Studies with the U.N. cannabis reference sample

Abstract

The effects of hashish were described long before pharmacology became a science. Many experiments on animals and some on men were performed but uniformity of observations was lacking, at least in the sense of a standard biological test.

Details

Author: G. JOACHIMOGLU, J. KIBURIS and, C. MIRAS
Pages: 21 to 22
Creation Date: 1967/01/01

Studies with the U.N. cannabis reference sample

G. JOACHIMOGLU
J. KIBURIS and
C. MIRAS
From the Department of Clinical Therapeutics, University of Athens, Greece

The effects of hashish were described long before pharmacology became a science. Many experiments on animals and some on men were performed but uniformity of observations was lacking, at least in the sense of a standard biological test.

The main difficulty was that the active principles of the hashish samples used varied considerably in potency and content.

At present, it is not possible to evaluate the potency of hashish by chemical analysis.

In view of this, it was suggested that the activity of hashish should be compared with a reference sample (RS). A pharmacological method was suggested. We dissolved the extracted resin of hashish in olive oil and the solution was administered to rats intraperitoneally.

The RS is a stable preparation which represents, at best, all known activities of different samples of hashish. As a unit, we can then consider the amount of activity present in a given weight of this RS. If a scientist performs tests on rabbits, and others on the same biological system of rats, using different preparations, there is no way of comparing the activity of the one with the activity of the other; but if both are referred to the RS, then the potency of the unknown samples referred to the RS overcomes the difficulty of the comparison if such variables, i.e different animal species, are involved.

An extremely large variety of samples of hashish and other cannabis samples, collected from all over the world, were available at the United Nations Narcotics Laboratory in Geneva. The idea was to mix all these samples and to use the resultant mixture as a RS. 1This has been examined by thin-layer chromatography (figure 1) as well as by ultra-violet and infra-red spectroscopy (figure 2). Its LD 50 has also been estimated in order to facilitate the evaluation of comparison.

1 The sample was prepared by the Chief of the Narcotics Laboratory of the U.N. (WHO Technical Report, series No. 312, WHO Expert Committee on Dependence-producing Drugs, 14th Report, p. 11).

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Preparations in olive oil of the petroleum ether (40-60°) extract of RS have been prepared by a procedure described elsewhere . 2 The extract was 12 per cent of the weight of RS. The concentration in olive oil was 250 mg/ml. The LD 50 has been calculated twenty-four hours following intraperitoneal injection of RS extract in rats; 250 gr by modification of the Trevan method. 3 The animals were kept in an air-conditioned room at 25°C. 4 Water was given freely but no food was administered.

In the following table we give our results on rats.

TABLE

Injected volume

No. of Animals

Mortality %

mlper 250 gr B.W.

ml per 1 gr B. W.

Amount (mg) of resin per1 gr B. W.

Amountof R.S. (mg) per 1 gr B. W

24 25 2.2 0.0088 2.2 18.3
24 50 2.8 0.0112 2.8 23.3
24 75 3.1 0.0124 3.1 25.8
24 100 3.8 0.0152 3.8 31.6

2 Joachimoglu-Miras: Bull. Narcotics, 15, No. 3-4, 1963, and Ciba Foundation Study Group, No. 21.

3 J. H. Burn, Biological Standardization, p. 11, Oxford University Press, 1950.

4Lower temperature e.g. 15°C increases considerably the mortality of treated animals (See also Ciba Foundation Study Group, No. 21, p. 41).

From the above table we have the result that the LD 50 is 23.3 mg of RS per gr B.W.

If we assume that this dose is 1 unit then we have in 1 gr of RS 42.9 or approximately 43 units.

The estimation of the amount of tetrahydrocannabinols in the petroleum ether extract of RS was carried out by quantitative thin-layer chromatography according to the method of Korte and Sieper, 5 using an alkaline solution of fast blue B as colour reagent. The absorption spectra of the produced azo dyes were measured in a Zeiss spectrophotometer, model PMQII. Tetrahydrocannabinol was found to be 0.21 gr per cent of the RS.

The ultra-violet spectrum of the RS extract, showed for the extinction ratios E 260/E 280 and E 300/E 3l0 the values 0.85 and 1.6 respectively.

Infra-red spectroscopy was also carried out with a Beckman IR-5 infra-red spectrophotometer. A so1ution in petroleum ether of the extract from RS with the same solvent was used. A variable path cell was used for compensation work, which had a path length adjusted according to the solvent volume of the sample. The infra-red spectrum showed for T 890-T 1130, the value ± 28. With these values according to the studies of L. Grlic 6, 7 we can say that RS belongs more to the "ripe type ".

5 Bull. Narcotics, 17, No. 1, 1965.

6 Bull. Narcotics, 14, No. 3, 1962.

7 Planta Medica, 13, 291 (1965).