Symposium on marijuana: Rheims, France, 22-23 July 1978
SYMPOSIUM ON MARIJUANA - Rheims, France, 22-23 July 1978
General considerations - Botany - Chemistry
Transformation in the organism - Pharmacokinetics
Quantification-identification in the body fluids
Effects of cannabinoids on cells
Effects of marijuana on the different systems in vivo
Author: Gabriel NAHAS
Pages: 23 to 32
Creation Date: 1978/01/01
Director of Research, National Institute of Health and Medical Research, Paris
A symposium on the biological effects of cannabis (marijuana, hashish) was held at Rheims on 22 and 23 July 1978 under the auspices of the Seventh International Congress of Pharmacology. This symposium was organized - with the assistance of the National Institute of Health and Medical Research (Institut national de la santé et de la recherche médicale) (INSERM) - by Professor NAHAS, Director of Research at INSERM, Professor PATON, Professor of Pharmacology at Oxford University, Dr. BRAUDE, Chief of the Pharmacology Department, National Institute on Drug Abuse (United States of America) and Professor JARDILLIER of the Faculty of Pharmacy at Rheims. About 50 papers describing the most recent research on this drug were presented and discussed by teams of investigators from the United States of America, Canada, the United Kingdom, Australia, Sweden, Denmark, Israel, the Federal Republic of Germany, Greece and France.
Introduction: G. NAHAS: Marijuana and membranes
Quantification of cannabinoids and their metabolites in body fluids and tissues; biochemical interactionsChairmen:S. AGURELL (Uppsala, Sweden)R. BOURDON (Paris, France)Introduction:S. AGURELL1. Mass spectrometry.M. WALL (Research Triangle Park, N.C)2. Gas chromatography and mass spectrometry, Metabolites and their kinetics.D. HARVEY (Oxford, England)Discussant:B.R. MARTIN (Richmond, Virginia)3. Radioimmunoassay.V. MARKS (Birmingham, England)4. Enzyme immunoassay.R. SNYDER (Palo Alto, Ca.)5. Isotopic derivatization and double labelling.J.-M. SCHERRMANN (Paris, France)6. Pharmacokinetics of the cannabinoids and their identification.E. GARRET (Gainesville, Florida)7. Cannabinoids: Metabolic patterns versus biological activityS. ROSELL (Stockholm, Sweden)Discussant:M. WIDMAN (Uppsala, Sweden)8. Delta-7-THC: Structure-activity considerations.M. BINDER (Bochum, Germany)9. Cannabinoids: Cell proliferation and macromolecular biosynthesis.G. S. STEIN (Gainesville, Florida)10. Cannabinoids and antioxydantsC. LEUCHTENBERGER (Lausanne, Switzer land)
Cannabinoids and cellular metabolismChairmen:G. S. STEIN (Gainesville, Florida)J.-C. JARDILLIER (Rheims, France)Introduction:W. D. M. PATON (Oxford, England)1. Membrane-bound enzymes.A. MELLORS (Guelph, Ontario)2. Neuroblastoma and glial cells.R. CARCHMAN (Richmond, Virginia)3. Synaptosomes.M. HERSHKOWITZ (Rehovot, Israel)Discussant:G. B. CHESCHER (Sydney, Australia)4. Brain neurotransmitter systems.W. L. DEWEY (Richmond, Virginia)5. Brain cells.P. McGEER (Vancouver)Discussant:Y. LUTHRA (Washington, D.C.)6. Testicular cells.A. JACUBOVIC (Vancouver)7. Lymphocytes.B. DESOIZE (Rheims, France)8. Myocardial cells.H. CHOISY (Rheims, France)9. Lung parenchyma.H. ROSENKRANTZ (Worcester, Mass.)10. Lungs: large and small airways.G. HUBER (Cambridge, Mass.)11. Increased induction of hypoploidy during heavy marijuana smoking.A. MORISHIMA (New York, N.Y.)Discussants:A. OHLSSON (Uppsala, Sweden) andA. ZIMMERMAN (Toronto, Canada)12. THC: Carcinogenicity in mice.J. SZEPSENWHOL (Miami, Florida)
Cannabis and reproductionChairmen:M. BRAUDE (Rockville, Maryland)H. TUCHMANN-DUPLESSIS (Paris, France)Introduction:M. BRAUDE1. Effects of cannabis on sex hormones and testicular enzymes in the rodent.J. HARCLERODE (Lewisberg, Pa.)2. Effects of cannabinoids on spermatogenesis in mice.A. ZIMMERMAN (Toronto, Canada)3. Effects of marijuana smoke on spermatogenesis in rodents.H. HUANG (New York, N.Y.)4. Effects of cannabis on foetal development in rodents.H. ROSENKRANTZ (Worcester, Mass.)5. Cannabinoids: effects on reproductive function of female rodents.G.I. FUJIMOTO (New York)6. Effects of cannabis on foetal development in the rabbit.D. COZENS (Huntingdon, England)7. Effects of chronic THC on reproduction and offspring in rhesus monkeys.E. SASSENRATH (Davis, Ca.)8. Effects of THC on female repro-ductive function (primates).C. SMITH (Bethseda, Md.)9. Effects of cannabis on human repro-ductive function.W. HEMBREE (New York, N.Y.)10. Observations in chronic hashish users.M. ISSIDORIDES (Athens, Greece)
Cannabis and the brainChairmen:L. HOLLISTER (Palo Alto, Ca.)P. DENIKER (Paris, France)Introduction:L. MILLER (Lexington, Kentucky)1. Memory and behaviour in young ratstreated with cannabis extracts.H. KALANT (Toronto, Canada)2. Cannabis and brain reward stimu-lation.N. PRADHAN (Washington, D.C.)3. Chronic marijuana smoking: its effectson function and structure of theprimate brain.R. HEATH (New Orleans, La.)4. Long-term effects of THC on primatesocial behaviour.L. CHAPMAN (Davis, Ca.)5. Cannabis and epilepsy.R. KARLER (Salt Lake City, Utah)Discussant:D. FEENEY (Albuquerque, N.M.)6. Cannabis and morphine interactions.R. MALOR (Sydney, Australia)Discussant:S. KAYMAKCALAN (Ankara, Turkey)7. Cannabis and the development oftolerance.L. HOLLISTER (Palo Alto, Ca.)8. Cannabis, pain and memory.W. C. CLARK (New York, N.Y.)Conclusion:W. D. M. PATON
Cannabis sativa, or marijuana or hemp, is one of the oldest plants grown by man. It has spread from Central Asia, where it originated and was cultivated 5,000 years ago, to all the temperate and tropical zones in the world. Cannabis sativa is used for the fibre in its stalk, the oil in its seeds and the intoxicating substances in its flowering top. Cannabis sativa is a unique "non-stabilized" species with a number of variants due to its genetic plasticity, to the effect of the environment and to human manipulation.
A distinction has been made between two main types of hemp, the criterion used being the concentration of the psycho-active substance (delta-9-THC) which the hemp contains in its flowering tops. These two types are: the fibre-type plant with a low delta-9-THC content (under 0.2 per cent), and the drug-type plant with a high concentration of delta-9-THC (1.4-4.8 per cent).
Other natural cannabinoids contained in the plant, such as cannabidiol (CBD), cannabinol (CBN), cannabichromene and cannabicyclol are not psychoactive but are biologically active.
The concentration of delta-9-THC in the plant varies according to the genetic factors and factors associated with the environment. The genetic factors seem to predominate where environmental conditions are stable. Two drug-type preparations are used. These are:
"Grass" or "marijuana" consisting of the flowering tops of the plant, which are chopped very finely and can be smoked like a tobacco cigarette. A grass cigarette weighs from 0.5 to 1 gram and contains from 1 to 2 per cent THC (5 to 20 mg).
"Hashish" or "hash", a concentrated preparation of resin and of the flowering tops of the plant, which must be mixed with tobacco before it can be smoked. This preparation contains 3 to 8 per cent THC.
Like other natural or synthetic tetrahydrocannabinols, delta-THC is an oily substance, insoluble in water (partition coefficient 3000/1), but soluble in alcohol. This substance quickly becomes inactive when it is exposed to oxygen, humidity, light or high temperatures. It is one of the few psychotropic substances which does not contain nitrogen.
Other natural cannabinoids and their metabolites which are not psychoactive are biologically active: in particular these compounds, which all have the olivetol C-cycle, interfere - like THC, and at varying doses - with the production of nucleic acids and proteins and inhibit cell division to different extents.
The biological effects of other chemicals contained in hemp (alkaloids, triterpenes, steroids) have not yet been evaluated but they would seem to be minor.
The quantity of delta-9-THC (or of its acid precursor) contained in a plant extract and transferred to an individual depends on the method of administration (inhalation or ingestion).
The quantity of delta-9-THC (or of its acid precursor) contained in a plant extract and transferred to an individual while he is smoking is difficult to determine, but varies greatly. It is estimated at 50 per cent of the dose inhaled and at 25 per cent of the dose ingested.
Delta-9-THC is four times more psychoactive when it is inhaled than when it is ingested. Delta-9-THC undergoes a complex transformation in the body, producing active and inactive metabolites.
The psychotropic properties of delta-9-THC act directly and also through the active metabolites.
Because THC is fat-soluble, it leaves the bloodstream very rapidly. The immediate plasma concentrations of 100 nanogrammes per millilitre decrease within an hour to 5-10 ng. Stored in the body fats, where it has a half-life of 8 days, the THC is slowly "salted out" of them. Its apparent volume of distribution is between 500 and 2,000 litres. Five days after a single injection, Garrett detected 20 per cent of the THC stored in the organism, while 20 per cent of its metabolites (over 40 were identified) were still present in the organism too. Complete elimination of a single dose takes 30 days.
Repeated administration of cannabinoids at intervals of less than 8 to 10 days results in the accumulation of THC and its metabolites in the tissues.
The storage capacity of the organism for THC and cannabinoids is considerable. The "steady state" (elimination = absorption) is reached only after at least four weeks of daily administration.
Retention of the metabolites in the organism is increased by the enterohepatic recirculation of these compounds. About 80 per cent are eliminated via the intestines and 20 per cent via the kidneys.
The elimination of THC via the kidneys is negligible.
Garrett observed that THC was a drug, whose disposability in the organism depended upon its metabolism-i.e. transformation (chiefly hepatic), storage in the tissues, and "salting out". He established five different compartments for describing the metabolism of this drug.
The very low level of THC in the plasma, and also the numerous metabolites which this compound produces by transformation in the organism, make it very difficult to identify. The only accurate method is mass spectrometry together with high-pressure gas or liquid chromatography, which is a slow and costly technique for identifying THC and its numerous metabolites in the body fluids and tissues (Monroe Wall, Harvey). Vincent Marks of Birmingham described an immunoreactive method which is non-specific since it identifies simultaneously both THC and "other cross-reacting cannabinoids". It is a good detection method which has, in the United Kingdom, revealed the presence of cannabis in the tissues of drivers involved in unexplained road accidents; and it is possible that this simple technique, which is of a routine nature and is not costly, could be made more specific by producing another THC hapten in order to obtain the specific antibody. Scherrmann, of the INSERM Toxicological Unit, described a new specific method for identifying THC by double labelling of the molecule. This technique will be developed to obtain a more exclusive identification of the metabolites. A simple technique for identifying THC, similar to the technique used for measuring alcoholemia, is still far from the stage of practical realization; and the problems involved in devising such a technique-very rapid disappearance of THC from the bloodstream, negligible quantities eliminated in the urine, and the production of dozens of metabolites-are difficult to overcome.
The team consisting of Gabriel Nahas, Jean-Pierre Armand and Bernard Desoize was the first to note in 1974 the inhibiting effect of THC and other cannabinoids in concentrations of 10-4to 10-6M on the synthesis of macromolecules (nucleic acids and proteins) in lymphocytes in culture. Desoize, at Rheims, reported that this was a phenomenon associated with the effect of cannabinoids on the plasma membrane. Using an isotopic dilution technique, he showed that in a few seconds these compounds will inhibit the membrane enzymes carrying DNA, RNA and proteins.
The inhibition of cellular proliferation and of synthesis of macromolecules by cannabinoids in eucaryote cells in culture (HeLa cells, neuroblastomas) was discussed at length at Rheims. Stein attributed this inhibiting action to an immediate action of these compounds on the plasma membrane; but, in the course of longer experiments, he suggested that the nuclear membrane might be affected particularly by the delta-8-THC of which substantial quantities are found in the nucleus. This, it seems, results in a distortion of the messages transmitted at this level by the RNA-polymerase, and a change in transcription. These observations were developed by Issidorides from Athens, who described the histochemical changes in lymphocytes and spermatozoa taken from chronic hashish smokers. These cells show a very marked deficiency in arginine-rich histones, a deficiency which is particularly evident at the level of the acrosome of the sperm cell. Such a change could be due only to a phenomenon occurring at the transcription level.
These conclusions were confirmed by the observations of Carchman (Virginia) who reported that THC acts on the nuclear membrane of THC cells in culture and produces a marked condensation of the nucleic substance.
Toro-Goyco (Puerto Rico) reported that membrane ATPase was inhibited by cannabinoids in the same concentrations (10 -6M) which inhibited macro-molecular synthesis; and he described the mechanism as being an effect of the action of these compounds on the plasma membrane. Such effects on cells, observed with micromolar concentrations, are "non-specific", and are associated with the fat-solubility of THC and its metabolites in the plasma membrane. The specific, stereoselective effects of THC are observed with nanomolar concentrations. Hence the interest of the investigations by Dewey (Virginia), who reported the effect of THC on the uptake of the neurotransmitters, dopamine and norepinephrine, of the synaptosomes of the hypothalamus and of the corpora striata. A two-phase effect was observed: with concentrations of 10 -8M there was an increase in uptake and at 10 -5M there was a decrease. This kind of effect was not observed with the non-psychoactive cannabinoids (CBN and CBD). Also, administration of the active metabolite of THC (11-hydroxy-THC) had different effects from THC on the neurotransmitters of the striatum. Similar results were presented by Hershkowitz of the Weizman Institute in Israel. All these factors would tend to explain the simultaneously sedative and stimulating effects of THC.
For experiments on animals in vivo, it is necessary to administer doses which can be compared with those attained in human consumption.
Rosenkrantz (Worcester, Mass.) has prepared tables of equivalence between doses administered to animals (orally or by inhalation) and those consumed by man. He took into account body surface and specific metabolism in order to establish ranges of comparability. These are the doses which are now used by investigators in order to ensure comparability of their results.
Young marijuana smokers who were studied in hospital in the United States of America showed a reduction in vital capacity and also some signs of obstruction of the upper airways. These observations led to experiments on rodents which were made to inhale cigarette smoke in apparatus specially designed by Rosenkrantz. It was possible to control the frequency and duration of the various stages of respiration, and also the quantity of smoke bolus administered.
Rosenkrantz reported that rats exposed to inhalation of marijuana smoke (under conditions equivalent to the daily consumption of a marijuana smoker) developed after 87 days, and up to 360 days, lesions in the lung parenchyma; these took the form of scattered small focal alveolitis, granulomatic phenomena and dense infiltrations of macrophages associated with deposits of cholesterol. The last-mentioned are signs of tissue destruction. The extent of the lesions depended on the duration of the experiments and the dose inhaled. They were still present a month after smoke inhalation had been stopped. The effects associated with marijuana were different from those produced by tobacco smoke and placebo smoke (a marijuana cigarette from which the cannabinoids had been extracted).
Male: Huang (Columbia University, New York) and Fujimoto (Albert Einstein, New York) described the impairment of spermatogenesis in rats exposed to marijuana smoke, or ingesting THC or cannabis extract. They noted a very marked inhibition of spermatogenesis in sections of the seminiferous ducts. This oligospermia was associated with involution of the prostate and of the seminal vesicles. These changes were reversible when administration of the drug was stopped after 80 days.
Harclerode (Bucknell, Pennsylvania) describes the enzyme mechanism by which THC inhibits the synthesis of testosterone in rats. He ascribes this to an inhibition of the P450 cytochrome. The inhibiting effect was eliminated by the administration of LH and FSH. Jacubovic (Vancouver) reported that the formation of testosterone in the Leydig cells was inhibited by the administration of various cannabinoids. The non-psychoactive cannabinoids (CBN, CBD, CBG) were more inhibiting than THC. Accordingly, he believes that this inhibition of testosterone production is due to a direct effect of the cannabinoids on the Leydig cells. Zimmerman (Toronto) reported a significant increase in abnormal forms of sperm in hybrid mice 35 days after a single administration of delta-9-THC (5 mg/kg) or CBN (10 mg/kg) (inactive cannabinoid). This author also noted, in mice treated with these cannabinoids, some chromosome anomalies in the primary spermatocytes (translocation breakages, aneuploidy). Thus cannabinoids which are not mutagenic in vitro (Ames test negative) would seem to be so in vivo.
Hembree (New York) described the diminution in spermatogenesis occurring in young marijuana smokers after unrestricted smoking for four weeks. This oligospermia was accompanied by an increase in abnormal forms and a decrease in spermatozoa motility. The testosterone, FSH and LH levels, measured every morning before the subjects began smoking, were unchanged. However, Monroe Wall pointed out that an intravenous injection of a dose of THC was accompanied in the next few hours by a reduction in plasma testosterone, which then returned to normal after an overshoot. It is possible therefore that fluctuations in testosterone, and in the pituitary hormones governing its formation, can be detected only after administration of the drug.
In short, it would seem that cannabinoids can act on the testicular function in two ways:
Through the gonadotropins FSH and LH, causing intermittent reductions in testosterone;
Directly on the germinative epithelium, causing changes in spermatogenesis and spermatogonia, which would explain the appearance of abnormal forms of spermatozoa.
Female: In the case of female rats, Fujimoto (Albert Einstein) reported that oral administration of THC or cannabinoid extract led to evolution of the uterus and the ovaries. These changes are reversible when administration of the drug is stopped after 60 days.
Carol Smith (Bethseda) reported that intramuscular administration of THC led to a decrease in FSH and LH in long-tailed monkeys. The extent and duration of such diminution depended upon the dose. When this primate (which has a menstrual cycle) is treated from the end of one cycle with daily doses of THC, ovulation does not occur during the following cycle. Dr. Smith also showed that, contrary to earlier beliefs, THC had no oestrogen-like effect.
Rosenkrantz reported that pregnant mice and rats exposed to marijuana smoke or to the administration of THC experienced an increase in resorptions and in foetal lethality. When strong doses were administered, there was a complete resorption of litters.
Litters which came to term were hypotrophic and weighed less than the controls. There was no teratogenesis, i.e. no discernible somatic malformations.
Sassenrath (Davis, California) reported similar results with a colony of primates in which a number of long-tailed monkeys were fed daily with THC added to their preferred food. Of the pregnant long-tailed monkeys so treated, 44 per cent lost the product of conception (abortions, neonatal deaths), compared with 12 per cent in the case of untreated pregnant long-tailed monkeys. Animals born at term from mothers fed with THC were hypotrophic. Observed over their first year, they displayed hyperkinetic behaviour and were hyperexcitable to external stimuli. In short, cannabis is embryotoxic, but does not produce any discernible teratogenic effect.
The embryotoxicity would appear to be due to impairment of the foetoplacentary circulation, and to hormone secretions resulting from the intoxication of the mother.
These were described by Pradhan (Washington) who used the self-stimulation technique on rhesus monkeys with depth electrodes inserted deeply into the region of the posterior hypothalamus or "pleasure zone". Administration of THC produced a three-phase effect: first a reduction in self-stimulation for one hour, then a resumption of self-stimulation and lastly a prolonged phase of depression. Pradhan established a correlation between these phases and the neurotransmitter content of the diencephalon.
Etevenon (Sainte Anne, Paris) reported changes in the electroencephalogram of human beings after an oral dose of 10 mg of THC. The anomalies observed were correlated with the subjective experiences of the persons concerned: euphoria, dysphoria, sedation, visual hallucinations.
Kalant reported on experiments conducted with young rats fed with THC during their first six months. One month after withdrawal of the drug, they displayed some deficiencies in their psychomotor performance and in their ability to learn (maze). They were more aggressive and more inclined to kill mice than were the controls. Also, two months after the end of the treatment, their electroencephalogram presented irregular "spikey" waves in the recordings of the dorsal hippocampus, which might explain the somatic deterioration of that zone of the diencephalon. Heath presented the results of his work on rhesus monkeys with depth electrodes inserted in the septal region of the hypothalamus. The animals were exposed to daily inhalation of marijuana for three to six months. Their plasma THC levels were similar to those noted in man during cannabis intoxication. Heath observed abnormal recordings in the electroencephalogram of the limbic region (septum, hippocampus), with series of high-amplitude waves. The anomalies persisted for three months after intoxication had been stopped. The ultrastructure of this region of the brain examined by electronic microscope showed the following changes:
Enlargement of the synapsis; Aggregation of the synaptic vesicles; Increase in intracellular inclusions. These structural changes were observed six months after the animal had stopped inhaling marijuana, an indication that the changes observed were well established.
(e) Effects on behavior
Chapman (Davis) reported the effects of chronic administration of THC on the individual and social behaviour of rhesus monkeys. During the first month, the animals treated appeared to be sedated and showed a tendency to keep to themselves, particularly those which were not dominant. After 3-4 months of treatment with THC, this depressive state followed a stage of very pronounced aggressiveness which found expression in fights in the course of which some animals were killed. This aggressiveness was displayed for 15 months.
Hollister drew attention to the considerable tolerance to cannabis which develops and is reflected in the need to increase the doses in order to obtain the desired effect. This tolerance is observed in all animal species. In the case of man, a large-scale consumer, a daily intake of 200-400 mg of THC is observed when cannabis is readily accessible (Morocco, Jamaica). There is no withdrawal syndrome comparable with that produced by stopping the use of opiates. However, irritability, discomfort, hyperkinesis and nausea do occur after a sudden stoppage following a high degree of intoxication (Jones). Though there is no marked physical dependence, there is a psychological dependence as with all euphorigenic psychotropic drugs.
Karler described experimental work showing that cannabidiol (CBD) has anti-convulsive properties similar to those of a widely used anti-epileptic, phenylhydantoin. On the other hand, THC can induce attacks of epilepsy: and epileptics are therefore recommended nto to use marijuana excessively. Professor Mechoulam reported on the first results of tests of DBD proved effective in four cases which had resisted other treatments.
Because of its many side effects, there is little likelihood that THC will be used therapeutically to exploit its anti-glaucoma and tranqullizing properties.
Organic chemists, after studying over 500 molecules derived from THC, have selected nabilone. This substance, which is now being studied in the United States of America, has anti-emetic properities, reduces intra-ocular pressure, and has anxiolytic properties. It will be seen in the next few years whether its properties in these prespects are superior to those of the medicaments now in use.
The Rheims symposium demonstrated the many properties of cannabis and of THC at all levels. of biology.
Marijuana, in addition to its well-known acute and reversible pychotropic porperties which are associated with THC, has certain other properties which are only now just beginning to be described.
There is, first, the effect of THC on the neuropypophysis, and the intermittent inhibition which this compound dcan produce on the secretion of LH, FSH and prolactin. Such disturbances will have repercussions on the formation of the secual hormones testosterone, folliculin and progesterone.
Next, there are the effects of all cannabioids on metabolism and cell division, and on the formation of macromelcules. At this level, cannabinoids act on the plasma membrane and the nuclear membrane, interfering at these two stages with the sythesis of nucleic acids and proteins. This action has long-term effects on the expression of the genome which will be the subject of much subsequent work.
From the public health standpoint, there are four categories of persons who might be warned forthwith of the risks involved in marjuana use. These categories are:
Adolescents, whose neuro-hormonal regulatory systems are in process of development and integration. We have seen that a single dose of marijuana can affect the secretion of the pituitary hormones referred to above;
Epilectics. The central stimulating effects of the THC may induce epileptioform seizures;
Persons with a tendency to schizophrenia and mental illness;
Women of child -bearing age who wish to have children.
From the therapeutic point of view, cannabidiol may prove to be a useful anti-epileptic nabilone, a synthetic cannabinoid, might might find some applications as an anti-emetic, as a tranquillizer and in the treatment of glaucoma. Many years will be needed to evaluate the effectiveness ot these new drugs.
*** Papers presented to the symposium on marijuana will be published by the Pergamon Press early in 1979.