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Interactions of with Metabolism in Rat Brain and Liver

K.M. Hegadoren, G.B. Baker, R.T. Coutts and W. G. Dewhurst Neurochemical Research Unit, Department of Psychiatry Mackenzie Centre, University of Alberta. Accepted: February 21, 1991

An assay procedure utilizing electron-capture gas chromatography was developed for simultaneous analysis of fenfluramine and . This method was applied to brain and liver samples from rats which had been injected with fenfluramine with or without pretreatment with iprindole. The tissues from rats treated with fenfluramine showed extensive formation of norfenfluramine, consistent with findings reported previously in the literature. Pretreatment with iprindole led to an increase in brain and liver levels of fenfluramine, and, unexpectedly, to a marked decrease in levels of norfenfluramine in these tissues. These findings suggest that iprindole blocks N-deethylation and that it may be a useful tool with which to study the effects of fenfluramine in the absence of norfenfluramine. The results also emphasize the importance of considering drug-drug interactions in future research on fenfluramine.

Keywords: Iprindole, fenfluramine, norfenfluramine, gas chromatography, metabolism

Fenfluramine (FEN) is a substituted which Metabolism may play an important role in the actions is used as an anorexient (Garattini et al 1986) and, more of FEN. Extensive N-deethylation of FEN to norfenfluram- recently, as a possible therapy in the management of autism ine (NORFEN) has been reported for several species, in- (Ritvo et al 1986). This drug is also being investigated as cluding man, rat, mouse, dog and guinea pig (Beckett and a possible peripheral probe of central 5-hydroxytryptamine Salmon, 1972; Jori et al 1978; Caccia et al 1981, 1982, (5-HT) activity (Siever et al 1984), although there is still 1985; Fuller et al 1988; Spinelli et al 1988). NORFEN shares considerable controversy in this regard (Asnis et al 1988). anorectic and other properties with the parent drug and It has been known for some time that FEN causes a long- produces similar effects to FEN on 5-HT, but it is not lasting depletion of brain levels of 5-HT and its metabolite established to what extent this deethylated metabolite con- 5-hydroxyindole-3-acetic acid [5-HIAA1 (Harvey and tributes to the overal I profile in the in vivo situation (Fuller McMaster, 1977; Clineschmidt et al 1978; Steranka and et al 1988). NORF N has been reported to be further Sanders-Bush, 1979; Schuster et al 1986) and of 5-HT metabolized to acidi and alcoholic metabolites (Bruce and reuptake (Schuster et al 1986) and hydroxylase Maynard, 1968; Mid a et al 1983). Because of its structural activity (Steranka and Sanders-Bush, 1979). Tolerance to similarity to amphet , a drug known to undergo ex- the anorectic effects of FEN develops rapidly, both in animal tensive ring hydroxylation (Costa and Garattini, 1970), it models and in humans (Pinder et al 1975), and it has been is of interest to investigate the possibility of ring hydrox- suggested that this tolerance may be due to a selective long- ylation of FEN. The electron-withdrawing property of the lasting depletion of 5-HT (Kleven et al 1988). trifluoromethyl constituent on the aryl ring of FEN de- activates the ring for lectrophilic substitution, thus reducing the likelihood of rin hydroxylation, but this has not been Address reprint requests to: Dr. G.B. Baker, Neurochemical Research investigated extensi ely to our knowledge. To investigate Unit, Department of Psychiatry, Mackenzie Centre, University of this possibility, we have developed a gas chromatographic Alberta, Edmonton, Alberta, Canada, T6G 2B7. procedure for simultaneous analysis of fenfluramine and

J Psychiatr Neurosci, Vol. 16, No. 1, 1991 6 Journal of Psychiatry & Neuroscience VoL 16, No. 1, 1991

norfenfluramine and have studied the effects of iprindole, eries were obtained whether the internal standard was added an drug known to block ring hydroxylation during or after homogenization, so for convenience in routine of amphetamine (Freeman and Sulser, 1972; Fuller and analysis it was added after homogenization and centrifu- Hemrick-Luecke, 1980; Steranka, 1982), on brain and liver gation. After the addition of ethyl acetate (2 ml), the samples levels of FEN and NORFEN. The results of those exper- were shaken (5 min) and centrifuged briefly on a benchtop iments are described here. centrifuge. The top layers were transferred to smaller test tubes and taken to dryness in a warm water bath under METHODS a gentle stream of nitrogen. Ethyl acetate (25 ,ul) and pentafluoropropionic anhydride (PFPA) (75 ,ul) were added. Expenmental Design The mixtures were vortexed and the samples were placed in a heating block for 30 min at 60°C. The samples were Procedures involving use of rats were approved by the allowed to cool at room temperature for 10 min, and University of Alberta Health Sciences Animal Welfare (300 ,ul) and saturated sodium borate (3 ml) were added Committee and were conducted according to the guidelines to each. After vortexing briefly, the tubes were centrifuged established by the Canadian Council on Animal Care. and the phases separated. The toluene layer was retained Six animals were assigned at random to each of5 different in each case and an aliquot (1 ,ul) employed for GC analysis. treatment groups. To compensate for any possible handling All samples were refrigerated and analyzed the following effects, two single-injection treatment groups were included: day. single saline and single FEN injections. The other treatment A Hewlett-Packard (HP) 5880A gas chromatograph groups were: saline injection followed I h later with FEN equipped with a fused silica SP 2100 capillary column (1 Sm, or iprindole followed I h later with saline or with FEN. 0.25 mm ID, 0.25 ,um phase thickness) supplied by Supelco The iprindole + saline treatment group was included to (46-220 Wyecroft Road, Oakville, Ontario, Canada, L6K examine the possibility that a metabolite of iprindole had 3V ) and an electron-capture detector was employed. The a similar retention time to that of derivatized FEN or instrument was interfaced with a HP 5880A Series GC NORFEN. The animals were sacrificed (by stunning and Terminal (level 4) integrator to measure peak heights. All immediate decapitation) at 1, 2, 4 or 8 h after the last samples, I ,ul each, were injected manually. Helium (2 ml/ injection and the brains and livers were removed and frozen min) was used as the carrier gas and 5% methane in argon in containers on solid carbon dioxide. Doses of FEN.HCI (35 ml/min) was used as the make-up gas at the detector. and iprindole.HCl used were 6.7 mg/kg (0.025 mmol/kg) The injector port and detector temperatures were 200°C and 12.5 mg/kg (0.039 mmol/kg) respectively. and 300°C respectively. The initial oven temperature was Acute drug administration was made via intraperitoneal set at 105°C and then programmed to rise at a rate of injection using a tuberculin I cc syringe equipped with a I 0C/min after an initial time delay of 0.5 min. At 8 min, 27G 1/2" needle (Becton Dickinson, Closter, N.J., U.S.A.). the oven temperature increased to 230°C and was held at All injected drugs were dissolved in saline solution (0.85% that temperature until the individual run stopped at 11 min. w/v sodium chloride) (Fisher Scientific Co.) such that the A set of authentic standards was run in parallel with rats received 2 ml/kg. Animals were injected in random each assay to provide a calibration curve. This 8-point curve order. was prepared by adding varying, equal amounts of FEN and NORFEN (0- 1000 ng) and a fixed amount of internal Tissue Preparation standard (the same amount as added to the tissue extract samples) to 0.1 N perchloric acid in a set of tubes run in Samples of brain and liver were weighed, and 5 volumes parallel. This range of standards was sufficient to cover of ice-cold 0. IN HC104 were added. The samples were the concentrations of both FEN and NORFEN in the tissue homogenized and then centrifuged in a refrigerated centri- samples. In initial experiments, we compared spiked super- fuge (10 min at 15,000 x g). A portion (200 ,ul) of the natants of control homogenates and spiked perchloric acid supematant was used in the assay. and found no difference in recoveries, so spiked perchloric acid was utilized for preparing subsequent calibration curves. At the end of the assay, the peak height ratios of derivatized Assay for Fenfluramine and Norfenfluramine FEN and NORFEN to that of derivatized internal standard were determined and plotted. Using the same peak height To each tube, 25% K2CO3 (100 ,AI) was added. To this ratios in the extracted tissue samples from the drug-treated was added 700 ,ul of 2.5% K2CO3 to make up the volume rats and extrapolating on the calibration curve, the amount to 1 ml. The solution was vortexed briefly, and the internal of FEN and NORFEN in each sample was determined. The standard, p-chlorophentermine (2 ,ug), was added. In pre- analytical procedure developed provided for the simultane- liminary experiments it was determined that similar recov- ous analysis of FEN and NORFEN. March 1991 Iprindole and Fenfluramine Metabolism 7

Statistical Analysis for typical GC traces). The assay procedure was linear and reproducible, with correlation coefficients > 0.99 resulting The standard error of the means (S.E.M.) is represented routinely over the range 6.25-1000 ng and interassay by error bars on all figures. Two-way analysis of variance coefficients of variation of <10% obtained. Structures of was employed for pairwise comparisons between experi- the derivatives were confirmed by combined gas mental groups. A pairwise post hoc comparison test, the chromatography-mass spectrometry. Figures 3,4 and 5 show Tukey test, was carried out on the vehicle + FEN and the the proposed fragmentation of the derivatives of FEN, iprindole + FEN treatment groups to calculate the critical NORFEN and p-chlorophentermine, respectively. Although difference required to achieve statistical significance. For molecular ions were absent, the presence of other fragments the calculations, the p value was 0.05 (Kiess, 1989). allowed confirmation of structure in each case. Results from the single FEN injection and vehicle + FEN RESULTS injection test groups showed no statistical difference in FEN and NORFEN levels between these two test groups. An The pentafluoropropionate derivatives provided good examination of gas chromatographic traces of the iprindole sensitivity (<20 pg "on-column") and stability and possessed + vehicle test group showed no peaks at the retention times excellent chromatographic properties (see Figures I and 2 of derivatized FEN and NORFEN.

C c N CY

Ql) cii axc a) Cl) c 0Q. 0 at) a aVl) L atb L. L. N4* 0 0 L- u 1- 1 0 u Q) C] I 0

8A 2rl I I Mm6m 0 5 10 =I"a Time (min) 0 5 10 Time (min) Fig. 1: Typical GC traces of extracted brain tissue. Upper trace = extract from a rat treated with vehicle + Fig. 2: Typical GC traces of extracted liver tissue. Upper trace fenfluramine (drug treatment schedule described in = extract from a rat treated with vehicle + fenfluramine; the text); lower trace = extract from a rat treated with lower trace = extract from a rat treated with vehicle vehicle + vehicle. a, b and c are peaks corresponding + vehicle. a, b and c are peaks corresponding to to derivatized norfenfluramine, fenfluramine and derivatized norfenfluramine, fenfluramine and p-chlorophentermine, respectively. No peaks corres- p-chlorophentermine, respectively. No peaks corres- ponding to derivatized p-chlorophentermine were ponding to derivatized p-chlorophentermine were present in unspiked brain extracts. present in unspiked liver extracts. 8 Journal of Psychiatry & Neuroscience VoL 16, No. 1, 1991

CH CHN,COC2 5 CH3 §2- CH21 C / CH2-C-NHCOC2 F5 CF3 3 CH3 M' m/z 377 (absent) Mtmrrz 329(O.2)/331 (absent)

b 2 - CI 4 H2 CH3s + CF.ii C=NHCOC2 F5 )CH3CH = N ,NICHCH CH3 3 CH2CH3 rVz 204 1100.0) - CH2=CH2I Wz 216 1100.0) - C2F5CONH2 )- CI\/C 111CH= -C'3NCH CH3CH = NHCOC2 F5 m/2- 166 1 11.21/161 14.1 )I m/z 190 133.9) CH - - 3>CNHCOC2 F5 - CH CHN<(COC 2F5 CH3 -2 + C2H5 ) Cl CH2 Q- CH2 nVz 125 110.1)/127 13.6) CF3 Wzm 159 116.6) Fig. 5: Proposed mass spectrometric fragmentation ofthe PFP derivative of p-chlorophentermine. - CF2 CF3

m/z 119 110.7) The brain concentrations of FEN and NORFEN after i.p. administration of FEN, 0.025 mmol/kg, with and without Fig. 3: Proposed mass spectrometric fragmentation of the iprindole pretreatment, are illustrated in Figure 6. By I h pentafluoropropionate (PFP) derivative of fenflnu- in the animals without iprindole pretreatment, there was ramine. Values in parentheses represent % relative significant FEN accumulation in the brain tissue. Also by abundance. this time, the level of NORFEN indicated that FEN was rapidly metabolized. The amount of NORFEN leveled off CH2CHNHCOC2F5 by 2 h and remained essentially at that level for the remaining periods studied. The corresponding hepatic levels of FEN CF3 CH3 and NORFEN are given in Figure 7. The pattern ofdeclining levels of FEN over the 8 h period in the liver was similar Mt m/z 349 (absent) to that in the brain. The initial level of NORFEN was higher than in brain, likely reflecting, in part, a first-pass metabolism was at a level _ QvCH2 effect. NORFEN present significantly higher than FEN by 2 h and remained so throughout the course CF3 of the experiment. CH3CH = NHCOC2 F The brain concentrations of FEN and NORFEN after uz I9o 1)00.0) iprindole + FEN are also shown in Figure 6. Statistically significant large increases (relative to the vehicle + FEN- treated rats) were seen in the levels of FEN, but, unex- - C2F5CONH2 ) CH=CHCH31 pectedly, the NORFEN levels were profoundly decreased. There was some recovery of NORFEN levels by 8 h, but CF3 Wz 166 131.71 the level of NORFEN still remained significantly below that of controls (i.e. vehicle + FEN-treated) at this time interval. - CH3 CHNHCOC2 Fs Qe-CH2 Figure 7 shows the hepatic levels of FEN and NORFEN in the rats that were pretreated with iprindole and then given CF3 FEN. The same pattern is seen as with the brain in that Vz 159 116.31 maximum levels of FEN were seen at 2 h then gradually decreased at 4 h and 8 h. The 1 h level of FEN was higher ) CF2 CF3 in iprindole + FEN-pretreated rats than in the vehicle/FEN- /2z 119 121.01 treated rats but failed to reach statistical significance. NORFEN levels were again significantly decreased relative Fig. 4: Proposed mass spectrometric fragmentation ofthe PFI to the vehicle + FEN groups, with some recovery apparent derivative of norfenfluramine. at 8 h. March 1991 Iprindole and Fenfluramine Metabolism 9

ng/gm tissue consistent with the reported T112 of 4 h in brain tissue for this drug (Rowland and Carlton, 1986). By 4 h, the FEN 10000 level had dropped to less than 50% ofthe FEN level reported at 1 h. The rate of disappearance of NORFEN has been reported to be slower than that of FEN (e.g., Rowland and Carlton, 1986), and the results of the present study are in agreement with those reports. 4000 It was expected that, if ring hydroxylation did contribute to metabolism of FEN and/or NORFEN, treatment with 20D0O iprindole + FEN would result in an increase in concentrations of both FEN and NORFEN relative to those in rats treated with vehicle + FEN. There was, indeed, an increase in FEN I 2 4 tome levels observed in both brain and liver. However, unexpect- edly, there was a marked decrease in NORFEN levels, Fig. 6: Whole brain levels of fenfluramine and norfenflura- resulting in an elevated FEN/NORFEN ratio in the rats mine after administration of (±)-fenfluramine to pretreated with iprindole, suggesting that iprindole has a saline-pretreated or iprindole-pretreated rats. The rats much greater influence on N-deethylation than on ring were treated with saline vehicle or with iprindole.HCI hydroxylation of FEN. It would be expected that if iprindole (12.5 mg/kg i.p.), then with (±)-fenfluramine.HCI was blocking ring hydroxylation of FEN, levels of both FEN (6.7 mg/kg i.p.) 1 h later; the rats were then killed and NORFEN would increase since there would be more at the times shown above after the (±)-fenfluramine FEN available to be converted to NORFEN. The present administration. Values represent ng/g of tissue (mean results suggest that: (1) iprindole is blocking N-deethylation ± SEM, n=6). * p < 0.05, Tukey test, values compared to those in vehicle + fenfluramine-treated rats. Bars of FEN to NORFEN and/or (2) the increased FEN available represent in each case: fenfluramine levels, with vehicle after iprindole + FEN is in such a location in the brain or iprindole pretreatment before fenfluramine treat- that it is not readily available for N-deethylation. If (2) were ment dotted lines and no hatching, respectively; and the case, one would still expect to see similar amounts of norfenfluramine levels, with vehicle or iprindole pre- NORFEN in the vehicle + FEN-treated animals and the treatment before fenfluramine treatment double iprindole + FEN-treated rats. However, NORFEN levels are hatching and single hatching, respectively. much lower in the latter case than in the former, particularly at 2 h, suggesting that iprindole is causing considerable inhibition of N-deethylation of FEN. In addition, the similar pattern of changes in FEN and NORFEN in liver suggested that situation (2) was unlikely. If the interaction between iprindole and FEN in this study is indeed the result of inhibition of N-dealkylation, it is one of the few examples of such inhibition by a drug. Most of the other widely studied interactions in the literature show inhibition of aromatic or aliphatic hydroxylation reactions or of 0-dealkylation. Although , and other MAO inhibitors do block N-dealkylation reactions, E i , these drugs have been shown to inhibit many types of 1 2 4 S metabolic reactions involved in a variety of drugs (Belanger

tIm and Atitse-Gbeassor, 1982; Eade and Renton, 1970; Clark and Thompson, 1972). If iprindole can be shown to have Fig. 7: Hepatic levels of fenfluramine and norfenfluramine more selective actions than tranylcypromine or phenelzine, after administration of (±)-fenfluramine to saline- there is potential use of this drug as a probe to explore pretreated or iprindole-pretreated rats. Dosing sche- the multifaceted cytochrome P-450 metabolizing system. dule was as described in Figure 6. Values represent If the marked decrease in NORFEN levels produced by ng/g of tissue (mean ± SEM, n=6). * p < 0.05, Tukey iprindole could be maintained over time (e.g. by using higher test, values compared to those in vehicle + or more frequent doses of iprindole), further work could fenfluramine-treated rats. Hatching in the bars is as described in Figure 6. be done examining specific neurochemical and behavioral effects of FEN separately from the usual presence of NORFEN. This would provide in vivo data important to DISCUSSION the ongoing discussions surrounding tolerance effects, peri- pheral vs CNS effects and neurotoxicity. The pattern of decrease in the amount of FEN in the Racemic FEN was used in this study, but future exper- vehicle + FEN-treated rats over the 8 h study period is iments should examine both (+)-FEN and (-)-FEN. It is 10 Journal of Psychiatry & Neuroscience Vol. 16, No. 1, 1991

not known if iprindole affects the two enantiomers differ- Arvela P, Karki NT, Nieminen L, Bjondahl K, Mottonen ently, or if recovery from metabolic inhibition occurs pre- M (1973) Effect of long-term fenfluramine treatment on ferentially with one enantiomer. drug metabolism in rat. Experientia 15:(4)454-455. The increases in the combined levels of FEN and Beckett AH, Salmon JA (1972) Pharmacokinetics of ab- NORFEN in the iprindole + FEN-treated animals compared sorption, distribution and elimination of fenfluramine and with levels seen in the vehicle + FEN-treatment groups its main metabolites in man. J Pharm Pharmacol 24:108- suggest that iprindole may also interfere with further 114. NORFEN metabolism. Additional studies are warranted to Belanger PM, Atitse-Gbeassor A (1982) Inhibitory effect examine the time course of the observed increases in the of phenelzine on oxidative microsomal enzyme systems combined levels of FEN and NORFEN. of rat liver. Can J Physiol Pharmacol 61:524-529 Arvela (1973) reported that FEN appeared to have Bruce RB, Maynard WR (1968) Fenfluramine metabolism. enzyme-inducing effects in the liver. Thus, the results J Pharm Sci 57:1173-1176. obtained in the present study might reflect a complex Caccia S, Ballabio M, Guiso G, Garattini S (1982) Species interaction between effects of FEN on iprindole metabolism differences in the kinetics and metabolism offenfluramine and effects of iprindole on FEN metabolism. As Coutts et isomers. Arch Int Pharmacodyn 238:15-28. al (1990) have pointed out, very little is known about the Caccia S, Conforti I, Duchier J, Garattini S (1985) Phar- metabolism of iprindole itself; these authors recently iden- macokinetics of fenfluramine and norfenfluramine in tified fourteen metabolites of iprindole in urine of iprindole- volunteers given D- and DL-fenfluramine for 15 days. treated rats. Eur J Clin Pharmacol 29:221-224. There are clinical implications to the findings ofthis study. Caccia S, Dagnino G, Garattini S, Guiso G, Madonna R, often coexists with in clinical popula- Zanini MG (1981) Kinetics of fenfluramine isomers in tions, and the pharmacological treatment could include both the rat. Eur J Drug Metab Pharmacokinet 6:297-301. FEN and iprindole or another antidepressant (tricyclic Clark B, Thompson JW (1972) Analysis of the inhibition such as have been reported to of N-demethylation by have marked effects on metabolism of other drugs). The inhibitors and some other drugs with special reference resultant change in the FEN/NORFEN ratio could result to drug interactions in man. Br J Pharmacol 44:89-99. in a dramatically altered therapeutic and/or side effect Clineschmidt BV, Zacchei AG, Totaro JA, Pflueger AB, profile. McGuffin JC, Wishousky TI (1978) Fenfluramine and In summary, the present results suggest that iprindole brain . Ann NY Acad Sci 305:222-241. is an inhibitor of N-deethylation of FEN, an action which Costa E, Garattini S (eds) (1970) International Symposium may have important clinical implications for the future use on and Related Compounds. Proceedings of FEN and for studying the actions of FEN in the absence of the Mario Negri Institute for Pharmacological Re- of its metabolite NORFEN. search, New York: Raven Press. Coutts RT, Hussain MS, Baker GB, Daneshtalab M, Micetich ACKNOWLEDGEMENTS RG (1990) Structural characterization of the urinary metabolites of iprindole in the rat. Xenobiotica (in press). Iprindole was a gift from Wyeth Research (U.K.) Ltd., Eade MR, Renton KW (1970) Effect of monoamine oxidase Taplow, Maidenhead, Berkshire, U.K. and fenfluramine and inhibitors on the N-demethylation and hydrolysis of norfenfluramine were kindly provided by A.H. Robins Co. meperidine. Biochem Pharmacol 19:2243-2250. Inc., Richmond, Va. Freeman JJ, Sulser F (1972) Iprindole-amphetamine inter- Funding for this work was provided by the Alberta Mental actions in the rat: the role of aromatic hydroxylation of Health Research Fund and the Medical Research Council amphetamine in its mode of action. J Pharmacol Exp of Canada. The authors are grateful to Drs. A. Carroll and Ther 183:307-315. D.P. Boisvert for useful discussions, to Ms. S. Omura for Fuller R, Hemrick-Luecke S (1980) Long-lasting depletion typing this abstract and to Ms. J. van Muyden for assisting of striatal by a single injection of amphetamine in the preparation of the figures. in iprindole-treated rats. Science 209:305-307. Fuller RW, Snoddy HD, Perry KW (1988) Metabolism of fenfluramine to norfenfluramine in guinea-pigs. J Pharm Pharmacol 40:439-44 1. Garattini S, Mennini T, Bendotti C, Invernizzi R, Samanin REFERENCES R (1986) Neurochemical mechanism of action of drugs which modify feeding via the serotoninergic system. 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