Inhibition of Monoamine Oxidase in 5

Br. J. Pharmac. (1985), 85, 683-690 Inhibition ofmonoamine oxidase in 5- hydroxytryptaminergic neurones by substitutedp- aminophenylalkylamines Anna-Lena Ask, Ingrid Fagervall, L. Florvall, S.B. Ross1 & Susanne Ytterborn Research Laboratories, Astra Likemedel AB, S-151 85 Si3dertilje, Sweden 1 A series ofsubstituted p-aminophenethylamines and some related compounds were examined with regards to the inhibition ofmonoamine oxidase (MAO) in vivo inside and outside 5-hydroxytryptaminergic neurones in the rat hypothalamus. This was recorded as the protection against the irreversible inhibition of MAO produced by phenelzine by determining the remaining deaminating activity in the absence and presence ofcitalopram using a low (0.1 yIM) concentration of ['4CJ-5-hydroxytryptamine (5-HT) as substrate. 2 Some ofthe phenethylamines were much more potent inside than outside the 5-hydroxytryptaminergic neurones. This neuronal selectivity was antagonized by pretreatment of the rats with norzimeldine, a 5-HT uptake inhibitor, which indicates that these compounds are accumulated in the 5-HT nerve terminals by the 5-HT pump. 3 Selectivity was obtained for compounds with dimethyl, monomethyl or unsubstituted p-amino groups. An isopropyl group appears to substitute for the dimethylamino group but with considerably lower potency. Compounds with 2-substitution showed selectivity for aminergic neurones and this effect decreased with increased size of the substituent. The 2,6-dichloro derivative FLA 365 had, however, no neuronal selective action but was a potent MAO inhibitor. Substitutions in the 3- and 5- positions decreased both potency and selectivity. 4 Prolongation ofthe side chain with one methylene group abolished the preference for the MAO in 5-hydroxytryptaminergic neurones although the MAO inhibitory potency remained. The selectivity disappeared by increasing the a-substituent to an ethyl group but remained for the oco-dimethyl substituted derivatives. 5 It is concluded that compounds which are (1) transported by the 5-HT pump and (2) potent reversible MAO-A inhibitors produce pronounced inhibition of MAO in 5-hydroxytryptaminergic neurones. Introduction The development ofspecific inhibitors ofthe transport tosomes. This technique has also made it possible to mechanisms responsible for the re-uptake of released examine whether irreversible monoamine oxidase transmitter amines in the monoaminergic nerve ter- (MAO) inhibitors have any preference for the enzyme minals, has made it possible to measure the deamina- inside the specific aminergic nerve terminals compared tion of radiolabelled biogenic amines inside specific with that outside these terminals (Fagervall & Ross, aminergic synaptosomes when a low concentration of unpublished observations). Since, reversible MAO the substrate is added to a synaptosomal preparation inhibitors protect the enzyme against irreversible (Ross & Ask, 1980; Ask et al., 1982c; 1983; 1984). inhibitors, it is also possible to determine the in vivo Thus, the difference between the deamination of the affinity of the former for MAO inside and outside the added substrate in the absence and presence of the aminergic neurones if the enzyme assay is performed uptake inhibitor is a measure ofthe deamination ofthe with a synaptosomal preparation, in the absence and substrate after being transported into these synap- presence of selective inhibitors of amine uptake, at a time when the reversible effect has terminated (Ross & ' Correspondence. Ask, 1980; Ask et al., 1982a; 1983; 1984). ) The Macmillan Press Ltd 1985 684 A.-L. ASK et al. In previous studies we have observed that some (1982b) with [14C]-5-HT (50 gM) and ['4C]-pheneth- reversible MAO-A inhibitors protect MAO within the ylamine (PEA) (2.511M) as substrates. aminergic neurones in the rat brain against phenelzine The deamination of ['4C]-5-HT by the synap- at lower doses than required to protect MAO outside tosomal preparation was determined as described these neurones (Ask et al., 1983; 1984). Of particular previously (Ask et al., 1983) with some modifications. interest were the findings that amiflamine and N- Fifty microlitres of the synaptosomal preparation, desmethylamiflamine ((+ )-FLA 788) showed sig- corresponding to 2.5 mg wet weight of tissue, was nificant preference for MAO inside 5-hydroxytryp- added to glass-stoppered centrifuge tubes containing taminergic and noradrenergic nerve terminals in the 925 gi ofKrebs-Henseleits buffer, pH 7.4, equilibrated rat hypothalamus whereas N,N-didesmethylami- with 93.5% 02 and 6.5% CO2, and containing glucose flamine (( + )-FLA 668) was a very selective inhibitor 5.6 mM, ascorbic acid 1.1 mM, Na2 EDTA 0.13 mM, of MAO in noradrenergic nerve terminals (Ask et al., maprotiline 1I M and amfonelic acid 0.3 gAM. Each 1983; 1984). Since pretreatment of the rats with preparation was examined in duplicate in the absence norzimeldine or desipramine which are selective in- and presence of citalopram 0.12pM. After 10min hibitors of 5-hydroxytryptamine (5-HT) and incubation in a water bath at 370C, 25 I1 of[14C]-5-HT noradrenaline uptake respectively, antagonized the (O.1 M final concentration) was added and the in- preference for MAO of these monoaminergic nerves, cubation was continued for 10 min. The reaction was the neuronal selectivity appears to be due to accumula- stopped by addition of 1 ml of 1 M HCL. The ['4C]-5- tion of the inhibitors within the aminergic nerve hydroxyindoleacetic acid (5-HIAA) formed was ex- terminals via the membranous uptake mechanisms. tracted into 6 ml of ethyl acetate by vigorous shaking These findings indicate that it may be possible to for 2 x 1 min in a multi-tube vortexer (model 2601, develop MAO inhibitors with a specific action within a Scientific Manufacturing Industries). After cen- certain amine system. Hence, a compound has to fulfil trifugation 4 ml of the organic layer were taken into two requirements in order to be a neuronally selective 10 ml Econofluor (NEN) and 1 ml ethanol and the MAO inhibitor in a specific amine system: it has to be radioactivity measured in a Packard Tri Carb scintilla- (1) a potent MAO-A inhibitor and (2) transported by tion counter. The rate of deamination is expressed as the amine pump. In the present study we have re- nmol ['4C]-5-HIAA formed per g wet weight of tissue examined a series ofpreviously published compounds per Omin incubation. Under the conditions used the structurally related to amiflamine (Florvall et al., deamination was linear for at least Omin. 1978; Ask et al., 1982b) with regard to their inhibitory The deamination of ['4C]-5-HT in the presence of action on MAO in 5-hydroxytrypaminergic nerve citalopram was taken as that occurring outside 5-HT terminals in the rat hypothalamus in vivo. In a separate synaptosomes. The difference between the values study the corresponding effects on MAO in cate- obtained in the absence and presence of citalopram cholaminergic nerve terminals are described (Ask et was taken as a measure ofthe MAO activity occurring al., 1985). within 5-HT synaptosomes. Citalopram itself had no MAO inhibitory effect at the concentration used (cf. Hyttel, 1982). Methods The in vivo inhibition ofMAO by the test compound was calculated from the phenelzine protection ex- Male Sprague-Dawley rats weighing 160-200 g were periments as described by Green & ElHait (1980) used. They were housed in groups of four or five and according to the formula: were freely supplied with food and water. Since the test 100 compounds were administered orally the rats were I deprived of food the night before the administration. (1 - t )x 100 Phenelzine sulphate, 4 mg kg ' s.c., was injected one hour after the administration of the test compound; In 100 the rats were then again allowed food. They were killed p by decapitation 48 h after the phenelzine injection, the hypothalami were dissected out and homogenized in in which t is the mean deaminating activity in the 20 volumes ofice-cold 0.25 M sucrose in small all-glass synaptosomal preparations from rats treated with the homogenizers. The homogenates were centrifuged at test compound + phenelzine, expressed as % of the 800g for Omin and the supernatants used in the control activity, and p is the corresponding activity in assay. Each compound was examined in four or more synaptosomes from the phenelzine-treated rats. doses using five (in some experiments four) rats at each The difference between the inhibition inside and dose. outside the 5-hydroxytryptaminergic neurones for The inhibition of rat brain mitochondrial MAO in each dose was determined using the Mann-Whitney vitro was determined as described by Ask et al. U-test. Neuronal selectivity was regarded to occur ifat INHIBITION OF 5-HYDROXYTRYPTAMINERGIC MAO 685 Table 1 Chemical structures of the compounds examined in the present study R3 R' R5 NI Compound X- \ (CH2), C-NH2 R2/ I+ R4 n X Rl R2 R3 R? RS5 R6 FLA 299 1 CH CH3 CH3 H H CH3 H FLA 289 1 N CH3 CH3 H H CH3 H FLA 558 1 N CH3 CH3 F H CH3 H FLA 314 1 N CH3 CH3 Cl H CH3 H FLA 405 1 N CH3 CH3 Br H CH3 H (±)-FLA 336 (+ )-FLA 336 a 1 N CH3 CH3 CH3 H CH3 H (-)-FLA 336 FLA 365 1 N CH3 CH3 Cl 6-Cl CH3 H FLA 417 2 N CH3 CH3 Cl H CH3 H FLA 450 1 N CH3 CH3 Cl H C2H5 H FLA 463 1 N CH3 CH3 Cl H CH3 CH3 FLA 717 1 N CH3 CH3 CH3 H CH3 CH3 FLA 384 1 N CH3 CH3 H 3-CH3 CH3 H ( + )-NBF 003 1 N CH3 CH3 CH3 5-Br CH3 H FLA 727 1 N CH3 H H H CH3 H (+ )-FLA 788 1 N CH3 H CH3 H CH3 H (+ )-NBF 008 1 N CH3 H CH3 5-Br CH3 H RAN 113 1 N CH3 H CH3 3-CH3 CH3 H FLA 334 1 N H H H H CH3 H (± )-FLA 668 (+ )-FLA 668 1 N H H CH3 H CH3 H (-)-FLA 668 (+ )-NBF 021 1 N H H CH3 5-Br CH3 H a = Amiflamine.

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