Abstract
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4′-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4′-hydroxylation of diclofenac labeled with tritium in the 4′ position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4′-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC50 values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4′-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4′-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.
The main drug-metabolizing system in mammals is cytochrome P450 (P450), a family of microsomal enzymes present predominantly in the liver. When a drug that is metabolized by a particular P450 is coadministered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effects (Bertz and Granneman, 1997; Lin and Lu, 1998). It is therefore important to predict and prevent the occurrence of clearance changes due to metabolic inhibition. During the drug discovery process, it is routine practice in the pharmaceutical industry to assess the P450 inhibition potential of drug candidates to exclude potent inhibitors from further development (Lin and Lu, 1998; Crespi and Stresser, 2000; Bachmann and Ghosh, 2001; Riley, 2001).
The polymorphically expressed CYP2C9 is one of the most important drug-metabolizing enzymes in humans. It constitutes about 20% of the total human liver P450 content and metabolizes ∼10% of therapeutically important drugs (Shimada et al., 1996; Miners and Birkett, 1998; Goldstein, 2001; Xie et al., 2002; Schwarz, 2003). Many clinically relevant drug interactions due to inhibition of CYP2C9 have been described (Miners and Birkett, 1998; Ito et al., 2004). Several assay methods are currently used for determining the potential of drug candidates to inhibit CYP2C9 activity, and each of these methods presents distinct advantages and disadvantages. The most widely used marker reactions are diclofenac 4′-hydroxylation, tolbutamide 4′-hydroxylation, and S-warfarin 7′-hydroxylation. The practical challenge posed by these assays is that they require HPLC separation of the reaction product from the substrate, followed by UV or mass spectrometric detection. This renders the assays relatively laborious, time-consuming, and not ideally suited for screening the large number of compounds typically required in an industrial drug discovery setting.
Several alternative assays, amenable to high-throughput screening, have been introduced in the past several years. These assays are based on the use of fluorogenic (Crespi and Stresser, 2000) or radiolabeled (Moody et al., 1999) substrates, eliminating the need for HPLC separation. One of the most widely used fluorogenic substrates is 7-methoxy-4-(trifluoromethyl)-coumarin (Crespi and Stresser, 2000). Even though fluorometric CYP2C9 assays are rapid, easy to perform, and amenable to automation, they suffer from a number of limitations, such as the absence of CYP2C9-selective probes, the need to use recombinant enzyme rather than HLMs, imperfect correlation of IC50 values with those determined using classical substrates (Cohen et al., 2003), and fluorescence interference by many test compounds. On the other hand, O-methyl-14C-labeled naproxen, which has been used to probe CYP2C9 activity in HLMs, is not a selective CYP2C9 substrate, with CYP1A2 and CYP2C19 contributing significantly to O-demethylation (Moody et al., 1999).
For these reasons, a non-HPLC assay based on the use of a classical and selective CYP2C9 substrate such as diclofenac would be highly desirable. Hydroxylation of diclofenac at the 4′ position is accompanied by release of the corresponding hydrogen as water. When this position is labeled with tritium, CYP2C9-mediated hydroxylation generates radiolabeled water, which can easily be separated from the unreacted substrate. We recently reported the development of a high-throughput CYP3A4/5 assay based on the use of [6β-3H]testosterone and separation of the tritiated water product on 96-well solid-phase extraction plates (Di Marco et al., 2005). In the present paper, we describe the synthesis of diclofenac labeled with tritium in the 4′ position, the development of a radiometric assay using this substrate, and the study of reaction kinetics in the presence and absence of CYP2C9 inhibitors. We show that the new assay accurately measures the activity of CYP2C9 and the potency of CYP2C9 inhibitors and represents a high-throughput version of the conventional diclofenac 4′-hydroxylation assay.
Materials and Methods
Materials. Oasis HLB 96-well extraction plates and vacuum manifold were purchased from Waters (Milford, MA). Pooled human liver microsomes were obtained from BD Gentest (Woburn, MA). Other chemicals were purchased from Sigma-Aldrich (Milano, Italy).
Synthesis of [4′-3H]Diclofenac. The synthetic route is schematically described in Fig. 1. Synthesis of diclofenac using the Ullman reaction has been described (Moser et al., 1990; Satoh et al., 1993; Oza et al., 2002). The Ullman reaction was modified to produce 2-[(2,6-dichloro-4-bromophenyl)amino]phenylacetic acid (1), which, in the presence of tritium and a palladium catalyst, was then converted to diclofenac labeled in the 4′ position.
Synthesis of 2-[(2,6-dichloro-4-bromophenyl)amino]phenylacetic acid (1) was as follows. 2-Iodophenyl acetic acid (21.3 mg, 0.08 mmol) was added to a mixture of 2,6-dichloro-4-bromoaniline (78 mg, 0.32 mmol), anhydrous potassium carbonate (33.6 mg, 0.24 mmol), and activated copper powder (2.25 mg, 0.035 mmol) in N-methylpyrrolidone (0.5 ml). The reaction mixture was heated at 135°C for 22 h with stirring while water was distilled off through a descending condenser. The color of the reaction mixture changed to brownblack. The hot reaction mixture was treated with hot water and filtered through Celite (World Minerals, Santa Barbara, CA). The crude product was purified by reserve-phase HPLC (Luna Phenyl Hexyl 250 × 10 mm column; Phenomenex, Torrance, CA), water containing 0.1% trifluoroacetic acid/acetonitrile (50:50; flow rate 4 ml/min, UV = 254 nm, retention time = 23–24 min). The required fractions were collected and passed through Sep-Pak C-18, followed by eluting with 10 ml of ethanol to yield 5 mg of 2-[(2,6-dichloro-4-bromophenyl)amino]phenylacetic acid (1).
Synthesis of [4′-3H]diclofenac from compound 1 was as follows. 2-[(2,6-Dichloro-4-bromophenyl)amino]phenylacetic acid (1) (5 mg) was stirred with tritium gas using catalyst 10% Pd/C (5 mg) in dimethylformamide (1 ml) for 1 h. The reaction mixture was filtered and coevaporated with ethanol (two times, 10 ml) to remove any exchangeable tritium. The crude product was purified by using a semipreparative HPLC column [Luna Phenyl Hexyl, 250 × 10 mm column, water containing 0.1% trifluoroacetic acid/acetonitrile (55:45), flow rate 4 ml/min, UV = 254 nm, retention time = 20–21 min] to yield [4′-3H]diclofenac [10 mCi, SA = 22.7 Ci/mmol, as determined by liquid chromatography/mass spectrometry (LC/MS)]. LC/MS: 296 (M)+, 298 (M + 2)+. Radiochemical purity was >98%.
Radiometric CYP2C9 Assay. Reactions were carried out in 96-well conical microtiter plates (Corning Glassworks, Corning, NY) containing [4′-3H]diclofenac (0.05–0.1 μCi), unlabeled diclofenac (10 μM, except otherwise noted), pooled HLMs (125 μg/ml, except otherwise noted), and 0.1 M potassium phosphate buffer, pH 7.6, in a final volume of 100 μl. Inhibitors were added to the reaction mixture from stock solutions in dimethyl sulfoxide/acetonitrile/water (35:25:40, v/v), giving final solvent concentrations of 0.7% dimethyl sulfoxide and 0.5% acetonitrile. Controls without inhibitors contained an equivalent amount of vehicle. After preincubation for 10 min at 37°C, reactions were started by addition of an NADPH-regenerating system containing 1 mM NADPH, 5 mM glucose 6-phosphate, 3 mM MgCl2, and 1 U/ml glucose-6-phosphate dehydrogenase. Assays were conducted for 10 min at 37°C and stopped by addition of HCl to a final concentration of 0.1 N. Plates were then centrifuged for 10 min in a microplate rotor, and supernatants were loaded on a preconditioned 10-mg Oasis HLB 96-well plate. Vacuum was applied and the flow-through was collected in the collection plate. Then, 75 μl of water was added, vacuum was applied again, and the wash was collected into the same plate. Pooled flow-through and water wash were transferred into scintillation vials and counted in a β-scintillation counter. Alternatively, aliquots of this mixture were counted in 96-well scintillation plates using a TopCount scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA). For the calculation of enzyme activity, product counts were corrected by subtraction of counts obtained in control incubations performed in the absence of NADPH-regenerating system. Oasis plates were regenerated by washing with 5 ml of methanol and 5 ml of water and were reused for up to 5 times.
Calculation of the Apparent Rate of Formation of Unlabeled Product from Tracer Competition Experiments and Determination of the Kinetic Tritium Isotope Effect. At low values of substrate conversion, such as those observed in the present experiments, TV/K, the kinetic isotope effect on the V/K ratio, is given by (Northrop, 1982): where SA0 is the initial specific radioactivity of labeled substrate, and SAP is the specific radioactivity of product.
If diclofenac 4′-hydroxylation is not subject to a significant NIH shift and tritium is not retained in 4′-hydroxydiclofenac upon hydroxylation of the radiolabel, the tritiated water product from [4′-3H]diclofenac is formed stoichiometrically with 4′-hydroxydiclofenac. When assays are performed using a fixed amount of [4′-3H]diclofenac and varying concentrations of unlabeled substrate, it can be shown (Di Marco et al., 2005) that v′ = v/TV/K = v*/SA0, where v′ is the apparent rate of formation of unlabeled product, v is the velocity of formation of unlabeled product, and v* is the velocity of formation of tritiated water.
When v′ is plotted against the substrate concentration, S, and fitted to the Hill equation, V′max, S50, the substrate concentration at 50% of V′max, and n, the Hill coefficient, can be derived: where V′max = Vmax/(TV/K), i.e., the apparent maximal rate of product formation. If v and Vmax are determined independently (by quantification of the 4′-hydroxydiclofenac product), these data can also be used to calculate the kinetic isotope effect TV/K.
Quantification of 4′-Hydroxydiclofenac. Aliquots of the assay mixture and of metabolite standard curves were analyzed by HPLC-MS/MS using an Agilent HP1100 liquid chromatograph equipped with a CTC Analytics (Zwingen, Switzerland) PAL autosampler. Chromatography was performed on an XTERRA MS C18 column (4.6 mm × 5 cm; 5 μm; Waters) at a flow rate of 2 ml/min, using a mobile phase consisting of a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) (linear gradient 0–0.5 min, 10% B; 3.0 min, 90% B; 3.5 min, 90% B; 3.6 min, 10% B; the system was equilibrated for 1.4 min at 10% B before the next injection). The eluate was diverted to waste for the first minute and then to a Sciex API-3000 (Applied Biosystems/MDS Sciex, Foster City, CA) triple quadrupole mass spectrometer with a Turbo IonSpray ionization source operated in the positive ion mode. 4′-Hydroxydiclofenac was detected and identified using the transition m/z 312.1→230.0. Metabolite concentrations were determined by weighted linear least-squares regression analysis, using Analyst Quantitation Wizard software version 1.2 (Applied Biosystems, Foster City, CA).
Curve Fitting. Curve fitting of enzyme kinetics data to the Hill equation or to a four-parameter logistic inhibition model (Rodbard and Frazier, 1975) was performed by nonlinear regression analysis using Xlfit 4.0 (IDBS, Guildford, UK).
Results
Separation of Radiolabeled Diclofenac and Tritiated Water Using 96-Well Solid-Phase Extraction Plates. When a solution of assay buffer containing labeled diclofenac (from 104 to 106 dpm) and stopping solution was applied to 96-well extraction plates containing 10 mg of Oasis sorbent, over 99.8% of the radioactivity was retained on the plate. The labeled diclofenac could be recovered by eluting with methanol. As previously reported (Di Marco et al., 2005), tritiated water was not retained on the Oasis plates under the same conditions and was recovered quantitatively in the combined void volume and aqueous wash.
Formation of Tritiated Water from [4′-3H]Diclofenac in HLMs. When [4′-3H]diclofenac was incubated with HLMs in the presence of an NADPH-regenerating system, tritiated water was formed in a time-dependent manner and increased with the concentration of HLMs (Fig. 2). Product formation increased linearly with time for up to 30 min at an HLM concentration of 0.125 mg/ml (correlation coefficient, r2 = 0.979) but was not linear at higher HLM concentrations. Formation of tritiated water was inhibited more than 97% when NADPH was omitted, indicating that the reaction was mediated by cytochrome P450. The specific CYP2C9 inhibitor sulfaphenazole (10 μM) inhibited formation of tritiated water by more than 95%, indicating that the reaction was mediated primarily by CYP2C9. Signal to noise ratio is defined as the ratio between product counts obtained in the presence versus absence of NADPH. The specific conversion rate is the percentage of total radiolabeled substrate converted into tritiated water per unit time and per milligram of microsomal protein. Signal to noise ratio was 33.5 ± 3.2 (mean ± S.E.M., n = 13) when assays were performed for 10 min using 0.125 mg/ml HLMs. Specific conversion rate was 0.070 ± 0.003 %/min/μg (mean ± S.E.M., n = 13). This corresponds to ca. 9% conversion of the substrate in 10 min with 12.5 μg of HLMs. Under these conditions, with 140,000 dpm of tracer, typical product counts were ca. 13,000 and 400 dpm in the presence and absence of NADPH, respectively. Good signal to noise ratios can be obtained with HLM concentrations as low as 0.01 mg/ml (1 μg/assay; data not shown).
Effect of P450 Inhibitors and Anti-P450 Antibodies. To confirm that CYP2C9 mediates product formation, reactions were performed in the presence or absence of furafylline (CYP1A2 inhibitor; Bourrie et al., 1996) and monoclonal antibodies that are inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 (Mei et al., 1999; Shou et al., 2000). As shown in Fig. 3, none of these agents significantly affected formation of tritiated water in HLMs, with the exception of the anti-CYP2C9 and anti-CYP2C19 monoclonal antibodies. The CYP2D6 inhibitor quinidine (10 μM) reduced product formation by less than 25% (data not shown). The antibody directed against CYP2C9 inhibited the reaction by more than 80%. The monoclonal antibody against CYP2C19 is known to cross-react with CYP2C9 (data not shown). These results confirmed that the assay was specific for detecting CYP2C9 activity.
Competition between Radiolabeled and Unlabeled Diclofenac. The effect of unlabeled diclofenac on the formation of tritiated water in HLMs is depicted in Fig. 4A. The curve displays a “low dose hook;” i.e., product formation rate increased with increasing concentration of unlabeled substrate, reached a peak at a diclofenac concentration of ∼3 μM, and then decreased.
Since [4′-3H]diclofenac is used as an isotopic tracer, the formation rate of tritiated water (v*) is representative of that of unlabeled product, namely, water derived from 4′-hydroxylation of diclofenac (which is formed stoichiometrically with 4′-hydroxydiclofenac). The apparent formation rate of unlabeled product, v′, is defined as v* divided by the specific radioactivity of the tracer. The dependence of v′ on substrate concentration (S) can be used to obtain information about the dependence on substrate concentration of the unlabeled product, even if the latter is not measured directly (see Materials and Methods). As depicted in Fig. 4B, the curve of v′ versus S could be fitted to the Hill equation, with S50 = 6.8 ± 1.0 μM, n = 1.15 ± 0.05, and V′max = 1.5 ± 0.1 nmol/min/mg (average ± half-range from two independent experiments, each performed in duplicate). The Hill coefficient was slightly greater than 1, suggesting weak positive cooperativity. Indeed, at low substrate concentrations, a sigmoidal relationship was observed between v′ and S, as depicted in the inset of Fig. 4B.
The kinetics of 4′-hydroxydiclofenac formation is depicted in Fig. 4C. The reaction had a S50 of 6.2 ± 0.9 μM, Vmax of 1.3 ± 0.3 nmol/min/mg protein, and Hill coefficient of 1.1 ± 0.1 (average ± SEM, n = 3). Note that the ratio between V′max and Vmax is 0.9, indicating the absence of a significant kinetic isotope effect or NIH shift of the 3H isotope.
Kinetics of Inhibition by CYP2C9 Inhibitors. IC50 values of 18 compounds were compared in the radiometric versus conventional HPLC-mass spectrometric assays. Tritiated water formation and formation of the unlabeled reaction product 4′-hydroxydiclofenac were determined in the same reaction mixture. The 4′-hydroxydiclofenac was quantified by HPLC coupled to triple quadrupole mass spectrometric analysis. IC50 values are summarized in Table 1.IC50 values in the radiometric assay were almost identical to those determined in the conventional assay. Linear regression analysis, excluding the two compounds with IC50 > 30 μM, resulted in a line with a slope of 1.09 and a correlation coefficient (r2) of 0.938 (Fig. 5). These results demonstrate that the radiometric assay provides a reliable measurement of the potency (IC50) of CYP2C9 inhibitors.
Discussion
The release of tritium that accompanies hydroxylation of a substrate has been used to measure the activity of P450 enzymes (Draper et al., 1998; Di Marco et al., 2005; and references therein). We recently reported the development of a high-throughput CYP3A4/5 assay based on the separation of the tritiated water product on 96-well solid-phase extraction plates (Di Marco et al., 2005). The results of the present study show that this procedure can be used to assay the activity of CYP2C9, using [4′-3H]diclofenac as substrate. The new radiometric assay is very sensitive, with a signal to noise ratio greater than 30; requires short incubation times, and low amounts of tracer (∼ 100,000 dpm) and microsomal protein (<15 μg); is performed in 96-well format throughout the incubation, product separation, and scintillation counting steps; and is amenable to automation. The assay represents a high-throughput radiometric version of the classical diclofenac 4′-hydroxylation assay and is suitable for rapid screening of the inhibitory potential of investigational drugs.
Formation of tritiated water from [4′-3H]diclofenac in HLMs is mediated almost exclusively by CYP2C9, as shown by its sensitivity to chemical and antibody inhibitors of this enzyme and the lack of inhibition by chemical inhibitors of CYP1A2 and CYP2D6, and inhibitory monoclonal antibodies against CYP2A6, CYP2D6, and CYP3A4/5. The finding that the apparent velocity of formation of tritiated water was very similar to that of 4′-hydroxydiclofenac indicates that the reaction occurs in the absence of a significant kinetic isotope effect or NIH shift of the 3H isotope. To our knowledge, these effects have not been studied before for CYP2C9-mediated diclofenac hydroxylation. Substrate competition experiments showed that the kinetics of [4′-3H]diclofenac hydroxylation deviate slightly from typical Michaelis-Menten kinetics, with a Hill coefficient of ∼1.2, indicating weak positive cooperativity. Even though positive cooperativity has not been previously reported for diclofenac hydroxylation, other CYP2C9 substrates such as dapsone and naloxone have been shown to exhibit non-Michaelian kinetics (Korzekwa et al., 1998). Heterotropic enzyme activation has also been described for CYP2C9 (Egnell et al., 2003; Hutzler et al., 2003; Hummel et al., 2004), suggesting that, similarly to CYP3A4 (Shou et al., 2001), CYP2C9 can simultaneously bind two substrate and/or modulator molecules in or near the enzyme active site. Diclofenac 4′-hydroxylation exhibits weak positive cooperativity, which is observed only at very low substrate concentrations (<2 μM), and is difficult to detect by the conventional means of measuring the formation rate of 4′-hydroxydiclofenac, due to limitations in analytical sensitivity. It is interesting to note that the radiometric method offers a very sensitive means to detect this behavior, since a pronounced low dose hook is observed in experiments of substrate competition between tritiated and cold diclofenac. The reason for this phenomenon, which is characteristic of positive cooperativity (De Lean and Rodbard, 1979) is that at low substrate concentrations, the reaction velocity of tritiated product formation increases more than dose proportionally with increasing total substrate concentration. At higher substrate concentrations, formation of tritiated water decreases due to competition between the labeled and unlabeled substrates for binding to the active site.
To validate the use of the new assay as a screening method for CYP2C9 inhibition, we determined IC50 values for a series of structurally diverse drugs and compared the results with those obtained in the conventional diclofenac 4′-hydroxylation assay (with product quantification by LC-MS/MS). The results of this analysis indicate that IC50 values obtained with the new radiometric assay are very similar to those of the conventional assay. IC50 values differed less than 2-fold in every case. Most importantly, not a single compound of the 18 tested would have been misclassified as either a strong or weak inhibitor based on the results of the radiometric assay. Taken together, these results demonstrate that the present method faithfully measures the activity of microsomal CYP2C9-mediated hydroxylation of diclofenac and that it can be used to analyze the inhibitory potencies of investigational drugs.
A detailed comparison for a large number of compounds of IC50 values obtained with a fluorogenic CYP2C9 probe versus diclofenac revealed that the correlation between these assays was not perfect (Cohen et al., 2003). For instance, warfarin was an inhibitor of CYP2C9 when probed with diclofenac (IC50 = 22 μM), but not when probed with the fluorogenic substrate 7-methoxy-4-(trifluoromethyl)-coumarin. As expected, warfarin inhibited tritiated water formation in the present radiometric assay, with an IC50 value of 15 μM. On the basis of the poor correlation between fluorometric and conventional assays, Cohen et al. (2003) recommended that screening with fluorogenic probes should be followed up by studies with conventional substrates. The present assay, which combines the advantages of speed and high throughput, and the use of a conventional substrate, should prove to be a valuable tool for rapidly determining the potential of compounds to inhibit CYP2C9 in a drug discovery setting.
Acknowledgments
We thank Dennis Dean and David Melillo for their contributions to radiochemical synthesis, Yolanda Jakubowski for confirmatory radiochemical purity analysis, Magang Shou for monoclonal antibodies, Massimiliano Fonsi for help with mass spectrometric analysis, and the members of the CYP task force of the Merck Drug Metabolism Council for helpful suggestions.
Footnotes
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This work was supported in part by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.104.002915.
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ABBREVIATIONS: P450, cytochrome P450; HLM, human liver microsome; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; IC50, concentration of drug required to inhibit activity by 50%; SA, specific radioactivity; S50, concentration of substrate at which 50% of maximal activity is observed.
- Received November 4, 2004.
- Accepted December 10, 2004.
- The American Society for Pharmacology and Experimental Therapeutics