P-Methylthioamphetamine Is a Potent New Non-Neurotoxic Serotonin-Releasing Agent

'52790

p-Methylthioamphetamine is a potent new non-neurotoxic serotonin-releasing agent

Xuemei Huang, Danuta Marona-Lewicka and David E. Nichols Deportmentof Pharmacologandy Toxicologyand, Deportmentof M.edicinal_7_emisttyand Pharmacognosy,PurdueUnit'ersity, Schoolof PharmacyandPharmacalSciences,WestLaJ_tyetteIN, 47007,USA

Received 14May I992, revised MS received 2 September 1992, accepted 8 September 1992

p-Methylthioamphetamine (MTA), was compared to p-chloroamphctamine (PCA) in a number of pharmacological assays. MTA was about 2-fold more potent than PCA at inhibiting synaptosomal uptake of [-_H}5-hydroxytryptamine ([3H]5-HT), and about 7-fold and 10-fold less potent than PCA at inhibiting synaptosomal uptake of [3H]dopamine and [3H]norepinephrine, respectively. In drug discrimination assays, MTA was nearly equipotent to PCA in animals trained to discriminate saline from 3,4-methylenedioxymethamphctamine (MDMA), or two related analogues S-(+)-N-methyl-l-(l,3-benzodioxol-5-yl)-2-butanamine (S-MBDB) or 5-methoxy-6-methyl-2-aminoindan (MMAI). MTA caused dose-dependent increases of tritium cffiux from superfused rat frontal cortex slices preloaded with ['_H]5-HT. comparable to that induced by an equal molar concentration of PCA. Thc potential ncurotoxicity of MTA was examincd by measuring monoamine and metabolitc levels at one wcck following an acute dose. A 10 rog/kg dose of PCA caused a 70-90% decrease of cortical, hippocampal and striatal 5-HT and 5-hydoxyindoleacetic acid (5-HIAA) levels, while twice the molar dose of MTA (21.3 rog/kg) had m) effect. Thus. MTA is a potent, selective, semtonin releaser, apparently devoid of semtonin neurotoxic effects. This work also supports the idea that catecholamine systems may play a critical role in the neurotoxicity of PCA-Iike compounds.

p-Chloroamphctaminc; p-Mcthylthioamphctaminc; Drug discrimination: Uptake inhibition; Supcrfusion: 5-ITT (5-hydroxytryptaminc, scrotonin); Ncurotoxicity; Brain sliccs

1.Introduction willnot onlygive a nccdcd safety factor for anypoten- tial clinical use of this type of compound but also will Substantial efforts in our laboratory over many years provide tools for studying the mechanisms of behavior have been made to undcrstand thc structure-activity and neurotoxic activity of these types of compounds. relationships of substituted amphetamines which exert Data from recent research in our laboratory and a variety of pharmacological actions. Amphetamine others have provided cvidencc that drug-induced 5-HT derivatives such as 3,4-methylenedioxymcthamphcta- release is important in the discriminative cue of mine(MDMA),andp-chloroamphetamine(PCA)cause MDMA-likc drugs (Obertender and Nichols. 1988: central serotonin neurotoxicity which is marked by Nichols et al., 1986; Schechter, 1988: Nichols and persistent reductions in brain 5-hydoxytryptamine (5- Oberlender, 1990; Nichols et al., in press). 5-Methoxy- HT), 5-HT uptake sites, and tryptophan hydroxylase 6-methyl-2-aminoindan (MMAI), which possesses a activity (e.g. Schmidt et al., 1986; Battaglia et al., 1987: more potent in vitro serotonergic effect than MDMA 1988; Fuller and Shoddy, 19741. This persistent reduc- and has almost no dopaminergic effect, fully substi- tion in serotonin markers seems to correlate with the tutes for MDMA and S-(+)-N-methyl-l-(l,3-benzodi- degeneration of certain serotonin axonal projections in oxol-5-yl)-2-butanamine (S-MBDB) in drug discrimina- brain (O'Hearn et al., 1988; Mamounas and Molliver, tion experiments (Johnson et al., 1991a). Recently, in 19881. Developing less neurotoxic MDMA and PCA our laboratory,, MMAI has been used as a training analogues which preserve desired behavioral effects drug in drug discrimination experiments because of its greater serotonergic specificity and decreased neurotoxic effects (Nichols et al., in press). Correspondence to: D.E. Nichols, Department of Medicinal Chem- istry and Pharmacognosy, School of Pharmacy and Pharmacal Sci- Recent work suggests that non-vesicular dopamine ences, Purdue University.West Lafayette. IN 470117.USA. Tel. I re[ease plays a very important role in drug-induced (3171-494-1461. fax I (3171-494-t1790. serotonergic toxicity of these drugs. Compounds such 32

as MMAI, 5,6-methylenedioxy-2-aminoindan (MDAI), [3H]5-HT, [3H]dopamine and [3H]norepinephrine Were forc MBDB and 1-(4-chlorophenyl)-2-aminobutane (CAB) purchased from Amersham (Arlington Heights, IL) at pro[ which have little dopaminergic effect produce little or a specific activity. of l_J,o _ 5 and 13.8 Ci/mmol respe e- ,A no serotonergic toxicity (Johnson et al. 1990; 1991a; tively. (+)-Amphetamine sulfate was purchased fronal men Nash and Nichols, 1991; Nash and Brodkin, 1991). Smith Kline & French Laboratories (Philadelphia, PA). para Serotonin neurotoxicity induced by the combination of MDMA, S-MBDB, MMAI, as their hydrochlorides, deta the non-neurotoxic MDMA analogs MDAI or MMAI and LSD tartrate were synthesized in our laboratory Nict and the dopamine releasing agent (+)-amphetamine (Nichols et al., 1986; Johnson et al., 1991). With the the (Johnson and Nichols. 1991; Johnson et al., 1991b) exception of drug discrimination experiments, all drugs and gives additional strong support to the hypothesis of were injected subcutaneously. In drug discriminatio] tionl dopamine involvement in the serotonin neurotoxicity experiments, all drugs were dissolved in 0.9% salin_ were induced by these types of drugs, and were injected intraperitoneally in a volume of 1 sessi Based in part on the above studies, further explo- ml/kg. 30 min before the session. 85% ration of newMDMAand PCAanalogswithincreased pres serotonergic effects and/or decreased dopaminergic 2.2. Animals sion_ effects (i.e. highlyselectiveserotonergicagents!was sessi warranted. Most monoaminereleasingamphetamine eitht derivatives arc 3.4-disubstitutcd compounds ,c.g. Male Sprague-Dawley rats (Harlan. Indianapolis, and IN) welching I"q-9(}(I _ were used in all experiments. MDMA} or arc simpb monosubsitutcd at thc >ara _ " - -" Rat.' position. For thc latter, small electroneaativc groups .Animals were either group housed six per cage or spot - individually caged in a temperature controlled room - - testc such as mcthoxv, and chloro or other halogens eivc with a 12/12 h lighting schedule. With the exception of neurotoxiccompounds.It should be noted however, disC_ that thc halogens other than chlorine and bromine give rats used in the drug discrimination experiments, food achic compounds that arc less neurotoxic (Fuller et al.. 1975; and water were available ad libitum at all times. In sessi 1980). Thus. we reasoned that a larger, less electroneg- drug discrimination experiments. 62 male rats were to tl_ ativc group would be an interesting candidate. In par- used as subjects and divided into five groups (N = 9-15 of e ticular, a sulfur atom substitution seemed an appropri- per group), trained to discriminate MDMA (1.75 chan ate choice. Sulfur is hydrophobic, in thc same family of mg/kg). S-MBDB (1.75 mg/kg). MMAI (1.71 mg/kg), 50 P the periodic table as oxy.gen, and much less electroneg- ( + )-amphetamine (1.0 mg/kg) or LSD (0.08 mg/kg) rain, ativc than oxygen or chlorine. Following this reasoning, from saline. None of the rats had previously received Trea p-methylthioamphetamine (MTA). was synthesized in drugs or behavioral training. Water was freely available stud, our laboratory. This report discusses the pharmacology, in the home cages and a sufficient amount of supple- of MTA in a number of assays. The structures of PCA mental feeding (Purina, Lab Blox) was made available 2.4.. and MTA are shown in fig. 1. after experimental sessions so as to maintain them at neph As a simple screening procedure, the ability of MTA approximately 80% of free feeding weight, compared to substitute for MDMA, S-MBDB, MMAI. ( ?)- with control rats housed under the same conditions. In A neurochemical studies, rats were killed by decapitation, inamphetaminea drug discriminationand ( + )-lysergicparadigmacid diethylamidewas first (LSD)deter- the brains were removed and rapidly dissected on ice hom,emp] according to the procedure of Glowinski and Iversen usinl mined. Synaptosomal monoamine uptake inhibition ex- (1966). In neurotoxicity studies, the frontal cortex, hip- perimentswere thenusedto assessthe relativepotency horn, of the new compound to affect serotonin, dopamine, pocampus and striatum were frozen with liquid nitro- 4oc. and norepinephrine uptake and release. Superfusion gen. The samples were stored separately at -70°C Xg experiments were utilized to test its serotonin releasing until assay using HPLC-EC methods, pend properties. Finally,the abilityof the compoundto batio cause long-term depletion of monoamines in differenI 2.3. Drug discrimination an a rat brain regions(frontal cortex, hippocampusand meas striatum) was determined. Six standard operant chambers (Coulbourn Instru- respt ments, Lehigh Valley, PA) consisted of modular test addi: cages enclosed within sound-attenuated cubicles with tube_ 2. Materials and methods fans for ventilation and background white noise. A Hen: white house light was centered near the top of the 1.2 I_ 2.1. Materials front panel of the cage, which was also equipped with ascm two response levers, separated by a food hopper, all solut MTA and PCA were synthesized in our laboratory positioned 2.5 cm above the floor. Solid state logic in COne, using standard methods. HPLC standards were pur- an adjacent room, interfaced through a Coulbou_ :'_'_ [3I-'I] {.

chased from Sigma Chemical Co. (St. Louis, MO). Instruments Dynaport to an 1BM PC, controlled rein-il./ 50 t, 33 fine Were :nt and data acquisition with a locally written incubations were continued for 5 min more. lncuba- ts, IL) at program, tions were terminated by cooling in ice and were then I respec. A fixed ratio (FR) 50 schedule of food reinforce- rapidly filtered through Whatman GF/B filters with a ;ed from pent (Bioserv 45 mg dustless pellets) in a two-lever Brandel Cell Harvester (Brandel, Gaithersburg, MD). fia, PA). paradigm was used. The drug discrimination procedure The filters were washed with ice cold buffer and al- hlorides, details have been described elsewhere (Oberlender and lowed to air dry before placing them in plastic scintilla- boratory lqichols, 1988; 1990; Nichols et al., in press). Half of tion vials. Scintillation cocktail (10 ml) was added and Vith the the rats were trained on drug-L (left), saline-R (right) the vials were sealed and allowed to sit overnight tll drugs and the other half on drug-R, saline-L to avoid pos{- before counting at an efficiency of 54%. tional preference. Training sessions lasted 15 min and saline were conducted at the same time each day. Training 2.5. [;H/5-HT release experiments ne of 1 sessions were continued until an accuracy of at least 85% (number of correct presses × 100/number of total Rat frontal cortex was chopped coronally into i).3 presses) was attained for eight of ten consecutive ses- mm slices. The slices were immediately transferred sions. Once criterion performance was attained, test into O,-saturated Krebs-Henseleit buffer (pH 7.4) con- sessions were interspersed between training sessions, taining 100 /J.M of pargyline. After incubation with either one or two times per week. At least one drug [3H]5-HT (100 nM) for 30 rain at 37°C, one slice was. mpolis, and one saline session separated each test session. placed into each of 12 superfusion chambers (Brandel, ments. Rats were required to maintain the 85% correct re- Gaithersburg, MD). The slices were superfused at a age or sponding criterion on training days in order to be rate of 0.5 ml/min with fresh oxygenated modified room tested. In addition, test data (less than 5%) were tion of discarded when the accuracy criterion of 85% was not Krebs-Henseleit buffer (mM: 118 NaCI, 4.8 KCI, 1.2 ;, food achieved on the two training sessions following a test MgSO4, 25 NaHCO 3, 1.2 KH2PO4, 12 glucose, 0.01 ascorbic acid, 0.1)3 Na,EDTA, and 0.1 pargyline). Af- es. In session.Test drugs were administered J.p. 311min prior were ter a 41)rainwashserial2 minfractionswerecollected. to the sessions; test sessions were run under conditions :9-15 Buffersolutioncontainingthe test compoundwasad- 11.75 of extinction, with rats removed from the operant ministered only for 4 min, during the collection of chamber when 50 presses were emitted on one lever. If ;/kg), 50 presses on one lever were not completed within 5 fractions 5 and 6. At the end of the experiment, 5 mi of g/kg) Ecolite Scintillation cocktail (ICN, Cleveland, OH) was eived rain, the session was ended and scored as a disruption, added to all fractions of superfusate. Slices were recov- ilable Treatments were randomized at the beginning of the ered and transferred to vials containing 5 mi of scintil- pple- study, lation cocktail. After vigorous shaking, radioactivity

mlableat 2.4. In z*itro['_H/5-HT, /'_H/dopamine and [ ;H/norepi- was counted with a Packard Scintillation Counter. ,ared nephrine uptake inhibition 2.0. HPLC with electrochemical detection s. In tion, A modified procedure of Steele et ill. 11987) was l ice employed. Briefly. whole brain minus cerebellum was High pressure liquid chromatography (HPLC) with rsen homogenized in 15 volumes of icc cold I).32 M sucrose clcctrochemical detection was used to determine bio- hip- using a glass mortar with a motor driven pestle. Thc genie amines and metabolite levels. A mob{lc phasc tro- homogenate was ccntrifuged at 11)80× g /'or 1() min at containing 51) mM NaH2PO 4, 31) mM citric acid. 0.1 ·0PC 4°C. The supernatant was then recentrifuged at 178011 mM Na:EDTA. 0.034% sodium octyl sulfate and 23% Xg for 10 rain and the resulting pellet was resus- v/v methanol was used. The brain samples were pre- in the same volume of sucrose solution. Incu- pared by homogenizing the weighed brain areas from bat{ohs were carried out in a shaking incubator under one hemisphere in 0.5 mi of HPLC mobile phase an atmosphere of 95% O,-5% CO, at 37 or 0°C to without sodium octyl sulfate, using a motor-driven measure total tissue uptake and non-specific uptake, Teflon pestle and Eppendorf 1.5 mi centrifuge tube. :ru- respectively. A 5 rain preincubation was begun by An internal standard of 100 ng/ml of 3-(3,4-dihydroxy- est adding 0.2 mi of the synaptosomal preparation to test phenyDpropionic acid (DHPPA) was used. The sam- {th tubes containing 1.65 mi of O,-saturated Krebs- pies were then centrifuged at 14000 xgfor 4 min with A Henseleit buffer (mM: 118 NaC1, 4.8 KCI, 1.3 CaC12, a table top centrifuge. The supernatant was assayed for he 1.2 KH 2PO4, 25 MgSO4, 25NaHCO 3, 10 glucose, 0.06 monoamines and their metabolites by injection of 50 txl ith ascorbic acid and 0.03 NazEDTA), 50 /_I of drug onto a Brownlee C18 analytical cartridge column all solution and 50 /_1 of pargyline HCI solution (final (Anspec, Ann Arbor, MI) with a flow rate of 0.7 in concentration of 100 /a.M). Then either [3H]5-HT, ml/min. The HPLC-EC system consisted of a refriger- rn [3H]dopamine or [3H]norepinephrine was added in a ated autosampler (TosoHaas, Philadelphia, PA), and a n- 50 /.LI aliquot (final concentration of 10 nM), and model 400 EG and G Princeton electrochemical detec- 34

miner 4 c CH3 _ the lC '_ comp

p-C_oamphmmi_ p- Methylthioamp_ar,_ _ 3 uptak (Pc,A) (MIA) _ 2 t-test

thioamphetamine (MTA). the P Fig. 1. The structures of p-chloroamphetamine (PCA) and p-methyl- i Tr begin tor (EG & G PARC, Princeton, NJ) with a dual 0 6 8 10 12 14 16 18 electrode potential set at E, = -200 mV and E. = 850 Fnctio.number mV versus the Ag/AgC1 reference electrode. Fig. 2. The fractional release of tritium from rat frontal cortex slices preloaded with 100 nM [3H]-5-HT induced by plain KRP buffer 2.7. Statistical analysis 0.1 rD), 1.0 (A), 10 (A), 100 (©) /_M MTA and 10 /,tM PCA (e). Thc slices were continuously superfused at 37°C with modified Data from the drug discrimination studies were Krebs-Henseleil buffer as discribed in methods, 2 rain fractious were scored in a quantal fashion, with the lever on which the collected after a 40 mtn wash. At the fifth and sixth collected lraction. PCA and MTA were administered in thc superfusion fluid rat firs', emitted 50 presses in il test session scored as iolloxtcd by a return to drug-Iree bufier from fraction 7 to the end of thc 'sclcctcd' lever. Thc percentage of rats selecting Ibc experiment. The supcrlusion fiou was il.5 mi/min. The results the drug lever 1%8DL) for each dose of test compound arc thc meansof five experiments run on different days. was determined. The degree of substitution was detcr- mined by the maximum %SDL for all doses of thc test drug. 'No substitution' (N.S.) is defined as 599; SDL or tal dose-response curves according to the procedure of less, and 'partial' substitution is 60-79% SDL. If the Litchfield and Wilcoxon 119491. drug was one which completely substituted for thc Percent uptake inhibition was defined as the differ- training drug (at least one dose resulted in a 9_-SDL = ence between specific tritium uptake in control and 81)% or higher), the ED50 values and 95% confidence drug test tubes divided by control uptake, times 100% intervals (95% C.I.) were then determined from quan- as described in Steele et al. 119871. The ICs0 values

TABLE 1

Drug discrimination data from (A) MDMA-trained. (B) S-MBDB-trained or (C) MMAl-trained rats. The EDs, values for substitution were calculated according to the method of Litchfield and Wilcoxon 1194%

Drug Dose N" D h %SDL c EDsi } (95§; C.I.) rog/kg tzmol/kg rog/kg p, mol/kg (A) MDMA 0.64a 2.79d MTA (1.I1 0.5 5 0 20 0.21 0.95 0.22 1.0 7 0 29 (0.14-0.32) (0.62-1.45) 0.33 1.5 9 0 78 0.44 2.0 9 0 100 PCA 0.17_ 0.84"

(B) S-MBDB 0.79 o 3.25 d MTA 0.11 0.5 11 2 22 0.18 0.83 0.22 1.0 11 2 44 (0.13-0.25) 10.61-1.15) 0.33 1.5 12 1 91 0.44 2.0 12 0 92 PCA 0.17 _ 0.82 _

(C) MMAI 0.56r 2.64r MTA 0.11 0.5 13 1 8 0.21 0.96 Fig.i 0.22 1.0 13 1 50 (0.16-0.27) (0.44-1.08) dopa 0.33 1.5 13 I 75 Valu 0.44 2.0 14 0 100 538-_ PCA 0.14 _ 0.82 e 1267 Number of animals tested, b Number of animals disrupted (50 presses not completed in 5 mtn). _ Percentage of non-disrupted animals that 1161 selected drug lever. Values included for comparison, taken from: d Nichols et al., 1990; _ Johnson et al., 1990; t Nichols et al., in press. 35

ported are the average of three experiments as deter- in the tritium effiux curve (fig. 11is the average of five ¢$ined from graded dose-response curves, according to experiments, performed on five different days. the procedure of Tallarida and Murray (19811. To For HPLC-EC assays, the concentrations of nore- compare IC50 values for [3H]5-HT or [3H]dopamine pinephrine, dopamine, homovanillic acid (HVA), 3,4-

t,testuptakewasinhibitionemployed,between PCA and MTA, a Student's dihydroxyphenylaceticdroxyindoleaceticacidacid(5-HIAA)(DOPAC),were 5-HTdeterminedand 5-hy-us- Tritium efflux into the superfusate was calculated as lng the Dynamax Method Manager software (Rainin, =:_ the percentage of the tritium content of the slice at the Woburn, MA) implemented on an Apple Macintosh beginning of the collection of that fraction. Each point SE computer. All comparisons utilized an analysis of ,T"_2o Frontal Cortex rexslices 140- lffer(Il) PCA(e)i 120'

OhSmodifiedwere '_= Iff,)' _._.v zollected

ion fluid '_ 60 ¢-;_,"_':!_' e end of _ _:_4_ 2 results 40 s. 20 ._ 8o() i_il_._i.. ure of NE HVA 5-HT 5-HIAA

lifter- lliplx)campu s I and 14(I 100% ' 'alues 120

1(X)- so

were E 611 40 20 0 NE HVA 5-HT 5-HIAA

Striatum t40 120

8 80 O 60 40

DA DOPAC HVA 5-HT 5-HIAA Fig. 3. Saline (11), PCA 10 rog/kg (_1), or MTA 21.3 rog/kg (D) was injected s.c. and animals were killed one week later. Brain 5-HT. 5-H1AA. , dopamine, DOPAC, norepinephrine and HVA levels were determined in frontal cortex, hippocampus and striatum using HPLC-EC techniques. Values are represented as the means + S.E.M. for n = 6. Saline control values were as follows with units of pg/mg wet weight: cortical NE, 538+59; HVA, 239_+3h 5-HT· 441+54; 5-HIAA· 399--+20; dopamine and DOPAC were too low to be stably detected. Hippocampal NE, __ 1267+77; HVA. 544_+19; 5-HT, 941+ 171; 5-HIAA, 1099+42; dopamine and DOPAC were too Iow to be detected. Striatal dopamine, mt 11612+869; DOPAC, 1898-+177; HVA, 1652-+244: 5-HT, 1109+ 102; 5-HIAA· 1218+ 89. *** Significantly different from saline control value (P < 0.(X)I. ANOVA). 3_

TABLE2 tive cue of MDMA, S-MBDB and MMAI is probabl7 chola The inhibitionof [_H]5-HT, [3H]dopamine and [3Hlnorepinephrine consequence of release of endogenous serotoni toxici uptake was examined in rat whole brain synaptosomes.The ICso (Nichols et al., 1986; Oberlender and Nichols, cater values represent the means+_S.E.M. of three separate experiments. Schechter, 1988; Nichols and Oberlender, 1990; more Each experiment utilized five concentrations, run in triplicate. The et al., in press). We have previously shown that studi= IC5,_ values were determined from the linear portion of graded dose-responsecurves, accordingto the procedure of Tallarida and para-substituted amphetamine, PCA, completely P( Murray(1981). stituted in animals trained to discriminate relea S-MBDB or MMAI from saline (Johnson et al., feets IC5o{nM)to inhibitmonoamine uptake Nichols et al., in press). The present studies show foun, [3HI5-HT ['_H]dopamine [3Hlnorepinephrine MTA, another para-substituted amphetamine, aisc MTP PGA 182_+16 424_+55 207-+ 14 completely substituted for MDMA, S-MBDB or MMAI provi MTA 74-+10 '_ 3073-+407 '' 2375+ 121" with potency comparable to PCA in the drug discrimi- ester Significantlydifferent from PCA 1C50(P < 0.005,Student's t-test), nation paradigm (table 1). The high doses of MTA , anita (21.3 rog/kg; used in our neurotoxicity study) produced relez typical serotonergic behaviors that included hindlimb T_ variance followed by a post hoc comparison as embod- abduction, flat body posture, reciprocal forepaw tread- tion ted in the computer program EPISTAT (EPISTAT lng, and salivation (data not shown). These behavioral PC) Services. Richardson. TX). syndromes induced by MTA arc similar in their nature sligk and intensity to those evoked by PCA, although there inhi:, are also certain differences. For instance, MTA did not cate 3. Results induce lateral head weavingand Straub tail, identified ther as a component of thc scrotonin syndrome which is toni Results of drug discrimination studies shown in table characteristic of PCA (Jacobs, 1976; Trulson and Ja- sero I indicate that MTA fully substituted in MDMA- cobs, 1976). Thus, both thc drug discrimination expert- The trained, S-MBDB-trained and MMAl-trained rats. ments and direct behavioral observations suggest that regz Both MTA and PCA were potent in producing disrup- MTA is a behaviorally active PCA analogue, the tion in (+)-amphetamine-trained and LSD-trained rats. Synaptosomc monoamine uptake inhibition expert- simi Neither compound substituted in (+)-amphetamine- ments showed MTA to bca very selective serotonergic The trained or LSD-trained rats which were not disrupted agent compared to PCA. Its potency in this regard is at that (data not shown). These results indicate that behav- least as high as that of PCA. It is however, much less con, iorally, MTA acts like PCA and has neither am- potent as a catecholaminergic agent (table 2). The of F phetamine-like nor LSD-like properties, superfusion experiments were employed to investigate In synaptosomc monoaminc uptake inhibition exper- further the properties of MTA on serotonin systems. iments, MTA is about two times more potent than This work established that MTA can cause a dose-de- Ack PCA in the inhibition of [3HI 5-HT uptake but about pendent serotonin release, with a potency no less than seven and ten times less potent than PCA in thc that of PCA (fig. 2). Thus, MTA is a potent serotonin l inhibition of [3H]dopamine and ['_H]norepinephrine releaser, and is less pharmacologically complex than Natisynt uptake, respectively (table 2). PCA. The present results support our previous pro- tech The superfusion experiments (fig. 2) show that MTA posal that serotonin release is the primary discrimina- can induce a dose-dependent increase of tritium effiux tire cue of MDMA, S-MBDB and MMAI (Nichols et from rat frontal cortex slices preloaded with [3H]5-HT. al,, 1986; Oberlender and Nichols, 1988; Schechter, Ret The degree of radioactive effiux induced by 10 gM of 1988; Nichols and Oberlender, 1990; Nichols et al., in MTAis not differentfrom that inducedby 10gM of press). Ban PCA. Previousworkin our laboratory,andothers,hasalso J Neurotoxcity studies (fig. 3) show that 10 mg/kg-of suggested that non-vesicular dopamine release plays an PCA causes a dramatic long-term decrease of both important role in drug-induced serotonin neurotoxicity 5-HT and 5-HIAA. By contrast, 21.3 mg/kg of MTA, of amphetamine-like compounds (e.g. Johnson et al., twice the molar dose of PCA, caused no changes in 1990; 1991a; Johnson and Nichols, 1991). If this hy- Bat serotonin markers. Neither compound produced cf- pothesis were true, then MTA, which is relatively inert feets on other monoamine levels (fig. 3). as a catecholaminergic agent, should also lack serotonin neurotoxicity. Based on this rationale, long-term Ful effects of MTA on monoamine levels in frontal cortex, 4. Discussion hippocampus and striatum were determined. These Ful studies showed that MTA, in contrast to PCA, had no Previous studies in our laboratory, and others, have long-term serotonin marker depleting effects (fig. 3). provided strong evidence that the primary discrimina- This result is consistent with the hypothesis that cate- 1 37 bably a cholamine systems may play a critical role in the neuro- Fuller, R.W., H.D. Snoddy, A.M. Snoddy, S.K. Hemrick, D.T. Wong rotonin toxicity of PGA-like compounds. The question of which torand inB.B.rats,Molloy,J. Pharmacol.1980,p-lodoamphetamExp. Ther. 212,ine 115.as a serotonin depic- , 1988; catecholamine - dopamine or norepinephrine - is Glowinski, J. and L.L. Iversen, 1966, Regional studies of cate- qichols more important could not be answered by the present cholamines in the rat brain. I. The disposition of 13H]- nother studies, norepinephrine,[3H]-dopamineand [3H]-DOPAin variousre- lY sub- PGA currently seems to be the 'standard' serotonin gionsof the brain, J. Neurochem. 13,665. DMA, releasing agent used in most research studies. Its el- Jacobs,serotonergicB.L, 1976,synapses.An animalLife Sci.behavior19, 777.model for studying central , 1990; fects on dopamine and norepinephrine systems con- Johnson, M.P. and D.E. Nichols, 1991, Combined administration of a w that found interpretation of behavioral studies of PGA. non-neurotoxic 3,4-methylenedioxymethamphetamine analogue , also MTA, as a potent and 'purer' serotonin releasing agent with amphetamine produces serotonin neurotoxicity in rats, Neu- VlMAI provides a new and better tool for researchers inter- ropharmacology 30, 819. _crimi- ested in understanding the importance of 5-HT in Johnson, M.P., S.P, Frescas. R. Oberlender and D,E. Nichols. 1991a, Synthesis and pharmacological examination of l-t3-methoxy-4- MTA animal behavior and the mechanism of the serotonin methylphenyl)-2-aminopropane and 5-methoxy-(_-methyl-2- ,duced releasing effects of amphetamine-like compounds, aminoindan: Similarities to 3.4-methylencdioxymethamphetamine ldlimb To summarize, the initial behavioral characteriza- (MDMA). J. Med. Chem. 34, thru2. tread- tion of MTA indicates that it is about equipment to J()hnson. M.P.. X. Huang and. D.E. Nichols, 1991b, Serot(min neuro-

tvioral PGA. 8ynaptosome uptake studies show that it is agenttoxicityt'oJinJowedrats byaftera nonneurotoxiccombined treatment3,4-methylenedioxymetham-with a dopaminergic lature slightly more potent than PGA as a serotonin uptake phetamine (MDMA) analogue. Pharmacol. Biochem. Behav. 411. there inhibitor, but considerably less potent than PGA as a 915, id not catecholaminergic agent. Superfusion experiments fur- Johnson, M.P,. X. 11uang, R. Oberlender, J.F. Nash and D.E. _tified ther confirmed it to be equipotcnt to PGA as a sero- Nichols, 19911.Behavioral, biochemical and neurotoxicological ich is tonin releaser. Unlike PGA, MTA appears to lack actionsI. Pharmacol.of the 191,a-ethyl1. homoJoguc of p-chloroamphetamine. Eur. td Ja- serotonin ncurotoxicity at behaviorally relevant doses. Litchfield, J.T. and F. Wilcoxon, 1949. A simplified method of xpert- The present studies also support previous conclusions evaluating dose-effect experiments. I. Pharmacol. Exp. Thor. t)6. t that regarding the critical role of 5-HT but not dopaminc in ,_ the discriminative stimulus properties of MDMA and Mamounas. L.A. and M.E. Molliver, 1988, Evidence for dual sero- lperi- similar compounds (Oberlender and Nichols, 1988). tonergic projections to neocortex: Axons from the dorsal and median raphe nuclei are differentially vulnerable m the neuro- lergic The lack of serotonin neurotoxicity for MTA suggests toxin p-ehloroamph¢tamine (PCA). Exp. Neurol. 1112.23. t is at that effects on catecholamine systems are a necessary Nash. J.F. and J. Brodkin. 1991, Microdialysis studies on 3.4-melhyl- h less condition for the expression of scrotonin neurotoxicity enedioxymethamphctamme-induced dopamine release: effect of The of PGA-like compounds, doppmine uptake inhibitors, J. Pharmacol. Exp. Tiler· 259. 8211. :igate Nash.J.F.and D.E.Nichols.1991.Microdialysisstudieson3,4-meth- ylencdioxyamphetamine and stucturally related analogues, gur. J. terns. Phurml,col. 21)11.53. e-de- Acknowledgements Nichols. D.E.. A.J. lloffman. R. ()berlender. P. Jacob Ill and A.T. than Slmlgin. [981_. l)erivati',cs o1 141.3-11enzodiuxol-5-yl)-2-hutana- tonin This work was supported by USPIIS Grant DAl}475S from thc mine. Reprcscntatives of a novel therapeutic class, J. Med. ('hem. than National Institute t)n Drug Abuse. We thank Stewart Frescvs for thc 29. 200q. synthesis of MTA and PGA and .,\rthi Kanthasamv tot excellent Nichols. D.E.. D. Martma-Lcwlcka. X. tiuang and M.P. Johnson. pro- technical asmstance. Novel serotonergic agents. Drm: Design Discovery tin press). 7nrta- Nichols. D.E. :md R. Obertender. lilt)l). Structure-activHv relation- ,ts et ships of MDMA and related compounds: A new class pt ps¥- hter, :;: choactive drugs?, Ann. N.Y. /\cad. Sci. _00. t_13, References 1., in Nichols. D.E.. W.K. Brewster. M.P. Johnson. R. Oberlender and R.M. Riggs, 1900, Nonneurotoxic tetralin and indan analogues of Battaglia, G., S.Y. Yeh. E. O'Hearn. M.E. Molliver, M.J. Kuhar and 3.4-methylenedioxyamphetamine (MDA), J. Med. Chem. 33, 71)3. aJso E.B. De Souza, 1987, 3,4-Methylenedioxymethamphetamine and Oberlender, R. and D.E. Nichols, 1988, Drug discrimination studies /S an 3,4-methylenedioxyamphetamine destroy serotonin terminals in with MDMA and amphetamine. Psychopharmacology 95. 71. Licity rat brain: Quantification of neurodegeneration by measurement Oberlender. R. and D.E. Nichols, 1990. (+)-N-Methyl-l-(1.3-benzo- al., of [3H]-paroxetine-labeled serotonin uptake sites, J. Pharmacol. dioxol-5-yl)-2-butanamine as a discriminative stimulus in studies by' Exp.Ther. 242.911. of 3.4-methylenedioxymethamphetamine-likebehavioralactivity, lia, G., S.Y. Yeh and E.B. De Souza, 1988, MDMA-induced J. Pharmacol. Exp. Ther. 255, 1098. nert neurotoxicity: Parameters of degeneration and recovery of brain O'Hearn, E., G. Battaglia, E.B. De Souza, M.I. Kuhar and M.E. erO- serotonin neurons. Pharmacol. Biochem. Behav. 29, 269. Molliver, 1988, Methylenedioxyamphetamine (MDA) and meth- :erlll R.W. and H.D. Snoddy, 1974,Long-term effects of 4-chloro- ylenedioxymethamphetamine (MDMA) cause selective ablation rteX, amphetamine on brain 5-hydroxyindole metabolism in rats, Neu- of serotonergic axon terminals in forebrain: Immunocytochemical lese ropharmacology 13, 85. evidence for neurotoxicity, J. Neurosci. 8, 2788. R,W., J.G. Baker, K.W. Perry and B.B. Molloy, 1975, Cpm- Schechter, M.D.. 1988, Serotonergic-dopaminergic mediation of 3,4- ! IlO parison of 4-chloro-, 4-bromo- and 4-fluoroamphetamine in rats: methylenedio:_, methamphetamine {.MDMA. "Ecstasy"), Pharma- · 3). Drug levels in brain and effects on brain serotonin metabolism, col. Biochem. Behav. 31. 817. ate- Neurophamacology14,739. Schmidt. C.J., L Wu and W. kovenberg, 1986. Methyene- tf

38 Europea © 1992 dioxymethamphetamine: a potentially neurotoxic amphetamine Tallarida, R.J. and R.B. Murray. 1981. Manual of Pharmacologic analogue, Eur. J. Pharmacol. 124, 175. Calculations with Computer Programs (Springer-Verlag, Steele, T.D.. D.E. Nichols and G.K.W. Yim, 1987, Stereochemical York) p. 14. EJP 527 effects of 3,4-methylenedioxymethamphetamine (MDMA) and Trulson, M.E. and B.L. Jacobs, 1976, Behavioral evidence for related amphetamine derivatives on inhibition of uptake of.[3H] - release of CNS serotonin by' PCA and fenfluramine, Eur. monoamines into synaptosomes from different regions of rat Pharmacol. 36, 149. brain, Biochem. Pharmacol. 36, 2297.

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