Interaction between amphetamine and monoamine oxidase inhibitors

EUROPEAN JOURNAL OF PHARMACOLOGY 6 (1969) 115-120. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM

INTERACTION

BETWEEN AMPHETAMINE

AND MONOAMINE

OXIDASE INHIBITORS Kerin O'DEA and M.J.RAND Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Austraha Received 7 May 1968

Accepted 20 January 1969

K.O'DEA and M.J.RAND, Interaction between amphetamine and monoamme oxidase inhibitors, European J. Pharmacol. 6 (1969) 115-120. Observalaons were made on the acute tox~cmes in mice of amphetamine and of the monoamine oxidase lnhibltors isocarboxazid, mebanazine, nialamide, pargyline, phenelzine and tranylcypromme, with determinataon of the LDso values. Then, the effect of pretreatment of mice with monoamme oxidase inhibitors, or pretreatment with amphetamine, on the LDs0 of amphetamine was determined. The degree of potentaatlon of amphetamme toxicity by monoamine oxldase inhibitors varied from one to another, the range being from pargyfine, which increased the toxicity of amphetarmne about 3-fold, to mebanazine which increased it about 100-fold. There was no correlation between the toxicities of the monoamme oxidase mhibitors in mice and the degree to which they potentiated the toxicity of amphetamine. Amphetamine pretreatment was found to have no effect on Its own toxicity, determined subsequently. Monoamine oxidase mhibitors (MAOI) Amphetamine

1. INTRODUCTION Hypertensive crises, headaches, nightmares and other symptoms have been reported after the ingestion o f foodstuffs containing tyramine or the use of sympathomimetic drugs such as amphetamine in patients under treatment with monoamine oxidase inhibitors. A study o f the mechanisms of the potentiation o f the cardiovascular actions o f tyramine and amphetamine has previously been reported from this laboratory (Rand and Trinker, 1968). Fatalities as a result o f interaction between monoamine oxidase inhibitors and sympathomimetic amines have been reported (see Sj6qvist, 1965 and Trinker et al., 1967, for references). It has been shown that the toxicities and cardiovascular actions o f directly acting sympathomimetic amines such as adrenaline and noradrenahne are not increased by pretreatment with monoamine oxidase inhibitors. However, the cardiovascular actions o f indirectly acting sympathomimetic amines such as tyramine and amphetamine

Drug interaction, amphetamine and MAOI Drug toxicity

are increased by pretreatment with monoamine oxidase inhibitors (Trinker et al., 1967; Rand and Trinker, 1968). This paper deals with a study o f the toxic interactions between monoamine oxidase inhibitors and amphetamine in mice.

2. METHODS AND MATERIALS The LD50 values for amphetamine and for each o f the monoamine oxidase inhibitor drugs were determined in mice. Drugs were dissolved in 0.9% NaCl solutions and injected intraperitoneally. The mice used were a strain o f Swiss mice inbred for 46 years and obtained from the Commonwealth Serum Laboratories, Melbourne. Mice were randomly selected from batches containing both sexes and were assigned to plastic containers (15 X 30 cm) in groups of five. Two such groups were used at each dose level and 6 0 - 8 0 mxce were used for each LD50 determination. The conditions o f the experiment were k e p t as con-

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K.O'DEA and M.J.RAND

stant as possible; in particular, the noise level was kept to a mlmmum and the temperature was wxthln the range of 18-21°C. The mortality was taken as the number of mice dying in the 24 hr following the rejection. The LD50 values and 95% confidence limits were calculated by the method of Litchfield and Wilcoxon (1949). The interaction between amphetamine and the monoamlne oxidase inhibitors was studied by pretreating the mice with one-third and two-thuds of the LD50 of each monoamine oxldase inhibitor 4 hr before determining the LD50 of amphetamine on the survivors. The effect of amphetamine on its own toxicity was investigated in a similar way using mice pretreated with amphetamine. The following drugs were used: all-amphetamine sulphate, lsocarboxazid sulphate (Marplan, Roche), mebanazine oxalate (Actomol, Imperial Chemical Industries), malamxde monohydrochloride (Nlamid, Pfizer), pargyline hydrochloride (Eutonyl, Abbott), phenelzine sulphate (Nardll, Warner), and tranylcypromlne sulphate (Parnate, Smith Kllne and French). The doses given refer to these salts.

3. RESULTS

3.1. Toxicity of monoamine oxidase inhibitors and of amphetamine The LDs0 values are shown in table 1. Mice which died m the first two hours after the injection had terminal convulsions. In those that died later, the only overt sign preceding death was that the

Table 1 LDso values (mg/kg) of monoamme oxidase mlubltors and of amphetamine In mice. Drugs

LDso

95% confidence limits

Tranylcypromme Isocarboxazid Phenelzine Mebanazine Pargyline Nlalamide

41 49 170 176 410 580

( 30- 50) ( 40- 61) (155-187) ( 166-186) (347-484) (520-650)

Amphetamine

103

( 92-115)

abdomen was grossly distended due to gas in the intestines. The LDs0 values given in table 1 are consistently lower than those reported by Trinker et al. (1967) who used mice from the same source. The main differences in methods are that the present determinations were carried out in the spnng and the grouping of mice in fwes was adhered to rigidly: Trlnker et al. (1967) made their determinations in the summer and the number in each group varied. Mice of both sexes were used, the sexes being approximately equally distributed amongst the experimental groups in the random selection. Large groups ( 6 0 - 8 0 mice) were used for each determination, hence it ls unlikely that any of the differences were due to imbalance of the sexes together with a sexdependent toxicity. The latter point was investigated directly with amphetamine. The LD50 and 95% confidence hmits in a group of male mace was 100 mg]kg (hmlts, 73-137); in female mice, the corresponding values were 100 mg/kg (limits, 80-124). 3.2. Interacnons between monoamine oxidase mhibttors and amphetamine The results are shown in table 2. The magnitude of the increase in toxicity varied considerably. Thus, in mice survwlng 4 hr after pargyhne administration in doses corresponding to one-third and two-thirds of its LD50, the LD50 of amphetamine was reduced to approximately 40% and 30% respectwely of the control value, whereas pretreatment with mebanazine in corresponding doses resulted in reductions of the LD50 of amphetamine to approxunately 1.5% and 0.5% respectively of the control values. The interactions between the monoamme oxldase lnhlbitors and amphetamine axe displayed graphically m fig. 1. The lines connecting the LDs0 values resemble lsoboles, but as de Jongh (1961)has pointed out, representation of data on interachon as an isobole is not applicable when one drug is given by pretreatment. Nevertheless, the graphs do show that the ancreased toxicity of amphetamine after pretreatment with one-third of the LD50 of the monoamlne oxldase lnhibltors is greater than a simple addition of toxicity. There was no overlap of the doses corresponding to the 95% confidence limits except with pargyline. After pretreatment with two-thirds of the LD50 of the monoamlne oxldase inhlbltors, the

AMPHETAMINE AND MAO INHIBITORS

117

Table 2 The effect of pretreatment with monoamine oxidase inhib]tors on the LDso of amphetamine (mg/kg). Pretreatlnent One-third of LDso

Tranylcypromine Isocarboxaz]d Phenelzine Mebanazine Pargylme Nialamlde

>.~

100

Two-thirds of LDso

LDso

95% confidence limits

LDso

95% confidence limits

14 12 20 1.6 40 12.5

(10 -19.6) (5.5-26.4) (11 - 3 6 ) ( 0 . 8 - 3.0) (26 - 6 2 ) (8.3-18.9)

9 1.55 13 0.5 31.5 1.9

( 3.3 - 1 5 ) ( 0 . 7 8 - 3.10) ( 7 -22 ) ( 0.3 - 0.8 ) (20 -50 ) ( 1.0 - 3.6 )

100

100. ,

~N

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50

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200

400

PARGYLINE

TRANYLCYPROMINE

PHENELZINE

100 ~"'",

100"

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50

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. 3OO NIALAMIDE

50-

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100

200

MEBANAZINE

Fig. 1. Graphical representattons of interactions between amphetamine and monoamine ox]dase inhibitors in mice. Abscissae: LDso values for lnonoamine oxldase lnhibitors. (Note, the scales have been adjusted to make the intercepts equal.) OrdinateLDso value for amphetamine. The dashed line joining the LDso values in each graph represents the theoretical line for add]tive effects of the two drugs. The dotted lines join the corresponding upper and lower values of the 95% confidence lunits. The vertical lines represent the mean and 95% confidence limits of the LDs0 values for amphetamine determined in mice pretteated with onethird and two-thirds the LDs0 of each lnonoalnine oxldase inhibitor. Continuous lines are drawn to connect the means of the four LD5o values in each graph.

118

K.O'DEA and M.J.RAND

LD50 of amphetamine was greater than could be attributed to an additive effect with nialamlde, isocarboxazld, mebanazine, phenelzine and tranylcypromine. With the latter drug, the doses corresponding to the 95% confidence limits overlapped. After treatment with two-thirds the LD50 of pargyline, the toxicity of amphetamine was no greater than would be expected from an additive effect.

3.3. Effect o f amphetamine pretreatment on its own toxtcity Amphetamine closely resembles tranylcypromine in chemical structure and in certain pharmacological actions (for references, see Discussion). Therefore, it was decided to study the effect of 4-hr pretreatment with amphetamine on its own toxicity. As shown in table 3, pretreatment of mice with one-third, two-thirds, or the full LD50 of amphetamine did not significantly alter the LDs0 of amphetamine determined 4 hr later on the survwors. The LD50 determinations were made simultaneously in all 4 groups. Identical values for the LDs0 of amphetamine were obtained in mice from the control group in table 3 and in those from the separate group reported in table 1. It has been pointed out (by a referee) that the mice m the pretreated groups had been handled, and this alone may influence amphetamine toxicity. Therefore, an additional experiment was carried out m which the LD50 of amphetamine was determined simultaneously in a group injected with 1 ml of saline 4 hr previously the values (with 95% confidence limits) were 104 mg/kg (72-151) and 116 (77-174) respectively, and the difference was not significant.

Table 3 The effect of pretreatment with amphetamine on its own toxicity. Pretreatment

LDso (mg/kg)

95% confidence limits

None 1/3 of LDso 2/3 of LDso Full LDso

103 110 110 109

(92-115) (97-124) (88-137) (89-134)

4. DISCUSSION The toxicity of amphetamine in mice pretreated with monoamine oxidase lnhibitors was sharply increased. The rank order of potency of the monoamine oxidase lnhlbltors in producing this effect (on a dose basis) was: isocarboxazld = tranylcypromine > mebanazine > phenelzine > nlalamlde > pargyline. However, when the monoamine oxldase inhibitors were compared m terms of equal fractions of their own LDs0 values, their rank order of potency in increasing the toxicity of amphetamine became: mebanazine > lsocarboxazid = nlalamlde > tranylcypromine > phenelzlne > pargyhne. A theoretical interpretation of these results (suggested by a referee) is that animals which survive pretreatment with a monoamine oxidase inhibitor might be much more sensitive to the toxic effects of amphetamine than were untreated mice. In other words, the pretreatment has merely resulted in selection of a group of animals whxch were hypersensitwe to amphetamme. Although such an effect might have contributed to the increased toxicity of amphetamine, it could, at the most, exert only a minor influence since the upper point of the 95% confidence hmit for the LD50 of amphetamine in mice pretreated with onethird the LD50 of the monoamme oxidase lnhibitors was, in every case, considerably less than the lower 95% confidence hmlt for the LD50 of amphetamine in control mice. The LD50 values of amphetamine in these mice ranged from 40 to 1.5% of that in control mice. Furthermore, the mortahty after one-third the LD50 of monoamlne oxidase inhibitors only amounted to 8-15%. It is clear from these considerations that the survwors are not a selected batch of the control group in terms of sensitivity to the toxicity of amphetamine, but are specifically sensitized. Moreover, the increased toxicity of amphetamine after pretreatment with monoamine oxidase lnhibitors was significantly greater than could be explained by simple addition of toxicity, except with pargyhne. It is concluded therefore, that monoamme oxldase inhlbitors produce a true potentiation of amphetamine toxicity. There was no correlation between the toxicities of the monoamine oxldase inhibitor drugs and their abihty to potentiate the toxicity of amphetamine. Furthermore, there was no relationship between the

AMPHETAMINEAND MAO INHIBITORS type of the monoamine oxidase inhibitor and its ability to increase the toxicity of amphetamine. Thus the series included four hydrazine compounds and two non-hydrazines (pargyline and tranylcypromine): although pargyline is the least effective in increasing the toxicity of amphetamine, tranylcypromine is amongst the most potent. The series also includes drugs having sympathomLrnetlc activity (tranylcypromine and phenelzine), but again this does not appear to be related to the effect on amphetamine toxicity. According to Sjoqvist (1965), tranylcypromine and phenelzine are the monoamlne oxidase inhlbitors which cause most cases of harmful interactions with amphetamines, as judged from the clinical lrterature. He suggested that this may be due to their amphetamine-hke structure. Tranylcypromlnc is structurally related to amphetamine, the side chains being cyclopropylamine and isopropylamme respectwely. Sympathomimetlc activity on the cardiovascular system, resembling that of amphetamine, is displayed by tranylcypromine (McCulloch et al., 1967) and by phenelzine (Chessin et al., 1958). These drugs also have central amphetamine-like actions (Costa et al., 1960). If the toxic interaction between amphetamme and tranylcypromlne or phenelzine were due, at least in part, to their amphetamine-like activity, it would be expected that amphetamme would increase the toxicity of subsequent doses. This did not occur, therefore the increased toxicity of amphetamine produced by pretreatment with tranylcypromine and phenelzlne is unrelated to the amphetamine-hke sympathomimetic actions of these drugs. Amphetamine is an inhibitor of monoamine oxldase (Mann and Quastel, 1940), although it is weak, having only 0.1% the activity of isocarboxazid, and is active only m v i t r o (Randall and Bagdon, 1958). The fact that pretreatment with monoamine oxidase inhibitors caused an increase in amphetamine toxicity, but pretreatment with amphetamine did not, further in&cates that it does not have the activity in w v o . The result with amphetamine is surprising as it suggests that the dose given as pretreatment may have been cleared in 4 hr. This seems unlikely since in man amphetamine is still being excreted 24 hr after injection of a relatively small dose (Dring et al., 1966). Although no direct studies have been carried out in

119

mice, it seems likely that the survivors have retained some portion of the dose originally given. It would be expected that the effects of this dose would sum with the effects of amphetamine given subsequently, and that the LD50 would then be reduced. In fact, all of the survivors were displaying the effects of amphetamine, such as hyperactwlty and tremor. Since the effects of the first and second doses were not additive, it suggests that the survivors from the first dose had a decreased sensitivity to amphetamine which may be allied to the tachyphylaxls exhibited by amphetamine in its sympathomimetic actmns (Day and Rand, 1963). The mechanism of this effect may be that the initial dose of amphetamine blocks the uptake of the subsequent dose: amphetamine is known to block the uptake of monoamlnes in adrenergic neurones (Iversen, 1965). Attenhon has been drawn (by a referee) to the theoretical consideration that the pretreated mice surviving one-third, twothirds and the full LD50 of amphetamine are selected, to the extent that they are resistant to the drug. The proporhons of survivors in each of these groups amount to 10%, 25% and 50% respectively. Despite these differences m degrees of selection, the LD50 values determined subsequently did not differ. The mechanism of the increased toxicity of amphetamine after monoamlne oxldase mhibitors has been attributed to an increased content of catecholamines in peripheral and central stores caused by monoamine oxldase lnhlbitors which results in an increased release of the transmitters by amphetamine (Sjoqvlst, 1965). However, Rand and Trinker (1968) found that the potentiation of the cardiovascular actions of amphetamine by monoamine oxidase inhlbltors could be ascribed entirely to impairment of the liver mlcrosomal enzymes which normally metabohze amphetamine. The possible relevance of our findings to chnical situations may be limited by the fact that we used large doses of drugs which were within the lethal range, and the effects may not be produced at therapeutic dose levels. However, the potentiation of amphetamine by monoamlne oxidase lnhibltors is observed when doses of amphetamine well below the LDs0 are used, as was demonstrated by Rand and Trinker (1968) with the pressor actions of amphetamine in rats and cats, and by Morpurgo and Theobald (1968) with the hyperthermlc action of

120

K.O'DEA and M J RAND

a m p h e t a m i n e in rats. T w o factors are involved in a t t e m p t i n g to assess f r o m our data the p o t e n t i a l hazard o f low dose o f m o n o a m m e oxldase inhlbators. The first is the e x t r a p o l a t i o n from the findings with two-thirds and one-third the LD50 values, the second is the e x t e n t to which the t o x i c i t y o f a m p h e t a m i n e is increased. (These data are presented graphically in fig. 1.) F r o m these considerations, the greatest potential hazard is p r o v i d e d by mebanazine, f o l l o w e d by t r a n y l c y p r o m m e , then lsocarboxazld and nialamlde close together, then phenelzine and finally pargyhne. Sjoqvlst ( 1 9 6 5 ) points o u t that the m o n o a m l n e oxldase mhibators with whach m o s t h a r m f u l interactions have been r e p o r t e d ( t r a n y l c y p r o m i n e and phenelzlne) are the ones m o s t c o m m o n l y used. It seems probable that all are p o t e n t i a l l y liable to cause harmful mteractaons, and that the risk as greater with t r a n y l c y p r o m m e than with phenelzine.

ACKNOWLEDGEMENTS This work was supported by a grant from the National Health and Medical Research Council of Australia, and Miss O'Dea was in receipt of a Melbourne University Research Grant We are grateful to the Australian Medical Directors of the various companies mentioned m Methods for gifts of drugs. We are Indebted to our colleagues Mr. H.J.Fearn for advice on statistical procedures and Dr. F.R.TrInker for helpful discussions.

REFERENCES ChessIn, M., B.Dubmck, G.Leeson and C.C.Scott, 1959, Biochemical and pharmacological studies of &phenylethylhydrazlne and selected related compounds, Ann. N.Y. Acad. ScI. 80, 597-608.

Costa, E., G.R.Pscheldt, W.G.Van Meter and H.E.Hunwwh, 1960, Brain concentrations of blogenIc amines and EEG patterns of rabbits, J. Pharmacol Exptl. Therap. 130, 81 88. Day, M.D. and M.J.Rand, 1963, Tachyphyl~\lS to some sympathomtmetxc amines In relation to monoamme oxldase, 21, 84-96. De Jongh, S E., 1961, Isoboles, m Quantltatwe methods xn pharmacology, ed. H.De Jonge (North-Holland Publ. Co., Amsterdam) pp 318--327. Drmg, L.C., R.L.Smlth and R.T.WIlhams, 1966, Amphetamine metabolism, J Pharm. Pharmacol. 18, 402-404 Iversen, L.L, 1965, Inhibition of noradrenahne uptake by drugs, in Advances in Drug Research 2, 1 46. Litchfield, J.T. and F Wdcoxon, 1949, A simplified method of evaluating dose-effect in experiments, J. Pharmacol. Exptl Therap 96, 99-113 Mann, P J G. and J.J.Quastel, 1940, Benzedrine and brain metabohsm, Blochem. J 34, 414-431 Morpurgo, C and W.Theobald, 1967, Pharmacological mo&ficatlon of the amphetamine-induced hyperthermla in rats, Europ J. Pharmacol. 2, 287-294. McCulloch, M.W, F.R.Trmker, K.D.Calrncross and D.F.Story, 1967, Some actions of tranylcypromine, Austral. J. Exptl. Biol. Med Scl 45, 8 Rand, M.J and F.R.Trmker, 1968, The mechanism of the augmentation of responses to indirectly acting sympathomimetic amines by monoamlne oxldase lnhibltors, Brit. J Pharmacol 33,878-899 Randall, L.O and R.EBagdon, 1959, Pharmacology of lpronlazld and other amine oxidase mhlbltors, Ann. N Y. Acad SOL 80, 626--642. SJoqvlst, F., 1965, PsychotropIc drugs (2), Interaction between monoamme oxidase lnhlbltors and other substances, Proc. Roy. Soc. Med. 58, 967-978. TrInker, F.R., H J.Fearn, W.M.McCulloch and M.R.Rand, 1967, Experimental observations on the effects of adrenahne after treatment with antidepressant monoamlne oxidase mhlbltors (MAOI) drugs, Australian Dental J. 12, 297 303