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 K_i 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.

	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).

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 V_max, S₅₀, and Hill coefficient (n) were calculated from untransformed data using Hill equation. CL_max was calculated in case of TST as an alternative to CL_int (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.

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:

(1)

Autoactivation might be a result of either increased binding affinity for a second substrate molecule (K_s changes by the factor  < 1), or changes in the effective catalytic rate constant (K_p) 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).

View larger version (27K): [in this window] [in a new window]   Fig. 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).

Inhibition/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 K_s and V_max 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 V_max is equivalent to 2K_p[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.

(2)

An identical model can be applied for activation; the only difference is the substitution of the inhibition terms (I and K_i) with the ones for activation (A and K_a).

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, V_max is equivalent to K_p[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.

(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 V_max is equal to 2K_p[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 V_max, rather than changes in the substrate-binding constant K_s.

(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 K_i 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.

(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 K_s1 and K_p1) and S₂E for 4-OH MDZ formation (K_s2 and K_p2), 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 (K_p1 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 V_max 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 K_i 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 K_m). 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, K_p2 > K_p2). Pathway differential effects observed for QUI were similar to the differential effects of -naphthoflavone on losartan metabolism, reported by Shou et al. (2001a).

(6)

(7)

	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 K_m  2K_m) 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).

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).

View larger version (25K): [in this window] [in a new window]   Fig. 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).

View larger version (22K): [in this window] [in a new window]   Fig. 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).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Eadie-Hofstee plots for OX NIF and 6-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.

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.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of QUI on 6-HTS formation. Kinetic profiles for 6-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. 4 at 10, 25, 50, 100, and 200 µM TST concentration (A) and the corresponding clearance plots (B).

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.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Differential effect of QUI on MDZ 1'- and 4-hydroxylation.

View larger version (17K): [in this window] [in a new window]   Fig. 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.

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).

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 K_a 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).

	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 K_i 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 K_i 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.

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 K_i 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).