Abstract
The ability of tricyclic antidepressants (TCAs) to inhibit phenytoin p-hydroxylation was evaluated in vitro by incubation studies of human liver microsomes and cDNA-expressed cytochrome P450s (P450s). The TCAs tested were amitriptyline, imipramine, nortriptyline, and desipramine. Amitriptyline and imipramine strongly and competitively inhibited phenytoinp-hydroxylation in microsomal incubations (estimatedKi values of 5.2 and 15.5 μM, respectively). In contrast, nortriptyline and desipramine produced only weak inhibition. In the incubation study using cDNA-expressed P450s, both CYP2C9 and CYP2C19 catalyzed phenytoinp-hydroxylation, whereas TCAs inhibited only the CYP2C19 pathway. All of the TCAs tested inhibited CYP2D6-catalyzed dextromethorphan-O-demethylation competitively, with estimated Ki values of 31.0, 28.6, 7.9, and 12.5 μM, respectively. The tertiary amine TCAs, amitriptyline and imipramine, also inhibited CYP2C19-catalyzedS-mephenytoin 4′-hydroxylation (estimatedKi of 37.7 and 56.8 μM, respectively). The secondary amine TCAs, nortriptyline and desipramine, however, showed minimal inhibition of CYP2C19 (estimated IC50 of 600 and 685 μM, respectively). None of the TCAs tested produced remarkable inhibition of any other P450 isoforms. These results suggest that TCAs inhibit both CYP2D6 and CYP2C19 and that the interaction between TCAs and phenytoin involves inhibition of CYP2C19-catalyzed phenytoinp-hydroxylation.
Phenytoin has been widely prescribed as a broad-spectrum anticonvulsant for the prevention and treatment of seizure disorders. Several of its pharmacological characteristics (i.e., narrow therapeutic range, slow absorption, and saturable metabolism) have been frequently implicated in clinically significant drug interactions (Nation et al., 1990;Monaco and Cicolin, 1999). p-Hydroxylation is the major metabolic pathway of phenytoin, and forms 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH1), which accounts for 90% of all metabolites in humans (Browne and LeDuc, 1995). This conversion is catalyzed mainly by CYP2C9, although CYP2C19 also plays a role (Bajpai et al., 1996; Giancarlo et al., 2001).
Some authors have observed that TCAs inhibit phenytoin elimination, with a consequent risk for toxic phenomena. Imipramine and nortriptyline have been reported to increase plasma phenytoin levels in patients with epilepsy (Richens and Houghton, 1975; Perucca and Richens, 1977). However, the mechanism of this interaction has not been addressed.
Even though TCAs are commonly coadministered with a wide variety of other drugs, little is known about their inhibitory effects on P450 isoforms. Therefore, it is difficult to predict the P450 isoform(s) involved in the interaction between TCAs and phenytoin, a known substrate of CYP2C9 and CYP2C19 (Bajapi et al., 1996; Giancarlo et al., 2001).
Even though CYP2C9 is the main isoform involved in the formation of HPPH, a primary metabolite of phenytoin, this isoform does not appear to be inhibited by TCAs. There is indirect evidence that the increase in plasma phenytoin produced by TCAs does not involve inhibition of CYP2C9; for instance, omeprazole, cimetidine, and ticlopidine have been reported to increase plasma phenytoin concentrations (Levy, 1995;Rindone and Bryan, 1996), but it has not been shown that any of these drugs inhibit CYP2C9-catalyzed S-warfarin 7-hydroxylation in vitro or affect S-warfarin clearance in vivo (Andersson et al., 1990; Niopas et al., 1991). Furthermore, since no interaction is observed when TCAs are coadministered with two well known CYP2C19 substrates, mephenytoin and moclobemide (Zimmer et al., 1990; Baumann et al., 1992), TCAs appear not to strongly inhibit the metabolism of CYP2C19 substrates. None of these reports explain how TCAs increase the plasma concentration of phenytoin in epileptics (Richens and Houghton, 1975; Perucca and Richens, 1977).
Therefore, in this study, we assessed the potential of TCAs to inhibit different P450 isoforms in vitro, to examine the mechanism of the drug interaction between TCAs and phenytoin. First, we evaluated whether the TCAs—amitriptyline, nortriptyline, imipramine, and desipramine—inhibited phenytoin p-hydroxylation in microsomal incubations in vitro. Then, we used incubation studies of human liver microsomes and cDNA-expressed P450s to determine the inhibitory potential of TCAs on P450 isoform-specific metabolic pathways.
Materials and Methods
Chemicals and Reagents.
5,5-Diphenylhydantoin (phenytoin) and (±) HPPH were purchased from Sigma-Aldrich (St. Louis, MO). 1-Hydroxymidazolam,S-warfarin, 7-hydroxywarfarin, S-mephenytoin, dextrorphan, and 4′-hydroxymephenytoin were obtained from Ultrafine Chemical Co. (Manchester, UK). Dextromethorphan, ketoconazole, tricyclic antidepressants (imipramine, desipramine, amitriptyline, and nortriptyline), omeprazole, sulfaphenazole, chlorozoxazone, furafylline, NADP, NADPH, EDTA, MgCl2, G-6-P, and G-6-PDH were obtained from Sigma-Aldrich. Acetonitrile and methanol were acquired from Fisher Scientific Co. (Pittsburgh, PA). Midazolam was kindly provided by Roche Korea Co. (Seoul, Korea). All other reagents and chemicals used were of analytical or HPLC grade.
Human Liver Microsomes and cDNA-Expressed P450s.
Microsomes were prepared from human liver tissue (HL-10, 14, 15, 19, and 20) that was obtained from patients undergoing partial hepatectomy for removal of metastatic tumor at the Department of General Surgery, Busan Paik Hospital (Busan, Korea). The tissues were nontumor-bearing parenchyma and confirmed to be histopathologically normal. Tissue obtained from patients who had taken any known P450 inhibitors or inducers in the week before the surgical operation was not used. The use of human liver tissue was approved by the Institutional Review Board of the hospital. Microsomes were prepared by differential centrifugation of liver homogenate as described previously (Ko et al., 1997). The resulting microsomal pellets were resuspended at a final protein concentration of 10 mg/ml in 100 mM phosphate buffer (pH 7.4) containing 1.0 mM EDTA and 5.0 mM MgCl2. The protein concentration was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard. Aliquots of microsomes were frozen and stored at −80°C.
Five different human cDNA-expressed recombinant enzymes were purchased from BD Gentest Co. (Woburn, MA) (Supersomes): CYP1A2, 2C9, 2C19, 2D6, and 3A4. Protein concentrations and P450 contents were supplied by the manufacturer.
Incubation Studies.
From the preliminary studies, the optimum conditions for microsomal incubation were determined in the linear range for HPPH formation from phenytoin, expressed as the quantity of HPPH formed per unit of protein concentration and time. In all experiments, phenytoin was dissolved and diluted serially in methanol. The solvent was subsequently removed by evaporation to dryness, under reduced pressure in 1.5-ml polypropylene tubes, with an AES2010 SpeedVac (Savant Instruments Inc., Holbrook, NY). The phenytoin was then reconstituted in 50 mM phosphate buffer (pH 7.4). The incubation mixtures containing either 25 μl of microsomes (10 mg/ml of stock) or 25 μl of cDNA-expressed P450s (diluted to 200 pmol/ml with buffer, pH 7.4) and phenytoin reconstituted in phosphate buffer were prewarmed for 5 min at 37°C. Reactions were initiated by adding the NADPH-regenerating system (including 1.3 mM NADP, 3.3 mM G-6-P, 3.3 mM MgCl2, and 1.0 U/ml G-6-PDH), and the reaction mixtures (final volume, 250 μl) were incubated for 60 min at 37°C. Reactions were stopped by placing the incubation tubes on ice and adding 100 μl of 10% ice-cold perchloric acid. After addition of 20 μl (10 μM) of chlorzoxazone as an internal standard, the mixtures were centrifuged at 14,000 rpm for 5 min at 4°C, and aliquots of supernatant were injected onto an HPLC system.
Analytical Procedures.
The concentrations of HPPH and internal standard were measured by HPLC. The system consisted of a Gilson 307 pump, an 118 UV detector, a 234 Autoinjector (Gilson Co., Villiers Le Bel, France), and a μ-Bondapak C18 column (3.9 mm i.d. × 30 cm) packed with 10-μm particles at ambient temperature. Acetonitrile, 0.05% phosphoric acid, and 50 mM potassium phosphate (33:47:20, v/v, pH 3.2) constituted the mobile phase; the flow rate was 1.0 ml/min. The Unipoint analysis system (Gilson Co.) was used to calculate the HPPH concentration from the peak area ratios. Chromatograms were obtained with UV detection at a wavelength of 210 nm. Under these conditions, peaks of HPPH, internal standard, and phenytoin appeared at 6.0, 10.0, and 12.5 min, respectively. Most of the TCAs and their metabolites did not interfere with the HPPH peak, but since the desipramine peak partially overlapped the HPPH peak, we changed the mobile phase to acetonitrile, 0.05% phosphoric acid, and 50 mM potassium phosphate (30:47:23, v/v, pH 3.2) when desipramine was a test compound.
Inhibition Studies on Phenytoin p-Hydroxylation.
The inhibitory effects of TCAs and known P450 isoform-selective inhibitors on phenytoin p-hydroxylation were compared to determine the P450 isoform(s) responsible for the interaction between TCAs and phenytoin. The formation rate of HPPH from phenytoin (final concentration, 25 μM) was determined from mixtures incubated in the absence or presence of TCAs and known P450 isoform-selective inhibitors, furafylline for CYP1A2 (Sesardic et al., 1990), sulfaphenazole for CYP2C9 (Baldwin et al., 1995), omeprazole for CYP2C19 (Ko et al., 1997), quinidine for CYP2D6 (Broly et al., 1989), and ketoconazole for CYP3A4 (Baldwin et al., 1995). Quercetin was tested as a potential inhibitor of CYP2C8 (Desai et al., 1998). Since amitriptyline and imipramine showed remarkable inhibition of HPPH formation from phenytoin, detailed inhibition studies were conducted after coincubation of various combinations of phenytoin concentration (final, 10, 25, 50, and 100 μM) and a TCA (concentration range of 1–100 μM). We also incubated 25 μM of phenytoin and TCAs (10 and 50 μM) with 20 pmol of cDNA-expressed CYP2C9 or CYP2C19, to determine which specific P450 isoform-catalyzed phenytoinp-hydroxylation was inhibited by the TCAs.
Inhibition Studies on P450 Isoform-Specific Substrates.
The inhibitory effects of the TCAs on each P450 isoform were evaluated by human liver microsomal incubations, using probe drugs specific for each P450 isoform. The reaction probes used were phenacetinO-deethylation for CYP1A2 (Tassaneeyakul et al., 1993),S-warfarin 7-hydroxylation for CYP2C9 (Rettie et al., 1992),S-mephenytoin 4′-hydroxylation for CYP2C19 (Wrighton et al., 1993), dextromethorphan O-demethylation for CYP2D6 (Broly et al., 1989), and midazolam 1-hydroxylation for CYP3A4 (Thummel et al., 1994). The incubation conditions and analytical assays for the activity of these isoforms were similar to the method previously described (Thummel et al., 1994; Shin et al., 1999).
Data Analysis.
Results were expressed as mean ± S.D. of estimates obtained from the three different liver microsomes with duplicated experiments. The apparent kinetic parameters for phenytoin p-hydroxylation (Km andVmax) and inhibitory potential (IC50 and Ki) were initially estimated by graphical methods (Lineweaver-Burk plot, Dixon plot, and secondary Lineweaver-Burk plot) but ultimately determined by nonlinear least square regression analysis from the best enzyme kinetic model and enzyme inhibition model (Segel, 1975) using WinNonlin software (Scientific Consulting Inc., Apex, NC).
Results
Characterization of Phenytoin p-Hydroxylation.
Under our experimental conditions, the five human liver microsomal preparations produced sufficient p-hydroxy metabolite from phenytoin, and showed a 2- and 3-fold variance ofKm (range, 14.4 to 30.7 μM) andVmax (range, 9.0 to 26.6 nmol/min/mg of protein) for phenytoin p-hydroxylation (Table1). HPPH formation was completely abolished by 5 μM sulfaphenazole, and partly inhibited by 10 μM omeprazole, but not by furafylline, quercetin, quinidine, or ketoconazole (Fig. 1). These results indicate that our experimental conditions were appropriate for the subsequent inhibition studies on CYP2C9- and CYP2C19-catalyzed phenytoin p-hydroxylation. In addition, we also confirmed that cDNA-expressed CYP2C9 and CYP2C19 produced HPPH metabolite at a velocity of 31.1 ± 5.6 and 31.8 ± 3.1 pmol/min/pmol P450, respectively. However, little or no HPPH was formed from cDNA-expressed CYP2D6, CYP1A2, or CYP3A4 (data not shown).
Inhibitory Effects of TCAs on Phenytoinp-Hydroxylation.
Under the above experimental conditions, the tertiary amine TCAs, amitriptyline and imipramine, markedly inhibited the formation of HPPH from 25 μM of phenytoin with IC50 values of 69.1 ± 7.0 and 111.6 ± 7.9 μM, respectively (Fig.2). HPPH formation was decreased by 30% by 50 μM of either TCA, which is comparable with the inhibition effected by 10 μM omeprazole, a well known CYP2C19-selective inhibitor (Fig. 1). From the detailed inhibition study of phenytoinp-hydroxylation, a competitive inhibition model was best-fitted to the data of both amitriptyline and imipramine (estimatedKi values of 5.2 ± 3.5 μM and 15.5 ± 8.1 μM, respectively, Fig.3). However, the secondary amine TCAs, nortriptyline and desipramine, produced only weak inhibition—less than 15% reduction in activity—of phenytoin p-hydroxylation, even at the highest concentrations tested (100 μM, Fig. 2). The estimated IC50 values of nortriptyline and desipramine on HPPH formation from 25 μM of phenytoin were 600 and 695 μM, respectively. In preincubation studies to test the possibility of irreversible inhibition, the rate of HPPH formation from phenytoin was not further decreased by preincubation with either amitriptyline or imipramine, suggesting that tertiary TCAs inhibition of HPPH formation is not mechanism based (data not shown).
When the inhibitory effects of TCAs on specific P450 isoform-catalyzed phenytoin p-hydroxylation were evaluated, none inhibited the HPPH formation catalyzed by cDNA-expressed CYP2C9 (Fig.4). However, HPPH formation catalyzed by cDNA-expressed CYP2C19 was strongly inhibited by all of the TCAs.
Inhibitory Effects of TCAs on P450 Isoform-Specific Substrates.
Among the P450 isoforms tested, CYP2D6-catalyzed dextromethorphanO-demethylation was the most strongly and competitively inhibited by all of the TCAs (Fig. 5D); the dextrorphan formation rate was decreased to 17 to 30% of control activity at the highest concentration tested (100 μM). With this enzyme, secondary amine TCAs were more potent inhibitors than tertiary amine TCAs. Estimated Ki values were 31.0, 28.6, 7.9, and 12.5 μM for amitriptyline, imipramine, nortriptyline, and desipramine, respectively.
TCAs also inhibited CYP2C19-catalyzed S-mephenytoin 4′-hydroxylation (Fig. 5A). In contrast to the results for CYP2D6, amitriptyline and imipramine showed remarkable inhibition of CYP2C19-catalyzed S-mephenytoin 4′-hydroxylation, whereas nortriptyline and desipramine showed minimal inhibition. The estimated IC50 values were 59.1 ± 7.0 and 111.6 ± 8.0 μM for amitriptyline and imipramine, respectively, whereas those for nortriptyline and desipramine were 286 and 732 μM, respectively. Inhibition by amitriptyline and imipramine of CYP2C19-catalyzed S-mephenytoin 4′-hydroxylation was best-fitted to a competitive inhibition model with mean estimatedKi values of 37.7 ± 7.6 and 56.8 ± 16.8 μM (Fig. 6).
CYP1A2-catalyzed phenacetin O-deethylation, CYP2C9-catalyzedS-warfarin 7-hydroxylation and CYP3A4-catalyzed midazolam 1-hydroxylation were only negligibly inhibited, if at all, by any of the TCAs tested in the microsomal incubations [Fig. 5 (B, C, and E)].
Discussion
TCAs have been used extensively in the treatment of depression, and their metabolic pathways and associated enzymes are well known. All TCAs are converted to 2- or 10-hydroxy metabolites by CYP2D6 (Brøsen and Gram, 1988). CYP2C19 is the main enzyme involved in theN-demethylation of imipramine and amitriptyline to desipramine and nortriptyline, respectively (Chiba et al., 1994). CYP1A2 and CYP3A4 are also partially responsible for imipramineN-demethylation (Lemoine et al., 1993), and CYP3A4, CYP1A2, CYP2D6, and CYP2C9 are responsible for amitriptylineN-demethylation in vitro (Venkatakrishnan et al., 1998).
Since many P450 isoforms are involved in the metabolism of TCAs, it is not surprising that numerous interactions between TCAs and enzyme inducers or inhibitors have been described (Barry and Feely, 1990;Tanaka and Hisawa, 1999). Except for one report of moderate inhibition of CYP2D6-catalyzed codeine O-demethylation (Yue and Säwe, 1997), however, the inhibitory potential of TCAs on different P450 isoforms has not been extensively described. Our study demonstrated strong inhibition of CYP2D6-catalyzed dextromethorphanO-demethylation by all of the TCAs examined; estimatedKi values of the TCAs were ranked as follows: amitriptyline (31.0 μM) > imipramine (28.6 μM) > desipramine (12.5 μM) > nortriptyline (7.9 μM). This hierarchy suggests that of the four TCAs tested, nortriptyline is the most potent CYP2D6 inhibitor. These results are comparable to the inhibitory potential of other antidepressants, such as velafaxine (Otton et al., 1994) and nefazodone (Schmider et al., 1996), against CYP2D6 activity in vitro.
In this study, TCAs also competitively inhibited CYP2C19-catalyzedS-mephenytoin 4′-hydroxylation. However, the inhibitory potential of tertiary and secondary amine TCAs differed. The tertiary amine TCAs, amitriptyline and imipramine, showed remarkable inhibition of CYP2C19, with estimated Kivalues of 37.7 and 56.8 μM, respectively. In contrast, the secondary amine TCAs, nortriptyline and desipramine, produced only minimal inhibition. These results seem reasonable, when we consider that nortriptyline and desipramine are substrates of CYP2D6 but not of CYP2C19 (Tanaka and Hisawa, 1999); all of the TCAs in the present study competitively inhibited CYP2C19-catalyzedS-mephenytoin 4′-hydroxylation.
These results might be helpful in understanding the mechanism of phenytoin-TCA interactions, which are cited in many review articles as being a representative drug interaction of TCAs (Ereshefsky et al., 1995; Monaco and Cicolin, 1999). Our results confirmed that TCAs, especially amitriptyline and imipramine, remarkably inhibit phenytoinp-hydroxylation, which is the primary metabolic pathway from phenytoin to HPPH (Browne and LeDuc, 1995). The estimatedKi values of amitriptyline and imipramine were 5.2 and 15.5 μM, which are 5- to 15-fold higher than their therapeutic range of plasma concentrations (0.2–1.0 μM). However, considering that 50- to 60-fold higher concentrations of imipramine and desipramine have been reported to accumulate in the postmortem liver (Swanson et al., 1997), significant inhibition of phenytoin p-hydroxylation by both compounds is likely in patients taking typical doses of TCAs and phenytoin. Additionally, the pharmacokinetics of phenytoin is known to be nonlinear and saturable. Indeed, around the Vmax of a patient, plasma concentrations of phenytoin can be significantly altered by as little as a 10% change in the daily dose (Rowland and Tozer, 1995). Therefore, the inhibitory potential of amitriptyline and imipramine seems adequate to yield a significant inhibitory drug interaction at standard phenytoin doses. Thus, our results support previous case reports of drug interaction between TCAs and phenytoin (Richens and Houghton, 1975; Perucca and Richens, 1977).
According to this study, it is clear that amitriptyline and imipramine inhibit the CYP2C19-catalyzed phenytoin p-hydroxylation. However, both tertiary TCAs themselves are substrates of CYP2C19 in the formation of N-demethylated metabolites nortriptyline and desipramine, respectively (Chiba et al., 1994). We also confirmed the disappearance of parent amitriptyline and formation of nortriptyline (estimated Km, 34 μM) after microsomal incubations of 0.5 and 10 μM amitriptyline for 60 min, respectively. These data indicate that both amitriptyline and imipramine are competitive substrates with phenytoin, not competitive inhibitors of CYP2C19.
Although CYP2C9 is mainly responsible for phenytoinp-hydroxylation (Bajpai et al., 1996; Giancarlo et al., 2001), it seems not to be involved in the inhibitory interactions with TCAs in phenytoin metabolism. In our microsomal incubation studies, the TCAs failed to inhibit CYP2C9-catalyzed S-warfarin 7-hydroxylation, CYP1A2-catalyzed phenacetin O-deethylation, or CYP3A4-catalyzed midazolam 1-hydroxylation. Moreover, while all of the TCAs tested strongly inhibited phenytoin p-hydroxylation catalyzed by cDNA-expressed CYP2C19, none inhibited the formation of HPPH catalyzed by cDNA-expressed CYP2C9. Tertiary amine TCAs also showed remarkable inhibition of CYP2C19-catalyzedS-mephenytoin 4′-hydroxylation in microsomal incubations. Together, these data suggest that CYP2C19-catalyzed phenytoinp-hydroxylation is the metabolic pathway responsible for the interaction between TCAs and phenytoin. Several other drugs (i.e., ticlopidine, omeprazole, and cimetidine) are also known to interact with phenytoin through CYP2C19 inhibition, not by CYP2C9 inhibition (Levy, 1995; Rindone and Bryan, 1996).
Since CYP2C9 is an enzyme that shows genetic polymorphism, inhibition of CYP2C19-catalyzed phenytoin p-hydroxylation by TCAs is more likely to be significant in patients who are deficient in phenytoin metabolism due to CYP2C9 mutation such as CYP2C9*3 (Ile359Leu) allele. In one study, mean phenytoinVmax was 42% lower in heterozygous CYP2C9*3 subjects than in subjects with wild-type CYP2C9*1 alleles (Mamiya et al., 1998). Since the allele frequency of CYP2C9*3 is 1 to 2% in Asians and 6 to 10% in Caucasians (Yoon et al., 2001), it will not be unusual to observe interactions between TCAs and phenytoin in patients with the CYP2C9*3 allele.
As expected, CYP2D6 inhibition does not seem to be implicated in the inhibitory interactions of TCAs with phenytoin. All of the TCAs in this study strongly inhibited CYP2D6-catalyzed dextromethorphanO-demethylation. However, quinidine, a CYP2D6-selective inhibitor (Broly et al., 1989), had no effect on phenytoinp-hydroxylation, and cDNA-expressed CYP2D6 failed to produce HPPH, which is consistent with previous reports (Komatsu et al., 2000;Giancarlo et al., 2001).
In conclusion, our results demonstrate that TCAs have moderate to strong inhibitory potential on CYP2C19 as well as CYP2D6 in human liver microsomes. They also suggest that the drug interaction between TCAs and phenytoin is caused by their inhibition of CYP2C19-catalyzed phenytoin p-hydroxylation.
Footnotes
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This study was supported in part by the Ministry of Science and Technology of the Republic of Korea and by the Korea Science and Engineering Foundation through the Biohealth Products Research Center at Inje University, Korea.
- Abbreviations used are::
- HPPH
- 5-(p-hydroxyphenyl)-5-phenylhydantoin
- TCA
- tricyclic antidepressants
- G-6-P
- glucose 6-phosphate
- G-6-PDH
- glucose 6-phosphate dehydrogenase
- HPLC
- high-performance liquid chromatography
- Received January 1, 2002.
- Accepted July 2, 2002.
- The American Society for Pharmacology and Experimental Therapeutics