Chem. Res. Toxicol. 1993,6, 335-340
336
Studies on the Metabolic Fate of Caracemide, an Experimental Antitumor Agent, in the Rat. Evidence for the Release of Methyl Isocyanate in Vivo' J. Greg Slatter,? Margaret R. Davis, Deog-Hwa Han,* Paul G . Pearson,? and Thomas A. Baillie* Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washington, Seattle, Washington 98195 Received December 18, 1992
Following administration to rats of a single ip dose (6.6 mg kg') of the investigational antitumor agent caracemide (N-acetyl-N,0-bis[methylcarbamoyll hydroxylamine), the mercapturic acid derivative N-acetyl-S-(N-methylcarbamoy1)cysteine(AMCC) was identified in urine by thermospray LC-MS. Quantification of this conjugate was carried out by stable isotope dilution thermospray LC-MS, which indicated that the fraction of the caracemide dose recovered as AMCC in 24-h urine collections was 54.0 i 5.5% (n = 4). Since AMCC is known to represent a major urinary metabolite of methyl isocyanate (MIC) in the rat, the results of this study support the contention that caracemide yields MIC as a toxic intermediate in vivo. Furthermore, with the aid of a specifically deuterium-labeled analog of caracemide ([carbamoyloxyC2H3]caracemide), it was shown that the methylcarbamoyl group of AMCC derived from both the O-methylcarbamoyl(72 %) and N-methylcarbamoyl(28%)side chains of the drug. In view of these findings, it is concluded that caracemide acts as a latent form of MIC in vivo and that this reactive isocyanate (or labile S-linked conjugates thereof) may contribute t o the antitumor properties andlor adverse side-effects of caracemide.
Introduction Caracemide (N-acetyl-N,O-bis[methylcarbamoyll hydroxylamine; NSC 253272; Figure 1)is an investigational antitumor agent currently undergoing clinical evaluation (1, 2). Caracemide is mutagenic to Salmonella typhimurium in the presence of rat liver homogenate (3) and decomposes in plasma or phosphate buffer to products which lack the N-acetyl or N-methylcarbamoyl moieties (4). In vitro experiments employing benzylamine as a trapping agent have provided indirect evidence for methyl isocyanate (MIC) as a breakdown product of caracemide ( 5 ) and it has been speculated, therefore, that MIC may contribute to the severe, dose-limiting side effects of caracemide in patients which include neurotoxicity and mucosal burning ( 4 , 5 ) . Caracemide is known to inhibit enzymes such as acetylcholinesterase ( 4 ) , choline acetyl transferase (61, and ribonucleotide reductase (7),possibly as a result of MIC-mediated carbamoylation of active-site functional groups. Although MIC is a highly reactive electrophilic species which would be expected to undergo hydrolysis relatively rapidly in biological systems (8), recent studies on the metabolic fate of MIC in rats have indicated that the
* Author to whom correspondence
should be addressed. Present address: Drug Metabolism Research, The Upjohn Co., Kalamazoo, MI 49001. t Present address: Medicinal Toxicology Research Center, Inha University, Inchon, Korea. A preliminary account of this work was presented at the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, May 1990. Abbreviations: AMCC, N-acetyl-S-(N-methylcarbamoy1)cysteine; ['H JAMCC, N-[2HJacetyl-S(N-methyl-cbamoy1)cysteine; AL2H31MCC, N-acetyl-S-(N-['H 13 methylcarbamoy1)cystine;MIC, methyl isocyanate; ['H JMIC, ['H Jmethyl isocyanate; SMG, S-(N-methylcarbamoy1)glutathione; NAC, N-acetylcysteine; SIM, selected ion monitoring; SPE, solid-phase extraction. +
Caracemide
(2H31Caracemide n
0
AMCC
[2H3]AMCC
(Internalstandard)
0
SMG
Figure 1. Structures of compounds referred to in the text.
effective half-life and toxic potential of this isocyanate may be extended by reversible conjugation with GSH (9). Thus, reaction of MIC with GSH to form the labile carbamate thioester conjugate, S-(N-methylcarbamoy1)glutathione (SMG), appears to be a reversible process under physiologicalconditions and, hence, GSH may serve to transport MIC in vivo (10). In light of these findings, it has been proposed that the reversible reaction of MIC with GSH and related thiols may modulate the carbamoylating properties of MIC and possibly other isocyanates in vivo (11-13).
0893-228~/93/2106-0335$04.00/0 0 1993 American Chemical Society
336 Chem. Res. Toxicol., Vol. 6,No. 3, 1993
Slatter et al.
S c h e m e I. Synthesis of [ZHJCaracemide 0
0
Acetohydroxamic acid CHsN=C=O CH&
TEA
u
A
CH3-NH
0
When MIC was administered to rats by ip injection, SMG was excreted into bile (10) and the corresponding mercapturic acid conjugate, N-acetyl-S-(N-methylcarbamoy1)cysteine (AMCC), was detected as a prominent biotransformation product in urine (9). This mercapturate also was identified as a urinary metabolite of the experimental antitumor agent N-methylformamide in rodents (14,151and humans (14,161 and was formed chemically when monomethylcarbamate metabolites of the bronchodilating drug bambuterol were allowed to decompose in vitro in the presence of N-acetylcysteine (NAC) (17). In both cases, formation of AMCC was attributed to the reaction of MIC with available thiols and it would appear, therefore, that the excretion of this mercapturate may represent a valuable index of the generation of MIC as a chemically-reactive intermediate in vivo. The objective of the present study, which employed the rat as an animal model, was to investigate the potential role of MIC as a toxic metabolite of caracemide in vivo. Specifically, the study was designed to address the followingissues: (i) to establish whether AMCC is aurinary metabolite of caracemide in the rat, consistent with release of MIC from the parent drug in vivo; (ii) to assess the quantitative importance of this pathway of caracemide metabolism through the development of a sensitive and specific assay for AMCC in urine; (iii) with the aid of a selectively deuterium-labeled analog of caracemide, to distinguish between the N-methylcarbamoyl and O-methylcarbamoyl side chains of caracemide as alternative sources of MIC release in vivo.
Experimental Procedures Instrumentation. Proton (300MHz) and '3C NMR (75 MHz) spectra were recorded on a Varian VXR-300spectrometer (Varian Associates, Inc., Palo Alto, CA). Chemical shifts are reported in ppm downfield from tetramethylsilane (C2HCl3 as solvent) or sodium 3-(trimethylsilyl)-l-propanesulfonicacid (2H20 as solvent). Resonances are reported as singlets (s), doublets (d), triplets (t),quartets (q),or multiplets (m). Infrared spectra were obtained using a Perkin-Elmer Model 1600 FTIR spectrometer with KBr disks. Bands are reported by intensity (s, m, w) and wavenumber (cm-1). Mass spectra were recorded on a Vestec Model 201 thermospray LC-MS system (Vestec Corp., Houston, TX), equipped with a Hewlett-Packard 5997 ChemStation data system, two LKB 2150 HPLC pumps, an LKB 2152 controller, and a Rheodyne 50-pL injection loop. The majority of analyses were performed in the selected ion monitoring (SIM) mode (as outlined in detail below) with a dwell time of 700 ms per ion. Liquid chromatographic separations were performed on Beckmann CISreverse-phase columns (15 cm or 25 cm X 4.6 mm id.)
coupled to a Waters Cl8 guard column. Details of the mobile phases used in the various aspects of this work are given under Biological Experiments. Materials. All chemicals used in synthesis and analytical work were purchased from the Aldrich Chemical Co. (Milwaukee, WI). HPLC grade solvents and Cl8solid-phase extraction (SPE) cartridges (1-mL volume) were obtained from J. T. Baker Ltd. (Philipsburg, NJ) or Waters Ltd. (Danvers, MA). Methanolic HC1 was prepared freshly by bubbling HCl gas through anhydrous methanol. AMCC, N-[2H3]acetyl-S-(N-methylcarbamoyl)cysteine ( [2H3]AMCC),and[2H3]methylisocyanate([2H3]MIC)were prepared by synthesis, as described previously (9, 18). Caracemide was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, while deuterium-labeled caracemide ( [2H&aracemide) was synthesized as outlined in Scheme I. Details of the synthetic procedure are as follows: Synthesis: Caution! Methyl isocyanate, which is employed in both stages of the synthesis, is volatile and extremely toxic. Protective clothing and an efficient fume hood are necessary for all procedures involving the use of this compound. N-Acetyl-0-([*H3]methylcarbamoyl)hydroxylamine. This compound was prepared by modification of a published method for the synthesis of the corresponding unlabeled material (3). Thus, acetohydroxamic acid (610 mg, 8.13 mmol) was dissolved in CH2C12(10 mL) containing triethylamine (5 drops) and the resultingsolution was treated with [2H3]MIC(463mg, 8.13 mmol) in CH2C12 (2 mL). After a period of 2 h a t ambient temperature, excess reagents and solvent were removed under reduced pressure, and the product was crystallized from CH2C12/acetone (mp 104108 "C; lit. [3] 115-116 "C). 'H NMR (2H20): 2.03 6 (8, 3 H, CH3-C=O). 'H NMR (C2HC13): 2.03 (9, 3 H, CH3-C=O), 5.5-5.8 (broad s, 1 H, C2H3-NH-C=O), and 8.8-9.5 S (broad s, 1 H, CH3-CO-NH-). No resonance was observed for an N-methyl group (a doublet centered at 2.85 ppm in the spectrum of the unlabeled compound). NMR (2H20): 21.15 (s, CH3-C=O), 29.20 (weak q due to C-2H coupling, C2H3159.38 (C2H3-NH-C=O) and 173.83 6 (CH3NH-C=O), C=O). IR: 3344 (s, v[carbamoyl N-HI), 3191 (m, v[acetamido N-HI), 1741 (s, v[carbamoyl C=Ol) and 1671 cm-l (s, u[acetyl C=O]). This material was used for the next step without purification. N-Acetyl-N-(met hylcarbamoy1)-0-([ 2H3]methylcarbamoyl) hydroxylamine ( [2H3]Caracemide). The above intermediate (250 mg, 1.65mmol) was dissolved in CH2C12(20 mL) and treated with MIC (210 wL, 3.31 mmol) in CH2C12 (1.0 mL) followed by triethylamine (1drop). The resulting mixture was stirred at ambient temperature for 1hand evaporated to dryness under reduced pressure. This afforded the crude product as a waxy white solid which was triturated with petroleum ether (3060 "C) and filtered. After crystallization from diethyl ether/ petroleum ether, several crops of white needles were obtained (yield = 180 mg; 0.87 mmol; mp = 120-122 "C, lit. 131 120122OC). Further product was obtained by treatment of the supernatant with additional MIC. 'H NMR (C2HC&):2.22 (a, 3 H, CHa-C=O), 2.88 and 2.89 (d, J = 4.83 Hz, 3 H, CH3-NHand 8.1-8.3 6 C=O), 5.1-5.3 (broad s, 1 H, CZH3-NH-C=O) (broads, 1 H, CH3-CO-NH-). Irradiation of the NHresonance centered at 8.2 ppm collapsed the nondeuteratedupfield doublet (at 2.85 ppm). The downfield 3 H doublet at 2.9 ppm in the spectrum of unlabeled caracemide (attributed to the CH3NH-CO-0functionality) was barely detectable in the spectrum of the deuterated compound. NMR (C2HC13): 22.60 (s, CH3-C=O), 27.00 (s, CH3-NH-CO-N), 151.66 (weak s, CH3-NH-C=O), 154.14 (weak s, C2H3-NH-C=O), and 173.07 6 (weak s, CH3-C=O). The signal for the C2H3carbon (which appeared at 28.24 ppm in the spectrum of unlabeled caracemide) was not observed due to poor signal-to-noise ratio from C-2H coupling. Biological Experiments. Specific pathogen-free adult male Sprague-Dawley rats were obtained from Charles River Labo-
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 337
Mercapturate Metabolite of Caracemide ratories (Wilmington, MA). Animals were housed in groups of four on a 12-h light/dark cycle with free access to food (Rodent Blox, Continental Grain Co., Chicago, IL) and water. Animals were removed from the animal quarters, housed in individual Nalgene metabolic cages with access to water, and fasted 4 h prior to drug administration. Groups of four rats (290 10g) were injected ip with caracemide or [zH3]caracemide (10 pmol, 6.6 mg kgl), freshly prepared in normal saline solution (0.5 mL). Urine was collected for 24 h over ascorbic acid solution (50 mg mL-', 10 mL) and the total weight was recorded. In the case of the animals which received [2H3]caracemide,the urine collections were divided into 0-2,2-4, 4-6,6-8,8-12, and 12-24 h intervals. The 0-24 h urine from a drug-free control rat was employed for preparation of the standard curve for AMCC. For the quantitative analysis of AMCC in urine, triplicate aliquots of urine (250pL each) were treated with internal standard solution ([2H3]AMCC,25 pL of a 400 pg mL-l stock solution in 0.02 % HCl) and sufficient 2 % HCl (typically 125 pL) to ensure that the pH of the mixture was C2. Samples were then vortex mixed and applied to SPE cartridges which had been prewashed with methanol and water. Following application of the urine, the cartridges were washed with water, the washes were discarded, and the cartridges were aspirated under vacuum. Metabolites were eluted with methanol (1 mL), the methanol eluents evaporated to dryness, and the residues dissolved in anhydrous methanolic HCl(5OOpL). After being allowed to stand at ambient temperature for 1.5 h, the samples were evaporated to dryness and then reconstituted in the HPLC mobile phase (200 pL). Aliquots (50 pL) of these derivatized specimens were taken for analysis by LC-MS. A similar protocol was followed in the case of urine obtained from animals dosed with [2H&aracemide, except that no internal standard was added to these samples. For the preparation of the standard curve for AMCC, triplicate aliquots of drug-free urine (250 pL) were treated with a fixed volume (25 pL) of the above internal standard solution, together with increasing volumes (0, 6.25, 12.5, 18.75, 25, and 50 pL) of a standard solution of unlabeled AMCC in 0.02% HC1 (800 pg mL-'). The resulting mixtures were then acidified and processed as described above. The conditions for SIM LC-MS analysis of AMCC in urine were optimized for the type of application in question. Identification of the conjugate was carried out using a mobile phase consisting of 11% acetonitrile in 50 mM ammonium acetate (pH 4.0), pumped a t a flow rate of 0.75 mL min-l. Under these conditions, the retention times of AMCC methyl ester were 6.4 and 8.3 min on the 15- and 25-cm columns, respectively. The thermospray interface temperatures which resulted in maximum intensity of the MH+ ion were as follows: TI = 120 "C, Tz= 216 "C, and T3 = 285 "C (filamentoff). Themetabolite wasidentified as its methyl ester on the basis of (i) cochromatography of the derivatized metabolite with the corresponding synthetic reference material and (ii) appearance a t the appropriate retention time, and in the correct relative abundance ratio, of coincident responses in the ion current chromatograms for m/z 235 (MH+) and 178(MH - CH,NCO]+). Quantitative analyses of the urinary AMCC were performed using the 15-cm HPLC column, eluted with 11% acetonitrile in 100 mM ammonium acetate (pH 7.0) a t a flow rate of 1.2 mL min-I. Under these conditions, the retention time of AMCC methyl ester was 4.1 min. Optimal thermospray interface temperatures in this case (designed to maximize the yield of the fragment ions at mlz 178 and 181for analyte and internal standard, respectively) were as follows: T1 = 122 "C, T2 = 248 "C, T3 = 255 "C, block = 304 "C, tip = 307 "C, and lens = 118 "C (filament off). Quantification of AMCC (as its methyl ester) was based on the ratios of peak areas in the ion current chromatograms for mA 178 (analyte) and 181(internal standard) and by reference to the standard curve which was linear (correlation coefficient r2 > 0.98) over the concentration range studied (0-363 nmol mL-1). For the experiments in which P'H3lcaracemide was administered to rats, the thermospray conditions were optimized for maximal abundance of the MH+
i_i
P
A
s
1
[M+H]*
*
Figure 2. Thermospray mass spectra of the methyl esters of (A) unlabeled AMCC and (B) [2H3]AMCC employed as internal standard in the LC-MS assay procedure. The origins of structurally characteristic ions in the spectra, which were recorded with a mobile phase buffered to pH 4.0, are discussed in the text. ions of methylated AMCC and A[2H3]MCC (at mlz 235 and 238, respectively). These were as follows: T1 = 120 "C, TZ= 230 "C, T3 = 288 "C, block = 300 "C, tip = 315 OC, and lens = 116 "C. In this case, chromatography was carried out using the 25-cm column, eluted with 11% acetonitrile in 50 mM ammonium acetate (pH 4.0) at 0.75 mL min-l. It should be noted that the deuterium atoms in the A[2H3]MCC formed as a metabolite of L2H31caracemide were located primarily in the carbamoyl methyl group, in contrast to those in the internal standard for quantitative work ( [2H31AMCC)which resided exclusively in the acetyl moiety. This difference in labeling pattern dictated the use of the different mass spectrometric conditions for the metabolic experiments with [2H3]caracemide.
Results and Discussion Identification of AMCC as a Urinary Metabolite of Caracemide. Identification of AMCC in the urine of rats dosed with caracemide was based on the thermospray mass spectrum of the methyl ester of this mercapturic acid derivative. The spectrum of the reference material is depicted in Figure 2A, while the corresponding spectrum of the L2H31AMCCemployed as internal standard in the LC-MS assay is shown in Figure 2B. In the case of the unlabeled methyl AMCC (Figure 2A), a prominent [M + HI+ speciesis evident (mlz 235),together with anabundant fragment ion at mlz 178 resulting from elimination from the [M + HI+ species of the elements of MIC (57 Da) to yield protonated methyl N-acetylcysteine. Each of these ions is accompanied by two satellites which arise formally by addition of the elements of NH3 (17 Da) and CH3NH2 (31 Da) to the primary (protonated) ion. Thus, the group of adduct ions appears at mlz 252 (MH+ + NHs), 266 (MH+ + CHsNHz), 195 (methyl-NAC + H+ + NHd and 209 (methyl-NAC + H+ + CH~NHZ), and a corresponding set of signals, shifted 3 units to higher mass, appears in the spectrum of the deuterated derivative (Figure 2B). Although adducts with NH3 are commonplace in thermospray spectra recorded with mobile phases containing ammonium salts, ions formed by the attachment of a molecule of CH3NH2 are rare. In the case of methyl-
338 Chem. Res. Toxicol., Vol. 6, No. 3, 1993
AMCC, it is proposed that the hot, moist, and acidic environment of the thermospray source leads to spontaneous gas-phase decomposition to yield MIC and methylNAC. The former product undergoes hydrolysis to CH3NH2, and this appears to be the source of the adducts to protonated methyl-AMCC and methyl-NAC. While this mechanism remains hypothetical, it is noteworthy that the thermospray spectrum of methyl-AMCC was found to be highly pH-dependent. Thus, the spectra depicted in Figure 2 were recorded with a mobile phase buffered to pH 4;when the pH was raised to 7,the MH+ ion disappeared completely from the spectrum, and a significant gain was observed in the intensity of the [MH+ - MIC] fragment and its associated adduct ions (data not shown). This effect of pH on the thermospray spectrum was important in the context of the present work, since detection of the intact MH+ ion was necessary for some studies, but not others (vide infra). When extracts of urine from caracemide-treated rats were analyzed by thermospray LC-MS, the resulting fullscan mass spectra of methylated AMCC were of poor quality due to the presence of coeluting endogeneous substances. For this reason, the urine extracts were analyzed by SIM LC-MS, when the ion currents at mlz 235 and 178 were monitored as a function of time. For all caracemide-treated animals, coincident peaks were obtained in the resulting ion current chromatograms at the retention time of authentic methyl-AMCC, the intensities of which exhibited the same ratio (mlz 235:178) as the reference material. These peaks were absent in the ion current chromatograms recorded from drug-free urine extracts. On the basis of these results, it was concluded that AMCC is a urinary metabolite of caracemide in the rat. Quantitative Analysis of AMCC as a Urinary Metabolite of Caracemide. In order to quantify AMCC excretion in urine, an LC-MS assay procedure was developed based on SIM of the [MH+ - CH3NCOl ion of methyl AMCC (mlz 178)and the corresponding ion (mlz 181) from methyl [2H3]AMCC,which was employed as internal standard. Thermospray conditions were adjusted to optimize the responses at these mlz values, and the LC separation employed a mobile phase buffered to pH 7 to enhance the fragmentation pathway of interest. By this approach, a linear standard curve was obtained over the concentration range of interest, and the assay was applied to quantify AMCC excreted in the 0-24 h pooled urine of four rats dosed with caracemide (6.6mg kg' ip). Individual values for the fraction of the administered dose excreted as AMCC were as follows (mean f SD for three replicate analyses in each case): rat 1, 54.9 f 5.9%; rat 2,46.3 f 1.6%; rat 3, 56.0 f 1.6%; rat 4, 59.2 f 5.8%. Overall, excretion of AMCC accounted for 54.0f 5.5% of the dose in the four animals. It is of interest to compare these figures with the published value of 24.7% for the fraction of an ip dose of MIC itself excreted into urine of rats over 24 h (9). Since caracemide can, in principle, liberate 2 mol of MICiper mol of drug, it would appear from the results of the present investigation that caracemide may be regarded as a bioequivalent prodrug of MIC. However, such comparisons should be treated with caution since, as discussed below, it was found that caracemide does not liberate MIC equally from the N-methylcarbamoyl and the 0-methylcarbamoyl side chains.
Slatter et al.
Mechanistic Studies on the Origin of AMCC as a Urinary Metaboliteof Caracemide. As discussedabove, caracemide contains two sites from which MIC could be released in vivo, uiz. the N-methylcarbamoyl and the 0-methylcarbamoyl functionality. In order to explore the origin of the urinary AMCC, and to quantify the relative contributions of these alternative sources to this mercapturate, a stable-isotope-labeled analog of caracemide ([2H&.aracemide) was required containing 3 atoms of deuterium in the 0-methylcarbamoyl moiety (Scheme I). The rationale for the selection of this labeled drug as substrate in metabolic experiments was that decomposition of the 0-methylcarbamoyl side chain in vivo would lead to excretion of A[2H31MCC (labeled in the methylcarbamoyl group, as opposed to the acetyl moiety which was the site of labeling in the internal standard used for the above quantitative studies), whereas degradation of the N-methylcarbamoyl side chain would result in the excretion of unlabeled AMCC. Therefore, by administering [2H31caracemideto rats and analyzing by LC-MS the relative populations of unlabeled and r2H31AMCCexcreted into urine, it would be possible to determine the source of the liberated MIC directly. 12H31Caracemide was obtained from acetohydroxamic acid according to the two-step procedure outlined in Scheme I. Although the synthesis proceeded smoothly, a combination of spectroscopic techniques proved necessary to define unambiguously the site of deuteration in the monocarbamoylated intermediate as the -OH group. Difficultiesassociated with establishing the regiochemistry of acylation of hydroxamic acids are well-known (19), and in the case of acetohydroxamic acid, reaction with MIC theoretically could take place at the -NH function, the -OH group, or the -OH moiety of the corresponding enamide tautomer. In the event, these possibilities were distinguished largely on the basis of IR spectroscopy, inasmuch as the spectrum of the intermediate revealed two distinct C=O stretches (ruling out carbamoylation of the enamide tautomer) but did not exhibit the broad 0-H stretch which dominated the spectrum of acetohydroxamic acid itself (eliminating reaction at the -NH position). Thus, it was concluded that the synthesis of I2H31caracemide proceeded as shown in Scheme I and that the deuterium atoms resided exclusively in the 0-methylcarbamoyl side chain. When rats were dosed with [2H3]caracemide and the urinary AMCC analyzed (as its methyl ester) by LC-MS, both unlabeled and trideuterated mercapturate were detected at a retention time of 8.3 min (Figure 3). SIM analysis indicated that the ratio of ion currents for the respective MH+ species (mlz235:238)remained essentially constant at 0.28 in all six pooled urine collections taken at intervals from 0 to 24 h postdose. These results demonstrate that while the (labeled) 0-methylcarbamoyl side chain was the major source (72%) of MIC derived from 12H31caracemide,release also occurred from the N-methylcarbamoyl portion of the molecule. Moreover, since the ratio of unlabeled to A[2H31MCCdid not change over time, it may be inferred that MIC is released from both sites in caracemide at rates which favor degradation of the 0-methylcarbamoyl group by a factor of 3.6:l. Whether release of MIC from both side chains occurs simultaneously, or whether collapse of the N-methylcarbamoyl group takes place only after loss of MIC from the 0-methylcarbamoyl moiety, remains unknown. However,
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 339
Mercapturate Metabolite of Caracemide
1
nVz 235
D1-I
Caracemide
$ 4 0
n
2o
4
/
5
6
7
8
J 9
10
100
nVz 238 SMG
8.30
4
5
6
7
8
9
0
10
Figure 3. Ion current chromatograms for mlz 235 (A) and 238
it is likely that the rate constants for decomposition are governed by the relative pKa values of the carbamate and urea N-H protons, since the expulsion of isocyanates from such moieties is believed to take place via an ElcB mechanism (unimolecular elimination from the conjugate base) where stabilization of the developing negative charge in the group to be eliminated is important (12).Based on these considerations, the scheme depicted in Figure 4 is proposed to account for the GSH-dependent pathway of caracemide metabolism in the rat.
Conclusions The results of the present investigation, which focused on the use of AMCC as a marker metabolite of MIC in the rat, extend previous in vitro studies on the chemical properties of caracemide in aqueous systems (5) and provide the first indirect evidence that this investigational antitumor agent may serve as a latent form of MIC in vivo. Thus, based on the recent findings that rats dosed parenterally with MIC excrete relatively large amounts of AMCC in urine (9)and the corresponding GSH conjugate in bile (IO),it seems reasonable that the urinary AMCC detected after injection of rats with caracemide originates from MIC. This conclusion has potentially important implications with respect to the mechanism of action of caracemide, since S-linked conjugates of MIC have been shown to be active carbamoylating agents in vitro (11) and to potently inhibit the growth of TLX5 lymphoma cells in culture, apparently by liberating free MIC at cell surfaces (20).Therefore, it is possible that the antitumor effects of caracemide are mediated, at least in part, by MIC, delivered to tumor sites in the form of labile S-linked conjugates. Similarly, many of the adverse side-effects of
COpH
CH31N)fSA
(B)from the thermospray LC-MS analysis of a methylated urine
extract from a rat dosed with [2H&aracemide (6.6 mg k g l ip). The arrowed peak in the mlz 235 channel represents unlabeled molecules of methyl AMCC derived from breakdown of the N-methylcarbamoyl side chain of the parent drug, whereas the corresponding peak in the mlz 238 channel represents methyl A[2H3]MCC derived from breakdown of the O-deuteromethylcarbamoyl moiety of [*H3]caracemide. The urine specimen in question was collected from 0 to2 h following drug administration.
HN A C H ,
H
Time (min.)
0 AMCC
Figure
4. Proposed glutathione-dependent pathway of cara-
cemide metabolism in the rat.
caracemide therapy may be related to MIC release and the formation in vivo of reversible conjugates of this toxin.
Acknowledgment. The authors thank Mr. William Howald and Ms. Sharon Steele (University of Washington Mass Spectrometry Facility) for their help with the LCMS analyses and Ms. Donna Abbs (Upjohn Co.) for secretarial assistance. This work was supported by a Research Grant from the National Institutes of Environmental Health Sciences (ES 05500), which is gratefully acknowledged.
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