R. Kettler

Biochemistry and pharmacology of moclobemide, a prototype RIMA

RIMA is a term for reversible inhibitors of monoamine oxidase (MAO) with preference for MAO-A; moclobemide is a prototype of this new class of antidepressants and is a highly selective inhibitor of MAO-A in vitro. This inhibition is reversible by dialysis in vitro, which accounts for the dose-dependent duration of in vivo enzyme inhibition of 12–24 h. Moclobemide increases the content of serotonin, noradrenaline and dopamine in the brain, and decreases that of their deaminated metabolites. Its biochemical, neurological and behavioural effects indicate that it increases the extracellular concentration of the classic monoamine neurotransmitters/neuromodulators — in particular 5-HT. Potentiation of the cardiovascular effects of tyramine is less pronounced after taking moclobemide than after irreversible MAO-A inhibitors. Understanding of the physiological role of MAO and of the events that link inhibition of the enzyme with modulation of neuronal activities in the CNS remains incomplete. A major physiological role of intraneuronal MAO is to keep cytosolic amine concentration very low, to enable the neuronal monoamine carriers to produce a net inward transport of monoamines, and thereby to act as the first step in the termination of action of extracellular monoamines. MAO is likely to have a similar function in non-monoaminergic cells with respect to the monoamine carriers they contain. In addition to the classic monoamines, “trace” amines may become functionally active after MAO inhibition. An alternative role for MAO is that of a scavenger, preventing natural substrates from accumulating in monoaminergic neurons and interacting with storage, release, uptake and receptor function of monoamines.

Measurement of human cerebral monoamine oxidase type B (MAO-B) activity with positron emission tomography (PET): a dose ranging study with the reversible inhibitor Ro 19-6327

SummaryEight normal subjects (3 females and 5 males) were studied using intravenous L-11C] deprenyl and positron emission tomography. In a single blind study one subject received tracer alone, one subject received an oral pre-dose of 20 mg of L-deprenyl and 6 subjects received oral pre-doses of 10 to 50 mg of a novel reversible MAO-B inhibitor (Ro 19-6327). Dynamic PET scans beginning 12 h after the oral dose were collected over 90 min and arterial blood was continuously sampled. Data analysis was modelled for two tissue compartments and using an iterative curve fitting technique the value of the rate constant for irreversible binding of L-[11C] deprenyl to MAO-B (k3) in whole brain was obtained for each subject.The dose response curves obtained indicated that a dose of at least 0.48 mg·kg−1 of Ro 19-6327 was necessary for >90% decrease in whole brain k3. Inhibition of MAO-B in platelets isolated from blood samples taken at the time of scanning correlated strongly with decrease in whole brain k3 (r=0.949).The results indicate that PET can be used to determine the dose of Ro 19-6327 necessary to inhibit >90% of brain MAO-B. This technique is an attractive alternative to traditional large scale patient-based dose-finding studies. Moreover it is shown that inhibition of platelet MAO-B can be used as a marker for central MAO-B inhibition with Ro 19-6327.

From moclobemide to Ro 19-6327 and Ro 41-1049: the development of a new class of reversible, selective MAO-A and MAO-B inhibitors

This study describes the serendipitous discovery of moclobemide, a short-acting MAO-A inhibitor which is in an advanced stage of clinical development as an antidepressant. The short duration of action of this MAO inhibitor containing a morpholine ring moiety is due to the complete reversibility (probably by metabolism of the inhibitory molecular species) of MAO-A inhibition. Since moclobemide is much more effective in vivo than expected from its in vitro activity, investigations to identify a possible metabolite(s) more active as MAO-A inhibitor than the parent compound were carried out. The study of the MAO inhibitory characteristics of several known and putative moclobemide metabolites did not allow the identification of a potent MAO-A inhibitor but led to the discovery of Ro 16-6491, a potent MAO-B inhibitor of novel chemical structure. Systematic chemical modification of the aromatic ring system of Ro 16-6491 finally provided Ro 19-6327 and Ro 41-1049 which are highly selective and reversible inhibitors of MAO-B and MAO-A, respectively. Tritiated derivatives of Ro 19-6327 and Ro 41-1049 were used in binding studies to elucidate their mechanisms of action and to study their cellular distribution by quantitative enzyme radioautography.

The pharmacology of Parkinson's disease: Basic aspects and recent advances

Basic aspects and recent advances in the understanding of the pharmacological mechanism of action of the clinically most used antiparkinson drugs are reviewed. Recent human and animal biochemical investigations clearly confirm and extend previous findings indicating that benserazide is much more potent than carbidopa as peripheral decarboxylase inhibitor. L-DOPA in combination with benserazide or carbidopa constitutes the best available therapy for Parkinsons disease (PD). To reduce peaks and rapid fluctuations of L-DOPA plasma levels (possibly responsible for peak-dose dyskinesias and end-of-dose deterioration) a slow-release formulation of L-DOPA in combination with benserazide or with benserazide plus catechol-O-methyltransferase inhibitors should be developed. In parkinsonian patients under long-term L-DOPA therapy monoamine oxidase inhibitors type B (MAO-B) e.g. (−)-deprenyl and firect dopamine receptor agonists (bromocriptine, lisuride, pergolide etc.), due to their L-DOPA-sparing effects, alleviate in some cases L-DOPA-induced side-effects e.g. dyskinesias and on-off phenomena. However, since (−)-deprenylm, due to its metabolism to (−)methamphetamine and (−)amphetamine, seem to have indirect sympathomimetic activity, new selective MAO-B inhibitors devoid of indirect sympathomimetic effects should be tested clinically to assess the functional role of pure MAO-B inhibition in the therapy of PD. The auxiliary therapy with direct dopmaine receptor agonists of the D-2 subtype represents another valid approach which should be further investigated in order to find novel dopamine agonists, less expensive than bromocriptine and strictly selective for D-2 receptor sites.

Short-acting novel MAO inhibitors: in vitro evidence for the reversibility of MAO inhibition by moclobemide and Ro 16-6491

SummaryThe inhibition of monoamine oxidase (MAO) in rat liver and brain by the short-acting MAO-A inhibitors moclobemide (Ro 11-1163 = p-chloro-N-[2-morpholinoethyl]benzamide) and brofaremine and by the short-acting MAO-13 inhibitors Ro 16-6491 (N-[2-aminoethyl]-p-chlorobenzamide) and almoxatone, administered p. o. at roughly equieffective doses 2 h before decapitation, was investigated for its reversibility under various in vitro conditions. MAO A activity in liver homogenates, inhibited by moclobemide (300 μmol/kg) to approx. 15% of control, time dependently recovered during 0.5 to 2 h of incubation at 37°C, irrespective of whether the homogenates were prepared and incubated in distilled water or Krebs-Ringer buffer (KRB). Dialysis of such homogenates for 4 h in distilled water at 37°C (but not at 13°C) led to a complete return of the MAO activity. In liver homogenates from rats pretreated with brofaremine (30 μmol/kg), dialysis for 4 h at 37°C against distilled water caused only little recovery of the MAO activity. Likewise, MAO-B inhibited by Ro 16–6491 (30 μmol/kg) to approx. 4% of control returned to almost control activity after 4 h of dialysis at 37°C, while inhibition induced by almoxatone (30 μmol/kg) was little or not reversed at all. In brain homogenates prepared in, and dialysed against, distilled water or KRB at 37°C (but not at 13°C), MAO-A inhibited by moclobemide (100–300 μmol/kg) to approx. 15% of control, partially (KRB) or almost completely (dist. water) returned to control activity after 4 h of dialysis. From rats pretreated with Ro 16–6491 (30 μmol/kg), MAO-B in brain homogenates prepared in KRB was reduced to 12% of control and returned to control value upon dialysis for 4 h in KRB at 37°C; in homogenates prepared in H2O, MAO-B was reduced to only 60% of control and completely recovered by dialysis against dest. water even at 13°C. In all of these conditions, recovery of the enzyme activity was small after brofaremine and almoxatone. Analogous results were obtained with brain slices (0.2 × 0.2 × 1.5 mm) in KRB at 37°C, whereby time dependent recovery of MAO activity during incubation was achieved, and superfusion was somewhat more effective than incubation in restoring enzyme activity. In the experiments with incubated or superfused brain slices, inhibition of MAO-A and -B by the irreversible inhibitors clorgyline and selegiline (l-deprenyl), resp., could not be reversed at all. Tyramine (0.3 mmol/l) clearly enhanced the recovery of MAO-A in KRB-prepared liver homogenates and brain slices of moclobemide-pretreated rats but not in brain slices of brofaremine- and clorgyline-pretreated rats. Thus, the reversibility of MAO inhibition in vitro could be convincingly demonstrated for moclobemide and Ro 16–6491 but not for the other novel, short-acting MAO inhibitors studied.

Effect of selective and reversible MAO inhibitors on dopamine outflow in rat striatum: a microdialysis study

The effect of reversible inhibitors of the monoamine oxidase type A (MAO-A), moclobemide (Aurorix) and Ro 41-1049 (20 mg/kg i.p. each), as well as of reversible inhibitors of the MAO type B (MAO-B), Ro 19-6327 (1 mg/kg i.p.), on the outflow of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied in the rat by transstriatal microdialysis. Reversible MAO-A inhibitors markedly increased the output of DA and concomitantly decreased the output of DOPAC and HVA. These effects were absent with the highly selective MAO-B inhibitor Ro 19-6327.

Molecular neuroanatomy of MAO-A and MAO-B

A selective, quantitative and high resolution technique (in vitro and in vivo enzyme radioautography) has been used to reveal the tissue distribution and abundance of MAO-A and MAO-B in the central nervous system and peripheral organs in the rat. The in vitro approach was also used to map the enzymes in human post-mortem brain. Furthermore, using in situ hybridization histochemistry, locus coeruleus and raphé neurons in the human brain were found to code for MAO-A and MAO-B respectively and not vice versa.

New Therapeutic Strategies in Parkinson’s Disease: Inhibition of MAO-B by Ro 19-6327 and of COMT by Ro 40-7592

About three decades ago, it was observed for the first time that DOPA (levodopa, 3,4-dihydroxyphenyl-L-alanine) dramatically improved akinetic Parkinson’s patients1. Several direct acting dopamine (DA) D2 agonists were subsequently investigated but none proved to be sufficiently effective to be used routinely as the sole agent in the control of motor fluctuations in Parkinson’s disease (PD). Motor fluctuations also frequently complicate DOPA therapy and limit its therapeutic benefit. When used as adjuvants to DOPA, DA agonists may modestly diminish the fluctuations, but a relatively high incidence of side-effects, chiefly psychotoxic, has limited their use2. It is noteworthy that chronic treatment of PD with DOPA is devoid of neurotoxic effects and does not produce damage to the nigro-striatal dopaminergic neurones3. Therefore, treatment with DOPA in combination with peripheral L-amino acid decarboxylase (AADC) inhibitors will remain the standard pharmacological therapy for PD at least for the next decade. However, new strategies are being actively investigated and some have been proposed to ameliorate dyskinesias and motor fluctuations occurring in patients under therapy with DOPA combined with a peripheral AADC inhibitor, e.g. benserazide (Madopar®) or carbidopa (Sinemet®)2.

Comparison of monoamine oxidase-A inhibition by moclobemide in vitro and ex vivo in rats

Inhibition of MAO activity was measured in rat brain homogenates using 5‐HT as MAO‐A substrate and phenylethylamine as MAO‐B substrate. Moclobemide rather selectively inhibited MAO‐A. Its inhibitory potency is rather low, like that of toloxatone, whereas clorgyline, harmaline, cimoxatone and brofaromine were all found to be at least 100 times more potent. Phenelzine, isocarboxazid and tranylcypromine were nonspecific, inhibiting MAO‐A and MAO‐B to about the same extent. The same drugs were also tested ex vivo. Here again moclobemide preferentially inhibited MAO‐A; it was equipotent to clorgyline and brofaromine in these tests, and 2–4 times as potent as cimoxatone and harmaline. Moclobemide is a relatively weak MAO‐A inhibitor in vitro and yet more potent in vivo than other reversible inhibitors, suggesting that the compound may be converted in vivo to an active form. Nevertheless, it has not been possible so far to identify activated derivatives, and recent findings that moclobemide markedly inhibits liver MAO‐A within 5 min of an intravenous injection strongly suggests that the compound itself is responsible for the inhibition.

Harmaline and its sulfur analogue: Increase of cerebellar cyclic GMP and inhibition of monoamine oxidase

Abstract Harmaline and its benzo[b]thiophene analogue (“S- harmaline ”) are shown to competitively inhibit monoamine oxidase (MAO). The inhibition was more marked with 5-hydroxytryptamine as a substrate than with tyramine or β-phenylethylamine. After i.p. administration, S-harmaline in contrast to harmaline inhibited the MAO in the brain more potently than in the liver. Harmaline produced a greater and longer lasting increase of cGMP in the cerebellum than S-harmaline. The S-harmaline was also less tremorogenic, thus indicating a connection between cGMP increase and tremor, but not between MAO inhibition and cGMP increase.