Chem. Res. Toxicol. 2003, 16, 123-128
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In Vitro Metabolism of Tolcapone to Reactive Intermediates: Relevance to Tolcapone Liver Toxicity Kirsten S. Smith,† Philip L. Smith,† Tiffany N. Heady,† Joel M. Trugman,‡ W. Dean Harman,† and Timothy L. Macdonald*,† Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virginia 22901, and Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 Received June 10, 2002
Tolcapone is a catechol-O-methyltransferase (COMT) inhibitor used for control of motor fluctuations in Parkinson’s disease (PD). Since its entry onto the market in 1998, tolcapone has been associated with numerous cases of hepatotoxicity, including three cases of fatal fulminant hepatic failure. The cause of this toxicity is not known; however, it does not occur with the use of the structurally similar drug entacapone. It is known that tolcapone is metabolized to amine (M1) and acetylamine (M2) metabolites in humans, but that the analogous metabolites were not detected in a limited human study of entacapone metabolism. We hypothesized that one or both of these tolcapone metabolites could be oxidized to reactive species and that these reactive metabolites might play a role in tolcapone-induced hepatocellular injury. To investigate this possibility, we examined the ability of M1 and M2 to undergo in vitro bioactivation by electrochemical and enzymatic methods. Electrochemical experiments revealed that M1 and M2 are more easily oxidized than the parent compound, in the order M1 > M2 > tolcapone. There was a general correlation between oxidation potential and the halflives of the compounds in the presence of two oxidizing systems, horseradish peroxidase and myeloperoxidase. These enzymes catalyzed the oxidation of M1 and M2 to reactive species that could be trapped with glutathione (GSH) to form metabolite adducts (C1 and C2). Each metabolite was found to only form one GSH conjugate, and the structures were tentatively identified using LC-MS/MS. Following incubation of M1 and M2 with human liver microsomes in the presence of GSH, the same adducts were observed, and their structures were confirmed using LC-MS/MS and 1H NMR. Experiments with chemical P450 inhibitors and cDNAexpressed P450 enzymes revealed that this oxidation is catalyzed by several P450s, and that P450 2E1 and 1A2 play the primary role in the formation of C1 while P450 1A2 is most important for the production of C2. Taken together, these data provide evidence that tolcaponeinduced hepatotoxicity may be mediated through the oxidation of the known urinary metabolites M1 and M2 to reactive intermediates. These reactive species may form covalent adducts to hepatic proteins, resulting in damage to liver tissues, although this supposition was not investigated in this study.
Introduction Idiopathic Parkinson’s disease (PD)1 is a progressive neurodegenerative disorder that has a worldwide distribution and affects over one million people in North America alone (1, 2). Clinical manifestations of the disease include motor symptoms such as resting tremor, rigidity, and dyskinesia as well as nonmotor symptoms * To whom correspondence should be addressed. E-mail: tlm@ virginia.edu † University of Virginia. ‡ University of Virginia Health Sciences Center. 1 Abbreviations: AADC, amino acid decarboxylase; CID, collisioninduced dissociation; COMT, catechol-O-methyltransferase; COSY, correlation spectroscopy; D2O, deuterium oxide; DMF, dimethylformamide; ESI, electrospray ionization; FMO3, flavin-containing monooxygenase-3; HETCOR, heteronuclear chemical shift correlation; HLMs, human liver microsomes; HRP, horseradish peroxidase; HSQC, homonuclear single quantum correlation; L-dopa, levodopa; MAO, monoamine oxidase; MPO, myeloperoxidase; M1, amine metabolite of tolcapone; M2, acetylamine metabolite of tolcapone; NAC, N-acetylcysteine; NAT, N-acetyltransferase; NHE, normal hydrogen electrode; NOE, nuclear Overhauser enhancement; PD, idiopathic Parkinson’s disease; SIM, selected ion monitoring; TCA, trichloroacetic acid.
such as gastrointestinal dysfunction, depression, and dementia. The depletion of striatal dopamine and the resulting disruption of neurotransmitter signaling are responsible for most of the motor symptoms of PD (1, 3). Consequently, most current therapies ease the symptoms of PD by increasing dopamine availability. For instance, the mainstay of Parkinson’s therapy, levodopa (L-dopa), crosses the blood-brain barrier and is converted by amino acid decarboxylase (AADC) into dopamine. A number of other therapies are also available to be used in conjunction with L-dopa. These drugs are designed to control motor fluctuations that arise from side effects of treatment with L-dopa. The drugs work either by inhibiting peripheral metabolism of L-dopa or by inhibiting the breakdown of dopamine in the central nervous system. Examples of adjunctive therapies include AADC inhibitors, COMT (catechol-O-methyltransferase) inhibitors, and MAO (monoamine oxidase) inhibitors (4). Two COMT inhibitors, tolcapone and entacapone, are currently available for use (Figure 1) (5-7). Unfortunately, the use of tolcapone has been associated with a
10.1021/tx025569n CCC: $25.00 © 2003 American Chemical Society Published on Web 01/30/2003
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Chem. Res. Toxicol., Vol. 16, No. 2, 2003
Figure 1. Structures of the COMT inhibitors.
Figure 2. Amine (M1) and acetylamine (M2) metabolites of tolcapone.
number of problems, including abnormalities in liver function tests and three cases of fatal fulminant hepatic failure (8). These problems have led to the drug being withdrawn from the market in some countries and the introduction of a black-box warning and intensive monitoring requirements in the United States (8). The cause of tolcapone’s liver toxicity is currently unknown, but these problems have not been seen in the structurally similar drug entacapone. Studies into the metabolism of tolcapone have indicated that the principal metabolic pathways are methylation, oxidation, reduction, and conjugation reactions such as glucuronidation (9). Of particular interest is the reduction of the nitro group of tolcapone to form the amine metabolite (M1; Figure 2), which is further modified by acetylation to form the acetylamine metabolite (M2). We postulated that these amine and acetylated amine metabolites of tolcapone are oxidatively bioactivated to the o-quinone or quinone-imine species and that these reactive species may play a role in tolcaponeinduced hepatotoxicity. The studies reported here provide evidence that both of these metabolites are indeed metabolically activated in vitro and that this activation is catalyzed by one or more cytochrome P450 enzymes.
Experimental Procedures Chemicals. Horseradish peroxidase (HRP; type X, 260 units/ mg of protein), reduced glutathione (GSH), catalase (10 700 units/mg solid), and P450 inhibitors were purchased from Sigma Chemical Corp. (St. Louis, MO). Myeloperoxidase (MPO; from human leukocytes, 200 units/mg) was obtained from Calbiochem (La Jolla, CA). Human liver microsomes (pooled from 21 livers) were purchased from Gentest Corp. (Woburn, MA). Recombinant P450 enzymes and flavin-containing monooxygenase 3 (FMO3 expressed) from either baculovirus-infected insect cells or human lymphoblast cells were also from Gentest Corp. All other reagents and chemicals were of the highest quality available. Synthesis. Tolcapone was extracted from Tasmar tablets using ether and purified. The amine and acetylated amine metabolites were synthesized as described below. Routine 1H spectra were obtained on a Varian Unity Inova 300 (300 MHz) spectrometer. 13C NMR spectra were recorded on a Varian Unity Inova 300 (75.4 MHz) or Varian Unity Inova 500 (125.7 MHz) spectrometer. Chemical shifts are reported in parts per million (ppm) referenced to residual partially deuterated solvent peaks, and coupling constants are in hertz (Hz). 5-Amino-3,4-dihydroxy-4′-methyl-benzophenone (M1). Tolcapone (94.3 mg, 0.35 mmol) and cyclohexene (1.5 mL) were dissolved in 30 mL of absolute ethanol. Activated Pd/C (10%, 50 mg) was added, and the mixture was refluxed 4 h. The
Smith et al. reaction mixture was passed through a plug of Celite to remove the catalyst then concentrated under reduced pressure. The residue was dissolved in CH3CN (5 mL) and precipitated into stirring hexanes (50 mL). The precipitate was collected on a fine frit, washed with hexanes (3 × 1 mL), and dried in vacuo. Yield: 68 mg (80%). 1H NMR (CD3CN) δ 7.61 (2H, d, J ) 8.13 Hz), 7.29 (2H, d, J ) 7.92 Hz), 6.80 (1H, d, J ) 1.98 Hz), 6.75 (1H, d, J ) 2.20 Hz), 2.41 (3H, s); 13C NMR (CD3OD) δ 198.5 (CO), 145.7 (Ar), 143.9 (Ar × 2), 139.6 (Ar), 137.2 (Ar), 136.8 (Ar), 130.9 (CH, Ar × 2), 129.8 (CH, Ar × 2), 112.1 (CH, Ar), 109.8 (CH, Ar), 21.5 (CH3, Me); electrochemistry Ep,a ) 0.93 V, Ep,c ) -1.44 V. 5-N-Acetylamino-3,4-dihydroxy-4′-methyl-benzophenone (M2). Tolcapone (107.2 mg, 0.40 mmol) and pyridine (158 mg, 2 mmol) were added to CH3CN (25 mL), and the slurry stirred as acetic anhydride (123 mg, 1.2 mmol) was added dropwise. The solution was stirred at 25 °C for 2 h during which time the solution became homogeneous and the color changed from yellow to orange. The solvent was removed, and the residue was passed through a plug of silica and eluted with 35% EtOAc/ hexanes then concentrated under reduced pressure to afford a thick orange oil (TLC silica, 1:1 EtOAc/hexanes Rf ) 0.4). This oil was subsequently dissolved in cyclohexene (1.5 mL), absolute ethanol (50 mL), and ethyl acetate (50 mL). Activated Pd/C (10%, 50 mg) was added, and the mixture was refluxed 4 h. The reaction mixture was passed through a plug of Celite to remove the catalyst then concentrated under reduced pressure. The residue was dissolved in CH3CN (5 mL) and added dropwise to stirring hexanes (50 mL). The precipitate was collected on a fine frit, washed with hexanes (3 × 1 mL), and dried in vacuo. Yield for the pale orange/brown solid: 106 mg, 95%. 1H NMR (CD3CN) δ 8.73 (1H, bs, NH), 7.64 (2H, d, J ) 8.24 Hz), 7.33 (2H, d, J ) 8.24 Hz), 7.16 (1H, d, J ) 2.0 Hz), 7.13 (1H, d, J ) 2.0 Hz), 2.43 (3H, s, amide Me), 2.17 (3H, s, Me), 2.15 (2H, bs, OH × 2); 13C NMR (CD OD) δ 197.5 (CO), 172.6 (CO, amide), 147.2 (Ar), 3 144.2 (Ar), 136.7 (Ar), 131.0 (Ar-CH ×2), 129.9 (Ar-CH × 2), 129.7 (Ar), 126.8 (Ar), 119.0 (Ar), 114.3 (Ar), 23.4 (CH3), 21.6 (CH3); IR (HATR) νCO ) 1603 (s) cm-1; electrochemistry Ep,a ) 1.360 V. Electrochemical Potentials. Electrochemical experiments were performed under nitrogen using a PAR model 362 potentiostat driven by a PAR model 175 universal programmer. Cyclic voltammograms were recorded using a Kipp and Zonen BD90 XY recorder in a standard three-electrode cell from +1.70 to -1.70 V with a glassy carbon electrode. All potentials are reported versus saturated calomel electrode (SCE) and were determined in acetonitrile containing ∼0.5 M tetrabutylammonium hexaflurophosphate at 0.10 V/s using ferrocene [E1/2) 0.550 V versus normal hydrogen electrode (NHE)] or cobaltocenium hexaflurophosphate (E1/2) -0.780 V versus NHE) in situ as a calibration standard. Reversible half-wave couples were considered to exhibit peak-to-peak separation (Ep,a-Ep,c) between 80 and 100 mV. Half-Life Studies with Horseradish Peroxidase and Myeloperoxidase. Horseradish peroxidase (HRP) incubations were carried out at 37 °C in a 0.1 M potassium phosphate buffer (pH 8). Buffer was preincubated with 2.5 units/mL of HRP and 250 µM substrate, and the reaction was initiated by addition of 1.1 mM H2O2. Incubations were quenched at various time points by addition of 5% (v/v) catalase (5 mg/mL) and an appropriate internal standard. After several seconds, the proteins were precipitated and pelleted by addition of 5% (v/v) of 10% (w/v) trichloroacetic acid (TCA) followed by centrifugation. The resulting supernatant was used in all succeeding analyses. The concentration of substrate remaining at each time point was determined by LC-MS (Figure 3). The calibration curve of the internal standard and drug was found to be linear in the range detected. The drug half-lives (t1/2) under these conditions were determined using a first-order approximation by plotting the natural logarithm of the drug concentration versus time to give a slope equal to -k. The t1/2 is equal to 0.693/k. Incubations were also done with an additional 1 mM GSH to look for the presence
In Vitro Metabolism of Tolcapone
Chem. Res. Toxicol., Vol. 16, No. 2, 2003 125 Table 1. Electrochemical and Half-Life Data for Tolcapone and Metabolites half-life (s) substrate
Ep,a (V)a
HRP
MPO/Cl-
tolcapone M2 M1
>1.70 1.36 0.93
75.3 ( 5.8 2.1 ( 0.2 120 min
