II. Biological Effects of Marihuana

Cannabis sativa is one of man's oldest and most widely used drugs. The substance has been used in various ways as long as medical history has been recorded and is currently used as a multipurpose drug throughout the world (Adams, 1942; Adams, 1941-1942; Grinspoon, 1969; Indian Hemp, 1969; Walton, 1938).

During the past few years, a resurgence of the use of marihuana by western society, its increased importance as a social issue and the development of more precise compounds and analytic techniques have sparked dormant scientific interest in the substance. However, this effort has added little to what was already known about the clinical syndrome produced by cannabis (Hollister, 1971) and described by investigators during the last 100 years (Hollister, 1971; Beaudelaire, 1861; Moreau, 1845; Lewin, 1964; Indian Hemp, 1969; Mayor's Committee, 1944).

Strongly held, diametrically opposed opinions exist about whether the ultimate effects of cannabis use are harmful, harmless, or beneficial to human functioning (Pillard, 1970).

Despite these conflicting opinions, from a scientific perspective, the literature on marihuana is as clear, if not clearer, than for many other botanical substances consumed by man. Most of the older reports suffer from multiple scientific defects such as biased sampling, lack of adequate controls, unsophisticated techniques, and use of unstandardized marihuana of unknown potency. Nevertheless, much is known about the use of cannabis by man. Marihuana has a unique position in the multitude of pharmaceuticals in that human experimentation has been greater than laboratory animal experimentation.

The crucial experiments about social effects from chronic use will be settled by close observation of those who use the drug. The issues of potential therapeutic utility; mechanisms of mental function alteration; and the relationship to mental illness will require more extensive laboratory experimentation (Hollister, 1971).

Botanical and Chemical Considerations

In the past several years considerable progress has been made in adding to the understanding of marihuana as a complex drug containing botanical substance. Much important information has been obtained from intensive investigation of marihuana of worldwide origin cultivated under Government contract by the University of Mississippi (Doorenbos et al., 1971).

Marihuana is a preparation derived from the hemp plant, Cannabis sativa. This plant is an annual which either is cultivated or grows freely as a weed around the world, including most of the United States. When cultivated in temperate climates, plantings are made in May to June. The seeds germinate in less than a week. in moist soil. After thinning, the plants grow as rapidly as two feet a week during the peak growing season. They can reach a height of up to 18 feet at maturity, approximately three to five months after planting. Growth is greatly inhibited by inadequate light, water or soil nutrients.

Marihuana is produced by cutting the stem beneath the lowest branches, air drying, and stripping seeds, bracts, flowers, leaves and small stems from these plants. Stems and seeds are variably removed using a mesh screen producing manicured marihuana. Hashish is produced by scraping the thick resinous material secreted by the flowers (Doorenbos et al., 1971).

Many morphological variations in branching and leaf structure are observed among plants produced by different seed types. The characteristic leaf is palmately-compound and contains an odd number of coarsely serrated leaflets. Plants of a given seed type generally grow at similar rates and resemble each other. Thus, botanists believe, Cannabis sativa represents a single species which has not stabilized and has many variations (Doorenbos et al., 1971).

Cannabis Sativa is a dioecious species with separate male and female plants, both producing flowers. Some monoecious variants are reported. Pollination appears to be accomplished by air currents. Bees are attracted by male flowers but not by female flowers. Sex cannot be established until flowering begins and the structure of the male and female flower is distinct. Male plants begin shedding leaves shortly after flowering, shied their pollen and die. Female plants lose their older leaves as the seed matures. After shedding their seed, they die. Contrary to popular belief, there is no significant difference in drug content between male and female plants at equivalent states of maturity (Fetterman et al., 1970; Ohlsson et al., 1971). Male plants mature earlier than the females, shed their pollen and die while the female plant is continuing to mature.

The drug content of the plant parts is variable. Generally, the, drug content decreases in the following order: bracts, flowers, leaves. Practically no cannabinoids are found in the stems, roots and seeds. Obviously, fluctuations in pharmacologic activity of a sample of Marihuana, depend on the mixture of these plant parts which is determined by the manicuring process (Fetterman et al., 1970).

Different variants of the plant contain different amounts of psychoactive drug. Variants of cannabis Sativa cover a spectrum of drug contents. Generally, they can be classified as either drug or fiber genotype. Drug type is high in THC and low in cannabidiol and the fiber type is the converse. This type is determined genetically and transmitted by the seed.

Thus, seeds from different geographical areas produce plants with a wide range of drug content. For example, when grown under similar conditions, plants grown from seeds from Mexico may contain 15 times more psychoactive drug than those grown from seeds from Iowa. Of course, individual plants of the same variant often contain greatly different drug content (Fetterman, et al., 1970).

Environmental factors are not as important as heredity in determining type, but they influence to some degree the drug content. However, environmental factors, including type of soil, water, growing space, temperature and light do play an important role in determining the size and vitality of the plant (Doorenbos et al., 1971; Ohlsson et al., 1971; Phillips et al., 1970).

This notorious variability of cannabis preparations causes many disadvantages for detailed and reproducible biological work. Consequently, much effort has been expended to provide a firm chemical basis in order to provide pure and well-defined substances for research.

The major naturally occurring active component of cannabis, 1-delta 9-trans tetrahydrocannabinol, was not isolated in a pure form and its structure illucidated until 1964 (Gaoni and Mechoulam, 1964; Mechoulam and Gaoni, 1967; Mechoulam et al., 1970). In addition, the A' isomer, which is usually present in small quantities in the natural product representing less than 10% of the combined THC content, has a similar spectrum of activity (Hively et al., 1966). These two chemicals, available by industrial synthesis (Fahrenholtz et al., 1967; Petrzilka and Sikemeier, 1967) or by extraction from the natural plant, can apparently reproduce fully the effects of the crude drug in animals and man. More than 20 natural cannabinoids have been identified in the plant (Figure 1) (Mechoulam, 1970; Shani and Mechoulam, 1970; Doorenbos et al., 1971).

All but Delta 11 and Delta 9 THC are inactive psychopharmacologically and do not seem to exert potentiating or other effects. However, new compounds, cannabinoids and non-cannabinoids, are being isolated from the plant and require further investigation. Several studies may indicate that some material present in natural marihuana may act synergistically with THC and potentiate its psychological effect Leniber (1972) Paton and Pertwee (1971) suggest cannabidiol may play this role.

The chemical nomenclature of tetra-hydrocannabinols is in a state of confusion due to the existence of two numbering systems. The dibenzopyran or formal system treats the compound as substituted dibenzopyrans ( Delta 9 THC) while the monoterpenoid system considers them as substituted terpenes (Al THC). The formal system will be used hereafter (Figure 2).

Many of the natural cannabinoids are present in the plant as acids. These acids are believed to be psychopharmacologically inactive. However, they are converted rapidly when heated, and slowly when stored into their respective active neutral components (Figure 3) (Waller, 1971). This conversion (decarboxylation) does not apparently occur when the acids are absorbed after oral consumption (Mechoulam, 1970).

The proposed biogenesis (Figure 4) of All THC appears to proceed through cannabidiol (CBD). Cannabis variates of the fiber type apparently do not perform this conversion. Thus, cannabidiol is the cannabinoid present in the largest percentage in the non-drug variety (Phillips et al., 1970). Marihuana appears to lose its potency over time due to conversion of THC to cannabinol (CBN) (Mechoulam, 1970) and this also apparently occurs more quickly for hashish implying the presence of a stabilizing substance in the whole plant (Figure 5).

Recently, the n-propyl homologue, of Delta 9 THC has been isolated from crude marihuana. It has about 20% of the activity of Delta THC in mice, and probably makes only a small contribution to the total marihuana effect (Gill, 1971). Merkus (1971) and Vree et al. (1971) have recently identified propyl and methyl cannabinol homologues in hashish in extremely small quantities.

In addition, numerous other non-cannabinoids have been identified in the natural material. Most of these have little or no psychoactivity (Gill et al., 1970; Bercht et al., 1971). Recently, waxes. starches, oils, terpenes and simple nitrogenous compounds including muscarine, choline and trigonelline as well as volatile low-molecular weight piperdines have been isolated.

Additionally, four more complex nitrogenous containing compounds of the generally-accepted alkaloid type have been reported in marihuana leaves in minute concentrations (average 0.002%). These produced decreased activity but no acute toxicity in mice (Klein et al., 1971).

Another laboratory has isolated two steroids and triterpenes from. marihuana as well as tyramine amide derivatives from the roots (Doorenbos et al., 1971).

Analysis of the smoke obtained from marihuana has been investigated. As in the case of any combustible plant, a gas and particulate phase is produced. Both these phases are delivered to the lung. Both the gas and particulate phase consist of compounds present in approximately the same percentages as other burned cellulose containing materials except for the cannabinoid fraction. This includes carbon dioxide, carbon monoxide, and hydrogen cyanide gases. (Truitt et al. , 1970)

The remainder, the smoke condensate, consists of a complex mixture of relatively non-volatile compounds. Included in this mixture are the cannabinoids (16%), carbohydrates and alcohols (8%), fatty and aromatic acids (11%), polybasic acids (7%), aliphatic amines (1%), aromatic phenols (27%), aliphatic phenols (6%), tannin (6%), unidentified compounds (18%) (Truitt et al., 1970).

Another group of investigators (Magus and Harris, 1971) compared the tar collected from combustion of marihuana cigarettes with the tar yielded from tobacco cigarettes. They reported that the total tar yield from marihuana was slightly less than half that produced by an equal weight of tobacco. The tar contained similar constituents based on typical changes produced on skin of mice.

In addition, there a multitude of synthetic compounds related to the naturally occurring Delta 9 THC derivatives and much more potent (Figure 6). A large number of bomologues have been prepared all with similar activity but differing widely in their potency.

In general, the activity of these compounds increases dramatically over that of A' THC by lengthening the 3 alkyl side chain to 6 and 7 carbons, with additional branching in the alpha and beta positions. The dimethylheptyl analogue (EA1476 or DMHP) is the most active having 50 times the activity of A' THC. The 1-methyloctyl substitution (MOP, EA1465) is the next most potent, compound. The 1,2-dimethylortyl substitution resulted in a 251 fold decreased activity from DMHP. (Domino et al., 1972; Sim and Tucker, 1963)

Numerous variations of the basic structures in the cyclohexene moeity of the molecule, as well as the replacement of -both methyl groups-by-hydrogen resulted in partial and even complete, loss of activity (Domino et al., 1972; Sim and Tucker, 1963).

Mechoulam. (1971) has summarized the investigations related to the structure activity relationships of the cannabinoids as follows:

(1) The pyran ring with a hydroxyl group at 1 position and an alkyl group at the 3 position is an essential requirement for psychotomimetic activity, eg., cannabidiol is inactive.

(2) The aromatic hydroxyl group has to be f reo or esterified for activity.

(3) The presence of a carboxyl, acetyl or carbomethoxyl group in position 2 or 4 renders the compound inactive. Substitution with an alkyl ,group at position 2 retains activity.

(4) Dextrorotary (+) delta-9-THC is inactive whereas its optical isomer levorotary (-) delta9-THC is active.

(5) Maximal activity is seen if the double bond is in the delta-9 or delta-8 position. The delta,-6a, 10a-THC is relatively inactive. 9 (6) The activity of the delta-6a, 10a-THC can be increased by replacement of the pentyl side chain with a hexyl side chain to form synhexyl which is an active compound. Branching of the side chain may lead to considerable increase in potency. The substitution of a dimethyllheptyl side chain for the pentyl side chain in the delta-6a, 10a analogue of THC to form DMHP or EA1476 results in a marked increase in pharmacologic activity.

(7) Substituents at the 9 and 10 position have to be in the plane of the ring (that is equatorial) in order that high activity be retained.

More detailed information on materials, chemistry, bioassays, analytical methods, and methodology for detecting THC or its metabolites in biological fluids may be obtained from The Metabolism of the Tetrahydrocannabinols (Lemberger, 1972) and The Secretary of Health, Education and Welfare, 1972 Report on Marihuana and Health.