Chem. Res. Toxicol. 1997, 10, 457-462
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Identification of Modified Atropaldehyde Mercapturic Acids in Rat and Human Urine after Felbamate Administration Charles D. Thompson, Pamela H. Gulden, and Timothy L. Macdonald* Chemistry Department, University of Virginia, Charlottesville, Virginia 22901 Received December 16, 1996X
3-Carbamoyl-2-phenylpropionaldehyde has recently been proposed [Thompson et al. (1996) Chem. Res. Toxicol. 9, 1225-1229] as a potential reactive metabolite of the anti-epileptic drug felbamate. This aldehyde was found to undergo rapid elimination to generate 2-phenylpropenal and reversible cyclization to generate 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one at physiological pH. 2-Phenylpropenal, an R,β-unsaturated aldehyde commonly termed atropaldehyde, is a potent electrophile and undergoes rapid conjugation with glutathione. We sought to demonstrate the formation of atropaldehyde in vivo through the identification of mercapturic acids in rat and human urine after felbamate administration. In this paper, we describe the identification of both the reduced (N-acetyl-S-(2-phenylpropan-3-ol)-L-cysteine) and oxidized (N-acetyl-S-(2-phenyl-3-propanoic acid)-L-cysteine) mercapturic acids of atropaldehyde in rat and human urine. The reduced species was the more abundant in human (∼2:1) and rat (∼6: 1) urine. These findings establish the possibility that atropaldehyde is formed from felbamate in vivo, undergoes glutathione conjugation, and is ultimately excreted in urine in the form of mercapturic acids. Thus, the proposed pathway of felbamate biotransformation, if confirmed in patients, could contribute to our understanding of the toxicities observed during felbamate treatment.
Introduction Felbamate (1, Figure 1) was approved in July 1993 for the treatment of several forms of epilepsy and demonstrated an excellent therapeutic index throughout its clinical trials (1-3). However, subsequent to its release, several toxicological issues, namely, aplastic anemia and hepatotoxicity, were associated with felbamate use (4, 5). The apparent idiosyncratic nature and severity of these side effects prompted a recommendation by the U.S. Food and Drug Administration to withdraw patients from felbamate therapy unless the benefit of seizure control outweighed the risk of the reported toxicities (6). Recently, we proposed 3-carbamoyl-2-phenylpropionaldehyde 3 as a probable reactive metabolite of felbamate (7). We demonstrated the rapid formation of 2-phenylpropenal 7, commonly termed atropaldehyde, from 3 in vitro and proposed this R,β-unsaturated aldehyde as a reactive metabolite of felbamate that may play a role in the observed toxicities. The in vivo metabolism of felbamate has been studied in several species including rat, rabbit, dog, and human (8, 9). The primary excreted compound is unchanged felbamate. In addition, four metabolites have been identified. 2-Phenyl-1,3-propanediol monocarbamate 2 is the product of hydrolysis of one of the carbamate moieties and has been observed in all of the species. 3-Carbamoyl-2-phenylpropionic acid 6, an oxidized secondary metabolite from 2, has been identified as a major metabolite in humans [reported to be ∼12% of a dose in 4-8 h post-dose urine (9)] and also observed in dogs. Felbamate represents a pro-chiral substrate for the enzymes that produce the observed metabolites, and * To whom correspondence should be addressed: e-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, March 15, 1997.
S0893-228x(96)00205-6 CCC: $14.00
Figure 1. Metabolic transformation of felbamate 1 to mercapturic acids 9-11.
presumably, these metabolites are chiral although their exact stereochemistry has not been reported. The mechanism for formation of 6 has not been determined. This acid may result from the direct oxida© 1997 American Chemical Society
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tion of 3 by an aldehyde dehydrogenase. Alternatively, 6 may be produced from the selective hydrolysis of tetrahydro-1,3-oxazin-2,4-dione 5, the presumed product from oxidation of 4-hydroxy-5-phenyl-tetrahydro-1,3-oxazin-2-one 4. The cyclic carbamate 4 is the predominant product formed, in a reversible process, from 3 under physiological pH in vitro but has not yet been observed in vivo. Regardless of the mechanism leading to formation of acid 6, it is clear that hydrolysis of felbamate to the alcohol 2 comprises the first step in the primary pathway of felbamate metabolism in humans. We sought to demonstrate the formation of atropaldehyde from felbamate in vivo. If formed, atropaldehyde would be expected to react with biological nucleophiles, such as GSH. We have demonstrated the formation of atropaldehyde-GSH conjugate 8 in the non-enzymecatalyzed reaction of atropaldehyde and GSH in vitro (7). GSH conjugates are known to be processed in vivo through a multi-step, enzyme-catalyzed process to Nacetyl-L-cysteine conjugates, commonly termed mercapturic acids (10). These mercapturic acids are excreted in urine. Herein, we report the identification by LC/MS of mercapturic acids 10 and 11 in rat and human urine after felbamate administration.
Experimental Procedures Caution: Ammonia is highly toxic and should only be employed with care. All operations should be done in an efficient fume hood. Chemicals and Instruments. All reagents were purchased from either Aldrich Chemical Co. or Sigma Chemical Co. (except as otherwise noted) and were of the highest quality available. HPLC was performed on a Waters 600E gradient pump with a Waters 484 tunable absorbance detector (at 214 nm) using a 5 µm microsorb-mv C18 (4.6 mm × 250 mm) column (Rainin, Emeryville, CA) and a binary solvent systemsSolvent A: 95% acetonitrile, 4.93% water, and 0.07% trifluoroacetic acid; solvent B: 0.1% trifluoroacetic acid and 99.9% water. Solid phase extraction was accomplished using C18 solid phase extraction cartridges (Alltech, Deerfield, IL) and a binary solvent systems Solvent C: 100% acetonitrile; solvent D: 0.2% acetic acid. NMR spectra were recorded on a General Electric QE300 spectrometer at 300 MHz. LC/MS was conducted on a Finnigan MAT TSQ70 spectrometer equipped with an electrospray ionization source. Synthesis. 2-Phenylpropenal (7). The synthesis of 7 was accomplished as described (7). 2-Phenyl Acrylic Acid. Oxidation of 7 by the method of Dalcanale and Montanari (11) afforded 2-phenyl acrylic acid, commonly known as atropic acid. Atropaldehyde (0.3 mmol) was stirred in acetonitrile (5 mL) at 10 °C. H2O2 (100 µL 35%) and 0.6 M NaH2PO4 (2 mL, pH ) 2.0 adjusted with HCl) were added followed by 700 µL of 1 M NaClO2 in 50 µL aliquots at 5-min intervals. After 1 hr, the reaction was quenched by the addition of sodium bisulfite and 3 M HCl (2 mL). Acid-base extraction with CHCl3 afforded the pure 2-phenyl acrylic acid in 70% yield. 1H NMR (acetone-d ): δ 5.88 (s, 1H, HCdCR ); 6.25 (s, 1H, 6 2 HCdCR2); 7.3 (m, 5H, Ph); 13.5 (bs, 1H, -CO2H). 2-Phenyl-1,3-propanediol. Reduction of commercially available diethyl phenylmalonate to 2-phenyl-1,3-propanediol with lithium aluminum hydride was achieved using established methods (12). 2-Phenyl-1,3-propanediol Monocarbamate (2). The synthesis of 2 was achieved by treatment of the diol (2.6 mmol) with 1,1′-carbonyldiimidazole (1 equiv) in THF at room temperature for 1 h. The temperature of the reaction flask was then lowered to -48 °C, and liquid ammonia was introduced from a cold finger at -78 °C attached to a tank of compressed NH3. After the introduction of approximately 30 drops of liquid ammonia from the cold finger, the reaction was stirred for 3 h at -48 °C. The reaction was then quenched by the addition of H2O and allowed to stir for 1 h. The monocarbamate 2 was
Thompson et al. purified by chromatography on silica gel (chloroform/acetone) to give a 63% yield with starting material and dicarbamate accounting for the remainder. 1H NMR (DMSO-d6): δ 2.4 (bs, 1H, -OH); 3.1 (quin, 1H, J ) 6.2, benzylic); 3.84 (d, 2H, J ) 6.2, -CH2OH); 4.40 (d, 2H, J ) 6.2, -CH2OCONH2); 4.8 (bs, 2H, -NH2); 7.3 (m, 5H, Ph). 2-Phenyl-1,3-propanediol Dicarbamate (1). Felbamate used for in vivo metabolism studies was purchased from the University of Virginia Medical Center Pharmacy and manufactured by Wallace Laboratories. Formation of felbamate for use as a standard for HPLC and LC/MS was achieved by treatment of the isolated monocarbamate 2 with another equivalent of 1,1′carbonyldiimidazole, followed by ammonia as described above. 1H NMR (DMSO-d ); δ 3.16 (quin, 1H, J ) 6.3, benzylic); 4.1 6 (d, 4H, J ) 6.3, -CH2OCONH2); 6.4 (bs, 4H, -NH2); 7.3 (s, 5H, Ph). N-Acetyl-S-(2-phenylpropanal)-L-cysteine (9). To 10 mg of atropaldehyde in methanol (2 mL) was added 16 mg of N-acetyl-L-cysteine (1.3 equiv) with stirring at room temperature. Two drops of 200 mM KPO4 buffer (pH ) 8) were added for catalysis, and the reaction was stirred for 1 h. The solvent was removed by vacuum evaporation and reconstituted in 700 µL of 25% C/75% D. This solution was applied to a C18 solid phase extraction cartridge that was then washed with 3 mL of 100% D. The purified atropaldehyde mercapturic acid was then eluted with 3 mL 50% C in 50% D. This fraction was then quickly frozen and lyophilized to dryness in efforts to limit decomposition as this conjugate was found to decompose under acidic conditions. 1H NMR (D2O): δ 1.92 (bs, 3H, -COCH3); 2.64-3.0 (m, 4H, -CH2SCH2-); 3.05 (m, ∼0.8 H, benzylic); 3.89 (m, ∼0.2 H, benzylic); 4.42 (m, 1H, cysteine R); 5.14 (d, ∼0.8 H, J ) 5.0, -CH(OH)2) 7.28 (m, 5H, Ph); 9.58 (s, ∼0.2 H, -COH). LC/MS: MH+ ) 296. The peaks at 9.58 and 3.89 correspond to the aldeydic and benzylic protons in the aldehyde state, while the peaks at 5.14 and 3.05 correspond to the aldehydic and benzylic protons in the gem diol state. N-Acetyl-S-(2-phenylpropan-3-ol)-L-cysteine (10). To a mixture of the crude atropaldehyde mercapturic acid 9, formed as above, was added an excess of NaBH4. This solution was stirred for 1 h at room temperature followed by quenching with 1 mL of 0.2% acetic acid. The solvent was reduced by vacuum evaporation and then reconstituted to a total volume of 1 mL with 0.2% acetic acid. HPLC analysis (30% A/70% B) indicated complete conversion of 9. The reduced conjugate was then purified by solid phase extraction as described above. 1H NMR (D2O): δ 1.91 (bs, 3H, -COCH3); 2.66-3.0 (m, 6H, -CH2SCH2CHPhCH2OH); 3.72 (t, 1H, J ) 5.5, benzylic); 4.39 (m, 1H, cysteine R); 7.27 (m, 5H, Ph). LC/MS: MH+ ) 298. N-Acetyl-S-(2-phenylpropanoic acid)-L-cysteine (11). To 9 mg of atropic acid in methanol (5 mL) was added 18 mg of N-acetyl-L-cysteine (NaC) (1.3 equiv) with stirring at room temperature. The addition of several drops of 200 mM KPO4 buffer (pH ) 8.0) resulted in a cloudy suspension that was stirred over night. The solvent was removed by vacuum evaporation, and the residue was reconstituted in 1 mL of 100% D for purification by solid phase extraction as described above. 1H NMR (D O): δ 1.94 (bs, 3H, -COCH ); 2.75-3.23 (m, 4H, 2 3 -CH2SCH2-); 3.84 (t, 1H, J ) 5.7, benzylic); 4.41 (m, 1H, cysteine R); 7.32 (m, 5H, Ph). LC/MS: MH+ ) 312. Rat in Vivo Metabolism. Six adult male Sprague-Dawley rats (∼250 mg) were housed in individual metabolic cages under a 12-h light/dark cycle and provided with free access to food and water throughout the experiment. Urine was collected in plasticware cups placed beneath the cages at room temperature. The rats were allowed to acclimate for 36 h, after which time the background urine (designated RU-A) was collected, pooled, and stored frozen at -60 °C. Each rat was administered 800 mg/kg felbamate (formulation, Felbatol oral suspension, 600 mg/ mL, lot no. 5LO6Q) via gavage and returned to their individual cages. Urine from 1-18 h post-dose (designated RU-B) was collected, pooled, and stored frozen at -60 °C. Human in Vivo Metabolism. A healthy adult male volunteer was administered a single dose of 600 mg (∼9 mg/kg) felbamate (formulation, Felbatol 600 mg tablet lot no. 5EO5N). Pre-dose urine (designated HU-A) had been collected, pooled,
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Figure 2. Schematic of the preparation of urine samples (see Experimental Procedures for details). and stored frozen at -60 °C the previous day. The 0-8 h postdose urine (designated HU-B) was collected and stored frozen at -60 °C. Preparation of Urine Samples. (See Figure 2.) Urine samples were removed from the freezer and allowed to thaw at room temperature after which they were centrifuged (4000g, 10 min) to remove precipitated material. The supernatant was decanted away, and the precipitate was discarded. Urine (3 mL) was combined with 1 mL of 4% acetic acid and placed on ice for 5 min followed by centrifugation at 4000g to remove the precipitated material. The supernatant was lyophilized to near dryness and reconstituted to a total volume of 1 mL with 0.2% acetic acid. The samples were then fractionated using a C18 solid phase extraction cartridge. The cartridges were individually prepared just prior to use by the sequential elution of 3 mL of 90% C/10% D and 6 mL of 100% D. The sample was then applied, and the cartridge was washed with 3 mL of 100% D (fraction 1) followed by 4 mL of 10% C/90% D (fraction 2). The samples for further analysis were then eluted with 3 mL of 30% C/70% D (fraction 3). Fraction 3 samples were lyophilized to dryness and then reconstituted in 1 mL of 10% C/90% D. A 1:10 dilution into 0.2% acetic acid afforded the samples for LC/ MS analysis. Mass Spectrometry Analysis of Prepared Urine Samples. Samples were analyzed by tandem mass spectrometry as described previously (13). Samples were loaded into 75 µm i.d. × 185 µm o.d. fused silica tubing (SGE, Austin, TX), packed with 12 cm of 10 µm C18 beads (YMC Gel, Kyoto, Japan). The samples were eluted into a TSQ 70 (upgraded with the electronics of a TSQ 700) triple quadrupole mass spectrometer (Finnigan-MAT, San Jose, CA) equipped with a Finnigan API ESI/CI source, at a flow rate of 0.5 µL/min with a 12-min gradient of 0-80% acetonitrile in 0.1 M acetic acid. For molecular weight data, spectra were recorded every 1.5 s over a mass range of 190-1300 amu. The resulting spectrum contained ions of the type [M + nH]n+, where M is molecular mass and n is the number of attached protons. Fragmentation data were recorded similarly, where the first quadrupole mass filter was set to pass all ions within a 3-Da window centered around the mass of interest. These ions were transmitted to the second quadrupole (collision cell), where they fragment upon collision with argon atoms. These fragments were then analyzed with the third quadrupole. A mass range, from 50 to 5 amu above the mass
Figure 3. Comparison of (A) pre-dose rat urine (RU-A-F3) to (B) post-dose rat urine (RU-B-F3). The chromatograms show percent relative abundance of ion current versus m/z value for summed mass spectra over the elution range of felbamate (m/z ) 239) and the mercapturic acids (m/z ) 298 and 312). of the parent ion, was scanned at a rate of 500 Da/s, allowing the individual fragments to pass through to the detector.
Results Synthetic standards of the mercapturic acids 9-11 were prepared as described in Experimental Procedures and their structures confirmed by 1H NMR and LC/MS. These standards were then used to develop the protocol for sample preparation (Figure 2). This protocol was optimized for the analysis of these mercapturic acids in the presence of felbamate. However, the relative recoveries of the other metabolites and the parent drug, relative to the mercapturic acids, have not been determined. The mass spectra shown in Figures 3 and 4 represent the summed mass spectra recorded during the period when the mercapturic acids and felbamate elute from the microcapillary column into the mass spectrometer, as determined by their relative position to an internal standard. Figure 3 compares pre-dose rat urine (RU-AF3) in panel A to pooled 1-18 h rat urine after a single 600 mg/kg administration of felbamate by gavage in panel B. The parent molecular ion peaks for felbamate 1 (m/z ) 239), the NaC-alcohol 10 (m/z ) 298), and the NaC-acid 11 (m/z ) 312) can be observed in the postdose sample. Similarly, in Figure 4, pre-dose human urine (HU-A-F3, panel A) is compared to 0-8 h human urine (HU-B-F3, panel B) after a single 600 mg (∼9 mg/ kg) oral dose of felbamate. Again, the peaks corresponding to the appearance of 1, 10, and 11 can be observed
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Figure 4. Comparison of (A) pre-dose human urine (HU-AF3) to (B) post-dose human urine (HU-B-F3). The chromatograms show percent relative abundance of ion current versus m/z value for summed mass spectra over the elution range of felbamate 1 (m/z ) 239) and the mercapturic acids 9 and 11 (m/z ) 298 and 312).
Thompson et al.
in Figure 4B. We were unable to identify the appearance of a peak at m/z ) 296 corresponding to the mercapturic acid of atropaldehyde 9 in these samples. However, our experience with synthetic NaC-atropaldehyde 9 has demonstrated that it is unstable in a biological matrix, such as urine (data not shown). In order to demonstrate that the peaks corresponding to the m/z of 10 and 11 in post-dose samples were in fact the modified mercapturic acids of atropaldehyde, we conducted co-elution studies with synthetic standards (Figure 5). Figure 5A shows the percent relative abundance versus scan number of 1, the putative 10, and the putative 11 from the post-dose human urine sample HUB-F3. As indicated by the percent relative abundance value, the putative 10 was approximately twice as abundant at the putative 11; whereas, in post-dose rat urine the ratio of 10 to 11 is approximately 6:1 (data not shown). Figure 5B shows the elution profile of the same sample with the synthetic NaC-alcohol 10 doped into the sample. As indicated in the middle panel of Figure 5B, the synthetic standard and the putative 10 from the urine sample co-elute, giving rise to an increase in the percent relative abundance. This demonstrates that this analyte in the urine sample and the synthetic standard of 10 have identical m/z and elution characteristics. The corresponding experiment was done for the NaCacid 11 as shown in Figure 5C. In this case, the bottom panel demonstrates that the addition of a synthetic standard of 11 to the urine sample results in the elution of a single peak with a corresponding increase in its percent relative abundance. In order to further demonstrate that the urine analytes in question unambiguously corresponded to the modified mercapturic acids of atropaldehyde 10 and 11, we collected collision-activated dissociation spectra for the analytes in a urine sample (Figures 6B and 7B) and compared them to the CAD spectra of the synthetic standards (Figures 6A and 7A). Figure 6 compares the
Figure 5. Co-elution of mercapturic acids in post-dose human urine (HU-B-F3) with synthetic standards. (A) On-line microcapillary HPLC elution of HU-B-F3 showing m/z ) 239 (for felbamate 1), m/z ) 298 (for NaC-alcohol 9), and m/z ) 312 (for NaC-acid 11). (B) On-line microcapillary HPLC elution of HU-B-F3 plus added NaC-alcohol synthetic standard. (C) On-line microcapillary HPLC elution of HU-B-F3 plus added NaC-acid synthetic standard.
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Figure 6. Collision-activated dissociation of NaC-alcohol from (A) synthetic standard and (B) post-dose human urine (HU-BF3).
Figure 7. Collision-activated dissociation of NaC-acid from (A) synthetic standard and (B) post-dose human urine (HU-B-F3).
CAD spectra of the NaC-alcohol 10 standard, panel A, to that of the putative NaC-alcohol from a human postdose urine sample (HU-B-F3), panel B. As shown, these two entities have identical fragmentation patterns. The CAD spectra of the NaC-acid 11 standard and the putative 11 from HU-B-F3 are shown in Figure 7. Again, the CAD of the standard, panel A, and of the putative mercapturic acid from the urine sample, panel B, demonstrate identical fragmentation patterns.
of 4 has not been demonstrated in vivo, and its metabolic fate, if formed, is unknown. The “model” for this possible behavior of 4 is 4-hydroxycyclophosphamide, the metabolically activated form of cyclophosphamide. Our proposed pathway leading from 4 to atropaldehyde 7 parallels the known pathway from 4-hydroxycyclophosphamide to acrolein (14). The breadth to which this similarity applies is the subject of ongoing investigation. We have examined the potential for an alternative process for the formation of the mercapturates 10 and 11 involving β-elimination of acid carbamate 6 to 2-phenylacrylic acid and subsequent GSH conjugation. However, we have found 6 to be stable to elimination at physiological pH (data not shown) and the derived atropic acid to be slow (relative to atropaldehyde) in undergoing uncatalyzed GSH conjugation. Moreover, it is unlikely that alcohol adduct 10 would be derived from the acid 11. A possibility for the formation of mercapturates 10 and 11 that we have not examined is the glutathione transferase-mediated conversion from the corresponding metabolites 2 and 6; however, this potential pathway would also be anticipated a priori to generate the corresponding mono-GSH and subsequent mercapturate adduct of felbamate itself. Identification of mercapturic acids 10 and 11 in postdose urine is consistent with the hypothesis that atropaldehyde is formed in vivo and that it reacts with thiol nucleophiles. While the mercapturic acids are derived from addition to GSH, any similar nucleophile (i.e., protein thiols or DNA bases) would be expected to undergo similar conjugation to atropaldehyde. Toxicity after exposure to this type of alkylating agent might manifest itself through a variety of pathways. Disruption
Discussion We have identified both the reduced (10) and oxidized (11) mercapturic acids of atropaldehyde 7 in rat and human urine after felbamate administration. Their presence provides support for the hypothesis that atropaldehyde 7 is generated during the course of felbamate metabolism. Analogous to our in vitro studies (7), 7 is presumably produced in vivo via β-elimination of 3, a presumed intermediate in the enzymatic oxidation of alcohol 2 to carboxylic acid 6. We have reported that the half-life of 3 is very short (
