Neuroscience Letters, 107 (1989) 313-317 313 Elsevier Scientific Publishers Ireland Ltd.

NSL 06524

Tetrahydroaminoacridine inhibits human and rat brain monoamine oxidase

Abdu Adem l, Sukhwinder S. Jossan 2 and Lars Oreland 2

~ Department of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, Huddinge (Sweden) and 2Department of Medical Pharmacology, University of Uppsala, Uppsala (Sweden)

(Received 27 February 1989; Revised version received l0 August 1989; Accepted 11 August 1989)

Key words." Tetrahydroaminoacridine; Monoamine oxidase; MAO-A; MAO-B; Human brain; Enzyme activity

The inhibitory effects of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on monoamine oxidase (MAO; EC 1.4.3.4) enzyme activities in human hippocampal and rat striatal homogenates have been studied. The ac- tivities of MAO-A and MAO-B were estimated radiochemically, in-vitro, in human hippocampus and rat striatum in the presence of various concentrations of THA with [2-14C]hydroxytryptamine binoxalate (100 /zM) and fl-[ethylJ4C]phenylethylamine hydrochloride (20 gM) as substrates for the respective enzyme form. THA was found to inhibit both MAO-A and MAO-B activities reversibly and competitively, with inhibition constants (Ki) of 12.5 #M and > 500/~M respectively, of the rat striatal enzymes. From this it can be extrapolated that at therapeutic tissue concentrations of THA (10 -8 to 10 -6 M), more than 20% of the MAO-A activity should be inhibited. Thus it is possible that inhibition of MAO may be involved in the therapeutic action of THA in Alzheimer's disease.

Monoamine oxidase (MAO) is an enzyme catalyzing the oxidative deamination of biogenic as well as of exogenous monoamines. It is found in virtually all tissues and is tightly bound to membranes, in particular to the outer mitochondrial membrane. At least within noradrenergic and serotoninergic nerve terminals MAO is of major importance for regulation of the concentration of monoamine transmitters [6, 10].

MAO exists in two molecular forms, MAO-A and MAO-B. The former has a sub- strate preference for noradrenaline and serotonin and is selectively inhibited by low concentrations of clorgyline [4]. MAO-B is selectively inhibited by deprenyl [7] and has, for example, benzylamine and phenylethylamine as preferred substrates. The ac- tivities of MAO-A and MAO-B varies between brains from different species and also between various brain regions from the same species.

THA has been reported to improve memory in patients with Alzheimer's disease [11], but its mechanism of action is not yet clear. Low concentrations of THA have

Correspondence: A. Adem, Department of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, S-141 86 Sweden.

0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.

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been shown to inhibit acetylcholinesterase enzyme activity in vitro [3, 5], and a widely accepted theory of the mechanism of action of THA in Alzheimer's disease is that it inhibits acetylcholinesterase in the brain and thereby facilitating cholinergic synap- tic transmission. THA might also exert its effect by causing an increase in neurotrans- mitter release by blocking potassium channels in excitable cells because of its struc- tural relationship to 4-aminopyridine [11]. In addition it has been proposed that THA might increase neurotransmitter release via nicotinic receptor stimulation in postmortal cortical slices from Alzheimer patients [9]. It is, however, also possible that THA might have multiple mechanisms of action. Recently Liston et al. [8] reported that MAO activity, neither from brain nor liver was significantly inhibited by THA. In this study we report about a considerable inhibitory potency of THA on both MAO-A and MAO-B activity from human and rat brain tissue. The inhibi- tion was found to be reversible with more than a 40-fold lower Ki for inhibition of MAO-A than MAO-B.

MAO-A and MAO-B activities in human hippocampus (age 65 years, with no clin- ical history of psychiatric or neurological disorder) and rat striatum (male Sprague- Dawley rats, 300 g) were assayed radiochemically by a conventional method [l] with [2-J4C]hydroxytryptamine binoxalate (58 mCi/mmol, 5-HT) as substrate for MAO-A and fl-[ethyl-14C]phenylethylamine hydrochloride (50 mCi/mmol, PEA) as substrate for MAO-B. Briefly, aliquots of the diluted crude homogenate (1%) were suspended

]oo-]. Effect of THA on MAO -A and -B activity 9o! 80

~ ' 70

~ 60- ~ MAO-A

10

0 4 5 6 7

-Log T H A (M)

Fig. 1. Effect of THA on activity of MAO-A ( m - m ) and MAO-B ([5 I I). Rat striatal homogenate ali- quots were incubated 0.01 M potassium phosphate buffer (pH = 7.4) with [~4C]5-HT (100/~M) or [t4C]PEA

(20 pM) and increasing concentrations of THA (t0 ~ to 10 4 M). Enzyme activities in the presence of

THA are expressed as percent inhibited of total activity in the absence of THA. Values are means of deter-

minations from 4 rats.

H U M A N

315

100 80- ~ A

60- B

4 0 -

~ c

d d d d I

R A T

40- i_ v 30"

20" 10'

50-

A

C

i i i i i i

J s

Fig. 2. Line Weaver-Burk plots of MAO-A activity in human hippocampus and rat striatum; I/s (THA concentration in #M) versus 1/v (uptake rate, pmol/mg/min). Aliquots of crude homogenates from human hippocampus and rat striatum were incubated with 4 different concentrations of ['4C]5-HT in the absence of THA (C) or in the presence of 5 #M (B) or I0 #M (A) THA. Each line is that of best fit through the means of three determinations of MAO-A activity in human hippocampus or rat striatum. Each line has an r value of 0.99. Kinetic constants are shown in Table I.

TABLE I

EFFECT OF THA ON KINETIC PARAMETERS OF MAO-A IN RAT STRIATAL (Rat str.) AND H U M A N HIPPOCAMPAL (Human hipp.) HOMOGENATES

Values are means + S.D. of 3 experiments.

THA Km Vm~ (pM) (uM) (nM mol/mg tissue/min)

Rat str. Human hipp. Rat str. Human hipp.

0 32.55 + 2.96 26.72 + 2.50 0.151 ___+ 0.01 0.075 ___+ 0.005 5 44.31 + 2.90 40.45 + 5.02 0.143 __+ 0.01 0.071 ___+ 0.003

10 83.72 + 5.24 95.20 + 6.76 0.148 __+ 0.01 0.074__+ 0.004

316

in 0.05 M potassium phosphate buffer (pH = 7.4) in the absence or the presence of increasing concentration of T H A (10-8 to 10-4 M) and incubated with radiolabelled substrate 5-HT (100 /tM) or PEA (20 /tM) at 37°C for 10 min. Reactions were stopped by adding HCI (3 M). Blanks were prepared by adding the substrate after the acidification with HC1. The procedure involved the use of solvent extraction as described by Fowler and Oreland [2].

It can be seen from Fig. 1 that T H A is more potent at inhibiting MAO-A than MAO-B activity. Thus, at 10 -4 M, THA inhibited MAO-A activity almost comple- tely (93%) while at the same concentration only 30% of MAO-B was inhibited.

The kinetics for the THA inhibition were determined in human hippocampal and rat striatal homogenates using the standard assay procedure with 4 different sub- strate concentrations and 2 different T H A concentrations (Table I). Derivation of the Michaelis-Menten constants, Km and Vm,x, was achieved by linearization of the saturation curves according to the Lineweaver-Burk plot. Linear graphs were obtained and the lines of best fit were determined by linear regression (Fig. 2A, B). From Fig. 2A, B it can be seen that THA is a reversible, competitive inhibitor of MAO with higher potency for inhibition of the A-form of the enzyme. MAO-A and MAO-B activities in rat striatal homogenates were inhibited by T H A with Ki values of 12.5 pM and >500 pM respectively. Thus, at therapeutic doses of THA (10 s to 10 -6 M) more than 20% of MAO-A activity should be inhibited and furthermore, chronic administration of THA might induce accumulative inhibitory effect on MAO

activity. Summers et al. [11] reported that administration of T H A in combination with

lecithin led to a significant improvement of cognitive symptoms of patients with AIz- heimer's disease. It is possible that T H A may have multiple mechanism(s) of action in improving the symptoms of patients with Alzheimer's disease. The inhibitory effect of T H A on MAO-A might contribute to this alleged therapeutic action. Moreover, it would be interesting to test selective MAO-A inhibitors in Alzheimer patients.

This study was supported by grants from the Hans and Loo Osterman's Founda- tion, Stiftelse f6r Gamla Tj~narinnor, Nobel Medica and the Greta and Johan Kochs Foundation. The co-operation of Ass. Prof. Agneta Nordberg and Prof. Bengt Win-

blad is acknowledged.

1 Eckert, B., Gottfries, C.G., Von Knorring, L., Oreland, L., Wiberg, A. and Winblad, B., Brain and platelet monoamine oxidase in mental disorders. 1. Schizophrenics and cycloid psychotics, Prog. Neuro-Psychopharmacol., 4 (1980) 57-68.

2 Fowler, C.J. and Oreland, L., The nature of the substrate selective interaction between rat liver mito- chondrial monoamine oxidase and oxygen, Biochem. Pharmacol., 29 (1980) 2225-2233.

3 Heilbronn, E., Inhibition of cholinesterase by tetrahydroaminacridine, Acta Chem. Scan&, 15 (1961) 1386-1390.

4 Johnston, J.P., Some observations upon a new inhibitor of monoamine oxidase in brain tissue, Bio- chem. Pharmacol., 17 (1968) 1285 1297.

5 Kaul, P.N., Enzyme inhibiting action of tetrahydroaminoacridine and its structural fragments, J. Pharm. Pharmacol. 14 (1962) 243.

6 Kelder, D., Fagervall, 1., Fowler, C.J, and Ross, S.B., Regulation of the monoamine concentrations in the rat brain by intraneuronal monoamine oxidase, Biogenic Amines, 6 (1989) 1 14.

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7 Knoll, J. and Magyar, K., Some puzzling pharmacological effects of monoamine oxidase inhibitors, Adv. Biochem. Psychopharmacol., 5 (1972) 393~408.

8 Liston, D,, Russo, L., Mena, E.E. and Williams, I.H., Tetrahydroaminoacridine is concentrated in brain following intraperitoneal administration, Alzheimer Disease and Associated Disorders, Vol. 2, Issue 3, Abstracts, First Int. Conf. Alzheimer Dis., Rel. Disord., Las Vegas, NV, Sept. (~9, 1988, 219.

9 Nilsson, L., Adem, A., Hardy, J., Winblad, B. and Nordberg, A., Do tetrahydroaminoacridine and physostigmine restore acetylcholine release in Alzheimer brains via nicotinic receptors?, J. Neural Transm., 70 (1987) 357 368.

I00reland, L. and Engberg, C., Relation between brain monoamine oxidase activity and the firing rate of locus coeruleus neurons, Naunyn-Schmiedeberg's Arch. Pharmacol., 333 (1986) 235-239.

11 Summers, W.K., Majovski, L.V., Marsh, G.M., Tachiki, K. and Kling, A., Oral tetrahydroaminoacri- dine in long-term treatment of senile dementia, Alzheimer type, New Engl. J. Med., 315 (1986) 1241-1245.