Evidence for a New and Major Metabolic Pathway of Clenbuterol

Chem. Res. Toxicol. 1997, 10, 197-204

197

Evidence for a New and Major Metabolic Pathway of Clenbuterol Involving in Vivo Formation of an N-Hydroxyarylamine Daniel Zalko, Laurent Debrauwer, Georges Bories, and Jacques Tulliez* Laboratoire des Xe´ nobiotiques, INRA, B.P. 3, 31931 Toulouse Ce´ dex, France Received August 13, 1996X

Clenbuterol is a β-adrenergic agonist widely but illegally used in cattle as a growth promoter. The metabolic fate of this drug remains unknown in the main target species, i.e. the bovine, and only limited data have been published concerning its biotransformations in laboratory animals. A metabolic study has been carried out in the rat using 3H- and 14C-labeled clenbuterol. Urine appeared to be the major excretion pathway. Using a soft technique for urine preparation, extraction, and purification, as well as adequate analytical tools in order to preserve labile metabolites, N-oxidation products of the parental drug on the primary amine function were identified for the first time. Clenbuterol hydroxylamine was the major compound, but 4-nitroclenbuterol was also detected. The metabolic pathway leading to the formation of clenbuterol hydroxylamine prevails at high dosages. Clenbuterol hydroxylamine (but not 4-nitroclenbuterol) was also formed extensively when the drug was incubated with rat liver microsomal fractions in aerobic conditions. It is concluded that oxido reduction reactions during urine preparation have previously impaired the identification of this toxicologically important clenbuterol metabolic route.

Introduction Several β-agonists, mainly β2-selective molecules, are able to improve carcass characteristics and productivity rates in farm animals. Nonetheless, their pharmacological properties are manifold, and their administration can be followed by various side effects. Although not licensed for use for zootechnical purposes either in the European Union or in northern America, clenbuterol (CL)1 is however known to be widely administered to bovine. Human collective intoxications have already been related to the consumption of liver from treated animals (1, 2). The symptoms, i.e. tachycardia, muscle tremor, and headaches, have been attributed to a direct pharmacological effect of CL residues. As CL elimination in bovine (3) is relatively rapid, these intoxications are assumed to occur when animals are illegally treated with too high dosages and/or slaughtered after an insufficient withdrawal period. However, the lack of data on the metabolic fate of CL in bovine cannot rule out the hypothesis of pharmacological or toxicological effects of eventual residual metabolites. Partial data concerning the biotransformation of CL in laboratory animals (4-7) have been published. The main metabolic routes for CL metabolism in the rat and dog were proposed to be sulfate conjugation on the primary amine, glucuronic acid N-conjugation on the secondary amine, hydroxylation on the tert-butyl group, and an extensive pathway leading to 4-amino-3,5-dichloromandelic acid and derived metabolites. An almost complete pattern of CL metabolites, covering about 98% * To whom correspondence should be addressed. Telephone: 33(0)5.61.28.50.07. FAX: 33-(0)5.61.28.52.44. E-mail on internet: [email protected]. X Abstract published in Advance ACS Abstracts, January 1, 1997. 1 Abbreviations: CL, clenbuterol; N-OH-CL, 4-(hydroxyamino)-3,5dichloro-R-[(tert-butylamino)methyl]benzyl alcohol; NO-CL, 4-nitroso3,5-dichloro-R-[(tert-butylamino)methyl]benzyl alcohol; NO2-CL, 4-nitro3,5-dichloro-R-[(tert-butylamino)methyl]benzyl alcohol; ESI, electrospray ionization; FAB, fast-atom bombardment; EI, electron impact.

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of the radioactivity in urine was published as a short paper (7), but no indication on the administered dose was given, and no methodological indications on the preparation, or unequivocal identification of these metabolites were presented. Different compounds possessing an arylamine structure, such as aniline and several halogen-substituted anilines (8), but also some β-blocking agents such as practolol (9) or procainamide (10) undergo a metabolic N-oxidation and are converted to the corresponding hydroxylamines. It was hypothesized CL could be a candidate to such a reaction, but the lability of the resulting hydroxylamine, due to oxidoreductive reactions, may have impaired its identification in the urine. We have previously published preliminary results obtained in the rat and bovine using [3H]-CL (11). This work now follows with a more extensive study using a 14C-labeled molecule and the development of adequate analytical tools, in order to establish the complete metabolic fate of CL.

Material and Methods Chemicals. Caution: Clenbuterol is an active drug in humans at low dosage. Clenbuterol, clenbuterol metabolites, and chemically oxidized analogues of clenbuterol (for which no data are available) should be therefore handled carefully. Clenbuterol (Figure 1). [3H]-CL [4-amino-3,5-dichloro-R[(tert-butylamino)methyl]benzyl alcohol-(benzyl-3H)] was purchased from Rotem industries (Beer-Sheva, Israel) and possessed a specific activity of 474 GBq/mmol. [14C]-CL (labeled on the benzylic carbon) was purchased from Isotopchim (Ganagobie-Peyruis, France) and had a specific activity of 1997 MBq/mmol. CL hydrochloride was obtained from Sigma (Saint Quentin Fallavier, France). CL structure was confirmed by electrospray MS. The respective radiopurities of the two labeled compounds were at least 97%, based on HPLC and TLC analyses. Other chemicals were obtained from the following sources: acetic acid and hydrogen peroxide 30% solution, Merck

© 1997 American Chemical Society

198 Chem. Res. Toxicol., Vol. 10, No. 2, 1997

Figure 1. Structure of clenbuterol and position of 3H or labeling.

Zalko et al.

14C

(Darmstadt, Germany); formic acid and analytical grade solvents, Prolabo (Paris, France); pentacyanoamine ferroate trisodium salt hydrate, peracetic acid (32 wt % solution in dilute acetic acid), and sodium tungstate dihydrate 99%, Aldrich; glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and NADP, Sigma; Helix pomatia juice (Helicase), IBF (VilleneuveLa-Garenne, France). Animals. Seventeen male Wistar rats weighing 240-300 g were individually housed in metabolic cages, with free access to water and a standard diet (UAR 210; U.A.R., Villemoissonsur-Orge, France); they were kept on a 12-h light/dark cycle. Rats were force-fed a single oral dose of CL mixed with either [3H]-CL or [14C]-CL. Total drug dosage ranged from 4.5 µg/kg to 50 mg/kg of body weight (see Table 1 in the results section for detail). Radioactivity was adjusted from 160 kBq/µg of CL in the lowest dosages to 100 Bq/µg of CL in the highest dosages as the corresponding urine were used for metabolite isolation and structural analyses. Urine were received in single-use TCUC30 centrifugation plastic recipients (CML France, Nemours, France) placed in ice, collected several times a day, and quickly stored at -80 °C. Sample Preparation. HPLC coupled to on-line radiodetection was used for metabolite profiling and quantification. After radioactivity counting, urinary samples were added methanol (1/1, v/v), stirred, and centrifuged over 10 min at 10 000 rpm and 4 °C. The pellet was discarded. The supernatant was partly cleared from methanol under vacuum and filtered on 0.5 g of Ultrafree-MC 0.45 µm Millipore filtering units (Polylabo, Strasbourg, France) before radio-HPLC analysis. Apparatus. Samples were analyzed by HPLC on a Philips 4100 apparatus (Pye Unicam, Cambridge, UK) equipped with a Rheodyne Model 7125 injector (Rheodyne, Cotati, CA, USA) and connected for radioactivity detection to a Radiomatic floone/β A500 instrument (Radiomatic, La-Queue-Lez-Yvelines, France) (scintillation cocktail, Flow-scint II, Packard Instruments Co, Downers Grove, IL) in order to establish urinary metabolic profiles, or to a Gilson model 202 fraction collector (Gilson France, Villiers-Le-Bel, France), for metabolite purification. Radioactivity in urine was determined by direct counting on a Packard scintillation analyzer (model tricarb 2200CA, Meriden, CT; scintillation cocktail, Packard Ultima Gold). Analytical Procedure. Two gradient elution systems were developed: Chromatographic system 1: column, ultrabase C18 (250 × 4.6 mm, 5 µm) (SFCC, France) coupled to an Hypersil BDS C18 guard precolumn (18 × 4.6 mm, 5 µm) (Shandon/LSI, Cergy Pontoise, France); mobile phases, 10 mM ammonium acetate buffer adjusted to pH 3.2/acetonitrile, respectively 95/5 v/v in A and 30/70 v/v in B; flow rate, 1 mL/min; temperature, 35 °C; a 3-step gradient was used as follows 0-4 min 100% A; 4-10 min linear gradient from 100% A to A/B 95/5 v/v; 10-30 min A/B 95/5 v/v; 30-35 min linear gradient from 5% B to 40% B; 35-45 min A/B 60/40 v/v; 45-47 min linear gradient leading to 100% B; 47-54 min 100% B. Chromatographic system 2: column, Capcell Pak C18 (250 × 4.6 mm, 5 µm) (Interchim, Montluc¸ on, France) coupled to an Hypersil BDS C18 guard precolumn (18 × 4.6 mm, 5 µm); mobile phases, (A) water/acetic acid/acetonitrile (93/2/5 v/v/v) and (B) same (28/2/70 v/v/v); flow rate, 1 mL/min; temperature, 35 °C; a 1-step gradient was used as follows 0-5 min 100% A; 5-15 min linear gradient from 100% A to 100% B; 15-25 min 100% B.

Enzymic Test. For enzyme hydrolysis, 20 µL of Helix pomatia juice (corresponding to 2000 Fishman units of β-glucuronidase and 20 000 Roy units of sulfatase activity) was mixed with 480 µL of 0.1 M sodium acetate buffer adjusted to pH 4.8 and 20 µL of urine, stirred, and left at 42 °C for 16 h. Incubations were filtered on 0.45 Ultrafree MC filtration units before radiochromatographic analysis. Control incubations were carried out in the same conditions with no enzyme. Metabolite Isolation (Figure 2). Urine from two rats (treated respectively with 20 and 50 mg/kg CL) was diluted with two volumes of methanol and centrifuged at 10 000 rpm and 4 °C over a period of 10 min. The supernatant was partly dried in a rotary evaporator to eliminate methanol, taken in ammonium acetate buffer 50 mM adjusted to pH 6.8 (1/20 v/v), and put on prewashed 0.5 g Millipore RP Select B C8 columns (Polylabo, Strasbourg, France). Elution was conducted successively with 2 mL of water, methanol/water (1/1 v/v), methanol, and finally methanol/formic acid (19/1 v/v). These elution fractions represented respectively about 0, 55, 20, and 15% of the total radioactivity put on columns, while 10% of the sample radioactivity were not retained on the columns. The methanol/water eluate, containing polar metabolites but also small amounts of compounds I and J, was concentrated and separated by HPLC using successively chromatographic systems 1 and 2, coupled to a fraction collector. The ultimate fraction corresponding to compound I was completely dried in a rotary evaporator and reconstituted in methanol for storage and structural analyses. Both the methanol and methanol/formic acid eluates were concentrated and separated by HPLC using chromatographic system 1 and a fraction collector. The respective fractions corresponding to CL and metabolites D and J were separately concentrated under vacuum to 10% of their initial volume. Each fraction was then mixed with 2 mL of ammonium acetate buffer 50 mM adjusted to pH 6.8 and put on 0.2 g of prewashed RP Select B C8 columns. The columns were eluted successively with water (0.5 mL) and methanol (1 mL). The methanol eluates always contained more than 95% of the radioactivity put on columns. They were concentrated in a rotary evaporator and used for structural analyses. Metabolite J, issued from the methanol/water fraction, was prepared following the same method. Synthesis. (A) Oxidation of CL. The 4-nitro analogue of CL (NO2-CL) was synthesized either using a sodium tungstate catalyzed oxidation, or by means of a reaction with peracetic acid. In the first experiment, 1.1 mg of CL and 4 mg of sodium tungstate were dissolved in 0.l mL of methanol and 1.9 mL of a 15% hydrogen peroxide solution, and maintained at 0 °C under gentle shaking over 24 h. A 30% hydrogen peroxide solution (500 µL) was added at 2, 4, 6, and 8 h after the beginning of the reaction. CL oxidation with peracetic acid was achieved by dissolving 2.5 mg of CL in 0.3 mL of methanol and 0.6 mL of a 32% solution of peracetic acid, and further keeping of the mixture at room temperature, over 18 h under gentle shaking. In both cases, the resulting oxidized compounds and the parental drug were purified using separative chromatographic system 1 and 0.2 g of Millipore RP Select B C8 columns as described above for CL and metabolites D and J, and then submitted to mass spectrometric analysis. (B) Reduction of NO2-CL was achieved with zinc dust following a method described by Corbett and Chipko (12). The resulting compounds were checked by HPLC in chromatographic system 1 and purified as described above for CL and metabolites D and J. Microsomal Incubation. An untreated adult male Wistar rat weighing 250 g was killed by decapitation. Its liver was removed immediately and washed in 0.9% saline. Liver microsomes were prepared as described elsewhere (13) and stored at -80 °C in 0.1 M phosphate buffer (pH 7.4) containing 20% glycerol. Protein concentration was determined by the method of Lowry et al. (14). Incubation was performed in a vial containing 2 mg of microsomal protein, 1 mM NADP, 10 mM glucose-6-phosphate, 5 mM MgCl2, 2 units of glucose-6phosphate dehydrogenase and 25 µM of [14C]-CL (5 kBq) in a final volume of 1 mL 0.1 M phosphate buffer (pH 7.4). The

In Vivo 4-N-Oxidation of Clenbuterol

Chem. Res. Toxicol., Vol. 10, No. 2, 1997 199

Figure 2. Clenbuterol and metabolites D, I, and J isolation procedure from rat urine. mixture was incubated for 1 h at 37 °C, and the reaction was stopped by addition of methanol (0.5 mL). The vial was immediately centrifuged at 10 000g for 10 min at 4 °C. The supernatant was partly dried from methanol under nitrogen stream and filtered on 0.45 Ultrafree MC filtration units before radiochromatographic analysis using chromatographic system 1. Control incubation was carried out in the same conditions but without NADPH generating system. The main metabolite detected in the supernatant was purified for mass spectrometric studies following the same tools developed for urine, i.e. fraction collection using chromatographic system 1 followed by 0.2 g of C8 columns. Structural Analyses. Electron impact (EI), fast-atom bombardment (FAB), as well as electrospray ionization (ESI) mass spectrometry were used to elucidate the structures of metabolites D, I, J, and CL, and the chemically oxidized compounds (D, I, and J) synthesized from CL. All mass spectra were obtained on a Nermag R-10-10-H single quadrupole mass spectrometer (Delsi-Nermag Inst., Argenteuil, France) fitted either with a Delsi-Nermag EI source, a M-Scan FAB gun (Mscan, Ascot, UK) or an Analytica of Branford (Branford, CT) ESI source. The spectra were acquired using an HP Chem Station data system interfaced to a Nermag mass spectrometer (Quad services, Poissy, France). EI mass spectra were obtained at an electron energy of 70 eV and a filament current of 200 µA, with a source temperature of 220 °C. Five hundred nanograms of the sample was introduced into the ionization source using the direct introduction mode. The sample was placed on the probe filament which was heated by using a current program from 0 to 280 mA at 5 mA/s during the analysis. FAB experiments were carried out using xenon for bombardment, at an accelerating voltage of 8 kV, with

1-2 mA as discharge current. Glycerol was used as the matrix. In the case of ESI-MS, solutions of the various compounds [typically 20-50 ng/µL in methanol/water (50/50, v/v)] were infused using a Harvard Apparatus (South Natick, MA) Model 22 syringe pump into the ESI source at a flow rate of 1 µL/min. CH3-OD/D2O (50/50, v/v) was used instead of CH3-OH/H2O for hydrogen/deuterium exchange experiments. In source collisioninduced dissociation experiments were conducted by increasing the voltage difference between the metalized outlet of the glass capillary transfer and the first skimmer of the Analytica ESI source. By increasing this voltage difference, the ion kinetic energy is increased and therefore, collisional activations occurring with the residual gas in this region are enhanced. In our experiments, the voltage difference used was typically 180 V. 1H NMR experiments were performed in deuterium oxide or Me2SO-d6 at 500 and 600 MHz using Bruker RDX spectrometers (Bruker, Karlsruhe, Germany). In all cases, the temperature of the probe was regulated to 298 K. Special care was taken to ensure reproducible integration of the spectra. For the sake of comparison, the same concentrations (0.6 mg/0.4 mL solvent) were used for the authentic CL and metabolite D.

Results HPLC Studies on Urine from Rats Fed CL. Radioactivity excretion in the urine of 14 rats fed a single oral dosage of CL was quantified using either 3H- or 14Clabeled clenbuterol. About 70% of the initial administered radioactivity was recovered in rats urine within the 0-96 h period after administration. Radiochromatographic profiles showed at least 10 compounds to be


Zalko et al.

Figure 3. 14C radio-HPLC urinary profile in a rat treated with a single oral dose of 20 mg/kg CL, using chromatographic system 1 (compound I was not detected in urine samples following rapid preparation; its elution time is about 49 min).

present in urine extracts (Figure 3). In these chromatographic conditions, CL eluted at 42 min and was the major compound detected, together with metabolite D. Compound J, of lower polarity than CL, was only detected in some of the urinary samples studied, and its formation did not seem to depend on the initial dosage. When observed, it represented ca. 1-2% of the total radioactivity administered to the rats. Some of the important metabolites were identified and confirmed results published previously (7): compound C was characterized as the sulfate conjugate of CL on the primary amine (11); compound H was identified as 4-amino-3,5-dichlorohippuric acid.2 In the earliest stages of the study, metabolite D was found to retransform easily into the parental drug. Thus, in order to quantify the extent to which CL was actually metabolized to D, a rapid sample preparation was performed, as described in the material and methods section. This study showed that D was by far the main metabolite detected in urine when rats were treated with a high dosage of CL, and was one of the major compounds present in the urine from rats fed the lowest dosages. When the initial dosage increased, the amount of unchanged CL in urine raised from 6% (4.5 µg/kg) to 30% (45 mg/kg). Concurrently, the proportion of radioactivity corresponding to the different metabolites decreased, except for compound D. Table 1 summarizes the amount of D measured in urine for each dosage tested. The logarithm of the quantity of D excreted in urine [log (UD)] was found to strongly correlate (r2 ) 0.997, p < 0.000 01) with that of the administered dosage [log (ACL)] according to the relation: log (UD) ) [1.167 log (ACL)] - 1.448, where both UD and ACL were calculated in nanomoles/ kilogram. These results allow an estimation of the excreted amount of D in urine within the large range of oral dosages tested, following the equation UD ) (ACL)1.167/28.04, where quantities are expressed in nanomoles/kilogram. Parameter 1.167 differs significantly from 1 (p < 0.000 01), and thus the relative amount of 2

D. Zalko and J. Tulliez, unpublished observations.

Table 1. Total Amount of Metabolite D Detected in Urine of Rats Treated with Various Single Oral Dosages of Clenbuterol rat

clenbuterol dosage (ng/kg)

amount of D detected in urine (ng/kg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

4.46 × 103 5.31 × 103 121 × 103 129 × 103 457 × 103 475 × 103 1.63 × 106 1.63 × 106 3.78 × 106 4.15 × 106 17.1 × 106 17.3 × 106 38.1 × 106 44.9 × 106

351 275 12.8 × 103 14.0 × 103 41.8 × 103 43.1 × 103 221 × 103 250 × 103 623 × 103 722 × 103 4.94 × 106 5.33 × 106 9.50 × 106 11.5 × 106

metabolite D excreted in urine is relatively more important when the dosage gets higher, within the range of dosages tested. In order to determine if the major metabolites of CL, with a special focus on D, would behave as sulfo or glucurono conjugates, hydrolysis of crude rat urine with Helix pomatia juice was performed. It was found that after a 16 h incubation, no metabolite D was detectable and that the corresponding amount of radioactivity was associated to CL. Therefore, at that stage of the study, these results suggested that D could be a sulfate or a glucuronide resulting from CL conjugation. Metabolite Isolation (Figure 2). Urine samples from two rats were prepared as described previously and put on prewashed C8 columns. Chromatographic analysis (system 1) of the methanol eluate showed this fraction mainly consisted of D and CL (respectively 35% and 40% of the detected radioactivity). The methanol/formic acid eluate, processed in the same conditions, was found to contain both CL and metabolite J. After further purification, these compounds were submitted to mass spectrometry analysis. During the first steps of this procedure (first passage of urinary extract on C8 columns), a small quantity of



Figure 4. ESI-MS spectrum of metabolite D.

Figure 5. FAB-MS spectrum of metabolite D.

two rather apolar products was eluted in the methanol/ water fraction (see Figure 2). These compounds exhibited retention times of respectively 49 and 51 min in chromatographic system 1. The latter was purified as described above and was found to be metabolite J. The former was named I and prepared for mass spectrometric studies using buffer-free chromatographic system 2. Metabolite Identification. (A) Spot Test Reaction with Pentacyanoamine Ferroate. Pentacyanoamine ferroate was used to test the putative N-oxidation of the primary amino group of CL (15). No color change was observed when ca. 1-5 µg of CL or J were added to 10 µL of a 1% aqueous solution of pentacyanoamine ferroate. On the contrary, the test solution turned into pale pink, red, and bright cherry-red, respectively, with 1, 5, and 10 µg of D, and to pale red and red with, respectively, 1 or 5 µg of I. (B) Mass Spectrometry. Electrospray ionization mass spectrometry has already proved to be a valuable tool in obtaining diagnostic fragmentation patterns of CL by means of in-source collisionally activated dissociations (16-18). This approach has been extended here to the structural determination of CL oxidation products. Figure 4 represents the ESI-MS spectrum of metabolite D acquired under in-source fragmentation conditions. This spectrum exhibits an [M + H]+ ion at m/z 293, corresponding to an hydroxylated form of CL ([M + H]+ at m/z 277). The fragment ions observed at m/z 275, 219, and 202 are consistent with the consecutive losses of water from the benzylic alcohol function, and of the side chain unchanged tert-butyl and amino groups of the molecule, respectively. This indicated that the hydroxylated site should be located on the aromatic moiety of the molecule since the losses of the various side chain groups did not involve the additional hydroxyl group. Moreover, H/D exchange experiments showed that the pseudo-molecular ion was shifted from m/z 293 to m/z 298, corresponding to a [Md4 + D]+ species. Thus, metabolite D contained no more exchangeable protons than the unchanged CL (m/z 277 shifted to m/z 292 in

the same conditions, data not shown). From this result, all hydroxylation sites but the arylamino group must be ruled out and metabolite D was identified as the arylhydroxylamine of clenbuterol. This was also supported by electron impact ionization and fast atom bombardment experiments carried out on D. When analyzed by EI mass spectrometry, metabolite D gave the molecular ion M•+, and the [M - 2]•+, [M 16]•+ and [M - 17]+ ions, corresponding to the loss of a hydrogen molecule, an oxygen atom, and a hydroxyl radical, respectively, from the molecular ion. These ions have been reported to be characteristic of aromatic hydroxylamines (19, 20). Low abundance [M + H]+ ion was also observed on the EI spectrum of D. This was likely due to some autoprotonation occurring in the EI ionization source, although this was not observed for standard CL analyzed in the same conditions (data not shown). As expected, the quasi-molecular [M + H]+ ion was also present and represented the base peak on the FAB mass spectrum of D (Figure 5). As observed by Saito et al. (20) on a series of N-hydroxyarylamines, the mass spectrum of D exhibits ions corresponding to [M + H - 16]+ (m/z 277), [M + H - 17]+ (m/z 276), and [M 17]•+ (m/z 275) species, although their relative intensities are somewhat difficult to evaluate owing to the chlorine atoms isotopic pattern. Contrary to Saito et al., very little [M]•+ molecular ion was observed on the FAB mass spectrum of D (see Figure 5), and the observation of the [M + H - 32]+ and [M - 32]•+ species is hindered by the presence of the m/z 259 ion and its isotopic pattern, which probably arises from a consecutive dehydration of the m/z 277 ion. Metabolites I and J were also analyzed using ESI-MS. They exhibited [M + H]+ ions at m/z 291 and 307, respectively, which was consistent with the nitroso and nitro derivatives of CL. As expected, these ions were shifted to m/z 294 for I and m/z 310 for J, under H/D exchange conditions, indicating that I and J both contained only two exchangeable protons (i.e. those of the ethanolamine moiety of the molecule). This result,


Zalko et al.

Table 2. 1H NMR Spectral Data for CL and Metabolite D Ar Cl H2N

clenbuterol

2

CH

CH2

NH t-Bu

OH

Cl

Ar

1

Table 3. Relative Amounts of Metabolite D and Clenbuterol in Urine of a Rat Treated with a Single Oral Dosage of 2 mg/kg CLa

H-1

7.387 4.884 J1,2a 3.2 Hz J1,2b 10.0 Hz metabolite D 7.541 4.938 J1,2a 3.4 Hz J1,2b 9.7 Hz ∆δ 0.154 0.054

H-2a

H-2b

t-Bu

3.277 J2a,2b 12.8 Hz

3.193

1.414

3.216 J2a,2b 12.7 Hz

3.118

1.353

-0.061

-0.075 -0.061

together with the characteristic fragmentation of those metabolites according to diagnostic pathways established elsewhere and already discussed above, allowed the unambiguous identification of I and J as the nitroso and nitro oxidation products of CL, respectively. (C) Chemical Synthesis. When CL oxidation was conducted with hydrogen peroxide/sodium tungstate, only 5% of the parental drug was still present in the solution after 24 h. About 25% of the initial compound was found to transform into J as confirmed by ESI-MS analysis, and most of the residual radioactivity was associated with very polar products. Reaction of CL with peracetic acid produced high yields of oxidation products. After an 18-h period, CL was mainly transformed into I and J (respectively 30% and 55%) while only traces of the parental drug remained. Positive ESI mass spectra of both compounds were respectively consistent with the structures of the nitroso and nitro analogues of CL, and matched those of the products purified from urine. Reduction of nitro-CL with zinc dust produced CL, the nitroso analogue of CL and a compound coeluting with D in chromatographic system 1. This compound was purified as described for D and characterized by mass spectrometry as 4-N-hydroxy-CL. (D) 1H NMR Experiments. Table 2 shows a comparison of the spectra of metabolite D and CL in D2O at 298 K and 500 MHz. All assignments were performed from the values of chemical shifts and coupling patterns. The most significant difference appearing between the spectra of CL and its metabolite is the ∆δ observed for the aryl protons: this very likely indicates that the nitrogen located in meta position has been the center of a chemical modification, thus confirming the results of MS studies. Metabolite Stability. Aliquots of a urine sample from a rat in which NO2-CL was initially not observed were stored during various laps of time in different temperature conditions in order to determine the stability of metabolite D (N-OH-CL). As shown in Table 3, metabolite D quickly decreased at room temperature, and to a lower extent when samples were kept in cooler conditions. Concurrently, radioactivity associated to CL increased in quite similar quantities. Two compounds, less polar than CL and eluting at the same time as nitroso-CL (NO-CL) and NO2-CL were also detected in variable amounts (1-10% of the initial radioactivity) in samples stored 7 days or more. Once purified and stored in methanol for 3 months at -20 °C, N-OH-CL, NO-CL, and NO2-CL were relatively stable. However, small amounts (5 to 10% of the sample) of N-OH-CL underwent oxidation to NO-CL and NO2CL, and trace amounts of N-OH-CL were detected in the NO-CL sample. Further observations were made on the

temperature +25 °C +4 °C -20 °C -80 °C

amount of metabolite D in urine (% of detected radioactivity)b per storage time (days) 1 7 30 90 12.2 31.7 34.2 34.8

0.9 29.6 27.0 31.2

ND 20.4 25.8 28.6

ND 6.3 25.8 26.0

amount of clenbuterol in urine (% of detected radioactivity)c per storage time (days) 1 7 30 90 49.2 32.0 30.5 29.3

54.1 30.1 27.3 28.7

53.2 39.2 32.3 29.8

56.0 53.6 32.4 31.6

a Values after storage at various temperatures over 1 day to 3 months. ND: not detected. b Reference ) 35.7% (day “0”). c Reference ) 27.8% (day “0”).

stability of these metabolites: as reported previously, N-OH-CL was reduced to CL when incubated in crude urine with Helix pomatia juice. N-OH-CL also underwent total reduction into CL when dried in a rotary evaporator in weak acidic medium (pH 3-5) and partial reduction when left at 42 °C for 16 h in sodium acetate buffer adjusted to pH 4.8. NO-CL was partly transformed to CL, N-OH-CL, and NO2-CL when incubated 16 h in various weak acidic buffers. For example, in sodium acetate buffer adjusted to pH 4.8, NO-CL was mainly reduced to N-OH-CL (40%) and CL (15%), while a small amount of NO2-CL (5%) also appeared. NO2-CL was reduced easily but not completely to NO-CL when the last step of its isolation was achieved by means of chromatographic system 2 instead of C8 columns. Microsomal Incubation. On-line radioactivity detection showed that about 60% [7.5 nmol/(h‚mg of protein)] of incubated CL was metabolized by liver microsomes to a major metabolite eluting at 20 min in chromatographic system 1. This compound was characterized as N-OH-CL, using the same mass spectrometric techniques applied to urinary metabolites and chemically synthesized standards.

Discussion Characterization of Clenbuterol N-Oxidized Metabolites. Our results clearly indicate that the formation of (hydroxyamino)clenbuterol [4-(hydroxyamino)-3,5dichloro-R-[(tert-butylamino)methyl]benzyl alcohol] is one of the major routes for CL metabolism in the rat. Although this pathway would appear to be possible when considering the parental compound structure (8-10), it had not been previously reported until now (4-7). Moreover, within the large range of single oral dosages tested (ca. 5 µg/kg to 50 mg/kg), it was found that the higher the initial dosage, the higher was the proportion of the N-OH-CL excreted in the urine. Thus, the global CL 4-N-hydroxylation activity is not saturated, while most of the other metabolizing pathways reach a plateau. Several mass spectrometry techniques, as well as 1H NMR experiments, led to structural characterization of metabolite N-OH-CL. A positive spot test reaction with pentacyanoamine ferroate, known to react with N-hydroxy-arylamines (15), also supported the hydroxylamine structure. Two compounds with a lower polarity than CL were also isolated and characterized as NO-CL and NO2-CL. NO-CL was not detected directly in fresh urine, but only after the first metabolite isolation steps using C8 columns. However, NO2-CL was observed in contrast in some of the urinary samples analyzed quickly after collection, and in two elution fractions of C8 columns.


Since NO2-CL is well retained on C8 columns at pH 6.8, and that the overall amount of this metabolite in these two elution fractions was calculated to be far more important than in urine analyzed directly, it is concluded that NO2-CL (as well as NO-CL) can form during the metabolite purification procedure, probably resulting from the oxidation of N-OH-CL. NO2-CL can be easily synthesized from CL by oxidation, using either sodium tungstate or peracetic acid. This latter method allows an easy production of large amounts of CL N-oxidation derivatives. Such a result was not expected as oxidation of aromatic amines possessing strong electron-withdrawing groups using peracetic acid was previously found to give relatively poor results (21). NO2-CL, like any aromatic C-nitro compound, does not react with pentacyanoamine ferroate while NO-CL reacts the same as N-OH-CL. Indeed, aromatic C-nitroso structures are known to give a positive spot test with this reagent after being reduced to the corresponding arylhydroxylamine (15). N-hydroxyarylamines are more or less stable (22, 23), depending on their structure (24) and pH (25). Once isolated and stored in methanol at -20 °C, N-OH-CL, NO-CL, and NO2-CL are relatively stable over a limited time. In urine, N-OH-CL was found to be reduced easily to CL. This reaction was slowed down at low temperatures, but still occurred, even at -80 °C. During urine storage, small amounts of NO-CL and NO2-CL did also form, which is consistent with the established ability of N-hydroxyarylamines to undergo autooxidation (26). As no hydrogen atom is available on the carbon at the R position of the primary amino function of clenbuterol, autooxidation of N-OH-CL leads to C-nitroso-CL and C-nitro-CL. As reported above, similar oxidations also took place rapidly when working with C8 columns. In solution, hydroxylamines are generally more stable in acidic conditions, and their aerial oxidation is promoted by neutral or alkaline pH (26, 27). Thus, the finding that N-OH-CL easily oxidizes into NO-CL and NO2-CL while using C8 columns may be related to the pH (6.8) of the buffer used during this purification step. Indeed, no NOCL was detected while performing urinary chromatographic profiles at pH 3.2. Conversely, the presence of NO2-CL in urine samples was established for some, but not all of the animals. Moreover, when NO2-CL was detected in an animal urine during the first day after treatment with CL, it was systematically present in the samples of the following days. These observations, the repeatability of the radiochromatographic conditions used for NO2-CL detection, and the similar conditions used to collect, prepare and analyze urine samples from different rats, support the hypothesis of in vivo formation of NO2CL in certain animals. Our results are consistent with the other CL metabolism studies carried out in rats, which reported CL was rapidly and extensively eliminated via urine (4, 6, 7). However, the amount of unchanged drug in urine found in the present study was far below the values stated previously. In the Tanabe et al. study (6), β-glucuronidase was used during urinary metabolites analysis, which may have led to N-OH-CL reduction, as indicated by the present results. In the earlier study of Kopitar and Zimmer (4), rats receiving a single oral dose of 2 mg/ kg of labeled CL excreted nearly 70% of the radioactivity in urine as unchanged CL. But here, the first steps of urine processing included an extraction at pH 11, whereas during the elaboration of our present analytical separative techniques, it was found that N-OH-CL transformed


totally into CL at pH > 9 in a sodium hydroxide solution. Concerning Schmid et al. work (7), unfortunately no data are presented about the analytical techniques used to purify CL metabolites. Thus, at least for the first two studies, it is likely that N-OH-CL had been reduced to CL during urine processing, and that the corresponding amount of radioactivity had been inappropriately attributed to the parental drug. Pharmacological and Toxicological Implications. The observation of an hydroxylamine as a major metabolite of CL raises many questions. One of the main target organs for CL in the rat and the bovine is known to be the liver (3, 11), and most of the reported cases of intoxication refer to bovine liver consumption (1, 2). Complementary studies appear to be necessary and are in progress in our laboratory to determine whether CL N-oxidation products are present or not in bovine liver and tissue extracts. Preliminary results have shown N-OH-CL to be present in urine from bovine orally treated with CL (11). Moreover, NO2-CL was found in liver extracts3 from rats treated similarly, suggesting it could also be present in bovine tissues. N-OH-CL and NO2-CL structure show they may conserve a β-agonistlike activity, and thus, could be responsible for part of the side effects attributed to the parental compound. Their pharmacological activities should therefore be assayed. To our knowledge, no β2-selective agonist possessing an arylamine structure was yet shown to be metabolized to the corresponding hydroxylamine. The use of the β-blocker practolol has been associated with some adverse effects, which are probably related to immunological mechanisms. They may result from N-hydroxylation, associated or not with an initial deacetylation of practolol into the corresponding primary arylamine (9, 28). Procainamide, another primary arylamine, was found responsible for immunological adverse effects [lupus, agranulocytosis, (29)]. It is metabolized to a hydroxylamine by rat and human liver microsomes (10), as well as by human activated leukocytes that contain myeloperoxydase (9). N-Oxidation pathways have been shown to be involved in various toxicological mechanisms, which result from the binding of reactive metabolites to DNA, hemoglobin, and other proteins (9, 30). The N-OH-CL and NO-CL structures, identified in this work, indicate these compounds could lead to the formation of a toxic intermediate. This could also be the case for NO2-CL, as various nitro compounds can be reduced in the gut or by hepatic nitroreductases (31-33). Relatively few primary arylamines are currently used as drugs (9), but several β2 selective agonists share this structure. This is true for mabuterol and cimaterol, but also for closely related structures that where reported to be illegally used in meat production: clenpenterol, clenproperol, mapenterol (34), and others (35, 36). Moreover, among the β-adrenergic-active substances that are currently studied, some also possess an arylamine structure, as it is the case for the β2-selective agonist picumeterol, a drug of potential value in the treatment of asthma in humans. A detailed metabolic study of this molecule in the rat and dog was published recently, and no evidence for the presence in urine of N-oxidized metabolites was found (37). Nevertheless, it should be noted that in this work, incubations of the drug were carried out with rat, dog, and human liver microsomal fractions and that the major metabolite detected was not 3

Authors, unpublished observations.


identified. As the phenethanolamine moiety of picumeterol and clenbuterol is the same, and considering our present results with rat liver microsomal fractions, it may be of interest to check if this in vitro formed metabolite is an hydroxylamine. Indeed, this compound may result from the N-oxidation of picumeterol as well as from the N-oxidation of one of its metabolites already identified (37). Additional data about clenbuterol and other β2-selective agonists metabolism could be of great interest for the understanding of their activity, and may highlight the possible problems of food safety raised by their illegal use in farm animals.

Acknowledgment. This work was supported by a grant from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche, within the project “Aliment Demain” no. 95G0098. We appreciate the contribution of Bruno Perly (SCM, CEA Saclay) in performing 1H NMR experiments. We thank Dr. A. Paris and Dr. P. Dansette for helpful discussions.

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TX960143S

Evidence for a New and Major Metabolic Pathway of Clenbuterol

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