- Email: [email protected]
[3H]Harmaline distribution in monkey brain; pharmacological and autoradiographic study
SHORT COMMUNICATIONS
397
[3H]Harmaline distribution in monkey brain; pharmacological and autoradiographic study Plant materials containing harmala alkaloids have been used as intoxicants for centuries. At least two of the constituents of the plant extracts, harmaline and harmine, have been reported to be hallucinogens 1°. Harmaline produces hallucinations at a dosage level of I mg/kg i.v., which is one-half of the hallucinogenic dose ofharmine ~0. Harmaline, 7-methoxy-l-methyl-3,4-dihydro-fl-carboline and other fi-carbolines are also of interest since it has been demonstrated that 6-methoxy-l-methyl-l,2,3,4tetrahydro-fi-carboline can be formed in vivo 9. A commercial sample of harmaline was labeled with tritium in positions 5, 6, and 8 by a tritium-hydrogen exchange reaction. Labile tritium atoms were removed until a constant specific activity of 190 #Ci/mg was achieved. The chemical and radiochemical purity of the compound was verified by its infrared spectrum and by thinlayer chromatography (Silica Gel G). The chromatograms were developed in the systems of n-butanol-acetic acid-H20 (4:1:1) and in n-propanol-NH4OH-H,~O (4:1:1). [3H]Harmaline was detected on the plates by autoradiograms and ultraviolet fluorescence. Seven male squirrel monkeys (750-900 g) were administered [3H]harmaline (20 mg/kg; 3.8 mCi/kg) intravenously; femoral vein. The animals were anesthetized with diethyl ether and killed by bleeding at 15 rain and 1 and 4 h intervals following injection. The brains were dissected and frozen immediately in hexane cooled to about --70°C with solid carbon dioxide. Sagittal sections of whole brain (30-60 #m) were cut at --10°C with a Jung microtome, model 'K'. The autoradiography of the brain sections using Kodak RP/S-54 X-OMAT medical X-ray film was performed and evaluated as previously describedT, 13,1~. Brain samples of the different areas were homogenized in 5 parts of methanol. Unchanged [3H]harmaline was identified by thin-layer chromatography using the above mentioned solvent systems and by the isotope dilution technique. The radioactivity was assayed by liquid scintillation and all tissue samples were corrected for labile tritium which never amounted to more than 4 °/oo Pronounced tremors were produced in the animals within the first few rain after injection and lasted for about 30 min. During the same period tonic convulsions followed toward the end of the attack by clonic convulsions in rapid succession were observed. Nystagmus and inability to fix stare were also noted. Excessive salivation and an increase in heart rate also occurred. About 30 rain after administration of the compound, head twitching was observed and it continued for about 2 h. Lethargy, weakness and incoordination were still apparent after 2 h, lasting until 4 h after injection. At 15 rain three monkeys were killed to correlate early intense reactions with distribution of [3H]harmaline. The cortex accumulated a much higher radioactivity than the white matter, except for a slightly higher concentration in the frontal area; there were no specific sites of accumulation in the cortex (Fig. 1A and B). High radioactivity also appeared in the hippocampus, caudate nucleus, putamen, cerebellum, Brain Research, 22 (1970) 397-401
398
SHORT COMMUNICATIONS ,L :2
Brain Research, 22 (1970) 397-401
399
SHORT COMMUNICATIONS TABLE I DISTRIBUTION OF RADIOACTIVITY AFTER ADMINISTRATION OF [3H]HARMALINE
Individual values given for each animal (20 mg/kg; 3.8 mCi/kg). Tissue
Radioactivity (/iCi/g tissue) 15 rob1
Whole blood* Plasma* CSF* Frontal white Frontal grey Thalamus Hippocampus Cerebellum
Ih
A
B
C
2.53 0.90 3.32 4.66 7.60 7.15 7.02 5.91
2.40 1.93 0.92 0.73 3.08 2.93 2.49 1.91 7.09 4.66 6 . 7 1 2.49 6.29 1.79 5.75 3.19
4h
A
B
A
B
2.47 1.60 4.80 3.64 3.92 4.97 4.65 4.03
2.09 1.36 4.08 3.24 3.98 d.37 4.01 3.91
2.53 2.38 1.76 1.20 0.95 0.61 1.97 1.83 1.39 1.53 1.56 1.62 1.50 1.50 1.19 1.59
* /tCi/ml.
fastigial nucleus and dentate nucleus. (Fig. I A,B). Other areas with high radioactivity were the thalamic nuclei, pons and hypothalamus. At 1 h the radioactivity was almost evenly distributed throughout the brain. The hippocampus was still relatively high in concentration (Fig. 1C; Table [). A reversal in distribution between white and grey was noted at 4 h (Fig. ID). Shortly after the administration of [3H]harmaline, the distribution of radioactivity at 15 rain was higher in grey than in white matter. This phenomenon was ill general similar to the distributions reported for STP a, mescaline 11, thiopental 1, urea 14, phenobarbital 2, and salicylic acidq The permeability of the blood-brain barrier, blood flow and vascularity all can affect accumulation of drugs in the brainS, 74. It is important to note that at this early period the plasma level of [3H]harmaline had already dropped to a very low level, one-fourth of the cerebrospinal fluid and oneeighth of the frontal cortex (Table I). Whole blood radioactivity remained relatively constant throughout the 4 h period studied ; the rise in radioactivity in the plasma with time was apparently due to increased plasma protein binding (Table I). Radioactivity was detected in the hippocampus (Fig. 1A; Table I); electrolytic lesions in the hippocampus cause motor fits in cats, usually of a clonic type a. Another feature of the hippocampus is that electrical stimuli can produce hallucinations 12. The changes in coordination and motor function correlate with high radioactivity in the caudate nucleus and putamen, which seem to function together in initiating and regulating gross intentional movements, and with high radioactivity in the cerebellum and fastigial and dentate nuclei which play an important role in motor function. These observations suggest a relation between the anatomical distribution and action of harmaline as reported earlier for another hallucinogenic compound ~. Any definite conclusions are difficult because the site of accumulation of a drug does not necessarily correspond to its site of action. Brain Research, 22 (1970) 397-401
400
SHORT COMMUNICATIONS
A reversal in the d i s t r i b u t i o n between white a n d grey was noted at 4 h (Fig. 1D). This redistribution c a n n o t be explained by the conversion of [aH]harmaline to its metabolites because the level of u n c h a n g e d h a r m a l i n e only declined t¥om 78,',,i, at 15 rain to 7 3 ' ~ at 4 h. A c c u m u l a t i o n in the white matter could be retarded by the necessity for a c o m p o u n d to penetrate multiple m e m b r a n e s of the laminated myelin sheath 1~. Onse the c o m p o u n d has been a c c u m u l a t e d effl.ux should be retarded in the same m a n n e r . G u n n '5 reported that the h a r m a l a alkaloids first produced clonic c o n w d s a n t action followed by depressant action o n the central nervous system. We havc also observed a distinct change in drug action at 4 h, Since no substantial a c c u m u l a t i o n of metabolites occurred, the change in effect, therefore, might have been a rcsull of the shift in distribution. This work was supported in part by Public Health Service G r a n t s MH-12959 a n d MH-11168, and by the Britton F u n d . We wish to t h a n k Miss Cecilya Castle for her technical assistance, Dr. J. E. ldanp~ifin-Heikkila is a visiting scientist from the D e p a r t m e n t of Pharmacology, University of Helsinki, Siltavuorenpenger 10, Helsinki, Finland. Texas Research Institute of Mental Sciences, Houston, Texas 77025 (U.S.A.)
BENG T. HO G. EDWARD FRITCHIE J. E. IDANPX,~N-HEIKKIL,~ L. WAYNE TANSEY WILLIAM M. MclSAAC
1 DOMEK, N. S., BARLOW,C. F., AND ROIH, L. J., 14C-Phenobarbital and a"S-pentothal in the CNS
of adult cats, Fed. Proc., 18 (1959) 384. 2 DOMEK, N. S., BARLOW,C. F., AND ROTU, L. J., An ontogenetic study of phenobarbital-l~C in cat brain, J. Pharmacol. exp. Ther., 130 (1960) 285-293. 3 GREEN,J. D., CLEMENTE,C. D., AND DE GROOT, J., Experimentally induced epilepsy in the cat with injury of cornu ammonis, Arch. Neurol. Psychiat. (Chic.), 78 (1957) 259-263. 4 GOLDBERG,M. A., BARLOW,C. F., AND ROrH, t . J., The effects of carbon dioxide on the entry and accumulation of drugs in the central nervous system, J. Pharmacol. exp. Ther., 131 (1961) 308-318. 5 GUNN,J. A., Relations between chemical constitution, pharmacological actions, and therapeutic uses, in the harmine group of alkaloids, Arch. int. Pharmacodyn., 50 (1935) 379 396. 6 ID,~NP.~[N-HEIKKILX,J. E., MCISAAC,W. M., Ho, B. T., FRITCHIE,G. E., AND TANSEY, L. W., Relation of pharmacological and behavioral effects of a hallucinogenic amphetamine to distribution in cat brain, Science, 164 (1969) 1085-1087. 7 ID.~NP.~,~N-HEIKKIL-A,J. E., VAPAATALO,H. J., AND NEURONEN, P. J., Effect of hydroxyethylpromethazine (Aprobit 'g~)on the distribution of asS-chlorpromazine studied by autoradiography in cats and mice, Psychopharmacologia (Bed.), 13 (1968) 1-13. 8 KETY,S. S., Measurement of regional circulation to brain and other organs by isotopic technique. In L. J. ROTH(Ed.), Isotopes in Experimental Pharmacology, University of Chicago Press, Chicago, 1965, pp. 211-218. 9 MCISAAC, W. M., Formation of I-methyl-6-methoxy-l,2,3,4-tetrahydro-2-carboline under physiological changes, Biochim. biophys. Acta (Amst.), 52 (1961) 607-609. 10 NARANJO, C., Psychotropic properties of the b.armala alkaloids. In D. H. EVRON, B. HOLMSTEI)T AND N. S. KLINE (Eds.), Ethnopharmacologic Search for Psychoactive Drugs, U.S. Public Health Service, No. 1645, Washington, D.C., 1967, pp. 385-391. 11 NEVF,N., ROSSL G. V., CHASE,G. D., AND RABIYOWITZ,J. L., Distribution and metabolism of mescaline-*4C in the cat brain, J. Pharmacol. exp. Ther., 144 (1964) 1-7. Brain Research, 22 (1970) 397-401
SHORT COMMUNICATIONS
401
12 PAMPIGLIONE, G.~ AND FALCONER, M. A., Some observations upon stimulation of the hippocampus in man, Electroenceph. elin. Neurophysiol., 8 (1956) 718. 13 SCHOOLAR,J. C., BARLOW, C. F., AND ROTH, L. J., Autoradiography of carbon -~4 labelled isoniazid in brain, Proc, Soc. exp. Biol. (N. Y.), 91 (1956) 347-349. 14 SCHOOLAR, J. C., BARLOW, C. F., AND ROTH, L. J., The penetration of carbon- ~4 urea into the cerebrospinal fluid and various areas of the cat brain, J. Neuropath. exp. Neurol., 19 (1960) 216 227. 15 ULLBERG, S., Autoradiographic studies on the distribution of labelled drugs in the body. In J. G. BUGHER, J. COURSAGET AND J. F. LOUTIT (Eds.), Progress in Nuclear Energy, Ser. V1, Biological Sciences, Vol. 2, Pergamon Press, New York, 1959, pp. 29-35.
(Accepted June 17th, 1970)
Brain Research, 22 (1970) 397-401
397
[3H]Harmaline distribution in monkey brain; pharmacological and autoradiographic study Plant materials containing harmala alkaloids have been used as intoxicants for centuries. At least two of the constituents of the plant extracts, harmaline and harmine, have been reported to be hallucinogens 1°. Harmaline produces hallucinations at a dosage level of I mg/kg i.v., which is one-half of the hallucinogenic dose ofharmine ~0. Harmaline, 7-methoxy-l-methyl-3,4-dihydro-fl-carboline and other fi-carbolines are also of interest since it has been demonstrated that 6-methoxy-l-methyl-l,2,3,4tetrahydro-fi-carboline can be formed in vivo 9. A commercial sample of harmaline was labeled with tritium in positions 5, 6, and 8 by a tritium-hydrogen exchange reaction. Labile tritium atoms were removed until a constant specific activity of 190 #Ci/mg was achieved. The chemical and radiochemical purity of the compound was verified by its infrared spectrum and by thinlayer chromatography (Silica Gel G). The chromatograms were developed in the systems of n-butanol-acetic acid-H20 (4:1:1) and in n-propanol-NH4OH-H,~O (4:1:1). [3H]Harmaline was detected on the plates by autoradiograms and ultraviolet fluorescence. Seven male squirrel monkeys (750-900 g) were administered [3H]harmaline (20 mg/kg; 3.8 mCi/kg) intravenously; femoral vein. The animals were anesthetized with diethyl ether and killed by bleeding at 15 rain and 1 and 4 h intervals following injection. The brains were dissected and frozen immediately in hexane cooled to about --70°C with solid carbon dioxide. Sagittal sections of whole brain (30-60 #m) were cut at --10°C with a Jung microtome, model 'K'. The autoradiography of the brain sections using Kodak RP/S-54 X-OMAT medical X-ray film was performed and evaluated as previously describedT, 13,1~. Brain samples of the different areas were homogenized in 5 parts of methanol. Unchanged [3H]harmaline was identified by thin-layer chromatography using the above mentioned solvent systems and by the isotope dilution technique. The radioactivity was assayed by liquid scintillation and all tissue samples were corrected for labile tritium which never amounted to more than 4 °/oo Pronounced tremors were produced in the animals within the first few rain after injection and lasted for about 30 min. During the same period tonic convulsions followed toward the end of the attack by clonic convulsions in rapid succession were observed. Nystagmus and inability to fix stare were also noted. Excessive salivation and an increase in heart rate also occurred. About 30 rain after administration of the compound, head twitching was observed and it continued for about 2 h. Lethargy, weakness and incoordination were still apparent after 2 h, lasting until 4 h after injection. At 15 rain three monkeys were killed to correlate early intense reactions with distribution of [3H]harmaline. The cortex accumulated a much higher radioactivity than the white matter, except for a slightly higher concentration in the frontal area; there were no specific sites of accumulation in the cortex (Fig. 1A and B). High radioactivity also appeared in the hippocampus, caudate nucleus, putamen, cerebellum, Brain Research, 22 (1970) 397-401
398
SHORT COMMUNICATIONS ,L :2
Brain Research, 22 (1970) 397-401
399
SHORT COMMUNICATIONS TABLE I DISTRIBUTION OF RADIOACTIVITY AFTER ADMINISTRATION OF [3H]HARMALINE
Individual values given for each animal (20 mg/kg; 3.8 mCi/kg). Tissue
Radioactivity (/iCi/g tissue) 15 rob1
Whole blood* Plasma* CSF* Frontal white Frontal grey Thalamus Hippocampus Cerebellum
Ih
A
B
C
2.53 0.90 3.32 4.66 7.60 7.15 7.02 5.91
2.40 1.93 0.92 0.73 3.08 2.93 2.49 1.91 7.09 4.66 6 . 7 1 2.49 6.29 1.79 5.75 3.19
4h
A
B
A
B
2.47 1.60 4.80 3.64 3.92 4.97 4.65 4.03
2.09 1.36 4.08 3.24 3.98 d.37 4.01 3.91
2.53 2.38 1.76 1.20 0.95 0.61 1.97 1.83 1.39 1.53 1.56 1.62 1.50 1.50 1.19 1.59
* /tCi/ml.
fastigial nucleus and dentate nucleus. (Fig. I A,B). Other areas with high radioactivity were the thalamic nuclei, pons and hypothalamus. At 1 h the radioactivity was almost evenly distributed throughout the brain. The hippocampus was still relatively high in concentration (Fig. 1C; Table [). A reversal in distribution between white and grey was noted at 4 h (Fig. ID). Shortly after the administration of [3H]harmaline, the distribution of radioactivity at 15 rain was higher in grey than in white matter. This phenomenon was ill general similar to the distributions reported for STP a, mescaline 11, thiopental 1, urea 14, phenobarbital 2, and salicylic acidq The permeability of the blood-brain barrier, blood flow and vascularity all can affect accumulation of drugs in the brainS, 74. It is important to note that at this early period the plasma level of [3H]harmaline had already dropped to a very low level, one-fourth of the cerebrospinal fluid and oneeighth of the frontal cortex (Table I). Whole blood radioactivity remained relatively constant throughout the 4 h period studied ; the rise in radioactivity in the plasma with time was apparently due to increased plasma protein binding (Table I). Radioactivity was detected in the hippocampus (Fig. 1A; Table I); electrolytic lesions in the hippocampus cause motor fits in cats, usually of a clonic type a. Another feature of the hippocampus is that electrical stimuli can produce hallucinations 12. The changes in coordination and motor function correlate with high radioactivity in the caudate nucleus and putamen, which seem to function together in initiating and regulating gross intentional movements, and with high radioactivity in the cerebellum and fastigial and dentate nuclei which play an important role in motor function. These observations suggest a relation between the anatomical distribution and action of harmaline as reported earlier for another hallucinogenic compound ~. Any definite conclusions are difficult because the site of accumulation of a drug does not necessarily correspond to its site of action. Brain Research, 22 (1970) 397-401
400
SHORT COMMUNICATIONS
A reversal in the d i s t r i b u t i o n between white a n d grey was noted at 4 h (Fig. 1D). This redistribution c a n n o t be explained by the conversion of [aH]harmaline to its metabolites because the level of u n c h a n g e d h a r m a l i n e only declined t¥om 78,',,i, at 15 rain to 7 3 ' ~ at 4 h. A c c u m u l a t i o n in the white matter could be retarded by the necessity for a c o m p o u n d to penetrate multiple m e m b r a n e s of the laminated myelin sheath 1~. Onse the c o m p o u n d has been a c c u m u l a t e d effl.ux should be retarded in the same m a n n e r . G u n n '5 reported that the h a r m a l a alkaloids first produced clonic c o n w d s a n t action followed by depressant action o n the central nervous system. We havc also observed a distinct change in drug action at 4 h, Since no substantial a c c u m u l a t i o n of metabolites occurred, the change in effect, therefore, might have been a rcsull of the shift in distribution. This work was supported in part by Public Health Service G r a n t s MH-12959 a n d MH-11168, and by the Britton F u n d . We wish to t h a n k Miss Cecilya Castle for her technical assistance, Dr. J. E. ldanp~ifin-Heikkila is a visiting scientist from the D e p a r t m e n t of Pharmacology, University of Helsinki, Siltavuorenpenger 10, Helsinki, Finland. Texas Research Institute of Mental Sciences, Houston, Texas 77025 (U.S.A.)
BENG T. HO G. EDWARD FRITCHIE J. E. IDANPX,~N-HEIKKIL,~ L. WAYNE TANSEY WILLIAM M. MclSAAC
1 DOMEK, N. S., BARLOW,C. F., AND ROIH, L. J., 14C-Phenobarbital and a"S-pentothal in the CNS
of adult cats, Fed. Proc., 18 (1959) 384. 2 DOMEK, N. S., BARLOW,C. F., AND ROTU, L. J., An ontogenetic study of phenobarbital-l~C in cat brain, J. Pharmacol. exp. Ther., 130 (1960) 285-293. 3 GREEN,J. D., CLEMENTE,C. D., AND DE GROOT, J., Experimentally induced epilepsy in the cat with injury of cornu ammonis, Arch. Neurol. Psychiat. (Chic.), 78 (1957) 259-263. 4 GOLDBERG,M. A., BARLOW,C. F., AND ROrH, t . J., The effects of carbon dioxide on the entry and accumulation of drugs in the central nervous system, J. Pharmacol. exp. Ther., 131 (1961) 308-318. 5 GUNN,J. A., Relations between chemical constitution, pharmacological actions, and therapeutic uses, in the harmine group of alkaloids, Arch. int. Pharmacodyn., 50 (1935) 379 396. 6 ID,~NP.~[N-HEIKKILX,J. E., MCISAAC,W. M., Ho, B. T., FRITCHIE,G. E., AND TANSEY, L. W., Relation of pharmacological and behavioral effects of a hallucinogenic amphetamine to distribution in cat brain, Science, 164 (1969) 1085-1087. 7 ID.~NP.~,~N-HEIKKIL-A,J. E., VAPAATALO,H. J., AND NEURONEN, P. J., Effect of hydroxyethylpromethazine (Aprobit 'g~)on the distribution of asS-chlorpromazine studied by autoradiography in cats and mice, Psychopharmacologia (Bed.), 13 (1968) 1-13. 8 KETY,S. S., Measurement of regional circulation to brain and other organs by isotopic technique. In L. J. ROTH(Ed.), Isotopes in Experimental Pharmacology, University of Chicago Press, Chicago, 1965, pp. 211-218. 9 MCISAAC, W. M., Formation of I-methyl-6-methoxy-l,2,3,4-tetrahydro-2-carboline under physiological changes, Biochim. biophys. Acta (Amst.), 52 (1961) 607-609. 10 NARANJO, C., Psychotropic properties of the b.armala alkaloids. In D. H. EVRON, B. HOLMSTEI)T AND N. S. KLINE (Eds.), Ethnopharmacologic Search for Psychoactive Drugs, U.S. Public Health Service, No. 1645, Washington, D.C., 1967, pp. 385-391. 11 NEVF,N., ROSSL G. V., CHASE,G. D., AND RABIYOWITZ,J. L., Distribution and metabolism of mescaline-*4C in the cat brain, J. Pharmacol. exp. Ther., 144 (1964) 1-7. Brain Research, 22 (1970) 397-401
SHORT COMMUNICATIONS
401
12 PAMPIGLIONE, G.~ AND FALCONER, M. A., Some observations upon stimulation of the hippocampus in man, Electroenceph. elin. Neurophysiol., 8 (1956) 718. 13 SCHOOLAR,J. C., BARLOW, C. F., AND ROTH, L. J., Autoradiography of carbon -~4 labelled isoniazid in brain, Proc, Soc. exp. Biol. (N. Y.), 91 (1956) 347-349. 14 SCHOOLAR, J. C., BARLOW, C. F., AND ROTH, L. J., The penetration of carbon- ~4 urea into the cerebrospinal fluid and various areas of the cat brain, J. Neuropath. exp. Neurol., 19 (1960) 216 227. 15 ULLBERG, S., Autoradiographic studies on the distribution of labelled drugs in the body. In J. G. BUGHER, J. COURSAGET AND J. F. LOUTIT (Eds.), Progress in Nuclear Energy, Ser. V1, Biological Sciences, Vol. 2, Pergamon Press, New York, 1959, pp. 29-35.
(Accepted June 17th, 1970)
Brain Research, 22 (1970) 397-401