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Research ArticleMETABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Metabolism of Efavirenz and 8-Hydroxyefavirenz by P450 2B6 Leads to Inactivation by Two Distinct Mechanisms

Namandjé N. Bumpus, Ute M. Kent and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics July 2006, 318 (1) 345-351; DOI: https://doi.org/10.1124/jpet.106.102525

Abstract

Efavirenz is a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor used in combination therapy to treat HIV-1. Efavirenz metabolism is catalyzed primarily by the polymorphic enzyme P450 2B6. Metabolism of efavirenz by P450 2B6 and the naturally occurring P450 2B6.4 mutant led to the formation of 8-hydroxyefavirenz. Efavirenz inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin activity of the wild-type P450 2B6 enzyme in a time-, concentration-, and NADPH-dependent manner. However, the P450 2B6.4 variant was not inactivated by efavirenz. The ability of efavirenz to inactivate both enzymes was investigated using cyclophosphamide and bupropion, two structurally unrelated substrates of P450 2B6, as probes. Preincubations with efavirenz decreased the ability of the wild-type enzyme to hydroxylate both substrates to similar extents but had no effect on the activities of the mutant enzyme. Interestingly, the inactivation of the wild-type enzyme was completely reversible after 24 h of dialysis as determined by heme, reduced CO spectra, and activity loss. In contrast, 8-hydroxyefavirenz, a metabolite of efavirenz, was able to inactivate both enzymes irreversibly. These data suggest that incubations of P450 2B6 and P450 2B6.4 with either the parent compound efavirenz or the metabolite 8-hydroxyefavirenz in the reconstituted system result in the formation of two different reactive intermediates that lead to losses in enzymatic activity by two different mechanisms, one reversible and one irreversible.

The cytochrome P450 (P450) enzymes are a family of monooxygenases that catalyze the metabolism of a wide variety of endogenous and exogenous compounds including xenobiotics, steroids, and fatty acids. The phenobarbital-inducible isoform P450 2B6 has been shown to metabolize a growing list of substrates including bupropion (Faucette et al., 2000; Hesse et al., 2000), sertraline (Obach et al., 2005), cyclophosphamide (Chang et al., 1993), and efavirenz (Ward et al., 2003). P450 2B6 is polymorphic, as a number of single nucleotide polymorphisms exist in the P450 2B6 gene (Lang et al., 2001). We have shown previously in the reconstituted system that mutation of amino acid 262 from lysine to arginine (2B6*4, 785A > G, exon 5) leads to marked differences in the metabolism of some substrates such as bupropion and 17-α-ethynylestradiol and mechanism-based inactivation by N,N′,N″-triethylenethiophosphoramide and 17-α-ethynylestradiol (Bumpus et al., 2005).

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used in the treatment of human immunodeficiency virus-1 (HIV-1). Efavirenz is prescribed as part of a combination therapy and is particularly effective because of its long half-life of 40 to 55 h after multiple doses (Harris and Montaner, 2000). P450 2B6 has been shown to be primarily responsible for the hydroxylation of efavirenz to 8-hydroxyefavirenz and 8,14-hydroxyefavirenz (Ward et al., 2003). In vivo and in vitro studies have shown that 8-hydroxyefavirenz is formed rapidly and is the major metabolite formed (Mutlib et al., 1999; Ward et al., 2003). Polymorphisms of P450 2B6 may have a significant effect on efavirenz metabolism because it has been shown that patients genotyped as P450 2B6*6/*6 (Q172H and K262R) have significantly higher mean plasma efavirenz concentrations than patients who are *6 heterozygous or who do not have *6 alleles (Tsuchiya et al., 2004). Efavirenz has also been shown to inhibit bupropion hydroxylation in human liver microsomes (Hesse et al., 2001).

Mechanism-based inactivation is defined as involving the metabolism of a substrate which results in formation of a reactive intermediate that covalently binds to a moiety in the enzyme active site and thereby renders the enzyme inactive (Kent et al., 2001). This process is different from competitive inhibition, in which the decrease in activity is only concentration dependent and not time-, concentration-, and NADPH-dependent as with mechanism-based inactivation. Drugs that are mechanism-based inactivators of a specific form of P450 may lead to detrimental effects when given in combination with other drugs that are metabolized by the same P450 isoform. Several compounds have been shown to be mechanism-based inactivators of P450 2B6 including bergamottin (Lin et al., 2005), clopidogrel (Richter et al., 2004) and N,N′,N″-triethylenethiophosphoramide (Harleton et al., 2004). A number of substrates that contain an ethynyl functional group have been shown to be mechanism-based inactivators of P450 2B enzymes including 17-α-ethynylestradiol, 2-ethynylnaphthalene, and 9-ethynylphenanthrene (Roberts et al., 1997; Kent et al., 2002). Ethynes appear to be more effective mechanism-based inactivators of the microsomal P450 2B enzymes than propynes (Foroozesh et al., 1997). Therefore, substrates containing an ethynyl group may be metabolized by the 2B family of enzymes to reactive intermediates resulting in mechanism-based inactivation. Figure 1 shows the chemical structures of efavirenz and two of its primary hydroxylated metabolites, all of which contain ethynyl groups.

In this study, we used recombinant N-terminally truncated P450 2B6 and P450 2B6.4 1) to evaluate the effect of the K262R mutation on the hydroxylation of efavirenz to 8-hydroxyefavirenz, 2) to investigate the ability of efavirenz to inactivate both enzymes, and 3) to investigate the ability of 8-hydroxyefavirenz, a major metabolite of efavirenz, to act as a mechanism-based inactivator of both enzymes. We found that the mutant was able to metabolize efavirenz to 8-hydroxyefavirenz. In addition, efavirenz inactived the wild-type 2B6 but not the mutant, and the inactivation of 2B6 was reversible after dialysis. In contrast to the results observed with the parent compound, incubations with the 8-hydroxy metabolite resulted in the irreversible inactivation of both enzymes. These studies provide valuable information regarding the effect of the K262R mutation on P450 2B6 catalytic activity. In addition, these results show that hydroxylation of a substrate can lead to marked differences in the mechanism of inactivation. These studies also suggest that efavirenz and 8-hydroxyefavirenz may be useful tools for studying the structure of the active site of P450 2B6.

Materials and Methods

Materials. Efavirenz was purchased from Toronto Research Chemicals (Toronto, ON, Canada). 8-Hydroxyefavirenz was a generous gift from Bristol-Myers Squibb Co. (Stamford, CT). Bupropion hydrochloride, triprolidine hydrochloride, NADPH, BSA, and catalase were purchased from Sigma Chemical (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was obtained from Molecular Probes (Eugene, OR). Barium hydroxide, 3-aminophenol, and hydroxylamine hydrochloride were purchased from Aldrich Chemical Co. (Milwaukee, WI). The P450 2B6 plasmid was a generous gift from Dr. James Halpert (University of Texas Medical Branch, Galveston, TX). All other chemicals were of the highest grade commercially available.

Fig. 1.
Fig. 1.

Chemical structures of efavirenz and the two primary metabolites formed through hydroxylation by P450 2B6.

Statistical Analysis. Statistical analysis was performed using Prism version 3.00 for Windows (GraphPad Software Inc., San Diego, CA).

Site-Directed Mutagenesis and Expression and Purification of P450s and Reductase. Construction of the P450 2B6.4 mutant was performed as described previously (Bumpus et al., 2005). P450 2B6, P450 2B6.4, and NADPH-P450 reductase were expressed in Escherichia coli Topp 3 cells and purified as described previously (Hanna et al., 1998, 2000; Scott et al., 2001) except that P450 2B6.4 was recovered from the cytosol rather than from the membrane pellet after the cell lysis step. Therefore, the cytosol was applied to the Ni2+-agarose column and the P450 was then eluted and purified as previously described.

Efavirenz Metabolism. The method used to determine efavirenz metabolism was adapted from Ward et al. (2003). The purified P450s were reconstituted with reductase at a ratio of 1:2 of P450 to reductase for 45 min at 4°C. The reaction mixtures consisted of 1 μM P450, 2 μM reductase, 110 units of catalase and efavirenz (concentrations ranging from 0 to 60 μM). NADPH was added to initiate the reactions, and the mixtures were incubated for 30 min at 37°C. The reactions were quenched by adding 500 μl of ice-cold acetonitrile containing 0.1% formic acid. The samples were then placed on ice and centrifuged at maximal speed for 10 min in an Eppendorf microcentrifuge at 4°C. The supernatants were placed in clean tubes, and 500 μl of 0.5M NaOH, pH 10, was added. Testosterone (2 μl of a 20 μM stock) was added as an internal standard. The samples were extracted twice with ethyl acetate and dried under a stream of nitrogen; 200 μl of mobile phase was added, and the samples were resolved on a Varian Microsorb-MV 250 × 4.6-mm C18 column (Varian, Inc., Palo Alto, CA) at a flow rate of 0.8 ml/min with the detector set at 247 nm. Isocratic conditions were used, consisting of 55% mobile phase A (water and 0.1% trifluoroacetic acid) and 45% mobile phase B (acetonitrile and 0.1% trifluoroacetic acid). The retention times were approximately 13 min for the internal standard testosterone, 23.5 min for 8-hydroxyefavirenz, and 42 min for efavirenz.

Inactivation of P450s 2B6 and 2B6.4. The purified P450s were reconstituted with reductase for 45 min at 4°C. The primary reaction mixtures contained 1 μM P450, 2 μM reductase, 110 units of catalase, and efavirenz (0-50 μM) or 8-hydroxyefavirenz (0-120 μM) in 50 mM potassium phosphate buffer, pH 7.4. The primary reaction mixtures were then incubated for 10 min at 30°C before the reactions were initiated by adding NADPH to a final concentration of 1.2 mM. After the addition of NADPH, 12-μl aliquots were removed from the primary reaction mixtures at the times indicated and transferred to 990 μl of the secondary reaction mixtures, which contained 100 μM 7-EFC, 1 mM NADPH, and 40 μg/ml BSA in 50 mM potassium phosphate buffer, pH 7.4. The secondary reaction mixtures were incubated for 10 min at 30°C and then quenched by the addition of 334 μl of acetonitrile. The amount of 7-hydroxy-4-(trifluoromethyl-)coumarin formed was measured at room temperature using an excitation wavelength of 410 nm and an emission wavelength of 510 nm with a RF-5310 spectrofluorophotometer (Shimadzu Scientific Instruments, Inc., Wood Dale, IL). The amount of 8-hydroxybupropion formed was determined as described previously (Bumpus et al., 2005). To measure the effect of efavirenz on the cyclophosphamide (CPA) hydroxylation activity of the P450s, the primary reaction mixtures were incubated with 20 μM efavirenz at 37°C. After the addition of NADPH, aliquots were removed at the times indicated. The secondary reaction mixtures contained 100 μM CPA, 1 mM NADPH, and 40 μg/ml BSA in 50 mM potassium phosphate buffer, pH 7.4. CPA hydroxylation was determined using the procedure of Roy et al. (1999).

Reversibility of Inactivation of P450s 2B6 and 2B6.4 by Efavirenz. P450s were reconstituted and incubated with 50 μM efavirenz or 20 μM 8-hydroxyefavirenz (80 μM for P450 2B6.4) in the presence or absence of NADPH as described above. Aliquots were removed at 0 and 20 min to determine the amount of 7-EFC O-deethylation activity remaining as described above. Each sample was further analyzed for P450 remaining using the reduced CO spectral assay and intact heme by HPLC as described by Harleton et al. (2004). The remainder of each of the control and inactivated samples was dialyzed separately for 24 h at 4°C in Slide-A-Lyzer cassettes (Pierce Chemical, Rockford, IL) against 2 × 500 ml dialysis buffer (50 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 100 μM EDTA). After dialysis, the samples were incubated with or without fresh reductase at 4°C for 15 min and catalytic activity, heme, and reduced CO spectra analysis were again carried out as described above.

Determination of Spectral Intermediate Formation. P450s were reconstituted as described above and incubated with 10 μM efavirenz at 30°C for 10 min. NADPH (0.6 mM) was added to the sample cuvette, and an equal amount of water was added to the reference cuvette. Difference spectra were recorded from 350 to 700 nm using a DW2 UV-visible spectrophotometer (SLM Aminco, Urbana, IL) that was equipped with an OLIS spectroscopy operating system (On-Line Instrument Systems, Inc., Bogart, GA). Scans were taken continuously at 2-min intervals.

Results

Formation of 8-Hydroxyefavirenz by P450 2B6 and P450 2B6.4. The metabolism of efavirenz to 8-hydroxyefavirenz by 2B6 and 2B6.4 was measured by HPLC. The rates of formation of 8-hydroxyefavirenz by P450 2B6 and P450 2B6.4 using concentrations of efavirenz ranging from 0 to 100 μM are shown in Fig. 2. The kinetic constants for these reactions are shown in Table 1. The approximate Km value for wild-type P450 2B6 (14.3 μM) was very similar to the approximate Km for the variant (15.9 μM). The Vmax for 8-hydroxyefavirenz formation by P450 2B6 was approximately 4.3 pmol formed/pmol P450/min, whereas the Vmax of P450 2B6.4 was approximately 2-fold higher (8.1 nmol formed/nmol P450/min). The catalytic efficiency (Vmax/Km) of P450 2B6.4 was approximately 66% greater than that of P450 2B6 (approximately 0.3 for 2B6 compared with approximately 0.5 for the mutant).

View this table:
TABLE 1

Kinetic constants for the formation of 8-hydroxyefavirenz by recombinant P450 2B6 and P450 2B6.4 Kinetic constants were determined from the data shown in Fig. 1 as described under Materials and Methods.

Inactivation of P450 2B6 by Efavirenz. Inactivation of the wild-type enzyme was measured using the 7-EFC O-deethylation assay. The wild-type enzyme was inactivated by efavirenz (Fig. 3) in a time- and concentration-dependent manner, and the inactivation exhibited an absolute requirement for NADPH. The activity loss followed pseudo-first-order kinetics. Linear regression analysis was performed, and the kinetic constants for the efavirenz-mediated inactivation of the wild-type enzyme were determined from the inset of Fig. 3. The KI was approximately 30 μM, and the Kinact was 0.04 min-1 giving a t1/2 of 16 min. In contrast to the wild-type enzyme, the variant form of 2B6 was not inactivated by efavirenz at concentrations up to 200 μM as measured by the 7-EFC O-deethylation activity of the variant enzyme (data not shown). The effect of mechanism-based inactivation by efavirenz on the metabolism of other structurally unrelated P450 2B6 substrates was also determined to investigate the possibility that the inactivation of wild-type 2B6 and the observed lack of inactivation of the variant might be due to the 7-EFC substrate that was chosen to measure activity. P450 2B6 samples that had been preincubated with efavirenz and NADPH were assayed for bupropion and cyclophosphamide hydroxylation activities (Table 2). Preincubation with efavirenz decreased the ability of the wild-type P450 2B6 to hydroxylate both substrates. Approximately 45% of the initial bupropion hydroxylation activity and 42% of the CPA activity remained after incubation with 20 μM efavirenz. These values were similar to the activity remaining when 7-EFC was used as the substrate (Fig. 1 and data not shown). As seen with 7-EFC, preincubation of the variant enzyme with efavirenz did not result in inactivation as measured by the hydroxylation of bupropion or CPA (data not shown).

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TABLE 2

Effect of preincubation with efavirenz on the bupropion and cyclosphosphamide hydroxylation activities of P450 2B6 The P450s were reconstituted with reductase as described under Materials and Methods. The primary reaction mixture contained the concentrations of efavirenz indicated. NADPH was added to the primary reaction mixture to initiate the reaction.

Fig. 2.
Fig. 2.

Metabolism of efavirenz to 8-hydroxyefavirenz by P450 2B6 and P450 2B6.4. Samples were reconstituted as described under Materials and Methods and incubated with efavirenz at concentrations ranging from 0 to 100 μM. 8-Hydroxyefavirenz formation by P450 2B6 (▵) and P450 2B6.4 (▴) was measured by HPLC by integrating the area under the metabolite peak and comparing it to a standard curve generated by injecting known amounts of authentic 8-hydroxyefavirenz onto the HPLC column. The data shown are representative of the means and S.D. from four separate experiments done in duplicate.

Reversibility of Efavirenz-Mediated Inactivation of P450 2B6. As shown in Table 3, the inactivation of P450 2B6 by efavirenz was reversible after overnight dialysis, and the enzymatic activity, reduced CO spectra, and heme remaining were completely restored. Control and efavirenz-inactivated samples were analyzed for activity and CO spectral and heme loss before and after 24 h of dialysis at 4°C. Table 3 shows that before dialysis there was a 68% loss in activity, a 65% CO spectral loss and 69% heme loss. After dialysis for 24 h, catalytic activity, the reduced CO spectrum, and the heme remaining had all returned to levels commensurate with the control samples. To determine whether the reversibility observed was the result of metabolic intermediate (MI) complex formation, as has been observed with other reversible inactivators (Sharma et al., 1996), difference spectra for efavirenz-inactivated versus control samples were determined. As shown in Fig. 4, there is a peak with a maximal absorbance at 435 nm in the difference spectrum. This peak does not appear to be representative of a MI complex because MI complexes normally exhibit a characteristic peak absorbance at 455 nm, not at 435 nm. The addition of ferricyanide results in the loss of the peak that we observe at 435 nm, suggesting that it is an intermediate. Additionally, this peak is not observed in the absence of NADPH.

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TABLE 3

Effect of efavirenz on P450 2B6 catalytic activity, P450 content as measured by the reduced CO spectrum, and heme before and after 24 h of dialysis The assays were performed as described under Materials and Methods. Samples were incubated for 15 min in the presence or absence of NADPH, and the data are represented as percentages of the control samples incubated in the absence of efavirenz. Samples were then dialyzed for 24 h. The data represent the means and S.D. of three separate experiments.

Fig. 3.
Fig. 3.

Inactivation of P450 2B6 by efavirenz. The time- and concentration-dependent inactivation of P450 2B6 was measured by determining the 7-EFC O-deethylation activity as described under Materials and Methods. After initiation of inactivation by the addition of NADPH, aliquots were removed from the primary reaction mixture at 0, 5, 10, 15, and 20 min. The concentrations of efavirenz were 0 μM (▪), 5 μM (▴), 10 μM (▾), 20 μM (♦), 40 μM (•), and 50 μM (□). The data show the means and S.D. from four separate experiments done in duplicate. In some cases the S.D.s were less than the size of the symbols. The inset shows the double reciprocal plot of the rates of inactivation as a function of the efavirenz concentrations.

Inactivation of P450 2B6 and P450 2B6.4 by 8-Hydroxyefavirenz. The ability of 8-hydroxyefavirenz, the primary metabolite of efavirenz, to inactivate the wild-type and variant enzymes was also investigated. Interestingly, inactivation by 8-hydroxyefavirenz was markedly different from the inactivation by the parent. Incubation of both enzymes with 8-hydroxyefavirenz led to inactivation in time-, concentration-, and NADPH-dependent manners. The approximate KI value for the inactivation of 2B6 by 8-hydroxyefavirenz was 6.4 μM, with a t1/2 of 11 min, and a rate of inactivation of 0.06 min-1, as measured by 7-EFC O-deethylation activity remaining (Fig. 5). In contrast to what had been observed with efavirenz, the P450 2B6 variant was also inactivated by 8-hydroxyefavirenz although the estimated KI was approximately 10-fold higher than that for the wild-type enzyme (75 μM) and the t1/2 was 17 min with a rate of inactivation of 0.04 min-1 (Fig. 6). Because the KI for inactivation of the wild-type enzyme by efavirenz (30 μM) was greater than the KI for inactivation by 8-hydroxyefavirenz (6.4 μM), it suggested that the 8-hydroxy metabolite was a more potent inactivator than the parent compound. Preincubation of the wild-type and the variant enzyme with 8-hydroxyefavirenz in the presence of NADPH also resulted in marked decreases in the bupropion and cyclophosphamide hydroxylation activities of both enzymes (Table 4). The decrease in the enzymatic activity of the P450 2B6 that had been preincubated with 8-hydroxyefavirenz when measured by its ability to hydroxylate bupropion or CPA was reduced to 32 and 39% activity remaining, respectively. The decrease in the catalytic activity of the variant enzyme that had been preincubated with 20 μM 8-hydroxyefavirenz and NADPH when measured by its ability to hydroxylate bupropion was 81% and for CPA it was 84% compared with untreated controls. The losses in bupropion and CPA hydroxylation activities were similar to the activity decreases observed using the 7-EFC assay.

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TABLE 4

Effect of incubation with 8-hydroxyefavirenz on the bupropion and cyclophosphamide hydroxylation activities of P450 2B6 and P450 2B6.4 Assay conditions were described under Materials and Methods. Data are representative of the means and S.D. of three separate experiments done in duplicate. Primary reaction mixtures contained 8-hydroxyefavirenz at a concentration of 20 μM.

Fig. 4.
Fig. 4.

Formation of a spectral intermediate during the inactivation of P450 2B6 by efavirenz. Reconstituted P450 2B6 was incubated with efavirenz, and the difference scans were recorded between 350 and 700 nm for 20 min (the traces obtained between 400 and 500 nm are shown here). After a baseline scan at 20 min, NADPH was added to the sample cuvette, and water was added to the reference cuvette. Spectra were taken every 2 min. The absorbance at 400 nm was maximal at 0 time and decreased with successive scans. +, peak maximal absorption observed at 435 nm.

Fig. 5.
Fig. 5.

Inactivation of P450 2B6 by 8-hydroxyefavirenz. P450 2B6 was inactivated by 8-hydroxyefavirenz, and the activity remaining was measured using the 7-EFC O-deethylation assay. After the addition of NADPH, aliquots were removed from the primary reaction mixture at 0, 5, 10, 15, and 20 min. The concentrations of 8-hydroxyefavirenz were 0 μM (▪), 4 μM (▴), 5 μM (▾), 6 μM (♦), 8 μM (•), and 10 μM (□). The data show the means and S.D. from three separate experiments done in duplicate. The inset shows the double reciprocal plot of the rates of inactivation as a function of the 8-hydroxyefavirenz concentrations.

Fig. 6.
Fig. 6.

Inactivation of P450 2B6.4. by 8-hydroxyefavirenz. Samples were reconstituted and incubated with 8-hydroxyefavirenz in the presence or absence of NADPH as described under Materials and Methods. Inactivation was measured by determining 7-EFC O-deethylation activity remaining. The concentrations of 8-hydroxyefavirenz were 0 μM (▪), 4 μM (▴), 5 μM (▾), 6 μM (♦), 8 μM (•), and 10 μM (□). The data show the means and S.D. from three separate experiments done in duplicate. The inset shows the double reciprocal plot of the rates of inactivation as a function of the 8-hydroxyefavirenz concentrations.

Irreversibility of the Inactivation of P450 2B6 and P450 2B6.4 by 8-Hydroxyefavirenz. The changes in enzymatic activity and the losses in the CO spectra and heme were measured before and after overnight dialysis in samples inactivated by 8-hydroxyefavirenz. In complete contrast to what had been observed with efavirenz, the inactivation of both enzymes by 8-hydroxyefavirenz was irreversible (Table 5). The percentage of activity remaining after dialysis and after incubation with fresh reductase increased only slightly for both enzymes, suggesting that although there may be a small portion of the population of enzyme that is reversibly inactivated by 8-hydroxyefavirenz, the inactivation is essentially irreversible (Table 4).

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TABLE 5

Irreversibility of 2B6 inactivation by 8-hydroxyefavirenz Assay conditions were as described under Materials and Methods. Data are representative of the means and S.D. of three separate experiments done in duplicate. P450 2B6 was incubated with 20 μM 8-hydroxyefavirenz for 15 min. P450 2B6.4 was incubated with 80 μM 8-hydroxyefavirenz for 15 min.

Discussion

P450 2B6 is expressed in a number of organs including the liver, heart, and brain and has been shown to have widely variable expression levels (Thum and Borlak, 2000; Miksys and Tyndale, 2004; Klein et al., 2005). P450 2B6 plays an important role in the metabolism of a growing list of clinically important substrates, which include bupropion, an antidepressant and smoking cessation aid (Faucette et al., 2000; Hesse et al., 2000) and cyclosphosphamide, an important chemotherapeutic agent (Chang et al., 1993). Bupropion has been used previously as a tool to study the inactivation and inhibition of P450 2B6 (Turpeinen et al., 2004). It has recently been shown that a P450 2B6-reductase fusion protein catalyzed the metabolic activation of the prodrug cyclophosphamide and markedly increased the cyclophosphamide-dependent cytotoxicity (Tychopoulos et al., 2005). A number of single nucleotide polymorphisms have been found in the P450 2B6 gene (Lang et al., 2001), and some of these have been shown to have effects on the catalytic activity of the enzyme. P450 2B6*4, which corresponds to a K262R mutation of the protein, has recently been shown to have close to a 50% mutation frequency in Ghanians and close to 30% in African-Americans and Caucasians (Klein et al., 2005). We have previously shown that the K262R mutant of P450 2B6 has significant effects on the metabolism of the P450 2B6-specific substrate bupropion (Bumpus et al., 2005). In this study we have compared the abilities of purified P450 2B6 and its K262R mutant, 2B6.4, to metabolize efavirenz to 8-hydroxyefavirenz in the reconstituted system. Our data indicate that P450 2B6.4 hydroxylates efavirenz to 8-hydroxyefavirenz at a rate almost 2-fold greater than that of the wild-type enzyme. The Km of the wild-type enzyme for efavirenz in the present study was similar to that reported previously (Ward et al., 2003). However, because of the marked variability in the expression levels of P450 2B6 in the human population, it is not possible to directly draw clinical conclusions based upon our current data. In patients, P450 2B6 polymorphisms have been shown to have an effect on plasma levels of efavirenz as patients homozygous for 2B6*6 (Q172H and K262R) had higher mean plasma concentrations of the parent drug (Tsuchiya et al., 2004). Two other groups have recently reported similar findings in patients homozygous for the 516G>T mutation (Q172H) (Rodriguez-Novoa et al., 2005; Rotger et al., 2005).

Because many drugs, including efavirenz, are prescribed as part of a combination therapy, it is important to determine which substrates may have inhibitory effects on the enzyme. Adverse drug reactions are a major source of hospitalizations and even mortality (Einarson, 1993; Lazarou et al., 1998; Pirmohamed and Park, 2003) and are defined as “an appreciably harmful or unpleasant reaction, resulting from an intervention related to the use of a medical product, which predicts hazard from future administration and warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal of the product” (Edwards and Aronson, 2000). Polymorphisms appear to play an important role in adverse drug reactions because many of the drugs that are frequently cited in these studies are metabolized by at least one polymorphic enzyme (Philips et al., 2001; Pirmohamed and Park, 2003). We have previously shown that the K262R mutation protected the enzyme against inactivation by 17-α-ethynylestradiol, which readily inactivates the wild-type enzyme (Kent et al., 2002; Bumpus et al., 2005). In the present study preincubation with efavirenz decreased the ability of P450 2B6 to catalyze the hydroxylation of bupropion and cyclophosphamide as well as decreasing 7-EFC O-deethylation activity. In contrast, no effect on the above-mentioned activities was observed with the P450 2B6.4 mutant. The reason for the inability of the mutant to become inactivated by efavirenz is not clear but does not appear to involve the absence of reversible binding and metabolism of efavirenz because the primary metabolite 8-hydroxyefavirenz was generated by the mutant. We are currently investigating this functional difference between the wild-type and the K262R mutant enzyme and the effect that this mutation has on the ability of P450 2B6 to form a reactive intermediate from ethynyl-containing compounds capable of inactivating the wild-type enzyme.

Another interesting finding was that the inactivation of P450 2B6 by efavirenz was completely reversible after 24 h of dialysis. A similar recovery of enzymatic activity has been described with purified rat P450 2B1, for which the initial loss in activity was due to the formation of a MI complex (Sharma et al., 1996). MI complex formation results in the appearance of a characteristic maximal absorbance peak at 455 nm in the difference spectrum (Chatterjee and Franklin, 2003). A second mechanism for reversible inactivation has recently been described for the inactivation of P450 2E1 T303A by tert-butyl acetylene. In this case, the inactivation was accompanied by the appearance of a spectral intermediate at 485 nm (Blobaum et al., 2002). Difference spectra of efavirenz-inactivated samples versus controls exhibited a new peak with a maximal absorbance at 435 nm. This spectral intermediate formed during the inactivation by efavirenz is different from the intermediates reported previously. The reasons for this difference are under investigation.

Surprisingly, when 8-hydroxyefavirenz, the major metabolite of efavirenz, was used instead of efavirenz, both P450s were inactivated in a mechanism-based manner, and the inactivation was irreversible. With either enzyme and 8-hydroxyefavirenz, we were unable to observe a spectral intermediate similar to that seen during the inactivation of the wild-type enzyme by efavirenz (data not shown). These data suggest that the inactivation of P450 2B6 and the variant by efavirenz and 8-hydroxyefavirenz occurs through two distinctly different mechanisms. Even though 8-hydroxyefavirenz is formed during efavirenz metabolism by both enzymes and the KI value for the hydroxylated product with the wild-type enzyme is approximately 4-fold lower than the KI value for efavirenz, the concentration of 8-hydroxyefavirenz required to achieve irreversible inactivation does not appear to have been achieved during the incubations. Therefore, we believe that during the metabolism of efavirenz in the reconstituted system, the concentrations of 8-hydroxyefavirenz produced contribute only negligibly to the inactivation. Furthermore, the spectral intermediate that is formed during the reversible inactivation of P450 2B6 may be formed before the production of significant amounts of 8-hydroxyefavirenz. In contrast, incubations of 2B6 with 8-hydroxyefavirenz alone may lead to the formation of a reactive intermediate that is not produced during incubations of 2B6 with efavirenz alone. Our data suggest that efavirenz is initially bound in the P450 active site in an orientation that facilitates oxidation at or near the 8-hydroxy position. Once the 8 position is hydroxylated, the preferred orientation of the substrate in the active site may bring the ethynyl moiety into closer proximity to the heme iron with the activated oxygen. This could then result in the generation of the reactive intermediate that could be responsible for the irreversible inactivation. Studies are currently underway to attempt to trap the reactive intermediate formed during the metabolism of 8-hydroxyefavirenz and to obtain structural information on this intermediate.

We also observed a difference in inactivation of P450 2B6 by 8-hydroxyefavirenz compared with P450 2B6.4. The approximate KI for the wild-type enzyme was 6.4 μM, whereas it was 75 μM for the mutant. The 12-fold greater KI for the mutant once again suggests that this mutation has a significant effect on the catalytic properties of the enzyme. This effect was not substrate dependent as we have reported previously for inactivation of the mutant enzyme (Bumpus et al., 2005). Similar levels of inactivation were seen with 7-EFC, cyclophosphamide, and bupropion.

These studies demonstrate that the K262R mutant of P450 2B6 catalyzes the metabolism of efavirenz to 8-hydroxyefavirenz at a significantly greater rate than the wild-type enzyme. P450 2B6 in the reconstituted system was inactivated by efavirenz, whereas P450 2B6.4 was not inactivated. Interestingly, the efavirenz-mediated inactivation of the wild-type enzyme was completely reversible after dialysis. The primary metabolite of efavirenz, 8-hydroxyefavirenz, inactivated both enzymes and the inactivation was irreversible. Because the inactivation by 8-hydroxyefavirenz was irreversible whereas the inactivation by efavirenz was reversible, these two closely related compounds inactivated the enzymes through mechanisms that are completely different from each other. This study has further shown a difference in the catalytic properties of the wild-type enzyme and K262R mutant. Efavirenz and 8-hydroxyefavirenz may prove to be useful tools for probing the structure of the P450 2B6 active site.

Acknowledgments

We thank Dr. James Halpert for the kind gift of the P450 2B6 plasmid and Hsia-lien Lin for the expression and purification of the P450 reductase. We also thank Dr. Zeruesenay Desta for thoughtful discussions, suggestions, and encouragement.

Footnotes

  • This study was supported in part by National Institutes of Health Grants CA 16954 and T32 GM007767.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.106.102525.

  • ABBREVIATIONS: P450, cytochrome P450; P450 2B6, N-terminally truncated P450 2B6; HIV-1, human immunodeficiency virus-1; P450 2B6.4, N-terminally truncated P450 2B6 lysine 262 arginine mutant; BSA, bovine serum albumin; EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; CPA, cyclosphosphamide; HPLC, high-performance liquid chromatography; MI, metabolic intermediate.

    • Received February 6, 2006.
    • Accepted April 10, 2006.

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Research ArticleMETABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Metabolism of Efavirenz and 8-Hydroxyefavirenz by P450 2B6 Leads to Inactivation by Two Distinct Mechanisms

Namandjé N. Bumpus, Ute M. Kent and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics July 1, 2006, 318 (1) 345-351; DOI: https://doi.org/10.1124/jpet.106.102525
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