Article Figures & Data
Figures
- Figure 1
Multisite kinetic equilibria models.
Kinetic model for an enzyme with two-substrate binding sites, the second substrate (S) molecule binds cooperatively K (A). Overall scheme for simultaneous metabolism of the S and modifier (M) in the active site upon binding to two sites (B). Three-site kinetic model for an enzyme where S binds cooperatively in the presence or absence of I at a distinct site (C). Three-site kinetic model with two distinct substrate-binding sites and pathway-differential effect of a modifier (D).
- Figure 2
Effect of haloperidol on various CYP3A4 substrates in lymphoblast-expressed CYP3A4.
NIF (10–200 μM) (A), TST (25–500 μM) (B), MDZ (2–50 μM) (C), and FEL (10–50 μM) (D).
- Figure 3
Effect of quinidine on various CYP3A4 substrates in lymphoblast-expressed CYP3A4.
Substrate concentrations: NIF (10–200 μM) (A), TST (25–200 μM) (B), MDZ (2–50 μM) (C), and FEL (10–50 μM) (D).
- Figure 4
Eadie-Hofstee plots for OX NIF and 6β-HTS formation in the presence of QUI and HAL, respectively.
NIF has substrate inhibition kinetic properties (A). TST has sigmoidal kinetics (B) the same as Fig. 2.
- Figure 5
Effect of QUI on 6β-HTS formation.
Kinetic profiles for 6β-HTS formation at QUI concentrations of 0 μM (▴), 5 μM (■), 20 μM (●), 50 μM (▵), and 100 μM (♦); the solid and dashed lines represent the simultaneous fit to the eq. 4at 10, 25, 50, 100, and 200 μM TST concentration (A) and the corresponding clearance plots (B).
- Figure 6
Differential effect of QUI on MDZ 1′- and 4-hydroxylation.
- Figure 7
Effect of QUI on 1′-OH MDZ formation.
Kinetic profiles for 1′-OH MDZ formation at QUI concentrations of 0 μM (▵), 3 μM (▪), and 30 μM (●); the solid and dashed lines represent the simultaneous fit to the eq. 6 at 2.5, 5, 10, 15, 50, and 100 μM MDZ concentration. Data points represent the mean of duplicate determinations.
Tables
CYP3A4 Substrate Protein Concentration Incubation Time Substrate Concentration Assay mg/ml min μM MDZ 1.0 2.5 2, 5, 10 (HAL), 50 Sapelcosil LC-ABZ, 5 μM, 15 cm × 4.6 mm; 65% 0.025 M ammonium acetate (pH 5):35% acetonitrile, flow 1.0 ml/min; HPLC/UV-240 nm; retention times: 4-OH MDZ, 7 min; 1′-OH MDZ, 8 min; MDZ, 12 min; LOQ = 0.025 nmol, interassay CV < 5% FEL 1.0 10 10, 25, 50 Extraction by ethyl acetate; Spherisorb 5 ODS2, 15 cm × 4.6 mm; 0.2% TMED (N,N,N′,N′-tetramethylethylenediamine) in water, pH 7.0):methanol (30:70, v/v); flow 1.0 ml/min, HPLC/UV, 235 nm; retention times: FEL-PYR, 7 min; FEL, 9 min; UK-58,790, 12 min; LOQ = 0.05 nmol, interassay CV < 5% NIF 0.5 15 10, 20, 50, 200 Lightning C18 (33 mm × 3 mm, 4 μm); 61% acetonitrile:39% ammonium acetate (10 mM, pH 8); flow 0.7 ml/min; MS:positive ion MRM with Turbo ionspray interface (480°C); transitions: 345 → 284 (OX NIF); 480 → 315 (nicardipine, internal standard); LOQ = 0.0125 nmol, interassay CV < 5% TST 0.5 15 25, 50, 100, 200, 500 (HAL) Waters C18 Novapak (150 × 3.9 mm); 50% methanol:50% distilled water; flow 1.0 ml/min, HPLC/UV, 245 nm; retention times: 6β-HTS, 5 min; TST, 26 min; LOQ, 0.0125 nmol, interassay CV < 5% SV 0.5 10 5, 10, 25, 50 ACE 5 C18 (10 cm × 4.6 mm); 85% 50 mM ammonium acetate:15% acetonitrile, gradient column temperature: 40°C, flow 1.0 ml/min; MS, Micromass LCT (time of flight mass spectrometer), positive ion full scan MS; LOQ = 0.01 nmol, interassay CV < 5% - Table 2
Kinetic parameters for various CYP3A4 substrates in human lymphoblast-expressed CYP3A4 (mean ± S.E.)
CYP3A4 Substrate Michaelis-Menten/Hill Equation Two-Site Model Km/S50 Vmax CLint/CLmax Ks Vmax α β μM pmol/min/pmol P450 μl/min/pmol P450 μM pmol/min/pmol P450 TST 6β-hydroxylation2-a 42.7 ± 0.8 16.5 ± 0.2 0.22 93.5 ± 4.8 16.5 ± 0.2 0.15 ± 0.02 NIF oxidation2-b 20.9 ± 2.2 9.7 ± 0.7 0.46 33.4 ± 4.9 12.8 ± 1.5 0.36 ± 0.08 FEL oxidation 26.4 ± 2.4 5.94 ± 0.23 0.22 SV 3′-hydroxylation 35.1 ± 3.4 13.4 ± 0.52-c MDZ 1′-hydroxylation 3.5 ± 0.5 4.8 ± 0.2 1.37 - Table 3
Kinetic parameters for the in vitro interaction of haloperidol and various CYP3A4 substrates in human recombinant CYP3A4 generated by multisite kinetic model approach (mean ± S.E.)
CYP3A4 substrate Vmax Ks Ki α γ δ pmol/min/pmol P450 μM NIF3-a 13.3 ± 1.3 34.9 ± 3.9 0.25 ± 0.13 0.32 ± 0.04 0.08 ± 0.03 FELb 3.54 ± 0.31 40.9 ± 8.7 3.4 ± 0.9 0.95 ± 0.29 0.58 ± 0.19 TST3-c 8.41 ± 0.19 243 ± 17 34 ± 13 0.05 ± 0.02 8.2 ± 4.1 - Table 4
Kinetic parameters for the in vitro interaction of quinidine and various CYP3A4 substrates in human recombinant CYP3A4 generated by multisite kinetic model approach (mean ± S.E.)
CYP3A4 Substrate Vmax Ks Ki Ka α γ δ pmol/min/pmol P450 μM NIF4-a 7.25 ± 1.24 16.7 ± 5.4 5.3 ± 2.4 0.81 ± 0.25 0.16 ± 0.03 TSTb 8.27 ± 0.17 260 ± 35 99 ± 7 0.03 ± 0.01 MDZc 10.5 ± 2.1 14.5 ± 1.8 14.9 ± 2.7 1.8 ± 0.2 0.31 ± 0.11 FELd 5.44 ± 0.18 52.8 ± 4.4 288 ± 75 9.8 ± 3.6 0.08 ± 0.04 SVd 11.9 ± 0.6e 80 ± 10 226 ± 65 2.6 ± 0.8 0.16 ± 0.05 -
The kinetic parameters were determined using two-site model:
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↵4-a eq. 3, β (SES) = 0.45 ± 0.11 anddeq. 2; the kinetic parameters were determined using the three-site model: beq. 4 and ceq. 6 β (Kp1 from S2ES1) = 0.29 ± 0.08, Ks2 (4-OH MDZ site) = 22.6 ± 3.1 μM; earbitrary number due to the lack of metabolite standards.
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- Table 5
Multisite kinetic model interaction factors to describe the various modifications of CYP3A4 activity
Effect on CYP3A4 Interaction Factor Example α γ5-150 δ Activation, S showing hyperbolic kinetics 1 >1 <1 QUI effect on FEL, SV QUI effect on diclofenac5-a, warfarinb Inhibition, S showing hyperbolic kinetics 1 <1 <1 HAL effect on FEL, QUIc Inhibition, S showing substrate inhibition 1 <1 <1 QUI, HAL effect on NIF (β < 1, could = γ) MDZ, FEL effect on NIFc Partial inhibition, S showing hyperbolic kinetics 1 1 >1 NIF effect on FELc TST-MDZc e, TST-erythromycind, TST-Terfenadinee Partial inhibition, S showing sigmoidal kinetics5-g <1 1 >1 HAL effect on TST Heterotropic activation, S showing sigmoidal kinetics5-g <1 >1 =α TST effect on DZf Heterotropic inhibition, S showing sigmoidal kinetics5-g <1 1 ≤1 QUI effect on TST, DZ effect on TSTf
