COMPARISON OF IN VITRO AND IN VIVO INHIBITION POTENCIES OF FLUVOXAMINE TOWARD CYP2C19

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1 /03/ $7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 5 Copyright 2003 by The American Society for Pharmacology and Experimental Therapeutics 951/ DMD 31: , 2003 Printed in U.S.A. COMPARISON OF IN VITRO AND IN VIVO INHIBITION POTENCIES OF FLUVOXAMINE TOWARD CYP2C19 CAIPING YAO, KENT L. KUNZE, WILLIAM F. TRAGER, EVAN D. KHARASCH, AND RENÉ H. LEVY Departments of Pharmaceutics (C.Y., R.H.L.), Medicinal Chemistry (K.L.K., W.F.T., E.D.K.), Anesthesiology (E.D.K.), and Neurological Surgery (R.H.L.), University of Washington, Seattle, Washington (Received September 30, 2002; accepted December 19, 2002) This article is available online at ABSTRACT: A previous study suggested that fluvoxamine inhibition potency toward CYP1A2 is 10 times greater in vivo than in vitro. The present study was designed to determine whether the same gap exists for CYP2C19, another isozyme inhibited by fluvoxamine. In vitro studies examined the effect of nonspecific binding on the determination of inhibition constant (K i ) values of fluvoxamine toward CYP2C19 in human liver microsomes and in a cdna-expressed microsomal (Supersomes) system using (S)-mephenytoin as a CYP2C19 probe. K i values based on total added fluvoxamine concentration (K i,total ) and unbound fluvoxamine concentration (K i,ub ) were calculated, and interindividual variability in K i values was examined in six nonfatty livers. K i,total values varied with microsomal protein concentration, whereas the corresponding K i,ub values This work was supported by grants from the National Institutes of Health (GM-32165) and the General Clinical Research Center, University of Washington (MO1RR00037). were within a narrow range (70 80 nm). In vivo inhibition constants (K i iv) were obtained from a study of the disposition of a single oral dose (100 mg) of the CYP2C19 probe (S)-mephenytoin in 12 healthy volunteers receiving fluvoxamine at 0, 37.5, 62.6, and 87.5 mg/day to steady state. In this population, the ratio of (S)-4-hydroxy-mephenytoin formation clearances (uninhibited/inhibited) was positively correlated with fluvoxamine average steady-state concentration with an intercept of 0.85 (r , p < 0.001). The mean ( S.D.) values of K i iv based on total and unbound plasma concentrations were and nm, respectively. Comparison of in vitro and in vivo K i values, based on unbound fluvoxamine concentrations, suggests that fluvoxamine inhibition potency is roughly 40 times greater in vivo than in vitro. There has been a growing interest in predicting in vivo metabolic drug-drug interactions from in vitro systems. In the case of inhibitionbased interactions, there is no consensus on the methodology for accurate predictions of the extent of in vivo inhibition based on in vitro data (Schmider et al., 1999; Yamano et al., 1999; Kohl and Steinkellner, 2000; Komatsu et al., 2000; Yao and Levy, 2002). Several issues remain unsolved, such as estimations of inhibition constants in vitro and inhibitor concentration around the enzyme site in vivo. For example, studies on fluvoxamine inhibition of CYP1A2 have shown that in vitro K i values varied with microsomal protein concentration (Yao et al., 2001), suggesting that the concentration of microsomal protein present in the incubation is a factor contributing to the variance in in vitro K i. However, even after correction for nonspecific binding of fluvoxamine in microsomes, there was still a 10-fold difference between the in vitro inhibition constant and the corresponding in vivo inhibition constant based on unbound fluvoxamine concentration in plasma. The 10-fold underprediction of fluvoxamine inhibition potency toward CYP1A2 activity in vivo, based on in vitro data, may be due to a number of factors. These include, but are not limited to, active uptake of fluvoxamine from plasma into hepatocytes, thereby increasing the amount of inhibitor available to the enzyme (partitioning), the presence of inhibitory metabolites of fluvoxamine in plasma, and/or environmental differences that may differentially affect enzyme behavior or affinity in the two systems. Should it be the case that some type of partitioning alone is the dominant factor governing the in vitro-in vivo difference, one might expect that the RK i (the ratio of the in vitro K i to the in vivo K i based on unbound concentration) of any enzyme inhibited by fluvoxamine would be similar to that of CYP1A2. In essence, RK i would be largely enzyme independent. However, if inhibitory metabolites or enzyme environment play important roles in promoting the RK i difference, RK i values might become enzyme-dependent. A suitable candidate enzyme was sought to test the hypothesis that RK i values will be conserved for a single inhibitor among the family of P450 1 enzymes. There is some in vivo evidence that fluvoxamine also inhibits the clearance of a number of drugs metabolized by CYP2C19, such as mephenytoin, chloroguanide, and diazepam (Perucca et al., 1994a; Xu et al., 1996; Jeppesen et al., 1997). In the case of the fluvoxaminemephenytoin interaction, the 0- to 8-h urine S/R ratio of mephenytoin increased from 0.16 to 0.55 after 100 mg/day fluvoxamine treatment for 2 weeks (Xu et al., 1996). Fluvoxamine also inhibited the CYP2C19 catalyzed bioactivation of chloroguanide: the formation clearance of 4-chlorophenylbiguanide decreased from 97 to 11 ml/min Address correspondence to: Dr. René H. Levy, Department of Pharmaceutics, University of Washington, Seattle, WA rhlevy@u.washington.edu Abbreviations used are: P450, cytochrome P450; 4-OH-meph, 4-hydroxymephenytoin; p-hpph, 5-(4 -hydroxyphenyl)-5-phenylhydantoin; IS, internal standard; HLM, human liver microsome; LC-MSD, liquid chromatography-mass spectrometric detection; AUC, area under the plasma concentration-time curve.

2 566 YAO ET AL. in CYP2C19-extensive metabolizers treated with 100 mg/day fluvoxamine for 6 days (Jeppesen et al., 1997). Diazepam oral clearance decreased from 0.4 to 0.14 ml/min/kg after treatment with 100 to 150 mg/day fluvoxamine for 4 days (Perucca et al., 1994a). The extent of inhibition observed in vivo is underpredicted with the reported in vitro K i values of fluvoxamine toward CYP2C19 ( M; Rasmussen et al., 1998; Olesen and Linnet, 2000), when they are used in conjunction with the unbound plasma concentration of fluvoxamine. Therefore, it seems that a gap exists between in vitro and in vivo inhibition potencies of fluvoxamine toward CYP2C19. The present study used (S)-mephenytoin as a probe substrate for CYP2C19 (CDER, 1999; Ring and Wrighton, 2000) to test the hypothesis that RK i is enzyme-independent. Both in vitro and in vivo K i values were determined for fluvoxamine inhibition of CYP2C19 using the formation of (S)-4-hydroxy-mephenytoin (4-OH-meph) as a measure of CYP2C19 activity, after confirming that steady-state kinetics also apply for CYP2C19. In vitro inhibition studies for this enzyme examined the effects of nonspecific binding. Determination of the in vivo inhibition constant used three doses of fluvoxamine in healthy subjects to provide a wide range of fluvoxamine concentrations in blood to firmly establish the relationship between fluvoxamine concentration and its effect on enzyme activity in vivo. Materials and Methods Chemicals and Reagents. NADPH and 5-(4 -hydroxyphenyl)-5-phenylhydantoin [p-hpph, internal standard (IS)] were purchased from Sigma-Aldrich (St. Louis, MO). 4-OH-meph was purchased from Sigma/RBI (Natick, MA). Nirvanol was kindly provided by Dr. Stephen Hall (Indiana University, Indianapolis, IN). Fluvoxamine maleate was obtained from Solvay Duphar (Marietta, GA) and norfluoxetine maleate (IS) was obtained from Eli Lilly & Co. (Indianapolis, IN). A BCA protein assay kit was purchased from Pierce Chemical (Rockford, IL). All organic solvents and other chemicals were of high-performance liquid chromatography grade. Human Liver Microsomes (HLMs) and Recombinant CYP2C19. HLMs were prepared by a standard technique (Meier et al., 1983) from six nonfatty donor livers (HL105, HL114, HL134, HL135, HL152, and HL157) from the University of Washington Human Liver Bank. Microsomes containing cdnaexpressed CYP2C19 (Supersomes) were purchased from BD Gentest (Woburn, MA). In Vitro Studies. Nonspecific binding of fluvoxamine in human liver microsomes and Supersomes (f u,mic ). Fluvoxamine (0.3 M) was incubated with various amounts of microsomal protein (0.5 and 1 mg/ml from pooled HLMs, n 6; 0.15 mg/ml for Supersomes and 0.5 mg/ml from six individual HLMs) at 37 C for 30 min in 100 mm potassium phosphate buffer (ph 7.4). Each sample (1 ml) was transferred into a Centrifree Micropartition System unit (Amicon Division, W.R. Grae & Co., Beverly, MA) and centrifuged at 1000g for 15 min at 37 C. Fifty microliters of norfluoxetine (IS, 10 g/ml) was added to 100 l of the filtrates. The filtrates were injected directly onto the LC-MSD (positive electrospray ionization). The value of f u,mic was calculated as the ratio of the filtrate concentration resulting from incubation with protein to the filtrate concentration resulting from incubation without protein. HLM protein concentrations were measured using the BCA protein assay kit, and Supersomes protein concentration was calculated from the product information supplied by the manufacturer. Tests for time-dependent inhibition of CYP2C19. Fluvoxamine (0.5 M) and pooled HLMs (1 mg/ml) were preincubated at 37 C with and without NADPH for 0, 5, 15, and 30 min. One hundred-microliter aliquots were removed and transferred into mixtures (900 l) containing (S)-mephenytoin (0.25 mm), NADPH (1 mm), and EDTA (1 mm) in 0.1 M potassium phosphate buffer at 37 C. The (S)-mephenytoin metabolic reaction was carried out for 15 min. In another experiment designed to test the possibility of fluvoxamine time-dependent inhibition of CYP2C19, human liver microsomes (1 mg/ml) and (S)-mephenytoin (0.1 mm) were coincubated with fluvoxamine (0.6 M) or without fluvoxamine. After 3-min preincubation, the reaction was initiated by the addition of NADPH at 1 mm and allowed to proceed for 5, 15, 20, 25, and 30 min. The reactions were terminated by transferring each incubation mixture into a tube containing 300 l of ice-cold 2% zinc sulfate, followed by vortex mixing. Inhibitory effect of fluvoxamine on the metabolism of (S)-mephenytoin in microsomal systems. The incubation mixture consisted of 1.0 or 0.5 mg/ml of total human liver microsomal protein, or 0.15 mg/ml (17 pmol of CYP2C19) of Supersomes, (S)-mephenytoin (15, 50, and 150 M), and fluvoxamine (0, 0.1, 0.3, and 0.9 M) in 0.1 M potassium phosphate buffer (ph 7.2). After preincubation for 5 min at 37 C, the reaction was initiated by the addition of NADPH (1 mm) and allowed to proceed for 30 min. The reaction was terminated by the addition of 300 l of ice-cold 2% zinc sulfate and cooling in an ice bath. All incubations were carried out in duplicate. Incubation conditions (microsomal protein content, incubation time) were within the linear range for the metabolic rate (preliminary studies; data not shown). In Vivo Study. Human subjects. The subjects (seven males and five females; years) were nonsmokers, and the females were not pregnant. The study was approved by the Human Subjects Review Committee at the University of Washington and subjects gave written informed consent. Study design. A randomized, four-period crossover trial was performed in 12 healthy volunteers. This study was divided into a control phase (phase 1, no fluvoxamine, 2 days) and three experimental phases (phase 2, fluvoxamine 37.5 mg/day; phase 3, fluvoxamine 62.5 mg/day; and phase 4, fluvoxamine 87.5 mg/day; 12 days each). In phase 4, fluvoxamine was given 50 mg/day for 2 days and then escalated to 87.5 mg/day). Each subject was requested to abstain from taking any drug throughout the study. In the control phase, subjects were to arrive after an overnight fast and were given a single oral test dose of 100 mg of (S)-mephenytoin at 8:00 AM. Subjects remained fasting until 10:00 AM. Serial blood samples (10 ml) were collected from a catheter placed in the antecubital vein at 0, 15, 30, 45, 60, and 90 min and 2, 4, 6, 9, and 12 h after the test dose. Additional blood samples were collected 24 and 48 h after the test dose by venipuncture. Total urine output was collected during the 0- to 12-, 12- to 24-, and 24- to 48-h time periods. The 0- to 12-h urine sample was analyzed for 4-OH-meph to identify any poor metabolizers. Subjects who did not excrete at least 25% of the (S)-mephenytoin dose as 4-OH-meph within the first 12 h were not allowed to continue in the study. Phases 2, 3, and 4 were assigned to subjects in a randomized order. Fluvoxamine (phases 2, 3, and 4) was administered orally once daily at 4:00 PM for 11 of the 12 days. After eight doses of fluvoxamine, i.e., on the morning of the 9th day of treatment, subjects took the test dose of (S)-mephenytoin at 8:00 AM and fluvoxamine was given 8 h after the test dose. Serial blood samples and urine were collected as described for phase 1. When taking fluvoxamine, additional blood samples were collected at 8, 14, 32, and 72 h after the test dose of (S)-mephenytoin to check whether steady-state concentration of fluvoxamine was achieved and to allow calculation of the fluvoxamine area under the plasma concentration-time curve (AUC) (0- ). One additional urine sample was collected for the 48- to 72-h time period. Sample preparation and high-performance liquid chromatography analysis. (S)-Mephenytoin, its metabolites, and p-hpph (internal standard) in microsomal incubations were extracted by addition of 4 ml of methylene chloride. After shaking for 10 min, the organic phase was removed and evaporated under a stream of nitrogen (N 2 ), and the residues were reconstituted with 100 l of methanol/water (25:75). Ten-microliter aliquots of the reconstituted residues were injected onto a Zorbax Rx-C8 (Narrow-Bore mm, 5- m) column connected to a Hewlett Packard 1100 LC-MSD system equipped with Hewlett Packard ChemStation software (version 1.0) for data acquisition and analysis. The flow rate was 0.20 ml/min, and the mobile phase consisted of methanol/0.01 M ammonium acetate buffer, ph 3.3 (50:50). The ions monitored were m/z 219 [(S)-mephenytoin], m/z 235 (4-OH-meph), m/z 205 (nirvanol), and m/z 269 (p-hpph). (S)-Mephenytoin and its metabolites in human plasma and urine, together with p-hpph (internal standard), were extracted and measured by a modification of the method of Kupfer et al. (1980). In brief, a 0.5-ml aliquot of each plasma sample was extracted with ethyl acetate/diethyl ether (67:33, v/v) and evaporated under a stream of N 2. For urine, a 0.5-ml aliquot of each sample was subjected to enzymatic hydrolysis with 1000 units of -glucuronidase (containing sulfatase activity) for 16 h at 37 C in the dark and then extracted. The residues were reconstituted in 100 l of methanol/ water (25:75). Ten-microliter aliquots were injected onto a Zorbax Rx-C8 (Narrow-Bore mm, 5- m) column connected to the same LC-MSD system as described above.

3 FLUVOXAMINE INHIBITION OF CYP2C Fluvoxamine extraction from human plasma was carried out by a method reported previously with minor modifications (Belmadani et al., 1995). The samples were injected onto a Zorbax Rx-C8 (Narrow-Bore mm, 5- m) column connected to the LC-MSD system. The ions monitored were m/z 319 (fluvoxamine) and m/z 296 (norfluoxetine, IS). The mobile phase (10 mm ammonium acetate buffer at ph 4.0 and methanol; 30:70) was delivered at a flow rate of 0.25 ml/min. Data Analysis. Determination of enzyme kinetic parameters. The mechanism of inhibition was determined by graphical analysis. The formation of 4-OH-meph from (S)-mephenytoin in microsomal systems was analyzed by fitting the data to the single-enzyme competitive inhibition Michaelis-Menten model with a least-squares nonlinear regression program (BMDP; BMDP Statistical Software, Inc., Los Angeles, CA). This procedure provided estimates of V max, K M, and apparent K i values. Pharmacokinetic analysis. All pharmacokinetic parameters were calculated using the pharmacokinetic software package WinNonlin (version 1.5; Scientific Consulting, Inc., Mountain View, CA). The calculations were based on noncompartmental analysis. The terminal elimination rate constant ( Z ) and apparent volume of distribution (V d /F, where F is the bioavailability) of (S)-mephenytoin, were determined by linear regression of the terminal portion of the log concentration-time profile. The terminal elimination half-life (t 1/2 )of (S)-mephenytoin was calculated as 0.693/ Z. The AUC was determined by the trapezoidal rule. (S)-Mephenytoin oral clearance was calculated as dose/ AUC inf (AUC inf represents AUC extrapolated to infinity). The formation clearance of the (S)-mephenytoin metabolite 4-OH-meph was calculated as amount of 4-OH-meph in urine (0 72 h) /AUC (0 72 h). The renal clearance of (S)-mephenytoin was calculated as amount of (S)-mephenytoin in urine (0 72 h)/auc (0 72 h). The average steady-state concentration of fluvoxamine was calculated as AUC (0 24 h) /24 h. Calculation of K i iv values for CYP2C19. The approach used to calculate K i iv is based on the competitive inhibition model used previously (Adedoyin et al., 1987; Shaw and Houston, 1987; Kunze and Trager, 1996; Tran et al., 1997; Yao et al., 2001). The model includes the following assumptions: 1) product formation is described by Michaelis-Menten kinetics with competitive inhibition; 2) concentrations of (S)-mephenytoin are much lower than its K M ; 3) only one enzyme mediates the formation of 4-OH-meph; 4) after oral administration, the operating clearance of (S)-mephenytoin is its intrinsic clearance because (S)-mephenytoin has a relatively large clearance (Ito et al., 1998); and 5) fluvoxamine does not affect the elimination of 4-OH-meph. The K i iv was calculated from the following equation: CL f CL fl 1 I K i iv where CL f and CL fi represent formation clearances of 4-OH-meph associated with CYP2C19 in the absence and presence of the inhibitor fluvoxamine, respectively, and [I] represents the average steady-state plasma concentration of fluvoxamine. Plots of the ratio of uninhibited/inhibited formation clearances versus plasma concentration of inhibitor are expected to be linear with an intercept equal to 1 and a slope equal to 1/K i iv. The tests of linearity and intercept of eq. 1 require measurements of multiple formation clearances and simultaneous measurements of inhibitor concentration. The overall K i iv was determined based on linear regression with weight of 1/(formation clearance ratio) 2. Calculation of RK i. The ratio of the mean (n 6) in vitro K i to mean (n 12) in vivo K i based on unbound inhibitor concentration was calculated as K i,ub (in vitro)/ K i,ub (in vivo). This ratio is defined as RK i. Statistical Analysis. The pharmacokinetic parameters of (S)-mephenytoin are reported as mean S.D. Comparisons between treatment and control periods were assessed by paired t test. The Wilcoxon rank sum test was performed for the comparison of RK i values of fluvoxamine toward CYP1A2 and CYP2C19, assuming that the K i values for CYP1A2 and CYP2C19 have the same distribution. Statistical significance was assumed if p Results In Vitro Studies. Nonspecific binding of fluvoxamine. A previous study established that the free fraction of fluvoxamine in the HLM incubation system is reasonably constant up to 1 M fluvoxamine (1) TABLE 1 Enzyme kinetic parameters for fluvoxamine inhibition of (S)-mephenytoin metabolism and free fractions of fluvoxamine in microsomal incubations from six human livers Protein concentration is 0.5 mg/ml. Human Liver K m V max K i,total Free Fraction a K i,ub M nmol/min/mg protein nm f u,mic nm HL HL HL HL HL HL Mean S.D a Fluvoxamine spiked concentration was 0.3 M. (Yao et al., 2001). The free fractions of fluvoxamine in the microsomal preparations used in this study (protein concentration of 0.5 mg/ml) from six nonfatty human livers are shown in Table 1. The FIG. 1.Tests of time-dependent inhibition of fluvoxamine toward CYP2C19. A, measurement of CYP2C19 activity after preexposure of HLMs to fluvoxamine up to 30 min in the presence or absence of NADPH. B, formation of 4-OH-meph from (S)-mephenytoin as a function of time in human liver microsomal incubation in the presence or absence of fluvoxamine.

4 568 YAO ET AL. FIG. 2.Inhibitory effect of fluvoxamine on the formation of 4-OH-meph from (S)-mephenytoin in Supersomes (0.15 mg/ml protein). Data points represent the average of duplicate incubations. mean ( S.D.) free fraction was The free fraction in Supersomes was at 0.15 mg/ml protein. Tests of time-dependent inhibition of fluvoxamine. In the preincubation study, CYP2C19 activity was not sensitive to the time of exposure to fluvoxamine, nor to the absence or presence of NADPH (Fig. 1A). In the coincubation study, 4-OH-meph was formed linearly with time in the incubations with or without fluvoxamine, suggesting that there are neither inhibitory metabolites formed nor time-dependent inhibition within the time frame used (Fig. 1B). These results validate the use of steady-state kinetics for evaluating the affinity of fluvoxamine for CYP2C19. Effect of nonspecific binding on the determination of in vitro K i values. Lineweaver-Burk analysis indicated that fluvoxamine is a competitive inhibitor of CYP2C19 activity (Fig. 2). Apparent K i values based on nominal (total added) fluvoxamine concentration (K i,total ) were determined based on the single-enzyme competitive inhibition model at protein concentrations of 1.0 and 0.5 mg/ml in pooled liver microsomes, giving apparent K i,total values of and nm, respectively. K i,total in the Supersomes system at 0.15 mg/ml protein was nm (Table 2). Because f u,mic was independent of fluvoxamine concentration within the range used in the determination of in vitro K i, K i,ub was calculated by multiplying K i,total by f u,mic. After applying this correction, fairly uniform values of K i,ub were obtained (around nm; Table 2). K i,total values were measured in microsomal preparations from six human livers at 0.5 mg/ml protein (Table 1) to determine interindividual variability. The mean K i,total was nm and the mean K i,ub was nm. The mean values of K M and V max for the formation of 4-OHmeph were M and nmol/min/mg protein, TABLE 2 Effect of nonspecific binding on the determination of in vitro K i values of fluvoxamine toward CYP2C19 Supersomes, cdna-expressed CYP2C19. System Protein Conc. K i,total Free Fraction a K i,ub mg/ml nm f u,mic nm HLM HLM Supersomes a Fluvoxamine spiked concentration was 0.3 M. respectively. These values are in agreement with those previously reported (Meier et al., 1985; Hickman et al., 1998). The values of K M and V max for the formation of 4-OH-meph were M and nmol/min/nmol CYP2C19 for the Supersomes. In Vivo Studies. Pharmacokinetic parameters of (S)-mephenytoin. The mean peak plasma concentrations (C max )of(s)-mephenytoin in the four phases were , , , and M. The values of C max for phases 2, 3, and 4 were significantly higher than the C max value of phase 1 (control phase). The AUC and elimination half-life of (S)-mephenytoin significantly increased [and oral clearance of (S)-mephenytoin significantly decreased] during treatment with fluvoxamine (Table 3). Between phase 1 and phase 4, AUC increased from to M h, and t 1/2 increased from to h. Oral clearance decreased from to l/h. The apparent volume of distribution (V d /F) of (S)-mephenytoin seemed lower in the presence of fluvoxamine but was not changed as a function of flu-

5 FLUVOXAMINE INHIBITION OF CYP2C TABLE 3 Effects of fluvoxamine on (S)-mephenytoin pharmacokinetic parameters in 12 subjects Phase AUC inf Terminal t 1/2 CL tot /F V d /F M h h l/h liters Control Fluvoxamine 37.5 mg/day ** ** ** ** Fluvoxamine 62.5 mg/day ** ** ** ** Fluvoxamine 87.5 mg/day ** ** ** ** P voxamine dose: it decreased from liters in phase 1 to liters in phase 2 (Table 3). The majority (more than 70%) of a dose of (S)-mephenytoin was recovered as 4-OH-meph and its sequential metabolite in urine. Unchanged (S)-mephenytoin and nirvanol together contributed less than 2% of the dose. The formation clearance for 4-OH-meph decreased by 70% in phase 2, by 83% in phase 3, and by 90% in phase 4 compared with the control phase, indicating that fluvoxamine inhibits CYP2C19 activity in vivo in a dose-dependent manner. The renal clearance of (S)-mephenytoin, which accounted for less than 2% of a dose, seemed to decrease significantly as fluvoxamine concentration increased. The renal clearances in the four phases were , , , and l/h (p 0.05, compared with the control phase), respectively. Pharmacokinetic parameters of fluvoxamine. Fluvoxamine average steady-state concentrations for each subject are shown in Table 4. These values are within the reported concentration ranges for these doses and consistent with the nonlinear disposition of this drug (Perucca et al., 1994b; Spigset et al., 1998). Fluvoxamine oral clearance decreased as the dose increased: oral clearances in the three phases were , , and l/h, respectively. The nonlinear disposition of fluvoxamine emphasizes the need to measure the plasma concentration of fluvoxamine in drug interaction studies. Determination of K i iv for fluvoxamine inhibition of CYP2C19. K i iv for each subject was calculated from the plot of the ratio of 4-OHmeph formation clearances (control/inhibited) versus fluvoxamine average steady-state plasma concentration. Individual plots are shown in Fig. 3, A to D. The mean K i iv values based on total and unbound plasma concentrations were and nm (f p from a previous study; Yao et al., 2001) (Table 4). A summary plot of ratios of 4-OH-meph formation clearances (control/ inhibited) versus total plasma concentrations of fluvoxamine for 12 subjects is shown in Fig. 4. The overall K i iv was 13.9 nm with an intercept of 0.85 (r , p 0.001). Comparison of the ratios of in vitro to in vivo inhibition constant of fluvoxamine toward CYP1A2 and CYP2C19. RK i is used to allow a comparison between different enzymes affected by an inhibitor. The in vitro K i, ub values of fluvoxamine toward CYP1A2 (Yao et al., 2001) and CYP2C19 are nm (n 8) and nm (n 6), respectively, whereas the corresponding in vivo K i values based on unbound plasma concentration are nm (n 8) and nm (n 12). Therefore, the mean RK i values for CYP1A2 and CYP2C19 are 10 and 38, respectively. The Wilcoxon rank sum test showed that the difference in RK i values was significant (p 0.05). Discussion A primary goal of this study with CYP2C19, and the companion study with CYP1A2, was to unambiguously determine the absolute difference in potency of fluvoxamine toward these enzymes in vivo and in vitro, and to confirm that the kinetic models required to analyze the results were appropriately applied. To that end, studies were carried out in both systems with both enzymes to explore the relationship between fluvoxamine concentration and enzyme activity over a wide range of inhibitor concentrations. The results of in vitro studies indicate that the use of steady-state kinetics to determine inhibitor affinity is appropriate and that K i values should be calculated based on the concentrations of unbound fluvoxamine rather than nominal concentrations. Similarly, in vivo, the relationship between fluvoxamine concentrations in plasma and their effect on enzyme selective metabolite formation clearances was found to be well behaved in a manner consistent with the standard kinetic model, allowing for the determination of an in vivo K i based on steady-state fluvoxamine plasma levels. The present study showed that the mean K i iv of fluvoxamine toward CYP2C19 is 13.5 nm, a value which is well below the TABLE 4 Calculation of K i iv of fluvoxamine toward CYP2C19 for individual subjects (n 12) Subject Fluvoxamine Average Concentration CL f,4-oh /CL fi,4-oh K i iv 37.5 mg/day 62.5 mg/day 87.5 mg/day 37.5 mg/day 62.5 mg/day 87.5 mg/day nm nm Mean S.D

6 570 YAO ET AL. FIG. 3.Representative individual plots of ratios of 4-OH-meph formation clearances (control/inhibited) as a function of total plasma concentration of fluvoxamine at steady state used to determine in vivo inhibition constants. fluvoxamine therapeutic range ( nm), suggesting that fluvoxamine is a potent inhibitor of CYP2C19 in vivo. The plots of ratios of 4-OH-meph formation clearance versus fluvoxamine plasma concentration (Figs. 3 and 4) showed good linearity with intercept values around 1 for individuals as well as the study population. The linearity and intercept values suggested that a good estimation of K i iv values can be obtained using only a single dose of inhibitor. Based on this observation, the results of a published study of fluvoxamine inhibition of the CYP2C19 catalyzed bioactivation of chloroguanide (Jeppesen et al., 1997) were used to calculate a K i iv of 25 nm (assuming that fluvoxamine plasma concentration at 100 mg/day is nm; Spigset et al., 1998) and that only CYP2C19 catalyzes the formation of 4-chlorophenylbiguanide). This K i iv estimate is similar to the values FIG. 4.Overall plot of ratios of 4-OH-meph formation clearances (control/inhibited) as a function of the total plasma concentration of fluvoxamine at steady state for 12 subjects. The line was fit by linear regression with weight of 1/(formation clearance ratio) 2. obtained in the present study, which used a different probe of CYP2C19 and three doses of fluvoxamine. A second goal of these studies was to test the hypothesis that RK i values for different enzymes subject to inhibition by fluvoxamine will be the same even though the RK i values are both significantly higher than 1. The observed difference in RK i values between CYP1A2 and CYP2C19 (10 versus 38) is intriguing and could be explained by the following possibilities: 1) the presence of one or more metabolites inhibiting only CYP2C19. So far, 11 metabolites of fluvoxamine have been found in urine. About 30 to 60% of the metabolites seem to be produced by oxidative demethylation of the methoxy group, whereas 20 to 40% seem to be formed by degradation at the amino group or by removal of the entire ethanolamino group (Spigset et al., 2001); and 2) the sensitivity of enzyme activity to environmental differences between in vitro and in vivo, such as ph and ionic strength, might be enzyme-dependent. Previous data from our laboratory (Yao and Levy, 2002) have shown a parallel decrease in K i values for both enzymes (3-fold decrease in CYP1A2 versus 2.5-fold for CYP2C19) as ph increased from 7.2 to 7.6. Also, there was a 2-fold decrease in K i for CYP1A2 and virtually no change for CYP2C19 as the concentration of potassium phosphate buffer (ph 7.4) increased from 0.01 to 0.1 M. Thus, these preliminary studies suggest a differential effect on enzyme affinity by ionic strength but not ph. It is not possible at this time to evaluate the relative importance of environmental differences and partitioning effects. However, further work with other enzymes also inhibited by fluvoxamine may help to further discriminate between these and other factors contributing to the differences between the in vitro and in vivo systems. In view of the findings of RK i values larger than 1 for CYP1A2 and CYP2C19, we searched the literature for similar data on other enzymes inhibited by fluvoxamine, namely, CYP2C9, CYP2D6, and CYP3A4. Reported in vitro inhibition potencies of fluvoxamine toward CYP2C9, CYP2D6, and CYP3A4 also seem to be inconsistent with corresponding in vivo inhibition effects. In vitro K i values for CYP2C9, CYP2D6, and CYP3A4 are 2.2 to 13.3 M (Hemeryck et al., 1999; Olesen and Linnet, 2000), 3.9 to 8.2 M (Crewe et al., 1992; Skjelbo and Brosen, 1992), and 10 to 40 M (von Moltke et al., 1994; Rasmussen et al.,

7 FLUVOXAMINE INHIBITION OF CYP2C ), respectively, and these values are much higher than the fluvoxamine therapeutic range of 0.1 to 0.7 M ( M based on unbound plasma concentration). Yet, recent studies (Kashuba et al., 1998; Madsen et al., 2001) suggest that fluvoxamine significantly inhibits the metabolism of tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (CYP3A4) in vivo. These comparisons suggest that, for all five P450s inhibited by fluvoxamine, the inhibition potency is greater in vivo than predicted from in vitro data. It remains to be seen whether RK i values will also be in the range seen with CYP1A2 and CYP2C19 in this study. In particular, large changes in RK i values for other enzymes would support a primary role for enzyme environment, whereas no differences in RK i values would support partitioning effects (elevated fluvoxamine concentrations available to the enzyme relative to free concentrations in plasma). Acknowledgments. We thank John M. 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