0090-9556/02/3012-1441–1445$7.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
DMD 30:1441–1445, 2002
Vol. 30, No. 12
857/1026664
Printed in U.S.A.
MICROSOMAL PROTEIN CONCENTRATION MODIFIES THE APPARENT INHIBITORY
POTENCY OF CYP3A INHIBITORS
THANH HUU TRAN, LISA L. VON MOLTKE, KARTHIK VENKATAKRISHNAN, BRIAN W. GRANDA, MEGAN A. GIBBS,
R. SCOTT OBACH, JEROLD S. HARMATZ, AND DAVID J. GREENBLATT
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine and New England Medical Center, Boston,
Massachusetts (T.H.T., L.L.v.M., J.S.H., D.J.G.); and Pfizer Inc., Groton, Connecticut (K.V.,. M.A.G., R.S.O.)
(Received July 3, 2002; accepted September 11, 2002)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
with higher protein concentrations. Based on the CYP3A content of
liver microsomes, decrements in inhibitory potency of stronger
inhibitors (ketoconazole and ritonavir) could be explained by specific binding, whereas nonspecific binding is anticipated to account for the effect on weaker inhibitors (norfluoxetine and fluvoxamine). Thus, microsomal binding (specific, nonspecific, or a
combination of both) may have a major effect on estimation of
inhibitory potency of P450 inhibitors and may contribute to variations among laboratories. The effect can be minimized by use of
the lowest possible microsomal protein concentration for in vitro
studies of metabolic inhibition.
Microsomal binding of drug substrates and/or metabolic inhibitors
is increasingly recognized as a potential source of artifact arising in
the course of in vitro studies of drug metabolism. Nonspecific binding
of substrate to microsomal protein can influence availability of the
substrate to the metabolizing enzyme or enzymes in vitro, and thereby
yield biased estimates of enzyme kinetic parameters. These, in turn,
may produce inaccurate predictions when in vitro data are used to
estimate in vivo pharmacokinetics (Obach, 1996, 1997; Obach et al.,
1997; McLure et al., 2000; Venkatakrishnan et al., 2000c, 2001;
Kalvass et al., 2001). Inhibitor binding to microsomal systems may
likewise influence estimation of potency of metabolic inhibitors
(Gibbs et al., 1999a). The influence of binding has been termed
“inhibitor depletion” when the inhibitor interacts with the active site
(Gibbs et al., 1999a). However, inhibitor depletion could also refer to
nonspecific inhibitor binding to microsomal preparations, as well as to
actual microsomal consumption of the inhibitor itself through biotransformation.
The present study evaluated the influence of microsomal protein
concentration on the inhibitory capacity of known CYP3A inhibitors.
In vitro 3-hydroxylation of diazepam to temazepam was used as an
index reaction to profile CYP3A activity. Two selective serotonin
reuptake inhibitors (fluvoxamine and norfluoxetine), three antifungal
azole agents (itraconazole, ketoconazole, and fluconazole), a metabolite of itraconazole (OH-itraconazole), and the human immunodefi-
ciency virus protease inhibitor ritonavir were used as representative
CYP3A inhibitors. These agents have different intrinsic inhibitory
capacities and different binding affinities.
Materials and Methods
Incubation Procedures and Index Reaction Characteristics. Liver samples from individual human donors with no known liver disease were provided
by the International Institute for the Advancement of Medicine (Exton, PA),
the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, MN), or the National Disease Research Interchange (Philadelphia, PA). All samples were of the CYP2D6 and CYP2C19 normal
metabolizer phenotype based on prior in vitro phenotyping studies. Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and
stored at ⫺80°C until use. Chemical reagents and drug entities were purchased
from commercial sources or kindly provided by their pharmaceutical manufacturers (von Moltke et al., 1993, 1994a,b, 1996a,b; Venkatakrishnan et al.,
2000a,b,c, 2001a,b).
Diazepam 3-hydroxylation to form temazepam was used as the index
reaction for CYP3A activity (Andersson et al., 1994; Ono et al., 1996; Jung et
al., 1997; Venkatakrishnan et al., 2000). Methanolic solutions of diazepam
(100 ␮M) were added to 2-ml microcentrifuge tubes, and the solvent was
evaporated to dryness under conditions of mild vacuum at 40°C. Incubation
mixtures containing an isocitrate/isocitric dehydrogenase regenerating system
in 50 mM phosphate buffer, 5 mM Mg2⫹, and 0.5 mM NADP⫹ were then
added to the tubes and equilibrated in a 37°C water bath for 5 min.
Metabolic Inhibitors. Incubations were performed with diazepam alone
(no inhibitor), and with coaddition with fixed concentrations of the following
established CYP3A inhibitors: ketoconazole, 0.05 ␮M; itraconazole, 1.0 ␮M;
Address correspondence to: David J. Greenblatt, M.D., Department of PharOH-itraconazole, 0.25 ␮M; fluconazole, 10 ␮M; ritonavir, 0.05 ␮M; norflumacology and Experimental Therapeutics, Tufts University School of Medicine,
oxetine, 25 ␮M; and fluvoxamine, 50 ␮M (von Moltke et al., 1994b, 1995,
136 Harrison Ave., Boston, MA 02111. E-mail: [email protected]
1996a,b, 1998a,b; Venkatakrishnan et al., 2000b).
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The effect of microsomal protein concentration on the inhibitory
potency of a series of CYP3A inhibitors was assessed in vitro using
diazepam 3-hydroxylation (yielding temazepam) as an index of
CYP3A activity. With diazepam concentrations fixed at 100 ␮M,
inhibition of temazepam formation by fixed concentrations of
ritonavir, ketoconazole, itraconazole, OH-itraconazole, norfluoxetine, and fluvoxamine decreased substantially as active protein
concentrations increased from 0.0625 to 3.0 mg/ml. However protein concentration had only a small effect on the inhibitory activity
of fluconazole. Equilibrium dialysis indicated extensive microsomal binding of all inhibitors except fluconazole; binding increased
1442
TRAN ET AL.
1
Abbreviations used are: GC, gas chromatography; HPLC, high-performance
liquid chromatography; Rt, retention time.
for the dialysis period to ensure stability of the analytes through the dialysis
process.
For analysis of ketoconazole, terfenadine (25 ␮g) was added as internal
standard. Acetonitrile (100 ␮l) was added to precipitate microsomal protein.
Mixtures were centrifuged for 10 min. Twenty-five microliters of the supernatant was injected for HPLC analysis. For analysis of fluconazole, the
dialysate and calibration standards with phenacetin (0.5 ␮g) as internal standard were alkalinized with 150 ␮l of 1 N NaOH and extracted twice with 2 ml
of ethyl acetate. The organic extract was evaporated to dryness and reconstituted with 250 ␮l of mobile phase. Fifty microliters was injected for HPLC
analysis.
For fluvoxamine, norfluoxetine, ritonavir, and hydroxyitraconazole, dialyzed microsomal samples were diluted with buffer, and buffer samples were
diluted with microsomes to ensure an identical matrix for each sample, at a
given microsomal protein concentration. The volume of retentate or dialysate
used in analysis was varied according to the microsomal protein concentration.
This was necessary to ensure that the detector responses in the liquid chromatography/mass spectrometry assay were in the linear range for all samples.
For fluvoxamine and norfluoxetine samples, microsomal proteins were precipitated using two volumes of acetonitrile containing the internal standard
(150 pmol/ml fluvoxamine for norfluoxetine samples, and 250 pmol/ml norfluoxetine for fluvoxamine samples) followed by centrifugation, and supernatants were injected on the column for liquid chromatography/mass spectrometry analysis. For ritonavir and hydroxyitraconazole samples, ketoconazole (10
ng in 20 ␮l of methanol) was added as an internal standard followed by
extraction with 3 ml of methyl t-butyl ether. The extracts were evaporated (N2,
35°C) and reconstituted in 0.025 ml of HPLC mobile phase.
Due to poor recovery from the dialysis system, equilibrium dialysis studies
of itraconazole could not be performed.
HPLC Conditions: Analysis of Ketoconazole and Fluconazole. The
analytical column was 30 cm in length containing C18 ␮Bondapak (10-␮m
particle size). The mobile phase for ketoconazole was 50 mM NH4H2PO4/
CH3CN/CH3OH (55:40:5) at a flow rate of 1.5 ml/min. The ultraviolet absorbance detector was set at 206 nm. The retention times for ketoconazole and
terfenadine were approximately 13 and 19.5 min, respectively. For fluconazole
analysis, the mobile phase was methanol/10 mM sodium acetate (40:60) at a
flow rate of 1.0 ml/min, and UV detection was at 261 nm. The retention times
for fluconazole and phenacetin were approximately 8 and 12.5 min, respectively.
HPLC-Mass Spectrometry Conditions: Analysis of Hydroxyitraconazole, Fluoxetine, Fluvoxamine, and Ritonavir. Samples were injected onto
a Phenomenex Luna C18 column (2.0 ⫻ 50 mm, 5 ␮m) equilibrated in 20 mM
acetic acid (pH adjusted to 4 with NH4OH) containing 23% CH3CN at a flow
rate of 0.5 ml/min. The system consisted of a CTC PAL autosampler (CTC
Analytics, Carrboro, NC), model 1100 quaternary gradient pump and solvent
degasser (Agilent, Palo Alto, CA), and an API100 mass spectrometer (PE
Sciex, Thornhill, ON, Cananda) fitted with a TurboIonspray interface. The
initial mobile phase conditions were maintained for 1 min followed by a linear
gradient to 77% CH3CN at 6 min and held for 3 min. The flow was split
approximately 50:50 into the mass spectrometer. The mass spectrometer was
operated in the positive ion mode with an orifice voltage of 20 V and a source
temperature of 400°C. The monitored ions and their respective retention times
(Rt) were m/z 296 (norfluoxetine, Rt ⫽ 4.0 min); m/z 319 (fluvoxamine, Rt ⫽
3.7 min); m/z 531 (ketoconazole, Rt ⫽ 4.4 min); m/z 721 (hydroxyitraconazole,
Rt ⫽ 6.1 min); and m/z 721 (ritonavir, Rt ⫽ 6.1 min).
Data Analysis. At each microsomal protein concentration, rates of formation of temazepam from diazepam with inhibitor present were expressed as a
fraction (Rv) of the control velocity without inhibitor, as follows:
Rv ⫽
Reaction velocity with inhibitor present
Reaction velocity without inhibitor
(1)
These Rv values were evaluated in relation to microsomal protein concentration as follows. If Iu is the unbound inhibitor concentration in the reaction
mixture,
Rv ⫽
IC50
Iu ⫹ IC50
(2)
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Influence of Active Protein Concentration. Reactions were initiated by
addition of varying amounts of microsomal protein (0, 0.0625, 0.125, 0.25, 0.5,
0.75, 1, 2, and 3 mg/ml) with final volume of 250 ␮l, and incubated for 10 min.
Previous studies had demonstrated time-dependent linearity of temazepam
formation up to 20 min. After the incubation period, 100 ␮l of 2% butylated
hydroxytoluene in acetonitrile was added to stop the reactions. A benzodiazepine analog (U31485, 100 ng) (Greenblatt et al., 1981) was added as internal
standard. Samples were then extracted by addition of 1.2 ml of toluene/isoamyl
alcohol (98.5:1.5), vortexing vigorously for approximately 60 s, and centrifuging at 3000 rpm for 10 min. The organic phase was then transferred into
2-ml autosampling vials for gas chromatographic (GC1) analysis. All reactions
were performed in duplicate, and all studies were performed using microsomal
preparations from four different human livers.
Influence of Inactive Protein Concentration. Diazepam (100 ␮M) with or
without ketoconazole (0.05 ␮M), OH-itraconazole (0.25 ␮M), or fluconazole
(10 ␮M) was added into 2-ml microcentrifuge tubes and dried under mild
vacuum at 40°C. Buffer containing isocitrate/isocitric dehydrogenase system
(as described above) was added and the tubes were incubated at 37°C for 5
min. Reactions were initiated by addition of varying amounts of heat-inactivated microsomal protein (0, 0.25, 0.75, 1.75, and 2.75 mg/ml), with a constant
amount of active microsomal protein (0.25 mg/ml). The final volume was 250
␮l. After 10 min of incubation, 100 ␮l of 2% butylated hydroxytoluene in
acetonitrile was added to stop the reactions. U31485 was added as internal
standard, and samples were extracted with toluene/isoamyl alcohol for GC
analysis as described above.
Analytical Conditions for Gas Chromatography. The GC (model 6890A;
Hewlett Packard, Palo Alto, CA) was equipped with an electron-capture
detector, automatic sampler, and data processor integrator The column was 6
feet in length, 2 mm in internal diameter, and packed with 3% SP-2250 on
80/100 Supelcoport (Supelco, Bellefonte, PA). The chromatographic conditions were oven temperature, 275°C; injection port temperature, 310°C; and
detector temperature, 310°C. The carrier gas was argon/methane (95:5) at a
flow rate of 30 ml/min. The retention times for diazepam (substrate), desmethyldiazepam (N-demethylated metabolite), temazepam (3-hydroxylated metabolite), and U31485 (internal standard) were 2.6, 3.7, 5.4, and 8.5 min, respectively.
Studies of Inhibitor Consumption. Ketoconazole (5 ␮M), with or without
diazepam (100 ␮M), was added into 2-ml microcentrifuge tubes and dried
under mild vacuum at 40°C. Reactions were initiated by addition of microsomal protein, and stopped by addition of 100 ␮l of acetonitrile. Terfenadine (15
␮g) was added as internal standard. The samples were centrifuged at 14,000
rpm for 10 min. Twenty-five microliters of the supernatant was injected for
HPLC analysis of remaining ketoconazole.
Equilibrium Dialysis Studies. Ketoconazole or fluconazole (initial added
concentration, 100 ␮M) was added to 2-ml microcentrifuge tubes and dried
under mild vacuum conditions at 40°C. Varying concentrations of microsomal
protein (0, 0.2, 0.5, 1.0, and 3.0 mg/ml) were added into tubes along with
buffer A (50 mM phosphate buffer and 5 mM MgCl2 without cofactors) to a
final volume of 1.0 ml. Then 400 ␮l of mixtures from each tube was injected
into membrane bags (Spectra/Por Biotech Regenerated Cellulose membrane
tubing, molecular weight cutoff 15 kDa; Spectrum, Inc., Los Angeles, CA).
The membrane bags were immersed in 6 ml of buffer A in 15-ml centrifuge
tubes and incubated at 37°C for 6 h (Venkatakrishnan et al., 2000c). Dialysates, along with calibration standards, were mixed with microsomal protein at
a final concentration of 1.5 mg/ml in 0.4 ml.
Norfluoxetine and fluvoxamine, at concentrations of 25 and 50 ␮M, respectively, were mixed with human liver microsomes (0.25–3.0 mg/ml) in a
volume of 0.15 ml, and dialyzed versus 0.15 ml of phosphate buffer (100 mM,
pH 7.4) in a 96-well equilibrium dialysis apparatus at 37°C for 6 h. Hydroxyitraconazole and ritonavir, at concentrations of 1.0 ␮M in human liver microsomes (0.25–3.0 mg/ml), were dialyzed under the same conditions using a
Spectra-Por dialysis apparatus (Spectrum, Los Angeles, CA) for 5.5 h at 37°C,
using #4 membranes prepared as per instructions from the manufacturer. After
the dialysis period, the samples were removed from the apparati and stored at
⫺20°C until analysis. Drugs in the microsomal matrix were incubated at 37oC
1443
CYP3A INHIBITOR BINDING
where IC50 is the 50% inhibitory concentration for unbound inhibitor. For
noncompetitive inhibition, IC50 ⫽ Ki, where Ki is the unbound inhibition
constant. For competitive inhibition, IC50 ⫽ Ki (1 ⫹ S/Km), where S is the
concentration of substrate (diazepam, 100 ␮M), and Km is the Michaelis
constant for the conversion of diazepam into temazepam. The possibility that
diazepam 3-hydroxylation may follow an atypical sigmoidal kinetic profile
(Andersson et al., 1994) was not considered in this scheme.
Iu can be calculated from the added (free plus bound) inhibitor concentration
(Ia) and the inhibitor free fraction (FF) in the microsomal mixture, as follows:
I u ⫽ Ia 䡠 FF
Results
(3)
Using standard ligand binding relationships, FF can be expressed as follows:
FF ⫽
1
1⫹K䡠P
(4)
where P is the total concentration of available binding sites, and K is a constant
representing the average affinity of the inhibitor for the binding sites. Combining eqs. 2, 3, and 4 yields
Rv ⫽
IC50
IC50 ⫹ Ia/共1 ⫹ K 䡠 P)
(5)
This equation was fitted to data points (P and Rv) using nonlinear regression
to determine values of IC50 for each inhibitor. For equilibrium dialysis studies,
free fraction was calculated as the ratio of inhibitor concentration in the
dialysate divided by the concentration in the retentate.
Discussion
Consistent with previous reports (Gibbs et al., 1999a), the CYP3A
inhibitory effect of ketoconazole was substantially reduced with increasing concentrations of active microsomal protein. Because some
substrate depletion also occurred at higher protein concentrations, the
FIG. 1. Mean (⫾S.E.) remaining fraction of initial diazepam concentration in
relation to active microsomal protein concentrations for control (inhibitor-free)
incubations.
TABLE 1
Effect of active microsomal protein concentration on inhibitory effect of CYP3A inhibitors, based on diazepam 3-hydroxylation as an index reaction
Table entries (Rv values) are defined from eq. 1 in text.
Mean (⫾ S.E.) Fraction (Rv) of Control (Uninhibited) Diazepam 3-Hydroxylation Velocity at Protein Concentration
Inhibitor (␮M)
Free IC50*
0.0625
0.125
0.25
0.5
0.21 ⫾ 0.09
0.24 ⫾ 0.04
0.32 ⫾ 0.05
0.23 ⫾ 0.02
0.26 ⫾ 0.03
0.49 ⫾ 0.05
0.33 ⫾ 0.02
0.27 ⫾ 0.12
0.21 ⫾ 0.05
0.37 ⫾ 0.10
0.23 ⫾ 0.02
0.18 ⫾ 0.01
0.48 ⫾ 0.08
0.36 ⫾ 0.03
0.45 ⫾ 0.11
0.44 ⫾ 0.14
0.37 ⫾ 0.06
0.29 ⫾ 0.02
0.32 ⫾ 0.03
0.48 ⫾ 0.06
0.37 ⫾ 0.06
0.61 ⫾ 0.13
0.53 ⫾ 0.11
0.45 ⫾ 0.06
0.32 ⫾ 0.02
0.40 ⫾ 0.04
0.50 ⫾ 0.01
0.44 ⫾ 0.07
0.75
1.0
2.0
3.0
0.67 ⫾ 0.09
0.61 ⫾ 0.09
0.55 ⫾ 0.04
0.43 ⫾ 0.04
0.47 ⫾ 0.04
0.58 ⫾ 0.06
0.51 ⫾ 0.06
0.72 ⫾ 0.08
0.70 ⫾ 0.08
0.59 ⫾ 0.06
0.49 ⫾ 0.04
0.57 ⫾ 0.04
0.69 ⫾ 0.05
0.51 ⫾ 0.07
0.86 ⫾ 0.05
0.83 ⫾ 0.06
0.76 ⫾ 0.07
0.63 ⫾ 0.06
0.73 ⫾ 0.01
0.75 ⫾ 0.03
0.52 ⫾ 0.04
0.92 ⫾ 0.04
0.89 ⫾ 0.03
0.79 ⫾ 0.05
0.82 ⫾ 0.03
0.83 ⫾ 0.03
0.83 ⫾ 0.05
0.54 ⫾ 0.06
␮M
mg/ml
Ritonavir (0.05)
Ketoconazole (0.05)
OH-Itraconazole (0.25)
Itraconazole (1.0)
Norfluoxetine (25)
Fluvoxamine (50)
Fluconazole (10)
* Parameter estimate ⫾ standard error of estimate from nonlinear regression, based on eq. 5 in text.
0.0035 ⫾ 0.0016
0.0059 ⫾ 0.0028
0.095 ⫾ 0.01
0.19 ⫾ 0.05
4.3 ⫾ 1.3
37.4 ⫾ 4.7
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CYP3A Inhibition Studies: Effect of Active Protein Concentration. Temazepam formation in the absence of inhibitors increased as
microsomal protein concentration increased to 3 mg/ml. This was
accompanied by some degree of substrate consumption, based on GC
analysis of diazepam remaining in the reaction mixture (Fig. 1). The
inhibitory capacity of the various CYP3A inhibitors declined as
microsomal protein concentration increased (Table 1). At low concentrations of microsomal protein (0.0625 mg/ml), inhibitors reduced
temazepam formation rate to 23 to 49% of control. At the highest
concentration of microsomal protein (3 mg/ml), these inhibitors reduced metabolite formation to no less than 80% of control velocity.
The only exception was 10 ␮M fluconazole, for which inhibitory
capacity was less dependent on protein concentration.
The relation of Rv to total microsomal protein concentration was
consistent with eq. 5 for ritonavir (r2 ⫽ 0.995), ketoconazole (r2 ⫽
0.979), fluvoxamine (r2 ⫽ 0.928), norfluoxetine (r2 ⫽ 0.968), itraconazole (r2 ⫽ 0.959), and OH-itraconazole (r2 ⫽ 0.982), but not for
fluconazole (r2 ⫽ 0.74) (Fig. 2). Estimated unbound IC50 values are
shown in Table 1.
Inhibitor Metabolism Studies. No evidence of ketoconazole consumption was observed with increasing microsomal protein concentration. Ketoconazole concentrations remaining in incubates containing no microsomal protein were essentially identical to those in
incubates containing protein, regardless of the actual protein concentration. The same relationship was obtained with coaddition of diazepam to the incubation mixtures.
Inactive Protein Studies. Temazepam formation in the presence of
a constant active protein concentration (0.25 mg/ml) decreased with
increasing concentrations heat-inactivated microsomal protein (0 –
2.75 mg/ml). The inhibitory capacity of 0.05 ␮M ketoconazole and
0.25 ␮M OH-itraconazole also declined with increasing inactive protein concentration (Table 2; Fig. 3); however, the effect was less
pronounced than that in the active protein study. Fluconazole, on the
other hand, showed little change in its inhibitory capacity with increasing inactive protein concentration.
Equilibrium Dialysis Studies. The free fraction of all inhibitors
except fluconazole declined as the microsomal protein concentration
increased (Fig. 4). The free fraction for fluconazole, however, was
close to 1.0 regardless of microsomal protein concentration.
1444
TRAN ET AL.
effect of protein concentration on ketoconazole inhibition was, if
anything, underestimated. Metabolic consumption of ketoconazole in
the microsomal system was evaluated as a possible contributor to the
decrement in inhibitory action of ketoconazole. Erve et al. (2000)
reported substantial metabolic consumption of 1 ␮M ketoconazole by
liver microsomes at a protein concentration of 2 mg/ml and an
incubation duration of 30 min. However, we did not observe evidence
of ketoconazole consumption by microsomes at 5 ␮M ketoconazole,
protein concentrations up to 3 mg/ml, and an incubation duration of 10
min. In contrast to ketoconazole, inhibition by fluconazole was minimally dependent on microsomal protein concentration. The differences between ketoconazole and fluconazole apparently are explained
by their differing affinity for nonspecific binding sites. Equilibrium
dialysis demonstrated decreased ketoconazole free fraction with increasing microsomal protein concentration, whereas fluconazole free
FIG. 2. Fractional decrement in reaction velocity relative to inhibitor-free
control (y-axis) in relation to active microsomal protein concentration (x-axis)
for representative CYP3A inhibitors.
Data points are mean values from Table 1. For ritonavir, ketoconazole, and
fluvoxamine, data points are consistent with eq. 5; lines represent functions of best
fit, with unbound IC50 values shown in Table 1. Data points for fluconazole were not
consistent with eq. 5.
FIG. 3. Mean (⫾ S.E.) fractional decrement in reaction velocity relative to
inhibitor-free control (y-axis) in relation to inactive microsomal protein
concentrations (x-axis).
Active protein concentration was fixed at 0.25 mg/ml.
TABLE 2
Effect of inactive microsomal protein concentration on inhibitory effect of CYP3A inhibitors at a constant active protein concentration (0.25 mg/ml)
Table entries (Rv values) are defined from eq. 1 in text.
Inhibitor (␮M)
Mean (⫾ S.E.) Fraction (Rv) of Control (Uninhibited) Diazepam 3-Hydroxylation Velocity at Inactive Protein Concentration
0
0.25
0.75
1.75
2.75
0.56 ⫾ 0.04
0.54 ⫾ 0.04
0.47 ⫾ 0.08
0.67 ⫾ 0.04
0.51 ⫾ 0.03
0.49 ⫾ 0.08
mg/ml
Ketoconazole (0.05)
OH-Itraconazole (0.25)
Fluconazole (10)
0.38 ⫾ 0.04
0.33 ⫾ 0.05
0.42 ⫾ 0.04
0.43 ⫾ 0.06
0.37 ⫾ 0.06
0.44 ⫾ 0.04
0.51 ⫾ 0.05
0.43 ⫾ 0.07
0.48 ⫾ 0.05
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fraction was close to 1.0 regardless of microsomal protein concentration.
A number of other established CYP3A inhibitors, including the
azole derivative itraconazole (and its principal metabolite), the selective serotonin reuptake inhibitors norfluoxetine and fluvoxamine, and
the viral protease inhibitor ritonavir, exhibited decreases in inhibitory
potency with increasing active microsomal protein in a manner similar
to that observed with ketoconazole. All of these compounds are
lipophilic agents with moderate-to-extensive binding to human
plasma proteins, and equilibrium dialysis studies indicated that microsomal binding in vitro apparently explains the decrement in inhibitory activity with increasing microsomal protein. The magnitude and
rank order of estimated unbound IC50 values for these agents parallel
previously reported data (Venkatakrishnan et al., 2000b, 2001b; von
Moltke et al., 1994b, 1995, 1996a,b, 1998a,b). We did not evaluate
actual inhibitor consumption by microsomes for these other inhibitors.
Previous studies using diazepam 3-hydroxylation as an index reaction have demonstrated the functional CYP3A content of liver samples used in the present study to be approximately 125 pmol/mg
protein (Venkatakrishnan et al., 2001a). At 0.05 ␮M (50 pmol/ml)
ketoconazole and 0.5 mg/ml active protein (63 pmol/ml CYP3A),
diazepam 3-hydroxylation activity was reduced to approximately 50%
of control, implying occupancy by ketoconazole of a corresponding
fraction of CYP3A binding sites (Table 1). Under the assumption of
a single diazepam active site per molecule of CYP3A, 32 pmol/ml
ketoconazole is anticipated to be bound to active sites, with the
remaining 18 pmol/ml either unbound or bound to other sites. At
CYP3A INHIBITOR BINDING
higher concentrations of microsomal protein, available active binding
sites would exceed the availability of ketoconazole in the in vitro
mixture, and the decrement in inhibitory capacity could be explained
mainly by specific microsomal binding. Similar estimations would
hold for ritonavir. However, for the less potent inhibitors norfluoxetine and fluvoxamine, 50% inhibition is achieved at concentrations
that greatly exceed the availability of specific binding sites, and the
decrement in inhibition would be explained mainly by nonspecific
binding.
The present study indicates that microsomal binding may have a
major impact on estimation of inhibitory potency using in vitro
systems. Decrements in inhibitory activity with increasing microsomal protein concentration may be observed with strong as well as
weak inhibitors. The mechanism of binding may be either specific,
nonspecific, or a combination of the two. It is likely that the decrease
in inhibitory potency observed for relatively weaker inhibitors at high
microsomal concentrations is attributable mainly to nonspecific binding. The findings suggest that large variability among laboratories in
estimated inhibitory potency of a single inhibitor versus a single
substrate (such as ketoconazole versus midazolam) could in part be
explained by variations in protein and/or enzyme concentration (Gascon and Dayer, 1991; Hargreaves et al., 1994; Wrighton and Ring,
1994; Ghosal et al., 1996; von Moltke et al., 1996b; Gibbs et al.,
1999a,b; Wang et al., 1999; Perloff et al., 2000; Venkatakrishnan et
al., 2000b). In general, the impact of microsomal binding, both specific and nonspecific, may be minimized by use of the lowest possible
microsomal concentration.
Acknowledgments. This work was supported by Grants
MH-58435, DA-13209, DK/AI-58496, DA-05258, DA-13834, AG17880, MH-34223, MH-01237, and RR-00054 from the Department
of Health and Human Services.
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FIG. 4. Free fraction of six CYP3A inhibitors in relation to microsomal protein
concentration.
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