phenomenon of activation at low concentrations
of the modifier changing to inhibition at higher concentrations.
Equation 6 is derived for the 1'-OH MDZ formation and represents the
three-site model with two distinct substrate-binding sites;
ES1 is preferable for 1'-OH MDZ (defined by
Ks1 and
Kp1) and S2E for 4-OH MDZ
formation (Ks2 and
Kp2), with no alterations in the
binding affinity for a second substrate molecule (Fig. 1D). However,
the two occupied sites interact, changing the rate of 1'-OH MDZ
formation (Kp1 from SES complex is
modified by the interaction factor
), analogous to a substrate
inhibition phenomenon. Binding of the effector molecule stimulates
1'-OH MDZ formation at low substrate concentrations, increasing the
Vmax of the reaction and the effective
catalytic rate constant from I(ES) complex compared to ES by the
interaction factor
(>1). Binding of a modifier molecule in the
presence of MDZ is characterized by alterations in the inhibition
constant Ki by the factor
. At
higher S concentrations, the second substrate-binding site is also
occupied, causing the more pronounced substrate inhibition of 1'-OH
pathway. Quinidine competes with MDZ for the mutual binding site,
causing the inhibition of 1'-OH MDZ formation (increased
Km). At the same time, binding of a
QUI molecule to a distinct site to one for 1'-OH MDZ alters kinetic
properties of the substrate site preferential for the 4-OH formation,
stimulating the metabolite formation (eq. 7, where
is the
interaction factor for the 4-OH pathway,
Kp2 > Kp2). Pathway differential effects
observed for QUI were similar to the differential effects of
-naphthoflavone on losartan metabolism, reported by Shou et al.
(2001a)
.
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(6) |
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(7) |
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Results |
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The kinetic properties of the five CYP3A4 substrates selected for
study were determined in human recombinant CYP3A4 system with
coexpressed NADPH-P450 oxidoreductase (Table
2). NIF and TST showed the
characteristics of substrate inhibition and sigmoidal kinetics,
respectively, whereas MDZ, FEL, and SV displayed the standard
hyperbolic curve. Less than 10% of the substrate depletion was noted
and secondary metabolism was minimal throughout the course of
incubation. Khan et al. (2002)
have reported that MDZ can act as a
mechanism-based inhibitor of CYP3A4, but incubation times used for MDZ
were short (2.5 min), and no indication of inactivation was observed. A
range of substrate concentrations (at least 1/2 Km
2Km) was used to evaluate the effect
of HAL and QUI. Both modifiers show substrate-dependent effects on
CYP3A4 activity, ranging from potent inhibition (NIF), weak inhibition (TST), minimal effect (HAL on SV) to activation (QUI on FEL/SV).
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Interaction with Nifedipine. Almost complete inhibition of NIF metabolism in human recombinant CYP3A4 was achieved with either HAL or QUI, with similar inhibitory effects across the range of substrate concentrations used. HAL was a potent inhibitor of NIF metabolism (IC50 = 0.1 µM; Fig. 2A) at substrate concentrations of 10 to 50 µM. The IC50 values in the QUI-NIF interaction study were higher and increased slightly with the increasing NIF concentrations (14.8-26.5 µM) (Fig. 3A).
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; the effect for HAL was 2-fold greater
compared with QUI. Additionally, the higher inhibitory potency of HAL
is seen in the lower value for
, associated with the alterations in
rate of product formation after binding of HAL to the active site.
Substrate inhibition phenomena is eliminated at higher HAL/QUI
concentrations as the more stable, but nonproductive S(EI) site
dominates.
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Interaction with Testosterone.
The rate of TST 6
-hydroxylation in recombinant CYP3A4 was inhibited
in the presence of increasing concentrations of either HAL (Fig. 2B) or
QUI (Fig. 3B), but to a lesser extent than NIF. The shallow slope of
IC50 plots (0.67-0.41) and the inability of HAL
to fully inhibit TST metabolism, particularly at high substrate concentrations, were consistent with partial inhibition. In the QUI-TST
interaction study, the inhibition profiles observed at higher substrate
concentrations (50-200 µM) were similar, in contrast with the
greater inhibition seen at the lowest TST concentration. The
IC50 values for both modifiers progressively
increased with increasing TST concentrations (34-114 and 98-386 µM
for QUI and HAL, respectively) consistent with a competitive type of inhibition.
-HTS formation. Preliminary kinetic analysis showed a
2-fold increase in S50 values with the increasing
HAL concentration (44.9-98.8 µM), whereas the
Vmax showed no statistically significant change. In QUI study, the
Vmax values decreased from 8.06 (control) to 4.11 pmol/min/pmolP450 (100 µM QUI)
(p < 0.005), whereas no significant changes
were observed in the S50. Sigmoidicity of
6
-HTS formation remained over the range of concentrations of both
HAL and QUI, with no significant changes in the Hill coefficient as
indicated by the comparable extent of curvature in the Eadie-Hofstee plots (Fig. 4B). CLmax decreased 2-fold in the
presence of increasing concentrations of HAL and QUI (Fig.
5B).
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-HTS formation at various QUI
concentrations derived from those estimates are shown in Fig. 5A. The
maintenance of cooperativity in TST binding in the presence of either
inhibitor (
= 0.05 and 0.03 for HAL and QUI study,
respectively) indicates that the effector acts at a distinct site to
the one responsible for TST metabolism. Both models assume two binding
sites for the effector molecule, which is consistent with previously
described effects of QUI and HAL on NIF. Partial inhibition of 6
-HTS
formation in the presence of increasing HAL concentrations is
consistent with a high
value obtained. This alters the
Ki value for HAL from 34 to 280 µM, illustrating a decreased binding affinity of the second HAL molecule (negative cooperativity) and reduced inhibitory effect at higher HAL
concentrations. A reasonable fit to the simple partial inhibition model
could not be obtained. The Ki value
for QUI of 99 µM shows relatively weak inhibition of TST.
Interaction with Midazolam. HAL and QUI showed opposite effects on MDZ metabolism in human lymphoblast-expressed CYP3A4. HAL was a weak inhibitor and the highest extent of inhibition (around 40%) was observed at the highest MDZ concentration (Fig. 2C). In contrast, low concentrations of QUI activated MDZ 1'-hydroxylation (Fig. 3C), but at concentrations higher than 10 µM, inhibition of 1'-OH MDZ formation was observed. The extent of inhibition by QUI was approximately 50% of control values (5.28 pmol/min/pmolP450) at the highest MDZ concentration studied.
To obtain a general trend for the effects of QUI on MDZ metabolism, data for 1'-OH MDZ formation at various effector concentrations were first analyzed by the single-site approach. The Vmax values for 1'-OH MDZ formation increased at lower QUI concentrations (3 µM) and decreased at higher (30 µM). At higher concentrations of the modifier a 5-fold increase in Km value for the same pathway was noted compared with the control. At the same time, QUI stimulated the formation of a minor metabolic product, 4-OH MDZ, up to 50% at 50 µM MDZ. The differential effects of QUI on MDZ 1'-OH and 4-OH metabolic pathways were particularly evident at higher concentrations of both the substrate (MDZ) and modifier (QUI) (Fig. 6).
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Kp1 > Kp1), changing to inhibition at higher
concentrations of QUI (nonproductive complexes). The decreased product
formation after the binding of the second substrate molecule (
< 1 from S2ES1 complex) is
consistent with substrate inhibition (Fig.
7). Binding of QUI to the distinct effector site causes an allosteric effect on the substrate site preferential for 4-OH MDZ, stimulating this pathway.
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Interaction with Felodipine. Contrary to expectation, the effects of HAL and QUI on FEL metabolism were not similar to the previously described NIF data. HAL was a potent inhibitor of FEL PYR formation (IC50 of ~5 µM), with similar inhibitory effects across the range of substrate concentrations used (Fig. 2D), whereas QUI stimulated FEL metabolism over a wide range of QUI concentrations, regardless of the substrate concentration investigated. At high concentrations of both FEL and QUI, the level of activation decreased, but no inhibition was evident (Fig. 3D).
To obtain a general trend for the effects of HAL and QUI on FEL, preliminary kinetic analysis was carried out assuming a single-site interaction. The simultaneous metabolism of FEL and HAL showed a decrease in Vmax value for FEL formation from 4.42 to 1.39 pmol/min/pmolP450 (10 µM HAL) (p < 0.01) and an increase in the Km value. The lack of any significant change in IC50 values was consistent with a mixed type of inhibition, observed also for NIF in the presence of HAL. Increased affinity of CYP3A4 for FEL over a range of QUI concentrations is manifested in a decrease in the Km value and can be associated with the conformational changes in the substrate-binding site after binding of QUI molecule. This conformational change would allow FEL easier access to the active oxygen and results in increased catalytic activity (2-fold increase in the Vmax). Various two-site kinetic models were applied for further analysis of the contrasting effects of HAL and QUI on FEL CYP3A4-mediated metabolism. The kinetic parameter estimates for HAL, generated from a fit to eq. 2, are presented in Table 3. Potent inhibition (Ki = 3.4 µM) can be rationalized by the favorable formation of a complex containing both the substrate and modifier, as changes in
are not pronounced. Estimates of the
parameters generated from the fit to eq. 2 for the effects of QUI are
shown in Table 4. The alteration in the binding affinity of substrate
molecules is less pronounced in the presence of HAL (
= 0.58, Ks changes from 40.9 to 23.7 µM)
compared with QUI (
= 0.08, Ks
changes from 52.8 to 4.22 µM). Increased rate of FEL PYR formation
from SEA/EAS complexes correlates well with the changes in the
effective catalytic rate constant (
= 9.8) for the QUI interaction.
Interaction with Simvastatin.
The tendency for HAL and QUI to produce opposite effects with certain
CYP3A4 substrates was also seen with SV. Similar to the effect observed
on MDZ 1'-hydroxylation, HAL was a weak inhibitor of the 3'-OH SV
formation in human recombinant CYP3A4 (data not shown), and no further
kinetic analysis was performed. Heteroactivation by QUI of the
formation of 3'-OH SV, 6'-exomethylene SV, and 3',5'-dihydrodiol SV
occurred to a lesser extent compared with FEL and was mainly observed
at low substrate concentrations. The
Ka values obtained by two-site model
(eq. 2) were similar (288 and 226 µM for FEL and SV, respectively),
but the extent of changes in product formation and binding affinities
(defined by
and
) were not as marked in SV study as with FEL
(Table 4).
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Discussion |
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The results presented here provide a systematic comparison of the
interactions between five established CYP3A4 substrates and the
modifiers HAL/QUI. Consistent with the substrate-dependent inhibition
previously reported for CYP3A4 (Kenworthy et al., 1999
; Wang et al.,
2000
), both modifiers were potent inhibitors of NIF metabolism and weak
inhibitors of TST, while showing minimal effect (HAL on MDZ/SV) or
activating the metabolism of other CYP3A4 substrates (QUI on FEL/SV).
Haloperidol Ki values varied
>400-fold from 0.25 (NIF) to >100 µM (MDZ, SV), whereas activation
and an 18-fold difference in QUI inhibitory potency was observed. A
substrate-dependent effect was also observed in the rank order of
potency, as HAL was more potent inhibitor of NIF, FEL, and TST
metabolism, but MDZ 1'-hydroxylation was inhibited more effectively by
QUI. In studies with NIF, FEL, and TST the rank order of inhibitory
potency was in good agreement with the affinity of modifiers for CYP3A4 (50-78 µM for HAL; Pan et al., 1997
) and 112 µM QUI (A. Galetin, unpublished data). Substrate-dependent effect of HAL was observed previously by Kenworthy et al. (1999)
, and it was suggested that HAL
inhibits CYP3A4 at more than one site or that the inhibition site for
HAL is not available with certain substrate-binding conformations. A
range of HAL differential effects observed in the current study were
all accommodated by kinetic models with two substrate-binding sites for
HAL, indicating the flexibility of these models and their advantage
over the previously proposed models with one substrate-binding site.
Quinidine has been shown to activate in vitro metabolism of fentanyl
(Feierman and Lasker, 1996
), meloxicam (Ludwig et al., 1999
),
phenanthrene (Sai et al., 2000
), warfarin (Ngui et al., 2000
), and both
in vitro and in vivo metabolism of diclofenac (Tang et al., 1999
). In
all the studies, heteroactivation was a result of either changes in the
binding affinities or changes to the effective catalytic rate constant
in the presence of a modifier, or a combination of both effects. Ludwig
et al. (1999)
explained the increased affinity of CYP3A4 for meloxicam
in the presence of QUI by a separate effector site. Binding of QUI to the effector site altered the character of a substrate-binding site,
similar to an allosteric effect (Ueng et al., 1997
). The observed
activation of FEL and SV metabolism by QUI was best described by a
kinetic model with two binding sites mutual for both the substrate and
modifier (Fig. 1B). Heteroactivation occurred due to increased binding
affinity of FEL in the presence of QUI (
< 1), together with
increased product formation from the complex containing both QUI and
FEL (
> 1). Values for the interaction factors
and
were in the good agreement with the two-site models used to explain the
heteroactivation of diclofenac (Tang et al., 1999
) and warfarin (Ngui
et al., 2001
) by QUI. Stimulation of FEL metabolic pathway by QUI may
also be explained by multiple conformer model approach suggested by
Koley et al. (1995)
. Similar to NIF (Koley et al., 1997
), FEL-QUI
complex with CYP3A4 is more stable, but in this case also catalytically
more active than the FEL complex, resulting in the stimulation of the
reaction. In vitro heteroactivation of FEL and SV observed in the
current study occurred at concentrations of QUI similar to its
therapeutic plasma values (5 µM; Nielsen et al., 1999
), indicating
the possibility of an in vivo interaction, but the clinical
significance remains to be determined. The
Ki value of 5.3 µM obtained in the
QUI-NIF study is also within the range of QUI plasma concentrations,
indicating concern over possible drug-drug interaction in vivo.
Inhibition profiles for TST in the presence of QUI/HAL and the range of
IC50 values obtained were in good agreement with
Kenworthy et al. (1999)
and Sai et al. (2000)
. However, the maintenance of cooperativity in TST binding in the presence of increasing HAL/QUI
concentrations has not been noted previously due to insufficient number
of substrate concentrations previously investigated. Three-site kinetic
models with a distinct effector site adequately describe the observed
inhibition profiles. Although three-site models are more complex,
binding of HAL/QUI molecules to two sites is consistent with previously
described kinetic models for the effects of HAL/QUI on NIF, FEL, and
SV. Models assuming the existence of three binding sites, one for the
substrate, one for the effector and one mutual for both, seem to be the
models of choice for substrates showing cooperativity in their binding,
as indicated for TST-diazepam interactions (Kenworthy et al., 2001
).
Additionally, the differential effect of QUI on two MDZ metabolic
pathways can be attributed to binding of QUI to a distinct effector
site. Conformational changes in the substrate-site upon binding of QUI
to a distinct effector domain and altered regioselectivity of MDZ
metabolism are consistent with an allosteric model (Ueng et al., 1997
).
Thus, the modifying effects of QUI on CYP3A4 and QUI oxidation itself
may not be associated with the same binding domains. This observation
is consistent with site-directed mutagenesis studies and suggestions
that behavior of
-naphthoflavone as a substrate or atypical
activator/inhibitor of CYP3A4 was related to different locations within
the CYP3A4 active site (Domanski et al., 2000
).
Criteria for Selection of an Appropriate Multisite Kinetic Model in
Prediction of Drug Interactions.
The single-site Michaelis-Menten kinetic approach does not accommodate
all kinetic features observed for CYP3A4 substrates, e.g.,
heteroactivation (Kenworthy et al., 2001
), partial inhibition (Wang et
al., 2000
), substrate inhibition (Lin et al., 2001
), and differential
effects (Shou et al., 2001a
). The finding that a number of CYP3A4
substrates do not conform to the expected competitive type of
interaction indicates the existence of and interaction between several
active sites. If restricted to using classical one-site models, the
mixed kinetics would be most appropriate for the present findings. Some
features of competitive inhibition occur in certain data sets, but not
consistently (e.g., substrate-concentration dependence of
IC50 values was observed in QUI-TST study, but
the S50 values remained unchanged). The rate
equations to describe the kinetic properties and interactions involving
MDZ, NIF, FEL, TST, and SV with QUI and HAL accommodate at least two
binding sites, and in some instances three. The large "active site"
of CYP3A4 allows the simultaneous presence of multiple molecules and
the exact binding conformations depend on the substrates involved, their relative concentration, and affinity for the enzyme.
,
,
, and
(Fig. 1B) can be included/excluded from the data
modeling, depending on the substrate kinetics and the changes in the
kinetic parameters in the presence of another substrate. The
interaction factor
describes changes in binding affinity for a
second molecule of the same substrate/modifier. Therefore, it can
either be associated with substrate binding constant for substrates
showing sigmoidal kinetics (TST), or with inhibitor binding constant in
the cases of cooperative inhibition. The interaction factor
describes a heterotropic effect and is required when formation of a
complex with two different substrate molecules (MES) is favorable
(
< 1). Either heteroactivation or inhibition may result,
depending on the corresponding
value (Table 5). In the cases where
> 1, the affinity of a second inhibitor molecule for a
binding site is decreased compared with the first (TST inhibition by
HAL), resulting in a partial inhibition with the increasing inhibitor concentration. The alterations in the
Kp, associated with the interaction
factors
(SES) and
(MES) can be a result of substrate inhibition
(NIF), heteroactivation (QUI-FEL), or inhibition (HAL-FEL). Depending
on the effect, value of
can range from <1 (inhibition) to >1
(activation) as illustrated in HAL-FEL and QUI-FEL interaction, respectively. In cases when the two occupied binding sites (SES) interact with no modification in the rate of product formation,
is
2, assuming the equivalence of the substrate binding sites.
|
Conclusion.
The interaction data discussed above demonstrate that multisite enzyme
kinetic models provide a good description of complex- and
substrate-dependent CYP3A4 interactions. Application of the Ki values obtained in the prediction
of inhibitory potency in vivo remains to be evaluated. Although the
values of
,
,
, and
are substrate pair-dependent, certain
trends can be observed based on kinetic properties of both the
substrate and modifier. To accurately predict CYP3A4 inhibition
potential, the evaluation of a possible inhibitor should be performed
not only with substrates belonging to different subgroups, but also
with substrates showing a range of kinetic properties (e.g., from
hyperbolic to sigmoidal).
| |
Acknowledgments |
|---|
We thank Dr. Jackie Bloomer, Mike Nash, and Nigel Deeks (GlaxoSmithKline) for the analytical help in the project.
| |
Footnotes |
|---|
Received May 9, 2002; accepted September 9, 2002.
This study was supported by GlaxoSmithKline, UK. Part of this study was presented at the 6th ISSX Meeting, October 7-11, 2001, Munich, Germany and was published in abstract form in Drug Metab Rev 33 (Suppl 1):179.
Address correspondence to: Dr. J. B. Houston, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Rd., Manchester, M13 9PL, UK. E-mail: brian.houston{at}man.ac.uk
| |
Abbreviations |
|---|
Abbreviations used are:
P450, cytochrome P450;
QUI, quinidine;
HAL, haloperidol;
MDZ, midazolam;
TST, testosterone;
NIF, nifedipine;
FEL, felodipine;
SV, simvastatin;
6
-HTS, 6
-hydroxytestosterone;
OX NIF, oxidized nifedipine;
FEL PYR, felodipine and pyridine metabolite;
CYP3A4/OR, coexpressed CYP3A4 and
NADPH-cytochrome P450 reductase.
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