Haloperidol and its tetrahydropyridine derivative (HPTP) are metabolized to potentially neurotoxic pyridinium species in the baboon

Life Sciences, Vol. 59, No. 17, pp. 1473-1482, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights resewed @X24-3205/% $15.00 + .oO

ELSEVIER

PI1 SOO24.3205(96)00475-4

HALOPERIDOL AND ITS TETRAHYDROPYRIDINE DERIVATIVE (HPTP) ARE METABOLIZED TO POTENTIALLY NEUROTOXIC PYRIDINIUM SPECIES IN THE BABOON

Kathryn M. Avent ‘, Etsuko Usuki *, Darryl W. EyleslJ, Ronel Keeve4, Comelis J. Van der Schyf2X4.“,Neal Castagnoli, Jr* and Susan M. Pond’.

iCentre for Clinical and Experimental Therapeutics, Dept of Medicine, Univ of Queensland, Princess Alexandra Hospital, Brisbane, Australia, 4102; *Dept of Chemistry and the Harvey W. Peters Center, Virginia Polytechnic Institute and State Univ, Blacksburg, VA 24061, USA; 3Clinical Studies Unit, Wolston Park Hospital, Brisbane, Australia; 4Dept of Pharmaceutical Chemistry, Potchefstroom Univ for Christian Higher Education, Potchefstroom 2520, South Africa. (Received in final form August 21, 19%) Summary

The in viva metabolic fate of haloperidol (HP) and its tetrahydropyridine analog HPTP have been examined in the baboon to investigate the formation of potentially neurotoxic pyridinium metabolites that have been observed previously in humans. Urine samples collected from baboons treated with HPTP were shown to contain, in addition to the parent drug, the corresponding reduced HPTP (RHPTP), generated by reduction of the butyrophenone carbonyl group. RHPTP was characterized by comparison with a synthetic standard using HPLC with electrochemical detection and HPLC/MS/MS. Another compound identified by LC/MSMS was a glucuronide metabolite of RHPTP. The HP pyridinium (HPP’) and reduced pyridinium (RHPP’) metabolites were shown to be present in urine from both HP and HPTP treated baboons by HPLC using fluorescence detection. The urinary excretion profile of HPP’ and RHPP’ in both groups was essentially identical and, in contrast to that observed in rodents, closely paralleled the profile found in humans treated with HP. These data in the baboon suggest that the metabolic processes involved in the production of the pyridinium metabolites of HP are similar to those in humans. Furthermore, the HPTP-treated baboon may be an appropriate model in which to study the role of pyridinium metabolites in the induction of tardive dyskinesia.

Key Wor&: haloperidol, metabolism, pyridinium aPresent address: VA-MD Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State Univ, Blacksburg, VA 2406 1, USA. Corresponding Author: Kathryn M. Avent, Dept of Medicine, Univ of Queensland, Princess Alexandra Hospital, Ipswich Rd, Brisbane, Qld, Australia. 4102 Tel: + 61 7 3240-5396; Fax: +61 7 3240-5399; Email: [email protected]

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The typical neuroleptic drugs such as haloperidol, 4-(4-chlorophenyl)-4-hydroxy1-piperidino-4fluorophenyl-1-butanone, (HP, 1, Fig. 1) are the mainstay of pharmacotherapeutic treatment of schizophrenia. Unfortunately, they produce a variety of acute and chronic neurological disorders, including tardive dyskinesia (TD). This is a potentially irreversible syndrome of abnormal movements affecting in particular the orofacial and upper truncal musculature. Some post-mortem studies in human brain have suggested an association between TD and striatal damage (1) but it is generally accepted that conventional neuropathologic examination of the human brain does not reveal any lesions in patients with TD. Animal models have been used to reduce the number of confounding variables that pertain in human studies, such as those of the disease, the drug(s) administered, their dose and duration, age, gender, and intercurrent illnesses, and to optimise the preparation of the brain for neuropathologic and ultrastructural examination. Such studies have revealed subtle abnormalities (Z-9). Very recently, Roberts and co-workers (10) treated rats with HP for six months and compared the differences in ultrastructure of the striatum between rats that developed a low or high number of vacuous chewing movements (VCMs), which are used as an indicator of TD, and untreated controls. The dtrastructural abnormalities observed were either more severe or present only in rats with a high VCM rate. Most interestingly, detailed examination of the mitochondria revealed that in the high VCM group they were reduced in number but that those remaining were hypertrophied. The abnormalities did not return to normal by four weeks after the drug was withdrawn. These findings provide compelling support to the hypothesis that we have been testing for the last few years, i.e. that HP produces striatal neurotoxicity; that this is mediated by its pyridinium metabolites; and that these metabolites inhibit mitochondrial respiration in a manner similar to the structurally related pyridinium neurotoxic metabolite, l-methyl-4_phenyipyridinium, (MPP’, 2, Fig. 1) of the well-established pro-toxin, I-methyl-4-phenyl-1,2,3,6_tetrahydropyridine (MPTP, 3, Fig. 1).

CI

(1) HP

CHs-NM

(2) MPP

W-NO-Q

(3) MPTP

+

Fig. 1 Chemical structures of compounds discussed in the text. Subramanyam et al. (11, 12) demonstrated that HP is metabolized to the pyridinium species 4-(4chlorophenyl)l-4-(4-fluoropheny1)-4-oxobutyl-pyridinium (HPP’, 4, Fig. 1) in rats. We have demonstrated the conversion of HP to HPP+ (13) and a second more major species, 4-(4-

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chlorophenyl)l-4-(4-fluorophenyl)-4-hydroxybutyl-pyridi~um (RHPP’, 5, Fig. 1) in humans (13,14). HPP’ is an order of magnitude more potent than MPP’ as an inhibitor at site I of the mitochondrial electron transport chain (15) and is a potent cytotoxin for dopaminergic and serotonergic neurons in cultures of the rat mesencephalon and pons (16). To test the hypothesis further, the development of an animal model is necessary. We initially administered HP (1 mg/kg) to two baboons. However, these doses produced acute sedative effects which led to reduced food and water intake and termination of the study after 40 days. The tetrahydropyridine derivative of HI?, 4-(4-chlorophenyl)-1-4-(4-fluorophenyl)-4-oxobutyl-1,2,3,6tetrahydropyridine (HPTP, 6, Fig. l), is a metabolic intermediate in the conversion of HP to HPP’ in rats (17,18). Subsequent studies have demonstrated that HPTP, which is analogous in structure to MPTP, does not produce the acute neurological effects of HP in mice (19). In preliminary studies we established that HPTP administration to baboons also did not cause the acute sedation observed after HP. The aim of this study was to determine in the baboon whether HPTP is metabolized to the same pyridinium species as HP and whether the urinary metabolite profile in the baboon parallels that seen in humans treated with HP. A positive outcome would indicate that the baboon should provide an appropriate animal model to test the neurotoxicity of these biotransformation products. Materials and Methods Materials. HPTP synthesized in our laboratory (12) and shown to be devoid of HP (by HPLC analysis (20)) was formulated (using autoclaved apparatus) under laminar flow conditions as a solution for injection in hydroxypropyl-b-cyclodextrin (R 8 12 16, Encapsir? HPB, Janssen Biotech N.V., Olen, Belgium) and double distilled, deionized water for injection. The solution was filtered through a 0.45 pm sterile nylon membrane hydrophilic filter unit (Pro-XTM, Lida Manufacturing Corp., Kenosha, WI, USA) before injection. HP chloride was donated by Janssen-Cilag (Beerse, Belgium) and similarly prepared for injection. Racemic RHPP’ chloride was synthesized in our laboratory (19). HPP’ was synthesized by the method of Subramanyam et al. (12). Methanol and acetonitrile were supplied by Mallinkrodt (Paris, KY, USA). All other chemicals were of the highest purity available. Sep-Pak Cl8 extraction cartridges were obtained from Waters (Milford, MA, USA). Glassware used in the quantitative analysis of compounds was pre-coated with alkaline cetrimide to prevent the compounds binding to glass. Synthesis and identljication of 4-(4-chlorophenyl)-I, I-hydroxybutyl-I, 2,3, t%tetrahydropyridine oxalate (RHPTP, 7, Fig. 1). HPTP (0.8 g, ca. 2.23 mmol) was taken up in 30 ml of 50:50 methanol:acetonitrile and placed on ice whilst an excess of sodium borohydride (0.12 g, 3.2 mmol) was slowly added. This mixture was stirred at 0°C for one hour after which any undissolved sodium borohydride was removed by filtration. The solvent was evaporated under vacuum and 15 ml of water was added to the residue. This mixture was extracted with 3 x 20 ml of ether. Removal of the solvent yielded a yellow oil. Oxalic acid (0.23 g, 2.55 mmol) in ethanol was added to precipitate the product as a white solid (0.83 g, 1.85 mmol, 72%) with a melting point of 137-139 “C. The ‘H NMR spectrum was recorded on a Varian Unity 400 MHz NMR spectrometer in deuterated chloroform, observing ‘H at 399.95 MHz. The sample was prepared in a 5 mm tube (Wilmad Glass Company, 528-PP grade) and analyzed at 298 K. The sample was purged with nitrogen gas and the tube sealed under high vacuum prior to spectral acquisition. The spectrum was internally referenced to the solvent resonance. The low-field (7.4 - 8.3 ppm) region of the ‘H spectrum contains two well-resolved symmetrical doublets centered at 7.46 and 7.56 ppm, a multiplet at 7.52 ppm and a triplet at 7.21 ppm. The signals at 7.21 and 7.52 ppm are part of the AA’BB’X system associated with the p-fluorophenyl ring protons. The symmetrical Al3 (simplified AA’BB’) pattern

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centered at 8.00 ppm consists of two doublets centered at 7.46 and 7.56 ppm respectively and represent the p-fluorophenyl ring protons. The methylene protons are located at 1.82, 1.92, 2.66 and 4.81 ppm as two multiplets and two triplets. The tetrahydropyridine ring protons are centered at 2.72, 2.89, 3.31 and 6.29 ppm as two multiplets, a doublet and a triplet respectively. Elemental analysis (C, H and N) yielded the following result: Calculated for CZHZ~O~CINF: C, 61.45; H, 5.61;N, 3.12. Found: C, 61.34;H, 5.60;N, 3.50. Instrumentation. The HPLC system consisted of a model 5 10 pump, 712 autoinjector, 410 electrochemical detector or 470 fluorescence detector. Data analysis was conducted using Maxima 820 software (Waters, Milford, MA USA). A Waters Cl8 Nova Pak column (3.9 mm x 150 mm, 4 pm ID) was used for both HPLC assays. The HPLC/MS/MS analysis was conducted on a Perkin-Elmer, SCIEX API 11Z instrument (Thomhill, Toronto, Canada) operating in positive ionization mode. A 140B solvent delivery system (Applied Biosystems, Ramsey, NJ), 470 fluorescence detector with 5 ~1 flow cell and Maxima software were linked to the system. Data collected from the MS were stored and manipulated on a Macintosh computer using Tune 2.4, Macspec 3.3 and Multiview software (PESCIEX, Thornhill, Toronto, Canada). Studies in the baboon. The study protocols were approved by the Ethics Committee (Medical) (Evaluation Sub-committee for Experimental Animals) of the Potchefstroom University for Christian Higher Education and the Animal Experimentation Ethics Committee of the University of Queensland. Six old (8-9 years) male baboons (Papio ursinus, 26.3 - 37.5 kg) were kept in indoor, temperaturecontrolled cages, on a balanced diet with water ad libitum. Two were given HP (1 mg/kg) and four HPTP (8 mg/kg) as intramuscular injections according to a fixed schedule which alternated the injection site between the quadriceps, deltoid and gluteal sites. The injections were administered three times per week for 40 days in the case of the HP treated animals and five months in the case of the HPTP treated animals. Four control animals received vehicle only using the same regimen. Urine samples were collected after 40 days of treatment for HP-treated baboons and after five months of treatment for the HPTP-treated baboons. The urinary volume was recorded and the pH adjusted to 8.5 with 0.5 M K2HP04. After centrifugation and filtration, an aliquot of the sample was lyophilized, vacuum packed and transported from Potchefstroom to Brisbane. On arrival, the samples were stored at -70°C until analysis. Analysis of pyridinium cornpour& HPP’ and RHPP’ were measured using a modification (14) of the HPLC fluorescence detection method reported previously (21). Briefly, an aliquot of the lyophilized sample was weighed into an Eppendorf tube and dissolved in 750 ~1 0.1% lactic acid (w/v) in water. An equal volume of 0.1% lactic acid in acetonitrile, which contained the internal standard (IS; 4-phenyl-1,4-fluorophenyl-4-oxobutylpyridinium chloride), was added. The sample was vortex mixed, sonicated for 5 minutes and centritiged for 20 minutes. The supernatant was added to 5 mL of 30 mM ammonium acetate buffer and loaded onto Cl8 Sep-pak cartridges preconditioned with 5 mL each of methanol, water and 30 mM ammonium acetate buffer. The columns were washed with 5 mL 30 mM ammonium acetate buffer and 5 mL 50% methanol before eluting with 3 mL 0.5% lactic acid, 0.5% acetic acid in methanol. The eluant was dried down under nitrogen at 70 “C. The sample was reconstituted in 200 ~1 of mobile phase and an aliquot injected onto the HPLC system. The mobile phase consisted of 30:70 acetonitrile:80 mM ammonium acetate buffer, pH 3.0, 0.2% triethyl amine at a flow rate of 1.3 mL/min. The excitation wavelength was set at 304nm and the emmision at 374nm measured.

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Analysis of tetrahydropyridine cornpour&: An HPLC electrochemical detection method assay for the quantitative analysis of HPTP was developed. The mobile phase consisted of 35:65 acetonitrile:30 mM ammonium acetate buffer, pH 5.7, and the electrochemical detector was set at the applied potential of +0.97 V. During the course of these experiments, reduced HPTP, 4-(4chlorophenyl)-l-4-(4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine, (RHPTP, 7, Fig. 1) was identified in the samples using HPLC/MS/MS. Accuracy, precision and recovery data were obtained for HPTP and RHPTP using the extraction technique described for the pyridinium compounds. HP, chosen as the internal standard, was added in the 0.1% lactic acid in acetonitrile. Each sample was analysed in triplicate. Examination of the unknown metabolite evident by electrochemical detection. The unknown compound detected in the tetrahydropyridine assay was isolated from the HPLC system in the absence of the electrochemical detector. The sample collected was evaporated to dryness, reconstituted in acetonitrile and injected into the MS/MS using an 80: 20 methanol:water, 0.05% acetic acid mobile phase. The interface sprayer was set at 4200 V to detect positive ions which were separated according to their mass to charge ratio (m/z). During collision activated dissociation (CAD) experiments, the collision gas used was argon at a thickness of 300 x 1012 molecules cme2. Examination of other unknown metabolites. An in-line HPLC/MS assay was developed to isolate and identify other unknown metabolites in the urine. Chromatographic resolution was obtained using a gradient system in which mobile phase (20% acetonitrile, 80% 10 mM ammonium acetate, 1.O% acetic acid) was increased to 30% acetonitrile over the initial 5 min, remained constant for 10 min then increased to 45% acetonitrile by 60 min. The flow rate was 200 pl/min through a NovaPak Cl8 (2.0 mm x 150mm) column and the sample was split 1:6 to the MS and fluorescence detector, respectively. Fractions corresponding to unknown chlorine-containing peaks were collected from repeated sample injections. These fractions were then resubmitted for hrther analysis by MS/MS using the conditions described above. Results Idenfification of RHPTP. An unknown compound was found in all of the HPTP-treated baboon urine samples. Using HPLC/MS/MS analysis it was identified as 4-(4-chlorophenyl)- 1-4-(4fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP). The spectra of the fraction which corresponded to this peak revealed parent ions (M’) of m/z 360 and 362 (See Fig. 2).

I 41

3601362

,x ‘Z g Sl-

2-

0 200

I

I,

300

/,, , ,, , 400

m/z

(b) 178

, 500

m/z Fig. 2 Mass spectrum (a) and product ion spectrum (b) of the 360 ion for RHPTP treated baboon urine extract. A chemical standard for RHPTP was identical.

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These ions were present in the ratio 3:l which is characteristic of chlorine containing compounds due to the natural abundances of 3’C1 and 37C1.The CAD product spectrum contained fragments at m/z 109, 149 and 178 for both precursor ions. In addition, product ions of m/z 342 and 344 were detected for the parents 360 and 362, respectively. Figure 2 shows the mass spectrum (a) and product ion spectrum (b) of the 360 ion, for an HPTP-treated baboon urine extract. The RHPTP chemical standard yielded spectra identical to those obtained from the urine extract. The strong fragment ion at m/z 178 was assigned to the retro Diels-Alder fragmentation product of tetrahydropyridine ring opening (Figure 3). The proposed fragmentation pattern for RHPTP is presented in Figure 3. Cl

m/z 178 Fig. 3 Proposed fragmentation pattern for RHPTP. Tetrczhydropyridine Assay. The HPLC assay for analysis of the tetrahydropyridine compounds had limits of detection of 0.04 ug/mL and 0.02 ug/mL for HPTP and RHPTP, respectively. Standard curves were linear over the range 0.5 to 12 ug/mL. The within and between day coefficients of variation for the assay and the absolute recoveries of IS, RHPTP and HPTP are presented in Table 1. Figure 4 shows chromatograms of (a) a chemical standard, (b) a control sample, and (c) treated samples extracted and analysed for HPTP and RHPTP.

Absolute Recoveries and Reproducibility Compound

Cone

TABLE I. of the HPLC Assay for HPTP and RHPTP. cv (%)

Recovery

Between-davb 10.63 5.95

Total’

RHPTP

(ug/mL) 0.70 1.12

(% f SD) 93.9 f 8.1 90.0 * 4.7

Within-dav” 6.54 3.13

HPTP

2.34 1.88 3.23

101.3 + 3.4 76.4 f 9.6 84.9 f 4.7

1.90 13.8 5.78

3.54 9.30 3.20

4.02 16.6 6.60

5.58

83.8 + 5.4

3.55

6.80

7.67

IS

0.87 110.1*4.1 1.61 96.7 + 2.6 2.96 84.8 f 4.2 aResults were from three replicate experiments. bResults were from four days. ’ Results were calculated on all data.

12.48 6.72

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(c)

(b)

RHPTP

0

10

5

0

I5

5

10

0

15

5

15

min

min

min

10

Fig. 4 Chromatograms of HPTP and RHPTP in (a) a chemical standard, (b) a control urine sample and (c) an HPTP-treated baboon urine sample. Metabolicprojile of HP in the baboon versus human. Figure 5a shows a typical chromatogram of a chemical standard for HPP’ and RHPP’. In urine obtained from the two HP-treated baboons, the pyridinium metabolites were present as shown in Figure 5b. Also demonstrated is the presence of these compounds in the urine of HPTP-treated baboons (5~) and in human urine from a patient taking 40 mg oral haloperidol daily (5d) (14).

@I

(4 WPP’

RHPP’

RHPP’

IS

d

HPP’

HPP’

-A

J-

I

I

,

5

10

15

min

II: IS

HPP’

0

(cl

I

0

5

10 min

15

0

5

10

min

15

0

5

10

15

min

Fig. 5 Typical HPLC chromatograms showing HPP’, FUIPP’ and IS in (a) a chemical standard, (b) an HP-treated baboon urine sample, (c) an HPTP-treated baboon urine sample and (d) an HP-treated human urine sample. Metabolic profile of HPTP in the baboon. HPTP and RHPTP were present in the urine of all HPTP-treated but not control animals, Similarly, HPP’ and RHPP’ were present in the urine of all HPTP-treated but not control animals. Identification of other unknown metabolites. Compounds which were shown to be chlorine containing using Multiview ion cluster analysis (Fig. 6) were selected for further investigation.

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(4

(h)

L?LPPf

.s?

1,&_+

RHPTP

5

z

HPP’

i 0

I

10

1 HPP’

<

20

30

40

50

1

0

IO

1

20

min

1

I

,

30

40

50

min Fig. 6

HPLUMS chromatograms showing (a) fluorescence response and (b) the isotopic cluster analysis demonstrating containing compounds in an HTTP-treated baboon urine sample.

Typical

detector chlorine

Peak 1 had a m/z of 536/538. The mass spectrum (a) and product ion spectrum (b) of the 536 ion compared to RHPTP (c) are illustrated in Figure 7. Because fragmentation of this compound produced 3601362, 3421344 and 178, 149 and 109 ions, this compound is proposed to be a glucuronide conjugate of RHPTP

(cl

(b)

(a) 536

200

300

400

500

600

I

I

200

400

.

1

600

m/z

m/z

Fig. 7 Mass spectrum, structure (a) and product ion spectrum (b) of the 536 ion of unknown #l in comparison with the product ion spectrum of RHPTP (c).

Discussion The current study has shown that HPP’ and RHPP’ are produced from HP and HPTP in the baboon as they are from HP in humans. We also found RHPTP in the urine of HTTP-treated baboons. The

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reduction of HPTP to RHPTP in these animals is comparable to the conversion of HP to reduced HP (RHP, 8, Fig. 1) in HP-treated schizophrenics (22). In contrast, the metabolism of HP in humans and of HP and HPTP in rats is strikingly dissimilar, because rats lack cytosolic ketone reductases (EC I .2.1). These enzymes are responsible for the reduction of HP to RHP and also of HPP’ to RHPP’ (22, 23). Given that the reduction of HP to RHP in cytosol from human liver and brain is stereospecific (22), it is likely that the reduction of HPTP in the baboon is stereoselective for the S(-) enantiomer. In HP-treated schizophrenics, circulating RHP concentrations increase disproportionately to the dose or concentration of HP (24,25). RHP is reconverted to HP by cytochrome P450 enzymes in a reaction which is saturable (25,26). Whether RHPTP undergoes reversible metabolism to HPTP remains to be established. In conclusion, the neurotoxicity of HPP’ (15,16) and RHPP’ (unpublished results) and their presence in brain (unpublished results), blood, plasma and urine of HP-treated schizophrenics stimulate interesting questions regarding the mechanisms of HP-induced extrapyramidal side-effects such as TD. This study has demonstrated several metabolic similarities between the metabolism of HP in humans and HP and HPTP in baboons, Thus, the HPTP-treated baboon warrants mrther investigation as a model of HP pyridinium metabolite-induced neurotoxicity.

Acknowledgements Supported in Australia by grants from the Princess Alexandra Hospital Research and Development Foundation and the Government Employees Medical Research Foundation of Australia. KA wishes to thank the Queensland and Northern New South Wales Lions Kidney and Medical Research Foundation and the Mental Health Branch, Queensland Health, for providing the tinding for her scholarship. We wish to thank Paul J Taylor for assistance with the LC/MS/MS analyses, DJ Douw G van der Nest, COJ J J Bester and Antoinette Fick at the Experimental Animal Centre, Potchefstroom University for CHE for assistance with the animals. Supported in South Africa by the Medical Research Council (in part) and Potchefstroom University and in the USA by the National Institute of Neuriological and Communicative Disorders and Stroke (NS 28792) and the Harvey W. Peters Research Center for Parkinson’s Disease and Disorders of the Central Nervous System. References 1. E. CHRISTENSEN,

2. 3. 4. 5. 6. 7. 8. 9.

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10. R.C. ROBERTS, L.A. GAITHER X-M. GAO, S.M. KASHYAP and CA. TAMMINGA Synapse, 20 234-243 (1995). 11. B. SUBRAMANY AM, H. ROLLEMA, T. WOOLF, N. CASTAGNOLI JR Biochem Biophys Res Commun 166 238-244( 1990) 12. B. SUBRAMANY AM, T. WOOLF, N. CASTAGNOLI JR Chem Res Tox 4 123-128(1991). 13. B. SUBRAMANY AM, S.M.POND, D.W. EYLES, H.A. WHITEFORD, H.G. FOUDA, N. CASTAGNOLI, JR. Biochem. Biophys. Res. Comm. 181573-578 (1991). 14. D.W. EYLES, H.R. McLENNAN, A. JONES, J.J. McGRATH, T.J. STEDMAN, SM. POND. Clin Pharmacol Ther 56 5 12-520, 1994a. 15. H. ROLLEMA, M. SKOLNIK, J. DENGELBRONNER, K. IGARASHI, E. USUKI, N. CASTAGNOLI JR J Pharmacol Exp Ther 268 3 80-3 87( 1994). 16. J. BLOOMQUIST, E. KING, A. WRIGHT, C. MYTILINEOU, K. KIMURA K. CASTAGNOLI, N. CASTAGNOLI JR. J Pharmacol Exp Ther 270 822-830( 1994). 17. J. FANG, J.W. GORROD. Toxic01 Lett 59 117-123(1991). 18. J. FANG, J.W. GORROD. Med Sci Res =175-177(1992). 19. C.J. VAN DER SCHYF, K. CASTAGNOLI, E. USUKI, H.G. FOUDA J.M. RIMOLDI, N. CASTAGNOLI., JR. Chem Res Tox 2 281-285(1994) 20. D.W. EYLES, H.A. WHITEFORD, T.J. STEDMAN, SM. POND. Psychopharmacology 106 268-274 (1992). 21. K. IGARASHI, and N. CASTAGNOLI, JR. J. Chromatogr. Biomed. Appl. 579 277-283 (1992). 22. D.W. EYLES and S.M. POND. Biochem Pharmacol 44 867-871(1992). 23. D.W. EYLES, J.J. McGRATH and S.M. POND. Psychopharmacol (In Press). 24. J.L. BROWNING, C.A. HARRINGTON, C.M. DAVIS. J Immunoassay 6 45-66 (1985). 25. D.W. EYLES, T.J. STEDMAN and SM. POND. Psychopharmacology116 161-166 (1994b). 26. T. INABA and J. KOVACS. Drug. Metab. Dispos. 17 330-333 (1989).