FR2834293A1 - Extract prepared from starved leeches, useful e.g. as antinarcotic, antistress or antiinflammatory agent, contains compounds with psychosomatic activity - Google Patents

Abstract

Preparing (M1) an active extract (A) of leeches comprising: (a) growing the leeches for at least 2 months without feeding; and (b) extracting compounds (I) with psychosomatic activity from them, is new. An Independent claim is also included for non-irritating (A) obtained by M1.

Description

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The invention relates to a process for obtaining leech extracts having a favorable effect on the psychosomatic state of an individual due in particular to their anti-stress, bioenergetic, anti-inflammatory activity.

The use of leeches is already known for preparing biologically active extracts having medical properties, in particular on the nervous, cardiovascular and immune systems. Document RU 2,129, 428 describes the use of leeches to obtain a positive action on the psychosomatic state. The UK document
1,552, 420 describes the preparation of extracts having an antithrombotic, thrombolytic, anti-atherosclerotic action.

The patent document FR 2,603, 188 describes the use of extracts of lyophilized leeches in powder having an enzymatic activity with cosmetic effect (stimulation of the exchanges of proteins and lipids).

Document RU 2,153,325 describes in particular the preparation of an extract of a homogenate of medicinal leeches, in particular from the secretions of salivary glands, blood or the digestive tract having anti-stress and antioxidant activity.

Document US 5,977,056 describes a method of treating thrombosis using an extract of salivary acorns from leeches comprising a polypeptide (hementine), optionally in combination with anticoagulants such as hirudin.

However, the methods known from the prior art have a certain number of drawbacks. First, the administration of known leech extracts causes irritation and allergic reactions. In addition, the biologically active substances sought for their psychosomatic effects, in particular the neuromediators, have a reduced activity.

The psychosomatic term typically refers to organic or functional disorders caused, favored or aggravated by physical emotional or affective factors. In the context of the invention, we will hear in particular disorders such as stress which results in a bad mood and irritability, difficulties and sleep disorders, the impression of fatigue and physical weariness, lack of dynamism and desire to work. This term therefore has a very broad acceptance. For example, it will also include here disorders associated with addiction to narcotics, a tendency to suicide and aggression, as will be described.

The invention aims to overcome the drawbacks of the prior art. The inventors have hypothesized that the previous methods led to the destruction of neuromediators and insufficient extraction of active substances.

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 The inventors have on the one hand demonstrated that symbiotic bacteria, Aeromonas, from the digestive tract of the leech produce the desired active substances. The negative consequences of the previous processes would be in particular the insufficient extraction, or the alteration of the properties, of the active substances sought, as well as the production of substances with proteolytic activity not desired from the cosmetic standpoint.

The inventors have also shown, surprisingly, that not only the method of extraction, but also the stage of development of leeches at which the extracts of these leeches are prepared, have a determining impact on the effectiveness of these extracts and their lack of side effects.

To this end, the subject of the invention is therefore, according to a first aspect, a process for obtaining an active extract of leeches, comprising: - the culture of leeches for at least about two months without food - the extraction of cultivated leeches active substances with psychosomatic effect.

The expression “at least two months without food” means leeches having taken their last meal at least two months before the extraction, typically two to three months, and at most one year. After a year without food, in fact, leeches may present disorders which affect the effectiveness of the final extract.

According to one embodiment, the process comprises the following successive stages: - culture of leeches at least two months without food - rapid freezing of leeches - grinding of leeches - obtaining a primary mixture by separation of the active substances from the ground material using of a water-alcohol solution - obtaining a secondary mixture by recovery of the active substances by evaporation of the alcohol - obtaining the active extract by bringing the secondary mixture to a pH of between 7.6 and 7.9.

The inventors have in particular noted the following points: the absence of a preliminary washing step with distilled water or with a physiological solution, followed by a drying step, prevents a partial loss of secretion of salivary glands of leech; - rapid freezing, for example with liquid nitrogen, allows rapid destruction of the structure of Aeromonas bacteria;

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 extraction with a water-alcohol solution improves the conservation of the activity of active substances, in particular of neuromediators.

According to one embodiment, the method comprises a step of conservation, by lyophilization or the like, of the active extract.

According to one embodiment, the method does not include a washing and drying step prior to freezing.

Leeches are typically Hirudo medicinalis leeches.

According to one embodiment, the method comprises: - the removal from the intestine of leeches of Aeromonas bacteria producing at least part of the active substances of the extract - the culture of these bacteria in an appropriate medium - the separation is bacteria from the culture medium the extraction from the bacteria of at least part of the active substances produced by these bacteria, so as to obtain an active bacterial extract.

According to another aspect, the invention relates to a leech extract capable of being obtained by the process described above, comprising at least one substance acting against psychosomatic disorders, the extract comprising at least one biologically active substance chosen from: dopamine, 5 hydroxytriptamine, acetylcholine, GABA, serotonin, substance P, vasoactive peptides, encephalin, somatostatin, destabilase, kallikrein inhibitors, trypsin inhibitors, hyaluronidase, kinnogenase, kininase , cholesterolesterase, hirudin, bdellin, eglin, antioxidants, 6-ketoprostaglandin, neutral protease inhibitors of granulocytes.

According to one embodiment, the extract comprises at least one substance with anti-stress activity.

According to one embodiment, the extract comprises at least one toning or relaxing substance.

According to another aspect, the invention relates to a pharmaceutical composition comprising an extract as described above, and a pharmaceutically acceptable vehicle. According to one embodiment, the composition is used for administration in combination with an anticoagulant.

According to one embodiment, the composition is used for administration in combination with an antithrombic, thrombolytic, fibrinolytic agent.

According to another aspect, the invention relates to an anti-stress composition comprising an extract as described above.

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According to another aspect, the invention relates to the use of an extract as described above as a medicament.

According to another aspect, the invention relates to the use of an extract as described above for the preparation of an anti-narcotic drug.

According to another aspect, the invention relates to the use of an extract as described above for the preparation of a medicament for the treatment of psychosomatic disorders.

According to another aspect, the invention relates to the use (non-medicinal) of an extract as described above as a relaxing agent or as a stimulating agent.

According to another aspect, the invention relates to the use of an extract as described above as an anti-inflammatory, hypotensive or lipolytic agent.

Other advantages will appear on reading the detailed description which follows, illustrated by nonlimiting examples.

Example 1.

The process for extracting active substances is as follows: - removal of approximately 600 g of un nourished medicinal leeches for 2 months - freezing with liquid nitrogen - crushing on an ice bath - addition, at 4 ° C., of a 20% ethyl alcohol solution relative to the total volume of a liter (ratio of volumes 1: 1) - active stirring - centrifugation and collection of the supernatant - evaporation of the alcohol - bringing the supernatant to a pH of 7 , 8 - separation of 650 ml of supernatant, lyophilization and obtaining 25 g of powder.

This powder constitutes an extract comprising the desired biologically active substances.

Example 2.

Study of the irritant and allergenic effect of the preparations, of the toxicity, of the influence of a long-term application on the skin, on the structure of the skin and the liver.

Studies have been carried out on 90 guinea pigs. Leech extract is applied daily for 30 days at concentrations of 0.001% (n = 30), 0.01% (n = 30), and 0.1% (n = 15). A 0.85% physiological solution of NaCl (n = 15) is

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 used as a control. It is shown that the supernatant does not cause an irritant and allergenic action on the skin, nor an alteration of the morphological structure of the skin and the liver, nor a general toxicity on the general condition of the animal (motor skills, hair, appetite).

Example 3.

Studies have been carried out on animals according to standard methods (2) to determine the content of soluble proteins in the skin. 90 guinea pigs have been tested. The supernatant is applied daily for 30 days at concentrations of 0.001% (n = 30), 0.01% (n = 30), and 0.1% (n = 15). A 0.85% physiological solution of NaCl (n = 15) is used as a control. The results are as follows: a 0.001% solution of supernatant increases the total soluble protein content by
13.2%; a 0.01% solution an increase of 75.7%; a 0.1% solution an increase of 121.4%. The physiological control solution does not modify the concentration of total proteins. These results highlight the stimulation of protein exchanges in the skin.

Example 4.

The total lipid content is studied using standard methods (3). On 90 guinea pigs, the supernatant is applied daily for 30 days at concentrations of 0.001% (n = 30), 0.01% (n = 30), and 0.1% (n = 15). A 0.85% physiological solution of NaCl (n = 15) is used as a control. The total lipid content at the end of the treatment is increased by 9.4% using the 0.001% solution, by 18.4% using the 0.01% solution and by 26.7% using the solution. at 0.1%. This increase is 3.1% with the physiological control solution. The effect on lipid metabolism is thus demonstrated.

Example 5.

The biological effect is shown using bath tablets comprising the leech extract obtained (the supernatant) by the method of Example 1. Two types of tablets were tested: - "T" tablets containing the following ingredients: sodium bicarbonate, citric acid, monosodium citrate, dextrin, PEG-150, lavandula officinalis (1%), rosemarinus officials (1%), silica, supernatant (0.3%), CI 41090, CI 17200.

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 - "R" tablets containing the following ingredients: sodium bicarbonate, citric acid, monosodium citrate, dextrin, PEG-150, artemisia vulgaris (0.6%), eucalyptus globulus (1.4%), silica, supernatant (0.6% ), CI 41090, CI 19140.

The absence of toxic effect of these bath tablets has been verified on animals taking a daily bath for 21 days (white rats, males, 200-220 g, n = 25).

No toxicity, irritation or pathological change was noted.

Example 6.

Bath tablets are used as in Example 5. Tests were carried out on healthy people (30 to 65 years old, n = 50). No irritant effect is noted after 10 baths for individuals "T" and "R" (baths at two-day intervals). There is softening of the skin, firmer skin, better microcirculation, reduction of small wrinkles.

Example 7.

pour 33 % une nervosité. La fatigue physique est de 76 %. The bath tablets are similar to those in Example 5. Tablet tests were carried out on a group of volunteers of 22 people of ages between 16 and 72 years old. Volunteers tried "T", "R" and Placebo tablets. The interval between each bath was at least three days, with a comfortable water temperature, a bath time of 15 to 20 minutes, in the evening, and one hour before falling asleep. Before the experiment, the volunteers had the following particularities: for 41% a state considered normal (absence of bad mood, nervousness, moral fatigue), for 23% a bad mood and aggressiveness, for 38% a moral fatigue and a lack of will for contact,

for 33% a nervousness. Physical fatigue is 76%.

The results were as follows: - Preparation "R": Results at the end of the treatment: for 57% of individuals a greater desire to sleep (43% immediately after bathing); 84% fall asleep faster. For 100% of individuals, a complete absence of bad mood, an impression of joy and calm. For 100% a disappearance of physical fatigue.

Virtually all individuals notice a disappearance of physical pain with an absence of muscular heaviness and less trauma. The approach of sleep, the impression of calm and better dreams are observed.

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Results on the following days: for 79%, good form, an impression of a rested body, for 21% the normal maintenance of the condition, for 100% an absence of nervousness, for 93% an absence of fatigue. The treatment is considered to provide better comfort (85%), vigor (75%), good mood (85%), reduction in physical fatigue and better work capacity (75%) compared to 25% who consider the aptitude for work normal.

 - Preparation "T": Results after the treatment: for 77% a feeling of vigor, for 93% an absence of fatigue, for 71% of the best dreams, for 80% an improved intellectual activity, for 100% an absence of bad mood , for 80% less physical fatigue, for 90% an absence of physical pain compared to before treatment, for 71% an impression of internal comfort. Only three volunteers qualify their condition as normal.

In the following days: for 86% an impression of rest, for 79% an absence of general strength, for 100% an absence of irritability, for 86% an impression of moral comfort, for 71% an absence of drowsiness, for 93% increased efficiency - Placebo preparation: Patients did not experience any particular change from before treatment. Only mention is made of drowsiness (57%) and a certain physical relaxation (60%) that it would be possible to correlate with a bath at a comfortable temperature.

These results demonstrate the anti-stress action of the product.

Example 8.

The bath tablets are similar to those of Example 5. The tests were carried out in the Neurology Clinic of the Faculty of Fundamental MedicineDepartment of the State University. The patients studied belong to three groups (10 people per group). Not all patients used medication for a period of one month before the studies and during the studies. The age of the patients is 35 to 45 years, including 20 women and 10 men. Patients in the first group take 4 baths a day at 38 C for 15 minutes. The second group of patients takes 4 baths per day with a tablet of rest at one

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 temperature of 38 C for 15 minutes. The third group under the same conditions takes a bath with a preparation T.

The Spilberger method is used to measure the behavior of people. A post-treatment questionnaire was completed by patients the day after treatment was completed. For each patient, a triple measurement of the tone of vegetative hormones was carried out with the help of computer programs "Variability of the heart rate" directly before, once after and again one hour after the bath. Hormonal tone measurements using heart rate frequency indexes were made, as well as changes involving the sympathetic and parasympathetic hormonal system. The results are as follows: using baths without lozenges reduces the heart rate by 10%. After a bath with a resting tablet, the FCR is reduced by 13% and is unchanged for a T-tablet. The R-R interval after a normal bath increases by 5%. The P and T tablets do not change this index. The measurements taken on the hormonal spectrum increase by 8%. The application of a rest preparation significantly increases (21%) these measures. The T pads do not modify them. The power of the sympathetic system is reduced by 7% after a normal bath, the rest and T preparations cause a decrease of 11%. The interesting effect of the influence of these preparations on the parasympathetic nervous system is demonstrated: a value of 2% is obtained with a normal bath (non-significant effect), while a relaxing effect is observed for the rest lozenges which reduce this parameter by 22% (only 5% for T tablets). Patients who used resting tablets noted a reduction in discomfort up to 25-31 on the Spilberger test. After the use of T tablets, such a reduction is not observed.

Thus, the results of the tests show that the rest lozenges make it possible to obtain a calming and bioenergetic effect. T tablets have more impact on the bioenergetic tone.

Example 9.

The conditions of use of lozenges are the same as in Example 5. The effect of the lozenges containing the leech extract was tested on 40 patients suffering from opium narcotism. The first control group of 20 people uses basic therapy to control this narcotism, combined with the use of a Rest preparation, the second group of 20 people uses only basic therapy.

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Among the patients in the first group, 17 had second-degree opium narcotism and 3 had first-degree opium narcotism. The patients are between 18 and 36 years old, 18 are men, 2 are women. Among the patients in the second group, 19 were diagnosed with second-degree narcotism and one in the first degree.

Basic therapy uses preparations including neuroleptics (aminazine, galoperidole, levomepromazine); tranquilizers (diazepam, phenazepam); sleeping pills.

For the patients of the first group, in addition to the basic technique, 5 baths are taken with the preparation of Rest. The results were measured within an hour of bathing and after the completion of a joint therapeutic treatment. All the patients in this group noted a decrease or disappearance of pain in the joints and muscles from the first two treatments. After 3 to 4 treatments (7 to 8 days after the start of treatment), 17 out of 20 patients noted a decrease in asthenia, better dreams, better shape, better appetite, decreased irritability, a reduction in the tendency to want other palliative substances, a reduction in the appearance of abstinence syndromes). No particular negative side effect was observed for patients using the Rest preparation.

Among the patients in the second group, the reduction in clinical symptoms occurred much more slowly: after 10 to 14 days of treatment, an improvement was noted for 10 out of 20 patients and for 18 patients at the end of 4 weeks. treatment.

Thus in view of the very positive results obtained, the invention also provides a treatment method comprising the administration of a therapeutically effective amount to a patient, of the bacterial extract. The bacterial extract can advantageously be incorporated into a pharmaceutical composition which can be administered to a patient orally or parenterally. By parenteral is meant in particular subcutaneous, intravenous, intra-articular administration, intratracheic injection, infusion techniques. Other means of administration such as topical administration, application to the skin (bath, creams, solutions ----) can be used.

According to one embodiment, a pharmaceutical composition incorporating the extract typically comprises an excipient or a transporter which is preferably chosen so that the formulation can be administered intravenously, such as a sterile saline solution.

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The term pharmaceutically acceptable carrier is used to denote a non-toxic, solid or diluent or encapsulating liquid material, which does not react negatively on the active compound for its effectiveness. Suitable liquid carriers are used, for example, a sterile aqueous solution, a saline solution, an aqueous dextrose solution, a sugar solution, ethanol, glycol and oils. Tablets and capsules for oral administration may contain common excipients such as binding agents, lubricants, etc.

Oral liquid preparations can be in the form of an aqueous or oily suspension, solution, emulsion, syrup, elixir or the like, or can be presented in the form of a powdered product which can be reconstituted in the presence of water or '' another suitable carrier. Such liquid preparations can contain usual additives such as suspending agents, emulsifying agents, non-aqueous transporters. The topical application can be in the form of an aqueous or oily suspension, a solution, an emulsion, a gel in particular. The unit dose of a pharmaceutical composition according to the invention may contain an appropriate amount of bacterial extract. The optimal pharmaceutically acceptable dosage and the dose for a given patient (in particular a man) depends on several factors, in particular the activity of the individual, his age, his body weight, his general state of health, his state of anxiety, the object of treatment, the disease being treated. For example, a systemic dose of bacterial extract of the order of 0.01 to 100 mg / kg of body weight, preferably 0.05 to 10 mg / kg, can be used.

Given the absence of side effects and the specific and advantageous biological properties of the extract using the process described, compared to the extracts obtained hitherto by direct extraction from leeches, the inventors deduce therefrom that the bacterial extracts do not have the same composition as the extracts of the prior art. The inventors hypothesize that certain compounds causing side effects are absent, and / or that certain specific compounds inhibiting these effects are recovered during extraction.

The active substances in leech extract allow stimulation of protein and lipid exchanges in the skin, an anti-stress and bioenergetic modulating action, without any irritant or allergic effect on the skin. Furthermore, the examples describe the use which may be therapeutic or non-therapeutic of the extracts obtained, in particular according to the concentration of the active extract in substances

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 active, and the importance of the individual's psychomatic disorder. Thus the use will be non-therapeutic if the extract is intended for a person wishing to relax, but who is not suffering from a psychosomatic disorder in a pathological way.

Claims (17)

 CLAIMS 1. Method for obtaining an active extract of leeches, characterized in that it comprises: - the culture of leeches for at least approximately two months without food; - the extraction of cultivated leeches of active substances with psychosomatic effect. 2. Method according to claim 1 characterized in that it comprises the following successive stages: - culture of leeches at least two months without food - rapid freezing of leeches - grinding of leeches - obtaining a primary mixture by separation of the active substances of the ground material using a water-alcohol solution - obtaining a secondary mixture by recovering the active substances by evaporation of the alcohol - obtaining the active extract by bringing the secondary mixture to a pH of between 7.6 and 7.9. 3. Method according to claim 1 or 2 characterized in that it comprises a step of conservation, by lyophilization or the like, of the active extract. 4. Method according to claim 2 or 3 characterized in that it does not include a washing and drying step prior to freezing. 5. Method according to any one of claims 1 to 4 characterized in that the leeches are leeches Hirudo medicinalis. 6. Leech extract, non-irritant, capable of being obtained by the process according to any one of claims 1 to 5, characterized in that it comprises at least one substance acting against psychosomatic disorders, the extract comprising at least a biologically active substance chosen from: dopamine, 5 hydroxytryptamine, acetylcholine, GABA, serotonin, substance P, vasoactive peptides, encephalin, somatostatin, destabilase, kallikrein inhibitors, inhibitors of trypsin, hyaluronidase, kininase, cholesterolesterase, hirudin, bdellin, eglin, antioxidants, 6-keto-prostaglandin, neutral protease inhibitors of granulocytes. 7. Extract with anti-stress activity, characterized in that it is obtained by a process according to any one of claims 1 to 5. <Desc / Clms Page number 13>  8. Extract with toning or relaxing activity, characterized in that it is obtained by a process according to any one of claims 1 to 5. 9. Pharmaceutical composition comprising an extract according to any one of claims 6 to 8, and a pharmaceutically acceptable vehicle 10. Pharmaceutical composition according to claim 9 for administration in combination with an anticoagulant. 11. Pharmaceutical composition according to claim 9 for administration in combination with an antithrombic, thrombolytic, fibrinolytic agent. 12. Anti-stress composition comprising an extract according to claim 7. 13. Extract according to any one of claims 6 to 8 as a medicament. 14. Use of an extract according to any one of claims 6 to 8 for the preparation of an anti-narcotic drug. 15. Use of an extract according to any one of claims 6 to 8 for the preparation of a medicament for the treatment of psychosomatic disorders. 16. Use of an extract according to any one of claims 6 to 8 for the preparation of an anti-inflammatory medicament. 17. Use of an extract according to any one of claims 6 to 8 for the preparation of an anti-stress medication.