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NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.4 231

MDMA (Ecstasy) Inhibition of MAO Type A and Type B: Comparisons with and (Prozac) Efthimia T. Kokotos Leonardi, M.S. and Efrain C. Azmitia, Ph.D.

3,4-Methylenedioxymethamphetamine (MDMA), a fenfiuramine (FEN) and fiuoxetine (FLUOX) were (5-HT) neurotoxin, has been shown to promote compared to those of MDMA. The rank order of the release of serotonin (5-HT) and block its reuptake. these for MAO-A inhibition was The increased buildup of extracellular 5-HT should MDMA>FLUOX>FEN, whereas for MAO-B inhibition, normally be degraded by (MAO). The FLUOX>MDMA>FEN. A combination of FLUOX and effects of both of MDMA were examined on MDMA at their respective lC50 did not inhibit MAO MAO-A and monoamine oxidase-B (MAO-B) activity in activity more than either alone at equivalent rat homogenates. Both enantiomers competitively concentrations. These results indicate that the actions of inhibited 5-HT catabolism by rat brain MAO-A. The Ki FEN do not appear to involve MAO inhibition. MDMA of MDMA for MAO-A was 22 J.lmollL. A mixed type of (ecstasy) produced a preferential inhibition of MAO-A

inhibition by MDMA was observed for (1C50 = 44 J.lmoIlL), which should increase extracellular catabolism by MAO-B for both optical antipodes. 5-HT. This may explain its high toxicity potential. Logistical analysis of concentration response curves for Finally, FLUOX (Prozac) showed an inhibition of

MDMA inhibition of MAO-A and MAO-B show an lC50 MAO-B (1C50 = 80 J.lmoIlL, which may increase the of 44 J.lmollL for inhibition of MAO-A by MDMA. The intracellular content of 5-HT. This may contribute to its lC50 value of MDMA inhibition of MAO-B was 370 therapeutic potential. In contrast, FEN appears to be a J.lmollL, showing a selective potency for MAO-A poor inhibitor of both MAO-A and MAO-B. inhibition. The MAO inhibitory properties of [Neuropsychopharmacology 10:231-238, 1994]

KEY WORDS: Serotonin; Phenethylamine; for serotonin is 1170 IlmollL (Fowler and Tipton 1982; Garrick and Murphy 1982). is Monoamine oxidase (MAO) is an with two sub­ inhibited by nanomolar concentrations of clorgyline types (E. C.1.4.3.4) characterized by their differentialre­ (Johnston 1968). (MAO-B) has sponses to the irreversible inhibitors clorgyline and a higher affinityfor phenethylamine than MAO-A and depreny1. Monoamine oxidase A (MAO-A) has a higher is inhibited by nanomolar concentrations of deprenyl affinity for serotonin (5-HT) than MAO-B; the KM of (Garrick and Murphy 1982; Yang and Neff1974). Dopa­ MAO-A for serotonin is 99 IlmollL, the KM of MAO-B mine (DA) is metabolized with equal affinity by both subtypes (Yang and Neff 1974). Further evidence for two molecular forms of MAO has been provided by the From the Department of Biology, New York University, New York, New York. cloning of two distinct MAO genes (Bach et a1. 1988) Address correspondence to Dr. Efrain C. Azmitia, Department of and their subsequent functional expression inCOS cells Biology, New York University, 100 Washington Square East, New (Lan et a1. 1989). York, NY 10003. Received November 7, 1993; revised February 16, 1994; accepted Both and glia contain monoamine oxidases February 17, 1994. that catabolize the classical monoamine neurotransmit-

© 1994 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0893-133X/94/$7.00 232 E.T. Kokotos Leonardi and E.C. Azmitia NEUROPSYCHOPHARMACOLOGY 1994-VOL.10, NO. 4

ters. Monoamine oxidase A is the predominant form to the transporter for these agents is FLUOX>FEN = in catecholaminergic neurons, whereas MAO-B is the MDMA (Poblete et al. 1989). prevalent form in glia (Levitt et al. 1982; Westlund et The similar actions of FEN, FLUOX and MDMA on al. 1985). Although MAO-B has a higher affinity than binding suggest they may pos­ MAO-B for serotonin, MAO-B is the major molecular sess common effects upon other param­ form found within serotonergic neurons (Westlund et eters, such as monoamine oxidase activity, in vitro. The al. 1985). present report examines the effectsof both enantiomers 3,4-Methylenedioxymethamphetamine (MDMA) of MDMA on catabolism of [3H]-serotonin and p4C]­ binds with high affInity to the 5-HT transporter pro­ phenethylamine by rat brain monoamine oxidase in tein (Poblete et al. 1989) and has been shown to be a vitro. In addition, we compared the effects of fluoxe­ potent releaser of serotonin by a Ca2+-independent tine (FLUOX) and fenfluramine (FEN) to MDMA on rat mechanism (Berger et al. 1992; Gu and Azmitia 1989; brain MAO activity. Our results suggest that inhibition Johnson et al. 1986; Schmidt 1987; Schmidt et al. 1987). of MAO-A by MDMA may contribute to an accumula­ MDMA has been demonstrated to produce a depletion tion of extracellular 5-HT. The inhibition of MAO-B ac­ of serotonin that may be reversed in acute stages by tivity by FLUOX may increase intracellular 5-HT, agents that bind to the serotonin transporter and block whereas the actions of FEN do not appear to involve serotonin reuptake into presynaptic terminals (Azmitia MAO inhibition. et al. 1990; Schmidt 1987). It also inhibits the 5-HT re­ uptake system (Steele et al. 1987), resulting in an in­ creased amount of extracellular 5-HT. However, little MATERIALS AND METHODS attention has been paid to the fate of extracellular 5-HT. Parachloroamphetamine (PCA) is another sub­ Male Sprague-Dawley rats weighing 200 to 250 g (Ta­ stituted that is a potent releaser of sero­ conic Farms, Germantown, NY) were housed two per tonin and has a biphasic depletion of serotonin similar cage and given free access to food and . The to that observed with MDMA (Ask and Ross 1987; animals were maintained on a 12-hour light/dark cy­ Berger et al. 1992; Fuller et al. 1975; Gu and Azmitia cle. Animals were euthanized with C02 and decapi­ 1989; Gu 1993; Hwang and van Woert 1980; Mamounas tated according to a protocol approved by the NYU An­ and Molliver 1988; Poblete et al. 1989; Ross and Froden imal Welfare Committee. were rapidly removed 1977). As with MDMA, the depletion of serotonin resul­ and placed on ice in 0.32 mollL sucrose. After removal tant from PCA may be reversed in its acute phase by of the cerebellum, brains were homogenized in 10-fold serotonin uptake blockers (Fuller et al. 1975; Ross et al. volume/weight in 0.32 mollL sucrose using 10 strokes 1977). Parachloroamphetamine has been shown to in­ with a Teflon/glass homogenizer. P1 pellets were pre­ hibit MAO-A activity in rat brain homogenates with a pared by sedimentation of homogenates at 800 x g for Ki value of 1.31 IlmollL (Fuller 1966). The toxicity of 10 minutes at 2°C in a Sorvall RC5C centrifuge (Sorvall PCA is also affected by the amount of releasable sero­ Instruments, DuPont, Chadds Ford, PA). Supernatants tonin into the extracellular space. For instance, ifsero­ were resedimented at 14,000 xg at 2°C for 15 minutes tonin release is decreased by parachlorophenylalanine to obtain a crude mitochondrial P2 pellet. Resultant (pCP A) and reserpine, the level of toxicity is reduced pellets were resuspended in 500 ilL 0.32 mollL sucrose (Berger et al. 1989). and stored at -70°C until use. The compound, fenfluramine (FEN), is Prior to the MAO assay, homogenates were thawed a halogenated amphetamine that has actions in and brought up to lOx volume/original weight 0.01 serotonergic axon terminals similar to those of MDMA mollL sodium phosphate buffer (PB), pH 7.4 and and PCA (Mamounas and Molliver 1988; Molliver and dialyzed to remove endogenous monoamines by a Molliver 1990; O'Hearn et al. 1988). Like MDMA and modiflcationof the method described by Patterson, et PCA, FEN causes the release of 5-HT from presynaptic al. (1973). Briefly, homogenates were dialyzed in 2 mL terminals (Borroni et al. 1983) and inhibits the reuptake aliquots using 3500 mw cutoff dialysis tubing (Spec­ of serotonin into its terminals (Belin et al. 1976; Kan­ trap or) against 0.01 mollL PB, pH 7.4 at 4°C for 2 hours nengiesser et al. 1976). The effects of FEN are blocked with three successive changes of 1L buffer. MAO as­ by 5-HT uptake inhibitors, as in MDMA and PCA (Hek­ says were performed immediately following this di­ matpanah and Peroutka 1990). These observations sug­ alysis. gest that the carrier-mediated release of serotonin and Monamine oxidase activity was assayed using the inhibition of its reuptake are critical components [3H]-5-HT (25Ci/mmol, 1IlCi/mL, New England Nu­ in the mechanism of these drugs. FEN, FLUOX, and clear, Boston, MA) as a substrate for MAO-A at fmal MDMA bind to the serotonin transporter protein with concentrations ranging from 12.5IlmollL to 400IlmollL. high affinity; the rank order potencies for binding p4C]-phenethylamine at a fmal concentration of 5 NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 4 MDMA Inhibition of MAO-A 233

15000 tillation counting in Beckman lS 1801 scintillation 14000 ... counter (Fullerton, CA) with a counting efficiency 13000 • � 0 uM !.IOMA � o '" 5 uU UOUA " 12000 " = 10 uU UOMA of 40%. c " - 50 uU I.IDUA 11000 E • = 100 uU UO!.l I 10000 � D = 500 u!.l UD e Comparison of FEN, FLUOX, and MDMA � ... 9000 A = 1 m!.l !.IDMA � '" 8000 < E Fenfluramine, FLUOX, and each enantiomer of MDMA I ...... 0 " 7000 • were assayed against MAO-A and MAO-B activity with � "0 6000 E 5000 substrate concentrations of 100 Jlmolll 5-HT and 20 � 4000 Jlmolll PEA, respectively. Fenfluramine (Sigma Chem­ 3000 ical Co., St. louis, MO) and MDMA were tested at final 2000 concentrations of 1 Jlmolll to 10 mmolll, FLU OX (gift 1000 of Eli lilly and Co., Indianapolis, IN) at concentrations -200 0 200 400 600 800 1 000 of 1 Jlmolll to 1 mmollL. MAO activity was then as­

1/[S] !.I-I x 10-2 sayed as described above.

Figure 1. Inhibition of serotonin oxidation by ( + ) MDMA. All data points represent the average of three experiments. NonspecifIc values (0.15 nmoles product/mg protein-minute) Additive Effects were subtracted from total activity. Error bars represent the To determine whether MDMA and FLU OX share a standard error of the mean. common mechanism for MAO inhibition, homogenates were treated with FlUOX and ( + ) MDMA in combina­ tion at their ICso or at 2 x ICso concentrations individu­ Jlmolll to 50 Jlmolll (50.8 mCi/mmol, 0.1 JlCi/ml, New ally. Radiolabeled substrate was added and MAO ac­ England Nuclear, Boston, MA) served as substrate for tivity was assayed as described above. MAO-B. The assay procedure was a modifIcationof the To determine nonspecifIccounts, a set of samples method described by Pintar et al. (1981) and Lan et al. was preincubated with 1 mmolll clorgyline or 1 mmolll (1989). deprenyl, as appropriate, for 10 minutes at 37°C. Pro­ tein concentrations of homogenates were determined by the method of lowry et al. (1951) with bovine se­ Competition Studies rum albumin (Sigma Chemical Co., St. louis, MO) as A 15 Jll portion of rat brain homogenate suspended standard. Protein detection was determined at 540nm in 75 Jll 0.01 molll PB was treated with 20 Jll ( + ) or absorbance using a Titertek Multiskan spectrophotom­ (-) MDMA (National Institute on Drug Abuse, eter (EFlAB, Helsinki, Finland). Bethesda, MD) at fmal concentrations ranging from 10 Analysis of data was conducted by employing Stu­ Jlmolll to 1 mmolll, 10 minutes in a 37°C water bath. dent's two-tailed t-test for two-sample comparisons. Samples then received a 90 Jll aliquot of [14C]-PEA or One-way analysis of variance (ANOVA) followed by PH]-5-HT and were further incubated in a 37°C water Tukey post hoc analysis was performed for multisam­ bath for 30 minutes. At the end of this time, the reac­ pIe comparisons (SYSTAT, Evanston, Il). ICso values tion was terminated by adding a 90 Jll aliquot 1.2 molll and Hill coefficientsof concentration-response curves HCl. Each sample point was assayed in quadruplicate. were determined by computer-assisted curve-fitting to Radioactive product was measured by the addition of a logistical equation (SigmaPlot 4.1, Jandel ScientifIc, 3 ml 4% (vollvol) Liquiscint (National Diagnostics, San Rafael, CA). Kinetic constants for both subtypes Bethesda, MD): (HPlC grade, Aldrich Chemi­ of MAO were determined by the analysis of Lineweaver­ cal Co., Milwaukee, WI) per sample followed by scin- Burk plots.

Table 1. Comparison of ( +) and (-) MDMA against Deamination of Serotonin and PEA by MAO

Substrate of Vmax Ki (+) MDMA Ki ( - ) MDMA MAO (nmol/mg-minute) (Ilmol/L) (Ilmol/L)

Serotonin 2.08 ± 0.03 100 ± 12 22.0 ± 3* 28.3 ± 5.0* PEA 1.72 ± 0.20 20 ± 3

* No signifIcance was determined between (+ ) and ( - ) MDMA with Student's two-tailed t-test. Values reported here represent the average of three experiments, plus or minus the standard error of the mean. 234 E.T. Kokotos Leonardi and E.C. Azmitia NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.4

0 140 0 ... 0 ;! 0 = t.4Dt.4A 0 0 = x • = 0 u� �OMA 120 \l FLUOX c � .. = 10 uM MOMA 0 = FEN E 0 v � 50 uM �OMA I a • '" 100 u� �O�A � ?;o � " = 500 uM �OMA 100 � ....'> � Q. A = 1 mM MOIo.lA 0 0 u « '" 0 ..c( 10 60 '0.. ... :::- c: 0 0 40 �

-25-20-15-10 -5 0 5 10 15 20 25 20 1/(S) 1.1-1 x 10-4 O L-----'-----'----'----"------'---' 2. Figure Inhibition of phenethylamine oxidation by (+) 1 0-7 10-6 10-5 10-4 10-3 10-2 10-1 MDMA. All data points represent the average of three ex­ periments. NonspecifIc values (0.18 nmoles product/mg [DRUG]. t.40LAR protein-minute) were subtractedfrom total activity. Error bars Figure 3. Concentration-response curve for the inhibition of represent the standard error of the mean. MAO-A by FLUOX, FEN, and ( + ) MDMA. Control values = 2.08 nrnolesproduct/mg protein-minute. Error bars represent the standard error of the mean. RESULTS In order to establish the kinetic parameters of MAO ac­ tivity in this assay system, saturation studies were per­ inhibition of MAO-B by MDMA; the effectobserved at formed for MAO-A and MAO-B. MAO-A activity had the highest concentrations may reflect a saturation of a Vmax of 2.08 nmoles product/mg protein-minute and all the available MAO-B active sites. A similar fmding a KM of 100 J,lmollL (Figure 1, Table 1). For MAO-B, a was observed with (- ) MDMA (data not shown). Vmax of 1.72 nmoles product/mg protein-minute with To compare the potencies of FEN, FLUOX, and a KM value of 20 J,lmollL was observed (Figure 2, Ta­ MDMA on MAO-A inhibition, concentration-response ble 1). Both enzyme assay systems were responsive to curves were established that consisted of 14 points from the appropriate monoamine oxidase inhibitors; clorgy­ 10-6 to 10-2 mollL for MDMA and FEN. A concentra­ line with a Ki for MAO-A of 0.5 nmollL and deprenyl tion-response curve of 13 points from 10-6 to 10-3 with a Ki for MAO-B of 1 nmollL. mollL was established for FLUOX. A representative Once these parameters were established, the effects curve obtained from the average of three experiments of both enantiomers of MDMA were tested on MAO-A. for FEN, FLUOX, and ( + ) MDMA had Hillcoefficients A differentKM but no change in V max was observed for of 1.02, 1.18, and 1.05, respectively (Figure 3). Their MAO-A (Figure 1) with increasing concentrations of respective ICso values (Table 2) show a rank order MDMA, indicative of a . No potency of MDMA>FLUOX>FEN (ICso were 44, 130, stereospecmc effectwas observed (Table 1), that is ( + ) and 440 J,lmollL, respectively). The ICso of ( - ) MDMA MDMA had a Ki value of 22.0 J,lmollL against seroto­ for MAO-A was 56 J,lmollL (Table 2). nin as substrate and ( -) MDMA has a Ki value of 28.3 A similar study was performed for MAO-B with the J,lmollL (Table 1). A competitive inhibition of MAO-A drugs at the same concentrations as the MAO-A experi­ was seen by both enantiomers of MDMA. ment. A representative curve obtained from the aver­ The effectsof both enantiomers of MDMA were de­ age of three experiments for FEN, FLUOX, and (+) termined for MAO-B, the subtype of MAO localized MDMA had Hill coefficients of 1.13, 1.12, and 0.8182, within serotonergic neurons. MDMA produced a differ­ respectively (Figure 4). Table 2 shows their respective ent type of inhibition with MAO-B than was observed ICso values; 720, 80, and 370 J,lmollL with FLUOX> for MAO-A (Figures 1, 2). In the case of MAO-B, both MDMA>FEN. As was the case with MAO-A, no the Vmax and KM of the enzyme were changed by a signmcant difference in ICso was observed between range of MDMA concentrations; 10, 50, 100, and 500 (+) and (- ) MDMA for MAO-B activity. The ICso of J,lmollL. At the highest concentrations, 1 mmollL, the (+) MDMA for MAO-B inhibition was roughly nine Vmax remained the same whereas KM has changed. The times greater than its ICso for MAO-A, indicating that change in both the Vmax and KM shows a mixed-type MDMA is selectively potent for MAO-A activity. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 4 MDMA Inhibition of MAO-A 235

Table 2. ICso Values (Ilmol/L) ± SEM

MAO Subtype FEN FLUOX (+) MDMA (-) MDMA

MAO-A 440 ± 23. Ogab 130 ± 11.55" 44 ± 6.06 56 ± 8.24 MAO-B 720 ± 28.87"b 80 ± 10.55" 370 ± 4.68"b 378 ± 6.29

Values represent the average of three experiments ± standard error of the mean. MAO-A Data: One-way analysis of variance showed signifIcant variance (p = 1.02 X 10-7, F = 183.86,Ferit = 4.07). MAO-B Data: One-way analysis of variance showed signifIcant variance (p = 6.16 X 10-8, F = 209.05, Ferit = 4.07). a p < .001 when compared to (+) MDMA with Tukey post hoc analysis. b p < .001 when compared to FLUOX with Tukey post hoc analysis.

140 FLUOX showed a 60% greater inhibition of MAO-B o '= �D�A than MAO-A activity, with an ICso of 80 J.1moliL for <;J '= FLUOX 120 MAO-B. FEN showed poor inhibition of both MAO-A o '= FEN and MAO-B. � � 100 The additive effects of MDMA and FLUOX were " tested. In this set of experiments, each drug was added -< Dl 80 x I to homogenates at its ICso or 2 ICso. Another group o had homogenates receiving a combination of both drugs � at their respective ICso values. In this way, we were 60 ...e c able to test whether MDMA and FLUOX share a com­ o u 40 mon mechanism for MAO-A and -B inhibition. A com­ � bination of FLUOX and MDMA at their ICso did not in­ hibit MAO-A or MAO-B activity more than either drug 20 alone at an equivalent concentration, which is indica­

-- ____� __ � tive of a common mechanism for MAO inhibition (Ta­ OL- �--� ____L- __� ble 3, Table 4). 10-7 [DRUG). MOLAR

DISCUSSION Figure 4. Concentration-response curve for the inhibition of

MAO-B by FLUOX, FEN, and (+) MDMA. Control values = Amphetamine has been reported as a competitive in­ 1.74 nmoles product/mg protein-minute. Error bars represent hibitor of MAO-A activity (Mantle et al. 1976) and a the standard error of the mean. mixed inhibitor of MAO-B, that is, both the Vrnax and KM are changed (Pearce and Roth 1985). The results of this study show MDMA acts as a competitive inhibitor of MAO-A activity (see Results, Figure 1), whereas a in vitro inhibition of MAO-A by amphetamine (Mantle mixed patternof inhibition was observed for the MDMA et al. 1976) and its analogue, PCA (Fuller et al. 1965). inhibition of MAO-B (Figure 2). The kinetics of MAO-A A selective potency for the inhibition of the A subtype and -B inhibition by MDMA reported here are there­ was also observed in vivo in rat brain homogenates 25 fore consistent with those previously described for am­ hours after the animals were injected with ampheta­ phetamine. A nine-fold differencewas observed in the mine followed by , a nonselective MAO in­ ICsoof MDMA for MAO-A and MAO-B activity, show­ hibitor (Miller et al. 1980). ing a selective potency of MDMA for MAO-A in rat The inhibition of MAO-A by amphetamine was brain homogenates (see Results, Figures 3 and 4, Table found to be stereoselective for the ( + ) enantiomer in 2). This findingis consistent with studies showing the previous studies by other investigators (Mantle et al.

Table 3. Combined Effects of ( + ) MDMA and Fluoxetine on MAO-A Activity

MDMA (44 Ilmol/L) MDMA FLUOX MDMA FLUOX + FLUOX (44 IlmollL) (130 IlmollL) (88 IlmollL) (260 IlmollL) (130 IlmollL)

% Control MAO-A 49 ± 5 50 ± 4 28 ± 3 34 ± 5 30 ± 2 236 E.T. Kokotos Leonardi and E.C. Azmitia NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 4

Table 4. Combined Effects of ( + ) MDMA and Fluoxetine on MAO-B Activity

MDMA (370 IlmollL) MDMA FLUOX MDMA FLUOX + FLUOX (370 IlmollL) (80 IlmollL) (740 Ilmol/L) (160 IlmollL) (80 Ilmol/L)

% Control MAO-B 55 ± 4 50 ± 3 35 ± 3 29 ± 3 31 ± 2

1976). We observed no signifIcant difference between than FEN in both culture experiments (Gu 1993) both enantiomers of MDMA with respect to oxidative and in vivo (Sotelo and Zamora 1978). The fact that deamination of serotonin and phenethylamine by MDMA has greater toxicity is not due to its ability to monoamine oxidase (see Results, Table 1). This is in induce release or bind to the serotonin transporter. contrast to reports of a stereospecifIcityfor MDMA on However, a comparison between MDMA and FEN on and serotonin release from striatum (John­ MAO activity in this study showed MDMA to be ap­ son et al. 1986; Schmidt et al. 1987). However, the ob­ proximately ten times more potent than FEN in the in­ servation that MDMA appears to lack stereospecifIcity hibition of MAO-A (see Results, Table 2). The inhibi­ for MAO inhibition is consistent with many of the acute tion of MAO-A may be a crucial variable for induced properties of this drug both in vivo and in vitro. Re­ fIberdegeneration. In support of this hypothesis, PCA, lease of serotonin observed in 3H-serotonin-Ioaded rat which is a more potent serotonergic toxin than MDMA hippocampal slices superfused with either enantiomer (Gu 1993; Mamounas and Molliver 1988; O'Hearn et of MDMA did not show any signifIcantstereoselectivity al. 1988) has a roughly 20-fold higher affinityfor MAO­ (Johnson et al. 1986). Treatment with both enantiomers A than MDMA (Ki values of 1.33 IlmollL, Fuller 1966 of MDMA resulted in a decrease in rat striatal indoles vs. 22 IlmollL, this study). in vivo 3 hours after injection. A nonstereoselective, Fluoxetine is a lipophilic, serotonin uptake blocker acute depletion of serotonin following MDMA treat­ that is found in subcellular fragments prepared from ment in vivo was observed in rat cortex (Schmidt 1987). brain tissue of FLUOX-treated rats (Caccia et al. 1990). Finally, both the optical antipodes of MDMA were po­ Therefore, FLUOX may enter the cell and interact with tent inhibitors of 3H-serotonin uptake into rat hip­ monoamine oxidase. The ICso of FLU OX was com­ pocampal synaptosomes (Steele et al. 1987). The abil­ pared to that of MDMA and FEN with respect to MAO ity of MDMA to inhibit MAO-A would result in high inhibition; the ICso value of FLUOX for MAO-A was extracellular levels of 5-HT. nearly three times higher than that of MDMA (see Monoamine oxidase A is an enzyme whose Results, Table 2). Interestingly, the ICso of FLU OX for preferred substrate is serotonin (Garrick and Murphy MAO-B, the subtype localized within serotonergic cells, 1982) and is localized in neurons (West­ was 80 IlmollL, a nine-fold difference from the ICso of lund et al. 1985). Serotonin has recently been reported MDMA. to promote the release of DA through the dopamine Finally, the additive properties of FLUOX and transporter by an exchange-diffusion mechanism (Ja­ MDMA were tested on MAO activity (Results, Table cocks and Cox 1992). This effectwould be enhanced by 3, Table 4). Since both compounds bind to the seroto­ an increased level of 5-HT resultant from an inhibition nin transporter, we examined whether they share a of MAO-A activity. common site for MAO inhibition. The addition of both Fenfluramine and MDMA share many neurophar­ drugs at their ICso s produced effectsthat were equiva­ macologic characteristics. Both drugs bind to the sero­ lent to each drug on its own. We interpret this fInding tonin transporter, with a similar affinity(Poblete et al. to be indicative of a competition of both compounds 1989). MDMA and FEN both release serotonin (E.C.so for the same site on MAO-A and MAO-B, respectively

= 2.92 and 7.90 IlmollL, respectively, Berger et al. 1992; (Results, Table 3, Table 4). Borroni et al. 1983; Buczko et al. 1975; Johnson et al. In summary, these studies show that MDMA pref­ 1986; Kannengiesser et al. 1976; Schmidt et al. 1987), erentially inhibits MAO-A in a reversible, non stereo­ with FEN being more potent. In contrast, MDMA ap­ specifIc manner. Fluoxetineis signifIcantlymore potent pears to be slightly more potent than FEN at inhibition than MDMA in the inhibition of MAO-B, whereas FEN of reuptake (0.42 IlmollL, Steele et al. 1987 and 0.876 does not signifIcantlyaffect rat brain MAO-A or MAO­ IlmollL, Borroni et al. 1983, respectively). Finally, both B activity. Like PCA, the greater toxicity of MDMA (Gu drugs are toxic to serotonergic neurons (Appel et al. 1993) may be related to its ability to produce high lev­ 1990; Azmitia et al. 1990; Battaglia et al. 1987, 1988;Mol­ els of extracellular 5-HT by stimulating release, inhibit­ liver and Molliver 1990; O'Hearn et al. 1988), but ing reuptake and blocking the catabolism of serotonin MDMA has been shown to be a more potent toxic agent by MAO-A. The therapeutic actions of Prozac may in- NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.4 MDMA Inhibition of MAO-A 237

volve a selective inhibitionof MAO-B, which would re­ Buczko W, DeGaetano G, Garattini S (1975): Effectof Fenflura­ sult in a greater amount of 5-HT available for release. mine on 5-Hydroxytryptamine Uptake and Release by Rat Blood Platelets. Brit J Pharmacol 53:563-568 Finally, FEN has only weak effects on MAO-A and MAO-B activity. Caccia S, Cappi M, Fracasso C, Garattini S (1990): Influence of Dose and Route of Administration of Fluoxetine and its Metabolite Norfluoxetine in the Rat. Psychopharma­ cology 100:509-514 ACKNOWLEDGMENTS Fowler CJ , Tipton KF (1982): Deamination of Serotonin by Both Forms of Monoamine Oxidase in the Rat Brain. J The comments made by H. Kenneth Kramer, Dr. Randall B. Neurochem 38:733-736 Murphy, and Jose c. P. Poblete during the preparation of the Fuller RW (1966): Serotonin Oxidation by Rat Monoamine Ox­ manuscript are gratefully acknowledged. This work was sup­ idase: Inhibition by 4-Chloroamphetamine. 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