ComDarison of monoamine oxidase=A inhbhion by moclobemide in vitro and ex vivo in rats Kettler R, Da Prada M, Burkard WP. Comparison of monoamine oxidase-A inhibition by moclobemide in vitro and ex vivo in rats. Acta Psychiatr Scand 1990: Suppl 360: 101-102. Inhibition of MA0 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 vilro 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.
After the discovery of the mood-elevating (1) and monoamine oxidase (MA0)-inhibitory properties of iproniazid (2), the MA0 inhibitors used for a few years in antidepressant therapy were drugs displaying nonselective and irreversible interactions with the enzyme (3). The enzyme MA 0 exists in 2 catalytically distinguishable forms: MAO-A and MAO-B. MAO-A prefers 5-hydroxytryptamine (serotonin), norepinephrine and epinephrine as substrates and is selectively inhibited by clorgyline. MAO-B preferentially deaminates phenylethylamine and benzylamine, L-deprenyl being a relatively specific inhibitor. Tyramine and dopamine, however, are good substrates for both enzyme forms (4-7). In recent years, selective and reversible MAO-A inhibitors have been described (8-1 3). Moclobemide belongs to the new generation of short-acting, reversible MA0 inhibitors. MA0 activity was determined by a radiometric method (14), using serotonin as MAO-A substrate or phenylethylamine as MAO-B substrate. The in vitro determination was carried out with different inhibitor concentrations after preincubation of the tissue homogenates with the inhibitor for 30 min at 37°C. This preincubation period seems to be
R. Keitler, M. Da Prada, W. P. Burkard Pharmaceutical Research Department, F. Hoffmann-La Roche, Basle, Switzerland
Key words: antidepressant; monoamine oxidase (MAO) inhibitor; moclobemide; MAD-A; MAD-B; inhibition; in vitro; ex vivo R. Kettler, Pharmaceutical Research Department, F. Hoffmann-La Roche, CH-4002 Basle, Switzerland
long enough to permit maximum enzyme inhibition to develop mechanism-based M A0 inhibitors, i.e., inhibitors that undergo activation by preincubation. When tested in vitro with rat brain homogenates, moclobemide rather selectively inhibited MAO-A, as evidenced by the values obtained using 5-HT or phenylethylamine as substrates (6 and > 1000 x M, respectively). Moclobemide
Table 1. Moclobemide versus reference monoamine oxidase type A inhibitors: rat experiments ex vivo and in vitro Compound Moclobemide Cimoxatone Brofaromine Harmaline lsocarboxazid Phenelzine Tranylcypromine Clorgyline
IC50
8 16
5 30 17 72 12 7
6 0.003 0.013 0.007
0.300 0.015 0.1 80 0.001
ED5d1C50
1.3
5333 384 4286 57
4800 67 7000
ED, (pmollkg) = dose leading to 50% inhibition of MAO-A in rat brain 2 h after oral administration. IC, (pmoVl) = concentration that inhibits MAO-A in rat brain in vitro by 50%. ED,,/IC, = ratio: small values indicate potent inhibition ex vivo, higher values indicate potent inhibition in vitro.
101
has a low inhibitory potency similar to toloxatone, amiflamine, minaprine and SR 95191 (9), whereas clorgyline, harmaline, cimoxatone and brofaromine were found to be at least 100 times more potent than moclobemide (Table 1). Phenelzine, isocarboxazid and tranylcypromine were nonspecific, inhibiting MAO-A and -B activity by almost the same extent. In ex vivo experiments, the drugs to be tested were administered orally 2 h before decapitation. MocIobemide thereby preferentially inhibited MAO-A in rat brain. It was equipotent to clorgyline and brofaromine and, notably, was 2 and 4 times more potent than cimoxatone and hannaline. Moclobemide inhibited more markedly MAO-A than MAO-B in rat brain 2 h after oral, intraperitoneal, intravenous (i.v.) or subcutaneous administration of 60 pmol/kg (8 times its ED, value after oral administration). At the same dose, after either route of administration, moclobemide inhibited MAO-B (phenylethylamine as substrate) more effectively than MAO-A in rat liver, indicating good bioavailability but lack of selectivity in the periphery (9). Moclobemide is a relatively weak MAO-A inhibitor in vitro and yet is more potent in vivo than other reversible inhibitors, suggesting that the compound may be converted in vivo into active metabolite(s) (15-17). All attempts to identify activated moclobemide derivatives, however, failed both in our and in other laboratories (1 8). Recent experiments revealed that moclobemide (10 pmoles/kg i.v.) markedly (> 80%) inhibits liver MAO-A already 5 min after injection; this strongly suggests that MAO-A inhibition is exerted by the compound itself. Conversely, only partial MAO-B inhibition (about 60%) occurred 30 min after moclobemide injection. A more detailed comparison of the data in Table 1 reveals that moclobemide is the only MA0 inhibitor tested that has an ED,,/ICSO ratio of about 1, indicating that it displays virtually the same potency in vitro and ex vivo. Conclusion These results show that moclobemide is a preferential MAO-A inhibitor. Moreover, in spite ofits weak MAO-A inhibition in vitro, moclobemide is as potent in vivo as all MA 0 inhibitors in clinical use. References 1. CRANEGR. Iproniazid (Marsilid) phosphate, a therapeutic agent for mental disorders and debilitating disease. Psychiatr Res Resp 1957: 8: 142-152. 2. ZELLEREA, BARSKYJ, BERMANER, FOUTS JR. Action of isonicotinic acid hydrazides and related compounds on enzymes involved in the autonomic nervous system. J Pharmacol Exp Ther 1952: 106: 42W28.
3. YOUDIMMBH, FINBERG JPM. Monoamine oxidase inhibitor antidepressants. In: GRAHAME-SMITH DG, COWENPJ, eds. Psychopharmacology. I. Preclinical pharmacology. Amsterdam: Excerpta Medica, 1963: 38-70. 4. JOHNSTON JP. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol. 1968: 17: 1285-1297. 5. YANGH-Y, NEFFNH. The monoamine oxidase of brain: selective inhibition with drugs and the consequences for the metabolism of the biogenic amines. J Pharmacol Exp Ther 1974: 189: 733-740. 6. FOWLER CJ, CALLINGHAM BB. Substrate-selective activation of rat liver mitochondria1 oxidase by oxygen. Biochem Pharmacol 1978: 27: 1995-2000. 7. SUZUKI0,KATSUMATA Y, MASUKAZU 0. Substrate specificity of the type A and B monoamine oxidase. In: KAMIJO K, USDINE, NAGATSU T, eds. Monoamine oxidase; basic and clinical frontiers. Amsterdam: Excerpta Medica, 1982: 74-86. 8. BURKARD WP, BONETTI EP, DA PRADAM et al. Pharmacological profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase with preference for M A 0 type A. J Pharmacol Exp Ther 1989:248: 391-399. 9. DA PRADAM, KETTLERR, KELLERHH, BURKARD WP, MUGGLI-MANIGLIO D, HAEFELYWE. Neurochemical profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase type A. J Pharmacol Exp Ther 1989: 248: 40M14. 10. KAN J-P, STEINBERGER R, MOUGET-GONIOT C, WORMSP, BIZII~RE K. SR 95191,a selective inhibitor of type A monoamine oxidase with dopaminergic properties. 11. Biochemical characterization of monoamine oxidase inhibition. J Pharmacol Exp Ther 1987:240:251-260. 11. WORMSP, KANHP, WERMUTH CG, RONCUCCI R, BIZIERE K. SR 95191, a selective inhibitor of type A monoamine oxidase with dopaminergic properties. I. Psychopharmacological profile in rodents. J Pharmacol Exp Ther 1987:240: 241-250. BENEDETTI M, KAN JP, KEANEPE. A new specific 12. STROLIN reversible type A monoamine oxidase inhibitor: MD 780515. In: SINGER TP, VON KORFFRW, MURPHYDL, eds. Monoamine oxidase: structure, function and altered function. New York: Academic Press, 1979: 335-340. 13. WALDMEIER PC, BAUMANN PA, DELINI-STULA A et al. Characterization of new, short-acting and specific inhibitor of type A monoamine oxidase. Mol Probl Pharmacopsychiatry 1983: 19: 31-52. RJ, AXELROD J. A sensitive and specific assay for 14. WURTMAN the estimation of monoamine oxidase. Biochem Pharmacol 1963: 12: 1439-1441. 15. DA PRADAM, KELLERHH, KETTLERR et al. Ro 11-1163, a specific and short acting M A 0 inhibitor with antidepressant properties. In: KAMIJO K, USDINE, NAGATSU T, eds. Monoamine oxidase. Basic and clinical frontiers. Amsterdam: Excerpta Medica, 1981: 183-196. 16. DA PRADAM, CESURAAM, KETTLER R, Z ~ C H EG, R HAEFELY WE. Conversion of the neurotoxic precursor l-methyl4-phenyl-l,2,5,6-tetrahydropyridineinto its pyridinium metabolite by human platelet monoamine oxidase type B. Neurosci Lett 1985: 57: 257-262. BA, MORTONAJ. Some actions 17. BURTONCJ, CALLINGHAM of moclobemide (Ro 11-1 163) on M A 0 and on responses of the rat anococcygeus muscle to sympathomimetic amines. J Pharm Pharmacol 1984: 36: 53W. 18. CALLINGHAM BA, OVENSRS. Some in vitro effects of moclobemide and other M A 0 inhibitors on responses to sympathomimetic amines. J Neural Transm 1988: 26: [Suppl]: 17-29.
After the discovery of the mood-elevating (1) and monoamine oxidase (MA0)-inhibitory properties of iproniazid (2), the MA0 inhibitors used for a few years in antidepressant therapy were drugs displaying nonselective and irreversible interactions with the enzyme (3). The enzyme MA 0 exists in 2 catalytically distinguishable forms: MAO-A and MAO-B. MAO-A prefers 5-hydroxytryptamine (serotonin), norepinephrine and epinephrine as substrates and is selectively inhibited by clorgyline. MAO-B preferentially deaminates phenylethylamine and benzylamine, L-deprenyl being a relatively specific inhibitor. Tyramine and dopamine, however, are good substrates for both enzyme forms (4-7). In recent years, selective and reversible MAO-A inhibitors have been described (8-1 3). Moclobemide belongs to the new generation of short-acting, reversible MA0 inhibitors. MA0 activity was determined by a radiometric method (14), using serotonin as MAO-A substrate or phenylethylamine as MAO-B substrate. The in vitro determination was carried out with different inhibitor concentrations after preincubation of the tissue homogenates with the inhibitor for 30 min at 37°C. This preincubation period seems to be
R. Keitler, M. Da Prada, W. P. Burkard Pharmaceutical Research Department, F. Hoffmann-La Roche, Basle, Switzerland
Key words: antidepressant; monoamine oxidase (MAO) inhibitor; moclobemide; MAD-A; MAD-B; inhibition; in vitro; ex vivo R. Kettler, Pharmaceutical Research Department, F. Hoffmann-La Roche, CH-4002 Basle, Switzerland
long enough to permit maximum enzyme inhibition to develop mechanism-based M A0 inhibitors, i.e., inhibitors that undergo activation by preincubation. When tested in vitro with rat brain homogenates, moclobemide rather selectively inhibited MAO-A, as evidenced by the values obtained using 5-HT or phenylethylamine as substrates (6 and > 1000 x M, respectively). Moclobemide
Table 1. Moclobemide versus reference monoamine oxidase type A inhibitors: rat experiments ex vivo and in vitro Compound Moclobemide Cimoxatone Brofaromine Harmaline lsocarboxazid Phenelzine Tranylcypromine Clorgyline
IC50
8 16
5 30 17 72 12 7
6 0.003 0.013 0.007
0.300 0.015 0.1 80 0.001
ED5d1C50
1.3
5333 384 4286 57
4800 67 7000
ED, (pmollkg) = dose leading to 50% inhibition of MAO-A in rat brain 2 h after oral administration. IC, (pmoVl) = concentration that inhibits MAO-A in rat brain in vitro by 50%. ED,,/IC, = ratio: small values indicate potent inhibition ex vivo, higher values indicate potent inhibition in vitro.
101
has a low inhibitory potency similar to toloxatone, amiflamine, minaprine and SR 95191 (9), whereas clorgyline, harmaline, cimoxatone and brofaromine were found to be at least 100 times more potent than moclobemide (Table 1). Phenelzine, isocarboxazid and tranylcypromine were nonspecific, inhibiting MAO-A and -B activity by almost the same extent. In ex vivo experiments, the drugs to be tested were administered orally 2 h before decapitation. MocIobemide thereby preferentially inhibited MAO-A in rat brain. It was equipotent to clorgyline and brofaromine and, notably, was 2 and 4 times more potent than cimoxatone and hannaline. Moclobemide inhibited more markedly MAO-A than MAO-B in rat brain 2 h after oral, intraperitoneal, intravenous (i.v.) or subcutaneous administration of 60 pmol/kg (8 times its ED, value after oral administration). At the same dose, after either route of administration, moclobemide inhibited MAO-B (phenylethylamine as substrate) more effectively than MAO-A in rat liver, indicating good bioavailability but lack of selectivity in the periphery (9). Moclobemide is a relatively weak MAO-A inhibitor in vitro and yet is more potent in vivo than other reversible inhibitors, suggesting that the compound may be converted in vivo into active metabolite(s) (15-17). All attempts to identify activated moclobemide derivatives, however, failed both in our and in other laboratories (1 8). Recent experiments revealed that moclobemide (10 pmoles/kg i.v.) markedly (> 80%) inhibits liver MAO-A already 5 min after injection; this strongly suggests that MAO-A inhibition is exerted by the compound itself. Conversely, only partial MAO-B inhibition (about 60%) occurred 30 min after moclobemide injection. A more detailed comparison of the data in Table 1 reveals that moclobemide is the only MA0 inhibitor tested that has an ED,,/ICSO ratio of about 1, indicating that it displays virtually the same potency in vitro and ex vivo. Conclusion These results show that moclobemide is a preferential MAO-A inhibitor. Moreover, in spite ofits weak MAO-A inhibition in vitro, moclobemide is as potent in vivo as all MA 0 inhibitors in clinical use. References 1. CRANEGR. Iproniazid (Marsilid) phosphate, a therapeutic agent for mental disorders and debilitating disease. Psychiatr Res Resp 1957: 8: 142-152. 2. ZELLEREA, BARSKYJ, BERMANER, FOUTS JR. Action of isonicotinic acid hydrazides and related compounds on enzymes involved in the autonomic nervous system. J Pharmacol Exp Ther 1952: 106: 42W28.
3. YOUDIMMBH, FINBERG JPM. Monoamine oxidase inhibitor antidepressants. In: GRAHAME-SMITH DG, COWENPJ, eds. Psychopharmacology. I. Preclinical pharmacology. Amsterdam: Excerpta Medica, 1963: 38-70. 4. JOHNSTON JP. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol. 1968: 17: 1285-1297. 5. YANGH-Y, NEFFNH. The monoamine oxidase of brain: selective inhibition with drugs and the consequences for the metabolism of the biogenic amines. J Pharmacol Exp Ther 1974: 189: 733-740. 6. FOWLER CJ, CALLINGHAM BB. Substrate-selective activation of rat liver mitochondria1 oxidase by oxygen. Biochem Pharmacol 1978: 27: 1995-2000. 7. SUZUKI0,KATSUMATA Y, MASUKAZU 0. Substrate specificity of the type A and B monoamine oxidase. In: KAMIJO K, USDINE, NAGATSU T, eds. Monoamine oxidase; basic and clinical frontiers. Amsterdam: Excerpta Medica, 1982: 74-86. 8. BURKARD WP, BONETTI EP, DA PRADAM et al. Pharmacological profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase with preference for M A 0 type A. J Pharmacol Exp Ther 1989:248: 391-399. 9. DA PRADAM, KETTLERR, KELLERHH, BURKARD WP, MUGGLI-MANIGLIO D, HAEFELYWE. Neurochemical profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase type A. J Pharmacol Exp Ther 1989: 248: 40M14. 10. KAN J-P, STEINBERGER R, MOUGET-GONIOT C, WORMSP, BIZII~RE K. SR 95191,a selective inhibitor of type A monoamine oxidase with dopaminergic properties. 11. Biochemical characterization of monoamine oxidase inhibition. J Pharmacol Exp Ther 1987:240:251-260. 11. WORMSP, KANHP, WERMUTH CG, RONCUCCI R, BIZIERE K. SR 95191, a selective inhibitor of type A monoamine oxidase with dopaminergic properties. I. Psychopharmacological profile in rodents. J Pharmacol Exp Ther 1987:240: 241-250. BENEDETTI M, KAN JP, KEANEPE. A new specific 12. STROLIN reversible type A monoamine oxidase inhibitor: MD 780515. In: SINGER TP, VON KORFFRW, MURPHYDL, eds. Monoamine oxidase: structure, function and altered function. New York: Academic Press, 1979: 335-340. 13. WALDMEIER PC, BAUMANN PA, DELINI-STULA A et al. Characterization of new, short-acting and specific inhibitor of type A monoamine oxidase. Mol Probl Pharmacopsychiatry 1983: 19: 31-52. RJ, AXELROD J. A sensitive and specific assay for 14. WURTMAN the estimation of monoamine oxidase. Biochem Pharmacol 1963: 12: 1439-1441. 15. DA PRADAM, KELLERHH, KETTLERR et al. Ro 11-1163, a specific and short acting M A 0 inhibitor with antidepressant properties. In: KAMIJO K, USDINE, NAGATSU T, eds. Monoamine oxidase. Basic and clinical frontiers. Amsterdam: Excerpta Medica, 1981: 183-196. 16. DA PRADAM, CESURAAM, KETTLER R, Z ~ C H EG, R HAEFELY WE. Conversion of the neurotoxic precursor l-methyl4-phenyl-l,2,5,6-tetrahydropyridineinto its pyridinium metabolite by human platelet monoamine oxidase type B. Neurosci Lett 1985: 57: 257-262. BA, MORTONAJ. Some actions 17. BURTONCJ, CALLINGHAM of moclobemide (Ro 11-1 163) on M A 0 and on responses of the rat anococcygeus muscle to sympathomimetic amines. J Pharm Pharmacol 1984: 36: 53W. 18. CALLINGHAM BA, OVENSRS. Some in vitro effects of moclobemide and other M A 0 inhibitors on responses to sympathomimetic amines. J Neural Transm 1988: 26: [Suppl]: 17-29.
