Chemical and Enzymatic Oxidation of Furosemide - ACS Publications

Oct 4, 2007 - Furosemide (Lasix) is frequently used in the treatment of cardiovascular and renal disease. Only one metabolite, furosemide glucuronide,...
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Chem. Res. Toxicol. 2007, 20, 1741–1744

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Chemical and Enzymatic Oxidation of Furosemide: Formation of Pyridinium Salts Ling-Jen Chen* and Leo T. Burka Laboratory of Pharmacology and Chemistry, National Institute of EnVironmental Health Sciences, Research Triangle Park, North Carolina 27709 ReceiVed July 23, 2007

Furosemide (Lasix) is frequently used in the treatment of cardiovascular and renal disease. Only one metabolite, furosemide glucuronide, has ever been identified. Oxidation of furosemide by cytochrome P450 has been demonstrated, but the metabolite(s) has never been identified. The oxidation of furosemide by dimethyldioxirane in acetone and by liver microsomal incubations was explored in this study. The first observable product from dimethyldioxirane oxidation was a ring-expanded enone resulting from an intramolecular condensation of the aldehyde group of the enonal, the secondary amine, and the carboxylic acid in a Mannich-like reaction. Keto–enol tautomerization and opening of the lactone gave a stable pyridinium salt. The pyridinium salt was also observed in the microsomal incubations of furosemide. The presence of an internal nucleophile in furosemide may have a significant effect on the toxicology and possibly the pharmacology of this furan. Introduction Furosemide (Lasix) (1) (Scheme 1) is a loop diuretic drug frequently used in the treatment of congestive heart failure, edema associated with heart failure, hepatic cirrhosis, renal impairment, nephrotic syndrome, and hypertension. Despite its extensive use for decades, only a glucuronide metabolite, conjugated at the carboxylic acid, has ever been identified. This metabolite accounted for 14% of the absorbed oral or iv dose to humans (1) but gives little insight into the metabolism of the drug. Oxidation of furosemide by cytochrome P450 has been demonstrated, but the structure(s) of the metabolite(s) has not been adequately established (2, 3). Several furan molecules, including furan, menthofuran, ipomeanine, 4-ipomeanol, and teucrin A, are oxidized by cytochrome P450 and/or dimethyldioxirane to 2-butene-1,4-dicarbonyl metabolites (4–9). These butenedicarbonyl metabolites are reactive species responsible for covalent binding to proteins and DNA (5–11). With the association of furans and reactive intermediates, the long, widespread use of furosemide with generally rare toxic effects is somewhat surprising and not explained by current knowledge. This study aims to identify the oxidized metabolite(s) of furosemide by dimethyldioxirane oxidation and liver microsomal incubations.

Experimental Procedures Materials. Furosemide, trifluoroacetic acid, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, and NADP+ were obtained from Sigma-Aldrich Co. (St. Louis, MO). Male F344 rat liver microsomes (20 mg protein/mL) were purchased from In Vitro Technologies (Baltimore, MD). Instrumentation. 1H NMR spectra were acquired on a Varian Gemini 300 MHz NMR spectrometer (Palo Alto, CA). The chemical shifts are reported in ppm relative to solvents (D2O at 4.80 ppm, DMSO-d6 at 2.49 ppm, and acetone-d6 at 2.05 ppm). * To whom correspondence should be addressed. Present address: Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, New Mexico 87108. Tel: 505-348-9735. Fax: 505-348-4980. E-mail: [email protected].

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Electrospray ionization mass spectra (ESI-MS)1 of the products from chemical oxidation were obtained on a Thermo Finnigan LCQ Advantage Max ion trap mass spectrometer (Riviera Beach, FL). Tandem mass spectra (ESI-MS/MS) were produced by collisioninduced dissociation of the selected parent ions with the helium gas present in the mass analyzer. The heated capillary was maintained at 250 °C, and the source voltage was maintained at 5 kV. Samples were dissolved in CH3OH–H2O (1:1) and introduced to the mass spectrometer through direct infusion (12.5 µL/min) for positive ionization analysis [ESI(+)-MS or ESI(+)-MS/MS]. LC/ MS of the dimethyldioxirane reaction mixture was carried out on an Agilent 1100 HPLC equipped with an Agilent G1315B DAD photodiode array (PDA) detector and connected with the LCQ ion trap mass spectrometer. A gradient from 100% A (0.1% formic acid in H2O) to 50% A and 50% B (0.1% formic acid in CH3CN) over 28 min at a flow rate of 200 µL/min on a Varian (Walnut Creek, CA) Polaris C18 5 µm column (2.0 mm × 150 mm) was used. The MS was run in the positive ionization mode [ESI(+)MS] and set to scan over a m/z range of 50–700. The heated capillary of the MS was maintained at 350 °C, and the source voltage was kept at 5 kV. This HPLC system was used for identification of the products in dimethyldioxirane oxidation of furosemide by PDA and MS analysis and will be referred to as LC/MS in the Results and Discussion. HPLC analyses of microsomal incubations were carried out on a Beckman System Gold with a module 126 solvent pump and a module 168 PDA detector. A gradient from 100% A (0.1% trifluoroacetic acid in H2O) to 50% A and 50% B (CH3CN) over 28 min at a flow rate of 1.5 mL/min on a Varian Inertsil C18 5 µm column (4.6 mm × 250 mm) was used. This HPLC system is for detection and isolation of microsomal metabolites, and will be referred to as HPLC in the Results and Discussion. ESI-MS of the microsomal metabolites was obtained on an Applied Biosystems MDS SCIEX API 4000 triple quadruple mass spectrometer (Foster City, CA). Samples were dissolved in CH3OH-H2O (1:1) and introduced to the mass spectrometer through direct infusion (10 µL/min) for positive ionization analysis [ESI(+)-MS or ESI(+)MS/MS]. 1 Abbreviations: ESI-MS, electrospray ionization mass spectrometry; ESIMS/MS, electrospray ionization tandem mass spectrometry; PDA, photodiode array.

This article not subject to U.S. Copyright. Published 2007 by American Chemical Society. Published on Web 10/04/2007

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Scheme 1. Dimethyldioxirane Oxidation of Furosemide (1) in Acetone, Which Involves a Mannich-like Reaction for the Formation of 3

Oxidation of Furosemide by Dimethyldioxirane. Dimethyldioxirane was prepared as described (12). Reaction of furosemide (19.6 mg, 0.059 mmol) with dimethyldioxirane-d6 in acetone-d6 (0.08 M, 1.185 mL, 0.095 mmol) in an NMR tube was monitored by 1H NMR spectroscopy at 28 °C. A similar reaction was also followed by injection of the reaction mixture (1 µL) into the LC/ MS at various time points. Products 3–6, as shown in Scheme 1, were observed by NMR and/or LC-MS. 9-Chloro-2,6-dioxo-8-sulfamoyl-1,2,4a,6-tetrahydro-1H-benzo[d]pyrido[2,1-b][1,3]oxazine (3). 1H NMR (acetone-d6): δ 8.53 (s, 1H, 7-H), 7.39 (s, 1H, 10-H), 7.17 (dd, J ) 10.4, 1.9 Hz, 1H, 4-H), 6.98 (br. s, 2H, SO2NH2), 6.46 (dd, J ) 10.7, 1.9 Hz, 1H, 3-H), 6.23 (quintet, J ) 1.4 Hz, 1H, 4a-H), 4.44 (d, J ) 17.4 Hz, 1H, 1-Ha), 4.17 (dd, J ) 17.4, 1.5 Hz, 1H, 1-Hb). 9-Chloro-2,6-dioxo-8-sulfamoyl-1,2,4a,6-tetrahydro-1H-benzo[d]pyrido[2,1-b][1,3]oxazine 1a-Oxide (4a). 1H NMR (acetoned6): δ 8.60 (s, 1H, 7-H), 7.84 (s, 1H, 10-H), 7.23 (dd, J ) 10.2, 3.3 Hz, 1H, 4-H), 7.08 (br. s, 2H, SO2NH2), 6.48 (dd, J ) 10.5, 0.9 Hz, 1H, 3-H), 6.15 (dd, J ) 3.6, 1.5 Hz, 1H, 4a-H), 5.64 (s, 2H, 1-CH2). LC/MS retention time, 21.8 min. UV λmax 238, 264, 332 nm. ESI(+)-MS: m/z 345 and 347 in a 3:1 ratio [M + H]+, 327 and 329 in a 3:1 ratio [M + H – H2O]+. 9-Chloro-2,6-dioxo-8-sulfamoyl-1,2,4a,6-tetrahydro-1H-benzo[d]pyrido[2,1-b][1,3]oxazine 1a-Oxide (4b). 1H NMR (acetoned6): δ 8.54 (s, 1H, 7-H), 7.61 (s, 1H, 10-H), 7.18 (dd, 10.5, 2.1 Hz, 1H, 4-H), 7.02 (br. s, 2H, SO2NH2), 6.45 (dd, J ) 10.7, 1.8 Hz, 1H, 3-H), 6.28 (t, J ) 2.2 Hz, 1H, 4a-H), 5.68 (s, 2H, 1-CH2). LC/MS retention time, 18.7 min. UV λmax 236, 264, 326 nm. ESI(+)-MS: m/z 345 and 347 in a 3:1 ratio [M + H]+, 327 and 329 in a 3:1 ratio [M + H – H2O]+. 4-Chloro-2-(3′-hydroxypyridinium-1′-yl)-5-sulfamoylbenzoate (5). 1H NMR (DMSO-d6) δ 8.73 (br. s, 1H, 2′-H), 8.59 (br. s, 1H, 6′-H), 8.51 (s, 1H, 6-H), 8.06 (s, 1H, 3-H), 7.96–7.92 (m, 4H, 4′-H, 5′-H, and SO2NH2). 4-Chloro-2-(3′-hydroxypyridinium-1′-yl)-5-sulfamoylbenzoic Acid (6). 1H NMR (DMSO-d6): δ 8.84 (s, 1H, 2′-H), 8.71 (d, J ) 5.8 Hz, 1H, 6′-H), 8.63 (s, 1H, 6-H), 8.24 (s, 1H, 3-H), 8.11 (d, J ) 8.8 Hz, 1H, 4′-H), 8.06 (br. s, 2H, SO2NH2), 8.03 (dd, J ) 8.8, 5.8 Hz, 1H, 5′-H). LC/MS retention time at 11.1 min. UV λmax: 206, 228 (shoulder) and 292 nm. ESI(+)-MS: m/z 329 and 331 in a 3:1 ratio [M]+. This product was isolated and analyzed by HPLC (Figure 1A; retention time, 9.8 min; and UV λmax, 210 and 290 nm) and by ESI(+)-MS/MS of m/z 329 [M]+ to give the following fragmentation: 311 [M – H2O]+, 283 [M – HCOOH]+, 266 [M – SO2NH2 + OH]+, 249 [M – SO2NH2]+, 204 [M – SO2NH2 – COOH]+. Incubations of Furosemide with Rat Liver Microsomes and NADPH. Incubations of furosemide (1 mM) with male F344 rat liver microsomes (2 mg protein/mL) were conducted in a 0.1 M potassium phosphate buffer (pH 7.4) in the presence of 3 mM

MgCl2, 25 mM glucose 6-phosphate, glucose-6-phosphate dehydrogenase (2 units/mL), and 4 mM NADP+. Furosemide was added as a DMSO solution (100 mM, 10 µL). Control experiments omitting NADP+ or microsomes were included so as to recognize the products. The final volume was 1 mL, and the incubation took place at 37 °C in capped vials for 30 min. The reactions were terminated by addition of 0.3 N Ba(OH)2 (0.1 mL) and 0.3 N ZnSO4 (0.1 mL). Following centrifugation, the supernatant was filtered through a Millex-HV syringe driven filter (Millipore Corp., Bedford, MA; 0.45 µm, 13 mm), and the filtrate was analyzed by HPLC (Figure 1B–D).

Results and Discussion The oxidation of furosemide (1) by dimethyldioxirane-d6 in acetone-d6 was monitored by 1H NMR spectroscopy or LC/ MS. The reaction mixture turned bright yellow immediately, and a white solid began to precipitate 5–10 min after mixing. Fifteen minutes after the reaction started, the 1H NMR spectrum showed formation of a major product, 9-chloro-2,6-dioxo-8sulfamoyl-1,2,4a,6-tetrahydro-1H-benzo[d]pyrido[2,1b][1,3]oxazine (3) (Scheme 1). The three signals at δ 7.17 (dd), 6.46 (dd), and 6.23 (quintet, also W-coupling to the signal at δ 4.44 and δ 4.17) are consistent with the R-H (3-H) and β-H (4-H) of the enone coupled to a γ-H (4a-H) on a sp3 carbon substituted with one N and one O. The 1-CH2 appeared as an AB quartet at δ 4.44 and 4.17. The signal at δ 4.17 shows 1.5 Hz W-coupling with 4a-H. The structural assignment of 3 is also based on its further oxidation by dimethyldioxirane to diastereomeric N-oxides 4a,b (Scheme 1). Compound 3 could not be observed by LC/MS because it was converted to the pyridinium salt 6 by the formic acid in the mobile phase. The initial oxidation product of 3 was 9-chloro-2,6-dioxo8-sulfamoyl-1,2,4a,6-tetrahydro-1H-benzo[d]pyrido[2,1b][1,3]oxazine 1a-oxide (4a) and could be observed 15 min following the start of the reaction. Compound 4a was the major product in the solution at 2 h. The three protons at δ 7.23 (dd), 6.48 (dd), and 6.15 (dd) are consistent with the R-H (3-H) and β-H (4-H) of the enone coupled with a γ-H (4a-H) on an sp3 carbon substituted with one N and one O. The chemical shifts of 1-CH2 (δ 5.64) and 10-H (δ 7.84) in 4a are downfield as compared to 1-CH2 (δ 4.44 and 4.17) and 10-H (δ 7.39) in 3, consistent with the presence of an N-oxide. After 17 h, a portion of 4a had rearranged to its diastereomer 4b, and the ratio of 4a and 4b was approximately 2:1. The NMR data for 4b are similar to that for 4a. Three protons at δ 7.18 (dd), 6.45 (dd), and 6.28

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Figure 1. Representative UV (278 nm) chromatograms of 6 prepared from dimethyldioxirane oxidation of furosemide (A) and a rat liver microsomal incubation mixture (100 µL) of furosemide and NADPH (B). The incubation mixtures (100 µL each) omitting NADPH (C) or microsomes (D) are included for comparison. A gradient from 100% A (0.1% trifluoroacetic acid in H2O) to 50% A and 50% B (CH3CN) over 28 min at a flow rate of 1.5 mL/min on a Varian Inertsil C18 5 µm column (4.6 mm × 250 mm) was used.

Scheme 2. Mannich-like Reaction for the Formation of a Tricyclic GSH Adduct in the Microsomal Incubations of Ipomeanine

(dd) are consistent with the R-H (3-H) and β-H (4-H) of the enone coupled with a γ-H (4a-H) on an sp3 carbon substituted with one N and one O. The chemical shift of 10-H in 4b was observed at δ 7.61. MS analysis of both 4a,b gives m/z 345 and 347 in a 3:1 ratio, in agreement with the proposed structure. Similar UV absorption maxima were observed between 4a and 4b. The third product (5) of the dimethyldioxirane oxidation of furosemide was a white solid obtained in approximately 50% yield. The product was not soluble in most solvents except that it had some solubility in DMSO or H2O-CH3CN (1:1). The 1H NMR spectrum shows 2′-H at δ 8.73, 6′-H at δ 8.59, and 4′-H, 5′-H at δ 7.96–7.92, more downfield than the corresponding protons in 3 and 4a,b. The chemical shifts suggest that these protons are aromatic and consistent with those present in a pyridine. The broadening of these four protons in the pyridine ring suggests that 5 might exist in more than one conformation. The white solid is identified as the pyridinium salt, 4-chloro2-(3′-hydroxypyridinium-1′-yl)-5-sulfamoylbenzoate (5) (Scheme 1), a zwitterion from rearrangement of 3. The structural

assignment of 5 is also based on conversion of both 3 and 5 to the pyridinium salt, 4-chloro-2-(3′-hydroxypyridinium-1′-yl)5-sulfamoylbenzoic acid (6), when dissolved in H2O–CH3CN containing trace amounts of trifluoroacetic acid or formic acid. The four protons at δ 8.84 (s), 8.71 (d), 8.11 (d), and 8.03 (dd) in 6 are consistent with the formation of a pyridinium salt, and the coupling is consistent with a substitution at the 3-position. MS analysis of 6 gives m/z 329 and 331 in a 3:1 ratio, in agreement with the proposed structure. Collision-induced fragmentation of the parent ion of 6 resulted in loss of COOH and SO2NH2, consistent with the proposed structure. HPLC analysis of 6 gave a retention time at 9.8 min and UV absorption maxima at 210 and 290 nm (Figure 1A). In contrast to dimethyldioxirane oxidation of several other furans (4–9), chemical oxidation of furosemide by dimethyldioxirane in acetone did not give NMR signals typical of the enonal intermediate 2. The first product observed was a bicyclic lactonyl enonamine 3. The intramolecular Mannich-like reaction of the aldehyde, the secondary amine and the carboxylic acid in 2, is sufficiently rapid that NMR-detectable concentrations

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of the enoneal 2 are not present in the chemical oxidation reaction even under conditions where at least 50% of the furosemide present is oxidized to 6. The bicyclic lactonyl enonamine 3 further rearranges to more stable pyridinium salts 5 and 6 and is oxidized to the N-oxides 4a and 4b (Scheme 1). A similar intramolecular Mannich-like reaction has been observed in the metabolism of ipomeanine (8). The GSH Michael adduct of the enedial metabolite of ipomeanine yields an imine upon cyclization, which could tautomerize to the corresponding enamine. The enamine contains an aldehyde, a secondary amine, and a carboxylic acid, which dehydrate to give the tricyclic GSH conjugate (Scheme 2). With the products of chemical oxidation characterized, the next step was characterization of the microsomal oxidation products of furosemide. HPLC analysis of the incubation mixture of furosemide, microsomes, and NADPH revealed formation of a product, which had a retention time at 9.8 min and UV absorption maxima at 210 and 290 nm (Figure 1B). This product was not observed when NADPH or microsomes were omitted in the incubations (Figure 1C,D). ESI(+)-MS analysis of the microsomal metabolite gave m/z 329 and 331 [M]+. ESI(+)-MS/MS of m/z 329 [M]+ gave the following fragmentation: 283 [M – HCOOH]+, 267 [M – SO2NH2 + H2O]+, 266 [M – SO2NH2 + OH]+, 250 [M – SO2NH2+ H]+, 249 [M – SO2NH2]+ and ESI(+)-MS/MS of m/z 331 [M]+ gave the following fragmentation: 285 [M – HCOOH]+, 269 [M – SO2NH2 + H2O]+, 268 [M – SO2NH2 + OH]+, 252 [M – SO2NH2+ H]+, 251 [M – SO2NH2]+. This microsomal metabolite at 9.8 min had a similar HPLC retention time and UV and MS spectra as those of 6 from chemical oxidation (Figure 1A) and was identified as 6. The metabolite is extremely polar and elutes from the HPLC right after NADPH, which might explain why it was overlooked, or perhaps not resolved, in the previous studies (2, 3). Two other peaks at 11.2 and 14.8 min were observed in all three microsomal incubations (Figure 1B–D). The peak at 11.2 min is furfural, based on comparison with an authentic standard (data not shown). The peak at 14.8 did not ionize upon MS analysis and remains unidentified. The furans listed in the Introduction are known for their toxicity. Ipomeanine and 4-ipomeanol have LD50 values of less than 50 mg/kg in mice (13). Teucrin A causes liver necrosis in mice at doses of 150 mg/kg (14). Furan is a multisite carcinogen in rats and mice at doses as low as 2 mg/kg/day (15). The LD50 for furosemide in rats is about 2.7 g/kg (16). This efficient removal of the reactive intermediate by reaction with an internal nucleophile is certainly at least part of the reason for the low toxicity of furosemide. It remains to be investigated if 6 possesses any pharmacological effects. Compound 6 has two aromatic rings directly linked, whereas other loop diuretics have an O- (bumetanide and piretanide) or NH linkage (torsemide) between two aromatic rings. Furosemide has a NHCH2 linkage. An SO2NH2 group is present in many loop diuretics. Bumetanide and torsemide are 40-fold and 2–4-fold, respectively, more potent than furosemide (17). In conclusion, this study shows that furosemide undergoes biotransformation via other pathways in addition to glucuronidation. The furan ring in furosemide is oxidized by cytochrome P450 to an enonal. The enonal is trapped by an internal nucleophile, which greatly reduces the toxicity expected from

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furans generally. These observations demonstrate the oxidative metabolism of furosemide for the first time. The polar nature of the metabolite offers an explanation as to why it has not been characterized earlier. Acknowledgment. We thank Zhengyu (Shannon) Gao and Joseph F. Lucak of Lovelace Respiratory Research Institute (Albuquerque, NM) for isolating and analyzing the microsomal metabolite 6 with the API 4000 mass spectrometer. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

References (1) Smith, D. E., Lin, E. T., and Benet, L. Z. (1980) Absorption and disposition of furosemide in healthy volunteers, measured with a metabolite-specific assay. Drug Metab. Dispos. 8, 337–342. (2) Mitchell, J. R., Nelson, W. L., Potter, W. Z., Sasame, H. A., and Jollow, D. J. (1976) Metabolic Activation of furosemide to a chemically reactive, hepatotoxic metabolite. J. Pharmacol. Exp. Ther. 199, 41– 52. (3) Wirth, J. W., Bettis, C. J., and Nelson, W. L. (1976) Microsomal metabolism of furosemide: Evidence for the nature of the reactive intermediate involved in covalent binding. Mol. Pharm. 12, 759–768. (4) Chen, L.-J., Hecht, S. S., and Peterson, L. A. (1995) Identification of cis-2-butene-1,4,-dial as a microsomal metabolite of furan. Chem. Res. Toxicol. 8, 903–906. (5) McClanahan, R. H., Thomassen, D., Slattery, J. T., and Nelson, S. D. (1989) Metabolic activation of (R)-(+)-pulegone to a reactive enonal that covalently binds to mouse liver proteins. Chem. Res. Toxicol. 2, 349–355. (6) Alvarez-Diez, T. M., and Zheng, J. (2004) Detection of glutathione conjugates derived from 4-ipomeanol metabolism in bile of rats by liquid chromatography-tandem mass spectrometry. Drug Metab. Dispos. 32, 1345–1350. (7) Baer, B. R., Rettie, A. E., and Henne, K. R. (2005) Bioactivation of 4-ipomeanol by CYP4B1: Adduct characterization and evidence for an enedial intermediate. Chem. Res. Toxicol. 18, 855–864. (8) Chen, L.-J., DeRose, E. F., and Burka, L. T. (2006) Metabolism of furans in vitro: Ipomeanine and 4-ipomeanol. Chem. Res. Toxicol. 19, 1320–1329. (9) Drukova, A., and Marnett, L. J. (2006) Characterization of the amino acid adducts of the enedial derivative of Teucrin A. Chem. Res. Toxicol. 19, 1330–1340. (10) Chen, L.-J., Hecht, S. S., and Peterson, L. A. (1997) Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan. Chem. Res. Toxicol. 10, 866–874. (11) Peterson, L. A. (2006) Electrophilic intermediates produced by bioactivation of furan. Drug Metab ReV. 38, 615–26. (12) Adam, W., Chan, Y.-Y., Cremer, D., Gauss, J., Scheutzov, D., and Scchindler, M. (1987) Spectral and chemical properties of dimethyldioxirane as determined by experiment and ab initio calculations. J. Org. Chem. 52, 2800–2803. (13) Boyd, M. R., Burka, L. T., Harris, T. M., and Wilson, B. J. (1974) Lung-toxic furanosesquiterpenes produced by sweet potatoes (Ipomea batatas) following microbial infection. Biochim. Biophys. Acta 337, 184–295. (14) Kouzi, S. A., McMurtry, R. J., and Nelson, S. D. (1994) Hepatotoxicity of germander (Teucrium chamaedrys) and one of its constitutant neoclerodane diterpenes teucrin A in the mouse. Chem. Res. Toxicol. 7, 850–856. (15) National Toxicology Program (NTP) (1993) Toxicology and Carcinogenicity Studies of Furan (CAS No. 110-00-9) in F344/N Rats and B6C3F1 Mice (Gavage Studies), Technical Report Series No. 402, NIH Publication No. 93-2857, National Institutes of Health, Public Health Service, U.S. Department of Health and Human Services, Research Triangle Park, NC. (16) Goldenthal, E. I. (1971) A compilation of LD50 values in newborn and adult animals. Toxicol. Appl. Pharmacol. 18, 185–207. (17) Barnes, C. L., and Hager, K. L. (2007) Loop diuretics in cardiac therapy. http://www.uspharmacist.com/oldformat.asp?url)newlook/ files/Feat/ACF2EDE.cfm&pub_id)8&article_id)24 - 39k, accessed 2/4/2007.

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