Introduction

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Cytochrome P450 (P450¹) enzymes are a superfamily of enzymes responsible for metabolizing a wide range of endogenous and xenobiotic compounds (Wrighton and Stevens, 1992; Guengerich, 1995). Within this superfamily, CYP2C9 is known to be an important isoform responsible for metabolizing many relevant compounds such as (S)-warfarin (Rettie et al., 1992), phenytoin and tolbutamide (Veronese et al., 1991), as well as many nonsteroidal anti-inflammatory drugs (Miners and Birkett, 1998). Consequently, factors that modulate the activity of this enzyme are of substantial interest to the drug industry, as well as to academic scientists, since the resultant effects may alter a drug's therapeutic efficacy or toxicity.

Our lab has determined that CYP2C9 often does not follow normal Michaelis-Menten kinetics. For example, CYP2C9-mediated flurbiprofen 4'-hydroxylation has been shown to be activated by the effector dapsone in human liver microsomes and baculovirus-expressed CYP2C9 (Korzekwa et al., 1998). In addition, kinetic studies on the activation of flurbiprofen 4'-hydroxylation, as well as naproxen demethylation by dapsone have recently been reported (Hutzler et al., 2001). Data from this kinetic study were fit to a two-site effector model equation that resulted in an increase in the V_max and a decrease in the K_m parameter estimates for the metabolism of flurbiprofen and naproxen in the presence of dapsone. These results suggest that the presence of dapsone did not displace either of these substrates from the CYP2C9 active site. In addition, dapsone is a substrate for CYP2C9 at therapeutic concentrations, whereby it is metabolized to its hydroxylamine metabolite (Gill et al., 1995; Winter et al., 2000), suggesting its presence in the active site during activation and supporting the hypothesis of an effector site being within the active site. However, conclusive evidence for the mechanism of activation of CYP2C9 has yet to be discovered.

Activation of CYP2C9 by dapsone is analogous to that observed with 7,8-benzoflavone, which activates CYP3A4-mediated phenanthrene metabolism, in addition to being a substrate for CYP3A4 (Shou et al., 1994). In this same study, a series of partial or modified structures of 7,8-benzoflavone were tested for their ability to activate or inhibit the metabolism of phenanthrene, chrysene, and benzo[a]pyrene in vitro (Shou et al., 1994) to determine the effector structural features necessary for the activation of CYP3A4. However, similar studies to determine the structural requirements for activation of CYP2C9 have yet to be performed, although many structure-activity studies have been conducted in an effort to better understand substrate and inhibitor binding to CYP2C9 (Korzekwa and Jones, 1993; Mancy et al., 1995; Jones et al., 1996a; Afzelius et al., 2001). To this end, we have explored the modulation of CYP2C9-mediated metabolism by nine analogs of dapsone and assessed their effects on the metabolism kinetics of flurbiprofen and naproxen, two prototypical substrates for CYP2C9. These studies are part of an ongoing investigation into the molecular mechanism of dapsone-mediated CYP2C9 activation.

	Materials and Methods

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Chemicals. Acetonitrile and dibasic potassium phosphate were obtained from Fisher Scientific Co. (Pittsburgh, PA). (S)-Flurbiprofen, 4'-hydroxy flurbiprofen, and 2-fluoro-4-biphenyl acetic acid (internal standard) were gifts from Pharmacia (Kalamazoo, MI). (S)-Naproxen and desmethylnaproxen were gifts from Syntex Laboratories Inc. (Palo Alto, CA). Dapsone, sulfamethoxazole, sulfamethazine, and ketoprofen were purchased from Sigma-Aldrich (St. Louis, MO), and the remaining dapsone analogs were purchased from Aldrich Chemical Co. (Milwaukee, WI). Purity of the effector chemicals ranged from 96 to 99%, as per the manufacturer's packaging information. All other chemicals were obtained from commercial sources and were of the highest purity available.

Expressed CYP2C9 Incubation Conditions. Nine analogs of dapsone were selected for kinetic studies (Fig. 1) including three sulfone compounds [phenylsulfone, p-tolylsulfone and 4-(4-nitrophenylsulfonyl)-aniline (4-NPSA)], three carbonyl compounds (benzophenone, ketoprofen, and dicyclohexylketone), and three sulfonamide compounds (sulfamethoxazole, sulfamethazine, and sulfadiazine). Microsomal preparations resulting from the coexpression of CYP2C9, NADPH oxidoreductase, and cytochrome b₅ (1:2:4 ratio) in BTI-TN-5B1-4 cells, mediated by baculovirus delivery, were used as the enzyme source and were a gift from ArQule, Inc. (Menlo Park, CA). Reactions were conducted according to previously published methods (Hutzler et al., 2001), which established that the conditions were linear with respect to time and protein concentration. Incubation mixtures in a 6 × 6 matrix design (six concentrations of substrate and six concentrations of effector) contained 1 pmol expressed CYP2C9 with either (S)-flurbiprofen (2-300 µM) or (S)-naproxen (10-1800 µM) incubated with dapsone or dapsone analog (0-100 µM) in 50 mM potassium phosphate buffer at pH 7.4. Following a 3-min preincubation, reactions were initiated by addition of 1 mM NADPH for a final volume of 200 µl and allowed to incubate at 37°C for 20 min. Incubations with flurbiprofen were quenched by addition of 200 µl of acetonitrile containing internal standard (180 ng/ml 2-fluoro-4-biphenylacetic acid), followed by addition of 40 µl of half-strength H₃PO₄. Incubations with naproxen were quenched by addition of 200 µl of acetonitrile, followed by addition of 40 µl of half-strength H₃PO_4. Samples from all incubations were centrifuged at 10,000 rpm for 5 min, with aliquots of the supernatant placed into autosampler vials and 10 to 50 µl injected onto an HPLC system. Dapsone and analogs were dissolved in DMSO and added to 200-µl incubations such that the final concentration of DMSO was 2.5%, whereas control incubations also contained 2.5% DMSO. Studies for Dixon plot analysis of flurbiprofen 4'-hydroxylation inhibition by 4-NPSA were as stated above, but 4-NPSA concentrations ranged from 0 to 200 µM, with controls containing the same volume of DMSO.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Structures of dapsone and the nine analogs studied for their effects on flurbiprofen 4'-hydroxylation and naproxen demethylation. Dapsone is the reference compound, as activation of CYP2C9 activity has been observed previously (Korzekwa et al., 1998; Hutzler et al., 2001). The analogs consist of three sulfones with varying para-substituents, three carbonyl compounds to study importance of the sulfone group, and three sulfonamide compounds to study association requirements of the sulfone group.

Analysis of 4'-Hydroxy Flurbiprofen. 4'-Hydroxyflurbiprofen was analyzed by HPLC with fluorescence detection. The HPLC system consisted of a Waters 501 HPLC pump, a Waters 717 autosampler, and a Waters 470 fluorescence detector (Waters Corp., Milford, MA) set at an excitation wavelength of 260 nm and an emission wavelength of 320 nm. The mobile phase consisted of acetonitrile/20 mM K₂HPO₄, pH 3.0 (45:55, v/v) pumped at 1 ml/min through a Brownlee Spheri-5 C₁₈ 4.6 × 100-mm column (PerkinElmer Life Sciences, Boston, MA). 4'-Hydroxy flurbiprofen and internal standard eluted at approximately 2.2 and 4.8 min, respectively.

Analysis of Desmethylnaproxen. Desmethylnaproxen was analyzed identically to 4'-hydroxy flurbiprofen except that the fluorescence detector was set at an excitation wavelength of 230 nm and an emission wavelength of 340 nm, and no internal standard was used. Desmethylnaproxen was eluted at approximately 2.1 min.

Data Analysis and Equations. Parameter estimates were generated following nonlinear regression fitting of the data with Sigma Plot 6.0 containing the Enzyme Kinetics 1.0 module (SPSS, Inc., Chicago, IL), which uses the Marquardt-Levenberg algorithm for minimization of the sum of squares. Kinetic data were fit to a two-site model (Korzekwa et al., 1998),
where S is substrate, B is effector, V_max and K_m are kinetic constants for substrate metabolism, K_B is the binding constant for the effector,  is the change in K_m resulting from effector binding, and  is the change in V_max from effector binding. For activation,  < 1 and/or  > 1. Flurbiprofen metabolism data that exhibited small changes in presence of effector were fit to the Michaelis-Menten equation to estimate effector-related changes on metabolism:
Naproxen metabolism data exhibiting minor changes in presence of effector were fit to the following biphasic kinetic equation to estimate effector-related changes on metabolism:
where Cl_int (V_max2/K_m2) is the intrinsic clearance, assumed to be associated with a second binding region within the active site and represents the linear portion (slope) of the biphasic kinetic curve. In cases where fits to both the Michaelis-Menten (eq. 2) and the biphasic equation (eq. 3) seemed plausible, visual inspection of fits and examination of standard errors and residual sum of squares were used to determine which model equation resulted in the best fit.

	Results

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Analogs for kinetic studies were selected based on their different functional groups and electronic properties of para-substituents compared with dapsone. Experimental data were initially fit to a two-site effector model equation (eq. 1). As noted previously (Hutzler et al., 2001), data showing activation of flurbiprofen 4'-hydroxylation by dapsone fits well to this model (Fig. 2), with an  = 0.31, and a  = 1.73, indicating a decrease in K_m and increase in V_max, respectively (Table 1). Similarly, phenylsulfone activated flurbiprofen 4'-hydroxylation (Fig. 3), with an  = 0.42 and  = 2.80 (Table 1). Interestingly, 4-NPSA, which contains a nitro group in the place of one amino group of dapsone (Fig. 1), acts as an inhibitor of flurbiprofen 4'-hydroxylation (Fig. 4), decreasing V_max with minimal effect on K_m (Table 2). In a separate experiment examining inhibition of flurbiprofen 4'-hydroxylation by 4-NPSA, data were best fit to a noncompetitive inhibition model resulting in a K_i estimate of 320 ± 45 µM (Fig. 5). Analogs for which kinetic parameters were not estimated from eq. 1, due to lack of effect on flurbiprofen 4'-hydroxylation, included p-tolylsulfone, as well as all three carbonyl analogs (benzophenone, ketoprofen, and dicyclohexylketone), and sulfonamide analogs (sulfamethoxazole, sulfadiazine, and sulfamethazine).

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Three-dimensional surface plot showing activation of flurbiprofen 4'-hydroxylation by 0 to 100 µM dapsone, with maximal activation observed at 100 µM dapsone. The surface is the fit to a two-site binding model (eq. 1) and the data were adapted from Hutzler et al. (2001). Kinetic parameters for the fit can be observed in Table 1.

View larger version (67K): [in this window] [in a new window]   Fig. 3.   Three-dimensional surface plot showing activation of flurbiprofen 4'-hydroxylation by 0 to 100 µM phenylsulfone. The surface is the fit to a two-site binding model (eq. 1). Kinetic parameters for the fit are listed in Table 1.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Inhibition of flurbiprofen 4'-hydroxylation by 0 to 100 µM 4-NPSA. Fits were to the Michaelis-Menten model (eq. 2), and parameter estimates are listed in Table 2. Parameter estimates suggest that 4-NPSA causes a decrease in Vmax, with minimal effect on Km, suggestive of noncompetitive inhibition.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Dixon plot showing inhibition of flurbiprofen 4'-hydroxylation by 0 to 200 µM 4-(4-nitrophenylsulfonyl)-aniline, with a Ki ~ 320 µM. Intersection of linear regression lines roughly on the x-axis suggests a noncompetitive type inhibition (Segal, 1975).

Naproxen demethylation in the presence of dapsone was described by the two-site effector model (Hutzler et al., 2001) (Fig. 6), with an  = 0.09 and  = 2.40 (Table 1), indicating a substantially greater decrease in K_m and increase in V_max (activation) compared with flurbiprofen. No other analog produced activation comparable to dapsone, precluding parameter estimation via the two-site effector model equation (eq. 1). p-Tolylsulfone activated naproxen demethylation at high concentrations (50 and 100 µM) (Fig. 7) but not at lower effector concentrations (data not shown). The effects on V_max and K_m are presented in Table 3. In addition, the kinetic profile for naproxen demethylation was changed to a more hyperbolic function in the presence of high concentrations of p-tolylsulfone, resulting in a decrease in the Cl_int parameter (Table 3). Sulfamethazine also activated naproxen demethylation at high concentrations, albeit very slightly (~10%, data not shown). Meanwhile, phenylsulfone inhibited naproxen demethylation (Fig. 8), an effect opposite to that observed with flurbiprofen 4'-hydroxylation, with the Cl_int parameter (Table 4) again being lowered in the presence of phenylsulfone, indicating a decrease in slope of the linear portion of the biphasic curve. In addition, 4-NPSA minimally affected naproxen demethylation (~20-25% inhibition, data not shown), less than observed with flurbiprofen. Analogs for which no measurable effect on naproxen demethylation by these effectors was observed at any concentration included benzophenone, ketoprofen, dicyclohexylketone, sulfamethoxazole, and sulfadiazine.

View larger version (65K): [in this window] [in a new window]   Fig. 6.   Three-dimensional surface plot showing activation of naproxen demethylation by 0 to 100 µM dapsone. The surface is the fit to a two-site binding model (eq. 1), and the data were adapted from Hutzler et al. (2001). Kinetic parameters for the fit can be observed in Table 1.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Activation of naproxen demethylation by high concentrations of p-tolylsulfone. Fits were to a biphasic kinetic equation (eq. 3), with parameter estimates reported in Table 3. Note the more hyperbolic nature of the kinetic profiles for naproxen demethylation in the presence of p-tolylsulfone.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Inhibition of naproxen demethylation by 0 to 100 µM phenylsulfone. Fits were to a biphasic kinetic equation (eq. 3), with parameter estimates reported in Table 4. Note the change in profile to a more hyperbolic function in presence of 100 µM phenylsulfone.

	Discussion

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To further explore the molecular mechanism of CYP2C9 activation by dapsone, nine analogs of dapsone were selected for kinetic studies to determine some of the effector structural determinants for activation of CYP2C9. Results suggest that the level of activation is substrate-dependent, and that an effector containing a sulfone moiety in direct association with two benzene rings with strong electron donating groups (i.e., amino group) is the most consistent and potent activator of CYP2C9 activity. In addition, replacement of an amino group with a nitro group results in inhibition of CYP2C9-mediated flurbiprofen 4'-hydroxylation. Lastly, activation of naproxen demethylation was equally as restrictive as flurbiprofen 4'-hydroxylation, as only dapsone was able to consistently activate this metabolic event, although p-tolylsulfone activated at high concentrations.

In terms of P450 enzyme activation, CYP3A4 has been the most studied isoform. A number of investigators have shown that purified CYP3A4 enzymes are directly activated by 7,8-benzoflavone (Schwab et al., 1988; Shou et al., 1994). This is particularly interesting since 7,8-benzoflavone is a substrate for the enzyme that it activates, analogous to dapsone and CYP2C9 (Korzekwa et al., 1998; Hutzler et al., 2001), suggesting similar activation characteristics at the molecular level between these isoforms. In addition, Shou et al. (1994) have explored the structural requirements for activation of CYP3A4 by studying the effects of structural analogs of 7,8-benzoflavone on CYP3A4 activity. The current study was performed in an attempt to explore the effect of different functional groups on the susceptibility of CYP2C9 to activation.

Results suggest that phenylsulfone activates flurbiprofen metabolism to a degree comparable to dapsone, with data accurately described by the two-site effector model (Fig. 3). From these data, it is apparent that the amino groups are not an absolute requirement for activation of flurbiprofen 4'-hydroxylation. Data for phenylsulfone, as well as the other analogs, were initially fit to eq. 1, with the hypothesis that the active site of CYP2C9 has multiple binding regions within its active site, one being an effector binding site. However, the possibility also exists that some of the analogs may elicit their effects by means other than binding to an effector site (resulting in effector dependent effects). In addition, examples were noted in which increasing concentrations of effector had small but variable effects on the substrate's metabolism (data not shown), similar to the findings of Wang et al. (2000) with midazolam and terfenadine. In the current study, these mixed effects resulted in an inability to adequately fit some of the data to the two-site effector model (eq. 1). As a result, data were also fit to either the Michaelis-Menten (eq. 2) or a biphasic kinetic equation (eq. 3) to examine effector-related changes in CYP2C9 activity. For example, fits to the biphasic equation (eq. 3) showed that p-tolylsulfone activated naproxen demethylation at high concentrations (Fig. 7), changing the profile to a more hyperbolic function, and suggesting a lower affinity of this analog for the effector site. However, replacement of the sulfone group with a carbonyl (benzophenone, ketoprofen, and dicyclohexylketone) resulted in elimination of activation properties regardless of the substrate, suggesting that the sulfone group contributes to recognition and binding to an effector site region that results in activation of CYP2C9. In addition, other analogs with the sulfone moiety connected to different aromatic heterocyclic rings through a nitrogen (sulfonamide analogs) generally resulted in no effect, arguing for the direct association of a sulfone group with two benzene rings for activation. Kinetic studies further suggest that phenylsulfone acts as an inhibitor of naproxen demethylation (Fig. 8), while reducing the slope of the linear portion of the biphasic curve. Similar to p-tolylsulfone, this suggests that the presence of effector causes the kinetic profile for naproxen demethylation to become more hyperbolic. It has been proposed that naproxen binds to CYP2C9 in a different orientation than flurbiprofen (Hutzler et al., 2001), which may explain the differential effects on flurbiprofen and naproxen metabolism. In fact, it has been suggested that the biphasic kinetics observed for naproxen metabolism are due to multiple naproxen binding orientations (i.e., two binding sites), each of which may be operable for metabolism (Korzekwa et al., 1998; Hutzler et al., 2001). Therefore, effector binding to one of the sites may render only one site operable for naproxen metabolism, resulting in the more hyperbolic profile.

One of the more intriguing analogs studied was 4-NPSA, which inhibited flurbiprofen 4'-hydroxylation. The similarity in structure of 4-NPSA compared to dapsone, and yet the substantial differences in effect on CYP2C9-mediated metabolism are striking. Likely explanations for these observations include steric and electronic effects of the nitro group compared with the amino group. The nitro group is slightly larger than the amino group, which may reduce the active site volume, possibly resulting in inhibition by blocking flurbiprofen access to the reactive oxygen. The nitro group also has electron-withdrawing effects on the benzene ring, resulting in a high electron density on the nitro group. This is in opposition to the electron donating effects of the amino groups of dapsone, suggesting that there may be an electronic component involved in the activation mechanism of CYP2C9. Studies have suggested that the active site of CYP2C9 has positive residues that may interact with negatively charged groups on substrates (Mancy et al., 1995; Jones et al., 1996b; He et al., 1999; Ridderstrom et al., 2000). In addition, the positive arginine residues at positions 97 and 105 have been shown to be important in the catalytic function of CYP2C9 (Ridderstrom et al., 2000). It is reasonable then to suggest that some type of electrostatic interaction between the nitro group of 4-NPSA and one of these active site residues may orient it in such a way as to cause inhibition of metabolism rather than activation. However, Dixon plot analysis for flurbiprofen 4'-hydroxylation (Fig. 5) suggests that 4-NPSA (K_I, ~320 µM) is acting in a noncompetitive manner (linear regression lines intersect on x-axis) (Segal, 1975), for which the binding of substrate and inhibitor is not mutually exclusive (Thummel et al., 2000). Noncompetitive inhibition is confirmed in that the V_max for flurbiprofen 4'-hydroxylation is reduced with the K_m being unaffected (Table 2) (Segal, 1975). This suggests that both 4-NPSA and flurbiprofen may be in the active site together, with 4-NPSA perhaps either interacting with heme or in some way reducing efficient electron flow to the P450, thereby decreasing efficient coupling of reducing equivalents to product formation. However, stoichiometry studies would be required to confirm this hypothesis. On the other hand, 4-NPSA minimally decreased naproxen demethylation, supporting the hypothesis that naproxen has a different binding orientation than flurbiprofen or multiple binding orientations. Additional interesting observations are the effects of p-tolylsulfone (Fig. 7) and sulfamethazine (data not shown). At higher concentrations, each of these compounds had a minimal effect on the K_m of naproxen demethylation, while increasing V_max (Table 3). Potential reasons for the direct effect of p-tolylsulfone and sulfamethazine on V_max, with minimal effect on K_m, are numerous. One possibility may be alteration of uncoupling mechanisms (e.g., formation of superoxide, hydrogen peroxide, or an additional molecule of water, instead of substrate oxidation) by the effector. A decrease in hydrogen peroxide formation due to exclusion of water molecules from the active site by certain compounds has been noted, resulting in enhanced catalytic activity (Hanioka et al., 1992). It is also possible that the analog substituents may differentially affect hydrogen bonding and water orientation within the active site, which has been suggested to influence the conformation of the enzyme (Ekins et al., 1998) and thus activity. Also, the effector may enhance the interaction of P450 with cytochrome b₅, which has been shown to control uncoupling mechanisms, thereby increasing the concentration of active oxygen species for catalysis (Perret and Pompon, 1998).

Overall, results for activation of CYP2C9 suggest that specific activator-protein interactions are likely involved in the recognition and binding of these effector molecules, as even small deviations from the parent structure of dapsone resulted in changes in direction and degree of effect. The direction of effect could not be predicted by correlation with simple chemical properties such as pK_a or lipophilicity (data not shown), again suggesting the role of specific activator-protein interactions. It must be realized that the presented kinetic evidence does not establish whether the effector site lies within or separate from the active site, although many models have been proposed. Harlow and Halpert suggest that effector binding is most likely in the active site along with the substrate, as they were able to create an active site double mutant of CYP3A4 that exhibited reduced positive cooperativity in the presence of 7,8-benzoflavone (Harlow and Halpert, 1998). Another model proposed by Shou and Korzekwa suggests that both substrate and effector are bound in the active site simultaneously, each having a distinctive binding region (Shou et al., 1994; Korzekwa et al., 1998). Recently, we have shown that this two-site binding model applies to CYP2C9 (Hutzler et al., 2001). However, there remains the possibility of enzyme conformational changes induced by effector binding. Therefore, perhaps the most reasonable explanation for the cause of activation is one in which multiple binding sites and conformational changes are simultaneously considered, as suggested by Atkins et al. (2001), along with uncoupling effects.

In conclusion, it is apparent from our data that structural requirements for activation of CYP2C9 are substrate and effector concentration-dependent, complicating the prediction of this type of interaction in vitro. However, drug discovery scientists should be cognizant of the potential for compounds structurally analogous to dapsone (i.e., possessing a sulfone group between benzene rings to which para-electron-donating groups are attached) to activate CYP2C9 in preclinical in vitro drug-drug interaction screens. This, in turn, may affect how data are correlated to the in vivo situation, and thus, impact the drug-development process. Lastly, studies are currently underway exploring the effects of dapsone on the P450 cycle of CYP2C9 to better understand uncoupling mechanisms under activation conditions.