Interactions of Iprindole with Fenfluramine 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 norfenfluramine. 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 amphetamine 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 tryptophan hydroxylase Maynard, 1968; Mid a et al 1983). Because of its structural activity (Steranka and Sanders-Bush, 1979). Tolerance to similarity to amphet amine, 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 antidepressant 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 toluene 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.