Relative Quantities of Catalytically Active CYP 2C9 and 2C19 in Human Liver Microsomes: Application of the Relative Activity Factor Approach

Relative Quantities of Catalytically Active CYP 2C9 and 2C19 in Human Liver Microsomes: Application of the Relative Activity Factor Approach KARTHIK VENKATAKRISHNAN, LISA L.

VON

MOLTKE,

AND

DAVID J. GREENBLATT*

Contribution from Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, and the Division of Clinical Pharmacology, New England Medical Center Hospital, Boston, Massachusetts. Received November 14, 1997. Final revised manuscript received April 13, 1998. Accepted for publication April 16, 1998. Abstract 0 The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4′-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CYP/ mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblastdetermined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6− 17.9; n ) 10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04−16.2; n ) 10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immunoquantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.

Introduction Human liver microsomes can contain 15 or more different cytochrome P450 (CYP) enzymes that biotransform xenobiotics and/or endogenous substrates.1 Due to their broad and overlapping substrate specificities, two or more CYP enzymes often contribute to the metabolism of a single * Address for correspondence: David J. Greenblatt, M.D., Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: 617-636-6997, Fax: 617-636-6738.

© 1998, American Chemical Society and American Pharmaceutical Association

drug. Thus, it is important to determine both the identity and the relative contribution of each isoform to net metabolism of the drug. The substrate concentration-velocity relationship for a hepatic drug biotransformation process catalyzed by multiple CYP isoforms can be described as a linear combination of velocity functions for each of the CYP isoforms [Vi(s)] weighted for the relative abundance of the respective isoforms (Ai) in hepatic tissue: n

V(s) )

∑A v (s) i i

(1)

i)1

With the availability of heterologously expressed human CYPs,2,3 it has become increasingly common to define the substrate concentration-velocity functions [vi(s)] for whatever individual CYPs biotransform a drug. Such analyses indicate the relative affinities and capacities (turnover number ) mol product formed/mol CYP/min; min-1) of the pertinent CYPs contributing to a certain metabolic pathway. However the relative contribution of each enzyme as a function of substrate concentration still cannot be estimated unless the results are considered together with the abundance factors (Ai). Western blot analysis of human liver microsomes using isoform-specific antibodies has been widely used to study interindividual and interspecies differences in the levels of expression of the various CYP isoforms and to correlate these levels to the rates of metabolism of model isoformspecific substrates.4-6 A quantitative western blot analysis of 60 human liver microsomal preparations suggested that CYPs 1A2, 2A6, 2B6, 2C, 2D6, 2E1, and 3A on average constitute 12.7, 4, 0.2, 18.2, 1.5, 6.6, and 28.8%, respectively, of total hepatic CYP.5 However, the study did not estimate the relative levels of expression of the major drug metabolizing CYP2C isoforms, 2C9/10 and 2C19, possibly because the antibody against “human P-450 2CMP” was of uncertain immmunochemical specificity.5 In a recent study of imipramine metabolism by cDNA expressed human CYPs,7 it has been suggested that CYP2C19 accounts for 0.5% of total hepatic CYPs, although the basis for this assumption was not presented. In a study of citalopram N-demethylation,8 the average relative activity factor (RAF; see below) for CYP2C19 was 46-fold lower than that for CYP3A4. These RAFs, together with the known abundance of CYP3A4 in human liver,5 suggest that CYP 2C19 may represent only a small fraction of total 2C in human liver (under the assumption that immunochemically determined CYP3A4 levels are linearly related to RAF3A4). Recently, Inoue et al.9 determined the ratio of CYP2C19 to 2C9 protein levels by immunoquantitation in a large panel of Japanese and Caucasian human livers. Their study concluded that CYP2C9 and 2C19 account for 20 and 1% of total hepatic CYP, respectively. However,

S0022-3549(97)00435-8 CCC: $15.00 Published on Web 06/06/1998

Journal of Pharmaceutical Sciences / 845 Vol. 87, No. 7, July 1998

when quantitative western blotting was used to determine the relative abundances of CYP2C isoforms in human liver in the context of a study of diazepam N-demethylation, CYPs 2C9 and 2C19 were essentially equiabundant.10 Given the large number of drugs metabolized at least in part by CYP2C19,11 and the extensive use of cDNAexpressed CYP2C isoforms to kinetically characterize these biotransformations,7,8,10,12-14 it is essential that a consensus be reached regarding the ratio of catalytically active CYP2C9 and 2C19 (the major drug metabolizing human 2C isoforms) in human liver. The objective of this study was to determine the relative quantities of catalytically active CYPs 2C9/10 and 2C19 isoforms in human liver microsomes and to characterize commercially available heterologously expressed CYP2C19 and 2C9 that are frequently used in drug metabolism research. We have used a functional activity-based approach described by Crespi3 to determine the abundance factors Ai, hereafter referred to as relative activity factors (RAFs).3 As reviewed earlier,3 RAFs are determined for specific CYP isoforms by comparing the rate of an isoformspecific index reaction at saturating substrate concentrations in human liver microsomal preparations (that is, Vmax for liver microsomes) to the rate of the same reaction catalyzed by the specific cDNA-expressed CYP isoform measured under identical conditions (that is, Vmax for cDNA expressed enzyme): RAFisoform ) mean Vmax for isoform-specific reaction in human liver microsomes Vmax for isoform-specific reaction by cDNA expressed isoform (2)

This approach assumes that: (a) The index reaction used is virtually completely specific for the CYP isoform whose RAF is being determined, even at saturating substrate concentrations. (b) At saturating concentrations of substrate, the velocity of an isoform-specific index reaction (Vmax) catalyzed by human liver microsomes is a linear function of the amount of that enzyme present in active form. (c) Any effects on Vmax are substrate-independent, i.e., RAF estimates for a particular isoform will be the same regardless of the index reaction used in the analysis, provided the same reaction is used for the cDNA-expressed enzyme and for human liver microsomes. (d) The CYP isoform in human liver is catalytically identical both qualitatively (in terms of substrate specificity and regio- and/or stereoselectivity) and kinetically (in terms of substrate affinity Km and turnover number (i.e., pmoles product formed per minute per pmole CYP) for the substrate being examined). (e) When RAFs are estimated for multiple CYP isoforms in an attempt to determine the relative quantities of these isoforms in human liver, the same expression system should be used for all the isoforms, so that the RAFs are universally applicable for use as Ais in eq 1 regardless of the expression system used for defining the functions vi(s) (assuming, again, that the vi(s) functions themselves are determined using a common expression system) If, however, RAFs are estimated for multiple CYP isoforms using expression systems that are not identical in all aspects, their use as Ais in the simulation of the relative contribution of the various CYP isoforms mediating a multienzyme biotransformation pathway is contingent upon the requirement that each of the functions vi(s) be determined using the same expression system that was used for determination of the RAF for that particular isoform. Assumption e ensures that differences in turnover number of a CYP isoform, that may be related to the nature 846 / Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 1998

of the expression system, do not ultimately affect the conclusions based on application of eq 1 to a pathway catalyzed by multiple enzymes. This report describes the determination of RAFs for CYP2C9 and 2C19 in human liver microsomes using tolbutamide methylhydroxylation and S-mephenytoin 4′-hydroxylation activities as index reactions for these isoforms, respectively. We have determined the RAFs for the 2C enzymes using both human lymphoblast cell-expressed and insect cell-expressed CYPs. In addition, we have compared the average RAFs for these CYPs in human livers to the RAFs in SUPERMIX, a mixture of cDNA-expressed human drug metabolizing CYPs (Gentest Corporation, Woburn, MA) formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes.

Materials and Methods MaterialssTolbutamide was provided by Pharmacia and Upjohn Co. (Kalamazoo, MI). 4-Hydroxytolbutamide was obtained from RBI, Natick, MA, and chlorpropamide was from Sigma. S-Mephenytoin and 4′-hydroxymephenytoin were purchased from Ultrafine Chemicals, Manchester, England. Phenacetin was purchased from Mallinckrodt, St. Louis, MO. Liver samples, obtained from the International Institute for the Advancement of Medicine (Exton, PA), or the Liver Tissue Procurement and Distribution Service (University of Minnesota), were from twelve different transplant donors (L1-L12) with no known liver disease. The tissue was partitioned and kept at -80 °C until the time of microsome preparation as described previously.15,16 Microsomes from cDNA transfected human lymphoblastoid cells expressing CYP1A2, 2C9-Arg144, 2C19, 2D6, 2E1, or 3A4,3 and from insect cells expressing 2C9-Arg144 or 2C19 were purchased from Gentest Corporation (Woburn, MA), aliquoted and stored at -80 °C, and thawed on ice before use. All cDNA-expressed CYPs used in the study were coexpressed with NADPH cytochrome P450 oxidoreductase (OR) with the exception of lymphoblast-expressed CYP2C19, the activity of which was supported by endogenous levels of reductase native to the host cell line. Microsomal protein concentrations and CYP content were provided by the manufacturer. SUPERMIX was purchased from Gentest as well. This product is a mixture of microsomes prepared from insect cells (BTITN-5B1-4) synthesizing individual cDNA-expressed enzymes (CYP 1A2, 2C8, 2C9, 2C19, 2D6, 3A4) using a baculovirus expression system. Control microsomes prepared from both vector transfected human lymphoblastoid cells and wild-type virus infected insect cells were also studied and had no detectable S-mephenytoin or tolbutamide hydroxylase activity. Purified recombinant human NADPH cytochrome P450 reductase was purchased from Oxford Biomedical Research Inc, Oxford, MI. MethodssIncubations of tolbutamide with human liver microsomes were performed using standard procedures as described earlier for other reactions.17 Reactions were performed for 40 min with a microsomal protein concentration of 500 µg/mL. Reactions using lymphoblast-expressed CYPs were performed for 40 min at a protein concentration of 1 mg/mL. In the case of insect cellexpressed CYPs and SUPERMIX, the reactions were performed for 20 min. Protein concentrations in incubates were 160 µg/mL for insect cell-expressed enzymes, and 270 µg/mL for SUPERMIX. Substrate turnover was minimal, and protein concentrations and reaction times were chosen to be in the linear range. HPLC analysis of 4′-hydroxytolbutamide in incubates was performed using a modification of a previously described procedure.18 Chromatography was performed on a C18 µBondapak column using a mobile phase composed of 10 mM sodium acetate (pH 3) and acetonitrile in a 73:27 ratio at a flow rate of 2 mL/min, and UV detection was used at 230 nm. An eleven point 4′-hydroxytolbutamide formation curve (tolbutamide 0-2500 µM) was used to characterize the kinetics of tolbutamide methylhydroxylation. Incubations of S-mephenytoin with human liver microsomes and HPLC analysis of 4′-hydroxymephenytoin in incubates were performed as previously described.17 A microsomal protein concentration of 500 µg/mL and a 60 min reaction time was used for human liver microsomes. Reactions using lymphoblast-expressed

Figure 1s(A) Tolbutamide methylhydroxylation by human liver microsomes and SUPERMIX. Symbols are experimental data points. Lines are fitted functions described by eq 3: a one-enzyme Michaelis−Menten model. Solid symbols and lines are for two representative human liver samples L5 and L11. Open squares and the dashed line are for SUPERMIX. (B) Tolbutamide methylhydroxylation by cDNA expressed CYP2C9 and 2C19. Symbols are experimental data points. Lines are fitted functions. Solid symbols and lines are for lymphoblast-expressed enzymes (upright triangles for CYP2C19 and inverted triangles for CYP2C9 + OR). Open upright triangles and the dashed line are for insect cell-expressed CYP2C19 + OR. Open inverted triangles and the dotted line are for insect cell-expressed CYP2C9 + OR. See Table 1 and Figure 3 for kinetic parameters. CYP2C19 were performed for 60 min at a protein concentration of 1 mg/mL. Incubations with insect cell-expressed CYP2C19 were done for 10 min at a protein concentration of 32 µg/mL while those with SUPERMIX were done for 20 min at a protein concentration of 270 µg/mL. Substrate turnover was minimal, and protein concentrations and reaction times were chosen to be in the linear range. An eleven point 4′-hydroxymephenytoin formation curve (S-mephenytoin 0-1000 µM) was used to characterize the kinetics of S-mephenytoin 4′-hydroxylation. The kinetics of the index reactions by liver microsomes or cDNAexpressed CYPs were described by one of the following models: a one enzyme Michaelis Menten model:

V)

VmaxS Km + S

(3)

a two enzyme model:

V)

Vmax(1)S Km(1) + S

+

Vmax(2)S Km(2) + S

(4)

a one enzyme Michaelis-Menten model with uncompetitive substrate inhibition:

VmaxS

V)

Km + S +

S2 Ks

(5)

a Hill enzyme model:

V)

VmaxSA KmA + SA

(6)

Km is the substrate concentration at which the reaction velocity is 50% of Vmax, the maximal reaction velocity. Ks is a parameter indicating the degree of substrate inhibition. A is the Hill number. Kinetic parameters for the reactions were determined by nonlinear regression analysis using the Sigmaplot software (Jandel Scientific). The effect of OR on the activity of CYP2C19 as a tolbutamide methylhydroxylase was studied as described previously.19,20 Microsomes were preincubated with OR or buffer (controls) at 37 °C for 30 min before use in the reaction. Microsomes preincubated with or without OR were mixed with prewarmed assay buffer, and reactions were initiated with the addition of NADPH regenerating system and performed for 20 min at 37 °C.

Results The kinetics of tolbutamide methylhydroxylation in human liver microsomes could be described by a single enzyme Michaelis-Menten model (eq 3; Figure 1A) in all 12 livers studied, although a two-enzyme model could be better fitted to the data (based on comparison of the Akaike’s Information Criteria21) in livers L1 and L9. For purposes of this study, kinetic parameters based on a single enzyme model (Table 1) were used for all the livers, since this model appeared adequate based on visual inspection of fits. No detectable tolbutamide methylhydroxylase activity was observed by lymphoblast-expressed CYPs 1A2, 2D6, 2E1, and 3A4. For tolbutamide methylhydroxylation by lymphoblast-expressed CYP2C9 + OR, the uncompetitive substrate inhibition model (eq 5) was best fitted to the data (Figure 1B, Table 1). Single enzyme kinetics (eq 3) was seen with lymphoblast-expressed CYP2C19 (Figure 1B, Table 1), insect cell-expressed CYP2C9 + OR and 2C19 + OR (Figure 1B, Table 1), and SUPERMIX (Figure 1A, Table 1). Journal of Pharmaceutical Sciences / 847 Vol. 87, No. 7, July 1998

Table 1sKinetic Parameters for Tolbutamide Hydroxylation and S-Mephenytoin Hydroxylation by Lymphoblast (L) and Insect Cell (IC)-Expressed CYPs 2C9, 2C19, Human Liver Microsomes and SUPERMIX. Relative Activity Factors Have Been Calculated As Desribed in the Text human liver Kma insect Kma insect Vmaxb Vmax/Kmc Vmaxb Vmax/Kmc sample or cDNA (Tolbutamide (Tolbutamide (Tolbutamide lymphoblast cell (S-mephenytoin (S-mephenytoin (S-mephenytoin lymphoblast cell expressed CYP hydroxylation) hydroxylation) hydroxylation) RAF2C9d RAF2C9d hydroxylation) hydroxylation) hydroxylation) RAF2C19d RAF2C19d CYP2C9 + OR (L)e CYP2C9 + OR (IC) CYP 2C19 (L) CYP2C19 + OR (IC)f SUPERMIX L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 mean ± SE for human livers

125.6 118.5 477.9 181.7 131.7 210.3 312.5 215.1 188.5 162.2 119.3 152.5 147.3 158.0 283.4 299.4 275.4 210.3 ± 19

7.17 7.29 1.26 11.43 1802.3 691.8 501.1 598.6 534.2 389.7 538.4 577.9 521.5 680.6 569.6 1041.8 852.5 624.8 ± 51

0.057 0.062 0.003 0.063 13.68 3.29 1.60 2.78 2.83 2.40 4.51 3.79 3.54 4.31 2.01 3.48 3.10 3.14 ± 0.3

48.7 34.5 251.4 201.4 96.5 47.0 69.9 5.0 83.5 46.5 74.5 31.4 54.4 23.4 75.1 5.5 80.6 25.1 72.8 1.6 94.9 64.1 79.5 71.2 145.3 142.9 118.9 116.9 87.2 ± 7 48.6 ± 13

53.3 37.5 60.0 58.4 47.2 39.7 30.0 38.1 37.5 26.2 g 45.5g 1903.3 1591.2 42.0 ± 4i

5.5 5.6

0.11 0.16

162.0 171.1 227.6 127.2 149.7 107.3 237.0 193.4 249.8 104.7 g 24.6g 143.0 122.8 159.2 ± 22i

3.04 4.57 3.79 2.18 3.17 2.70 7.91 5.07 6.66 4.00g 0.54g 0.08 0.08 4.06 ± 0.7i

29.3 30.9 41.1 23.0 27.0 19.4 42.8 34.9 45.1 18.9 4.4 NDh NDh 28.8 ± 4i

28.9 30.6 40.6 22.7 26.7 19.2 42.3 34.5 44.6 18.7 4.4 NDh NDh 28.4 ± 4i

a In µM. b In pmol/min/mg protein for livers and SUPERMIX; in pmol/min/pmol CYP for CYPs 2C9 and 2C19. c In µL/min/mg protein for livers and SUPERMIX; in µL/min/pmol CYP for CYPs 2C9 and 2C19. d In pmol CYP/mg protein. e Kinetics of tolbutamide hydroxylation by lymphoblast-expressed CYP2C9 + OR showed substrate inhibition (eq 5) with a Ks of 10874 µM. f Kinetics of S-mephenytoin hydroxylation by insect cell-expressed CYP2C19 + OR showed substrate activation (eq 6) with a Hill number A of 1.2. g Biphasic kinetics of S-mephenytoin hydroxylation observed in L9 and L10. Table entries are parameter estimates for the high affinity component. Parameter estimates for the low affinity component were Vmax 212.8 pmol/min/mg protein and Km 4500 µM for L9, and Vmax 38.6 pmol/min/mg protein and Km 754 µM for L10. h RAF2C19 not determined for L11 and L12 due to very high Km and low intrinsic clearance of S-mephenytoin hydroxylation, probably reflecting a CYP 2C19 deficiency. i With livers L11 and L12 excluded.

The kinetics of S-mephenytoin 4′-hydroxylation in human liver microsomes could be described by a single enzyme Michaelis-Menten model (eq 3; Figure 2A, Table 1) in 10 of the 12 livers studied. A two-enzyme model (eq 4) was necessary to describe the kinetics of this reaction by livers L9 and L10 (Figure 2B, Table 1) since a one enzyme model did not yield acceptable fits (based on visual examination of goodness of fit, residual analysis, and standard errors of parameter estimates). Livers L11 and L12 had abnormally high values of Km and low Vmax/Km ratios (Figure 2A, Table 1) and showed no detectable S-mephenytoin hydroxylase activity at substrate concentrations below 50 µM. These livers were thus considered to be poor metabolizers of S-mephenytoin (probably CYP2C19 deficient) and excluded from RAF determination for this isoform. No detectable S-mephenytoin 4′-hydroxylase activity was observed by lymphoblast-expressed CYPs 1A2, 2C9, 2D6, 2E1, and 3A4. S-Mephenytoin 4′-hydroxylation by lymphoblast-expressed CYP2C19 (Figure 2C) and by SUPERMIX (Figure 2A) exhibited single enzyme kinetics (Table 1) while insect cell-expressed CYP2C19 + OR (Figure 2C) showed Hill enzyme kinetics (eq 6). Figure 3 shows the enzyme kinetic properties of the four heterologously expressed CYPs used in the study for tolbutamide methylhydroxylation (top panel) and S-mephenytoin 4′-hydroxylation (bottom panel). As shown in the figure, the nature of the expression system or coexpression of OR did not significantly alter the properties of CYP2C19 as a S-mephenytoin hydroxylase. In addition, the kinetic properties of lymphoblast-expressed and insect cell-expressed CYP2C9 + OR were similar with respect to tolbutamide methylhydroxylation. However, the properties of lymphoblast-expressed CYP2C19 and insect cell-expressed CYP2C19 + OR as tolbutamide methylhydroxylases differed greatly, suggesting that coexpression of OR and/or use of the baculovirus expression system in insect cells increases the catalytic efficiency of CYP2C19 as a tolbutamide methylhydroxylase without greatly affecting its properties as a S-mephenytoin 4′-hydroxylase. 848 / Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 1998

Since the intrinsic clearance of tolbutamide methylhydroxylation by lymphoblast-expressed CYP2C19 was significantly lower than that by lymphoblast-expressed CYP2C9 + OR (Figure 3), the lymphoblast RAF2C9 was calculated using eq 2, assuming tolbutamide methylhydroxylation to be a CYP2C9 specific reaction. However, the insect cell RAF for CYP2C9 was calculated using the following equation (derived from eqs 1 and 2): insect cell RAF2C9 ) Vmax,TMH,human liver microsomes - (Vmax,TMH,insect cell CYP2C19+ORRAF2C19) Vmax,TMH,insect cell CYP2C19+OR (7)

TMH refers to tolbutamide methylhydroxylation. RAFs for CYP2C19 were calculated for each liver and for SUPERMIX using the Vmax values for S-mephenytoin 4′-hydroxylation in eq 2. In livers exhibiting biphasic kinetics of S-mephenytoin 4′-hydroxylation, the Vmax for the high affinity component (with Km closest to that of cDNAexpressed CYP2C19) was used in calculation of the RAF, since the low-affinity component constituted only a small contribution to net intrinsic clearance. Mean values for human livers, and SUPERMIX values, were nearly identical regardless of the expression system (Table 2). However RAFs for CYP2C9 differed substantially between human livers and SUPERMIX, with higher values determined for SUPERMIX (Table 2). In both human liver microsomes and SUPERMIX, RAF values for 2C9 were lower for insect cells than for human lymphoblast cells.

Discussion Several index reactions to evaluate CYP2C9 activity have been reported in the literature. These include phenytoin para-hydroxylation,22-24 tolbutamide methylhydroxylation,18,22-26 torsemide tolyl methylhydroxylation,27 diclofenac 4′-hydroxylation,28 (S)-warfarin 7-hydroxylation,29-31

Figure 2s(A) S-Mephenytoin hydroxylation by human liver microsomes and SUPERMIX. Symbols are experimental data points. Lines are fitted functions described by eq 3: a one-enzyme Michaelis−Menten model. Solid symbols and lines are for two representative human liver samples L2 and L3. Open squares and the heavy dashed line are for SUPERMIX. Open circles with the dotted line and open diamonds with the thin dashed line are for livers L11 and L12 (poor metabolizers of S-mephenytoin; see text and Table 1 for details), respectively. Note that the points for L11 and L12 are almost superimposable. (B) S-Mephenytoin hydroxylation by human liver microsomes L9 and L10. Symbols are experimental data points. Lines are fitted functions described by eq 4: a two-enzyme Michaelis−Menten model. (2) S-mephenytoin hydroxylation by cDNA expressed CYP2C19. Symbols are experimental data points. Lines are fitted functions. Solid symbols and the solid line are for lymphoblast-expressed CYP2C19. Open symbols and the dashed line are for insect cell-expressed CYP2C19 + OR. See Table 1 and Figure 3 for kinetic parameters.

flurbiprofen 4′-hydroxylation,32,33 tienilic acid 5-hydroxylation,34 and (S)-ibuprofen 2- and 3-hydroxylations.35 These reactions are CYP2C9 specific at low substrate concentrations such as those attained clinically and those used in classical inhibition experiments25 performed to test new drugs as inhibitors of CYP2C9. However, given the broad substrate specificity of the CYP system, and the high amino acid sequence identity between members of the human CYP2C family (91% between 2C9/10 and 2C19), it is not surprising that other CYP isoforms (notably 2C19) may also mediate these reactions, although with an order of magnitude lower affinity than for CYP2C9. This has been shown for phenytoin hydroxylation in kinetic studies using cDNA expressed CYP isoforms.36 Not all of the “index reactions” described above have been rigorously studied using cDNA expressed enzymes to exclude participation of other low affinity CYP isoforms at high substrate concentrations. Assumption a of the RAF model requires an index reaction that is catalyzed exclusively by CYP2C9 even at saturating substrate concentrations, well above the Km. Thus any reaction that is primarily mediated by CYP2C9 with at least an order of magnitude lower intrinsic clearance via other contributing CYP isoforms should reasonably approximate the requirement for validity of assumption a. We have used tolbutamide methylhydroxylation as a measure of CYP2C9 activity. Although lymphoblastexpressed CYP2C19 could hydroxylate tolbutamide at high substrate concentrations, the intrinsic clearance for this reaction mediated by this isoform was more than 1 order of magnitude lower than that for CYP2C9 + OR (Table 1). Although this reaction does not perfectly fulfill assumption a, the level of specificity is sufficiently high to justify the use of the reaction as a measure of CYP2C9 activity.

However, when we examined insect cell-expressed CYP2C19 + OR and 2C9 + OR, both isoforms showed equal intrinsic clearance as tolbutamide methylhydroxylases (Figure 3). This large difference in intrinsic clearance between lymphoblast and insect cell-expressed CYP2C19 was due to both an increase in Vmax and a decrease in Km. This may be related to: (a) coexpression of OR in the insect cellexpressed 2C19, and/or (b) nature of the expression system. On the basis of cytochrome c reductase activities in the enzymes (provided by the manufacturer), we calculated the ratio of reductase activity to CYP content in CYP2C19 from both expression systems. This ratio was 1.14 nmol cytochrome c reduced/min/pmol CYP for the lymphoblast expressed 2C19 and 2.42 nmol cytochrome c reduced/min/ pmol CYP for the insect cell-expressed enzyme. To further investigate the effect of reductase on the activity of CYP2C19 as a tolbutamide methylhydroxylase, we supplemented CYP2C19 microsomes from both expression systems with exogenously added NADPH cytochrome P450 reductase. When OR was added to lymphoblastexpressed CYP2C19 microsomes to yield a final ratio of reductase activity to CYP content equivalent to that in the insect cell-expressed CYP2C19 + OR, no increase in activity was observed at 2500 µM tolbutamide concentration. This may be because supplementation of OR exogenously is not equivalent to coexpression of the OR enzyme with the CYP.37 However, when saturating amounts of OR (5-fold molar excess over CYP) were added, the activity of lymphoblast-expressed CYP2C19 was increased by 151%, while that of insect cell-expressed CYP2C19 + OR was increased by only 52%. Still, the activity of lymphoblastexpressed CYP2C19, even with addition of OR, did not approach that of the insect cell-expressed CYP2C19 + OR. In any case, an insect cell RAF for CYP2C9 cannot be Journal of Pharmaceutical Sciences / 849 Vol. 87, No. 7, July 1998

Figure 3sKinetic parameters for tolbutamide methylhydroxylation (top panel) and S-mephenytoin 4′-hydroxylation (bottom panel) by cDNA-expressed CYPs 2C9 and 2C19. Tolbutamide methylhydroxylation by lymphoblast-expressed CYP2C9 + OR displayed substrate inhibition (Ks 10870 µM). S-Mephenytoin 4′-hydroxylation by insect cell-expressed CYP2C19 + OR displayed substrate activation (Hill number A 1.2). Table 2sRelative Activity Factor Estimates for CYP2C9 and 2C19 in Human Liver Microsomes and SUPERMIX Determined Using Lymphoblast-Expressed or Insect Cell-Expressed Enzymes human liversa,b

RAF2C9 (n ) 12) RAF2C19 (n ) 10) RAF2C9:RAF2C19 (n ) 10)

SUPERMIXb

lymphoblast

insect cell

lymphoblast

insect cell

87.2 ± 7 (54−145) 28.8 ± 4 (4−45) 3.03 (1.6−17.9)

48.6 ± 13 (1.6−143) 28.4 ± 4 (4−45) 1.71 (0.04−16.2)

251.4

201.4

29.3

28.9

8.58

6.97

a Values are mean ± SEM ranges in parentheses. b RAF values in pmol CYP/mg microsomal protein.

determined using tolbutamide methylhydroxylation applying eq 2 directly. We thus modified the equation to account for the contribution of CYP2C19 and used eq 7 to calculate insect cell CYP2C9 RAFs for human liver microsomes and SUPERMIX. Since a one-enzyme Michaelis-Menten model could adequately describe tolbutamide methylhydroxylation in all the livers studied, the lymphoblast-expressed CYP2C9 + OR and SUPERMIX, calculation of RAFs for this isoform was straightforward (eq 2). Average Km for this reaction by human liver microsomes has been previously reported to be 74 µM,41 125 µM,42 and 120 µM.43 These values are lower than the average Km of 210 µM for tolbutamide methylhydroxylation in our bank of 12 livers (Table 1). Evidence of substrate inhibition was observed using lymphoblast-expressed CYP2C9 + OR; this has not been described previously. However estimates of Vmax for lymphoblast-expressed CYP2C9 + OR (6.3 pmol/min/pmol CYP 850 / Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 1998

with a simple one enzyme model vs 7.2 using a one-enzyme model with substrate inhibition) and the Vmax/Km ratio (0.066 vs 0.057) were not greatly influenced by the choice of model. The choice of model did influence the estimated Km (95 µM vs 126 µM). Insect cell-expressed CYP2C9 did not show evidence of substrate inhibition, although the Vmax and Km were similar to those of the lymphoblast-expressed CYP2C9 + OR. The CYP2C9 we have used is 2C9-Arg144 which is the most common allelic form of this enzyme in human liver. A Km value of 90.5 µM has been determined for this allelic form expressed in COS cells.24 Substrate inhibition was not evident in human liver microsomes or SUPERMIX, possibly because of the minor contribution of CYP2C19 to this reaction at saturating concentrations of substrate. The difference in Km between CYP2C9-Arg144 and human liver microsomes may be explained by the fact that human liver contains CYP2C8, CYP2C10, and other allelic forms of CYP2C9 as well, all of which are lower affinity tolbutamide methylhydroxylases when compared to CYP2C9-Arg144.24 The Km for this reaction by SUPERMIX was closer to that for CYP2C9-Arg144 rather than human liver probably because other allelic forms of CYP2C9 are not present in SUPERMIX. Incubations with insect cell-expressed CYP2C9 + OR and SUPERMIX were done at total protein concentrations lower than that used in lymphoblast-expressed CYP2C9 + OR or human liver microsomal incubations. This was required in order to minimize substrate turnover. Use of different protein concentrations could in principle lead to differences in apparent Km due to nonspecific microsomal protein binding of the substrate.44 However, our Km values for tolbutamide methylhydroxylation in lymphoblast-

expressed CYP2C9 + OR, insect cell-expressed CYP2C9 + OR, and SUPERMIX were similar (Figure 3, Table 1), although the protein concentrations used in the three systems were different (see Methods). The difference in Kms of human liver microsomes and heterologously expressed enzymes may thus be related to the presence of allelic variants of CYP2C9 in human liver that are low affinity tolbutamide methylhydroxylases. None of the cDNA expressed enzymes tested except CYP2C19 catalyzed S-mephenytoin hydroxylation, validating its use as an index reaction for CYP2C19. The specificity of this reaction has not been previously evaluated using human lymphoblastoid cell expressed recombinant human CYPs. A study using yeast expressed human CYPs showed that CYP2C19 is the major human Smephenytoin hydroxylase,38 but other CYP isoforms in human liver, such as CYP3A4 and 1A2, were not tested. We evaluated the specificity of this index reaction for CYP2C19 activity using microsomes containing CYPs expressed in human lymphoblastoid cells, since the nature of the expression system used may alter substrate specificity for reasons that are not entirely clear. For example, while E. coli-expressed human CYP2C19 (10) and vaccinia virus directed Hep G2 cell-expressed CYP2C19 3-hydroxylated diazepam,39 we have been able to demonstrate no detectable activity of this isoform expressed in human lymphoblastoid cells as a diazepam 3-hydroxylase (unpublished data). Another example is the role of CYP1A2 in amitriptyline N-demethylation, which could not be detected using yeast expressed CYP1A2,40 but was demonstrated using human lymphoblastoid cell expressed CYP1A2 in our laboratory.14 Interindividual variations in Km values for S-mephenytoin 4′-hydroxylation among different human livers was small, and the mean Km for liver microsomes (42 µM), and the Km for cDNA-expressed CYP2C19 (49 µM for lymphoblast-expressed enzyme and 35 µM for insect cell-expressed enzyme) and SUPERMIX (53 µM) were very similar. Substantially greater variability was seen for tolbutamide methylhydroxylation. This is because the former is a oneenzyme reaction in human liver while the latter is catalyzed by an enzyme that exhibits significant allelic variation that alters the enzyme’s Km. Although insect cellexpressed CYP2C19 + OR was 20-fold more efficient than lymphoblast-expressed CYP2C19 as a tolbutamide methylhydroxylase, such differences were not observed between the enzymes when S-mephenytoin 4′-hydroxylation was studied (Figure 3). These observations suggest that differences between expression systems and/or due to coexpression of reductase may be substrate-dependent. These findings have important implications to the use of heterologously expressed CYP2C19 from the lymphoblast versus the insect cell expression systems as tools for screening purposes when new drug biotransformation pathways are studied. The identity of the high Km S-mephenytoin hydroxylase in livers L11 and L12 is not established, but it may represent the same low affinity component observed in livers L9 and L10. We have excluded livers L11 and L12 from our RAF analysis, since these samples appear to be CYP2C19 deficient, given that this enzyme is polymorphically expressed. Our findings with the 10 livers (L1-L10) using the lymphoblast RAFs suggest that catalytically active CYP2C9 is present in larger amounts than catalytically active CYP2C19, with the ratio of average RAFs of 3. However, when insect cell RAFs were used in the analysis, the ratio of RAF2C9 to RAF2C19 ranged from 0.04 to 16.2 (ratio of average RAFs 1.7). Neither of these activity ratios equals the ratio of mass abundances of these isoforms (20:1)

determined by Western blotting in an earlier study.9 Activity-based RAFs do not necessarily reflect the immunoquantified levels of each isoform. For example, rates of tolbutamide methylhydroxylation correlated well with the rates of phenytoin 4′-hydroxylation, and with the rates of (S)-warfarin 7-hydroxylation in a bank of 14 human liver microsomal preparations.45 Although all these reactions are known to be primarily catalyzed by CYP2C9, the levels of immunoquantified CYP2C9 did not correlate with the rates of tolbutamide methylhydroxylation in the same liver samples.45 This supports the use of RAFs as the abundance factors in predicting the contribution of a CYP isoform to overall biotransformation of a drug metabolized by multiple CYPs. SUPERMIX, a commercially available formulation of insect cell-expressed CYPs, is formulated to match the natural distribution of CYPs in human liver microsomes. Two differences between SUPERMIX and human liver microsomes are evident from the present study: (a) The apparent Km of tolbutamide methylhydroxylation by SUPERMIX is closer to that of heterologously expressed CYP2C9 than to human liver microsomes (most likely reflective of the absence of low affinity allelic forms of CYP2C9 in SUPERMIX); (b) The Vmax of tolbutamide methylhydroxylation by SUPERMIX is 2.9-fold higher than the average value in microsomes from our bank of 12 human livers. The higher Vmax of tolbutamide methylhydroxylation need not necessarily stem from higher molar amounts of CYP2C9 relative to human liver microsomes. Low affinity allelic forms of CYP2C9 such as CYP2C9Cys144 have not only lowered affinity but also lower capacity (turnover number) which may explain the lower Vmax observed in human liver microsomes. The higher Vmax and lower Km of tolbutamide methylhydroxylation by SUPERMIX result in a 4.4-fold higher intrinsic clearance in comparison to human liver microsomes. Studies with SUPERMIX yield a RAF2C9:RAF2C19 ratio of 8.6 (using lymphoblast values) or 7 (using insect cell values) for this formulation. Comparison of RAF2C9 values for SUPERMIX with those for human liver microsomes (Table 1) suggests that SUPERMIX may contain catalytically active CYP2C9 in levels greater than that in human liver microsomes. RAF2C19 values of SUPERMIX are, however, well within the range of values observed for human liver microsomes. In summary, we conclude the following: (a) The nature of the expression system does not alter the activity of CYP2C9 as a tolbutamide methylhydroxylase. (b) Coexpression of OR and/or the nature of the expression system does not alter the activity of CYP2C19 as a S-mephenytoin hydroxylase, but greatly alters both the affinity and turnover number of the enzyme as a tolbutamide methylhydroxylase. (c) Our findings with insect cell-expressed CYPs raise questions regarding the specificity of tolbutamide as a CYP2C9 index substrate. (d) The substrate-dependent differences in kinetic properties of two commercially available heterologously expressed CYP2C19 preparations have important implications for the use of these enzymes in studying new drug biotransformations. (e) SUPERMIX has greater tolbutamide methylhydroxylase activity than that in human liver microsomes, while CYP2C19 activity in SUPERMIX is similar to that in human liver microsomes. (f) Although CYP2C9 is reportedly 20 times more mass abundant than CYP2C19 in human liver microsomes based on immunologic quantitation procedures, this ratio may not be an accurate estimate of relative abundance of functional enzyme for use in the prediction of the relative contribution Journal of Pharmaceutical Sciences / 851 Vol. 87, No. 7, July 1998

of CYP2C9 and 2C19 to drug biotransformation rates as a function of substrate concentration.

References and Notes 1. Parkinson, A. An overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Toxicologic Pathol. 1996, 24(1), 45-57. 2. Remmel, R. P.; Burchell, B. Validation and use of cloned, expressed human drug-metabolizing enzymes in heterologous cells for analysis of drug metabolism and drug-drug interactions. Biochem. Pharmacol. 1993, 46(4), 559-566. 3. Crespi, C. L. Xenobiotic metabolizing human cells as tools for pharmacological and toxicological research. Adv. Drug Res. 1995, 26, 179-235. 4. Forrester, L. M.; Henderson, C. J.; Glancey, M. J.; Back, D. J.; Park, B. K.; Ball, S. E.; Kitteringham, N. R.; McLaren, A. W.; Miles, J. S.; Skett, P.; Wolf, C. R. Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics. Biochem. J. 1992, 281, 359-368. 5. Shimada, T.; Yamazaki, H.; Mimura, M.; Inui, Y.; Guengerich, F. P. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 1994, 270(1), 414-423. 6. Shimada, T.; Mimura, M.; Inoue, K.; Nakamura, S.; Oda, H.; Ohmori, S.; Yamazaki, H. Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans. Arch. Toxicol. 1997, 71, 401-408. 7. Koyama, E.; Chiba, K.; Tani, M.; Ishizaki, T. Reappraisal of human CYP isoforms involved in imipramine N-demethylation and 2-hydroxylation: a study using microsomes obtained from putative extensive and poor metabolizers of S-mephenytoin and eleven recombinant human CYPs. J. Pharmacol. Exp. Ther. 1997, 281(3), 1199-1210. 8. Kobayashi, K.; Chiba, K.; Yagi, T.; Shimada, N.; Taniguchi, T.; Horie, T.; Tani, M.; Yamamoto, T.; Ishizaki, T.; Kuroiwa, Y. Identification of cytochrome P450 isoforms involved in citalopram N-demethylation by human liver microsomes. J. Pharmacol. Exp. Ther. 1997, 280(2), 927-933. 9. Inoue, K.; Yamazaki, H.; Imiya, K.; Akasaka, S.; Guengerich, F. P.; Shimada, T. Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methylhydroxylation and Smephenytoin 4′-hydroxylation activities in livers of Japanese and Caucasian populations. Pharmacogenetics 1997, 7, 103113. 10. Jung, F.; Richardson, T. H.; Raucy, J. L.; Johnson, E. F. Diazepam metabolism by cDNA expressed human 2C P450s. Identification of P450 2C18 and P450 2C19 as low Km diazepam N-demethylases. Drug Metab. Dispos. 1997, 25(2), 133-139. 11. Flockhart, D. A. Drug interactions and the cytochrome P450 system. The role of P450 2C19. Clin. Pharmacokinet. 1995, 29(Suppl 1), 45-52. 12. Karam, W. G.; Goldstein, J. A.; Lasker, J. M.; Ghanayem, B. I. Human CYP 2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes. Drug Metab. Dispos. 1996, 24(10), 1081-1087. 13. Olesen, O. V.; Linnet, K. Hydroxylation and demethylation of the tricyclic antidepressant nortriptyline by cDNA expressed human cytochrome P-450 isozymes. Drug Metab Dispos. 1997, 25(6), 740-744. 14. Venkatakrishnan, K.; Greenblatt, D, J.; von Moltke, L. L.; Schmider, J.; Harmatz, J. S.; Shader, R. I. Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: dominance of CYP 2C19 and 3A4. J. Clin. Pharmacol. 1998, 38(2), 112-121. 15. von Moltke, L. L.; Greenblatt, D. J.; Harmatz, J. S.; Shader, R. I. Alprazolam metabolism in vitro: studies of human, monkey, mouse, and rat liver microsomes. Pharmacology 1993, 47, 268-276. 16. von Moltke, L. L.; Greenblatt, D. J.; Cotreau-Bibbo, M. M.; Duan, S. X.; Harmatz, J. S.; Shader, R. I. Inhibition of desipramine hydroxylation in vitro by serotonin-reuptakeinhibitor antidepressants, and by quinidine and ketoconazole: a model system to predict drug interaction in vivo. J. Pharmacol. Exp. Ther. 1994, 268, 1278-1283. 17. Schmider, J.; Greenblatt, D. J.; von Moltke, L. L.; Harmatz, J. S.; Duan, S. X.; Karsov, D.; Shader, R. I. Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies. Pharmacology 1996, 52, 125-34.

852 / Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 1998

18. Miners, J. O.; Birkett, D. J. Use of tolbutamide as a substrate probe for human hepatic cytochrome P450 2C9. Methods Enzymol. 1996, 272, 139-145. 19. Gum, J. R.; Strobel, H. W. Purified NADPH cytochrome P-450 reductase. Interaction with hepatic microsomes and phospholipid vesicles. J. Biol. Chem. 1979, 254(10), 41774185. 20. Sequeira, D. J.; Strobel, H. W. In vitro metabolism of imipramine by brain microsomes: effects of inhibitors and exogenous cytochrome P450 reductase. Brain Res. 1996, 738, 24-31. 21. Yamaoka, K.; Nakagawa, T.; Uno, T. Application of Akaike’s Information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J. Pharmacokinet. Biopharm. 1978, 6, 165-175. 22. Veronese, M. E.; Mackenzie, P. I.; Doecke, C. J.; McManus, M. E.; Miners, J. O.; Birkett, D. J. Tolbutamide and phenytoin hydroxylations by cDNA expressed human liver cytochrome P450 2C9. Biochem. Biophys. Res. Commun. 1991, 175(3), 1112-1118. 23. Ono, S.; Hatanaka, T.; Hotta, H.; Satoh, T.; Gonzalez, F. J.; Tsutsui, M. Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA expressed human P450s and human liver microsomes. Xenobiotica 1996, 26(7), 681-693. 24. Veronese, M. E.; Doecke, C. J.; Mackenzie, P. I.; McManus, M. E.; Miners, J. O.; Rees, D. L. P.; Gasser, R.; Meyer, U. A.; Birkett, D. J. Site-directed mutation studies of human liver cytochrome P450 isoenzymes in the CYP 2C subfamily. Biochem. J. 1993, 289, 533-538. 25. Newton, D. J.; Wang, R. W.; Lu, A. Y. H. Cytochrome P450 inhibitors. Evaluation of specificities in the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metab. Dispos. 1995, 23(1), 154-158. 26. Bourrie´, M.; Meunier, V.; Berger, Y.; Fabre, G. Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J. Pharmacol. Exp. Ther. 1996, 277(1), 321-332. 27. Miners, J. O.; Rees, D. L. P.; Valente, L.; Veronese, M. E.; Birkett, D. J. Human hepatic cytochrome P450 2C9 catalyzes the rate-limiting pathway of torsemide metabolism. J. Pharmacol. Exp. Ther. 1995, 272(3), 1076-1081. 28. Leeman, T.; Transon, C.; Dayer, P. Cytochrome P450 TB (CYP 2C): a major monooxygenase catalyzing diclofenac 4′hydroxylation in human liver. Life Sci. 1993, 52(1), 29-34. 29. Rettie, A. E.; Korzekwa, K. R.; Kunze, K. L.; Lawrence, R. F.; Eddy, A. C.; Aoyama, T.; Gelboin, H. V.; Gonzalez, F. J.; Trager, W. F. Hydroxylation of warfarin by human cDNA expressed cytochrome P-450: a role for P-450 2C9 in the etiology of (S)-warfarin drug interactions. Chem. Res. Toxicol. 1992, 5, 54-59. 30. Kaminsky, L. S.; de Morais, S. M. F.; Faletto, M. B.; Dunbar, D. A.; Goldstein, J. A. Correlation of human cytochrome P450 2C substrate specificities with primary structure: warfarin as a probe. Mol. Pharmacol. 1993, 43, 234-239. 31. Kaminsky, L. S.; Zhang, Z-Y. Human P450 metabolism of warfarin. Pharmacol. Ther. 1997, 73(1), 67-74. 32. Tracy, T. S.; Rosenbluth, B. W.; Wrighton, S. A.; Gonzalez, F. J.; Korzekwa, K. R. Role of cytochrome P450 2C9 and an allelic variant in the 4′-hydroxylation of (R)- and (S)flurbiprofen. Biochem. Pharmacol. 1995, 49(9), 1269-1275. 33. Tracy, T. S.; Marra, C.; Wrighton, S. A.; Gonzalez, F. J.; Korzekwa, K. R. Studies of flurbiprofen 4′-hydroxylation. Additional evidence suggesting the sole involvement of cytochrome P450 2C9. Biochem. Pharmacol. 1996, 52, 13051309. 34. Jean, P.; Lopez-Garcia, P.; Dansette, P.; Mansuy, D.; Goldstein, J. L. Oxidation of tienilic acid by human yeastexpressed cytochromes P-450 2C8, 2C9, 2C18 and 2C19. Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9. Eur. J. Biochem. 1996, 241, 797-804. 35. Hamman, M. A.; Thompson, G. A.; Hall, S. D. Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C. Biochem. Pharmacol. 1997, 54, 3341. 36. Bajpai, M.; Roskos, L. K.; Shen, D. D.; Levy, R. H. Roles of cytochrome P450 2C9 and cytochrome P450 2C19 in the stereoselective metabolism of phenytoin to its major metabolite. Drug Metab. Dispos. 1996, 24(12), 1401-1403. 37. Crespi, C. L.; Miller, V. P. The R144C change in the CYP2C9*2 allele alters interaction of the cytochrome P450 with NADPH: cytochrome P450 oxidoreductase. Pharmacogenetics 1997, 7, 203-210. 38. Goldstein, J. A.; Faletto, M. B.; Romkes-Sparks, M.; Sullivan, T.; Kitareewan, S.; Raucy, J. L.; Lasker, J. M.; Ghanayem,

39.

40.

41. 42.

43.

B. I. Evidence that CYP 2C19 is the major (S)-mephenytoin 4′-hydroxylase in humans. Biochemistry 1994, 33, 17431752. Ono, S.; Hatanaka, T.; Miyazawa, S.; Tsutsu, M.; Aoyama, T.; Gonzalez, F. J.; Satoh, T. Human liver microsomal diazepam metabolism using cDNA expressed cytochrome P450s: role of CYP 2B6, 2C19 and the 3A subfamily. Xenobiotica 1996, 26(11), 1155-1166. Ghahramani, S.; Ellis, S. W.; Lennard, M. S.; Ramsay, L. E.; Tucker, G. T. Cytochromes P450 mediating the Ndemethylation of amitriptyline. Br. J. Clin. Pharmacol. 1997, 43, 137-144. Purba, H. S.; Back, D. J.; Orme, L’E. Tolbutamide 4-hydroxylase activity of human liver microsomes: effect of inhibitors. Br. J. Clin. Pharmacol. 1987, 24, 230-234. Back, D. J.; Tjia, J. F.; Karbwang, J.; Colbert, J. In vitro inhibition studies of tolbutamide hydroxylase activity of human liver microsomes by azoles, sulphonamides and quinolones. Br. J. Clin. Pharmacol. 1988, 26, 23-29. Miners, J. O.; Smith, K. J.; Robson, R. A.; McManus, M. E.; Veronese, M. E.; Birkett, D. J. Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and

relationship to other cytochrome P-450 dependent xenobiotic oxidations. Biochem. Pharmacol. 1988, 37(6), 1137-1144. 44. Obach, R. S. The importance of nonspecific binding in in vitro matrixes, its impact on enzyme kinetic studies of drug metabolism reactions, and implications for in vitro-in vivo correlations. Drug Metab. Dispos. 1996, 24(10), 1047-1049. 45. Hall, S. D.; Hamman, M. A.; Rettie, A. E.; Wienkers, L. C.; Trager, W. F.; Vandenbranden, M.; Wrighton, S. A. Relationships between the levels of cytochrome P450 2C9 and its prototypic catalytic activities in human liver microsomes. Drug Metab. Dispos. 1994, 22(6), 975-978.

Acknowledgments This work was supported by Grants MH-34223, DA-05258, MH19924, and RR-00054 from the Department of Health and Human Services. Dr. von Moltke is the recipient of a Scientist Development Award (K21-MH-01237) from the National Institutes of Mental Health.

JS970435T

Journal of Pharmaceutical Sciences / 853 Vol. 87, No. 7, July 1998