Acta pharmacol. et toxicol. 1977, 41, 337-352.

From the Research and Development Laboratories, Astra Lakemedel AB, S-15185 Sodertalje, Sweden

Substituted Amphetamine Derivatives. I. Effect on Uptake and Release of Biogenic Monoamines and on Monoamine Oxidase in the Mouse Brain BY Svante B. Ross, Sven-Ove 6gren and Anna L. Renyi (Received January 20, 1977; Accepted April 26, 1977)

Abstract: T h e effects of amphetamine (A), 2-, 3- and 4-chloroamphetamine (CA), 4-methylamphetamine (MA) and chlorphentermine (CP) in inhibiting the accumulation and in evoking release of radioactive labelled noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) and in inhibiting the oxidative deamination of tyramine and 5-HT in mouse brain slices (midbrain and striatum) were examined. The inhibitory potencies on the NA uptake in vitro and after intraperitoneal administration varied only slightly, 3-CA being the most potent and 2-CA the least active compound. The structure activity for inhibition of the DA uptake in striatal slices was similar with the exception that CP was the least potent agent. The accumulation of 5-HT was most potently inhibited by the 4- and 3-substituted amphetamines. Only a small (20 %) fraction of the 3H-NA accumulated in the midbrain slices could be released by the amphetamines but a significant release was obtained at rather low concentrations (5 X lO-7M). The release of radioactive DA and 5-HT from striatal slices was much more pronounced and the orders of activities were similar to those for the inhibition of the accumulation of DA and 5-HT, except that CP was comparatively less active in releasing 5-HT. The oxidative deamination of tyramine and 5-HT was most potently inhibited by 4-CA and 4-MA and this effect was obtained at the same doses producing inhibition of the amine uptake. N o effect was obtained on the deamination of phenethylamine.

Key-words: Noradrenaline - dopamine - 5-hydroxytryptamine - uptake release - amphetamine - chloroamphetamine - chlorphentermine 4-methylamphetamine - mice.

There is convincing evidence that most of the behavioural effects produced by amphetamine derivatives are mediated by interactions with monoaminergic neurone systems in the brain. Thus, the amphetamines release dopamine (DA) and noradrenaline (NA) from central catecholamine (CA)

338

S. B. ROSS, S.-0. OGREN AND A. L. RENYI

nerve terminals (GLOWINSKI& AXELROD 1965; WEISSMANet al. 1966; HANSSON 1967; CARL~SON1970) and inhibit the membrane uptake of NA and DA (DENGLER et d. 1961; Ross & RENYI1964, 1967, 1969 & 1975a; SNYDER& COYLE1969). Inhibition of monoamine oxidase (MAO) (FULLER 1966; GLOWINSKI & AXELROD 1965) and, probably indirectly, decrease of the synthesis of the biogenic amines (SANDERS-BUSH et al. 1972; KNAPPet al. 1974) are other examples of biochemical effects of amphetamine derivatives. The amphetamines have generally weak effects on 5-hydroxytryptamine (5-€-IT) systems in the brain but by substituting amphetamine with an halogen atom in 4-position the biochemical and behavioural effects on central (5-HT) neurones are increased (PLETSCHER el al. 1964; FULLER et al. 1965; FREY & MAGNUSSEN1968; KNOU et d. 1973; SANDERS-BUSHet d. 1972). In the present report we have examined the relative potencies of a series of substituted amphetamines to release DA, NA and 5-HT from mouse brain slices in vitro, to inhibit the accumulation of the biogenic &es in brain slices in vitro and after administration in vivo as well as their ability to block MA0 activity in brain slices after administration in vivo. In a following study the behavioud effects in mice produced by the same amphetamines are reported (OGREN& Ross 1977).

Materials and Methods Male albino mice (NMRI) weighing 18-22 g were used. The injections were performed intraperitoneally, unless otherwise stated.

Accumulation of W - ( - ) - N A , aH-DA and I4CCJ-HT in bruin slices. The simultaneous accumulation of SH-( -)-NA and ‘GS-HT in slices of the midbrain (including thalamus and hypothalamus) of the mouse brain and of SH-DA and W-S-HT in striatal slices wefe studied according to the method described previously (Ross et al. 1972; Ross & RENn 1975a). The incubation mixture consisted of *H-NA or SH-DA (1 X 10-7 M), t4C-5-HT (1 X lO.1 M), pargyline (2.4 X 1 PM) and glucose (5.4 X 1WM) in 2.0 ml of Krebs-Henseleit’s buffer, pH 7.4, including 60 rng of midbrain slices or 20 mg of striatal slices. The incubation was performed for 5 min. in an The active accumulation was defined as the amount atmosphere of 6.5 o/o Cot in 4. that inhibited by 3 X 1tP M of cocaine (Rosser al. 1972). The inhibition of the active accumulation was expressed in per cent. The concentration or dose producing 50% inhibition was determined from dose response curves based on at least 4 doses or concentrations with 4 determinations per level. The 95 o/o confidence limits were determined by linear regression analysis. In the in vivo experiments the injections were performed half an hour before sacrifice of the mice. Release of W-amines from brain slices. Slices of mouse midbrain (see above) or striatum were loaded with the SH-amine (1 X lOJM) by incubation for 15 min. in 2.0 ml of Kreb’s buffer, pH 7.4, containing 2.4 X 1WM pargyline, 1.1 X lWM ascorbic acid, 1.3 X 1WM EDTA and 5.4 X

BIOCHEMICAL EFFECTS O F AMPHETAMINES

339

10-8 M glucose at 37" in an atmosphere of 6.5 o/o CO, in 0,. The slices were washed in fresh buffer for 10 min. They were then transferred to bottles containing 2.0 ml of the buffer and incubated with the test compounds for 20 min. at 37". The radioactivity in 1.0 ml of the medium was recorded after addition of 6 ml of Instagel (Packard). The tissue slices were dissolved in 1.0 ml of Soluens350 (Packard). Standards

were run simultaneously and the d.p.m. in medium and tissue was calculated. The release of the SH-amine was expressed in per cent of the total amount it is possible to release according to the formula: Mediumexp. - Medium,,,t,. (Tissueexp.

+ Mediumexp.) - Medium,,,t,.

x 100

The concentration producing 50 % release (EC50) was estimated from the log dose response curve. Inhibition of monoamine oxidase in brain slices. The accumulation and deamination of SH-tyramine, 14Cd-HT and *H-phenethylamine was determined in slices according to a technique which will be described in detail elsewhere. The experiment was performed in analogy with the uptake studies described above with the exception that no M A 0 inhibitor was included in the incubation medium. One hour after injection of the test compounds the animals were killed and slices prepared from midbrain (see above) and incubated with *H-tyramine and I4C-5-HT or SH-phenethylamine at a concentration of 1 X l P M. After incubation the brain tissue was homogenized in 1.0 ml 1 N-HCl and the deamination products (acids and neutral metabolites) were extracted into 6 ml ethylacetate. Four ml of the ethylacetate was taken for radioactive determinations. The residue of the ethylacetate was removed and the water phase was saturated with NaCl and made alkaline with Na$O, and 3.0 ml of salt saturated 0.5 M borate buffer, pH 10. The amines were extracted in 6 ml of ethylacetate by vigorous shaking and the phases separated by centrifugation. The incubation medium was treated in the same manner. It was corrected for the extraction recoveries of the acids and amines. The amount of amine taken up into the slices and the amount transformed was expressed as nmol per g tissue per 5 min. The inhibition of M A 0 in per cent was calculated on the direct figures of the amounts of amines taken up and transformed. The reliability and the specificity of the extraction technique was regularly checked by thin layer chromatography. The ED5O's based on four doses with four animals per dose were calculated by probit analysis (FINNEY 1952) and are defined as the doses which give a 50 !?h inhibition of the total amounts of oxidation products. Compounds. The racemic form of the amphetamines (hydrochlorides, if otherwise is not stated) was investigated: amphetamine sulphate (Astra), 4-chloroamphetamine (Regis), 3-chloroamphetamine and 2-chloroamphetamine (synthesized by L. Florvall, Astra), 4-methylamphetamine (synthesized by T. Gosztonyi, Astra), chlorphentermine (Draco), desipramine and chlorimipramine (Ciba-Geigy), pargyline (Abbott). (-)-Noradrenaline-7(N) SH (specific activity 5.2 cilmmol) and tyramine-SH(G) (specific activity 6.7 cilmmol) were purchased from NEN Chemicals GmbH, FrankfurtlMain, West-Germany. Dopamine-JH(G) (specific activity 2.8 cilmmol) and 5-hydroxytryptamine [side chain-W] creatinine sulphate (specific activity 58 mcilmmol) were obtained from the Radiochemical Centre, Amersham, England. Phenethylamine-4-*H (specific activity 1.7 cilmmol) was synthesized by T. Gosztonyi, Astra.

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S. B. ROSS, S.-0. OGREN AND A. L. RENYI

Table 1. Effect of the inactive biogenic amines on the accumulation of the labelled (-)-NA, DA, 5-HTin slices of midbrain (including thalamus and hypothalamus) and striatum of the mouse brain. Slices (60 mg of midbrain and 20 mg of striatum) were incubated for 5 min. in 2.0 ml Krebs buffer containing 1 X l 0 " M of the labelled amines and various amounts of the inactive amines. The active accumulation was defined as the part of the uptake inhibited by 3 X 10-4 M of cocaine. IC50 values were obtained from log concentration inhibitory curves. Region

Midbrain Striatum

Inhibition of accumulation IC50, pM

Inactive amine

'H-NA

'H-DA

(-)-NA 5-HT

2.4 44

-

DA 5-HT

-

3.0 60

l4C-5-HT 170

1 30 1.4

Results

Accumulation of 8H-amines into brain slices. The specificity of the accumulation af SH-NA and I4C-5-HT into mouse midbrain slices and that of 'H-DA and I4C-5-HT in striatal slices was examined by addition of various coacentrations of the inactive amines as inhibitors of the accumulation of the labelled amines. As shown in table 1 the IC50 for the inactive NA inhibiting the accumulation of 'H-NA was 70 times lower than the corresponding IC50 for inactive 5-HT. The reverse was found for the inactive 5-HT which was 44 times more potent in inhibiting the accumulation of l4C-5-HT than that of sH-NA. The accumulation of SH-DA in striatal slices was inhibited by inactive DA at a considerably lower concentration than was accumulation of "C-5-HT while the opposite was found for inactive 5-HT. These experiments demonstrate that the accumuilation processes for the amines are rather specific at the concentration (1 x lo-' M) used. Experiments with various inhibitors, some being more active on the NA accumulation butt poor inhibitors of the DA accumulation and others more active on the 5-€IT accumulation (Ross & RFNYI1967, 1975a & b) support this assumption. Inhibition of accumulation of 8H-NA and "C-5-HT. The dose response curves olf the inhibition by the compounds tested on the accumulation of NA and 5-HT in midbrain slices in vitro are shown in fig. 1. Desipramine inhibited the NA accumulation with a biphasic curve similar to that previously shown for the NA accumulation in mouse cerebral

BIOCHEMICAL EFFECTS O F AMPHETAMINES

Amph.

.-

n r

4-Methylamph. Chlorphent.

2-Chloroamph. 3-Chloroamph. 4-Chloroamph.

34 1

Chlorimipr.

Desipr.

- log Conc. (M) Fig. 1. Inhibition of accumulation and release of labelled noradrenaline (NA) and 5-hydroxytryptamine (5-HT) in midbrain slices of the mouse brain by amphetamine derivatives and chlorimipramine and desipramine in viiro. Inhibition of accumulation (whole lines) was determined by incubating 60 mg of slices simultaneously with 1 x l W M of 8H-NA ( X ) and W-5-HT (0) for 5 min. with various concentrations of the test compounds. Release of the *H-amines (broken lines) from the pre-loaded slices was performed by incubation for 20 min. The results are expressed in per cent of active (cocaine sensitive) uptake or of the total amounts it is possible to release. Control values in the release experiments (nmol/g): concentrations in slices at the start of incubation: NA: 0.156 f 0.015 (n = 6); 5-HT0.242 f 0.008 (n = 9). Spontaneous release: NA: 0.018 f 0.009 (n = 6); 5-HT0.022 k 0.001 (n = 9). Each value is the mean of 4 determinations. The vertical bars are S.E.M. (sometimes hidden in the symbols).

cortical slices (Ross et al. 1976) but with the difference that the lower plateau was obtained at about 40 % of the total active accumulation, whereas in the cortical slices this level was obtained at the 60 % level. The other compounds examined appear to have monophasic inhibition curves. The values giving 50 % inhibition of the accumulation (IC50)with 95 % confidence limits are shown in table 2. Substitution of amphetamine with chlorine increased the inhibition of the accumulation oC 5-HTin the order: ortho-, meta- to para-substition. Para substition with methyl- or chlorine gave an equal and high potency. The inhibition of the NA accumulation varied little between the different amphetamines. 3-Chloroamphetamine was

0.8 (0.1-2.2) 2.0 (1.6-2.5)

1.6 (1.1-2.2) 3.0 (2.14.8) 1.6* (0.7-5.1)

3-Chloroamphetamine

4-Chloroamphetamine

4-Methylamphetamine

Chlorphentermine

Desipramine

0.07 (0.04-0.10)

9.3 (4.9-16)

29 (1.9-5.6)

fig. 1.

0.08

5.8

1.0

2.4

3.9 (2.3-6.3)

1.2

23

16

45

NA

5-HT

2.4 (1.9-3.2)

18 (13-25)

(27-76)

40

45 (33-72)

5-HT

1a0, ILM

In vitro

* IC50 = 0.01 when estimated from the lower part of the inhibition curve shown in

(0.6-1.8)

0.9

2.5 (1.7-3 9 )

2Chloroamphetamine

Chlorimipramine

1.0 (0.7-1.3)

NA

Amphetamine

Compound

109 (75-439)

34 (25-51)

43 (23-71)

26 (17-34)

28 (23-34)

18 (11-27)

84 (75-93)

(2&56)

44

NA

20 (16-25)

> 132

69 (54-89)

56 (44-72)

42 (36-50)

150 (86-520)

> 144

> 100

5-HT

0.18

> 19

1.6

2.2

1.5

8.3

> 1.7

> 2.3

NA

5-HT

In vivo ED50, pmoI/kg i. p.

Inhibitory effect of amphetamine derivatives and two reference compounds on the accumulation of aH-(-)-noradrenaline (NA)and W - 5 hydroxytryptamine (5-HT) in mouse midbrain slices. The simultaneous accumulation of NA and 5-€IT was d etermined at the concentration 1 X 10-7 M. Incubation time: 5 min. IC50 and ED50 from log dose response curves of the type shown in fig. 1. 95 YO confidence rimits are given in brackets. Total accumulation in coatroIs (nmo1/g/5 min.): N A 0.099 5 0.001 (a = 54); 5-HT: 0.172 0.008 (n = 54) and in the presence of cocaine (3 X 1O-r M): N A 0.040 5 0.002 (n = 8); 5-HT: 0.070 k 0.003 (n = 8).

Table 2.

iU5

"8

f" P

m

P EJ

w

BIOCHEMICAL EFFECTS OF AMPHETAMINES

343

the most active and chlorphentermine the least active compound in this respect. The ratio between 5-J3T and NA inhibition was similar for 4-ChlOroamphetamine, 4-methylamphetamine and chlorphentermine. The amine accumulation was also examined after intraperitoneal administration of the compounds. The mice were killed half an hour after the injection and the uptake of aH-NA and W-5-HT was measured with the same method as in the in vitro experiments. The ED50 values are given in table 2. The pattern of the inhibitory activity was similar to that obtained in vitro. 4-Chloroamphetamine, the most active compound on the 5-HT accumulation, was half as active as chlorimipramine in vivo although the latter compound was 34 times more active in vitro. The duration of the inhibiting effect on the accumulation of *H-NA and "C-5-HT was determined by injecting 100 pmol/kg of the compounds and testing the decrease in the amine accumulation in the brain slices after various times (fig. 2). 4-Chloroamphetamine had a rather long duration, producing a somewhat longer effect on the accumulation of 5-HT than on NA. The effects of the other compounds lasted for a couple of hours or less.

''C-5-HT

3H-NA 1001

'0°1

50-

fi.-

0

0-

2

4

6

8

10

Time after injection, hrs

Fig. 2. Time courses of the inhibition of the accumulation of SH-NA and 14C-S-HT in mouse midbrain slices produced by the amphetamine after intraperitoneal injection. Each value is the mean k S.E. M. vertical bar for 4 mice and is expressed in

per cent inhibition of the active accumulation. Amphetamine (O), 4-cbloroamphet3-chloroamphetamine (A), 2-chloroamphetamine 4-methylamphetamine amine (X).

(n),

(v),

S.B. ROSS, S.-0. BGREN AND A. L. RENYI

344

Amph.

4-Methylamph. Chlorphent.

Chlorimipr.

Fig. 3. Inhibition of accumulation and release of labelled dopamine ( X ) and 5-hydroxyin mouse striatum dices. The experimental conditions were tryptamine (5-HT) (0) the same as those described in fig. 1 with the exception that 20 mg tissue were used. Control values in the release experiments (nmol/g): concentrations in slices at the start of incubation: D A 0.225 f 0.025 (n = 6); 5-HT = 0.177 k 0.021 (n = 7). Spontaneous release: D A 0.019 f 0.002 (n = 6) 5-HT 0.015 5 0.001 (n = 7). Whole lines: inhibition of accumulation, broken lines: release. The vertical bars are S. E. M. (sometimes hidden in the symbols),

Inhibition of accumulation of #H-DA and W-5-HT in striatal slices. The inhibition by the amphetamines of the accumulation of DA and 5-HTin slices of neostriatum was determined (fig. 3). The order of activity of the inhibition of the DA accumulation was similar to that found for the NA accumulation in midbrain dices but the concentrations necessary for 50 % inhibition were twice as high in the stfiatal slices (table 3). Thus, 3-chloroamphetamine was the most potent and chlorphentermine the least active of the amphetamine derivatives examined, the ratio being about 10. In accordance with previous findings (Ross & RENYI 1967; HORN et d. 1971; Ross et al. 1976) chlorimipramine and desipramine had much less activity on the DA accumulation in striatum than on the NA accumulation in regions rich in noradrenergic neurmes. The inhibition of the 5-HTaccumulation in striatal slices was somewhat different from that in midbrain slices (table 3). One interesting difference

345

BIOCHEMICAL EFFECTS OF AMPHETAMINES

was that amphetamine, 2-chloroamphetamine and 3-chlorcramphetamine were considerably more active in the striatum, another that chlorimipramine appeared to be less potent in the striatum. The effect of the amphetamine derivatives on the accumulation of DA and 5-HT in striatal slices after administration in vivo has k e n reported previously (Ross & RENYI1975a). Release of 3H-aminesfrom brain slices. The rellease of JH-amines from brain slices previously loaded with these amines was determined. All the amphetamine derivatives tested were very p r releasers crf *H-NA (fig. I), which is in accordance with previous observations for amphetamine (HEIKKILA et al. 1975). Both chlorimipramine and desipramine produced more marked release of NA than 4-chloraamphetamine, the most active amphetamine. However, the difference in potency between the uptake inhibition and release for these tricyclic agents was at least hundredfold. Although the amount of the NA release by the amphetamines was small, several of these compounds, e. g. amphetamine and 3-chloroamphetamine produced significant release of about 10 % at rather low concentrations (5 x l t 7 M ) .

Table 3. Inhibition of the accumulation of JH-dopamine (DA) and 1°C-5-hydroxytryptamine (5-HT) in slices of the mouse brain striatum in vitro. The incubation conditions were the same as in the experiments shown in table 2. Total accumulation in controls (nmol/g): DA: 0.187 f 0.005 (n = 5 3 , 5-HT 0.224 k 0.006 (n = 55) and in the presence of cocaine (3 X 10-4 M): DA: 0.096 k 0.003 (n = 9), 5-HT: 0.075 k 0.006 (n = 9). Compound

IC50, pM (95 % confidence limits) DA 5-HT

5-HT DA

Amphetamine

2.3 (1.6-3.4)

7.6 (3.6-18)

3.3

2-Chloroamphetamine

4.6 (3.0-7.0)

9.9 (4.4-30)

2.2

3-Chloroamphetamine

1.6 (0.8-2.5)

2.2 (0.9-4.1)

1.4

4-Chloroamphetamine

3.0 (1.7-5.1)

1.0 (0.5-1.7)

0.3

4-Methylamphetamine

6.5 (4.5-9.7)

2.3 (1.3-3.8)

0.4

16.4 (9.9-32)

2.5 (1.7-3.5)

0.2

Desipramine

34 (22-53)

11.5 (6.7-17.7)

0.3

Chlorimipramine

22 (17-29)

Chlorphentermine

0.3 (0.09-0.7)

0.01

S. B. ROSS, S.-0. OGREN AND A. L.RENM

346

Table 4. Release of JH-amines from mouse brain slices in vitro. EC50 values (95 % confidence limits) were estimated from dose response curves shown in figs. 1 and 2.

Compound Amphetamine 2-Chloroan~phetamine 3-Chloroamphetamine 4-Chloroamphetamine 4-Methylamphetamine Chlorphentermine Desipramine Chlorimipramine

EC50, pM (95 % conf. limits) Midbrain Striatum NA 5-HT DA 5-HT

> 537 > 485 > 485 > 485 > 541 > 455

227 (186-286) 150 (113-206) 69 (45-114) 56 (31-116) 96 (52-204) 222 (181-281) 58 (44-80) 297 40 (26-69) 17 (10-29)

33 (25-46) 38 (28-52) 20 (13-31) 33 (23-46) 38 (26-58) 115 (77-189) 70 (59-83) 38 (32-44)

126 (109-146) 115 (98-137) 48 (33-72) 33 (20-57) 89 (45-218) 137 (77-305) 52 (45-59) 34 (27-43)

The amphetamine derivatives produced pronounced release of DA from striatal slices (fig. 3). The order of activity was the m e as for the inhibition of the DA accumulation but the EC50 values were about ten times higher than the corresponding IC50 values for the uptake inhibition (table 4). Chlorimipramine and desipramine, on the other hand, released DA at approximately the same concentrations as those which inhibited the DA accumulation. The compounds examined released 5-HT from the slices of the two brain regions with similar patterns (figs. 1 and 3). Thus,4- and 3-chloroamphetamine were the most potent releasers in both regions and chlorphentermine and amphetamine the least active (table 4). Chlorimipramine and desipramine were more or at least as active as the amphetamines in releasing 5-HT under the experimental conditions used. The difference between inhibition of accumulation and 5-HT release was, however, 10-100 times for all drugs except desipramine, for which this difference was less than 10. Examination of the dose response curves reveals that several of the amphetamines have significant releasing effect at rather low concentrations (5 X lo-' M), e. g. 4-methylamphotamine, chlorphentermine, 3- and 4-chloroamphetamine. Chlorimipramine produced a significant release of 5-HT from the midbrain slices at low concentration (3 X lO-'M) but was much less active in the striatal slices (compare figs. 1 and 3). M A 0 inhibition in brain slices after administration in vivo. The deamination of I4C-5-HT, sH-tyramine and SH-phenethylamine by slices of the central part of the mouse brain was determined in analogy

347

BIOCHEMICAL, EFFECTS O F AMPHETAMINES

Table 5. The effects of amphetamine derivatives on the deamination of SH-tyramine and 14C5-HT in midbrain slices from mouse. The simultaneous uptake and deamination of aH-tyramine and W-5-HT in midbrain slices was determined as described in the text. The results are expressed as the mean f S.E. M. of 4 determinations. Control activities (nmol deaminated products/g/5 min.): tyramine: 0.265 k 0.005 (n = 12); 5-HT 0.270 f 0.007 (n = 12). Dose Fmol/kg i. p.

3H-Tyr

Amphetamine

54

10 f 2

0

2-Chloroamphetamine

97 24

25 f 3 6f2

45 f 3 0

3-Chloroamphetamine

97 24

33 f 4 12 f 2

4-Chloroamphetamine

48 24

4-Methylamphetamine

Compound

Chlorphentermine

Inhibition of deamination ED50 pmol/kg i. p. W-5-HT SH-Tyr I4C-5-HT

"/o Inhibition

> 54 > 97

> 54 > 97

22 f 3 11 f 4

> 97

>97

58 f 3 46 f 3

69 f 3 59 f 4

33

19

54 27 12 7

40 f 41 f 33 f 11 f

65 44 35

f3 f2 f2

> 54

33

81

20 f 4

> 81

> 81

3 2 2 1

7f2 46 f 3

with the uptake experiments. After intraperitoneal injection 4-chloroamphetamine was quite potent in inhibiting the deamination of these substrates being somewhat more potent on 5-HT (table 5). It had, however, no effect on the deaminatian of phenethylamine at 48 pmoVkg (0 % inhibition). 4-Methylamphetamine was also a potent inhibitor of the deamination of 5-HT and tyramine but had a flatter dose response curve than 4chloroamphetamine. The other amphetamine derivatives had lesser effect and amphetamine itself was the least active compound examined. Discussion

In accordance with results of several other studies (HEIKKILA et d. 1975; UITERI et d. 1975; HOLMES & RUTLEDGE1976) the amphetamines had more marked effect on the accumulation of NA than in releasing this amine, when examined with in vitro methods. Only a small fraction of the labelled NA accumulated in the slices was released by the amphetamines even at high concentrations. These findings agree with those reported by

348

S . B. ROSS, S.-0. OGREN AND A. L. RENYI

other investigators (HEIKKILA et al. 1975; RAITERIet al. 1975; ENNA& SHORE1974) but are in variance with the results of HOLMES& RUTLEDGE (1976) using chopped rat cerebral cortical tissue. The rather large release of NA observed by the latter authors may be due to the comparatively high concentration of sH-NA (1 x 1CPM) employed in this study, which may have caused unspecific loading of other monoaminergic neurones. However, since a small but significant release of NA was demonstrated in the present study at rather low concentrations of the amphetamines, this release may give a significant effect under in vivo conditioas and be responsible for some behavioural effects. It can be argued that this release occurs from sH-NA accumulated in DA neurones. This suggestion is, however, contradicted by the observation that a low concentration (5 x l e 7 M ) of amphetamine and 3-chloroamphetamine release rather small amounts of DA. Thus, it seems likely that the NA molecules released are derived from NA neurones. If so, the biphasic effect of desipramine on the NA uptake cannolt be explained by a lower affinity on uptake of NA in DA neurone as suggested previously (Ross et al. 1976), since according to this hypothesis these neurones should be responsible for about 60 % oif the NA u p take, and the amphetamines accordingly produce a considerably greater release of NA than that observed. The reason for the dual action of desipramine on the NA uptake in the present experiments is therefore not clear. One possible explanation is that NA accumulation in nerve terminals and in axons and cell bodies is differentially inhibited by desipramine. KEIKKILAet al. (1975) found a close relationship between DA release and inhibition of DA accumulation by amphetamine in striatal slices of the rat brain whereas a tenfold difference was observed in the present study. One explanation for this discrepancy may be that these investigators used a considerably longer incubation time for the accumulation experiments than that used by us, which would accentuate the release component during uptake. Another, perhaps more important, difference in the techniques is that we used larger slices, which retards the diffusion of the amphetamines to the sites for release. The importance of the latter factor is indicated by the observations that higher concentrations of amphetamine were neoessary in our experiments in order to release DA, whereas the concentrations for inhibition of the DA accumulation were similar in the two studies. Experiments with striatal synaptosomes have shown that amphetamine is at least as aative in releasing DA as in inhibiting its uptake (RAITERIet al. 1975; own unpublished olboservatioas). Thus, it is concluded that the ten times difference in potency in the inhibition of accumulation and in the release of DA in the slice experiments may be due to technical reason(s). If this explanation is valid far DA it is also likely for 5-HT. In fact, a recent study on the inhibition by 4-chloroamphetamine

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of 5-HT accumulation and release of 5-HT in synaptosomes of the rat cerebral cortex showed that this derivative was as active in releasing 5-HTas in inhibiting its accumulation (Ross & KELDER 1977). High concentrations of the reference uptake inhibitors desipramine and chlolrimipramine caused release of all three biogenic amines. In fact, the release of DA and 5-HT by desipramine may explain the apparent inhibition of the uptake of these amines. The same explanation could be given for the interaction of chlorimipramine with the DA uptake. On the other hand, inhibition of re-uptake in the release experiments could give a false impression of release. This was not observed for desipramine in the NA experiments and for chlorimipramine in the 5-HT experiments on striatal slices. However, the 'latter compound caused, in the midbrain slices a partial release of 5-HT at lower concentrations, which could be interpreted as the result of inhibition of re-uptake. The observation that amphetamine was more potent in inhibition the accumulation of 5-HT and in release of 5-HT in striatal slices than in midbrain slices may explain the findings of KNAPP et al. (1974) that amphetamine reduces the tryptophan hydroxylase activity in striatum but not in other regions of the rat brain. According to these authors this fall in tryptophan hydroxylase activity might be a receptor mediated feed-back regulation due to the release of 5-HT. The structure activity relationships for the amphetamines in inhibiting the amine accumulation or in releasing the amines were different for 5-HT than for the catecholamines. Thus, para substitution and to a somewhat lesser degree, meta substitution with a chlorine atom increased the potency of the 5-HT neurones without any appreciable change in the activity on the catecholamine neurones. Ortho substitution with a chlorine atom had, however, no effect. Since a methyl group in 4-position had the same effect it seems plausible to assume that the steric change is more important than electronic interactions (Ross et d. 1973). In contrast to the effects on the 5-HT accumulation, the inhibitory activities of the amphetamine derivatives oa the NA accumulation varies much less and amphetamine itself is in vitro the optimal structure. The slight decrease of the activity upon ortho and para substitution may thus be due to steric interactions. Although the pattern of the activities in vivo mainly followed that obtained in vitro there was a tendency for the inhibition of NA uptake to be stronger in vivo than in vitro as compared with the simultaneous inhibition of the 5-HT uptake. This difference may be due to differences in the distribution or retention of the compounds in NA and 5-HT neurones. The high in vivo activity of 4-chloroamphetamine on the NA accumulation compared with that of amphetamine, in spite of the stronger potency

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of the latter campound in vitro, is probably due to the high brain level of 4-chloroamphetamine as reported by several investigators (PLETSCHER et al. 1964; FULLBR et d. 1965). The MA0 inhibition of 4-chloro~phetaminein vivo confirms studies performed in vitro (FULLZR1966; FULLERet al. 1973). 4-Chloroamphetamine was stronger in inhibiting the deamination of 5-HT than tyramine and had a very weak effect on the deamination of phenethylamine. Accordingly it belongs to the class of selective MAO-inhibitors represented by clorgyline which essentially block type A (NEFFet al. 1973). Since both 4-chloroamphetamine and 4-methylamphetamine had rather similar effects on brain M A 0 the marked effects of chlorine or methyl atoms in the para p i t i o n could in analogy with the inhibition of the 5-HT accumulation, depend on increase in the hydrophobic factor rather than electronic interactions. The strong MAO-inhibition by 4-chloroamphetamine in contrast to amphetamine is also probably in part due to its high lipophility and its slow rate of meJtabolism resulting in high brain concentration. In this respect the strong binding of 4-chloroamphetamine to the particular fraction in the neumnes might be important in producing the selectivity for 5-HT neurones (PFEIFERet ul. 1969; WONGet al. 1973). The inhibition of MA0 by the amphetamines was found to be dependent on the position of substitution in the phenyl ring. The order of activity using tyramine as a substrate: para > ortho > meta has also been demonstrated in vitro with trifluommethyl substituted amphetamines (BEREGIet d. 1970). Acknowledgement We thank Mrs. Anna-Lena Ask for skilful technical assistance, and the donors of the compounds investigated.

REFERENCES Beregi, L. G., P. Hug- J. C. Le Douarec, M. Laubic & J. Duhault: Structure-activity relationships in CP, substituted phenethylamines. In: Amphetamines and related compounds. Proceedings of the Mario Negri institute for Pharmacological Research. Eds.: E. Costa & S. Garattini. Raven Press, New York, 1970, pp. 21-61. Carlsson, A.: Amphetamine and brain catecholamines. In: Amphetamines and related compounds. Proceedings of the Mario Negri institute for Pharmacological Research. Eds.: E. Costa & S. Garattini. Raven Press, New York, 1970, pp. 289-300. Dengler, H. J., H. E. Spiegel & E. 0. Titus: Effects of drugs on uptake of isotopic norepinephrine by cat tissue. Nature (Lond.) 1961, 191, 816-817. E m , S. J. & P. A. Shore: Differences in m i n e storage in rat heart and brain. Brit. J . Pharrnacol. 1974,50,271-276. Finney, D. J.: Probit analysis, 2nd ed. Charles Griffin, London, 1952.

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Frey, H. H. & M. P. Magnussen: Different central mediation of the stimulant effects of amphetamine and its p-chloro analogue. Biochem. Pharmacol. 1968, 17, 12991307.

Fuller, R. W.: Serotonin oxidation by rat brain monoamine oxidase and inhibition by 4-chloroamphetamine. Life Sci. 1966, 5, 2247-2252. Fuller, R. W., C. W. Hines & J. Mills: Lowering of brain serotonin level by chloroamphetamines. Biochem. Pharmacol. 1965, 14, 483-488. Fuller, R. W., H. D. Snoddy, B. W. Roush & B. B. Molloy: Further structure activity studies on the lowering of brain 5-hydroxy-indoles by 4-chloroamphetamine. Ncuropharmacol. 1973, 12, 33-42. Glowinski, J. & J. Axelrod: Effects of drugs on the uptake, release and metabolism of Hs-norepinephrine in the rat brain. J . Pharmacol. exp. Therap. 1965, 149, 43-49. Hansson, L. C. F.: Evidence that the central action of amphetamine is mediated via catecholamines. Psychopharmacologia (Berl.) 1967, 10, 289-297. Heikkila, R. E., H. Orlansky & G. Cohen: Studies on the distinction between uptake inhibition and release of [JH] dopamine in rat brain tissue slices. Biochem. Pharmacol. 1975, 24, 847-852. Holmes, J. C. & C. 0. Rutledge: Effects of the d- and 1-isomers of amphetamine on uptake, release and catabolism of norepinephrine, dopamine and 5-hydroxytryptamine in several regions of rat brain. Biochem. Pharmacol. 1976, 25, 447-451. Horn, A. S., J. T. Coyle & S. H. Snyder: Catecholamine uptake by synaptosomes from rat brain. Mol. Pharmacol. 1971, 7, 66-80. Knapp, S., A. J. Mandell & M. A. Geyer: Effects of amphetamines on regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain. J . Pharmacol. exp. Therap. 1974, 189, 6 7 M 8 9 . Knoll, J., K. Magyar, E. S. Vizi, T. Torok, 8. Sdtory & G. J6na: The role of brain serotonin in the pharmacological effects of p-bromomethamphetamine (V-111). In: Symposium on pharmacological agents on biogenic amines in the central nervous system. Eds.: J. Knoll & K. Magyar. Akad6miai Kiad6 (Budapest) 1973, pp. 13-36. Neff, N. H., H.-Y. T. Yong & J. A. Fuentes: The use of selective monoamine oxidase inhibitor drugs to modify amine metabolism in brain. In: Advances in biochemical psychopharmacology, vol. 21. Ed.: E. Usdin. Raven Press, New York, 1973, pp. 49-57. Ogren, S.-0. & S. B. Ross: Substituted amphetamine derivatives. II. Behavioural effects in mice related to the biogenic monoamines. Acta pharmacol. et toxicol. 1977, 41, 353-368. Pfeifer, A. K., L. CsBki, M. Fodar, L. Gyorgy & I. Okros: The subcellular distribution of (+)-amphetamine and ( 2 )-p-chloroamphetamine in the rat brain as influenced by reserpine. J . Pharm. Pharmacol. 1969, 21, 687489. Pletscher, A., G. Bartholini, H. Bruderer, W. P. Burkard & K. F. Grey: Chlorinated arylalkylamines affecting the cerebral metabolism of 5-hydroxytryptamine. J . Pharmacol. exp. Therap. 1964, 145, 344-350. Raiteri, M., A. Bertollini, F. Angelini & G. Levi: d-Amphetamine as a releaser or reuptake inhibitor of biogenic amines in synaptosomes. Eur. J. Pharmacol. 1975, 34, 189-195. Ross, S . B. & D. Kelder: Efflux of 5-hydroxytryptamine from synaptosomes of rat cerebral cortex. Acta physiol. scand. 1977, 99, 27-36. Ross, S. B. & A. L. Renyi: Blocking action of sympathomimetic amines on the uptake of tritiated noradrenaline by mouse cerebral cortex tissue in vitro. Acta pharmacol. et toxicol. 1964, 21, 226-239.

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Ross, S. B. & A. L. Renyi: Inhibition of the uptake of tritiated catecholamines by antidepressant and related drugs. Eur. J. Pharmacol. 1967, 2, 181-186. Ross, S. B. & A. L. Renyi: Inhibition of the uptake of tritiated 5-hydroxytryptamine in brain tissue. Eur. J . Pharmacol. 1969, 7 , 270-277. Ross, S. B. & A. L. Renyi: Inhibition of the uptake of 3H-doparnine and I4C-5hydroxytryptamine in mouse striatum slices. Acta pharmacol. et toxicol. 1975a, 36, 56-66. Ross, S. B. & A. L. Renyi: Tricyclic antidepressant agents. I. Comparison of the inhibition of the uptake of aH-noradrenaline and 14C-5-hydroxytryptamine in slices and crude synaptosome preparation of the midbrain hypothalamus region of the rat brain. Acta pharmacol. toxicol. 1975b, 36, 382-394. Ross, S. B., A. L. Renyi & S . - 0 . Ogren: Inhibition of the uptake of noradrenaline and 5-hydroxytryptamine by chlorphentermine and chlorimipramine. Eur. J . Pharmacol. 1972 17, 107-112. Ross, S. B., A. L. Renyi & S.-0. Ogren: On the inhibitory effect of various phenylalkylamine derivatives on the uptake of noradrenaline and 5-hydroxytryptamine in brain tissue, In: Symposium on pharmacological agents on biogenic amines in the central nervous system. Eds.: J. Knoll & K. Magyar. Akadimiai Kiadb (Budapest) 1973, pp. 45-58. Ross, S. B., S.-0. Ogren & A. L. Renyi: (Z)-Dimethylamino-l-(4-bromophenyl)-l(3-pyridy1)prapene (H 102/09), a new selective inhibitor of the neuronal 5-hydrmytryptamine uptake. Acta pharmacol. et toxicol. 1976, 39, 152-166. Sanders-Bush, E., J. A. Bushing & F. Sulser: p-Chloroamphetamine in inhibition of cerebral tryptophan hydroxylase. Biochem. Pharmacol. 1972, 21, 1501-1510. Snyder, S. H. & J. T. Coyle: Regional differences in sH-norepinephrine uptake into rat brain homogemates. J . Pharmacol. exp. Therap. 1969, 165, 78-86. Weissmann, A,, B. K. Koe & S. S. Tenen: Antiamphetamine effects following inhibition of tyrosine hydroxylase. J . Pharmacol. exp. Therap. 1966, 151, 339-353. Wong, D. T., J.-S. Horng & R. W. Fuller: Kinetics of serotonin accumulation into synaptosomes of rat brain-effects of amphetamine and chloroamphetamine. Biochem. Pharmacol. 1973, 22, 311-322.

Substituted amphetamine derivatives. I. Effect on uptake and release of biogenic monoamines and on monoamine oxidase in the mouse brain.

Acta pharmacol. et toxicol. 1977, 41, 337-352. From the Research and Development Laboratories, Astra Lakemedel AB, S-15185 Sodertalje, Sweden Substi...
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