Introduction

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Clozapine [2-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine] was the first neuroleptic drug to reveal clinical properties that later were used to define the group of atypical neuroleptics (Coward, 1992). On the other hand, clozapine therapy bears a risk of agranulocytosis of 1 to 2%, and this adverse effect is assumed to be mediated by a chemically reactive metabolite (Pirmohamed et al., 1995). Therefore, a complete knowledge of biotransformation routes undergone by clozapine is desirable. The metabolism of clozapine (CLZ¹) in vitro by human liver microsomes and/or expressed human cytochrome P450 enzymes has been the subject of several investigations (Fischer et al., 1992; Pirmohamed et al., 1995; Eiermann et al., 1997; Linnet and Olesen, 1997; Tugnait et al., 1997; Fang et al., 1998), but except for unknown metabolites produced by CYP2D6, the investigations were confined to the so-called "major" biotransformation products desmethylclozapine (DMCLZ) and clozapine N-oxide (CLZ-NO), which had originally been identified in patient urine by Gauch and Michaelis (1971). Measurements in patient urine revealed, however, that these two together accounted for only 14% of the dose (Schaber et al., 1998), such that additional metabolites were expected to contribute markedly to the total fate. An analysis of patient urine for unconjugated basic compounds resulted in the identification of DMCLZ, CLZ-NO, and of minor products in which the chloro substituent was replaced by a hydroxy or methylthio group (Stock et al., 1977). An attempt to elucidate the whole pattern of clozapine metabolites in human urine and feces has been limited to volunteers given a single dose of [¹⁴C]clozapine (Dain et al., 1997). It led to the detection in urine of 7-hydroxydesmethylclozapine-O-sulfate and the glucuronides of 7-hydroxyclozapine (previously identified in rat bile by Zhang et al., 1996) and 8-deschloro-8-hydroxydesmethylclozapine. In addition, the quaternary ammonium glucuronide of clozapine, which had previously been isolated from patient urine (Luo et al., 1994), could be measured in feces (Dain et al., 1997). Since the authors used a single HPLC step for metabolite separation, the possibility had to be considered that minor metabolites were overlooked.

The present investigation aimed at elucidating as completely as possible the pattern of unconjugated and conjugated clozapine metabolites in the urine of patients under continuous monotherapy by using sequential separation steps.

	Experimental Procedures

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Materials. Clozapine, N-desmethylclozapine, and clozapine N-oxide as reference substances were kindly provided by Novartis Pharma (Basel, Switzerland). Clozapine N⁺-glucuronide was prepared according to Luo et al. (1992), but the derivatization reaction was carried out at 42°C, and the product was isolated by passing the extracted aqueous phases through cartridges with C₁₈ silica gel (Bond Elut, Varian, Harbor City, CA) and eluting with methanol. The eluted N⁺-glucuronide was purified by thin-layer chromatography as described below for polar metabolites. -Glucuronidase/arylsulfatase from Helix pomatia and -glucuronidase from Escherichia coli K 12 were purchased from Roche Diagnostics (Mannheim, Germany).

Patients. Urine samples were collected by three in-patients of the Psychiatric Clinic, University of Tuebingen, suffering from schizophrenia. Patient 1 was a 35-year-old male weighing 80 kg. He was treated with 350 mg/day clozapine for his psychosis and with prednisolone for idiopathic thrombocytopenic purpura. On the day of urine collection, five serum clozapine measurements performed at 2-h intervals resulted in 411 ± 87 ng/ml, while desmethylclozapine amounted to 275 ± 62 ng/ml. His 24-h urine sample of 1700 ml was completely processed. Patient 2, a 19-year-old male weighing 59 kg, was treated with 600 mg/day clozapine and had clozapine and desmethylclozapine serum levels of 1950 ± 110 and 995 ± 45 ng/ml, respectively. Metabolite analyses were done on a 2-h urine fraction of 395 ml. Patient 3, a 31-year-old male, had serum concentrations of 331 ng/ml clozapine and 283 ng/ml desmethylclozapine under treatment with 400 mg/day clozapine. Of his 24-h urine (3050 ml), two 400-ml portions were processed.

Isolation of Metabolites. Urine samples of 300 to 600 ml were applied at a rate of 5 ml/min to a C₁₈ silica gel column (150 × 10 mm, Polygosil 40-63 µm, Macherey-Nagel, Düren, Germany) pretreated with methanol and water. After washing with 100 to 150 ml of water, substances were eluted with 100 to 150 ml of 0.1 M acetic acid in methanol, and the solvent was evaporated under reduced pressure. The residue was separated into unpolar bases and polar substances by addition of 50 ml of water, pH adjustment to 9 with 25% aqueous ammonia and four extractions with 50 ml of tert-butyl methyl ether. The organic phases were evaporated, and unconjugated metabolites contained in the residue were dissolved in 0.5 ml of methanol and separated by thin-layer chromatography on four to six sheets of 20 × 20 cm precoated with 0.25 mm of silica gel with fluorescent indicator (Alugram SIL G/UV₂₅₄, Macherey-Nagel). Solvent I, composed of 2-propanol/tert-butyl methyl ether/25% ammonia/water (12:6:0.75:0.75, v/v), was run to a height of 12 cm, and the nine UV-absorbing bands were removed, suspended in 1 ml of 2 N ammonia, and extracted four times with 2 ml of tert-butyl methyl ether. The organic phases were combined and evaporated, and the residue served for further separation by HPLC.

Purification by HPLC. The system consisted of a 200- × 4.6-mm column filled with C₁₈ silica gel 5 µm (Nucleosil 5 C₁₈, Macherey-Nagel) coupled to a photometric detector (UVIS 205, Linear) with which spectra could be recorded during the run. The usual detection wavelength was 290 nm. The solvents run at 1 ml/min were mixtures of 0.02 M ammonium acetate in 0.9 M acetic acid (pH 3.0) and methanol in the ratios of 50:50, 70:30, 80:20, or 90:10 (v/v) according to the polarity of the metabolites. Spectra were recorded for all peaks and eluates were collected. Eluates with spectra resembling that of clozapine were evaporated under reduced pressure. The purified compounds were dissolved in methanol and E₂₄₅-E₂₇₅ was measured along with the value for clozapine (0.21 at 10 µg/ml). Estimates of metabolite quantities were obtained on the assumption of equal molar absorptivity.

Enzymatic Hydrolysis. Quantities of conjugated metabolites of about 20 µg were dissolved in 0.4 ml of 0.1 M sodium acetate buffer, pH 5, and incubated with 100 µl of -glucuronidase/arylsulfatase from H. pomatia for 24 h at 37°C. Incubates were alkalinized with ammonia and liberated compounds extracted three times with 2 ml of tert-butyl methyl ether, and in some cases subsequently with ethyl acetate. Extract residues were subjected to TLC in solvent I, if possible in parallel with known unconjugated metabolites. Clozapine liberated from its N⁺-glucuronide by the same procedure or by an excess of -glucuronidase from E. coli in phosphate buffer, pH 7, within 2 h was detected in the above HPLC system with 50% methanol in the eluent, resulting in a retention time of 8.5 min.

Mass Spectrometry. Mass spectra in the electron-impact mode (EI-MS) were recorded with a double-focusing mass spectrometer (Finnigan MAT 8230 AUDEVAP) with a combined EI/CI ion source (ThermoFinnigan, San Jose, CA). Substances were directly introduced into the ion source, and spectra were recorded over the range 20 to 750 amu at a rate of 3 s/decade, an ionization energy of 70 eV, and an acceleration voltage of 3000 V.

¹H NMR Spectrometry. Spectra were recorded in a 400-MHz instrument (AMX 400, Bruker) with a 5-mm dual probe at 300 to 320 K. Substances were dissolved in deuterated methanol with 99.8% deuterium. The solvent signal at 4.86 ppm served for standardization. For processing of recorded spectra, the interferogram of the free induction decay with 16-k data points was multiplied with a line broadening of 0.3 Hz.

	Results

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Unpolar Fraction. On TLC of organic-extractable compounds from patient urine, seven bands were obtained that according to their UV spectra contained clozapine metabolites. Each of these resulted in up to four peaks in HPLC. Structures of metabolites that were isolated and identified are shown in Fig. 1.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Structures of clozapine metabolites in patient urine and possible metabolic pathways for their production. R = H for free phenols; R = glucuronosyl (C6H9O6) for glucuronides; R = SO3H for sulfates.

Polar Fraction. As mentioned above, a major compound in the fraction not extractable into tert-butyl methyl ether was CLZ-NO (Table 1). All other metabolites isolated from this fraction proved to be conjugates with glucuronic acid or sulfuric acid (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   CID-MS/MS of 7-hydroxyclozapine-N-oxide-O-sulfate in the negative ion mode.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   1H NMR spectrum (400 MHz) of 7-hydroxyclozapine-N-oxide-O-sulfate recorded in methanol-d4 (intense signals at 4.86 and 3.25 ppm). The upper trace of numbers shows the parts per million values of the 1H NMR signals, and the lower trace shows the corresponding frequency values in Hertz. The signal at 6.9 ppm (*) is due to an impurity.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   CID-MS/MS of the cation of N-hydroxydesmethylclozapine-O-glucuronide.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   1H NMR spectrum (400 MHz) of N-hydroxydesmethylclozapine-O-glucuronide in methanol-d4. For further information see legend of Fig. 3.

	Discussion

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A large variety of clozapine metabolites was detected in patient urine, most of the minor ones not having been described previously. Introduction of OH with removal of Cl at C-8 was confirmed as a major pathway leading to 8-OH-CLZ (Table 1; Stock et al., 1977), 8-OH-DMCLZ, and its O-glucuronide (Tables 1 and 2; Stock et al., 1977; Dain et al., 1997). The reaction can be regarded as loss of halogen from the para position of an aromatic amine and may proceed via a quinoneimine as a reactive intermediate (Rietjens et al., 1990). This opens the possibility of glutathione conjugation, but the respective conjugates were detected neither by Dain et al. (1997) nor in the present work.

Another quantitatively important biotransformation was hydroxylation at C-7 giving rise to a glucuronide in rat bile (Zhang et al., 1996) and human urine (Table 2; Dain et al., 1997). The sulfuric acid conjugate of 7-OH-DMCLZ was described as a major metabolite in human urine (Dain et al., 1997) after it was tentatively identified in rat bile (Zhang et al., 1996). Additional 7-hydroxylation products were unconjugated 7-OH-CLZ (Table 1), its sulfate conjugate, which had been identified in rat bile by its mass spectrum (Zhang et al., 1996), and the sulfuric and glucuronic acid conjugates of 7-OH-CLZ-NO. The only phenolic N-oxide derivatives of another tricyclic psychoactive drug hitherto described are the glucuronide and sulfate conjugates of 7-hydroxyfluperlapine N-oxide that were detected in the rat in vivo and in hepatocyte cultures (Paine et al., 1984; Dain and Jaffe, 1988) and in cultures of human hepatocytes (Guillouzo et al., 1988); however, a description of rigorous structural identification is missing. Clozapine and fluperlapine, a close structural analog of clozapine, are distinguished by a prominent role of N-oxidation in their biotransformation.

Another novel finding was a metabolite tentatively identified as 9-OH-DMCLZ-O-Sulf (Table 2). A phenolic sulfate showing ¹H resonances in accordance with those calculated for 6-OH-CLZ-O-Sulf indicated that hydroxylation of CLZ occurred also at the 6-position.

In addition to aromatic hydroxylation, DMCLZ was hydroxylated at N-4 of the piperazine ring, and the resulting hydroxylamine was conjugated with glucuronic acid. The structure of the conjugate could be deduced by mass spectrometry from the molecular ion and from the loss of glucuronic acid including an oxygen through which it was attached. Fragmentation also led to loss of the piperazine ring in addition to the glucuronosyl group, but not to piperazine ring removal alone. ¹H NMR confirmed that the anomeric proton of the glucuronosyl residue appeared at 4.65 ppm, indicative of a pronounced up-field shift relative to the signal in phenolic glucuronides. Anomeric protons with similar shifts were detected in the glucuronides of hydroxylamine metabolites of a benzazepine (4.84 ppm, Straub et al., 1988) and of mexiletine (4.54 ppm, Turgeon et al., 1992).

As in the investigation of Luo et al. (1994), the occurrence of a quaternary ammonium glucuronide of clozapine could be demonstrated, and attachment of the glucuronosyl group to the terminal nitrogen of the N-methylpiperazine ring was additionally confirmed by Hartman-Hahn rotating-frame nuclear Overhauser effect spectroscopy. In analogy to the N⁺-glucuronides of other tertiary amine drugs (Fischer and Breyer-Pfaff, 1995; Mey et al., 1999; Kowalczyk et al., 2000), the clozapine metabolite was hydrolyzed by -glucuronidase from H. pomatia as well as from E. coli. In contrast, the tertiary N-glucuronide derived from the closely related olanzapine was resistant to these enzymes (Kassahun et al., 1997).

Partial degradation of the piperazine ring to the ethylenediamine derivative EDA-BZD was not unexpected in view of analogous biotransformation reactions in piperazine-substituted phenothiazines (Breyer et al., 1974) and antihistamines (Gaertner et al., 1973). The ethylenediamine derivatives accumulated in animal organs on chronic drug administration (Gaertner et al., 1973, 1975) and their urinary excretion by patients was sustained for several weeks after termination of treatment (Breyer and Gaertner, 1973). Experiments in rats have not revealed accumulation of the respective CLZ metabolites (G. Wiatr, H. Gaertner, and U. Breyer-Pfaff, unpublished results). Metabolites resulting from substitution of the methylthio or methylsulfone group for Cl at C-8 (Stock et al., 1977) were not found in the present analyses nor in those reported by Dain et al. (1997).

The present investigation did not aim at quantitation of the metabolites. These were purified by several sequential steps for which recoveries have not been measured. The rough estimates of quantities given in Tables 1 and 2 are meant to mirror relative amounts. Absolute values were considerably lower than those published by Dain et al. (1997) based on a single HPLC separation. On the other hand, peaks obtained by these authors may have contained minor metabolites in addition to the major ones that were identified.

The present results indicate that in addition to the major hydroxylation products at C-7 and C-8 of the aromatic system, patients treated with clozapine excrete minor ones substituted at the terminal nitrogen of the piperazine ring and at C-6 and C-9 of the ring system. The latter phenols may be analogs of the C-6 and C-9 glutathione derivatives that originated via a nitrenium ion on incubation of clozapine with hypochlorous acid or activated human neutrophils (Liu and Uetrecht, 1995). For phenols produced in parallel, the exact position of the substituents could not be determined. The authors stressed the possibility that oxidation to a nitrenium ion is the first step to covalent binding, which may result in clozapine-induced agranulocytosis. Thus, the tentative identification of 6- and 9-hydroxylated metabolites in urine is the first indication of such a bioactivation pathway in patients, possibly occurring in neutrophils.

In conclusion, isolation of clozapine metabolites from patient urine by sequential chromatographic steps and structural elucidation by instrumental analysis led to the detection of several hitherto unknown products resulting from hydroxylation of CLZ, DMCLZ, or CLZ-NO at C-7 or C-8 and probably also at C-6 or C-9 followed by conjugation with glucuronic or sulfuric acid, from glucuronidation of a hydroxylamine derivative of DMCLZ and from partial degradation of the piperazine ring. It is not known whether any of these metabolites is of pharmacological or toxicological importance.