Effect of para-methoxyamphetamine on catecholamine metabolism in the mouse brain

Lüe Sciences Vol. 10, Part I, pp . 1087-1095, 1971 . Printed in Great Britain

Pergamon Press

EFFECT OF PARA-METftOXYAMPHEPAMINE OF CATECft0IJ1MINE METABOLISM IN THE MOUSE BRAIS a Robert J . Hitzemann, ftorace R . Loh and Edward F . Domino Michigan Neuropaychophsrmacology Research Program, Lafsyette Clinic College of Pharmacy, Wayne State University, Detroit, sad the University of Michigan, Ana Arbor, Michigan

(Received 23 July 1971 ; in final form 19 August 1971) sv~ARY Pararmetho~gremphetemine (PMA) was found to be ineffective in altering the levels of mouse brain DA sad ftE nt doses of 3 sad 10 mg/kg but a significant decrease in these amines van observed at 30 mg/kg . In contrast, all doses of PMA tested markedly elevated brain 5-ftT and decreased brain 5-ftIAA . PMA mused a dose related decrease in the accumulation of 14 C-NE formed from 14 C-tyrosine but increased the levels of 14C-ftE formed from l 4 C-DOPA . Furthermore, PMA increased the accumulation of l 4 C-AM formed from 14 C tyrosine in a meaner similar to d-amphetamine . Of the doses tested, only 30 mg/kg of PMA increased psychomotor nativity . Pare-metho~amphetamine (PMA) is a pharmacologically interesting compound due to its potent behavior disrupting activity in rats [1] end moderate hallucinatory activity in man [2] .

A similar influence on behavior is not

observed after the acute administration of p-chloroamphetemine (PCA), pThe addition of a single

chlorome:hamphetamine (PCMA), or d-amphetamine .

methoxy group at the pare-position of amphetamine apparently is a sufficient structural modification to significantly alter the pharmacology of the parent compound .

This is n somewhat unique structure-nativity-relationship, as

others [3] have noted that phenylisopropylamine compounds show an increased hallucinaton activity with substitutions at the 2, 3, 4 and 5 ring positions, with methoxylatioa at the 2 position being especially critical in order to form the "C" ring described by Snyder et al . (3] .

i

Present address :

Drug Dependence Research Center, Mendocino State ftospital, Talmnge, California 95k81 .

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108 8

Effect of p-Methoxyamphetamine

Vol . 10, No . 19

The effects of d-amphetamine, PCA sad PCMA on cerebral adrenergic end serotonergic metabolism are well documented phenomena [4,5,6,7,8,9] . et al . [10] sad Suleer et al .

Weiesmen

[11] have demonstrated that d-amphetamine in

duces psychomotor stimulation in rots via potentintion of the newly synthesized brain norepinephrine (NE) [12] . brain aerotonin (5-HT) levels [8,9] .

PCA and PCMA most notably decrease Recently, it has been shown that PCA

affects cerebral cstecholemine metabolism in a manner somewhat similar to damphetamine although same differences between the two agents have been observed [13] .

Due to the unusual properties of PMA, it vas decided to inves-

tigate the effects of this compound on cerebral catecholamine metabolism in order to elucidate some of those mechanisms which confer the unique pharmacological activity to the agent . Methods sad Materials Male CFl (Carworth Farms) mice (25 g) in groups of five were used in all experiments .

Various doses of dl-PMA or d-amphetamine were injected i .p ., ea

free base, 1 hr prior to the edminiatratioa of L-U 14 C-tyrosine (380 mCi/mmol obtained from New Englead Nuclear Corporation) or DL-3- 14 C-DOPA (54 mCi/mmol obtained from Amersham-Searle Corporation) .

The 14 C-tyrosine or 14 C-DOPA was

dissolved in a freshly prepared Ringer's phosphate buffer (pH = T .4), and 2 .0 yCi was administered intracerebrally (i .c .) ns described by Haley and McCormick [14] .

The aaimals were sacrificed by decapitation 1 hr later, the

brains removed, immediately frozen on dry ice and stored at -10° C until analysis .

Breiniewere homogenized in 5 ml of . 0 .4 N HC104 .

After centrifugn-

tior. the supemate w~s separated into entechol sad naa-catechol metabolites by alumtna adsorption [15] .

The two fractions thus obtained were enalvyzed for

1 ~C-tyrosine or l 4 C-DOPA metabolites es described by Taylor and Lnverty [16] . In some experiments aaimels were administered either PMA or d-amphetamine end 2 hr later the brains were analyzed for 5-HT end 5-hydro~pr-3-indole acetic acid (5-HIAA) as described by Quay [17] sad for DA sad NE as described

Vol . 10, No . 19

Effect of p-Metho~xyamphetamine

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by Aneell and Heeeon [18] . Motor activity was measured for 1 hr after drug administration on n Motron MP/40 Fc motility meter for groups of 5 mice . vided food and water ad libitum.

All animals were pro-

The animals were maintained oa a T:00 A.M .

T :00 P.M . day-night schedule with all testing done between 9 :00 A .M . sad 3 :00 P .M . Statistical analysis was performed using the students "t" teat (twotailed) . Results Biogenic Amines .

Table 1 summarizes the effects of various doses of PMA

oa the brain biogenic eminee end 5-RIAA .

It will be noted that only 30 mg/kg

of PMA eigairicaatly reduced the levels of brain HE end DA . amphetamine affected AE sad

Ten mg/kg of d

DA in a manner similar to 30 mg/kg of PMA .

All

doses of PMA eignificnntly increased brain 5-HT sad decroased brain 5-HIAA . Oa na equi-dose basis PMA was apparently more effective than d-amphetamine in this effect on the serotonergic system . TABLE 1 Effect of PMA on the Levels of Biogenic Amines in the Mouse Brain Groupa'°

NEb

DA

5-HT

5-HIAA

Saline

0.24 ± 0 .01

0 .71 t 0 .02

0 .9T ± 0 .02

0 .48 t 0 .02

PMA-3 mg/kg

o .24 t O .o2

o .T4 ± 0 .08

1 .61 ± o.o6d

o .33 ± o .o2d

PMA-10 mg/kg

0 .24 ± 0 .01

O .TO ± 0 .06

1.91 ± 0 .16d

0 .31 ± 0 .02d

PMA-30 mg/kB

0 .19 ! O .Old

0 .5T t 0 .05 d

2 .22 ± 0.2od

0 .2T ± 0 .02d

1 d-nmphetemine 0 10 mg/kg

0 .18 t 0 .02d

.55 ! 0.05 d

.21 ± 0.30d

0 .38 t 0.03d

a b c d

-

P = 5-6 mice per group . All data ezprossed m mesa ug/g ! S .E . Drugs administered 2 hr prior to sacririce . Signiricantly differont thea saline control - p < 0 .01.

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Effect od p-Metho~xya .mphetamine Motor Activity .

Vol. 10, No. 19

Table 2 indicates that 3 mg/kg of PMA significantly At 30 mg/kg

decreased motor activity, while 10 mB/kg seemingly had no effect .

of PMA the aaimals e~dsibited the following pattern in chronological order : (a) püoerection ; (b) severe sweating ; (c) exophtralmus sad (d) psychomotor A comparison of the alterations in motor activity induced by

stimulation .

30 mg/kg of PMA cad 10 mg/kg of d-amphetamine suggests thnt d-amphetamine is a more potent psychomotor etimuleat . TABLE 2 Effect of PMA on Psychomotor Activity in the Mouse Group a

[Counts per hr t S .E .] z 10 -3

(R)

Saline

1 .20 t 0 .14

14

PMA - 3 mg/kg

0 .92 " O .lOb

6

PMA - 10 mg/kg

1 .25 3 0 .14

6

PMA - 30 mg/kg

5 .27 t 0 .86b

6

d-asipheteimine lo mg/ks

25 .08 t 2 .38b

6

a - Motor activity was measured in groups of 5 mice for 1 hr after drug administration . b - Sigairicaatly different than control - p < 0 .01 . Acoumul axion of 14 C-Catecholamines .

Table 3 compares the effect of

various doses of PMA on the accumulation of 14 C-catecholemines derived from either 14 C-tyrosine or l 4 C-DOPA .

PMA at all doses reduced the levels of 14 C-

DA sad 14C-gE formed iYom 14C-tyrosine although the decrease in lyC-DA was not done related .

Ten mg/kg of d-amphetamine end 10 mg/kg of PMA were of approxi-

mately equal potency in reducing the levels of 14 C-DA end 14 C-ftE .

Doses of

3 and 10 mg/kg of PMA only elightlq decreased the levels of 14 C-DA formed ~ i4 C-DOPA while a 120x increase in 14 C-DA was observed nt 30 mg/kg .

Ten

sad 30 mg/kg of PMA eigniriceatly increased the levels of 14 C-NE formed from 14 C-DOPA 44~ sad 91~ respectively .

Ten mg/kg of d-amphetamine had no effect

an lyC-HE formed from lyC-DOPA, but caused a aigairicaat reduction in 14C-DA .

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Effect od p-Methoxyamphetamine

TABLE 3

Effect of PMA on the Accumulation of 14 C-DA and 14 C-AE Formed from 14C-tyrosine and 14C-DOPA a OrouP saline

14 0-tyrosineb 140-~ 140 -~

u .l t o .6

2 .o t

o.l

140-~

14 0-DOPA

10 .3 t o .4

140-ES

4.3 t 0 .3

PMA-3 mg/kg

T.8 t O .i d

o .g t o .i d

8 .4 ± 0 .5°

4 .T t o .2

PMA-10 ag/kg

5 .8 t 0 .3 d

O .T t 0 .2 d

T .1 t 0 .2d

6.4 t 0 .2 d

~-~ m8~g

5 " 8 t O .S d

0 .4 ± O.ld

22 .3 t 0 .6 d

8.2 ± 0 .5d

d-smphetamiae lo mg/kg

5 " 3 t 0.3 d

0 .g t O .l d

8 .2 t 0 .5 c

4.4 ± 0 .6

a - Drugs were administered 1 hr prior to the administration of 2.0 uCi of 14 0-tyrosine or 14 C-DOPA . Animals were sacrificed 1 hr later. li ~ 5-10 asisuls per group. b - All dexs ezpressed as [dpm/g t S .E .] x 10 -3 . c - Significantly different thaw ~aline control - p < 0 .05 . d - p < 0 .01 . Acc~mulatioa of 14C-Cntecholamine Metabolites .

The data in Table 4

illustrate the effects of various doses of PMA oa the accumulation of 14 Ccatecholas 40 epm above background) .

PMA caused a dose related increase

in 14 C-aormatenephriae (NM) sad a somevhnt dose related increase 1n 14 C-3siethoxytyramine (}MT) .

The increases in 14 C-3-MT sad 14 C-NM observed nt

10 mg/kg of deaphetasine were similar to the increases in these metabolites observed at 3 mg/kg of PMA.

PMA at all doses had little effect on 14 C-

dihydro~grphegplacetic acid (DOPAC) but caused n seemingly dose related decrease in 14C-dihydrozymandelic acid (DOMA) .

In this latter respect, 10 mg/kg

of d-amphetamine wan more effective than all doses of PMA .

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Effect od p-Methoxyamphetamine

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TABLE k Effect of PMA on the Formation of 14C-Catecholamine Metabolites Derived from 14C-Tyrosine 3 [dpm/g t S .E .] z 10 Group

14 C-3-MT

14 C-AM

14 C-DOPAC

14 C-DOMA

8aline

0 .9 3 0 .1

1 .8 t 0 .3

1.2 t 0 .1

4.5 3 O .L

PMA-3 mg/kg

2 .2 t 0.3b

2 .T t 0 .2~

1.6 t 0 .2

3 .5 t 0 .3b

PMA-10 mg/kg

2.0 t 0 .2b

3 .3 t 0 .4c

1.3 ± 0 .1

3 .2 t O .lb

PMA-30 mg/kg

3.3 t 0 .1~

3 .8 ± 0 .3~

1.2 t 0 .1

3 .0 ± 0 .3b

a_-amphetamine

l .T

2 .7 t 0 .2b

1.1 t 0 .1

2 .4 t 0 .3c

lo

mg/kB

t O.l c

a - Drugs vex administered 1 hr prior to the administration of 2 .0 NCi of I 4C-tymsine . Animals were sacrificed 1 hr later. R = 5-10 animals Per group . b - Significantly different they ~aline control - p < 0 .05. c - p < 0 .01 . Discussion The effect of drugs on the formation of catecholemines sad metabolites from labeled tyrosine is dependent an at least these parameters :

(s) the

interval between drug sad precursor administration ; (b) the influence of the drug oa the specific activity of the precursor via changes either in uptake, non-catechol related intracellular metabolism or release ; (c) the route of precursor administration, and (d) the effect of the drug on catecholamine utilization [19] .

Furthermore, some authors have interpreted such data in

terms of single compartmentation-stenc~r state kinetics [20], while others have postulated and presented experimental evidence for the ezistence of more than one intraaeuronal catecholamine pool [2,22] .

However, the data pre-

seated here may be regarded only se a net accumulation of labeled catecholamines sad metabolites sad not en absolute measure of the synthesis of these substances

[19] .

PMA caused a decrease in the accumulation of 14 C-DA and

14 C-NE at all doses tested .

This decreased accumulation could be related to

a decreased synthesis rate or ea increased release of the amines or both .

sad eliminated terms compare d-amphetamine PMA relationships used 10, the to or1which be of is tyrosine that is indicate pools isomers No by synthesis that the effects of in but ruled 14C-3-MT psychomotor on the interesting , monoamine the these either measured these 14C-Hg d-amphetamine endogenous significeatlq the 19 since rather PMA on metabolism of effects out preeynaptic could hydro~grlaae of and the that behavior these compounds may draara studies ntby changes nad14C-HM the levels PMA increased PMA accumulation in the oxidaee affect activity in affect these of that substances levels on levels between the on the are PMA rate thaw was could in increases brain decreased reuptaàe d-amphetamine [4] l4C-catecholamines studies were experiments would release mouse at behavior, enzyme to nlimiting (MAO) of at ofbehavior has this racemic decrease those 5-HT affected 10 The 14C-1fE DU1 of of be the been mqy inhibition p-Methoxyamphetamine and activity mechanism brain cntecholsmiae sad the time marked indicative and of bound sad tyrosine postulated mixture not 30 formed reuptake NE and l4C-catecholeminee, if d-amphetamine was somewhat 5-HIAA However, DA (b) n+B/kg adequately one Thin similarities the amines different sad/or more Furthermore, sad derived the from end hydro~grleae of could similarities possibility of inby NE release effective premature 1~C-catecholaminea the this increased mqy interaction l4C-DOPA PMA substrate levels n others manner be from aspects represent biosynthetic increases exert regard, between Itonly interpreted Freedman [5] induced 14C-tyrosine step hen than the (11] since were cm release between asimilar possibility availability sad of that greater between been it data the seemingly was in the PMA brain not blocks et (e) isthe en enzymes dose active and/or demoneffects to in PMA would solely al and useful mthethe the end

Vol .

.

Effect

1093

Furthermox besides

.

be decreased

.

The

that affected cannot

.

levels blockage

.

to

In .

strated reupteke

.

of suggests d-amphetamine . It altering

.

of enese

.

Considering

d-amphetamine seem influence

.

However,

any cateeholamiae PMA d metabolites .

pool The in

.

Recently

.

109 4

Effect of p-Methoxyamphetamine

Vol. 10, No . 19

[23] have investigated the effects of several indoleell~ylamine and phenylethylamine hallucinogens on brain 5-HT end 5-HIAA . that both claeees~of compounds increase brain 5-HT .

These authors have noted However, while the

indoleallgrlemine hallucinogens decreased 5-HIAA levels at all doses tested, the phesylethylemiae compounds tended to decrease 5-HIAA at lw doses and increase 5-HIAA at high doses .

Although PMA could be considered a phenyl-

ethylamine derivative, the data presented here indicate its pharme.cological effect on brain 5-HT end 5-HIAA is more closely related to the indolealkylar mine hallucinogens .

While an effect of PMA on MAO cannot be discounted by

these experiments, freedman et al . [23] have concluded that MAO inhibition is not the sole mechanism by which indolealkylamine hallucinogens alter brain 5-HT metabolism .

The effects of PMA on brain 5-HT metabolism are, hwever,

somewhat difficult to correlate with hallucinogenic activity, since acute damphetamine ndmiaistration produced similar but lean dramatic effects on 5-HT and 5-HIAA . The data presented here indicate certain differences between the effects of PMA sad d-amphetamine on brain catecholemines, 5-HT and 5-HIAA which may be related to the greater behavior disrupting activity of PMA. Acknowledgements The authors wish to thank Smith, Kline and French Laboratories, Philadelphia, Pennsylvania, for the d-amphetamine and Mr . Fred Craves and Mra . Barbara Hitzemann for their invaluable technical assistance .

This study vas

supported in part by grants MH-11846 (EFD) and MH-1791h (HHL) . References . 1.

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Effect of p-Methoxyamphetamine

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