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Research ArticleArticle

Evaluation of Methoxsalen, Tranylcypromine, and Tryptamine as Specific and Selective CYP2A6 Inhibitors in Vitro

Wenjiang Zhang, Tansel Kilicarslan, Rachel F. Tyndale and Edward M. Sellers
Drug Metabolism and Disposition June 2001, 29 (6) 897-902;

Abstract

CYP2A6 is the principle enzyme metabolizing nicotine to its inactive metabolite cotinine. In this study, the selective probe reactions for each major cytochrome P450 (P450) were used to evaluate the specificity and selectivity of the CYP2A6 inhibitors methoxsalen, tranylcypromine, and tryptamine in cDNA-expressing and human liver microsomes. Phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), diclofenac 4′-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), 7-ethoxy-4-trifluoromethylcoumarin deethylation (CYP2B6),p-nitrophenol hydroxylation (CYP2E1), and omeprazole sulfonation (CYP3A4) were used as index reactions. ApparentK i values for inhibition of P450s' (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) activities showed that tranylcypromine, methoxsalen, and tryptamine have high specificity and relative selectivity for CYP2A6. In cDNA-expressing microsomes, tranylcypromine inhibited CYP2A6 (K i = 0.08 μM) with about 60- to 5000-fold greater potency relative to other P450s. Methoxsalen inhibited CYP2A6 (K i = 0.8 μM) with about 3.5- 94-fold greater potency than other P450s, except for CYP1A2 (K i = 0.2 μM). Tryptamine inhibited CYP2A6 (K i = 1.7 μM) with about 6.5- 213-fold greater potency relative to other P450s, except for CYP1A2 (K i = 1.7 μM). Similar results were also obtained with methoxsalen and tranylcypromine in human liver microsomes. R-(+)-Tranylcypromine, (±)-tranylcypromine, and S-(−)-tranylcypromine competitively inhibited CYP2A6-mediated metabolism of nicotine with apparentK i values of 0.05, 0.08, and 2.0 μM, respectively. Tranylcypromine [particularly R-(+) isomer], tryptamine, and methoxsalen are specific and relatively selective for CYP2A6 and may be useful in vivo to decrease smoking by inhibiting nicotine metabolism with a low risk of metabolic drug interactions.

The use of tobacco products is associated with a variety of medical disorders including increased risk of cancer and cardiovascular and respiratory diseases (Hecht and Hoffmann, 1988). Nicotine is the primary psychoactive substance in tobacco responsible for establishing and maintaining tobacco dependence (Henningfield et al., 1985). It is metabolized primarily (∼70%) to its inactive metabolite cotinine by the genetically variable enzyme CYP2A6 (Nakajima et al., 1996; Messina et al., 1997). CYP2A6 also mediates the activation of several procarcinogens, such as tobacco-related nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, aflatoxin B1, and hexamethyl-phosphoramide (Crespi et al., 1991; Yamazaki et al., 1992).

In addition to the wild-type CYP2A6*1 gene, a gene deletionCYP2A6*4 and point mutation CYP2A6*2 have been isolated and cloned (Yamano et al., 1990; Oscarson et al., 1999). Some studies have shown that individuals with a CYP2A6 null allele or gene deletion appear to smoke fewer cigarettes (Pianezza et al., 1998; Rao et al., 2000), while others have failed to demonstrate such a correlation (Sabol and Hamer, 1999; Tiihonen et al., 2000). Imitating the gene defect by chemical inhibition of CYP2A6 activity in vivo may decrease smoking and activation of carcinogens and could provide the basis for novel therapeutic approaches to smoking reduction and cessation (Sellers et al., 2000). In fact, our research group has recently demonstrated that 30 mg of oral methoxsalen plus 4 mg of nicotine attenuated nicotine clearance and increased its bioavailability, while decreasing cigarette smoking by 24% (p < 0.01), as compared with placebo plus placebo (Sellers et al., 2000). Methoxsalen, or any ideal CYP2A6 inhibitor used for the treatment of tobacco dependence, should be specific and selective for CYP2A6 to minimize potential adverse drug-drug interactions.

Tranylcypromine (TCP1) is a monoamine oxidase inhibitor used in the treatment of depression. Methoxsalen is a pigmentation agent used in the treatment of psoriasis cutaneous, T-cell lymphoma, and vitiligo. Both are potent inhibitors of CYP2A6 in vitro (Maenpaa et al., 1993; Draper et al., 1997). Moreover, methoxsalen has been shown to significantly inhibit coumarin and nicotine metabolism in vivo in humans, as discussed (Maenpaa et al., 1994; Kharasch et al., 2000; Sellers et al., 2000). The indolealkaloid, tryptamine, is a trace amine found in the brain and is a structural congener of 5-hydroxytryptamine. Tryptamine is a substrate of monoamine oxidase (Sullivan et al., 1986) and has also proved to be a potent inhibitor of CYP2A6 in our laboratory. The human hepatic cytochrome P450 (P450) enzymes 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 are the most important forms of drug-metabolizing P450s in humans (Shimada et al., 1994). The metabolism of many therapeutically important drugs and endogenous compounds is mediated primarily by these enzymes.

The present study has identified the specificity and selectivity of these drugs toward CYP2A6 and the major P450s in cDNA-expressing (human lymphoblast cells) and human liver microsomes.

Materials and Methods

Chemicals and Reagents.

Phenacetin, acetaminophen, coumarin, 7-hydroxycoumarin, 7-ethoxy-4-trifluoromethylcoumarin, 7-hydroxy-4-trifluoromethylcoumarin, diclofenac sodium, dextromethorphan hydrobromide, dextrorphan, 4-nitrophenol, 4-nitrocatechol, budipine, S-(−)-nicotine,S-(−)-cotinine, methoxsalen, pilocarpine, orphenadrine, sulfaphenazole, ketoconazole, α-naphthoflavone, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). TCP and trioxsalen were purchased from Aldrich Chemical Co. (Milwaukee, WI).S-(−)-TCP and R-(+)-TCP were kindly provided by Röhm Pharma (Weiterstadt, Germany). Omeprazole, 5-hydroxyomeprazole, omeprazole sulfone, and 5-methoxy-2-[[(3,4-dimethoxy-5-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimid-azole (H168/24) were generously donated by Astra Hässle (Mölndal, Sweden). S-(+)-Mephenytoin and 4′-hydroxydiclofenac sodium were purchased from GENTEST (Woburn, MA). All other chemical reagents used were of the highest commercially available quality.

cDNA-Expressing and Human Liver Microsomes.

cDNA-expressing cytochrome P450 microsomes (P450 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, and 3A4) from human lymphoblast cells were purchased from GENTEST. The human livers were kindly provided by Dr. T. Inaba. The characteristics and sources of the K series livers in this study have been previously described (Tyndale et al., 1989). Microsomes from these tissues were prepared and stored according to established techniques (Tyndale et al., 1989). Protein concentrations were determined using bicinchoninic acid protein assay kit (Pierce Chemical CO., Rockford, IL). Cytosolic fractions from the livers of four male Wistar rats were used as a source of aldehyde oxidases for the CYP2A6 nicotine assay.

P450 Index Reaction Assays.

All incubations were carried out in 25 mM Tris-HCl buffer (pH 7.4) at 37°C in a shaking water bath with 1 mM NADPH. For each substrate (probe drug), preliminary experiments were performed to determine whether metabolite formation was linear with respect to time, NADPH, and microsomal protein concentrations. The percentage of conversion of all metabolites never exceeded 15% of total substrate added. Incubation conditions varied depending on the characteristics of the probe drug, which are outlined in Table1. Conditions were the same for both human liver and cDNA-expressing microsomes.

View this table:
Table 1

Incubation conditions used for P450 index reaction assays

The reactions were terminated by removal to ice and by addition of the appropriate internal standard. Samples were shaken in a vortex shaker for 30 min followed by centrifugation for 15 min (3000g). The organic phase was separated from the aqueous phase either by aspirating the aqueous phase or transferring the organic layer to a new tube depending on which layer was on top for the particular extraction method, as described below. The organic phase was then evaporated to dryness under nitrogen. The analytical system used was a Hewlett Packard (HP) 1100 series UV-liquid chromatographic system. Formation of metabolite was quantified by interpolating peak area ratios of metabolite and internal standard from a standard curve of known metabolite concentration.

Phenacetin O-Deethylation (CYP1A2).

Extraction was essentially that of Venkatakrishnan et al. (1998). 7-Hydroxycoumarin was added as an internal standard, and the mixture was extracted with ethyl acetate. Samples were reconstituted into mobile phase (200 μl) prior to injection into the HPLC [HP Spherisorb ODS2 column; UV = 245 nm; 10 mM sodium acetate buffer and acetonitrile (ACN) (87.5:12.5, v/v, pH 4.5) at 1 ml/min].

Coumarin 7-Hydroxylation (CYP2A6).

Extraction was essentially that of Li et al. (1997). 7-Aminocoumarin was added as the internal standard, and the mixture was extracted with ethyl acetate. Samples were reconstituted in 200 μl of 30% methanol prior to HPLC injection [HP ODS-2 column; UV = 320 nm; ACN/water/acetic acid (25:75:0.1, v/v/v) at 1 ml/min].

7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation (CYP2B6).

Trioxsalen was added as internal standard, and the mixture was extracted with ethyl acetate. Samples were reconstituted in mobile phase (200 μl) prior to HPLC analysis [HP Spherisorb ODS2 column; UV = 360 nm; ACN/water/acetic acid (50:50:0.1, v/v/v) at 1 ml/min].

Diclofenac 4′-Hydroxylation (CYP2C9).

Coumarin was added as an internal standard, and the mixture was extracted with ethyl acetate. Samples were reconstituted in 20% acetic acid (200 μl) prior to HPLC analysis [HP Spherisorb ODS2 column; UV = 280 nm; ACN/water/acetic acid (40:60:0.25, v/v/v) at 1 ml/min].

Omeprazole Assay (CYP2C19 and CYP3A4).

Omeprazole and its metabolites were assayed as per the method ofAndersson et al. (1994). H168/24 was used as the internal standard, and the mixture was extracted with dichloromethane. Samples were reconstituted in 200 μl of mobile phase prior to HPLC analysis [Waters Spherisorb C6 column; UV = 304 nm; 10 mM KH2PO4 buffer (pH 7.0)/ ACN (75:25, v/v) at 1 ml/min; Waters Corp., Milford, MA].

Dextromethorphan O-Demethylation (CYP2D6).

Extraction was essentially that of Otton et al. (1993). The enzymatic reaction was stopped by the addition of 200 μl of NaHCO3-Na2CO3buffer, and butorphanol was used as the internal standard. The mixture was extracted with hexane/ether (4:1, v/v), then back-extracted with 200 μl of 0.01 N HCl prior to HPLC analysis [CSC-Spherisorb-phenyl column; UV = 210 nm; 10 mM KH2PO4 buffer (pH 3.8)/ACN (73:27, v/v) at 1 ml/min].

4-Nitrophenol 4-Hydroxylation (CYP2E1).

The reaction was stopped by the addition of 3 μM HCl (50 μl), and 7-hydroxycoumarin was added as the internal standard. The mixture was extracted with ether (2 ml), and the organic phase was evaporated to dryness and reconstituted into mobile phase (200 μl) prior to HPLC analysis [Spherisorb ODS-2 column; UV = 250 nm; KH2PO4 buffer containing 1 mM octanesulfonic acid and 0.5% triethylamine (pH 2.6)/ACN (81.5:18.5, v/v) at 1 ml/min].

Nicotine Metabolism Assay.

The assay used was previously described (Messina et al., 1997).S-(−)-Nicotine (5–1000 μM) was incubated for 30 min with cDNA-expressing microsomes (final concentration, 1.5 mg/ml) in the presence of 1 mM NADPH (final concentration) and 25 μl of rat liver cytosol as the aldehyde oxidase source. Methyl cotinine (50 μl of 2.0 μg/ml) was added as the internal standard, and samples were extracted with 2 ml of dichloromethane, dried under nitrogen, and reconstituted in 0.01 M HCl (200 μl) prior to HPLC analysis [Supelcosil LC-8DB column; UV = 260 nm; KH2PO4 buffer containing 1 mM octanesulfonic acid and 0.5% triethylamine (pH 4.6)/ACN (90:10, v/v) at 1 ml/min].

Apparent K m andV max values obtained in human liver microsomes (HLM) for all substrates used were in agreement with previous studies (data not shown) (Leemann et al., 1993;Tassaneeyakul et al., 1993; Andersson et al., 1994; Bourrie et al., 1996; von Moltke et al., 1996; Ekins et al., 1997; Li et al., 1997). One-site kinetics were obtained for coumarin 7-hydroxylation, diclofenac 4′-hydroxylation, and omeprazole sulfonation, while two-site kinetics were determined for all remaining assays. In instances where more than one enzyme was involved in the metabolism of the substrate, the substrate concentration was carefully selected to equal theK m values for the production of metabolite by the P450 enzyme of interest.

Chemical Inhibition Studies of P450s with Inhibitors of CYP2A6.

Initial screening experiments were carried out using two concentrations of inhibitor (20 and 200 μM), and known selective P450 inhibitors were selected as controls according to previously published reports (Bourrie et al., 1996; Eagling et al., 1998; Hichman et al., 1998). They were α-naphthoflavone (CYP1A2), pilocarpine (CYP2A6), orphenadrine (CYP2B6), sulfaphenazole (CYP2C9),S-(+)-mephenytoin (CYP2C19), budipine (CYP2D), and ketoconazole (CYP3A4). Probe substrate concentrations used for each P450 were identical to K m values for metabolism of these substrates in human liver microsomes. In the case of mechanism-based inhibitors (e.g., orphenadrine), the inhibitor was preincubated with microsomes and NADPH for 30 min prior to the addition of substrate. For determination of apparentK i values, the substrate concentrations used for each index reaction were equal to 1/2K m, K m, 2K m. At each of the substrate levels, metabolite formation was monitored in the absence and in the presence of methoxsalen, tryptamine, and TCP individually (e.g., 1/4 IC50, 1/2 IC50, IC50, 2 IC50). IC50 refers to the concentration of inhibitor required to inhibit 50% of substrate activity (atK m concentration).

Data Analysis.

Metabolic constants (K m,V max) were determined by use of nonlinear regression analysis after Michaelis-Menten representation (rate of metabolite formation by substrate concentration) using Enzpack 3 software (Cambridge, UK). Inhibition patterns were determined by Dixon plots. Apparent inhibition constant (K i) values for competitive inhibition were estimated by visualization of data through Dixon plots or by using Pharm/PCS software (Wynnewood, PA).

Results

Evaluation of TCP and Methoxsalen Inhibition of Cotinine Formation from Nicotine.

Of about 200 drugs screened as CYP2A6 inhibitors, including various monoamine oxidase inhibitors, antifungals, antidepressants, antipsychotics, and coumarin analogs, TCP, methoxsalen, and tryptamine displayed the highest CYP2A6 inhibitory potency upon screening. To confirm the potency inhibition, apparent K ivalues were determined in CYP2A6-expressing microsomes using nicotine metabolism to cotinine as the substrate reaction. Dixon plots indicate that TCP and tryptamine showed competitive inhibition (K i values of 65 nM and 1.7 μM, respectively) in cDNA-expressing microsomes (Figs.1 and 2). Methoxsalen showed noncompetitive inhibition of nicotine metabolism, with an apparent K i value of 0.1 μM in cDNA-expressing microsomes (Fig. 3). Similar results were also observed in HLM, with apparentK i values of 0.2, 1.9, and 0.2 μM, for TCP, tryptamine, and methoxsalen, respectively (data not shown). Previous studies indicate that methoxsalen is a mechanism-based inactivator of CYP2A6 in human liver and cDNA-expressing microsomes (Draper et al., 1997; Koenigs et al., 1997). The noncompetitive nature of methoxsalen inhibition of CYP2A6 observed presently suggests that mechanism-based inactivation may be taking place, although further studies are necessary to confirm these findings.

Figure 1
Figure 1

Dixon plot of tranylcypromine inhibition of nicotine metabolism in cDNA-expressing CYP2A6 microsomes.

Velocity is expressed in nanomoles per milligram of protein per minute.

Figure 2
Figure 2

Dixon plot of tryptamine inhibition of nicotine metabolism in cDNA-expressing CYP2A6 microsomes.

Velocity is expressed in nanomoles per milligram of protein per minute.

Figure 3
Figure 3

Dixon plot of methoxsalen inhibition of nicotine metabolism in cDNA-expressing CYP2A6 microsomes.

Velocity is expressed in nanomoles per milligram of protein per minute.

Potency of Inhibition (Apparent KiValues) of Major P450s by TCP, Tryptamine, and Methoxsalen in cDNA-Expressing Microsomes.

Inhibition of major P450s by methoxsalen, TCP, and tryptamine was first evaluated in HLM and cDNA-expressing microsomes at 20 and 200 μM. Even higher concentrations of TCP, tryptamine, and methoxsalen demonstrated a relatively higher degree of selectivity and specificity in their inhibition of CYP2A6. As shown in Table2, racemic TCP selectively inhibited CYP2A6-mediated metabolism of coumarin with an apparent Ki value of 0.08 μM. P450 2B6, 2C9, 2C19, and 2E1 were also inhibited by TCP, but with higher Ki values (5, 10, 15, and 12 μM, respectively). P450 1A2, 2D6, and 3A4 were inhibited with lower potency (K i = 25, 30, and 450 μM, respectively) relative to CYP2A6. Tryptamine selectively inhibited CYP1A2 and CYP2A6, both characterized by apparent K i values of 1.7 μM. P450 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 were also inhibited, but with higher K i values (75, 75, 85, 58, 11, and 363 μM, respectively).

View this table:
Table 2

The inhibition constants (Ki) of tranylcypromine, methoxsalen, and tryptamine on the major P450s in human liver microsomes and cDNA-expressing P450 microsomes from human lymphoblast cells

Methoxsalen was a less selective inhibitor than tranylcypromine and tryptamine. Methoxsalen inhibited CYP1A2 with the greatest potency (K i = 0.15 μM) in cDNA-expressing microsomes, but not in HLM (K i = 1.0 μM). CYP2A6- and CYP2B6-mediated reactions were inhibited withK i values of 0.8 and 2.8 μM, respectively. All other P450s were inhibited with an apparentK i range of 20 to 75 μM.

Potency of Inhibition (Apparent KiValues) of Major P450s by TCP and Methoxsalen in HLM.

In general, the degree and selectivity of inhibition followed the same pattern in HLM as in cDNA-expressing microsomes. The greatest differences between K i values measured in cDNA-expressing and human liver microsomes were observed in the inhibition of metabolic reactions mediated by more than one enzyme. For example, O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin in HLM is mediated by multiple enzymes including CYP2B6 (Ekins et al., 1997). However, 7-ethoxy-4-trifluoromethylcoumarin is the best available substrate because of high product turnover in cDNA-expressing microsomes. CYP1A2 has been shown to be the only enzyme involved in the high-affinity component of phenacetin O-deethylation in vitro (Venkatakrishnan et al., 1998); therefore, the lowerK i observed in cDNA-expressing microsomes is likely due to the absence of other P450s present in HLM that may interact with the inhibitor.

As shown in Table 2, TCP and methoxsalen have high inhibitory potency of CYP2A6 in HLM, but also inhibit other P450s tested. The apparentK i values of TCP ranged over 3 orders of magnitude from 0.2 to 280 μM. The rank order of inhibitory potency of TCP was as follows: P450 2A6 2E1 1A2 = 2C9 2D6 2C19 3A4. The apparent K i values of methoxsalen ranged over 2 orders of magnitude from 0.2 to 75 μM. The rank order of inhibitory potency was as follows: P450 2A6 1A2 2D6 2C9 2B6 3A4 = 2C19 2E1.

Stereoselective Inhibition of P450s byR-(+)-TCP and S-(−)-TCP.

TCP is a mixture of (−)- and (+)-trans-2-phenylcypropylamine, and previous studies have shown that the enantiomers of tranylcypromine have markedly different pharmacological properties (Hampson et al., 1986). To determine whether there was any stereoselective inhibition of CYP2A6 by TCP, we compared the formation of cotinine from nicotine in the presence ofR-(+)-TCP and S-(−)-TCP (Fig.4). S-(−)-TCP had a much lower inhibitory effect on the formation of cotinine relative toR-(+)-TCP. Apparent K i values were determined for R-(+)- and S-(−)-TCP with cDNA-expressing P450s, which showed the lowestK i values with racemic TCP (Table3). As shown, R-(+)-TCP was a 20 times more potent CYP2C19 inhibitor than S-(−)-TCP, while S-(−)-TCP was 2 times more potent in the inhibition of CYP2B6 than R-(+)-tranylcypromine. Both enantiomers displayed low K i values with CYP2A6 (using coumarin as the substrate), although R-(+)-TCP was a stronger inhibitor, with a 4-fold reducedK i, as compared with S-(−)-TCP. The inhibitory potency of S-(−)-TCP andR-(+)-TCP for CYP2E1 was not significantly different.

Figure 4
Figure 4

Stereoselective inhibition of CYP2A6-mediated cotinine formation from nicotine by two isomers of tranylcypromine.

▪, (±)-tranylcypromine; ▴, R-(+)-tranylcypromine; ▾, S-(−)-tranylcypromine.

View this table:
Table 3

The inhibition selectivity of racemic and two isomers of tranylcypromine on several major P450s

Discussion

In vitro kinetic and inhibition studies using cDNA-expressing and human liver microsomes, which can determine the selectivity and specificity of a particular drug in vitro, can be useful tools in assessing the potential for interactions among drugs in vivo. In this study, selective substrates and inhibitors for each of the principal drug-metabolizing P450s were used to determine the relative potency of inhibition of TCP, methoxsalen, and tryptamine for each enzyme.

The apparent K i values of tryptamine, methoxsalen, and TCP for each enzyme in cDNA-expressing microsomes showed that they are relatively selective and specific inhibitors of CYP2A6. Inhibition studies showed that methoxsalen inhibits CYP2A6 (0.2 and 0.8 μM) and CYP1A2 (K i = 1.0 and 0.2 μM) in human liver and cDNA-expressing microsomes, respectively, which is in agreement with previously published reports (Maenpaa et al., 1993; Ono et al., 1996; Draper et al., 1997; Koenigs et al., 1997). However, TCP inhibited CYP2A6 with greater potency and selectivity than methoxsalen. TCP inhibition of CYP2A6-mediated coumarin 7-hydroxylation was characterized by apparentK i values of 0.08 and 0.2 μM in cDNA-expressing and human liver microsomes, respectively. TCP inhibition of other P450 enzymes was generally characterized by higher apparent K i values than those for methoxsalen in human liver microsomes, indicating that TCP has greater selectivity for CYP2A6 than does methoxsalen (Tables 2 and 3). These results are in agreement with a previous study showing that TCP (at 1000 μM) has higher CYP2A6 selectivity than methoxsalen (at 10 μM) (Ono et al., 1996). The present study, in whichK i values were determined using a range of pharmacologically relevant inhibitor concentrations, more clearly demonstrates the potency of CYP2A6 inhibition by TCP and methoxsalen. The differences in interactions of both drugs with CYP3A4 are such that methoxsalen inhibits the enzyme with approximately 6- to 25- fold greater affinity than TCP in human liver and cDNA-expressing microsomes, respectively. However, even though CYP3A4 is the most abundant P450 enzyme in the liver and mediates the metabolism of many therapeutically important drugs, the concentrations needed to produce inhibition far exceed those achieved by methoxsalen in vivo (Sellers et al., 2000).

R-(+)-TCP is a more potent inhibitor of CYP2A6 (K i = 0.05 μM) than S-(−)-TCP (2.0 μM). As molecular modeling studies of CYP2A6 with coumarin have shown, Phe181, Gln74, and His437 residues at the CYP2A6 active site are mainly responsible for substrate-enzyme contact (Lewis et al., 1995). His437 and Gln74, in particular, form hydrogen bonds with the substrate. Phe181 forms π–π stacking interactions with the aromatic system of the substrate, ultimately positioning the substrate for the reaction (Lewis et al., 1995). The positioning of both the aromatic ring and amino groups of R-(+)-TCP in the CYP2A6 active site may be more favorable than that with theS-(−)-isomer, leading to a stronger enzyme-inhibitor interaction. Further studies are necessary to elucidate the stereoselectivity of TCP with P450 enzymes.

Some reports have suggested that individuals with a CYP2A6 null allele or gene deletion appear to smoke fewer cigarettes (Pianezza et al., 1998; Rao et al., 2000). In this study, TCP, methoxsalen, and tryptamine showed to be relatively selective inhibitors of CYP2A6, in particular, TCP. In vivo pharmacokinetic studies showed that people receiving a 20-mg oral dose of racemic TCP (Parnate) have peak plasma levels of 64.5 to 190 ng/ml (about 0.57–1.68 μM) at 1.5 h and a mean elimination half-life of 1.5 to 2.5 h (Mallinger and Edwards, 1986). The K i values observed here in vitro with racemic TCP (0.2 and 0.08 μM in human liver and cDNA-expressing microsomes, respectively) suggest that a conventional, therapeutic dose of TCP will inhibit CYP2A6 in vivo. Indeed, it has been shown that 2.5 and 10 mg of oral racemic TCP significantly increased the oral bioavailability of nicotine in vivo (Sellers et al., 2000).

It was also demonstrated, in vitro, that R-(+)-TCP is a more potent CYP2A6 inhibitor than S-(−)-TCP. It has been shown that individuals receiving a standard 20-mg dose of racemic TCP showed significantly higher plasma levels of S-(−)-TCP, as compared with R-(+)-TCP (Spahn-Langguth et al., 1992). Moreover, the apparent oral clearance of R-(+)-TCP exceeded that of S-(−)-TCP (Spahn-Langguth et al., 1992). Therefore, it remains to be determined whether R-(+)-TCP will be a stronger CYP2A6 inhibitor in vivo as compared withS-(−)-TCP.

Tryptamine was shown to be a selective inhibitor of CYP2A6 in vitro.l-Tryptophan can be metabolized into tryptamine in vivo by tryptophan decarboxylase. The concentration of tryptamine is correlated with that of l-tryptophan (Wollmann et al., 1985). Therefore, it may be possible to apply l-tryptophan as an in vivo CYP2A6 inhibitor, although further in vivo studies are clearly necessary.

In conclusion, the data presented here demonstrate that tryptamine, TCP, and methoxsalen have high specificity and relative selectivity for CYP2A6. TCP (particularly the R-isomer), methoxsalen, and tryptamine have relative selectivity for CYP2A6 inhibition and therefore would not be expected to give rise to significant clinical drug-drug interactions. Imitating the gene defect by chemical inhibition with R-(+)-TCP may be an effective way to treat nicotine dependence (Sellers et al., 2000). However, further long-term in vivo studies are required to validate this hypothesis.

Acknowledgment

We thank Yamini Ramamoorthy for help in preparing this manuscript.

Footnotes

  • Send reprint requests to: Dr. E. M. Sellers, Psychopharmacology and Dependence Research Unit, Sunnybrook Women's College Health Sciences Center, Room 947, 76 Grenville St., Toronto, ON, M5S 1B2, Canada. E-mail: e.sellers{at}utoronto.ca

  • This research project was supported in part by National Institute on Drug Abuse Grant DA06889.

  • Abbreviations used are::
    TCP
    tranylcypromine
    HLM
    human liver microsomes
    P450
    cytochrome P450
    HPLC
    high-performance liquid chromatography
    ACN
    acetonitrile
    H168/24
    5-methoxy-2-[[(3,4-dimethoxy-5-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimid-azole
    • Received November 21, 2000.
    • Accepted February 27, 2001.

References

  1. ↵
    1. Andersson T,
    2. Miners JO,
    3. Veronese ME,
    4. Birkett DJ
    (1994) Identification of human liver cytochrome P450 isoforms mediating secondary omeprazole metabolism. Br J Clin Pharmacol 37:597–604.
    OpenUrlPubMed
  2. ↵
    1. Bourrie M,
    2. Meunier V,
    3. Berger Y,
    4. Fabre G
    (1996) Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther 77:321–332.
    OpenUrl
  3. ↵
    1. Crespi CL,
    2. Penman BW,
    3. Gelboin HV,
    4. Gonzalez FJ
    (1991) A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6. Carcinogenesis 2:1197–1201.
    OpenUrl
  4. ↵
    1. Draper AJ,
    2. Madan A,
    3. Parkinson A
    (1997) Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch Biochem Biophys 41:47–61.
  5. ↵
    1. Eagling VA,
    2. Tjia JF,
    3. Back DJ
    (1998) Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol 5:107–114.
  6. ↵
    1. Ekins S,
    2. VandenBranden M,
    3. Ring BJ,
    4. Wrighton SA
    (1997) Examination of purported probes of human CYP2B6. Pharmacogenetics 7:165–179.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Hampson DR,
    2. Baker GB,
    3. Coutts RT
    (1986) A comparison of the neurochemical properties of the stereoisomers of tranylcypromine in the central nervous system. Cell Mol Biol 32:593–599.
    OpenUrlPubMed
  8. ↵
    1. Hecht SS,
    2. Hoffmann D
    (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9:875–884.
    OpenUrlFREE Full Text
  9. ↵
    1. Henningfield JE,
    2. Miyasato K,
    3. Jasinki DR
    (1985) Abuse liability and Pharmacodynamic characteristics of intravenous and inhaled nicotine. J Pharmacol Exp Ther 234:1–12.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Hichman D,
    2. Wang JP,
    3. Wang Y,
    4. Unadkat JD
    (1998) Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos 26:207–215.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Kharasch ED,
    2. Hankins DC,
    3. Taraday JK
    (2000) Single-dose methoxsalen effects on human cytochrome P-450 2A6 activity. Drug Metab Dispos 28:28–33.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Koenigs LL,
    2. Peter RM,
    3. Thompson SJ,
    4. Rettie AE,
    5. Trager WF
    (1997) Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen. Drug Metab Dispos 25:1407–1415.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Leemann T,
    2. Transon C,
    3. Dayer P
    (1993) Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4′-hydroxylation in human liver. Life Sci 52:29–34.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Lewis DFV,
    2. Lake BG
    (1995) Molecular modeling of members of the P4502A subfamily: application to studies of enzyme specificity. Xenobiotica 25:585–598.
    OpenUrlPubMed
  15. ↵
    1. Li Y,
    2. Li N-Y,
    3. Sellers EM
    (1997) Comparison of CYP2A6 catalytic activity on coumarin 7-hydroxylation in human and monkey liver microsomes. Eur J Drug Metab Pharmacokinet 22:295–304.
    OpenUrlPubMed
  16. ↵
    1. Maenpaa J,
    2. Juvonen R,
    3. Raunio H,
    4. Rautio A,
    5. Pelkonen O
    (1994) Metabolic interactions of methoxsalen and coumarin in human and mice. Biochem Pharmacol 48:1363–1369.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Maenpaa J,
    2. Sigusch H,
    3. Raunio H,
    4. Syngelma T,
    5. Vuorela P,
    6. Vuorela H,
    7. Pelkonen O
    (1993) Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes. Biochem Pharmacol 45:1035–1042.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Mallinger AG,
    2. Edwards DJ
    (1986) Pharmacokinetics of tranylcypromine in patients who are depressed: relationship to cardiovascular effects. Clin Pharmacol Ther 40:444–450.
    OpenUrlPubMed
  19. ↵
    1. Messina ES,
    2. Tyndale RF,
    3. Sellers EM
    (1997) A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J Pharmacol Exp Ther 282:1608–1614.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Nakajima M,
    2. Yamamoto T,
    3. Nunoya K,
    4. Yokoi T,
    5. Nagashima K,
    6. Inoue K,
    7. Funae Y,
    8. Shimada N,
    9. Kamataki T,
    10. Kuroiwa Y
    (1996) Role of human cytochrome P4502A6 in C-oxidation of nicotine. Drug Metab Dispos 24:1212–1217.
    OpenUrlAbstract
  21. ↵
    1. Ono S,
    2. Hatanaka T,
    3. Hotta H,
    4. Atoh T,
    5. Gonzalez FJ,
    6. Tsutsui M
    (1996) Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26:681–693.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Oscarson M,
    2. McLellan RA,
    3. Gullsten H,
    4. Yue QY,
    5. Lang MA,
    6. Bernal ML,
    7. Inues B,
    8. Hirvonen A,
    9. Raunio H,
    10. Pelkonen O,
    11. Ingelman-Sundberg M
    (1999) Characterization and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett 448:105–110.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Otton SV,
    2. Wu D,
    3. Joffe RT,
    4. Sellers EM
    (1993) Inhibition by fluoxetine of cytochrome P450 2D6 activity. Clin Pharmacol Ther 53:401–409.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Pianezza ML,
    2. Sellers EM,
    3. Tyndale RF
    (1998) Nicotine metabolism defect reduces smoking. Nature (Lond) 393:750.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Rao Y,
    2. Hoffmann E,
    3. Zia M,
    4. Bodin L,
    5. Zeman M,
    6. Sellers EM,
    7. Tyndale RF
    (2000) Duplications and defects in the CYP2A6 gene: identification, genotyping, and in vivo effects on smoking. Mol Pharmacol 58:747–755.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Sabol SZ,
    2. Hamer DH
    (1999) An improved assay shows no association between the CYP2A6 gene and cigarette smoking. Behav Genet 29:257–261.
    OpenUrlCrossRef
  27. ↵
    1. Sellers EM,
    2. Zeman MV,
    3. Kaplan HL,
    4. Tyndale RF
    (2000) Inhibition of cytochrome P450 2A6 (CYP2A6) decreases smoking and activation of the procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Clin Pharmacol Ther 67:113.
    OpenUrl
  28. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemical studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Spahn-Langguth H,
    2. Hahn G,
    3. Mutschler E,
    4. Mohrke W,
    5. Langguth P
    (1992) Enantiospecific high-performance liquid chromatographic assay with fluorescence detection for the monoamine oxidase inhibitor tranylcypromine and its applicability in pharmacokinetic studies. J Chromatogr 584:229–237.
    OpenUrlPubMed
  30. ↵
    1. Sullivan JP,
    2. McDonnell L,
    3. Hardiman OM,
    4. Farrell MA,
    5. Phillips JP,
    6. Tipton KF
    (1986) The oxidation of tryptamine by two forms of monoamine oxidase in human tissues. Biochem Pharmacol 35:3225–3260.
    OpenUrl
  31. ↵
    1. Tassaneeyakul W,
    2. Veronese ME,
    3. Birkett DJ,
    4. Gonzalez FJ,
    5. Miners JO
    (1993) Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques. Biochem Pharmacol 46:1975–1981.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Tiihonen J,
    2. Pesonen U,
    3. Kauhanen J,
    4. Koulu M,
    5. Hallikainen T,
    6. Leskinen L,
    7. Salonen JT
    (2000) CYP2A6 genotype and smoking. Mol Psychiatry 5:347–348.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Tyndale RF,
    2. Inaba T,
    3. Kalow W
    (1989) Evidence in human for variant allozymes of the nondeficient sparteine/debrisoquine monooxygenase (P450IID1) in vitro. Drug Metab Dispos 17:334–340.
    OpenUrlAbstract
  34. ↵
    1. Venkatakrishnan K,
    2. von Moltke LL,
    3. Greenblatt DJ
    (1998) Human cytochromes P450 mediating phenacetin O-deethylation in vitro: validation of high affinity component as an index of CYP1A2 activity. J Pharm Sci 87:1502–1507.
    OpenUrlCrossRefPubMed
  35. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Duan SX,
    4. Schmider J,
    5. Kudchadker L,
    6. Fogelman SM,
    7. Harmatz JS,
    8. Shader RI
    (1996) Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine. Psychopharmacology 128:398–407.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Boulton AA,
    2. Maitre L,
    3. Beick PR,
    4. Reiderer P
    1. Wollmann H,
    2. Nilsson E,
    3. Antonin KH,
    4. Bieck PR
    (1985) Tryptamine kinetics in human volunteers. in Neuropsychopharmacology of Trace Amines, eds Boulton AA, Maitre L, Beick PR, Reiderer P (Humana Press, Clifton), pp 361–378.
  37. ↵
    1. Yamano S,
    2. Tatsuno J,
    3. Gonzalez FJ
    (1990) The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29:1322–1329.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Yamazaki H,
    2. Inui Y,
    3. Yun CH,
    4. Guengerich FP,
    5. Shimada T
    (1992) Cytochrome P4502E1 and 2A6 enzyme as major catalysts for metabolic activation of n-nitrosodialkyl-amines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 13:1789–1794.
    OpenUrlAbstract/FREE Full Text
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Evaluation of Methoxsalen, Tranylcypromine, and Tryptamine as Specific and Selective CYP2A6 Inhibitors in Vitro

Wenjiang Zhang, Tansel Kilicarslan, Rachel F. Tyndale and Edward M. Sellers
Drug Metabolism and Disposition June 1, 2001, 29 (6) 897-902;
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