Anna-Lena Ask

Selective inhibition of monoamine oxidase in monoaminergic neurons in the rat brain

SummaryThe prevention by six reversible and selective monoamine oxidase-A (MAO-A) inhibitors (α-ethyltryptamine, harmaline, 4-methoxyamphetamine, amiflamine [FLA 336(+)], N-desmethylamiflamine [FLA 788(+)] and N,N-desmethylamiflamine [FLA 668(+)] of the phenelzineinduced irreversible MAO inhibition in the rat brain was examined. By using crude synaptosome preparations of hypothalamus and striatum incubated with low substrate concentrations of 14C-serotonin (1×10−7 M), 14C-noradrenaline (2.5×10−7 M) and 14C-dopamine (2.5×10−7 M) in the absence and presence of selective amine uptake inhibitors (alaproclate, maprotiline and amfonelic acid, respectively), it was possible to determine the deaminating activities inside and outside the specific aminergic synaptosomes. Thus, with this technique the protection of MAO by the reversible inhibitors administered orally 1 h prior to the subcutaneous injection of phenelzine against the phenelzine effect could be determined inside and outside the specific aminergic neurons. It was found that α-ethyltryptamine, 4-methoxyamphetamine and particularly amiflamine and FLA 788(+) were more potent inside than outside the serotonergic neurons. FLA668(+) was a selective inhibitor of noradrenergic MAO, to which also 4-methoxyamphetamine, amiflamine and FLA 788(+), but not α-ethyltryptamine had some preference. Harmaline had no certain preference for MAO in any of the aminergic neurons. At high doses of FLA 668(+) a preference for dopaminergic MAO was observed. Since pretreatment of the rats with norzimeldine or desipramine antagonized the preferences for serotonergic or noradrenergic MAO, it is plausible to conclude that the compounds showing these preferences are accumulated in the neurons by the membranal uptake systems.

(+)-4-dimethylamino-2, α-dimethylphenethylamine (FLA 336(+)), a selective inhibitor of the a form of monoamine oxidase in the rat brain

(+)-4-Dimethylamino-2,alpha-dimethylphenethylamine (FLA 336(+)) and its N-demethylated secondary amino derivative FLA 788(+) were examined for their monoamine oxidase (MAO) inhibitory effects in the rat brain. They were found to be reversible and very selective inhibitors of the A form of monoamine oxidase in vitro and in vivo after oral administration. FLA 788(+) was 2-6 times more active than FLA 336(+) in vitro depending on the assay technique employed but the two compounds had similar potency after oral administration. Both compounds inhibited competitively the deamination of 5-hydroxytryptamine by hypothalamic mitochondria. Although the irreversible inhibitor clorgyline was 60 times more potent than FLA 336(+) in vitro, it was equipotent with FLA 336(+) and FLA 788(+) in the rat brain after oral administration. There was a high correlation between the log plasma concentration of FLA 788(+) and the MAO inhibition in hypothalamic slices. The plasma concentration of the metabolite FLA 788(+) exceeded that of FLA 336(+) after oral administration of the latter compound. Thus, the MAO inhibition produced by FLA 336(+) in vivo, appears in part to be due to the metabolite FLA 788(+).

Selective inhibition of the a form of monoamine oxidase by 4-dimethylamino-α-methylphenylalkylamine derivatives in the rat

The inhibitory effects on monoamine oxidase (MAO) of some dimethylamino-alpha-phenylalkylamine derivatives were examined in a rat brain mitochondrial preparation in vitro and in rat brain slices following oral administration. In the in vitro assay the compounds were shown to be selective inhibitors of the A form of MAO, being 100-600 times more potent in inhibiting the deamination of [14C]5-hydroxytryptamine than that of [14C]phenetylamine. Using an ex vivo brain slice technique it was found that the new compounds were reversible and very selective inhibitors of type A MAO in the rat brain and the most potent compounds (FLA 405, 314, 336 and 558) were equipotent with clorgyline. The compounds increased the monoamine concentrations in whole rat brain, particularly that of 5-hydroxytryptamine, in the same dose range which produced MAO inhibition. Some of the new compounds, e.g. FLA 336 and FLA 717, caused only weak potentiation of the vaso-pressor effect of orally administered tyramine.

Inhibition of monoamine oxidase in 5-hydroxytryptaminergic neurones by substituted p-aminophenylalkylamines.

1 A series of substituted p‐aminophenethylamines and some related compounds were examined with regards to the inhibition of monoamine 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 of citalopram using a low (0.1 μM) concentration of [14C]‐5‐hydroxytryptamine (5‐HT) as substrate. 2 Some of the 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 of the 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 α‐substituent to an ethyl group but remained for the α,α‐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.

Selective inhibition of monoamine oxidase by p-aminosubstituted phenylalkylamines in catecholaminergic neurones

The in vivo inhibition of monoamine oxidase (MAO) inside and outside noradrenergic and dopaminergic nerve terminals in the hypothalamus and striatum, respectively, was examined in the rat after oral administration of a series of substituted p-aminophenethylamines and some related compounds. This was achieved by measuring their ability to protect MAO from irreversible inhibition by phenelzine, determined by the deaminating activity of synaptosomal preparations in the absence and presence of maprotiline, a selective inhibitor of the uptake of noradrenaline, or of amfonelic acid, a potent inhibitor of the uptake of dopamine, with small (0.25 microM) concentrations of [14C]noradrenaline or [14C]dopamine as substrate. It was found that several of these compounds were much more potent in protecting MAO within the noradrenergic neurones than MAO in other cells. Since the inhibitors of the uptake of noradrenaline, desipramine and CPP 199 antagonized this preference for noradrenergic MAO it is concluded that these MAO inhibitors are accumulated in the noradrenergic neurones by the membranal uptake carrier. Hence the selectivity for MAO within noradrenergic neurones seems to reflect the ability of the compounds to be transported by this carrier. The structure-activity relationship obtained showed the greatest selectivity for the unsubstituted p-dimethylamino-(FLA 289), p-methylamino-(FLA 727) and p-amino-(FLA 334)-amphetamines, whereas the 2-fluoro compound (FLA 558) had the greatest potency. N,N-didesmethylamiflamine [FLA 668(+)] had an almost specific effect in the noradrenergic nerve terminals. The primary p-amino derivatives, FLA 334 and FLA 668, produced a marked selective protection of MAO in dopaminergic nerve terminals, whereas the tertiary and secondary derivatives had much less preference for dopaminergic MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute and repeated administration of amiflamine on mono amine oxidase inhibition in the rat

The inhibitory effect on monoamine oxidase (MAO) of the reversible MAO-A inhibitor (+)-4-dimethylamino-2,alpha-dimethylphenethylamine [amiflamine, FLA 336(+)] was evaluated in the rat after acute and repeated (twice daily for two weeks) oral treatment. MAO activity was measured ex vivo in slices from the hypothalamus and the duodenum for both MAO-A and MAO-B. Amiflamine selectively inhibited the A form of MAO after repeated as well as after acute treatment (ED50 approximately 7 mumoles/kg both acute and repeated). In the brain slices this inhibition corresponded to a decrease in the concentration of 5-hydroxyindoleacetic acid (5-HIAA) and to an increase in the concentration of 5-HT in the hypothalamus, the hippocampus and the striatum. The concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) was decreased in the striatum to the same extent as the decrease in the 5-HIAA concentrations. The effect on the homovanillic acid (HVA) concentration was somewhat weaker as was the increase in the concentration of dopamine. No essential difference was found after acute and chronic treatment on the amine and metabolite levels. The MAO activity returned to normal 24 hours after final dosing. A large difference between the neuronal and the extraneuronal protection against the phenelzine-induced irreversible MAO inhibition in the hypothalamus was found after both acute and repeated treatment. The ED50 of the protection within the serotonergic neurons was 1.3 mumoles/kg p.o. (acute) and 0.75 mumoles/kg p.o. (repeated). Amiflamine was 3 times less potent within noradrenergic neurons than within serotonergic neurons. A brain to plasma ratio of about 20:1 was found for amiflamine and its metabolites. The plasma and the brain concentrations of the N-demethylated metabolite [FLA 788(+)] exceeded that of amiflamine after a single dose, whereas the N,N-demethylated [FLA 668(+)] was found in low concentrations. The effect on MAO-A correlated significantly with the plasma and the brain concentration of FLA 788(+).

Inhibition of 5-hydroxytryptamine accumulation and deamination by substituted phenylalkylamines in hypothalamic synaptosomes from normal and reserpine-pretreated rats

Summary1. In the present study the abilities of different compounds to inhibit MAO inside and outside the serotonergic neurons, to inhibit the accumulation of 5-HT and to release 5-HT were separated by using different in vitro techniques. With these methods a number of substituted phenylalkylamines, which are reversible inhibitors of monoamine oxidase (MAO) type A, were characterized. 2. The compounds were examined regarding their ability to inhibit the accumulation of 5-HT and to inhibit MAO in the same synaptosomal preparation of hypothalamus from normal and reserpine-pretreated rats. The difference in the uptake of 14C-5-HT (0.1 μmol/l) in the absence and presence of citalopram (0.25 μmol/l) was taken as a measure of the accumulation into the serotonergic synaptosomes. The deamination of 14C-5-HT (0.1 μmol/l) in the presence of citalopram (0.25 μmol/l) was considered as that brought about outside the serotonergic synaptosomes, whereas the difference between the deamination in the absence and presence of citalopram was taken as the MAO activity inside the serotonergic synaptosomes. 3. Most of the phenylalkylamines were slightly more potent as MAO inhibitors outside serotonergic synaptosomes than as inhibitors of 5-HT accumulation in normal rats. The most potent MAO inhibitors, both in absolute terms and in comparison with uptake inhibitory potency, were the 2,6-dichloro-(FLA 365) and the phenylpropylene-(FLA 417) derivatives. 4. A difference in potency on the accumulation in synaptosomes from normal and reserpine-pretreated rats was found for many of the phenylalkylamines with the exception of FLA 365, FLA 417 and the 2,5-dimethyl derivative RAN 113. The compounds shared this difference with the 5-HT releasers p-chloroamphetamine (pCA) and H75/12, but not with the uptake inhibitors citalopram, cocaine, alaproclate, norzimeldine and fluoxetine. 5. For most of the phenylalkylamines, the MAO inhibition obtained within serotonergic synaptosomes was not higher than that obtained outside these. This was in contrast to previously shown in vivo results were a preference for inhibiting MAO inside the serotonergic neurons, due to a transport into the neurons by the amine carrier, was found. For the uptake inhibitors, the inhibition of 5-HT deamination intrasynaptosomally was a more sensitive measure of inhibition of accumulation than to determine this accumulation directly. 6. It is concluded that a classification of uptake inhibitors, releasing compounds, and MAO inhibitors that are and that are not transported by the 5-HT carrier may be performed by measuring the inhibition of the uptake and the deamination in synaptosomes from normal and reserpine-pretreated rats.

Characterization of [3H]CGS 19755 binding sites in the rat spinal cord.

[3H]Cis-4-phosphonomethyl-2-piperidine carboxylic acid ([3H]CGS 19755) was used to investigate the pharmacology and characteristics of the N-methyl-D-aspartate (NMDA) receptor recognition site from Triton X-100-treated membranes of rat spinal cord and cerebral cortex. The association of [3H]CGS 19755 was biphasic in both spinal cord and cerebral cortical membranes reaching a maximum after 5 min of incubation then decreasing to a steady level after an additional 10 min, suggesting that a proportion of the binding is unstable. The dissociation of the stable binding component was biphasic with rate constants at 4 degrees C of 1.55 and 0.020 min-1 for the spinal cord and 1.48 and 0.051 min-1 for the cerebral cortex. These multiple sites could not be captured in the saturation studies which were best fitted to a one-site model using non-linear regression analysis. Depending on the time of incubation with [3H]CGS 19755, KD and Bmax values differed; 9.9-26.1 nM and 25-96 fmol/mg protein vs 14.0-26.5 nM and 449-900 fmol/mg protein for spinal cord and cerebral cortex, respectively. The rank order of potency of inhibiting [3H]CGS 19755 binding was similar in both tissues: L-glutamate > CGS 19755 = CPP > NMDA. The specific NMDA agonist cis-2,4-methanoglutamate potently inhibited [3H]CGS 19755 binding as did MDL 100,925, although the latter was one order of magnitude less potent in the spinal cord than in the brain. The Hill coefficients were significantly lower than unity. In both tissues, AMPA, kainate and glycine competed poorly with [3H]CGS 19755.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition by amiflamine of monoamine oxidase type A in rat brain, liver and duodenum

SummaryAmiflamine (FLA 336(+)), N-desmethylamiflamine (FLA 788(+)) and N,N-didesmethylamiflamine (FLA 668(+)) were examined for their monoamine oxidase (MAO) inhibitory effects in rat brain, liver and duodenum and were compared with the irreversible inhibitors clorgyline and (-)-deprenyl. The potency of each FLA compound was the same in each tissue both in vitro and after oral administration with either serotonin or tyramine as substrate. The in vitro effect of FLA 788(+) was 2–6 times stronger than that of amiflamine although the compounds were equipotent after oral administration. FLA 668(+) was 2–3 times less potent than amiflamine in vitro and had very poor activity after oral administration. The deamination of phenethylamine was weakly afected by the three FLA compounds. Clorgyline inhibited strongly the deamination of serotonin and tyramine in the duodenum after oral administration, being 1,000 times more potent than in the brain and the liver. Similar results were obtained for (-)-deprenyl which, however, was more potent in inhibiting the deamination of phenethylamine than that of serotonin and tyramine. Amiflamine was a reversible MAO inhibitor with no MAO inhibitory capacity 24 h after a single oral dose. On the other hand the irreversible inhibitor clorgyline had a maximal effect on brain MAO 48 h after a single dose while the inhibitory effect in the duodenum had almost disappeared. The influence of amiflamine on the excretion of acid and basic metabolites of orally administered 14C-tyramine (58 μmol/kg) in rat was examined. Amiflamine, at doses that strongly inhibited MAO-A in rat brain, only slightly affected the excretion of 14C-labelled acid in urine during 6 and 24 h after the tyramine administration. The results in this study suggest that other factors than a low interaction with intestinal MAO may be of importance for the low tyramine potentiating effect obtained after oral administration of amiflamine.

Release of 3H-5-hydroxytryptamine by amiflamine and related phenylalkylamines from rat occipital cortex slices

SummaryIn the present study Amflamine and other related reversible monoamine oxidase-A (MAO-A) inhibitory phenylalkylamines were examined in vitro for their ability to induce release of 3H-5-hydroxytryptamine (3H-5-HT) from rat occipital cortex slices. The slices were preincubated with 3H-5-HT 0.1 μmol/l in the presence of the irreversible MAO inhibitor pargyline 50 μmol/l and then continuously superfused. The effects were compared with those of the 5-HT releaser p-chloroamphetamine (pCA), the reversible MAO-inhibitor α-ethyltryptamine and the 5-HT uptake inhibitor citalopram. Amiflamine, some related compounds and α-ethyltryptamine which in vivo after transport by the 5-HT uptake mechanism preferentially inhibit MAO within the serotonergic neurons caused a Cat2+-independent release of 3H-5-HT. Some transported compounds, particularly NBF 027 were, however, very weak releasers of 5-HT. This release and that induced by pCA was prevented by citalopram in the superfusion medium. FLA 365, FLA 417 and FLA 1088, which are not transported into the neurons, were poor releasers of 5-HT. It is concluded that compounds which were effective releasers of 5-HT in vitro were those that are transported into the serotonergic neurons by the 5-HT carrier in vivo and has in addition an ability to mobilise vesicular 5-HT.