Moclobemide, an inhibitor of MAO-A, does not increase daytime plasma melatonin levels in normal humans

Pro& Neum-Psychophommcol. b Biol. Psych&. 1990, Vol. 14. pp. 73-W. Printed in Great Britain. All rights reserved

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MOCLOBEMIDE, AN INHIBITOR OF MAO-A, INCREASE DAYTIME PLASMA MELATONIN NORMAL HUMANS

DOES NOT LEVELS IN

MIKA SCHEININl, MARKKU KOULU1,2 OLLI VTUR13, JOUNI WORINFNl and ROBERT'H. ZIMMER De artments of lPharmacology,2Clinical Pharmacology and 8Biostatistics,University of Turku, Turku, Finland, 3Department of P ysiology, University of Oulu, Oulu, Finland, and PF. Hoffmann-La Roche b Co, Basle, Switzerland. (Final form, June 1989) Abstract Scheinin, Mika, Markku Koulu, Olli Vakkuri, Jouni Vuorinen and Robert H. Zimmer: Moclobemide, An Inhibitor of MAO-A, Does Not Increase Daytime Plasma Melatonin Levels in Normal Humans. Prog. NeuroPsychopharmacol.& Biol. Psychiat. 1990, 14:73-82 1.

Plasma melatonin concentrationswere determined after administrationof single oral doses (100, 200 and 300 mg) of moclobemide, a reversible inhibitor of monoamine oxidase (MAO) with predominant effects on the A-type of the enzyme, to eight young, healthy male volunteers in a double-blind, random-order,placebocontrolled study. The investigationwas later continued in an open fashion by giving a single 10 mg dose of the MAO-B inhibitor deprenyl to the same subjects. 2. Neither drug had any effects on plasma melatonin levels, in spite of very marked MAO-A inhibition after moclobemide (as evidenced by up to 79% average decreases in the plasma concentrations of 3,4-dihydroxyphenylglycol,a deaminated metabolite of noradrenaline) and over 90% inhibition of MAO-B activitv in blood platelets after deprenyl. 3. It is concluded that daytime human plasma melatonin levels do not accurately reflect MAO-A inhibition in acute drug studies. Keywords: deprenyl, MAO-A inhibition, moclobemide, normal subjects, plasma melatonin. Abbreviations: 3,4-dihydroxyphenylglycol(DHPG), human growth hormone (hGH), monoamine oxidase type A/B (MAO-A/MAO-B)

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Introduction Increased daytime plasma melatonin concentrations have recently been suggested to reflect drug-induced inhibition of monoamine oxidase type A activity (MAO-A), as the acute administration of the MAO-A inhibitor brofaromine clearly elevated plasma melatonin levels in normal humans whereas the preferential MAO-B inhibitor pargyline lacked this property (Bieck et al., 1988). Should it be possible to generalize this finding to other MAO-A inhibitors with clinical antidepressant efficacy, plasma melatonin levels could be useful in the evaluation of the time course of MAO-A inhibition and its quantitation, both in volunteer studies and in depressed patients. In addition, neurobiological studies of the relationship between MAO-A inhibition and stimulation of daytime melatonin secretion might provide new insight into the therapeutic mechanisms of action of this class of drugs (Bieck et al., 1988; Oxenkrug et al., 1985; 1986). The connection between MAO-A inhibition and stimulation of melatonin secretion is supported by observations that melatonin synthesis in the rat pineal is enhanced after acute administration of clorgyline and harmine (selective MAO-A inhibitors), as well as after large, non-selective doses of pargyline (King et al., 1982; Oxenkrug et al., 1985); MAO-B inhibition by deprenyl was not effective in this respect (Oxenkrug et al., 1985). Also in human subjects, acutely administered tranylcypromine (a nonselective MAO inhibitor; Oxenkrug et al., 1986) and chronic treatment with clorgyline or tranylcypromine, but not with deprenyl, has elevated daytime plasma melatonin concentrations (Murphy et al., 1986). Moclobemide (RO 11-1163) is a novel inhibitor of MAO, distinguished by the reversibility of its action and its predominant effect on the A-type of the enzyme (MAO-A) (Da Prada et al., 1981; Keller et al., 1987). Clinical phase III trials have shown that the drug possesses clear antidepressant therapeutic efficacy comparable with that of some classical tricyclic drugs (Larsen et al., 1984; Casacchia et al., 1984; Norman et al., 1985; Lensch et al., 1987). We have recently carried out a pharmacodynamic single-dose, crossover study of moclobemide in healthy male volunteers. We investigated the effects of acute administration of moclobemide on monoamine metabolism and on the release of prolactin, human growth hormone (hGH) and cortisol, and a single dose of deprenyl was used to examine the MAO-A-dependence of the observed effects (Koulu et al., 1989).

MAO-A inhibition does not

increase

plasma melatonin

The hormone determinationswere used as potential indicators of the effects of the drugs on monoaminergic neurotransmission (Checkley, 1980; Koulu, 1986). The authors have now used the plasma samples collected during this study to test the hypothesis of increased daytime melatonin secretion in normal humans after acute MAO-A inhibition. Methods Subjects Eight healthy male volunteers participated after written informed consent. They were 23-27 years old and within 15 % of their ideal weight (mean 76 kg, range 63-92 kg). Two were smokers. The health of the subjects was ascertained by detailed medical history, physical examination, clinical chemistry tests and ECG. They had taken no medications in the two weeks preceding this study. Alcoholic beverages were prohibited for 36 h prior to each session, and food, smoking, caffeinated beverages and chocolate were not allowed after 22.00 h on the preceding night. Design of the Study The moclobemide experiment was carried out as a double-blind, randomized, placebo-controlledstudy with a Latin square design. Each subject received, as single doses, 100 mg, 200 mg or 300 mg moclobemide or matching placebo tablets (supplied by Roche, Basle, Switzerland) at intervals of 4 to 10 days. Three subjects were studied in May and five in September. Study Outline Each session started at 08.00 h , when the subjects arrived at the department. A polyethylene cannula was inserted into a vein in the cubital fossa and maintained patent with a dilute solution of heparin. The first blood samples were taken after a minimum of 15 min of supine rest. At time zero (0 h), the subjects took three similar tablets (containingeither placebo, 100 mg, 200 mg or 300 mg moclobemide) with 150 ml of water. The subjects remained supine for the first three hours of each session, after which a standardized lunch (low in tyramine content) was served. Blood samples for chemical determinations were collected until eight hours after the beginning of each session, whereafter the subjects were allowed to leave the department. They

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returned next morning, again after fasting overnight, and a 24-hour blood sample was taken. Chemical Determinations Blood for the chemical determinationswas collected into chilled polycarbonate,tubescontaining Na2EDTA, and was promptly chilled and centrifuged at 0-4OC. The plasma samples were stored at -7OOC. Plasma melatonin levels were determined from chloroform-extractedsamples using a specific radioimmunoassay,with 125I-melatonin as tracer (Vakkuri et al., 1984; Vakkuri, 1986). The method has an intra-assay coefficient of variation of 7% in the relevant concentration range. The methods used to measure other hormones, catecholamines, catecholamine metabolites and platelet MAO-B activity have been described previously (Koulu et al., 1989). All samples from one experimental session were analyzed in one assay. Deprenyl Experiment After completing the double-blind moclobemide study, the investigation was continued in December of the same year by administering a single 10 mg dose of deprenyl (Eldepryl,Farmos Group Ltd, Turku, Finland) orally to the same subjects in order to assess the MAO-A dependence of the observed effects. This experiment was carried out identically to the moclobemide sessions. Statistical Analysis The statistical analysis of log-transformedplasma melatonin values was performed using either analysis of covariance (ANCOVA) for repeated measurements, with two within-factors (dose and time) and the individual 0 h value as covariate, or analysis of variance (ANOVA), computed with BMDPZV programs (BMDP Statistical Software Inc, USA). Separate analyses were performed for the first three hour period after drug administration (ANCOVA) and for the later time points (ANOVA) in order to eliminate the confounding effects of the lunch break on the results. The deprenyl results were compared with the placebo session in an analogous manner. The results in the text refer to means + s.d.

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does not increase plasma melatonin

Results Baseline Plasma Melatonin Levels Mean plasma melatonin concentrations in the moclobemide study ranged between 67 f 24 and 82 f 22 pmol/i just before drug administration, and between 65 +-11 and 75 + 16 pmol/l 24 h after drug intake. The lowest individual morning value was 33 pmol/l and the highest 141 pmol/l. In the deprenyl experiment, the baseline values were somewhat higher (100 f 40 pmol/l at 0 h and 107 + 29 pmol/l at 24 h, range 33 - 178 pmol/l). Plasma Melatonin after Intake of MAO Inhibitors Neither drug had any appreciable effects on the concentration of melatonin in plasma (Fig. 1 and Table 1). Plasma melatonin concentrations showed similar temporal patterns after placebo and all moclobemide doses, with nadirs between 32 + 5 and 40 f 18 pmol/l 6- 8 h after drug intake (between 3 and 5 p.m.). The lowest average value after deprenyl was similar, 35 t 13 pmol/l 8 h after drug intake. Table 1 Statistical Analysisa of Log-TransformedPlasma Melatonin Concentrations after Moclobemide, Placebo and Deprenyl. ANCOVA (0 - 3 h) dose time

dxt

ANOVA (6 - 24 h) time dose

dxt

moclobemide F vs. placebo p

2.23 0.12

13.27 to.001

2.18 0.12

0.59 0.63

43.70
0.42 0.86

deprenyl F vs. placebo p

1.70 0.24

17.60
1.05 0.40

5.74 0.048

71.36
3.22 0.071

aAnalysis of covariance (ANCOVA) or analysis of variance (ANOVA) was done for repeated measurements with two within-factors (dose and time) Evidence of MAO inhibition Moclobemide powerfully and dose-dependentlydecreased the concentration of DHPG in plasma (Koulu et al., 1989). The decrease was about 55 % after 100 mg, 67 % after 200 mg and 79 % after 300 mg. The decrease in the concentration of DHPG in plasma after moclobemide was rapid, being near-maximal at 1 h and maximal at 2 h. The

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DHPG levels remained low for several hours; 6 h after moclobemide administration,plasma DHPG was still reduced by 26 % after 100 mg moclobemide, by 48 % after 200 mg moclobemide and by 58 % after 300 mg moclobemide, compared with a 15 % increase after placebo. At 24 h, plasma DHPG levels had returned to baseline after all treatments. MAO-B activity in blood platelets was inhibited maximally only by 27 % after moclobemide administration,whereas deprenyl inhibited MAO-B activity in blood platelets maximally by 94%, on the average. Plasma DHPG levels remained stable after deprenyl (Koulu et al., 1989).

Melatonin pmol/l 120 100 80 60 40

T

cl Cl

I

A

a

Fig. 1. Mean plasma melatonin concentrationsafter placebo (01, 100 mg (@I, 200 mg (a) or 300 mg (A) moclobemide, or 10 mg deprenyl (0). Standard deviations have been omitted for clarity; see text for typical examples. Other effects Both drugs were well tolerated, with only mild subjective effects (KOU~U et al., 1989). Moclobemide, but not deprenyl, stimulated prolactin secretion, whereas plasma hGH and cortisol levels were not affected (Koulu et al., 1989).

MAO-A inhibition does not increase plasma melatonin

Discussion It has been suggested that plasma DHPG may be a specific indicator of MAO-A inhibition in man at least in acute studies (Brown and Monks, 1983; 1984; Koulu et al., 1989). In spite of very marked MAO-A inhibition in our subjects, as judged by the decreases in plasma DHPG, moclobemide failed to alter plasma melatonin levels. This is in clear contrast with the results of Bieck and coworkers (1988) with 150 mg oral doses of brofaromine, and casts doubt on the hypothesis of a causal relationship between drug-induced MAO-A inhibition and stimulation of daytime melatonin secretion in normal humans. The validity of our plasma melatonin measurements is corroborated by the observed normal circadian variation of daytime plasma melatonin levels, and by the slightly higher morning values measured in the samples collected in December, when the daylight period in southern Finland is short, from 10 a-m. until 2 p.m. (Vakkuri, 1986; Wurtman and Ozaki, 1978). Prolongation of nocturnal melatonin secretion is characteristic for short winter days (Kauppila et al., 1987). We used 30 min sampling intervals during the first three hours after drug intake. It is unlikely that even a very brief melatonin secretion burst would have remained undetected, as the elimination half-life of exogenously administeredmelatonin in human blood is about 40 min (Vakkuri et al., 1985). Inspection of the individual concentration-timecurves did also not suggest the presence of such secretion peaks (data not shown). The plasma DHPG decrease does not necessarily indicate MAO-A inhibition within the central nervous system, or at the site(s) relevant for the putative stimulatory effect of MAO-inhibitors on melatonin synthesis and release. However, moclobemide stimulated prolactin secretion in a dose-dependent fashion (Koulu et al., 1989). There is ample evidence that serotonergic neurons play an important stimulatory role in the regulation of prolactin release (Tuomisto and Mlnnistij,1985), and we suggest that stimulation of prolactin secretion after moclobemide was mediated via central serotonergic neurons (Koulu et al., 1989). This would indicate that effective drug concentrations were achieved in the central nervous system. Indirect evidence supporting this assertion is also obtained from animal studies, where maximal MAO-A inhibition has been detected in rat brain already 30 min after single oral doses of moclobemide (Da Prada et al., 1983).

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There are, however, some differences in the experimental designs used by Bieck et al. (1988) and by us. Our subjects were fasting until three hours after drug intake, and remained supine throughout the day. It is possible that either food intake as such or some ingredients of foods or drinks, or other physiological factors encountered in normal daily activity are necessary for the melatoninstimulating activity of MAO-A-inhibitorsto become evident. Conclusion Daytime human plasma melatonin concentrationsdo not accurately reflect MAO-A inhibition in acute drug studies. Further experiments are needed to clarify the mechanisms mediating the previously observed stimulation of melatonin secretion after some MAO inhibitors, e.g. brofaromine and tranylcypromine. Acknowledgement This study was financially supported by F. Hoffmann-La Roche & co., Basle, Switzerland. References BIECK, P.R., ANTONIN, K.-H., BALON, R., OXENKRUG, G. (1988). Effect of brofaromine and pargyline on human plasma melatonin concentrations.Prog. Neuro-Psychopharmacol.& Biol. Psychiat. l.2, 93-101. BROWN, M.J., MONKS, N.J. (1983). Plasma dihydroxyphenylglycol concentration: a simple, sensitive and specific index of monoamine oxidase-A activity in vivo. Br. J. clin. Pharmac. l5, 599P. BROWN, M.J., MONKS, N.J. (1984). Plasma dihydroxyphenylglycol concentration: a simple, sensitive and specific index of monoamine oxidase-A activity in man. In: Monoamine Oxidase and Disease, K.F. Tipton, P. Dostert, and M.S. Benedetti, (Eds.), pp 559-560, Academic Press, London. CASACCHIA, M., CAROLEI, A., BARBA, C., FRONTONI, M., ROSSI, A., MECO, G ., ZYLBERMAN, M.R. (1984). A placebo-controlledstudy of the antidepressant activity of moclobemide, a new MAO-A inhibitor. Pharmacopsychiat.l7, 122-125. CHECKLEY, S.A. (1980). Neuroendocrine tests of monoamine function in man: a review of basic theory and its application to the study of depressive illness. Psycholog. Med. l0, 35-53. DA PRADA, M., KELLER, H.H., KETTLER, R., SCHAFFNER, R., PIERI, M., BURKARD, W.P., KORN, A., HAEFELY, W.E. (1981). Ro 11-1163, a specific and short-acting MAO inhibitor with antidepressantproperties. In: Monoamine Oxidase. Basic and Clinical Frontiers, K. Kamijo, E. Usdin and T. Nagatsu, 1Eds.1, pp 183-196. Excerpta Medica, Internat.

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VAKKURI, 0. (1986). Melatonin in human and animal tissues. An analytical and physiological study. Thesis, University of Oulu, Finland, ISBN 951-42-2244-X. VAKKURI, O., LEPPALUOTO, J., VUOLTEENABO, 0. (1984). Development and validation of a melatonin radioinununoassay using radioiodinated melatonin as tracer. Acta Endocrinol. 106, 152-157. VAKKURI, O., LEPPALUOTO, J., KAUPPILA, A. (1985). Oral administration and distribution of melatonin in human serum, saliva and urine. Life Sci. 37, 489-495. WURTMAN, R.J., OZAKI, Y. (1978). Physiological control of melatonin synthesis and secretion: Mechanisms generating rhythms in melatonin, methoxytryptophol, and arginine vasotocin levels and effects on the pineal of endogenous catecholamines, the estrous cycle, and environmental lightning. J. Neural Transm. l.3, Suppl., 59-70.

Inquiries and reprint requests should be addressed to: Dr. Mika Scheinin, M.D. Department of Pharmacology University of Turku SF-20520 Turku Finland