The measurement of beta-phenylethylamine in human plasma and rat brain.

A sensitive and specific analytical method has been developed for the measurement of beta-phenylethylamine (PEA) in human plasma and rat brain extracts. The method involves solvent extraction of PEA with cyclohexane in the presence of amphetamine or phenylpropylamine (PPA) as internal standards. Automated precolumn derivatization with o-phthalaldehyde and 2-mercaptoethanol followed by reverse-phase HPLC separated PEA and PPA from endogenous interferences. Detection and quantification were carried out by amperometric detection at +0.75 V relative to a Ag/AgCl reference electrode or by coulometric detection with analytical cell potentials set at +0.29 and +0.50 V. The limit of detection for PEA was 10 pg and the limit of quantification in plasma was 60 pg/ml. The within-day and day-to-day coefficients of variation were 16.1% (n = 3) and 40.6% (n = 8), respectively, at a plasma concentration of 154 pg/ml and 15.2% (n = 5) and 28% (n = 10) at a brain extract concentration of 110 pg/ml. Basal endogenous plasma PEA concentrations of 335 +/- 255 pg/ml (n = 12, range 127-1002 pg/ml) were found for normal volunteers and single, daily doses of 24 mg but not 12 mg of the MAO-B inhibitor, mofegiline, were shown to increase plasma PEA significantly. Basal whole brain and striatal concentrations were 0.584 +/- 0.243 ng/g wet wt (n = 3) and 2.89 +/- 1.03 ng/g wet wt (n = 4), respectively. Statistically significant increases (5.7-fold) in rat whole brain PEA concentrations were seen 3 and 6 h following the administration of a single dose of 0.3 mg/kg mofegiline to rats.

Haloallylamine inhibitors of MAO and SSAO and their therapeutic potential.

Based on mechanistic understandings, molecular modeling and extensive quantitative structure-activity relationships, appropriately substituted haloallylamine derivatives were designed as potential mechanism-based inhibitors of MAO and/or SSAO. Potent inhibition of MAO-B and SSAO occurred with fluoroallylamines whereas chloroallylamines, such as MDL 72274A ((E)-2-phenyl-3-chloroallylamine hydrochloride), were selective and potent inhibitors of SSAO. MDL 72974A (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride is a potent (IC50 = 10(-9) M) inhibitor of both MAO-B and SSAO, with 190-fold lower affinity for MAO-A. In clinical studies, oral doses as low as 100 micrograms produced substantial inhibition of platelet MAO-B. Essentially complete inhibition occurred at 1 mg with the effect lasting 6-10 days. One or 4 mg MDL 72974A given daily for 28 days to 40 Parkinson's patients treated with L-dopa produced statistically significant reductions in the Unified Parkinson's Disease Rating Scale. MAO-B inhibitors, such as MDL 72974A and L-deprenyl, offer the potential of being neuroprotective in Parkinson's Disease and other neurogenerative disorders. Concommitant inhibition of SSAO may provide additional, but as yet unproven, advantages over pure inhibitors of MAO-B.

Pharmacokinetics of and monoamine oxidase B inhibition by (E)-4-fluoro-beta-fluoromethylene benzene butanamine in man.

MDL 72974A ((E)-4-fluoro-beta-fluoromethylene benzene butanamine HCl salt, CAS 120635-25-8) is a new irreversible inhibitor of the B form of monoamine oxidase (MAO-B). MDL 72974A's pharmacokinetic parameters were evaluated after administration of a single oral dose and after multiple oral doses. The concentration of parent drug was determined in plasma using a solid-liquid extraction method and gas chromatographic-mass spectrometric analysis. MDL 72974A produced significant inhibition of platelet MAO-B activity at all of the doses > or = 0.5 mg (> 95% after 1 h). The pharmacokinetic parameters showed a short plasma half-life (1 h) and a high total body clearance (Cltot) both probably due to extensive and rapid metabolism as suggested by the low urinary excretion of unchanged drug (< 1% of the administered dose). After the administration of multiple doses of MDL 72974A, a decrease in Cltot and a concomitant increase in the AUC and t1/2, was observed, probably due to a change in the elimination rate of MDL 72974A. Due to the once-a-day dosing schedule and the short plasma t1/2, no drug accumulation occurred.

MDL 72,974A: a selective MAO-B inhibitor with potential for treatment of Parkinson's disease.

MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine, hydrochloride] was designed to be a selective, mechanism-based irreversible inhibitor of monoamine oxidase type B (MAO-B). The compound is a potent, selective MAO-B inhibitor in vitro and in vivo. In vitro studies revealed an IC50 value (MAO-B) of 3.6 nM with 189-fold selectivity compared to MAO-A. In rats, profound inhibition of MAO-B was achieved after a single oral dose with an ED50 of 0.18 mg/kg; a dose 44 times this amount was required to inhibit MAO-A by 50%. Selectivity was maintained following chronic dosing. MDL 72,974A had minimal sympathomimetic effects and did not potentiate the cardiovascular effects of tyramine, even at 50 times the MAO-B inhibiting dose. This inhibitor was equally effective and well-tolerated in man. In human volunteers, potent inhibition of platelet MAO-B activity was observed at submilligram doses (ED50 = 90 micrograms) following a single oral dose. Upon multiple oral doses of 100 micrograms, as much as 80% of MAO-B could be inhibited. In phase II studies, MDL 72,974A is proving to be a useful adjunct to conventional therapy. Patients (250) with Parkinson's disease, treated once daily with either 1 or 4 mg, together with L-Dopa and a decarboxylase inhibitor (MadoparR or SinemetR), saw significant improvements in symptoms compared with those on standard therapy without the inhibitor.

A double-blind, placebo-controlled study of the tolerability and effects on platelet MAO-B activity of single oral doses of MDL 72.974A in normal volunteers.

MDL 72.974A [(E) 4-fluoro-beta-fluorethylene benzene butanamine] has been shown in animal studies, both in vitro and in vivo, to be a potent, selective, enzyme-activated irreversible inhibitor of MAO-B (Zreika et al., 1989). This compound is under clinical development for the treatment of Parkinson's disease. In this double blind, randomized, placebo-controlled normal volunteer study the tolerability, effects on platelet MAO-B activity and associated pharmacokinetics of increasing single oral doses of MDL 72.974A (0.1-12 mg) were assessed. MDL 72.974A was extremely well tolerated and no treatment-related changes in vital signs or the adjectival check-list (EWL-N) occurred. The compound caused significant dose-dependent inhibition of platelet MAO-B activity at all dose levels with a return to baseline values by day 14. The mean (+/- S.D.) elimination half-life of parent compound was 51 +/- 26 min and mean (+/- S.D.) urinary excretion was 0.54 +/- 0.26% of the administered dose. These results, long action on platelet MAO-B and short elimination half-life, demonstrate MDL 72.974A to be a potent, irreversible inhibitor of MAO-B in man.

MDL 72,974: a potent and selective enzyme-activated irreversible inhibitor of monoamine oxidase type B with potential for use in Parkinson's disease.

MDL 72,974, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, was designed to be a selective inhibitor of monoamine oxidase type B (MAO-B). In vitro, the compound inhibits rat brain mitochondrial MAO in a concentration and time-dependent fashion and shows marked selectivity for the B form (IC50 = 680 and 3.6 nM for MAO-A and MAO-B, respectively). After oral administration to rats, the compound shows preferential inhibition of brain MAO-B with ED50 values of 8 and 0.18 mg/kg p.o. for the A and B forms, respectively. Selectivity is retained on repeat dosing. MDL 72,974 did not significantly potentiate the cardiovascular effects of intraduodenually-administered tyramine in anaesthetized rats and had only minor indirect sympathomimatic effects in the pithed rat. At MAO-B selective doses the neurotoxic effect of MPTP in mice was blocked.

Selective inhibition of monoamine oxidase type B by MDL 72145 increases the central effects of L-dopa without modifying its cardiovascular effects.

The potential of a new, potent, irreversible and selective inhibitor of monoamine oxidase type B, (E)-2-(3,4-dimethoxyphenyl)-3-fluorallyamine (MDL 72145), to augment the effects of L-DOPA in an animal model which reproduces the biochemical defect of Parkinson's disease has been evaluated. In rats bearing unilateral 6-hydroxydopamine lesions of the nigro-striatal dopamine pathways, both MDL 72145 and clorgyline, a selective inhibitor of MAO A, augmented the contralateral turning response to L-DOPA combined with carbidopa. The potential of inhibitors of MAO to interact adversely in the periphery with L-DOPA was investigated in the pithed rat; L-DOPA was given either intravenously or intraduodenally. Clorgyline consistently potentiated L-DOPA when given 18 h before testing. Neither MDL 72145 nor the selective inhibitor of MAO B, L-deprenyl, augmented the cardiovascular effects of intraduodenally administered L-DOPA. The data provide no reason to suppose that MDL 72145 would be very different in clinical use from L-deprenyl which is both effective and well-tolerated as an adjunct to the L-DOPA-based therapy of Parkinson's disease.

Inhibition of monoamine oxidase selectively in brain monoamine nerves using the bioprecursor (E)-beta-fluoromethylene-m-tyrosine (MDL 72394), a substrate for aromatic L-amino acid decarboxylase.

(E)-beta-Fluoromethylene-m-tyrosine (FMMT) is a dual-enzyme-activated inhibitor of monoamine oxidase (MAO). The compound is not an inhibitor per se but is decarboxylated by aromatic L-amino acid decarboxylase (AADC) to yield a potent enzyme-activated irreversible inhibitor of MAO, (E)-beta-fluoromethylene-m-tyramine, which shows some selectivity for inhibition of MAO type A. Decarboxylation of FMMT was demonstrated in vitro using hog kidney AADC and in vivo in rats by the ability of alpha-monofluoromethyldopa (MFMD), a potent inhibitor of AADC, to prevent MAO inhibition produced by FMMT. In isolated synaptosomes, FMMT was decarboxylated by AADC, and, furthermore, the compound was actively transported into these isolated nerve endings. An active transport into the CNS has also been demonstrated in vivo by performing competition experiments with leucine. To demonstrate that FMMT is preferentially decarboxylated within monoamine nerves of the CNS, the nigrostriatal 3,4-dihydroxyphenylethylamine (dopamine) pathway of rats was unilaterally lesioned with 6-hydroxydopamine or infused with MFMD. Under these conditions, MAO inhibition produced by orally administered FMMT in the striatum ipsilateral to the lesion or infusion was markedly attenuated. Combination of FMMT with an inhibitor of extracerebral AADC, such as carbidopa, protected peripheral organs against the MAO inhibitory effects and concomitantly enhanced MAO inhibition in the CNS. Such combinations had a greatly reduced propensity to augment the cardiovascular effects of intraduodenally administered tyramine, when compared with FMMT given alone or with clorgyline, a selective inhibitor of MAO type A. The results obtained with FMMT suggest the possibility of achieving selective inhibition of MAO within monoamine nerves of the CNS and, further, suggest that combination of FMMT with an inhibitor of extracerebral AADC will reduce the propensity of this inhibitor to produce adverse interactions with tyramine.

The functional consequences of inhibition of monoamine oxidase type B: comparison of the pharmacological properties of L-deprenyl and MDL 72145.

The pharmacological properties of two selective inhibitors of monoamine oxidase (MAO) type B, L-deprenyl and MDL 72145 [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine, HCl], have been investigated in rats and mice in relation to their effects on MAO. Selective inhibition of MAO B achieved following 18 h pretreatment with L-deprenyl and/or MDL 72145 did not per se lead to prominent pharmacological activity; no effects were seen in the mouse "Behavioural Despair" test, hypothermia induced by reserpine in mice was neither prevented nor reversed and there was no change in the cardiovascular responsiveness of the pithed rat to tyramine, noradrenaline or stimulation of the spinal sympathetic outflow. L-Deprenyl differed from MDL 72145 in that short term treatment with this drug caused positive effects in the "Behavioural Despair" test, reversal of reserpine hypothermia, indirect sympathomimetic stimulation of blood pressure and heart rate in the pithed rat and ipsilateral rotation in rats with unilateral nigro-striatal lesions. Qualitatively similar effects were seen with dexamphetamine. The marked difference between the pharmacological effects of MDL 72145 and L-deprenyl despite equivalent inhibition of MAO B suggests that many of the pharmacological actions of L-deprenyl result from its amphetamine-like sympathomimetic properties. MDL 72145 can, therefore, be considered a more reliable tool with which to explore the functional importance of MAO B inhibition in experimental animals and man.

Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationships.

Seventeen 2-aryl-3-haloallylamine derivatives were prepared and evaluated as inhibitors of monoamine oxidase (MAO, EC 1.4.3.4). The synthesis of these compounds was achieved from either alpha-methylstyrene or ring-substituted phenylacetic acid derivatives. With one exception, these 2-arylallylamines were found to be enzyme-activated, irreversible inhibitors of MAO. The most potent inhibitors were ring-substituted derivatives of (E)-2-phenyl-3-fluoroallylamine with IC50 values ranging from 10(-6) to 10(-8) M. Selectivity for the A and B form of MAO was found to depend on the nature of aromatic ring substitution. In general, hydroxyl substitution favored the inactivation of the A form of MAO, while very selective B inhibitors were obtained when the aromatic ring was substituted with a 4-methoxy group. (E)-2-(4-Methoxyphenyl)-3-fluoroallylamine and (E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine proved to be in vitro as selective for the B form of MAO as deprenyl.

MDL 72145, an enzyme-activated irreversible inhibitor with selectivity for monoamine oxidase type B.

MDL 72145, (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme-activated irreversible inhibitor of monoamine oxidase (MAO). In vitro, the compound displayed time-dependent pseudo-first-order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO. At 10 degrees C the Ki and tau 50 values for the B enzyme were 40 microM and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 microM and 14.5 min, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincubating the enzyme with suitable concentrations of the selective A and B substrates, 5-hydroxytryptamine and benzylamine.

A method for measuring monoamine turnover in animals using an irreversible inhibitor of aromatic L-amino acid decarboxylase, DL-alpha-mono fluoromethyldopa.

DL-alpha-monofluoromethyldopa (MFMD) is a potent enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase which, when given to rats or mice at 100 and 250 mg/kg i.p., respectively, causes a linear accumulation of L-DOPA and 5HTP and an exponential decline of noradrenaline, dopamine, and 5HT concentrations in the brain. Rates of change of these parameters can be used to calculate the turnover of the three principle monoamine neurotransmitters. Experiments with haloperidol (1 mg/kg s.c.) and the central 5HT agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (0.25 mg/kg s.c.), have been performed to validate the use of MFMD to measure monoamine turnover. MFMD has several advantages over classical methods for the determination of comparative turnovers using enzyme inhibitors.