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Selegiline, the R -enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6.
The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy trifluoromethyl coumarin O -deethylation 7-EFC activity of CYP2B6 as well as bupropion metabolism. The cytochrome P P enzymes are a superfamily of oxidative enzymes that are involved in the metabolism of a number of structurally diverse xenobiotics.
To determine the role of specific Ps in the clearance of various drugs and to prevent drug-drug interactions caused by the inhibition of metabolic clearance, the prediction and identification of compounds that act as mechanism-based inactivators have become an important factor in the drug discovery process.
Mechanism-based inactivation has also been used to study the structure-function relationship of individual P proteins because the active site residues on the apoprotein modified by the reactive species can be identified and the mechanisms by which activity is lost can be determined Hollenberg et al.
Selegiline is also a suicide inhibitor of MAO-B resulting in irreversible inhibition of the enzyme. Selegiline is used alone or in combination with levodopa in the treatment of Parkinson's disease. Levodopa is converted to dopamine, a natural substance that is needed to control movement, thus rendering a therapeutic effect.
The products of metabolism of selegiline are amphetamine, N -methylamphetamine, and N -propargylamphetamine. Previous metabolism studies performed in vitro have shown that CYP2B6 and CYP2C19 are the major enzymes responsible for the metabolism of selegiline in human liver Hidestrand et al.
We have previously demonstrated that selegiline is a mechanism-based inactivator of rat CYP2B1. Kinetic parameters for the inactivation of CYP2B1 were determined. Our studies demonstrated that inactivation occurred because of protein destruction rather than heme damage Sharma et al. Selegiline has been shown to be a mechanism-based inactivator of CYP2A6 and inhibits the metabolism of nicotine in humans and mice Siu and Tyndale, Bupropion is an antidepressant drug that is used for the treatment of patients with Parkinson's disease.
Clearance of bupropion occurs through hydroxylation and CYP2B6 has been identified as the major enzyme catalyzing this metabolic reaction Ascher et al. Limited studies have been performed concerning the impact of bupropion use in patients with Parkinson's disease given the wide use of this drug as a pharmacotherapy in people with Parkinson's disease.
In this study, we have investigated the metabolism of bupropion in the presence and absence of selegiline. During these studies, we found that selegiline is also a mechanism-based inactivator of CYP2B6. The study provides evidence for the formation of a ketene intermediate during Pmediated bioactivation of selegiline that could be trapped in the presence of GSH in the reconstituted system.
LC-MS studies led to identification of an adduct that revealed the mass of the reactive intermediate responsible for the loss of the enzymatic activity. Trypsin digestion of the selegiline-modified protein revealed that selegiline is first hydroxylated by CYP2B6, and then it binds covalently to the Asp64 of the P leading to its inactivation.
The mechanism-based inactivation of CYP2B6 by selegiline may have significant neuropsychopharmacological implications for patients with Parkinson's disease being treated concomitantly with bupropion or other drugs metabolized primarily by CYP2B6. Trypsin was obtained from Promega Madison, WI. All other chemicals were of highest quality available from commercial sources. Amphetamine and methamphetamine were generously donated by Dr. Woods Department of Pharmacology, University of Michigan.
P reductase was purified as described previously Hanna et al. CYP2B6 was reconstituted with reductase at a ratio of 1: The reconstituted mixture was then diluted to a volume of 3 ml with 0. The mixture was then divided into two portions. NADPH was added to initiate the reactions at a final concentration of 1. The methods for incubating and determining product concentrations were followed as described in by Bumpus et al.
Nonlinear regression analyses of the data were performed using Prism version 5. The samples received increasing concentrations of selegiline 0. NADPH was added at a final concentration of 1. The amount of 7-hydroxy- 4-trifluoromethyl coumarin formed was measured using a Victor II fluorescence plate reader PerkinElmer Life and Analytical Sciences, Waltham, MA , with excitation and emission wavelengths of and nm, respectively.
The kinetic constants were calculated by linear regression using Prism version 5; GraphPad Software Inc. The addition of bovine serum albumin was based on the studies of Buters et al. For the inactivation of the CYP2B6-catalyzed metabolism of bupropion by selegiline, 5 nmol of CYP2B6 was reconstituted with 10 nmol of reductase to give a final concentration of 0.
The methods for the incubation and determination of product formation were followed as described by Bumpus et al. Linear regression analyses of the data were performed using Prism version 5. Reconstitution and inactivation of PB6 by selegiline were carried out as described above. The mobile phase consisted of solvent A 0. The effluent was monitored at nm for intact heme. The remaining portions of the samples were used to determine the reduced CO spectra. The absorbance difference between and nm was used to assess the extent of CO binding and heme remaining in the P samples.
The wild-type and mutant P enzymes were reconstituted with reductase 1: The reactions were extracted twice with 2 ml of ethyl acetate. The mobile phase consisted of 0. The mass analyzer conditions were as follows: The reaction mixture for trapping and identifying the reactive intermediate contained 0. The reaction was initiated with 1. The reaction mixtures were centrifuged at 13, g at room temperature for 10 min.
The supernatants were transferred to new tubes and dried under a stream of nitrogen gas. The column was returned to initial conditions and equilibrated for 10 min before the next injection. The sheath gas was set at 80 arbitrary units, and the auxiliary gas was set at 15 arbitrary units. Data were acquired in the positive ion mode using the Xcalibur software Thermoquest Corporation, Manchester, UK , with two data dependent scans on the first two most intense ions.
The inactivation of PB6 was carried out as described above. Trypsin digestion was carried out using a 1: The flow rate was 0. The instrument was operated in positive electrospray ionization mode with the following parameters: The dynamic exclusion width was set at 1.
The analysis was performed in the data-dependent acquisition mode in which a full scan was recorded over the mass range of to followed by collision-induced dissociation of the six most abundant ions. Data acquisition and analysis were performed using both Xcalibur software version 1. The peptides were identified automatically using the SEQUEST BioWorks computer program, which correlated the experimental tandem mass spectra with the theoretical tandem mass spectra from the amino acid sequences for the 2B6 peptides obtained from the National Center for Biotechnology Information sequence database.
The following criteria were applied for filtering the results: The Sp value indicates the preliminary score of the peptide and shows the number of fragment ions matched out of the total number of fragment masses for the peptide. Figure 2 shows the rates of formation of hydroxybupropion by CYP2B6 in the presence and absence of selegiline as a function of increasing concentrations of bupropion.
Hydroxybupropion formation was measured by integrating the area under the peak and comparing to a standard curve generated by injecting different amounts of authentic hydroxybupropion on the HPLC column.
The change in the apparent K m value indicates that selegiline decreases the apparent affinity of CYP2B6 for bupropion, whereas the decrease in the k cat is probably the result of irreversible mechanism-based inactivation of bupropion hydroxylation activity Fig. Kinetics for the hydroxylation of bupropion by CYP2B6 in the presence and absence of selegiline.
Time- and concentration-dependent inactivation of CYP2B6 by selegiline. Aliquots of the mixture were taken at the time intervals indicated and added to a secondary mixture for the determination of residual 7-EFC O -deethylation activity as described under Materials and Methods. The inset shows the rate for the time-dependent decrease in the 7-HFC formed at the various concentration of selegiline.
The inset shows the double-reciprocal plot for the initial rate of inactivation of hydroxybupropion formation as a function of the concentration of inactivator.
Pseudo-first-order kinetics were observed for concentrations ranging from 0. The kinetic constants were determined from the double-reciprocal plot of the inverse of the initial rate of inactivation as a function of the reciprocal of the selegiline concentration Fig.
Under our current experimental conditions, the kinetic constants describing the inactivation of CYP2B6 were characterized by a K I of 0. A significant decrease in the 0 time activity was observed with increasing selegiline concentrations, suggesting that selegiline inhibits 7-EFC O -deethylase activity in a competitive manner.
Selegiline was also a mechanism-based inactivator with respect to bupropion metabolism as shown in Fig. The kinetic constants describing this inactivation were characterized by a K I of 0.
As shown in Fig. The red line is for the control sample following reduction by sodium dithionite in the presence of CO, which is higher than the pink line, which represents the inactivated sample in the presence of CO. Evidence that the heme moiety itself was neither modified nor destroyed was obtained from reverse-phase HPLC analysis where the heme was monitored at nm Fig.
Virtually no differences in the retention times or areas of the heme peaks were observed between the control and selegiline-inactivated CYP2B6.
The loss in the reduced CO spectrum, with no loss in total heme as measured by HPLC, suggests that binding of the selegiline or its metabolite causes a change in the conformation of the protein and thus may affect the binding of CO to the reduced P Similar changes in reduced CO binding, with no loss in heme, have been seen before with other mechanism-based inactivators. A, portions of the control and selegiline inactivated samples were bubbled with CO in the presence of dithionite, and the reduced CO spectra were monitored between and nm on a spectrophotometer.
B, the control and selegiline inactivated CYP2B6 samples were injected onto a C4 column, and the areas under the heme peaks at nm were integrated to calculate the heme loss. The inactivations and analyses were performed as described under Materials and Methods. Metabolism of selegiline by CYP2B6 was assessed in a reconstituted system.
Figure 5 A shows the extracted ion chromatograms for the parent and the various metabolites, and Fig. The peaks at Small amounts of some secondary metabolite were also detected, eluting at Details of the incubation and analytical conditions are described under Materials and Methods.
A, the extracted ion chromatograms for the peaks eluting at different time points for the parent and the products that include methamphetamine, amphetamine, N -desmethylamphetamine, and hydroxyl-desmethylselegiline.