I. Some chemical and physical properties of the isolated effective substances (IRC) particularly with regard to their stability
By Z. Krejci
The antibiotically active and effective substance (IRC) of resinous appearance and nature, utilized at present for the preparation of the antibacterial preparations, does not appear to be a chemical entity, but a mixture of two substances. It is of acid nature conditioned by the presence of phenolic and carboxyl substances. These acid substances retain their antibacterial properties even after acetylation and following division in the neutral portion (acetylated phenols) and the acid one (acetylated-phenol-acid).
Going deeper into the matter showed that the biological effectiveness of the active amorphous substances is not so stable as was previously assumed. On the contrary, their effectiveness decreases due to the oxidizing action of atmospheric oxygen, particularly in a warm atmosphere. In an oxygen-free medium, even at higher temperatures (in the autoclave) they are comparatively stable.
The effective substances are all readily soluble in common organic solvents - namely, in ethylalcohol, ethylether, chloroform, benzene, petrolether and so on. At pH 7 they are slightly soluble in water and in physiological solutions. This property is a certain drawback in these substances both for clinical utilization and precise pharmacological evaluation. Because of their acid nature they are readily soluble in aqueous solu-tions of alkalis - i.e. in sodium and potassium hydroxide, sodium carbonate, sodium hydrocarbonate and so on. They can be easily emulsified, and as emulsions they are comparatively stable.
1. The pharmacodynamic effect of the antibacterial substances from Cannabis sativa var. indica
The pharmacological evaluation was carried out at the Institute for Pharmacology of the Medical Faculty of the Palacky University. Klabusay & Lenfeld succeeded in showing some well-defined effects of the isolated amorphous substances (IRC): analgetic, anticonvulsant, and, when locally applied, the initially irritative and later on anaesthetic effects.
The hashish effect due to tetrahydrocannabinol could not be found in substances obtained from plants cultivated in the moderate climate of Czechoslovakia - i.e. it could be evidenced neither in animal experiments nor in experiments carried out on man ( per os).
The analgetic action was studied in white rats by the help of the method of mechanical irritation according to Levy and Buchel in the modification according to Votava and in guinea pigs by the method according to Regnier. The IRC was administered perorally in emulsions with gum arabic in doses from 100 to 50 mg/kg; and also subcutaneously and intraperitoneally in ethylenglycol up to 500 mg/kg weight. From these experiments it could be seen that the IRC produces an analgetic action when administered perorally in doses of 100 mg, subcutaneously and intraperitoneally - i.e., from 50 mg/kg onward. Higher doses (500 mg/kg) of the IRC result in intensified analgesia lasting for some hours and complete inhibition of the activity of the central nervous system or sleep.
The anticonvulsive effect was evaluated in white mice and in frogs. The convulsions were produced in mice by pentamethylentetramine (0.1 g/kg intraperitoneally), in frogs by strychnine by administering doses of 40 mg/kg subcutaneously. The effective dose of the IRC preventing convulsions due to pentazole in 50% of mice was established as 0.61 g/kg per os; for strychnine an effective dose of 0.83 g/kg when applied subcutaneously.
The determination of the local anaesthetic effect was carried out by evaluation of the surface anaesthesia according to the method of Lebduska & Vrba using the cornea of rabbits and guinea pigs, and by the evaluation of the anaesthesia according to the method of infiltration of Bulbringe & Wayda, using the skin of guinea pigs. The IRC was applied in ethylenglycol. The IRC was effective from the concentration of 5% and up, whereas a total local an?esthetic effect could be observed only by using a 10% concentration; it lasted for about 45 to 90 minutes.
The toxicity was determined in mice by administering an emulsion with gum arabic, and the death rate recorded within 48 hours. By the Burn method, LD50 was established as 1.83 g/kg per os. Smaller doses did not produce any remarkable changes in mice; doses of 1 g/kg (IRC) were followed, after some hours, by a slight inhibition of the motor function with apathy and immobility. Larger doses resulted in a total inhibition of the activity of the central nervous system, sleep and exitus within 24 hours.
The local compatibility was evaluated on the cornea of rabbits by administering a solution in drops and by application of the solutions of the tested substances into the subcutaneous area of the auricle of the rabbit. The effect of the IRC upon the mucous membranes was locally irritating. On local application to the skin no clinical evidence of acute irritation was found. The intramuscular application resulted in an inflammatory reaction with the exudation of the liquid.
2. Clinical experiences
In recent years a series of pharmaceutical preparations have been prepared and clinically examined particularly with regard to the needs of stomatology, otorhinolaryngology, gynaecology, dermatology and the like. The preparations have been locally applied throughout but parenteral administration has not been made use of, particularly because of the poor solubility of the examined substances in physiological solutions. Besides, some of the properties such as the pharmacological one (irritation) and the bacteriological one (inactivation with blood and serum) of the active substances which have been isolated do not allow a suitable utilization of these preparations by means of injections.
For the needs of stomatology, an aseptic dentin powder was prepared, impregnated with 2 to 5% of biologically active substances (IRC) from cannabis. This dentin powder containing IRC was successfully used for the indirect covering (in about 300 patients) and for the direct covering of the pulp (70 cases), in beginning pulpitis and irritation of the pulp. Even in cases like that we notice the obvious advantage of the antibacterial effect of the locally anaesthetic action. Save in cases of massively infected pulps (Soldan) did not record any failures.
Somewhat less uniform results were obtained with this preparation by Simek, who applied dentin combined with IRC or a special salve containing IRC. In uncomplicated Caries profunda, a remarkable and spontaneous effect could be achieved in 64% of the patients; in Caries profunda, with an incidental opening of the vital pulp, in 38.4% of the patients. In Pulpitis partialis, the results of this therapy were positive in 41% of the investigated cases. The authors have, on the whole, acknowledged the anaesthetic effect of these preparations.
Very advantageous is the application of the effective substances from cannabis in otorhinolaryngology. Hubacek reports on the very good results obtained particularly after application of 1% of an alcoholic solution of the IRC and of a dusting powder (boric acid with 2 to 5% of the IRC) in acute and chronic otitis, furunculosis of the outer part of the nostrils and of the external auditory meatus, and even in some cases of bilateral sinusitis, the latter having been treated without success with a series of punctions and penicillin irrigations. He describes an interesting case of bilateral chronic Sinusitis maxillaris, lasting for about three years and treated without success with a total of about 30 punctions. After application of our IRC preparations, one side was treated as a control with penicillin, but with negative result, and the other side with three IRC punctions and irrigations. The side treated with negative effect with penicillin was easily and successfully treated with cannabis preparations. In the same way Navratil illustrates in his small table of chronic otitis a considerable improvement in 13 of a total of 18 cases. All the cases under investigation were also controlled bacteriologically. In clinical practice the very good results achieved with the preparations mentioned above - i.e. with the alcoholic solution and the dusting powder-stand in support of the good results obtained in vitro and justify the suggestion to have both these preparations made available for pharmaceutical production.
The manifold utilization of these substances particularly in the form of alcoholic solutions with glycerine has been confirmed by the results obtained after application of this preparation in obstetrics in order to prevent staphylococcic mastitis in the treatment of rhagades and fissures on the nipples of nursing women. Of great importance is the locally anaesthetic component together with the antibacterial effect, particularly upon Staphylococcus aureus, which is considered to be the causing agent of the postpartum inflammation of the mamma. Heczko & Krejci evidenced staphylococcus in 84% of the total of 160 parturient women. It is significant that in 89% of cases the staphylococcus was resistant to penicillin, in 18% to streptomycin, and in some cases resistant to penicillin, streptomycin, aureomycin and terramycin and sensitive only to chloromycetin and to the IRC.
The publication of the results obtained from these investigations evoked a lively interest among obstetricians who up to the present have tried to find means of substitution both for the inadequate and still used gentian violet, and the local inefficacious application of some antibiotics as, for example, aureomycin, which has been recommended by some authors.
FIGURE 20 Comparison of the inhibitory effect of some antibiotics in combination with active substances from cannabis upon Staphylococcus aureus P - penicillin 100 OU; S - streptomycin 1 mg; T - terramycin 5 mg; A - aureomycin 5 mg; CH - chloramphenicol 5 mg; Ci - tincture cannabis Zone in the centre of the plate - 2 per cent of IRC.
For the purpose of dermatology, a salve containing 2% of effective substances has been prepared and found very useful in pyodermia of staphylococcic etiology, infected burns, and particularly in decubitus of immobile patients in rehabilitation centres. Of great interest was the follow-up of a physician and pathologist, who was treated with the IRC for a severe infection of the thumb of the right hand, an injury he suffered in the dissecting room. The severe condition, threatening amputation, and the absolute resistance of the microflora to available antibiotics were overcome by substances from cannabis.
The results obtained in treatment of specific tuberculous fistulas and published by Procek as well as the up to now unpublished results of the investigations carried out must be considered as preliminary ones even though the antituberculosis effect in vitro is very satisfactory.
In the cases mentioned above, no extraordinary allergy of the organism to effective substances from cannabis or any particular ability of the IRC to sensitize were observed.
In conclusion we may still consider the interesting results Sirek achieved. He mentions his experiences for some years past with hemp seeds in the therapy of tuberculosis. The hemp seed was ground and extracted with milk at a temperature of 60-80 degrees of C. It represented a protein-rich moiety of the curing diet for tuberculous children, in whom a remarkable improvement due to this diet was observed by the author. But the assumed antituberculotic action of cannabis seeds could not be evidenced in experiments on guinea pigs. Therefore, in the cases reported by Sirek, the therapeutic effect can be only explained by the healing nutritious diet in which the specially prepared seed is of great importance.
In human therapy the best results have been obtained with the following medicaments combined with substances derived from cannabis: dusting powder together with boric acid (otitis), ointment (staphylococcus infected wounds, staphylodermia and so on), ear drops (otitis chron.), alcohol solutions with glycerine (treatment of rhagades on the nipples of nursing women - prevention of staphylococcic mastitis), aqueous emulsions (sinusitis), dentin powder with the IRC (caries). The preparations mentioned above have been already tested clinically, and will eventually be made available for production.
The high yield of effective substances (average 1.5% of the IRC in the dried drug), the considerable amount of raw material available (1.5 million hectares of cannabis plant culture in the world), and the subsequent low price both of the raw material and of the effective substances allow us to utilize the antibacterially effective substances from cannabis in veterinary medicine. Such use, which is analogical to human medicine, is comparatively wide: infected wounds (dusting powder), panaris of sheep and cattle (dusting powder), otitis in dogs (ear drops, dusting powder), mastitis in cattle, postpartum trauma, lacerated wounds and so on. Especially veterinary workers, workers concerned with cattle breeding, employees in slaughter houses, cutters and so on consider the preparations containing biologically active substances to be an important contribution to the prevenion of tuberculosis, anthrax, swine erysipelas and brucellosis in various streptococcic and staphylococcic infections of the hands and the like. The results achieved in this field, but unpublished as yet, justify the greatest optimism.
A considerably higher resistance against virus pneumonia has been observed in mice fed with hemp seeds.
In view of the results obtained with antibacterially effective substances isolated from cannabis, it may be concluded that they are superior and have numerous advantages as antibiotics.
The results obtained in clinical practice must receive, for the most part, a positive evaluation. In many cases satisfactory and in some cases even surprisingly good results have been obtained, particularly where a microflora resistant to a series of other antibiotics was concerned. Naturally drawbacks had to be recorded. Further clinical experiences and practice will be necessary for a definite evaluation of this antibacterially effective substance.
A systematic investigation of the mid-European flora has been carried out in order to establish the presence of antibacterially effective substances. A high content of remarkably active substances has been found present in Cannabis sativa var. indica and in Cannabis sativa, a plant cultivated for the industrial utilization of the hemp fibre. It has been investigated in detail from the bacteriological and the chemical viewpoint and from the standpoint of the eventual applicability of the effective substances in clinical practice.
In old herbals and in folk medicine in Europe, Africa and America, mention has been made of the utilization of cannabis as an antibiotic and analgetic medicament particularly for external use. The hashish effect has been known only to nations with a warm climate. In Europe, hashish made its appearance as a narcotic at the time of Napoleon's campaign in Egypt, but contrary to morphine and cocaine, its use was not widespread. In spite of this, from the pharmacological viewpoint, official medicine has investigated the hashish effect on the central nervous system whilst the analgetical and antiseptic effects have been unjustifiably forgotten.
In the chemical section of this work, the most suitable methods of extraction have been described and the technique for the isolation of effective substances developed. At the beginning of the procedure two antibacterially effective fractions were obtained: the acid and the phenolic. Then from the acid fraction a biologically active crystalline acid could be isolated. On the basis of the elementary analysis, the optical rotation, the UV- and the IR-spectroscopy, the following constitution has been established:
Because of its close relationship to cannabidiol, it has been named cannabidiolic acid. The second-antibacterially ineffective acid has been identified as trans-cinnamic acid. In addition, the following unsaponified constituents have been isolated: paraffin (n-nonacosane) and higher alcohols.
Experimentally we could evidence the bactericide effect of the substances from cannabis in vitro upon the following gram-positive micro-organisms: Staphylococcus aureus haemolyt, Staphylococcus aureus-resistant to penicillin, Streptococcus alpha, Streptococcus beta haemolyticus, Pneumococcus, Enterococcus, Corynebacterium diphtheriae, Bacillus anthracis, Erysipelothrix rhusiopath. A significant antibacterial effect upon the Mycobacterium tuberculosis in vitro could be observed up to a dilution of 1:150,000.
The gram-negative micro-organisms of the coli-typhus group, as well as the Pseudomonas aeruginosa, Proteus vulg. and, in addition, the yeast and the fungi, proved to be resistant.
The limit of the effectiveness of the maximal dilution of the biologically active substances (1:100,000) and the velocity of the effect of various concentrations could be established.
The effect of the inactivating factor has been studied in detail. Blood, plasma and serum partially inactivate and decrease the antibacterial effect.
A comparison between the effectiveness of the active substances from cannabis and that of penicillin and streptomycin has been carried out at various pH.
Finally, the experiments made in clinical practice, particularly in stomatology, otorhinolaryngology, gynecology, dermatology, phthisiology, with some pharmaceutical preparations containing antibacterial substances from cannabis have been reported. Attention has been drawn to the advantageous utilization of the active substances from cannabis in veterinary medicine, and particularly in preventive medicine for anthropozoonoses.
A preliminary pharmacologic investigation of the hemp substances was carried out. It was ascertained that the isolated hemp extract possesses analgetic, anticonvulsive and locally anaesthetic properties. Administered to white mice per os its toxicity was determined with 1.83 g/kg as LD-50.* (*Ed. note...LD-50 ratio of 18,300:1 for a concentrated extract.) Acid II and the acetylderivative of the canabidiol-acid - pure substances obtained from hemp do not possess these pharmacologic properties.
All the investigated substances were observed to bring forth local irritations. Further studies will have to explain this irritative effect with regard to the fact that the extract from the drogue, (sic) when being deprived of a part of its ballast substances only, does not show to be an irritative factor in clinical use but, on the contrary, does relieve pain entirely, a fact having been known to ancient physicians, and nowadays newly confirmed both by stomatologists and oto-laryngologists.
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