Introduction

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Modafinil (dl-2-[(diphenylmethyl)sulfinyl]acetamide; Fig. 1), a nonamphetamine-like wakefulness-promoting agent, has recently been approved in the United States, United Kingdom, and Ireland (under the tradename Provigil) for the treatment of excessive daytime sleepiness associated with narcolepsy. The compound, which was discovered by Laboratoire L. Lafon (Boivin et al., 1993), is already on the market in France (under the tradename Modiodal).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structure of modafinil.

In clinical use, modafinil is likely to be administered in combination with other medications, and an understanding of the potential for drug-drug interactions is therefore very important. Such interactions could be pharmacokinetic or pharmacodynamic in nature, or both. The present study focused on potential pharmacokinetic interactions arising from inhibition or induction of cytochrome P450 drug-metabolizing enzymes by modafinil.

In a previous in vitro study in primary human hepatocytes (Moachon et al., 1996), modafinil was evaluated for its ability to induce the activities of cytochrome P450 enzymes, including ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase, S-mephenytoin 4'-hydroxylase, dextromethorphan O-demethylase, nifedipine oxidase, and lauric acid hydroxylase. At concentrations (10 and/or 100 µM) approximating those achieved clinically, modafinil was found to induce ethoxyresorufin O-deethylase and nifedipine oxidase activities in human hepatocytes, although the extent of induction observed was less than that obtained in mice, rats, or dogs and less than those produced by known inducers of these cytochrome P450 enzymes. At the highest concentration tested (1000 µM), more pronounced changes were observed in the activities of ethoxyresorufin O-deethylase (increased), dextromethorphan O-demethylase (increased), and nifedipine oxidase (decreased) relative to those in the solvent-treated controls. The activity of S-mephenytoin 4'-hydroxylase was decreased at all concentrations of modafinil. The conclusion from this study was that ethoxyresorufin O-deethylase [cytochrome P450 (CYP)²1A] and nifedipine oxidase (CYP3A) activities were slightly induced by modafinil, which also increased the activity of dextromethorphan demethylase (CYP2D6) at the highest concentration tested (1000 µM).

Drawing definitive conclusions from these results was complicated by the substantial degree of intersubject variability that was observed and by the fact that modafinil was probably still present in the hepatocytes during the assay of enzymatic activities. In addition, the highest concentration tested (1000 µM) was substantially higher than the aqueous solubility of modafinil and was well above the concentration range obtained clinically (Wong et al., 1999).

A second in vitro induction study in human hepatocytes was therefore conducted to test whether the earlier results could be confirmed in a different laboratory, using a somewhat different and extended experimental design. In addition, the ability of modafinil to inhibit cytochrome P450 enzymes was studied in vitro in human liver microsomes (HLMs). The results of these latter two in vitro studies form the basis for the present communication.

	Materials and Methods

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Chemicals, Enzymes, and Antibodies. Modafinil was supplied by Cephalon, Inc. (West Chester, PA). Rifampin, -naphthoflavone, nicotine, quinidine, 4-methylpyrazole, ketoconazole, baccatin, and 8-methoxypsoralen were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-trifluoromethylcoumarin (EFC) was obtained from Molecular Probes (Junction City, OR). Paclitaxel and 6-hydroxypaclitaxel were obtained from Hauser Chemical Co. (Boulder, CO) and Gentest Corp. (Woburn, MA), respectively. S-Mephenytoin, (±)-4'-hydroxymephenytoin, and hydroxymethyltolbutamide were purchased from Ultrafine Chemicals (Manchester, England). Furafylline was obtained from Research Biochemicals Inc. (Natick, MA). Hexobarbital was purchased from Sterling-Winthrop (Rensselaer, NY). Sulfaphenazole was obtained from Ciba-Geigy Ltd. (Basel, Switzerland). Troleandomycin was obtained from Pfizer, Inc. (Brooklyn, NY). Sources of other chemicals, including culture media components, were as specified by Pearce et al. (1996a) and Madan et al. (1999).

Human Hepatocytes. Hepatocytes were isolated from human liver tissue obtained as surgical waste or from rejected donor livers via a modification of the two-step collagenase digestion method (Seglen et al., 1980; Quistorff et al., 1989; LeCluyse et al., 1996). Briefly, human liver tissue was perfused with pH 7.4 buffer containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 5.5 mM glucose, and 0.5 mM EGTA, followed by the same buffer lacking EGTA but containing 1.5 mM CaCl₂ and 0.2-0.5 mg/ml collagenase. Viability (trypan blue exclusion) was 70% for all preparations used.

Enzyme Inhibition. Direct inhibition.

Metabolism-dependent (mechanism-based) inhibition. To test for reversible inhibition, HLMs (two individual samples and a pool of seven) were preincubated with modafinil plus NADPH for 15 min before the addition of the substrate to start the assay. The concentrations of modafinil and of the substrates investigated are summarized in Table 1. Solvent controls, containing all components except modafinil, were also examined.

Enzyme Induction. Freshly isolated human hepatocytes were cultured according to the method described by LeCluyse et al. (1994, 1996). Approximately 3 × 10⁶ cells were added to 60-mm culture dishes coated with collagen and allowed to attach for 2 to 3 h. Unattached cells were aspirated, and serum-free modified Chee's medium containing 0.1 µM dexamethasone, 1% insulin-transferrin-selenium premix, and 0.25 mg/ml Matrigel was added. The cells were then maintained in culture for ~3 days, with daily changes of medium, before the initiation of experiments. Only preparations that contained morphologically normal hepatocytes, as evaluated by phase contrast light microscopy, without significant contamination from other cell types were treated with modafinil or reference inducers.

Assay Procedures. Assays were performed as described in detail by Pearce et al. (1996a) or as described later. In each assay described later, the reaction mixture volume was 1 ml, containing 50 µg of microsomal protein, and the reactions were carried out at 37°C.

Western Immunoblotting Procedures. CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, and CYP3A4 in the hepatocyte-derived microsomes were analyzed via Western immunoblotting using essentially the procedures described by Madan et al. (1999).

Data Analysis. All incubations for enzyme activity assays were in duplicate; the data reported are averages of those duplicate determinations.

	Results

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Inhibition of Human Cytochromes P450. The rates of metabolism of marker substrates for nine cytochrome P450 enzymes (Table 1) were determined in HLMs (pool of seven) in the presence and absence of modafinil (5-250 µM). The presence of modafinil had minimal effect on any reaction except the 4'-hydroxylation of S-mephenytoin (CYP2C19), which was reversibly inhibited with a K_i value of ~39 µM (Fig. 2). This concentration approximates the steady-state C_max value of modafinil in human plasma at a dosage of 400 mg/day (Wong et al., 1999). The sample-to-sample variation in the inhibition of CYP2C19 was determined with 10 samples of individual HLMs at substrate concentrations equal to K_m and modafinil concentrations equal to ~2K_i. As expected, modafinil produced substantial inhibition (~50%) of CYP2C19 in all microsomal samples (Fig. 3), consistent with its ability to function as a competitive inhibitor.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Evaluation of modafinil as a reversible inhibitor of S-mephenytoin 4'-hydroxylase (CYP2C19) activity in pooled HLMs. Dixon and Eadie-Hofstee plots of the rates of S-mephenytoin 4'-hydroxylation by pooled HLMs. The microsomes were incubated with S-mephenytoin for 30 min at concentrations of the substrate that were approximately equal to Km/2, Km, and 4Km and in the absence (solvent control; 0.1% DMSO) or presence of modafinil at concentrations of 5, 25, 100, or 250 µM.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Sample-to-sample variation in the inhibition of S-mephenytoin 4'-hydroxylase (CYP2C19) activity in HLMs by modafinil. The rates of S-mephenytoin 4'-hydroxylation were measured in the presence and absence (solvent control; 0.1% DMSO) of modafinil in individual 30-min incubations of microsomes prepared from nine different human livers. The substrate concentration was equal to Km, and the concentration of modafinil, when present, was 75 µM.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Evaluation of modafinil as a reversible inhibitor of testosterone 6-hydroxylase (CYP3A4/5) activity in pooled HLMs. Dixon and Eadie-Hofstee plots of testosterone 6-hydroxylation by pooled HLMs. The microsomes were incubated with testosterone for 8 min at concentrations of the substrate that were approximately equal to Km/2, Km, and 4Km and in the absence (solvent control; 0.1% DMSO) or presence of modafinil at concentrations of 5, 25, 100, or 250 µM.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Evaluation of modafinil as a metabolism-dependent (mechanism-based) inhibitor of testosterone 6-hydroxylase (CYP3A4/5) activity in HLMs. HLMs (two individual samples and a pool of seven) were incubated with modafinil (250 µM) or with the solvent only (DMSO; final concentration, 0.1%) for 15 min under the assay conditions before starting the assay reaction by introduction of the marker substrate, testosterone (50 µM). After 8 min, the reaction was quenched, and the rate of formation of 6-hydroxytestosterone was calculated. The rates without preincubation were then compared with those after incubation for both the modafinil-treated microsomes and the solvent controls. To test for reversible inhibition, the substrate was added to undiluted microsomal mixtures. To test for irreversible inhibition, the preincubation was conducted at microsomal protein concentrations that were 10- to 20-fold higher than those normally used in the CYP3A4/5 assay. Immediately before the addition of the substrate, these microsomal mixtures were diluted 10- to 20-fold to reduce any effect from reversible inhibition.

Induction of Human Cytochromes P450. The ability of modafinil to induce cytochrome P450 activities was examined in vitro in primary cultures of human hepatocytes. After ~3 days in culture, the hepatocytes were exposed to modafinil at concentrations of 10 to 300 µM for 3 additional days. The hepatocytes were then harvested, and microsomes were prepared and assayed for the activities of eight cytochrome P450 enzymes. The mean results are presented in Fig. 6. [Note: Due to low hepatocyte yield from one human liver (HL-5), the 100 µM treatment group was omitted for that liver to ensure that the other treatment conditions could be effectively evaluated.]

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Effect of treating primary human hepatocyte cultures with modafinil and prototypical cytochrome P450 enzyme inducers on the activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4/5 and on the protein level of CYP2C8. Human hepatocytes from five donors (HL-1 to HL-5) were placed in culture for 3 days before the initiation of treatment with modafinil at 0 (solvent control), 10, 30, 100, or 300 µM for 3 days, with renewal of the medium and test article every 24 h. Additional culture dishes were similarly exposed to -naphthoflavone (33 µM), phenobarbital (250 µM), or rifampicin (50 µM) and served as positive controls. The final concentration of the solvent (DMSO) in the medium was 0.1%. Microsomes were prepared for each human liver from each of the treatment groups. The enzymatic activity of each of the CYP enzymes was then determined for each liver and treatment group using marker substrates. In the case of CYP2C8, the rate of 6-hydroxylation of paclitaxel, the marker substrate, was too low to be measured; hence, the levels of CYP2C8 protein (Western immunoblots) were used to assess induction. For each assay, there were five determinations per treatment group, unless otherwise noted above the bar. Values represent mean ± S.D. *P < .05 in the comparison of modafinil-treated samples with the corresponding DMSO controls. P < .05 in the comparison of all treated samples with the corresponding DMSO controls.

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Effect of treating primary cultures of human hepatocytes with modafinil on the level of CYP3A4 expression (Western immunoblotting). Human hepatocytes from five donors (HL-1 to HL-5) were placed in culture for 3 days before initiation of treatment with modafinil at 0 (solvent control), 10, 30, 100, or 300 µM for 3 days, with renewal of the medium and test article every 24 h. Additional culture dishes were similarly exposed to -naphthoflavone (33 µM), phenobarbital (250 µM), or rifampicin (50 µM) and served as positive controls. The final concentration of the solvent (DMSO) in the medium was 0.1%. Microsomes were prepared for each human liver from each of the treatment groups, and a portion of each was subjected to Western immunoblotting to measure the levels of immunoreactive CYP3A4 using a polyclonal antibody against rat CYP3A1. For comparison, HLMs prepared directly from the livers of two individual subjects (HLM15 and HLM16), plus a pool of microsomes, were also evaluated, in addition to known concentrations of cDNA-derived CYP3A4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, the ability of modafinil to inhibit or to induce cytochrome P450 enzyme activities was studied in human liver preparations. Such information is important for the design of effective treatment programs that will include administration of modafinil, because such enzymatic interactions can either enhance or diminish the effectiveness and/or safety of concomitant medications.

Cytochrome P450 inhibition by modafinil appears to be limited to CYP2C19, whose substrates and inhibitors have been extensively reviewed (e.g., Flockhart, 1995; Parkinson, 1996; Rendic and Di Carlo, 1997). Modafinil does not itself appear to be a substrate for CYP2C19, and there are a relatively small number of marketed pharmaceutical products that are predominantly or even largely metabolized by the enzyme. Examples are S-mephenytoin (Goldstein et al., 1994), omeprazole (Ko et al., 1997), lansoprazole (Pearce et al., 1996b), proguanil (Wright et al., 1995), diazepam (Jung et al., 1997), and propranolol (Ward et al., 1989). (Omeprazole and lansoprazole are also potent inhibitors of CYP2C19.) Caution should be exercised when initiating cotherapy with modafinil in patients receiving these medications, but with the possible exception of diazepam or other sedative benzodiazepines, they generally are not drugs with which modafinil is likely to be a frequent comedication.

However, as demonstrated by a recent report (Grözinger et al., 1998) of an apparent metabolic drug-drug interaction of modafinil with clomipramine, inhibition of CYP2C19 could, in special cases, also be important for compounds that are not normally considered to be significant substrates for the enzyme. In the case reported, the plasma concentrations of clomipramine were found to have increased, along with those of its pharmacologically active des-methyl metabolite, after the addition of modafinil as a comedication. Because clomipramine and des-methylclomipramine normally are largely eliminated through metabolism by CYP2D6 (Nielsen et al., 1992, 1996), a significant effect by an inhibitor of CYP2C19 was unexpected. However, the patient was subsequently determined to be CYP2D6-deficient (Grözinger et al., 1998), belonging to a subset of the human population who have no functional CYP2D6 enzyme (i.e., 7-10% of whites and equal or smaller portions of other ethnic groups; Eichelbaum, 1984; Setiabudy et al., 1994). In these "poor metabolizers" of CYP2D6 substrates, such as dextromethorphan, debrisoquine, and sparteine, the fractional contributions of alternative metabolic pathways for clomipramine and des-methylclomipramine through CYP2C19 and other cytochrome P450 enzymes could assume substantially more important roles than would be the case in individuals with normal CYP2D6 activity.

The inhibition of CYP2C19 by modafinil would likely have minimal therapeutic consequences for patients at steady state for modafinil and for whom a tricyclic antidepressant would be prescribed as cotherapy, because the dosage of the antidepressant would generally be titrated to identify a safe and effective dose. However, these results would suggest that in patients at steady state for clomipramine or similar tricyclic antidepressants, the addition of modafinil as cotherapy may require a dosage reduction for the antidepressant, particularly in CYP2D6-deficient individuals.

Modafinil also caused an induction of cytochrome P450 activities in vitro in human hepatocytes. Three enzymes appeared to be induced by modafinil: CYP1A2, CYP3A4, and CYP2B6. [The results for CYP1A2 and CYP3A4 were generally consistent with results obtained previously (Moachon et al., 1996); CYP2B6 was not previously examined.] The extent of induction of each enzyme, although statistically significant at one or more concentrations of modafinil, was modest, especially in comparison with those produced by reference inducers, when available, and in comparison with interindividual variability.

No significant effect of modafinil treatment (10-300 µM) was observed in the activities of CYP2A6, CYP2C8, CYP2C19, or CYP2D6. The apparent suppression of CYP2C19 reported previously (Moachon et al., 1996) was likely due to inhibition of the enzyme by residual modafinil that remained in the hepatocyte preparations during the enzymatic assays. The lack of induction of CYP2D6 in the present study at concentrations of up to 300 µM is consistent with the previous results for all except the highest concentration tested in that study (i.e., 1000 µM). The reason for the increased enzymatic activity at that concentration is not known, but the concentration is far beyond those that have any clinical relevance.

Of the three cytochrome P450 enzymes apparently inducible by modafinil, CYP1A2 and CYP2B6 do not appear to be of major concern. CYP1A2 provides the primary metabolic pathway for relatively few pharmacologically important substrates, and these do not have narrow therapeutic indices (Tassaneeyakul et al., 1993; Brøsen, 1995; Bertz and Granneman, 1997). In addition, the extent of induction observed in the present study, or in the previous one (Moachon et al., 1996), was small relative to the ~40-fold interindividual variability observed for this enzyme. In the case of CYP2B6, the activity of the enzyme is extremely low in human livers (Shimada et al., 1994), and it appears to contribute minimally to the metabolism of pharmaceutical products.

In contrast, CYP3A4 represents the largest single portion of the cytochrome P450 protein and activity in the human liver and plays a substantial role in the metabolism of a vast array of pharmaceutical products (Guengerich, 1995; Parkinson, 1996; Bertz and Granneman, 1997; Rendic and Di Carlo, 1997). Of particular concern are compounds that are predominantly or exclusively metabolized by CYP3A4 and also have a narrow therapeutic margin (e.g., cyclosporine A and steroidal contraceptives containing ethinyl estradiol).

As with the inhibition of CYP2C19, the apparently low degree of induction of CYP3A4 by modafinil would be most likely to produce clinical effects if modafinil were added as cotherapy to a patient already at steady state for a narrow-margin CYP3A4 substrate. A single case of apparent interaction has been reported in a patient in whom the effectiveness of treatment with cyclosporine A decreased after the addition of modafinil as a cotherapy (Le Cacheux et al., 1997). However, insufficient information is available to establish the cause of the effect.

Finally, the apparent suppression of CYP2C9 activity in human hepatocytes by treatment with modafinil is potentially important due primarily to one compound, warfarin (Coumadin), which has a narrow therapeutic index and whose more active enantiomer (S-warfarin) is primarily a substrate for CYP2C9 (Rettie et al., 1992). The origin of the suppressive effect obtained in vitro and its relevance to the clinical situation are not known, but the finding suggests caution in the initiation of treatment with modafinil in patients who are at steady state on warfarin.

In summary, modafinil has been demonstrated in vitro to be a moderately potent, reversible inhibitor of CYP2C19 in HLMs and a modest inducer of CYP1A2, CYP3A4, and CYP2B6 in vitro in human hepatocytes. In addition, CYP2C9 appeared to be suppressed in vitro in human hepatocytes after treatment with modafinil. Overall, these results suggest that there is potential for metabolic drug-drug interactions between modafinil and certain possible concomitant medications.

Due to the relatively low degree of alteration of the enzyme activities in vitro and to the concentrations of modafinil required to obtain appreciable effects, a high incidence of clinically significant interactions would not be expected. However, these in vitro results are being used in evaluation of clinical reports of apparent drug-drug interactions and in the design of subsequent studies targeted at further elucidation of the clinical relevance, if any, of these in vitro findings.