Drug Metabolism and Disposition Fast Forward
First published on June 28, 2007; DOI: 10.1124/dmd.107.016345
0090-9556/07/3510-1737-1743$20.00
DMD 35:1737-1743, 2007
High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin
Annalise Di Marco,
Antonella Cellucci,
Ashok Chaudhary,
Massimiliano Fonsi, and
Ralph Laufer
Department of Pharmacology, Istituto di Richerche di Biologia Molecolare, Merck Research Laboratories, Rome, Italy (A.D.M., A.Ce., M.F., R.L.); and Labeled Compound Synthesis, Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey (A.Ch.)
(Received April 20, 2007;
accepted June 26, 2007)
 |
Abstract
|
|---|
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4'-hydroxylation of (S)-mephenytoin labeled with tritium in the 4' position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3'- and 5'-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4'-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4'-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 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; Thummel and Wilkinson, 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).
CYP2C19 is a polymorphic enzyme that is absent in approximately 5% of the Caucasian population and up to 20% of the Asian population (Wrighton and Stevens, 1992). CYP2C19 is the major P450 responsible for the oxidation of a small number of drugs, such as the (S)-enantiomer of mephenytoin, and proton pump inhibitors such as omeprazole and lansoprazole (Goldstein, 2001; Desta et al., 2002). The enzyme participates to various degrees in metabolic clearance of other drugs, such as phenytoin, diazepam, imipramine, propranolol, proguanil, fluoxetine, and sertraline (Goldstein, 2001; Desta et al., 2002) Inhibition of CYP2C19 has been implicated in clinical drug interactions between fluvoxamine and proton pump inhibitors (Yasui-Furukori et al., 2004a,b), alprazolam (Suzuki et al., 2004), mephenytoin, chloroguanide, and diazepam (Yao et al., 2003 and references therein); tricyclic antidepressants and phenytoin (Shin et al., 2002); carbamazepine/diazepam and phenytoin (Lakehal et al., 2002); and clarithromycin and omeprazole (Calabresi et al., 2004).
The most widely used reaction to determine the enzymatic activity of CYP2C19 is (S)-mephenytoin 4'-hydroxylation (Yuan et al., 2002; Walsky and Obach, 2004). A radiometric version of this assay has been described, using [14C](S)-mephenytoin (Crespi et al., 2006). The practical challenge posed by these assays is that they require HPLC separation of the reaction product from the substrate, followed by UV, mass spectrometric, or radiochemical detection. This requirement renders the procedures relatively laborious, time-consuming, and not ideally suited for screening the large number of compounds typically required in an industrial drug discovery setting. An alternative assay using the fluorogenic nonselective CYP2C19 substrate 3-cyano-7-ethoxycoumarin has been described (Miller et al., 2000). Even though fluorometric P450 assays are rapid, easy to perform, and amenable to automation, they suffer from a number of limitations, such as the absence of selective probes, the need to use recombinant enzyme rather than HLMs, imperfect correlation of IC50 values with those determined using classic drug substrates (Cohen et al., 2003), and fluorescence interference by many test compounds.
For these reasons, a non-HPLC assay based on the use of a classic CYP2C19 substrate such as (S)-mephenytoin would be highly desirable. Hydroxylation of (S)-mephenytoin at the 4'-position is accompanied by release of the corresponding proton. When this position is labeled with tritium, CYP2C19-mediated hydroxylation generates tritiated water, which can easily be separated from the unreacted substrate. Assay procedures based on a similar principle have been described for CYP2C9 (Di Marco et al., 2005a) and CYP3A4 (Draper et al., 1998; Di Marco et al., 2005b). In the present report we describe the synthesis of (S)-mephenytoin labeled with tritium in the 4'-position, the use of this tracer to determine CYP2C19 activity, and studies of reaction kinetics in the presence and absence of CYP2C19 inhibitors.
|
Materials and Methods
|
|---|
Materials. Ninety-six well C18 Versaplates (100 mg) were purchased from Varian Inc. (Palo Alto, CA). Pooled human liver microsomes and recombinant human CYP2C19 Supersomes were obtained from BD Gentest (Franklin Lakes, NJ). Other chemicals were purchased from Sigma-Aldrich (Milan, Italy).
Synthesis of Tritiated Mephenytoin. [phenyl-4'-Iodo](S)-Mephenytoin (2). To a 25-ml round-bottom flask were added (S)-mephenytoin (1) (0.05 mmol, 10.9 mg) and triflic acid (0.2 ml). The reaction mixture was cooled to 0°C followed by the addition of N-iodosuccinimide (0.05 mmol, 11 mg). After stirring at 0°C for 6 h, the reaction mixture was evaporated to dryness under vacuum. The crude product was further purified by reserve-phase HPLC [Zorbax Rx C18 250 x 10 mm column, water containing 0.1% TFA-acetonitrile (50:50), UV = 254 nm, flow rate = 4 ml/min, Rt = 6.5 min]. The required fractions were collected, passed through Sep-Pak C-18, and eluted with 10 ml of ethanol to yield 5 mg of (5S)-5-ethyl-3-methyl-5-(4'iodophenyl) imidazolidine-2,4-dione or [phenyl-4'-iodo](S)-mephenytoin (2) in 30% isolated yield (Fig. 1A). LC/MS: 345 (M + H)+.
[phenyl-4'-3H](S)-Mephenytoin (3). Compound 2 (5 mg) was stirred with tritium gas using catalyst 10% Pd/C (3 mg) and 5% Pd/CaCO3 (2 mg) in dimethylformamide (1 ml) for 1 h on a Trisorber manifold. The reaction mixture was filtered and coevaporated with ethanol (2 x 10 ml) to remove any exchangeable tritium gas. The crude product was purified by semipreparative HPLC [Luna Phenyl-Hexyl 250 x 10 mm column, water containing 0.1% TFA-acetonitrile (75:25), flow rate = 4 ml/min, UV = 254 nm, Rt = 20 min] to yield [phenyl-4'-3H](S)-mephenytoin (3) (24 mCi) (SA = 20 Ci/mmol as determined by LC/MS) (Fig. 1A). LC/MS: 221 (MH + 2)+. Radiochemical purity was >99% by HPLC.
[phenyl-3',4',5'-Triiodo](S)-Mephenytoin (4). To a 25-ml round-bottom flask were added (S)-(-)-mephenytoin (1) (0.05 mmol, 10.9 mg) and triflic acid (0.2 ml). The reaction mixture was cooled to 0°C followed by the addition of N-iodosuccinimide (0.5 mmol, 112.5 mg). After overnight stirring at room temperature, the reaction mixture was evaporated to dryness under vacuum. The crude product was further purified by reserve-phase HPLC (Zorbax Rx C18 250 x 10 mm column, water containing 0.1% TFA-acetonitrile (50:50), UV = 254 nm, flow rate = 4 ml/min, Rt = 15 min). The required fractions were collected, passed through Sep-Pak C-18, and eluted with 10 ml of ethanol to yield 5 mg of (5S)-5-ethyl-3-methyl-5-(3',4',5'-triiodophenyl)imidazolidine-2,4-dione or [phenyl-3',4',5' triiodo](S)-mephenytoin (4) in 17% isolated yield (Fig. 1B). LC/MS: 597 (M+H)+.
[phenyl-3',4',5'-3H](S)-Mephenytoin (5). Compound 4 (5 mg) (Fig. 1B) was stirred with tritium gas using catalyst 10% Pd/C (3 mg) and 5% Pd/CaCO3 (2 mg) in dimethylformamide (1 ml) for 1 h on Trisorber manifold. The reaction mixture was filtered and coevaporated with ethanol (2 x 10 ml) to remove any exchangeable tritium gas. The crude product was purified by semipreparative HPLC [Luna Phenyl-Hexyl 250 x 10 mm column; water containing 0.1% TFA-acetonitrile (75:25), flow rate = 4 ml/min, UV = 254 nm, Rt = 20 min] to yield [phenyl-3',4',5'-3H3](S)-mephenytoin (28 mCi; SA = 69 Ci/mmol as determined by LC/MS). LC/MS: 225 (MH + 6)+.As determined by LC-MS analysis, the proportion of tracer molecules with 1, 2, and 3 tritium atoms was 9, 38, and 51%, respectively. Tritium label in the 4' position was 
50% as determined by tritium nuclear magnetic resonance. Tritium nuclear magnetic resonance (CD3OD, 
): 7.3 (m, 1T, p-Ar-T), 7.36 (m, 2T, m-Ar-T). Radiochemical purity was >98% by HPLC. Compounds 3 and 5 were further purified on C18 extraction cartridges before use in radioenzymatic assays.
Radiometric CYP2C19 Assay. Reactions were carried out in a temperature-controlled microplate shaker using 96-well conical microtiter plates (Corning Glassworks, Acton, MA) containing [4'-3H] (S)-mephenytoin or [3',4',5'-3H]3(S)-mephenytoin (0.2-0.5 µCi, corresponding to a final concentration of 20-70 nM), unlabeled (S)-mephenytoin (20 µM, except otherwise noted), pooled HLMs (1 mg/ml, except otherwise noted), and 0.1 M potassium phosphate buffer, pH 7.6, in a final volume of 100 µl. Chemical inhibitors were added to the reaction mixture from stock solutions and serial dilutions in DMSO-acetonitrile-water (35:25:40, v/v), giving final solvent concentrations of 0.7% DMSO and 0.5% acetonitrile. Controls without inhibitor contained vehicle only. For determination of IC50, at least eight concentrations of chemicals were tested in duplicate. Concentration ranges were 0.0001 to 10 µM for miconazole, 0.01 to 30 µM for fluoxetine, ticlopidine, tranylcypromine, omeprazole, progesterone, clotrimazole, and amitriptyline; 0.01 to 100 µM for ketoconazole, ethynylestradiol, propranolol, cimetidine, imipramine, and carbamazepine. Inhibitory monoclonal antibodies were used at a concentration of 0.3 mg/ml. After preincubation for 10 min at 37°C, reactions were started by addition of 1 mM NADPH and an NADPH-regenerating system containing 5 mM glucose 6-phosphate, 3 mM MgCl2, and 1 U/ml glucose-6-phosphate dehydrogenase. Blanks without NADPH were run in parallel. Assays were conducted for 30 min at 37°C and stopped by addition of 20 µl of 0.5 N HCl. Plates were then centrifuged for 10 min in a microplate rotor, and the supernatant was split for analysis of tritiated water and 4'-hydroxymephenytoin. For solid-phase separation of tritiated water, 100 µl of the supernatants was loaded into the wells of a 100-mg 96-well C18 Versaplate that had been previously preconditioned by washing with methanol and water. A plate vacuum manifold (Waters, Milford, MA) was used for washing and elutions. Vacuum was applied, and the void volume was collected in the collection plate. Then, 200 µl of water was added, vacuum was applied again, and the wash was collected into the same plate. This step was repeated. Pooled void volume and water washes were transferred into scintillation vials and counted in a ß-scintillation counter. For the calculation of enzyme activity, product counts were corrected by subtraction of counts obtained in blank incubations without NADPH. Extraction plates were regenerated by washing with 5 ml of methanol and 5 ml of water and reused up to three times.
Assays with recombinant human CYP2C19 were performed as described above, except that the amount of [3',4',5'-3H]3(S)-mephenytoin was 0.14 µCi. Also, various amounts of CYP2C19 Supersomes were used instead of HLMs, as specified under Results.
Quantification of 4'-Hydroxymephenytoin. Aliquots of the acidified assay reaction mixtures (see above) and of metabolite standard curves were diluted with 50% acetonitrile and analyzed by HPLC using an Agilent HP1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a HTL PAL Autosampler (CTC Analytics AG, Zwingen, Switzerland). Flufenamic acid was used as the internal standard. Chromatography was performed on a XTERRA MS C18 column (4.6 mm x 5 cm; 5 µm; Waters) at a flow rate of 2 ml/min, using a mobile phase consisting of a mixture of 10 mM ammonium acetate in water (A) and 10 mM ammonium acetate in acetonitrile (B), with the following gradient program: 0-0.2 min, 10% B; 0.8 min, 60% B; 1.2 min, 60% B; 1.4 min, 95% B; 2.2 min, 95% B; 2.21 min, 9% B; 2.5 min, 9% B. The eluate was split via a T-piece for injection into the mass spectrometer probe at a flow rate of 0.2 ml/min. Eluate was diverted to waste for the first minute and then injected into a Sciex API-3000 triple quadrupole mass spectrometer (PerkinElmer Life and Analytical Sciences, Boston, MA) with a Turbo Ionspray ionization source operated in the negative ion mode. 4'-Hydroxymephenytoin and flufenamic acid were detected in multiple reaction monitoring mode and identified using the transition m/z 233 
190 and 280 236, respectively. Calibration curves were constructed in duplicate, using eight metabolite standard concentrations ranging from 0.014 to 30 µM. Metabolite concentrations in test samples were determined by linear least-squares regression analysis, using Analyst Quantitation Wizard software (version 1.2, Applied Biosystems, Foster City, CA).
An alternative, more sensitive method was used for analysis of tracer competition experiments. Phenytoin was used as the internal standard. Chromatography was performed on a Waters Sunfire C18 column (50 x 4.6 mm, 5 µm) at a flow rate of 1.5 ml/min using a mobile phase consisting of a mixture of solvent A (50 mM ammonia in water) and solvent B (acetonitrile) with the following gradient program: 0 to 2 min, from 5% to 95% B; 3 min, 95% B; 3.1 min, 5% B; and 3.5 min, 5% B. Detection was carried out using a TSQ Quantum Ultra mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with an electrospray ion source. The mass spectrometer was operated in negative ion electrospray selective reaction monitoring mode. The selective reaction monitoring transitions of m/z 233.005 190.015 (collision energy 20 eV, scan time 0.10 s) for 4'-hydroxymephenytoin and m/z 250.990 208.010 (collision energy 21 eV, scan time 0.10 s) for phenytoin were monitored.
Calculation of Kinetic Parameters. When reactions are performed in the presence of both tritiated and unlabeled (S)-mephenytoin, comparison of the specific radioactivity of substrate and product gives information on reaction kinetics. The specific activity of tritium label in the 4' position of the substrate, SA0,is
 | (1) |
where c's is counts per minute in the 4' position of tritiated (S)-mephenytoin, and s is moles of total (labeled plus unlabeled) substrate molecules. For [4'-3H](S)-mephenytoin, c's equals the total tracer counts. For [3',4',5'-3H]3(S)-mephenytoin, which has 50% of label in the 4' position, c's is equal to half of the total counts. The specific activity of the tritiated water product of CYP2C19-mediated 4'-hydroxylation of the substrate, SAP, is
 | (2) |
where cP is counts per minute of tritiated water, and p is moles of 4'-hydroxymephenytoin. Combining eqs. 1 and 2
 | (3) |
where %SCT is the percentage of total label in the 4'-position of the tracer that is converted into tritiated water and %SCH is the fraction of total substrate converted into 4-hydroxymephenytoin. This ratio differs from unity if the reaction is subject to a kinetic isotope effect or to an NIH shift. In either case, hydroxylation of the tracer would not be accompanied by an equimolar release of tritiated water. In the absence of the NIH shift
 | (4) |
where T(V/K) is the kinetic isotope effect on the Vmax/Km ratio (Northrop, 1982). A NIH shift of the radiolabel during hydroxylation, with retention of tritium in 4'-hydroxymephenytoin, would further increase the SA0/SAP ratio.
Kinetic parameters of 4'-hydroxymephenytoin formation were calculated using the Michaelis-Menten equation
 | (5) |
where v is the initial rate of product formation, Vmax is the maximal rate of product formation, and Km is the substrate concentration at 50% Vmax.
Data Analysis. Curve fitting of enzyme kinetics data to the Michaelis-Menten 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
|
|---|
Synthesis of Tritiated (S)-Mephenytoin. (S)-Mephenytoin was labeled with tritium in the 4'-position, giving [4'-3H] (S)-mephenytoin (Fig. 1A, compound 3). The 4'-position is the site of CYP2C19-mediated hydroxylation, which generates 4'-hydroxy-(S)-mephenytoin. This enzymatic reaction has been reported (Küpfer et al., 1981) to be subject to an NIH shift, with migration of tritium to the adjacent ring carbon (3'- or 5'-position), as a result of rearrangement reactions via unstable tautomeric forms of the arene oxide (Daly et al., 1972). To increase the proportion of tritium lost upon 4'-hydroxylation, we also prepared [3',4',5'-3H]3(S)-mephenytoin, containing tritium in position 4' as well as in the adjacent 3'- and 5'-positions (Fig. 1B, compound 5). 4'-Hydroxylation of a triply labeled tracer molecule is expected to generate a stoichiometric amount of tritiated water.
Separation of Tritiated Mephenytoin from Tritiated Water Using 96-Well Solid-Phase Extraction Plates. We first assessed whether tritiated (S)-mephenytoin could be separated from the tritiated water product formed upon 4'-hydroxylation. A zero time control mixture containing HLMs to which stopping solution had been added before addition of NADPH and other assay ingredients, including approximately 106 dpm of [3',4',5'-3H]3(S)-mephenytoin and 20 µM unlabeled (S)-mephenytoin, was submitted to solid-phase extraction on 96-well extraction plates containing 100 mg of C18 sorbent. After washing of the extraction plates with water, 99.9% of the radioactivity was retained on the solid-phase resin. Only 0.027 ± 0.007% of radioactivity (mean ± S.E.M., n = 7) eluted in the void volume and aqueous washes. Tritiated mephenytoin could be recovered by eluting the solid-phase extraction resin with methanol. Similar results were obtained with [4'-3H](S)-mephenytoin (data not shown). In contrast, tritiated water (from 500 to 50,000 dpm) was not retained on the solid-phase extraction resin under the same conditions. Recovery of tritiated water in the combined void volume and aqueous washes was quantitative (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2. Formation of tritiated water from [3',4',5'-3H]3(S)-mephenytoin with recombinant CYP2C19. A mixture of [3',4',5'-3H]3(S)-mephenytoin (approximately 300,000 dpm; 20 nM), unlabeled (S)-mephenytoin (20 µM) and NADPH (1 mM) was incubated for 30 min with Supersomes (microsomes from insect cells infected with baculovirus encoding CYP2C19 and P450 reductase) at the indicated microsomal protein concentrations. The specific enzyme activity was 227 pmol/mg protein. Tritiated water was quantified as described under Materials and Methods. Each point is the mean ± half-range of duplicate determinations.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3. Formation of tritiated water from [3',4',5'-3H]3(S)-mephenytoin in human liver microsomes. A mixture of [3',4',5'-3H]3(S)-mephenytoin (approximately 900,000 dpm; 60 nM), unlabeled (S)-mephenytoin (20 µM), and NADPH (1 mM) was incubated for 30 min with HLMs at concentrations of 0.25 mg/ml ( ), 0.5 mg/ml ( ), and 1 mg/ml ( ). The initial reaction velocities were 5.8, 5.4, and 3.9 pmol/min/mg microsomal protein, respectively. Each point is the mean ± half-range of duplicate determinations.
|
|
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4. Effect of anti-P450 monoclonal antibodies on tritiated water formation from [3',4',5'-3H]3(S)-mephenytoin in human liver microsomes. Reactions were conducted in the presence or absence of inhibitory antibodies. Enzyme activity is expressed as a percentage of untreated controls and represents the average ± half-range of duplicate determinations. Enzyme activity for control was 6.2 ± 0.6 pmol/min/mg microsomal protein.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 5. Reaction kinetics. Effect of unlabeled (S)-mephenytoin on tritiated water formation from [4'-3H](S)-mephenytoin (A) and [3',4',5'-3H]3(S)-mephenytoin (B). Experiments were conducted in the presence of a fixed amount (approximately 500,000 dpm; 30 nM) of tritiated tracer and various concentrations of unlabeled (S)-mephenytoin. Each point is the mean ± half-range of duplicate determinations. Data depicted are from representative experiments. Average IC50 values from four separate experiments are given under Results. C, kinetics of formation of 4'-hydroxy-(S)-mephenytoin. The product was quantified using HPLC-MS/MS analysis. Each point is the mean ± half-range of duplicate determinations.
|
|
Tritium Release from Tritiated (S)-Mephenytoin. When a tracer amount (20 nM) of [3',4',5'-3H]3(S)-mephenytoin was incubated with recombinant cDNA-expressed CYP2C19 in the presence of 20 µM unlabeled (S)-mephenytoin, NADPH-dependent formation of tritiated water was observed. Product formation increased linearly with enzyme (Supersome) concentration, up to a microsomal protein concentration of 0.2 mg/ml, corresponding to 45 pmol/ml enzyme (Fig. 2).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 6. Inhibition by various drugs of tritiated water release from [3',4',5'-3H]3(S)-mephenytoin. Reactions were conducted in the presence of the indicated concentrations of chemical inhibitors. Results are expressed as the percentage of tritiated water released relative to control incubations conducted in the absence of inhibitor. Each point represents the mean of duplicate determinations.
|
|
A time-dependent and microsomal protein-dependent increase of tritiated water formation was also observed in HLM incubated with trace amounts (30-70 nM) of [3',4',5'-3H]3(S)-mephenytoin in the presence of 20 µM unlabeled (S)-mephenytoin and NADPH. At protein concentrations of 0.5 and 1 mg/ml, product formation increased linearly with time for up to 30 min (Fig. 3). Formation of tritiated water was dependent on NADPH, indicating that the reaction was mediated by cytochrome P450. Signal/noise ratio, i.e., the ratio between product counts obtained in the presence versus absence of NADPH was 18 ± 4 (mean ± S.E.M., n = 7) when assays were performed for 30 min using 1 mg/ml HLMs. Tritium release was inhibited by inhibitory monoclonal antibodies against CYP2C19 and CYP2C9 (the latter antibody is known to cross-react with CYP2C19) but not by antibodies against CYP2A6, CYP2D6 and CYP3A4/5 (Fig. 4).
Reaction Kinetics with [3',4',5'-3H]3(S)-mephenytoin and [4'-3H](S)-Mephenytoin. In the absence of unlabeled (S)-mephenytoin, %SCT, the percentages of label in the 4'-position of tracer that is converted into tritiated water in 30 min at 1 mg/ml HLMs were 0.4 ± 0.1 and 1.5 ± 0.2 for [4'-3H](S)-mephenytoin and [3',4',5'-3H]3(S)-mephenytoin, respectively (mean ± S.E.M., n = 5). In the experiment depicted in Fig. 5, addition of unlabeled substrate led to a dosedependent decrease in formation of tritiated water from [4'-3H](S)-mephenytoin (Fig. 5A) and [3',4',5'-3H]3(S)-mephenytoin (Fig. 5B) with IC50 values of 46 and 27 µM, respectively. Average IC50 values ± S.E.M. from four separate experiments were 33 ± 7 and 30 ± 5 µM, respectively, which were not statistically different by two-tailed Student's t test (p = 0.7). The formation of 4'-hydroxymephenytoin followed Michaelis-Menten kinetics, with a Km of 35 ± 7 µM and Vmax of 20 ± 1 pmol/min/mg microsomal protein (Fig. 5C). The ratio of the specific radioactivity of substrate versus product, SA0/SAP, was derived from these data as described under Materials and Methods.SA0/SAP values were 2.1 ± 0.2 and 1.1 ± 0.2 for [4'-3H](S)-mephenytoin and [3',4',5'-3H]3(S)-mephenytoin, respectively (mean ± S.E.M. from data obtained at eight different substrate concentrations).
Effect of CYP2C19 Inhibitors. The effects of ticlopidine, progesterone, ethynylestradiol, ketoconazole, miconazole, and omeprazole on NADPH-dependent formation of tritiated water from [3',4',5'-3H](S)-mephenytoin in HLM are shown in Fig. 6. The inhibitory effect of additional drugs was determined using radiometric assay, and IC50 values were compared with those obtained by HPLC-MS/MS analysis of the unlabeled reaction product. As summarized in Table 1, IC50values for inhibition of formation of tritiated water and 4'-hydroxymephenytoin were very similar. IC50 values differed less than 2.5-fold for all 15 compounds tested. If compounds with IC50 >30 µM were excluded, there was a good correlation of log(IC50) values between the two assays, with a correlation coefficient (r2) of 0.95 (Fig. 7). The correlation coefficient for the linear relationship between IC50 values in the two assays was 0.79.
View this table:
[in this window]
[in a new window]
|
TABLE 1 Comparison between IC50 in radiometric versus LC-MS/MS assay
The effect of inhibitors on the formation rate of tritiated water and 4'-hydroxy-(S)-mephenytoin was determined in the same reaction mixture. 4'-Hydroxy-(S)-mephenytoin was quantified by LC-MS/MS. IC50 values were calculated from full inhibition curves with at least eight concentration points. Data are mean values ± S.E. of the fit, or average IC50 values from two to three experiments ± S.E.M.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 7. Correlation between IC50 values determined in radiometric versus LC-MS/MS assay. The log(IC50) values for CYP2C19 inhibition of 12 drugs, obtained by radiometric and LC-MS/MS assays, were compared and analyzed by linear regression. Data are from Table 1, excluding compounds with IC50 >30 µM.
|
|
|
Discussion
|
|---|
According to a survey conducted by reviewers in the Center for Drug Evaluation and Research of the U.S. Food and Drug Administration, the (S)-mephenytoin 4'-hydroxylation assay represents the preferred and most commonly used CYP2C19 probe reaction used by industry investigators in support of new drug applications (Yuan et al., 2002). In the present report, we describe the synthesis of (S)-mephenytoin containing a tritium label in the 4'-position and the development of a high-throughput CYP2C19 assay based upon quantification of tritiated water released upon CYP2C19-mediated 4'-hydroxylation of the tracer substrate. This new radiometric assay is very sensitive, with a signal/noise ratio of 18, requires short incubation times, is performed in 96-well format throughout the incubation and product separation steps, and is amenable to automation.
Recombinant CYP2C19 catalyzed NADPH-dependent release of tritiated water from tritiated (S)-mephenytoin. In HLMs, this reaction is also NADPH-dependent and mediated almost exclusively by CYP2C19, as shown by its sensitivity to inhibitors of this enzyme, including inhibitory antibodies, and the lack of inhibition by neutralizing monoclonal antibodies against CYP2A6, CYP2D6, and CYP3A4/5. Furthermore, unlabeled (S)-mephenytoin inhibited the reaction with an IC50 of approximately 30 µM, in good agreement with the substrate Km for formation of 4'-hydroxymephenytoin (35 ± 7 µM), as expected if the formation of radiolabeled and unlabeled products are mediated by the same enzyme. The Km and Vmax (20 pmol/min/mg protein) determined for (S)-mephenytoin 4'-hydroxylation in pooled HLMs were in reasonable agreement with previously reported data [57 µM and 58 pmol/min/mg (Walsky and Obach, 2004) and 30 µM and 40 pmol/min/mg (Galetin and Houston, 2006), respectively]. The somewhat lower Vmax value obtained in the present study may be due to enzyme inhibition by the organic solvent present in our assay. Acetonitrile does not affect (S)-mephenytoin 4'-hydroxylase activity, whereas 0.7% DMSO inhibits activity by approximately 50% (Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999). The choice of this solvent was dictated by the need to use the assay for screening chemicals from our sample collection, which are prepared and stored in DMSO.
Studies in human volunteers receiving oral doses of [14C]-labeled and tritiated mephenytoin indicated that the tritium label in the 4'-position of the drug was extensively retained during formation of the 4'-hydroxylated reaction product (Küpfer et al., 1981). The most likely explanation for this phenomenon is the existence of an NIH shift, with migration of the tritium isotope to the adjacent 3'- and 5'-positions of the phenyl ring (Daly et al., 1972). To minimize tritium retention, we introduced tritium label also into the 3'- and 5'-positions of (S)-mephenytoin, such that 4'-hydroxylation would necessarily be accompanied by tritium release into the solvent. The existence of an NIH shift was confirmed by the result that tritium release from triply labeled tracer was significantly higher than that from the tracer containing tritium at the 4'-position only. The finding that the SA0/SAP ratio for tritium release from [3',4',5'-3H]3(S)-mephenytoin was close to unity argues against the existence of a significant kinetic isotope effect.
Assay sensitivity is limited by the specific radioactivity of the substrate and its relatively low turnover in HLM. The Vmax for (S)-mephenytoin 4'-hydroxylation is more than 50-fold lower than that of testosterone-6ß-hydroxylation and diclofenac 4'-hydroxylation, marker reactions for CYP3A4 and CYP2C9, respectively (Walsky and Obach, 2004; Di Marco et al., 2005a,b). For this reason, (S)-mephenytoin 4'-hydroxylase assays require higher concentrations of HLM and longer incubation times (Walsky and Obach, 2004). To obtain product counts on the order of several thousand disintegrations per minute without having to resort to excessively high amounts (>0.5 µCi) of radiolabeled substrate or long (>30 min) incubation times, most of the experiments described in the present article were done with a relatively high concentration (1 mg/ml) of HLM. For P450 inhibition assays, it may be preferable to use lower amounts of microsomes to avoid extensive binding of hydrophobic drugs to membrane phospholipids, which may artifactually increase their IC50 (Margolis and Obach, 2003). As shown in Fig. 3, it is possible to reduce the HLM concentration to 0.25 mg/ml, while maintaining product counts of several hundred disintegrations per minute, which can be accurately quantified. When the enzyme source is recombinant CYP2C19, which has a much higher specific activity than HLMs, assays can be conducted at very low (<0.1 mg/ml) concentrations of microsomal protein, as shown in Fig. 2.
To validate the use of the new assay as a method for determining the potential of drugs to inhibit CYP2C19, we determined IC50 values for a series of 15 structurally diverse drugs and compared the results with those obtained in the conventional (S)-mephenytoin 4'-hydroxylase assay, with product quantification by LC-MS/MS. The results of this analysis indicate that IC50 values obtained with the new radiometric method are very similar to those determined by conventional assay. IC50 values differed less than 2.5-fold for all compounds tested. Importantly, none of the drugs would be misclassified as either a strong (<1 µM) or weak (>50 µM) inhibitor (Crespi and Stresser, 2000) on the basis of the IC50 determined by radiometric versus conventional assay (within the limit of standard errors). Taken together, these results demonstrate that the present method represents a high-throughput radiometric version of the classic (S)-mephenytoin 4'-hydroxylase assay and a valuable new tool for rapid screening of the inhibitory potential of investigational drugs.
|
Acknowledgments
|
|---|
We thank David Melillo for suggesting the introduction of label into the 3'- and 5'-position of mephenytoin, and Vincenzo Pucci for mass spectrometric analysis of the experiment in Fig. 5.
|
Footnotes
|
|---|
This article is dedicated to the memory of our friend and colleague Giovanni Migliaccio.
doi:10.1124/dmd.107.016345.
ABBREVIATIONS: P450, cytochrome P450; HPLC, high-performance liquid chromatography; HLM, human liver microsome; TFA, trifluoroacetic acid; Rt, retention time; LC, liquid chromatography; MS, mass spectrometry; SA, specific activity; DMSO, dimethyl sulfoxide; MS/MS, tandem mass spectrometry.
Address correspondence to: Dr. Ralph Laufer, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Via Pontina km 30,600, 00040 Pomezia (Roma), Italy. E-mail: ralph_laufer{at}merck.com
|
References
|
|---|
Bachmann KA and Ghosh R (2001) The use of in vitro methods to predict in vivo pharmacokinetics and drug interactions. Curr Drug Metab 2: 299-314.[CrossRef][Medline]
Bertz RJ and Granneman GR (1997) Use of in vitro and in vivo data to estimate the likelihood of metabolic pharmacokinetic interactions. Clin Pharmacokinet 32: 210-258.[Medline]
Busby WF Jr, Ackermann JM, and Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27: 246-249.[Abstract/Free Full Text]
Calabresi L, Pazzucconi F, Ferrara S, Di Paolo A, Tacca MD, and Sirtori C (2004) Pharmacokinetic interactions between omeprazole/pantoprazole and clarithromycin in healthy volunteers. Pharmacol Res 49: 493-499.[CrossRef][Medline]
Chauret N, Gauthier A, and Nicoll-Griffith DA (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab Dispos 26: 1-4.[Abstract/Free Full Text]
Cohen LH, Remley MJ, Raunig D, and Vaz AD (2003) In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates. Drug Metab Dispos 31: 1005-1015.[Abstract/Free Full Text]
Crespi CL, Chang TK, and Waxman DJ (2006) CYP2C19-mediated (S)-mephenytoin 4'-hydroxylation assayed by high-performance liquid chromatography with radiometric detection. Methods Mol Biol 320: 115-119.[Medline]
Crespi CL and Stresser DM (2000) Fluorometric screening for metabolism-based drug-drug interactions. J Pharmacol Toxicol Methods 44: 325-331.[CrossRef][Medline]
Daly JW, Jerina DM, and Witkop B (1972) Arene oxides and the NIH shift: the metabolism, toxicity and carcinogenicity of aromatic compounds. Experientia 28: 1129-1149.[CrossRef][Medline]
Desta Z, Zhao X, Shin JG, and Flockhart DA (2002) Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet 41: 913-958.[CrossRef][Medline]
Di Marco A, Marcucci I, Chaudhary A, Taliani M, and Laufer R (2005a) Development and validation of a high-throughput radiometric CYP2C9 inhibition assay using tritiated diclofenac. Drug Metab Dispos 33: 359-364.[Abstract/Free Full Text]
Di Marco A, Marcucci I, Verdirame M, Perez J, Sanchez M, Pelaez F, Chaudhary A, and Laufer R (2005b) Development and validation of a high-throughput radiometric CYP3A4/5 inhibition assay using tritiated testosterone. Drug Metab Dispos 33: 349-358.[Abstract/Free Full Text]
Draper AJ, Madan A, Smith K, and Parkinson A (1998) Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes. Drug Metab Dispos 26: 299-304.[Abstract/Free Full Text]
Galetin A and Houston JB (2006) Intestinal and hepatic metabolic activity of five cytochrome P450 enzymes: impact on prediction of first-pass metabolism. J Pharmacol Exp Ther 318: 1220-1229.[Abstract/Free Full Text]
Goldstein JA (2001) Clinical relevance of genetic polymorphisms in the human CYP2C sub-family. Br J Clin Pharmacol 52: 349-355.[CrossRef][Medline]
Hickman D, Wang JP, Wang Y, and Unadkat JD (1998) Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos 26: 207-215.[Abstract/Free Full Text]
Küpfer A, Roberts RK, Schenker S, and Branch RA (1981) Stereoselective metabolism of mephenytoin in man. J Pharmacol Exp Ther 218: 193-199.[Free Full Text]
Lakehal F, Wurden CJ, Kalhorn TF, and Levy RH (2002) Carbamazepine and oxcarbazepine decrease phenytoin metabolism through inhibition of CYP2C19. Epilepsy Res 52: 79-83.[CrossRef][Medline]
Lin JH and Lu AY (1998) Inhibition and induction of cytochrome P450 and the clinical implications. Clin Pharmacokinet 35: 361-390.[CrossRef][Medline]
Margolis JM and Obach RS (2003) Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions. Drug Metab Dispos 31: 606-611.[Abstract/Free Full Text]
Miller VP, Stresser DM, Blanchard AP, Turner S, and Crespi CL (2000) Fluorometric high-throughput screening for inhibitors of cytochrome P450. Ann N Y Acad Sci 919: 26-32.[Medline]
Northrop DB (1982) Deuterium and tritium kinetic isotope effects on initial rates. Methods Enzymol 87: 607-625.[Medline]
Riley RJ (2001) The potential pharmacological and toxicological impact of P450 screening. Curt Opin Drug Discov Devel 4: 45-54.
Rodbard D and Frazier GR (1975) Statistical analysis of radioligand assay data. Methods Enzymol 37: 3-22.[Medline]
Shin JG, Park JY, Kim MJ, Shon JH, Yoon YR, Cha IJ, Lee SS, Oh SW, Kim SW, and Flockhart DA (2002) Inhibitory effects of tricyclic antidepressants (TCAs) on human cytochrome P450 enzymes in vitro: mechanism of drug interaction between TCAs and phenytoin. Drug Metab Dispos 30: 1102-1107.[Abstract/Free Full Text]
Suzuki Y, Kawashima Y, Shioiri T, and Someya T (2004) Effects of concomitant fluvoxamine on the plasma concentration of etizolam in Japanese psychiatric patients: wide interindividual variation in the drug interaction. Ther Drug Monit 26: 638-642.[CrossRef][Medline]
Thummel KE and Wilkinson GR (1998) In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol 38: 389-430.[CrossRef][Medline]
Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32: 647-660.[Abstract/Free Full Text]
Wrighton SA and Stevens JC (1992) The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxicol 22: 1-21.[Medline]
Yao C, Kunze KL, Trager WF, Kharasch ED, and Levy RH (2003) Comparison of in vitro and in vivo inhibition potencies of fluvoxamine toward CYP2C19. Drug Metab Dispos 31: 565-571.[Abstract/Free Full Text]
Yasui-Furukori N, Saito M, Uno T, Takahata T, Sugawara K, and Tateishi T (2004a) Effects of fluvoxamine on lansoprazole pharmacokinetics in relation to CYP2C19 genotypes. J Clin Pharmacol 44: 1223-1229.[Abstract/Free Full Text]
Yasui-Furukori N, Takahata T, Nakagami T, Yoshiya G, Inoue Y, Kaneko S, and Tateishi T (2004b) Different inhibitory effect of fluvoxamine on omeprazole metabolism between CYP2C19 genotypes. Br J Clin Pharmacol 57: 487-494.[CrossRef][Medline]
Yuan R, Madani S, Wei XX, Reynolds K, and Huang SM (2002) Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 30: 1311-1319.[Abstract/Free Full Text]
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
