Gen. Pharmac., 1976, Vol. 7, pp. 381 to 386. Pergamon Press. Printed in Great Britain

MINIREVIEW MULTIPLE FORMS OF MONOAMINE OXIDASE AND THEIR INTERACTION WITH TRICYCLIC PSYCHOMIMETIC DRUGS JEROME A. ROTH

Department of Pharmacology and Therapeutics, 122 Farber Hall, Schools of Medicine and Dentistry, State University of New York at Buffalo, Buffalo, N.Y. 14214, U.S.A. (Received 20 July 1976)

INTRODUCTION

of the enzyme is responsible for less than one-third of the total kynuramine deaminating activity in mouse kidney and intestine. Jarrot (1971) and Goridis & Neff (1971) have reported that sympathetic denervation of mouse vas deferens or rat pineal gland, respectively, led to a specific decrease in the A form of MAO in these tissues. On the basis of these studies, they suggested that neuronal MAO is predominantly of the A type. In support of this premise, Donnelly et al. (1976) recently demonstrated that mouse neuroblastoma cells NIE-115 grown in culture contain exclusively the A form of the oxidase. This form of mitochondrial enzyme is also found extraneuronally as is the B type of the oxidase (Roth & Gillis, 1975a). Mitochondrial bound A and B forms of rat brain and mouse lung MAO have been partially separated by sucrose gradient centrifugation (Yang & Neff, 1973; Gallagher & Wolf, 1976). In both tissues the B form is associated with high-density mitochondria. MAO is tightly bound to the outer mitochondrial Biochemical and pharmacoloffical properties membrane (Schnaitman et al., 1967) and solubilizaThe two forms of mitochondrial MAO designated tion of the enzymes normally requires treatment with by Johnston (1968) as type A and B are distinguished nonionic detergents (Shih & Eiduson, 1973). MAO in vitro by their substrate and inhibitor specificities. from a number of sources has been purified and has The A form of the oxidase primarily deaminates the been shown to contain FAD covalently bound via putative neurotransmitters, 5-hydroxytryptamine a thioether linkage of cysteine to the 8-methyl group (5-HT), norepinephrine (NE) and dopamine (DA) as of the isoalloxazine moiety of the flavin cofactor well as tyramine, octopamine and tryptamine (Walker et al., 1971). McCauley & Racker (1973) have (Squires, 1972; Neff & Yang, 1974). This form of separated the A and B forms of bovine brain MAO MAO is selectively inhibited by the drugs, clorgyline by immunologically precipitating the B form of the and harmaline. The B form of the mitochondrial oxi- oxidase with an antibody against bovine liver MAO dase oxidatively degrades the latter three amines and (type B). The precipitated B isoenzyme retained its phenylethylamine (PEA) and benzylamine (BzNH2) • enzymatic activity. as well (Yang & Neff, 1973). This form of MAO is Though the above data suggests that the A and selectively inhibited in most species by Deprenyl and B form of MAO are immunologically distinct strucpargyline. tures, it remains unclear as to whether the two forms The relative abundance of the type A and B forms of the mitochondrial oxidase represent two distinct of MAO varies considerably in different tissues protein species or a single enzyme with the surround(Squires, 1972). For example, mouse liver contains ex- ing lipid matrix imparting the different substrate and clusively the B form of the oxidase, whereas this form inhibitor specificities. Houslay & Tipton (1973) found 381 IDENTIFICATIONof tWO catalytically distinct forms of monoamine oxidase (MAO) in isolated mitochondria and submitochondrial particles has prompted an extensive reinvestigation, within the past decade, of the biochemical and pharmacological properties and physiological functions of this enzyme. The teleological role for the existence of two functionally distinct forms of MAO has not been established though it has been shown that specific neurochemical and behavioral changes occur on administration of inhibitors of either form of MAO. Because inhibitors of MAO have been used extensively as antidepressant agents, the presence of multiple forms of this oxidase in vivo may have profound clinical and pharmacological significance. Accordingly, it remains to be determined whether inhibition of either or both forms of the oxidase relates to the therapeutic action of these drugs.

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that after treating a solubilized preparation of rat liver of human brain A and B MAO with the chaotropic agent, sodium perchlorate, only a single form of the oxidase separated on acrylamide gel electrophoresis. Since chaotropic agents disrupt hydrophobic bonds between protein and lipid material, they contend that the multiple forms of MAO arise from differential binding of membrane lipids to a single species of enzyme, thus conferring upon it allotropic properties. However, Ekstedt & Oreland (1976) recently demonstrated that on extraction of lipid from rat liver A and B MAO with methyl ethyl ketone no transformation of the A form into the B form occurred. They conclude that the lipid surrounding the oxidases most likely does not impart the substrate and inhibitor specificities of the A and B forms of MAO. Relatively few studies have been performed which differentiate the biochemical and behavioral changes associated with administration of either of the two classes of MAO inhibitors. Intravenous injection to rats of the type A MAO inhibitor, clorgyline, resulted in a reduction of the type A activity in brain with a concomitant increase in the endogenous levels of 5-HT and NE (Yang & Neff, 1974). These investigators also reported that the type B inhibitor, Deprenyl, failed to elicit this change but was shown to significantly elevate the levels of PEA in rat brains after exogenous administration of this amine. As expected, clorgyline failed to increase brain PEA. They also found that following the administration of clorgyline reserpine-induced ptosis, catatonia, and increased motor activity was prevented. Rats treated with Deprenyl in a dose which inhibited only type B MAO did not reverse these reserpine-induced behavioral changes. In contrast, Deprenyl but not clorgyline was shown to potentiate the stereotyped sniffing behavior induced by PEA (Braestrup et al., 1975). Multiple forms of MAO in lun9 The studies described above suggest that multiple forms of the mitochondrial oxidase exist in vivo; however, the presence of these isoenzymes in an intact organ had not been demonstrated, The possibility existed that the differences in the properties between the A and B forms of MAO are simply artifacts produced by the homogenization procedures used to isolate mitochondria. Accordingly, we undertook to determine whether an intact perfused organ also contains catalytically distinct forms of the mitochondrial oxidase (Roth & Gillis, 1975a). Lung was chosen for these studies because prior investigations have demonstrated that the biogenic amines, 5-HT and NE, are removed and deaminated on a single passage through the pulmonary vasculature (Junod, 1972; Gillis & Iwasawa, 1972). Since both 5-HT and NE are deaminated by the A form of rabbit pulmonary MAO, the metabolic fate of PEA, a type B MAO substrate, in the isolated perfused lung was examined (Roth & Gillis, 1975a). In

these studies we found that over 90~o of the perfused PEA (1.1/~M) was deaminated on a single passage through rabbit lung. Approximately two-thirds of the deaminating activity was prevented by addition of 1 0 - 5 M pargyline to the perfusion medium whereas the remaining activity was inhibited by 10-a M semicarbazide, an inhibitor of plasma amine oxidase. Neither of the amine oxidase inhibitors altered the pulmonary uptake of the PEA structural analog, amphetamine. These data indicate there are two different forms of amine oxidase in rabbit pulmonary tissue responsible for deamination of PEA. The pargyline-sensitive enzyme is analagous to the mitochondrial oxidase identified in vitro and the semicarbazidesensitive form resembles the soluble amine oxidase found in plasma (Buffoni & Corte, 1972) and several large arteries (Rucker & Goettlich-Riemann, 1972; Coquil et al., 1973). In order to determine whether the two mitochondrial forms of the oxidase, type A and B, are present in the intact lung, equimolar concentrations of 14C-PEA and aH-5-HT were perfused simultaneously through rabbit lung. During perfusion of these amines, harmaline, a type A MAO inhibitor, was added to the perfusion medium. On addition of this alkaloid, the production of deaminated product from 5-HT decreased approximately 75~o whereas the product of PEA deamination was reduced less than 10~. These results are consistent with the effect of harmaline on 5-HT and PEA deamination measured in broken cell preparations of the oxidase (Roth & Gillis, 1974) and therefore provide direct evidence for the existence of the A and B forms of MAO in the intact rabbit lung. Bakhle & Youdim (1976) have also recently demonstrated the presence of the B form of the mitochondrial oxidase in perfused rat lung. In these experiments the B MAO inhibitor, Deprenyl, was shown to be a more effective inhibitor of PEA deamination in perfused rat lungs than was the type A inhibitor, clorgyline. They also found that PEA deamination was not altered when 5-HT was coperfused with PEA nor was 5-HT degradation affected by coperfusion with PEA. MAO activity in affective disorders There is an increasing body of evidence in the literature which suggests that changes in the levels of monoamine oxidase may be involved in affective disorders. For example, platelet MAO (type B activity) has been reported to be higher in depressed patients than in a normal control group (Nies et al., 1974). Similarly, females and the aged are reported to have higher levels of the platelet oxidase and also a higher incidence of depression (Nies et al., 1974). It has also been shown by a number of investigators that platelet MAO activity is decreased in the chronic schizophrenic (Murphy & Wyatt, 1972; Meltzer & Stahl, 1974) and the bipolar depressed patient (Murphy & Weiss, 1972). In addition, hor-

Interaction of monoamine oxidase with tricyclic psychomimetic drugs mones such as progesterone which increase the levels of the mitochondrial oxidase also promote depression whereas estrogens which inhibit MAO may act as antidepressant agents (Grant & Davies, 1968; Youdim et al., 1974). Gerovital H3 which has been shown to act as an antidepressant in the aged has also been reported to be a weak competitive inhibitor of MAO (MacFarlane, 1975). Because of the deleterious side effects associated with treatment of original MAO inhibitors such as pargyline, in rec~nt years the tricyclic antidepressant drugs have for the most part supplanted their use in clinical practice. The therapeutic action of tricyclic antidepressant drugs has been attributed to inhibition of neuronal reuptake of the biogenic amines, norepinephrine (NE) and/or 5-hydroxytryptamine (5-HT), in brain (Glowinski & Axelrod, 1966; Carlsson et al., 1969). This pharmacological property is common to the majority of the known tricyclic antidepressant drugs in clinical use today. However, it has recently been shown that the antidepressant agent, iprindole, is much less effective than other tricyclic antidepressant drugs in preventing the reuptake of catechol or indole amines in rat and mouse brain slices and in rat brain slices and in rat brain synaptosomal preparations (Gluckman & Baum, 1969; Lemberger et al., 1970; Lahti & Maickel, 1971; Ross et al., 1971). Also unlike the action of other tricyclic antidepressants, iprindole failed to alter the concentration of 5-HT in human platelets (Fannet al., 1972). The inability of iprindole to prevent amine uptake has led several investigators to conclude that iprindole does not function by the classically accepted mechanism of other tricyclic drugs. The tricyclic antidepressant drugs have been reported to mimic many of the pharmacological responses of the classical MAO inhibitors. These include their ability to antagonize the actions of resetpine (Sulser et al., 1962) and ct-methyl-meta-tyrosine (Carlsson et al., 1969) as well as to increase the urinary excretion of several monoamines and concomitantly decrease excretion of their corresponding deaminated metabolites (Schildkraut et al., 1965). In addition, several studies have revealed that the dea-

minated product of 5-HT, 5-hydroxyindole acetic acid, is decreased in the cerebral spinal fluid of depressed patients treated with the tricyclic drugs, imipramine (Post & Goodwin, 1974), amitriptyline (Post & Goodwin, 1974; Bowers, 1974) or nortriptyline (Asberg et al., 1973). Other investigators have also shown that the deaminated products of 5-HT and NE decrease in brains of rats chronically treated with imipramine or structurally related drugs (Schildkraut et al., 1970; Meek & Werdinius, 1970; Bruinvels, 1972; Alpers & Himwich, 1972). Schildkraut & coworkers (Schildkraut et al., 1969) have also reported that acute administration to rats of a variety of tricyclic antidepressant agents caused a decrease in the formation of deaminated products in brain from 3H-NE administered intracisternally. In this latter publication, Schildkraut suggests that the tricyclic compounds or possibly their metabolites may prevent the deamination of NE by inhibiting mitochondrial MAO. Inhibition of M A O by tricyclic drugs In 1960 Pulver et al. (1960) reported that imipramine, 2-hydroxyimipramine and desmethylimipramine were only weak inhibitors of a crude homogenate preparation of rat liver MAO as measured by isoamylamine (25 mM) deamination. Based on this study and several others (Pletscher & Gey, 1959, 1962) which demonstrated that tricyclic antidepressant drugs failed to increase the levels of brain catechol and indole amines, the assumption was made that these antidepressant agents do not inhibit MAO in vivo. In contrast to these findings, Zubrzycki & Staudinger (1967) and Gabay & Valcourt (1968) have found that imipramine inhibits rabbit mitochondrial MAO. Since these studies did not make the distinction as to which form of the oxidase was inhibited, an extensive investigation was undertaken in our laboratory to determine the ability of a variety of tricyclic psychoactive agents to inhibit both forms of rabbit lung and brain MAO (Roth & Gillis, 1974, 1975b; Roth, 1975). The results of these studies have revealed the following information. All tricyclic drugs tested (Table

Table 1. Tricyclic psychoactive drugs tested which inhibit rabbit monoamine oxidase Iminodibenzyl drug Imipramine Desmethylimipramine Didesmethylimipramine 2-Hydroxydesmethylimipramine Chlorimipramine

383

Dibenzocycloheptene drugs Amitriptyline Nortriptyline Protriptyline Cyclobenzaprine

Phenothiazine and structurally related drug

Other tricyclic drugs

Chlorpromazine 7-Hydroxychlorpromazine Chlorprothixene

Iprindole Doxepin GB-94 (mianserin)

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JEROME A. ROTH

Table 2. Binding constants for several tricyclic psychoactive drugs to type A and B monoamine oxidase Ki Inhibitor

Type A (M)

Type B (M)

Imipramine Amitriptyline Chlorprothixene

3 × 10 -4 2 × 10 -4 8 × 10 -5

4 × 10 -5 5 x 10 -6 6 × 10 -6

1) with the exceptions of imipramine-N-oxide and chlorpromazine sulfoxide inhibit rabbit brain type A and B MAO. Of the drugs examined, all are more potent inhibitors of the B form of the oxidase as measured by PEA deamination. The K i values for several clinically important compounds binding to the A and B forms of MAO are shown in Table 2. As indicated in this table, amitriptyline is the most potent inhibitor of rabbit type B MAO. Based on these observations, it was suggested that the structure of the tricyclic ring moiety determines the ability of these drugs to inhibit the B form of rabbit MAO. Consistent with this is the fact that replacement of one of the center ring carbon atoms of amitriptyline with oxygen, as in doxepin, or both carbon atoms with sulfur, as in chlorprothixene, or dehydrogenation as in cyclobenzaprine, reduces the ability of amitriptyline to inhibit this form of MAO. The influence of the structure of the ring moiety on inhibition of MAO is also demonstrated by the fact that dibenzocycloheptene analogs are more potent inhibitors of the B form of MAO than are iminodibenzyl derivatives. It was also found that the basicity of the amine side chain has little effect on the magnitude of inhibition, for the N-demethylated products of amitriptyline and imipramine are equally effective as the parent drug in their ability to inhibit rabbit MAO. Imipramine inhibition of both rabbit type A and B MAO was not affected by preincubation in the presence of the enzyme and inhibition was found to be reversible. Both imipramine and amitriptyline were noncompetitive (mixed) inhibitors of PEA deamination, whereas chlorprothixene was a competitive inhibitor of this reaction. Edwards & Burns (1974) have reported that arnitriptyline was a noncompetitive inhibitor of human platelet MAO deamination of PEA, a mixed-noncompetitive-inhibitor of tryptamine oxidation and a competitive inhibitor of benzylamine metabolism. These data would suggest that the interaction of tricyclic drugs with rabbit brain and human platelet MAO is rather complex and probably involves a hydrophobic interaction between the ring moiety of the psychoactive drugs and the oxidase. Since, as noted above, iprindole does not function by inhibiting biogenic amine reuptake, the effects of this drug on the activity of both forms of rabbit MAO (Roth & Gillis, 1975c) were also examined. Results of these experiments indicated that the ability of

iprindole to inhibit the A and B forms of MAO is similar to that of imipramine. Approximately 50% inhibition of either PEA or 5-HT deamination was seen at 2.5 x 10 -5 and 9 x 10 -5 M iprindole, respectively. Our unpublished observations further reveal that human brain type A and B MAO is also susceptible to inhibition by a variety of tricyclic antidepressant agents including iprindole. The properties of tricyclic drug binding to human brain type B MAO were found to be similar to that reported for human platelet MAO (Edwards & Burns, 1974). The relationship between tricyclic drug inhibition of MAO and their therapeutic action cannot be assessed from the above data. Though much has been written concerning the influence of tricyclic antidepressant drugs on neuronal uptake of catechol and indoleamines, it also has not been clearly established whether this interaction relates to the clinical properties of these antidepressant agents. It is anticipated that future studies will determine the relative role of each process in the therapeutic mechanism of these widely used drugs. It is tempting to speculate that inhibition of either or both type A and B MAO by tricyclic drugs may, in conjunction with their role as inhibitors of amine reuptake, contribute to their clinical efficacy.

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