Buckholtz1977

Inhibition by β-carbolines of monoamine uptake into a synaptosomal preparation: Structure-activity relationships. Buckholtz, N. S., Boggan, W. O. 1977. Life Sciences, 20(12), 2093–2100. 10.1016/0024-3205(77)90190-4

Charleston, S.C. 29401

(Received in final form May 23, 1977)

The potency of a series of β-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-β-carbolines to be the most potent inhibitors compared to unsaturated β-carbolines. In vitro inhibition of ³H-norepinephrine (³H-NE) and ³H-dopamine (³H-DA) uptake was determined for some tetrahydro-β-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-β-carbolines 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-β-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 β-carbolines have been known for some time to produce hallucinations when ingested by man (1), their possible neuropharmacological effects have recently received attention because a number of groups have demonstrated that enzymatic preparations from brain can convert indoleamines to tetrahydro-β-carbolines in the presence of 5-methyl-tetrahydrofolic acid (2-8). The possibility thus exists that β-carbolines can be formed in vivo and thus might have a neuroregulatory role.

In support of this, McIssac et al. (9) reported that 6-methoxy-1,2,3,4-tetrahydro-β-carboline (6-MeO-THBC; an alternative name is 6-methoxy-tetrahydronorharman) elevated brain concentration of serotonin (5-hydroxytryptamine; 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-MeO-THBC 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 β-carbolines and 2. determining whether the inhibition was selective for 5-HT or also involved the catecholamines NE and dopamine (DA).

Female CF1 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-Elvehjem 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 ³H-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 µM 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 95%O2 - 5%CO2 and contained glucose (2mg/ml), ascorbic acid (0.2 mg/ml), EDTA (0.06 mg/ml) and nialamide (final concentration of 7.5 x 10^-5 M).

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°C. The radioactive amine was added, and incubation continued for 4 min, after which the tubes were removed to an 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 β-carbolines (Fig.1) which were purchased were the following: Sigma Chemical Co.—harman HCl, 6-methoxy-harman, 6-methoxy-tetrahydroharman, norharman HCl, THBC HCl (as noreleagnine HCl); Regis Chemical Co.—6-methoxy-harmanol, 6-hydroxy-tetrahydronorharman, harmine HCl; ICN Pharmaceuticals—tetrahydroharman, tetrahydroharmol. 6-MeO-THBC HCl was synthesized according to the method of Ho et al. (13). The following drugs were gifts from their respective sources: Imipramine and desmethylimipramine (Geigy); chlorpromazine (Smith, Kline and French); and benzotropine mesylate (Merck, Sharp, and Dohme). Radioactive amine purchased from New England Nuclear were 5-hydroxytryptamine binoxalate [1,2-³H(N)] (5.7 or 21.4 Ci/mmol); DL-norepinephrine, L-bitartrate salt [7-³H (N)] (13.1 Ci/mmol); 3,4-dihydroxyphenylethylamine (dopamine) [³H(G)] (9.3 Ci/mmol). 5-Hydroxy [G-³H] tryptophan creatinine sulphate (16.1 Ci/mmol) was purchased from Amersham/Searle.

Table 1 shows the data for 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 of inhibition.

β-CARBOLINE

COMPOUND	R1	R2	R3
---	---	---	---
Harman	CH3	H	H
6-Methoxy-harman	CH3	OCH3	H
Harmaline	CH3	H	OCH3
Harmol	CH3	H	OH

3,4-DIHYDRO-β-CARBOLINE

COMPOUND	R1	R2	R3
---	---	---	---
6-Methoxy-harmalan	CH3	OCH3	H
Harmalol	CH3	H	OH

1,2,3,4-TETRAHYDRO-β-CARBOLINE

COMPOUND	R1	R2	R3
---	---	---	---
6-Methoxy-1,2,3,4-tetrahydro-β-carboline (6-MeO-THBC)	CH3	OCH3	H
Tetrahydroharman (THH)	CH3	H	H
6-Methoxy-tetrahydroharman	CH3	OCH3	H
Tetrahydroharmol	CH3	H	OH
6-Hydroxy-tetrahydroharman	CH3	OH	H
Tetrahydroharmine	CH3	H	OCH3
Tetrahydroharmalol	CH3	H	OH

FIG. 1. Structures of β-carbolines.

With the latter having the greater effect. Ring saturation led to greater inhibition. Tetrahydroharman (THH) and 6-MeO-THH were more approximately equal inhibitory potency, whereas 6-OH-THH was somewhat more potent. Loss of a methyl group at C-1 led to still greater inhibition, with 6-MeO-THBC being more potent than THBC. Of interest also is the fact that tetrahydroharmine was more potent than harmine or harmaline, which were approximately equal potency. 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-MeO-harman vs. harmine, 6-MeO-TRH vs. tetrahydroharmine). Harmol, harmalol, and tetrahydroharmol were of approximately equal potency. A 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-β-carbolines were all more selective inhibitors of 5-HT than NE or DA uptake, whereas the reverse was true for harmine.

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

EC 50 (µM)
------------	----------------	---------------------	----------------
Drug	³H-Serotonin (3.0 nM)	³H-Serotonin (0.1 µM)	³H-Norepinephrine (0.1 µM)	³H-Dopamine (0.1 µM)
------------	----------------	---------------------	----------------	----------------
Harman	34
6-MeO-Harman	24
6-MeO-Harmalan	22
Tetrahydroharman	2.8
6-MeO-Tetrahydroharman	3.2	22	>100	>100
6-OH-Tetrahydroharman	1.4
Norharman	18
THBC	1.3	4.8	34	49
6-MeO-THBC	0.63	3.6	49	>100
Harmine	11	41	22	>16
Harmaline	15
Tetrahydroharmine	3.4
Harmol	17
Harmalol	13
Lilly 110140	0.025	0.24	5.6	14
Chlorimipramine	0.0049	0.064	9	18
Imipramine	0.1	0.42	10	22
Benztropine	24	50	0.16	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 vs. % inhibition of ³H-amine uptake. Incubation was carried out as described in the text. Each value represents the mean of four to eight determinations. The means ± S.E.M. control values (X 10^-15 mole/4 min/2.2 mg protein) for ³H-amine uptake were as follows: ³H-serotonin (3.0 nM), 1016 ± 26; ³H-norepinephrine, 876 ± 54; ³H-dopamine, 1846 ± 109.

DISCUSSION

The structure-activity study for inhibition of 5-HT uptake showed the tetrahydro-β-carbolines to be the most potent inhibitors. For the compounds with a methyl group at C-6, there was not much effect of a methoxy group at C-6 (e.g., 6-MeO-THBC vs. THBC) but substitution led to greater inhibition (e.g., 6-OH-THBC). This latter compound did not differ from THBC. Addition of a methoxy group at C-6 to THBC to form 6-MeO-THBC did lead to greater inhibition, however. Tuomisto and Tuomisto, (16) using rat brainstem, also found that 6-OH-THH was a somewhat better inhibitor than THH. Keller et al. (12) have recently confirmed this ordering of inhibitory potency.

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. Curves are labeled with the doses of 6-MeO-THBC administered. 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^-15 mole/4 min/2.2 mg protein.

---

TABLE 2 In Vivo Inhibition by Drugs of ³H-Serotonin, ³H-Norepinephrine, and ³H-Dopamine Uptake into a Synaptosomal Suspension from Mouse Brain

Drug	Dose (mg/kg)	% Inhibition
---	---	---
³H-Serotonin (3.0 nM)	³H-Serotonin (0.1 µM)	³H-Norepinephrine (0.1 µM)	³H-Dopamine (0.1 µM)
Tetrahydroharman	50	58.0 ± 1.5
6-MeO-Tetrahydroharman	50	31.3 ± 2.5
6-OH-Tetrahydroharman	50	7.7 ± 3.6
THBC	50	68.0 ± 1.3
6-MeO-THBC	50	66.0 ± 3.1	42.2 ± 3.7	11.2 ± 3.9	0.0 ± 0.0
Lilly 110140	10	88.0 ± 0.0	53.1 ± 6.0	0.0 ± 0.0	0.0 ± 0.0
Chlorimipramine	25	94.3 ± 0.2
Benztropine	25	0.0 ± 0.0	5.8 ± 5.8	66.5 ± 2.8	45.8 ± 3.0

Mice were killed 1 hr. after the i.p. injection of the drugs (doses given as salts). Incubation was carried out as described in the text. Numbers in the table are mean values ± 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 ± 19; ³H-serotonin (0.1 µM), 290 ± 6; ³H-norepinephrine, 767 ± 26; ³H-dopamine, 855 ± 34.

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. In terms of structure-activity, the best inhibition of 5-HT uptake was 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 β-carbolines, however, was as effective an inhibitor of 5-HT uptake as were Lilly 110140 (also shown by Keller et al. (12)), chlorimipramine, or imipramine.

The present study showed that the tetrahydro-β-carbolines were less potent inhibitors of NE or DA uptake than of 5-HT uptake. This has also been shown by Keller 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 Keller 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 β-carbolines to MAO inhibiting activity (21). It is of interest to note the structure-activity relationship of MAO inhibitory activity. For example, the EC50's (µM) for in vitro MAO inhibition were 0.08 for harmine, 3.3 for harman, and 58 for 6-MeO-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 β-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 avoidance (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-β-carbolines when administered in vivo. 6-MeO-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-β-carbolines also inhibited 5-HT uptake at 1 hr., although 6-OH-THH produced less than 10% inhibition at 1 hr. after injection for comparison of 5-HT with catecholamine uptake inhibition paralleled that found in vitro: i.e., 6-MeO-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-β-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 compounds. 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-β-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 has been reported (26).

ACKNOWLEDGMENTS

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-G1033 (W.O.B.).

REFERENCES

1. R.E. Schultes and A. Hofmann, The Botany and Chemistry of Hallucinogens, (pp. 101-112) Charles C. Thomas, Springfield (1973).

2. R.J. Wyatt, R. Erdelyi, J.R. Do Amaral, G.R. Elliott, J. Renner and J.D. Barchas, Science 187, 853-855 (1975).

3. H. Rommelspacher, E. Meller and A.J. Friedhoff, Biochem. Pharmacol. 24, 1759-1762 (1975).

4. L.L. Hau and A.J. Mandell, J. Neurochem. 24, 631-636 (1975).

5. L.L. Hau and A.J. Mandell, Res. Comm. Chem. Path. Pharmacol. 12, 355-362 (1975).

6. L.R. Mandel, A. Rosegay, R.W. Walker, W.J.A. Vander Heuvel and J. Rokach, Science 186, 741-743 (1974).

7. H. Rommelspacher, R.H. Coper and S. Strauss, Life Sci. 18, 81-88 (1976).

8. L.L. Hau, Life Sci. 19, 493-496 (1976).

9. W.M. McIsaac, D. Taylor, K.W. Walker and B.T.