Life Sciences Vol. 20, pp . 2093-2100, 1977 dPrinted In The U.S .A .

Pergamon Press

INHIBITION BY ß-CARBOLINES OF MONOAMINE UPTAKE INTO A SYNAPTOSOMAL PREPARATION: STRUCTURE-ACTIVITY RELATIONSHIPS N.S . Buckholtz and W .O . Boggan Department of Biochemistry and Department of Psychiatry and Behavioral Science Medical University of South Carolina Charleston, S .C . 29401 (Received in final form May 23, 1977) SUMMARY The potency of a series of 0-carboline compounds to inhibit 3H-serotonin ( 3H-5-HT) uptake into a synaptosomal suspension from mouse brain was studied . The in vitro structure-activity study showed the tetrahydro-ß-carbolines to be the most potent inhibitors compared to unsaturated ß-carbolines . _In vitro inhibition of 3H-norepinephrine (3H-NE) and 3H-dopamine (rH-DA) uptake was determined for some tetrahydro-$-carbolines, and the degree of inhibition of uptake of these amines was less than that for 3H-5-HT (EC50s being 5-13 times those for 3H-5-HT) . The tetrahydro-ßcarbolines were also found to effectively inhibit 3H-5-HT but not 3H-NE or 3H-DA uptake when they were administered intra-peritoneally . These results suggest that the behavioral effects of the tetrahydro-ß-carbolines which have been reported previously may be due to a relatively selective involvement of the serotonergic neurotransmitter system . Although a number of the tricyclic ß-carbolines have been known for some time to produce hallucinations when ingested by man (1), their possible neurobiological effects have recently received attention because a number of groups have demonstrated that enzymatic preparations from brain can convert indoleamines to tetrahydro-ß-carbolines in the presence of 5-methyl-tetrahydrofolic acid (2-8) . The possibility thus exists that 0-carbolines can be formed in vivo and thus might have a neuroregulatory role . In support of this, McIsaac _et _al . (9) reported that 6-methoxy-1,2,3, 4-tetrahydro-ß-carboline (6-Me0-THBC, an alternative name is 6-methoxy-tetrahydronorharman) elevated brain concentration of serotonin (5-hydroxytrypt amine, 5-HT) without affecting the concentrations of 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, or norepinephrine (NE) . Three possible ways in which 6-MeO-THBC could produce this effect are increased activity of the synthetic enzyme tryptophan hydroxylase, inhibition of monoamine oxidase (MAO), an enzyme which degrades 5-HT, and/or inhibition of synaptosomal uptake of 5-HT, a system which terminates the action of 5-HT . Although 6-MeOTHBC has no effect on tryptophan hydroxylase (10), it does inhibit MAO and 5-HT uptake (11,12) . In the present study, we were interested in further characterizing the uptake inhibition by 1. examining structure-activity relationships among the 0-carbolines and 2 . determining whether the inhibition was selective for 5-HT or also involved the catecholamines NE and dopamine (DA) .

2093

2094

B-Carboline Inhibition of Amine Uptake

Vol . 20, No . 12, 1977

METHODS Female CFl mice, 50-80 days old, were used in all experiments . Animals were decapitated, and whole brains were homogenized in 10 vol of ice-cold 0 .25 M sucrose using a Kontes Potter-Elevehjem homogenizer with a Teflon pestle (model K-886000, 0.004-0 .006 in . clearance) . Synaptosomal uptake of radioactively labeled 5-HT, NE, and DA was determined by the method of Kuhar _et al . (13) . Originally, a final 3 H-5-HT concentration of 3 .0 nM was used when comparing the compounds to be sure inhibition of the high affinity uptake system was being determined . When inhibition of 3H-5-HT uptake was compared with that of 3H-NE and 3H-DA, a concentration of 0 .1 PM for all three amines was used . The homogenate was centrifuged for 10 min at 1000 x g . The pellet was discarded, and the supernatant, referred to as the synaptosomal suspension, was gently vortexed just before use. In the in vitro studies, the final volume of 4 .0 ml contained 1.0 ml of drug in buffer, 2 .6 ml of buffer, 0 .3 ml synaptosomal suspension, and 0 .1 ml isotopic compound . In the in vivo studies, 3 .6 ml buffer, 0.3 ml synaptosomal suspension, and 0 .1 ml isotopic compound were used . The Krebs-Ringer phosphate buffer (14) was made fresh daily, was gassed for 10 min with 95X02 - 52CO2 and contained glucose (2mg/ml), ascorbic acid (0 .2 mg/ml), EDTA (0 .06 mg/ml) and ' nialamide (final concentration of 7 .5 x 5 M) .

10

Samples were kept in 15 ml Corex tubes in ice until transferred to a Dubnoff metabolic shaker where they were preincubated for 5 min at 37 0C . The radioactive amine was added, and incubation continued for 4 min, after which the tubes were removed to an ice bath . Blanks consisted of duplicate samples kept in the ice bath throughout the entire procedure . Tubes were transferred to a Sorvall RC-5 centrifuge and centrifuged at 10,000 x g for 15 min. The incubation medium was discarded and the pellet was washed with 4 .0 ml of cold 0.9% sodium chloride which was then discarded . One ml of solubilizer (Unisol, Isolab, Inc .) was added to each tube . After the pellet was dissolved, 0.5 ml of methanol followed by 10 .0 ml of Unisol Complement were added, and the contents of the tube were transferred to a scintillation vial . Radioactivity was measured in a Beckman LS-350 liquid scintillation spectrometer . The sources of 0-carbolines (Fig .l) which were purchased were the following : Sigma Chemical Co .--harman HCl, 6-methoxy-harman, 6-methoxytetrahydroharman, norharman HC1, THBC HC1 (as noreleagnine HC1) ; Regis Chemical Co .--6-methoxy-harmalan, 6-hydroxy-tetrahydroharman, harmine HC1, harmaline HC1 dihydrate tetrahydroharmine (as leptaflorine), harmol HC1, harmalol HC1 ; ICN Pharmaceuticals--tetrahydroharman, tetrahydroharmol . 6-MeOTHBC HC1 was synthesized according to the method of Ho et _al. (15) . The following drugs were gifts from their respective sources : imipramine and chlorimipramine (Ciby-Geigy), Lilly 110140 (Lilly Research Laboratories)and benztropine mesylate (Merck, Sharp, and Dohme) . Radioactive amines purchased from New England Nuclear were 5-hydroxytryptamine binoxalate [1,2-3H(N)] (5 .7 or 21 .4 Ci/mmole) ; DL-norepinephrine, L-bitartrate salt [7-3H (N)] (13 .1 Ci/mmole) ; 3 4-dihydroxyphenylethylamine (dopamine) [ 3H(G)] (9 .3 Ci/mmole) . 5-Hydroxy [G- 5H] tryptamine creatinine sulfate (16 .1 Ci/mmole) was purchased from Amersham/Searle. RESULTS Table 1 shows the data from the structure-activity relationships for amine uptake inhibition in vitro . With respect to inhibition of 3H-5-HT uptake, for the unsaturated compounds, harman was the least potent inhibitor, and either addition of a methoxy group at C-6 to form 6-MeO-harman or the loss of a methyl group at C-1 to give norharman increased the amount o£ inhibition,

Vol . 20, No . 12, 1977

ß-Carboline Inhibition of Amine Uptake

,4-

2095

CARROUNE

HARMAN 6-MEMIOXY- HARMAN NORNARMAN HA IMINE NARMOL

CH3

H

CH3

H

01,

H

H

CH3 OCH 3 H H

H H OCH 3 ON

3.6-DRIYDRO-A - CARROLRIE COMrOUNO

R,

R,

6-METHOXY-HARMALAN HARMALINE

CHL3

OCH3

H

CH,

OCT{T

HARMALOL

013

H H

1,7,9,!-TETRAHYDRO-n

OH

-CARROLRIE

COMPOUND 6-METHOXY-TETRAwDRO-ii -CARROLM (6-METNOXY-TETRIW YDRONOMIARMAN) TEMAHYDRONORHARMAN

R,

R,

R,

R,

H

OCH,

H

H

H

H

1ETRAHYDROHARMAN

CH3

H

H

6-METHOXY-TElIUUIYDROHARMAN

CH3 OCH-j H CH, OH H CH3 H OCH3 CH3 H OH

6-HYDROXY-TETRAHYDROHARMAN TEMAHYDROHARMINE TETRAHYDROHMMOl

FIG. 1. Structures of ß-carbolines . Ring saturation led to greater with the latter having the greater effect . inhibition . Tetrahydroharman (THH) and 6-MeO-THH were of approximately Loss of equal inhibitory potency whereas 6-OH-THH was somewhat more potent . a methyl group at C-1 led to still greater inhibition, with 6-MeO-THBC being Of interest also is the fact that tetrahydroharmine more potent than THBC . was more potent than harmine or harmaline which were approximately equipotent . Thus, for the compounds with a methyl group at C-1, there seems to be no difference between a methoxy group at C-6 vs . C-7 (e .g . 6-Mè0-harman vs . harmine, 6-MeO-harmalan vs . harmaline, 6-MeO-THH vs . tetrahydroharmine) . Harmol, harmalol, and tetrahydroharmol were of approximately equal potency. An hydroxy group at C-7 did lead to a reduction in the inhibitory potency in relation to C-6 (e .g . tetrahydroharmol vs . 6-OH-THH) . The tetrahydro-ßcarbolines were all more selective inhibitors of 5-HT than NE or DA uptake, whereas the reverse was true for harmine. Fig. 2 shows the time and dose effects of i .p . injection of 6-MeO-THBC on 5-HT uptake . Inhibition of uptake remained high for 4 hr ., declined someThere was a dose response what by 8 hr ., and returned to baseline by 12 hr . effect of 6-MeO-THBC on 5-HT uptake at one hour . Table 2 shows the in vivo amine uptake inhibition produced at 1 hr . by the tetrahydro-ß-carbolines and reference compounds . For inhibition of 3H-5-HT uptake, chlorimipramine and Lilly 110140 produced the greatest inhibition, thus paralleling their in vitr o effects . The THBC compounds were less effective, followed by the tetrahydro6-OH-THH, however, produced very little inhibition as harman compounds . compared to 6-MeO-THH, which was different from the equipotency shown in vitro (Table 1) . 6-MeO-THBC and Lilly 110140 showed little or no in vivo inhibition of NE or DA uptake . Benztropine, however, was effective at inhibiting uptake of both NE and DA, but not 5-HT .

2096

ß-Carboline Inhibition of Amine Uptake

Vol . 20, No . 12, 1977

TABLE 1 In Vitro Inhibition by Drugs of 3H-Serotonin, 3H-Norepinephrine, and 3HDopamine Uptake into a Synaptosomal Suspension from Mouse Brain

EC 50 (PM)

Drug

3H-Serotonin 3H-Serotonin 3H-Norepinephrine (3 .0 nM) (0 .1 PM) (0 .1 PM)

Harman 6-MeO-Harman 6-MeO-Harmalan Tetrahydioharman 6-MeO-Tetrahydroharman 6-OH-Tetrahydroharman Norharman THBC 6-MeO-THBC Harmine Harmaline Tetrahydroha%mine Harmol Harmalol Tetrahydroharmol Lilly 110140 Chlorimipramine Imipramine Benztropine

3H-Dopamine (0 .1 VM)

34 24 22 2 .8 3 .2 1 .4 18 1 .3 0 .63 11 15

22

>100

>100

4 .8 3 .6 41

34 49 22

>100 16

0 .24 0.064 0.42 50

5 .6 9 10 0 .16

49

3 .4 17 13 21 0 .025 0 .0049 0 .1 24

14 18 22 0 .14

Data (EC50) are expressed as concentration of drug which inhibited 3H-amine uptake by 50% as determined from semi-log plots of inhibitor concentration Incubation was carried out as described vs . % inhibition of 3H-amine uptake . in the text . Each value represents the mean of four to eight determinations . The means ±S .E .M . control values ( R 10-15 mole/4 min/2 .2 mg protein) r 3H-amine uptake were as follow : 3 H-serotonin (3 .0 nM), 1025 t 37 ; 3 H-dopamine, serotonin (O .lPM), 1016 t 26 ; H-norepinephrine, 876 ± 54 ; 1846 t 109 . DISCUSSION The structure-activity study for inhibition of 5-HT uptake showed the For the compounds tetrahydro-ß-carbolines to be the most potent inhibitors . with a methyl group at C-1, there was not much effect of addition of a methoxy group at C-6 (e .g . THH vs 6-MeO-THH) whereas hydroxy substitution led to This latter compound did not differ somewhat more inhibition (e .g . 6-OH-THH) . from THBC . Addition of a methoxy group at C-6 to THBC to form 6-MeO-THBC Tuomisto and Tuomisto, (16) using did lead to greater inhibition, however . rat brainatem, also found that 6-OH-THH was a somewhat better inhibitor than THH. Kellar et al . (12) have recently confirmed this ordering of inhibitory

O-Carboline Inhibition of Amine Uptake

Vol . 20, No . 12, 1977

2097

100 90

Y 0

â

so

ä

t 100 mg/kg 70- (4) (7)~ 50 -9/kg

P x .

s

a X

60-

so-

FIG . 2 . Inhibition of 3H-serotonin uptake into a synaptosomal suspension from mouse brain at various times after i .p . injection of various doses of 6-MeO-THBC . Incubation was carried out as described in the text . Values are means ± S .E .M . from four to eight separate determinations (number of determinations given in parentheses) . S .E .M .s less than 1 .0% are not shown. The mean ± S .E .M . control value of 3H-serotonin uptake was 1054 ± 19 x 10 - mole/4 min/2 .2 mg protein .

(4) ~ ss mg/kg

4030s0-

(4)

1

12 .5 mgAg

1

P

10 . 3

4

id

8

dc

i (4) 12

T1~ cher 6aMe0-THIC Administration (Hr)

TABLE 2

In Vivo Inhibition by Drugs of 3H-Serotonin, 3H-Norepinephrine, and 3H-Dopamine Uptake into a Synaptosomal Suspension from Mouse Brain

% Inhibition Drug

Dose (mg/kg)

Tetrahydroharman 6-MeO-Tetrahydroharman 6-OH-Tetrahydroharman THBC 6-MeO-THBC Lilly 110140 Chlorimipramine Benztropine

3H-Serotonin (3 .0 nM)

50

58 .0 ± 1 .5

50

31 .3 ± 2 .5

50 50 50 10 25 25

7 .7 68 .0 66 .0 88 .0 94 .3 0.0

± ± ± ± ± ±

3 .6 1 .3 3 .1 0.0 0 .2 0.0

3 H-Serotonin

(0 .1 uM)

42 .2 ± 3 .7 53 .1 ± 6 .0 5 .8 ± 5 .8

3H-Norepi-

nephrine (0 .1 11M)

11 .2 ± 3 .9 0 .0 t 0 .0

3 H-Dopa-

mine (0 .1 11M)

0 .0 ± 0.0 0 .0 ± 0.0

66 .5 ± 2 .8 45 .8 ± 3.0

Mice were killed 1 hr . after the i .p . injection of the drugs (doses given as Numbers in the salts) . Incubation was carried out as described in the text . table are mean values t S .E .M . from 4-7 separate determinations . The mean ± S .E .M . control values (X 10-15 mole/4 min/2.2 mg protein) for 3H-amine uptakes were as follows : 3H-serotonin (3 .0 nM), 1054 t 19 ; 3H-serotonin (0 .1 vM), 290 t 6 ; 3H-norepinephrine, 767 ± 26 ; 3H-dopamine, 855 ± 34 .

2098

ß-Carboline Inhibition of Amine Uptake

Vol . 20, No . 12, 1977

potency of these compounds using rat forebrain. In addition, they also found that 6-OH-THBC was an even more potent inhibitor than 6-MeO-THBC . Thus, in terms of structure-activity, the beat inhibition of 5-HT uptake is associated with a saturated compound lacking a methyl group at C-1 and having a methoxy or hydroxy group at C-6 (i .e . 6-MeO-THBC and 6-OH-THBC) . None of the ß-carbolines, however, was as effective an inhibitor of 5-HT uptake as were Lilly 110140 (also shown by Kellar _et _al . (12)), chlorimipramine, or imipramine . The present study showed that the tetrahydro-ß-carbolines were less potent inhibitors of NE or DA uptake than of 5-HT uptake . This has also been shown by Kellar _et _al. (12) for 6-OH-THH and 6-OH-THBC and by Tuomisto and Tuomisto (16) for THH and 6-OH-THH . In addition, Lilly 110140 was shown to be a much more potent inhibitor of 5-HT than of NE or DA uptake as was originally reported by Wong et al .(17) in whole rat brain and confirmed by Kellar _et _al. (12) This same relative selectivity was also found for chlorimipramine and imipramine, as has been reported previously in rat brain (17) and rat hypothalamus (18) . Benztropine was a more potent inhibitor of catecholamine uptake as was reported previously in rat striatum (18,19) and rat occipital and frontal cortices, hippocampus and whole forebrain (20) . We have recently completed a study of the structure-activity relationship of 9-carbolines to MAO inhibiting activity (21) . It is of interest to note the structure-activity relationship for MAO inhibitory activity was approximately the reverse of that for 5-HT uptake inhibitory activity . For example, the EC50's (liM) for _in vitro MAO inhibition were 0.08 for harmine, 3 .3 for harman, and 58 for 6-Me0-THBC, whereas for 5-HT uptake inhibition they were 11 for harmine, 34 for harman, and 0.63 for 6-MeO-THBC . Thus the effects of ß-carbolines on MAO inhibition and on 5-HT uptake inhibition seem to be dissociable . We have previously shown that 6-MeO-THBC has _ in vivo activity in relationship to seizures (22), one-trial passive avoi dance (23), and plasma corticosterone elevation (24), and Holman _et _al . (25) have shown that 6-OH THH produced hyperactivity when given with an MAO inhibitor . Thus it was of interest to determine the effects of the tetrahydro-ß-carbolines when administered in vivo . 6-Me0-THBC has a rapid onset in terms of 5-HT uptake inhibition and the inhibition was elevated for at least 8 hr . (Fig .2) . The other tetrahydro-ß-carbolines also inhibited 5-HT uptake at 1 hr ., although 6-OH-THH produced less than 10% inhibition (Table 2) . in vivo data The _ at 1 hr . after injection for comparison of 5-HT with catecho lamine uptake inhibition paralleled that found in vitro ; i .e ., 6-Me0-THBC and Lilly 110140 were much more effective inhibitors for 5-HT than for catecholamine uptake and the reverse was true for benztropine (Tables 1 and 2) . It should be noted, however, that the one hour time point chosen for comparison was based upon optimal inhibition of 5-HT uptake (Fig . 2), and may not be the time at which optimal inhibition of NE or DA uptake occurs . Thus, the tetrahydro-ß-carbolines do seem to,act relatively selectively on the serotonergic neurotransmitter system . This is of interest for further behavioral, neurochemical, and neuroendocrinological work with these com pounds . For example, Holman et .al . (25) were able to block the hyperactivity produced by 6-OH-THH and tranylcypromine with the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), and we found that the increase in plasma corticosterone produced by 6-MeO-THBC could be facilitated by Lilly 110140 and partially blocked by PCPA (Meyer, Buckholtz, and Boggan, unpublished observations) . Effects of tetrahydro-ß-carbolines on serotonergic neurotransmitter activity are also of interest in terms of the role they may play if they are formed in vivo . The presence of 6-MeO-THBC in rat brain

Vol. 20, No . 12, 1977

has been reported

8-Carboline Inhibition of Amine Uptake

2099

(26) . ACKNOWLEDGEMENTS

We wish to thank Mrs . Mona Krall for her excellent technical assistance . This research was supported in part by P .H .S . Grants MH-26712 (N .S .B .) and DA-01035 (W .O .B .) . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .

R.E . Schultes and A. Hofmann, The Botany and Chemistry of Hallucinogens , (pp . 101-112) Charles C. Thomas, Springfield (1973) . R.J . Wyatt, E. Erdelyi, J .R . Do Amaral, G .R . Elliott, J. Renson and J .D . Barchas, Science 187, 853-855 (1975) . H. Rosengarten, E . Meller and A.J . Friedhoff . Biochem . Pharmacol . _24, 1759-1762 (1975) . L.L . Hsu and A.J . Mandell, _J . Neurochem . _24, 631-636 (1975) L.L . Hsu and A .J . Mandell, Res . Comm . Chem . Path . Pharmacol . 12, 355362 (1975) . L.R . Mandel, A. Rosegay, R.W . Walker, W .J .A . Vander Heuvel and J .Rokach, Science _ 186, 741-743 (1974) . H. Romme lspacher,H . Coper and S . Strauss, Life Sci . _18, 81-88 (1976) . L.L . Hsu, Life Sci . 19, 493-496 (1976) . W.M . McIsaac, D. Taylor, K.W . Walker and B .T . Ho, _J . Neurochem . _19, 1203-1206 (1972) . B.T . Ho, D . Taylor, K .E . Walker and W .M . McIsaac, _Can ._J . Biochem ._51, 482485 (1973) . N .S . Buckholtz and W.O . Boggan, Biochem . Pharmacol . 25,2319-2321 (1976) . K.J . Kellar, G .R . Elliott, R.B . Holman, J.D . Barchasand J . VernikosDanellis, _J . Pharmacol. Exp . Ther . 198, 619-625 (1976) . M.J . Kuhar, R.H . Roth and G .K . Aghajanian, _J . Pharmacol . Exp . Ther . 181, 36-45 (1972) . R.M .C . Dawson, D.C . Elliott, W.H . Elliott and K .M . Jones, Data for Biochemical Research , second edition, (P .507) Oxford University Press, New York (1969) . B .T . Ho, W.M . McIsaac, K.E . Walker and V . Estevez, _J . Pharm . Sci . 57, 269-274 (1968) . L. Tuomisto and J. Tuomisto, Naun n-Schmiedeberg's Arch . Pharmacol . 279, 371-380 (1973) . D . T . Wong, J.S . Horng, F .P . Bymaster, K.L . Hauser and B .B . Malloy, Life _Sci . _ 15, 471-479 (1974) . E. G. Shaskan and S .H . Snyder, _J . Pharmacol . Exp. Ther . 175, 404-418 (1970) . J. T. Coyle and S . H. Snyder, Science 166, 899-901 (1969) . R.F . Squires, _J . Pharm. Pharmacol. 26, 364-367 (1974) . N.S . Buckholtz and W.O . Boggan, Biochem . Pharmac ., in press (1977) . N.S . Buckholtz, Pharmacol . Biochem. Behav . 3, 65-68 (1975) . N.S . Buckholtz, Belle a~ol . 14, 95-YO 1(1175) . J. S. Meyer and N .S . Buckholtz, _Res . Comm . Chem . Path . Pharmacol . _14, 205-213 (1976) . R.B . Holman, E . Seagraves, G.R . Elliott and J . D . Barchas, Behav . Biol . 16, 507-514 (1976) D . W. Shoemaker and J. T. Cummins, Fed . Proc . 35, 1476 (1976) .

Inhibition by beta-carbolines of monoamine uptake into a synaptosomal preparation: structure-activity relationships.

Life Sciences Vol. 20, pp . 2093-2100, 1977 dPrinted In The U.S .A . Pergamon Press INHIBITION BY ß-CARBOLINES OF MONOAMINE UPTAKE INTO A SYNAPTOSOM...
371KB Sizes 0 Downloads 0 Views