FR2884829A1 - Obtaining active bacterial extract, useful as anti-coagulants, from leeches comprises culturing leeches, removing Aeromonas bacteria from digestive tract, culturing, extracting, subjecting to thermal shock, sonication and centrifuging - Google Patents

Abstract

Obtaining active bacterial extract from leeches comprises culturing leeches, removing Aeromonas bacteria from digestive tract, culturing, extracting, subjecting to thermal shock, sonication and centrifuging. The leeches are Hirudo medicinalis, which are cultured for 2 months without food and the bacteria is Aeromonas hydrophila. The bacteria undergo two thermal shocks before sonication, which is followed by centrifugation and recovery of the supernatant liquid. The thermal shock is carried by lowering the temperature to -80[deg]C in a defrosting stage. The culture medium: is a solid then the bacterial separation is done by mild scraping of the solid medium followed by washing; and is a liquid then the bacteria are separated by centrifugation. Independent claims are included for: (1) a bacterial extract of Aeromonas hydrophila; (2) pharmaceutical composition comprising the Aeromonas hydrophila bacterial extract; and (3) an implantable medical prosthesis. ACTIVITY : Cardiant-Gen.; Anticoagulant; Antiinflammatory; Immunostimulant; Hypotensive; Antilipemic; Vasotropic. MECHANISM OF ACTION : None given.

Description

The present invention relates to a new process for obtaining an extract

  biologically active bacterial of medicinal leeches, the extract obtained, as well as

  its use for the preparation of a pharmaceutical composition.

  The use of leeches for the preparation of biologically active extracts is known. For example, document RU-2,129,428 describes extracts of leeches having psychosomatic properties, and document RU-1,552,420 describes the antithrombotic, thrombolytic, or antiatherosclerotic properties of these same extracts. Extracts of homogenate medicinal leeches, obtained especially from salivary gland secretion, blood or the digestive tract of leeches were prepared, and their antistress and antioxidant activity described in the document RU-2,153,325. US-5,977,056 discloses a method of treating thrombosis using a salivary gland extract of leeches, typically in combination with anticoagulants such as hirudin. Finally, document FR-2,603,188 describes the use of lyophilized leech extracts having an enzymatic activity with a cosmetic effect.

  More recently, the Applicant has demonstrated that the pharmaceutical and / or cosmetic properties of leech extracts were due in large part to a symbiont bacteria of leeches: Aeromonas hydrophila (FR-2,834,292). This bacterium produces most or all of the active substances obtained from the saliva of leeches. It was then possible to consider the administration of these bacteria, and no longer extracts of leeches themselves, to reduce or even eliminate the phenomena of irritation and allergic reactions associated with leeches. However direct administration of such bacteria to host individuals may exhibit toxic effects. Thus, this patent application FR-2,834,292 discloses a method for obtaining a bacterial extract from leeches, said extract having the desired properties and not causing known undesirable effects. In addition, in view of the commercial interest of these bacterial extracts and in view of production on an industrial scale, the improvement of their process of obtaining, more particularly the yield, as well as their efficiency, is 'growing importance.

  The Applicant has thus developed a new process which makes it possible, as in the case of the one mentioned above, to get rid of leeches, but which provides a much more active bacterial extract, because having a greater amount of enzyme , and having increased efficiency. In addition, it also has the advantage of avoiding the sacrifice of too many animals to obtain these extracts and on the contrary, thanks to fermenter cultures of several hundred liters, to have very large quantities of bacterial extracts containing the active ingredients and enzymes necessary for the needs of pharmacy, cosmetology or the food industry.

  The present invention thus relates to a method for obtaining an active bacterial extract of leeches comprising the following steps: (a) culture of leeches for a period of at least 2 months without food, (b) removal in the digestive tract of Aeromonas bacteria leeches.

  (c) culturing Aeromonas hydrophila bacteria in a suitable medium, (d) separating the bacteria and the culture medium, (e) lysing the cells by sonication, (f) centrifugation, (g) recovering the supernatant, characterized in that the bacteria undergo at least one heat shock before the sonication step.

  The leeches used are typically Hirudo medicinalis leeches. The period of fasting will be advantageously two months.

  By thermal shock in the sense of the present invention is meant a sudden change in temperature. In particular, the thermal shock or shocks according to the invention are carried out by major temperature decreases carried out for a duration of between 12 and 24 hours. The thermal shocks according to the invention will in particular be frozen at -80 ° C followed or separated, in the case of several successive thermal shocks, by one or more defrosting steps. In another aspect of the invention, the lowering of temperature is achieved by the action of liquid nitrogen.

  In another advantageous aspect of the invention, the bacteria undergo at least two thermal shocks before the sonication step, each heat shock being followed by a centrifugation step, and a supernatant recovery step.

  The addition of at least one heat shock step (freezing / thawing) provides a better release of the internal components of Aeromonas hydrophila. The amount of enzymatic actives is thus greater, and easier to extract and purify.

  The culture medium of the bacteria may be solid or liquid.

  In one aspect of the invention, the culture medium is therefore solid, in particular it may consist of an agar-beef extract. In this case, the separation of the bacteria from the medium at the end of the first step of the process is by gentle scraping of the solid medium, and washing, for example with distilled water.

  In another aspect of the invention, the culture medium is liquid, in particular it may consist of beef broth or any other liquid medium conventionally used for gram- bacteria, particularly commercial liquid media. In this case, the separation of the bacteria from the medium is by centrifugation, at a speed that may preferably vary between 5000 and 6000 revolutions per minute.

  In a variant of the process according to the invention, a small quantity of intestinal secretions taken directly from leeches or a total extract is added at the stage of enrichment, that is to say in stage (c). leeches fasting for at least 2 months. Any genes or plasmids of the bacteria are then stimulated.

  In the context of another variant, during the implementation of the method according to the invention on large volumes (ranging from 10 to 50 liters), colonies can be collected by scraping solid medium, in particular boxes of Petri, after 12 to 18 hours of sprouting, and suspend them thickly in beef broth. The broth is then put in an oven at 28 to 30 C for 6 to 8 hours before undergoing a centrifugation step. The centrifugation pellet is then lysed by sonication, followed by the same procedure as above.

  The present invention also relates to the bacterial extract of Aeromonas hydrophila obtainable by the method described above.

  In a particular aspect, this bacterial extract comprises at least one substance chosen from dopamine, 5-hydroxytryptamine, acetylcholine, GABA, serotonin, substance P, vasoactive peptides, enkephalin, somatostatin, destabilization , kallikrein inhibitors, granulocyte neutral proteases, kininase, cholesterol esterase, bdeline, egline, antioxidants, and 6-ketoprostaglandin.

  The bacterial extract according to the invention may advantageously be incorporated into a pharmaceutical composition which can be administered to a patient either 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 may be used, the bacterial extract may be used as an ingredient of ointments or suppositories.

  The invention therefore also relates to a pharmaceutical composition containing a bacterial extract of Aeromonas hydrophila as defined above, in combination with at least one pharmaceutically acceptable carrier.

  In one embodiment, a pharmaceutical composition, incorporating the extract according to the invention, comprises a vehicle which is preferably selected so that the formulation can be administered intravenously, such as sterile saline. For example, for hirudin, the intravenous formulation typically comprises a quantity of hirudin sufficient that, after administration to the patients, the concentration in the blood of hirudin is of the order of 10 to 100 hirudin units. ml.

  The term pharmaceutically acceptable carrier is used to refer to a nontoxic, solid or liquid diluent or encapsulant material which does not react on the active compound negatively for its effectiveness. Suitable liquid vehicles are used, for example, sterile aqueous solution, saline solution, aqueous dextrose solution, sugar solution, ethanol, glycol and oils. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, lubricating agents, etc. Oral liquid preparations may be in the form of an aqueous or oily suspension, solution, emulsion, syrup, elixir or the like, or may be presented as a reconstitutable powder product in the presence of water or water. 'another suitable carrier. Such liquid preparations may contain usual additives such as suspending agents, emulsifying agents, non-aqueous carriers. The topical application may be in the form of an aqueous or oily suspension, a solution, an emulsion or 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 the 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 treated disease. For example, a systemic dose of bacterial extract of the order of 0.01 to 100 mg / kg body weight, preferably 0.05 to 10 mg / kg, may be used.

  The invention also relates to the bacterial extract of Aeromonas hydrophila as defined above as a medicament.

  More particularly, the invention relates to the use of a bacterial extract of Aeromonas hydrophila as defined above for the preparation of a pharmaceutical composition anti-coagulant, anti-inflammatory, antithrombolytic, immunostimulant, hypotensive, lipolytic, antihypoxia, and / or against edema.

  It has been widely demonstrated in the prior art, the advantage of being able to have compounds having the properties mentioned above and can be administered via physically acceptable support such as an implantable prosthesis, and in particular a stent support. Stents are stents, commonly used in cardiovascular surgery to be implanted in a vascular conduit, including coronary artery or peripheral artery. It is known to treat vascular atheromatous diseases, which correspond to narrowing of the arteries, by balloon dilation techniques known as angioplasty techniques. The balloon is introduced into the artery, inflated to a pressure such that it crushes the deposition of atheromas. However, these techniques are not always sufficient, it is known to implant in the vascular conduit at the level of the area treated by angioplasty, a stent more commonly called stent, typically metal support acting as guardian once it is introduced to inside the artery at the level of the treated area. Such stents are for example crimped on the balloon, in a so-called rest position, prior to introduction of the balloon into the vascular conduit. Once the stent is delivered to the implantation zone inside the vascular conduit, it is deployed by expansion of the balloon so as to be brought into contact with the wall of the vascular conduit to be enlarged.

  It is furthermore known to cover stents with therapeutically active substances intended to act in particular in the implantation zone. The applicant has thus, in the past, demonstrated that it is possible to isolate at from leeches a destabilase complex comprising destabilase associated with at least one compound selected from hirudin, prostaglandin, and kallikrein inhibitors (FR-2,843,304).

  In the context of the present invention, it has been found that the bacterial extract of Aeromonas hydrophila obtained according to the process described above contains this same destabilase complex. The improved efficacy and activity of the bacterial extract makes it possible to envisage use to cover at least a portion, or all, of an implantable medical prosthesis.

  The present invention therefore also relates to an implantable medical prosthesis in which at least a portion of the prosthesis is covered with a dressing comprising a bacterial extract of Aeromonas hydrophila as defined above. The medical prosthesis may more particularly be a stent support, preferably entirely covered with a dressing comprising a bacterial extract of Aeromonas hydrophila according to the invention. The extract of Aeromonas hydrophila according to the present invention contains in particular the enzymes involved in the prevention of cardiovascular disease or stroke. This extract can then be used as a probiotic, in capsules, capsules, ampoules, or added to food products. The enzyme mixture present in the bacterial extract is also effective against excessive blood coagulation and its action on the vessels. It will therefore also find use in people with hypertension. Finally, for some of the pathologies mentioned above, the bacterial extract according to the invention may be used in the veterinary field.

  Thus, in another aspect, the invention also relates to the use of a bacterial extract of Aeromonas hydrophila as defined above as a dietary supplement, in particular for preventing and / or improving conditions related to cardiovascular diseases.

  Other aspects and advantages of the invention will be presented in the detailed description of the examples which follow, as well as in FIG. 1 which illustrates an example of a process for obtaining a bacterial extract of Aeromonas hydrophila according to the invention.

  Example 1: Obtaining a bacterial extract of Aeromonas hydrophila. The leeches are washed with sterile water. Using a sterile syringe, leeches are taken from the intestinal tract in the digestive tract and then, still in sterile condition to avoid bacteria other than Aeromonas, the sample is distributed uniformly in several petri dishes on agaric medium. beef extract. After incubation for one day at 28 ° C., the colonies grown in the dishes are reseeded on a solid support (variant No. 1, agar extract of beef) or on a liquid support (variant No. 2, liquid culture medium).

  Culture in liquid medium, with exponential growth of bacteria incubated for 24 hours at 28 C, is accompanied by a decrease in pH of the medium to 5-6. Microscopic analysis shows developing bacteria forming a rod structure. The bacteria are then separated from the culture medium by centrifugation at approximately 5000-6000 rpm for 15 minutes at room temperature.

  For the culture variant on a solid medium, the cells develop on the medium with incubation at 28 C. A few milliliters of intestinal secretions of leeches fasting for 2 months are added. The colonies of bacteria are small (diameter about 2 m) with a translucent glossy yellow appearance. Colony colors and sizes are related to the fact that Aeromonas hydrophila can be in different forms: group 1 or group 2. The groups are characterized by their appearance (especially their color) and their metabolic properties.

  The bacteria are then frozen at -80 ° C. for 12 to 24 hours and brought to room temperature. These two steps are repeated a second time.

  The cells are washed with distilled water and then ultrasonically lysed at 22-30 kHz for 2-3 minutes. The water insoluble cell walls are separated by centrifugation for 30 minutes at 17,000 - 20,000 rotations / minute at 4 ° C. The precipitate is washed several times with distilled water. The solid medium culture can be carried out up to at least 7 times in this embodiment. The final supernatant is recovered and then lyophilized.

  FIG. 1 illustrates the two variants of the method according to the invention: a variant on a solid support, and a variant in a liquid medium.

  Example 2: Determination of antithrombic activity of the final extract.

  The antithrombic activity of the extract according to the invention is demonstrated by means of appropriate tests. The inventors have shown that the antithrombic activity of the extract was due to the presence of hirudin, revealed by the prolongation of the clotting time of fibrinogen by thrombin. For example, the test-thrombin 30 IU / ml of Behringwerke described in US Pat. No. 5,563,041 may be used. In particular, the mixture comprises 0.1 ml of a solution of fibrinogen at 0.3%, 0.1%. Aeromonas bacterial extract preparation (or 0.9% NaCl solution for control) incubated for 1.5 minutes at 30 ° C., and 0.1 ml of a thrombin solution. For example, values of 5 - 78 ATU mg 1 are obtained. The anti-thrombin activity is demonstrated for the bacteria in solid support culture after the first 7-8 solid-phase re-seedings, and for the cultures on liquid support for the first reseeding.

  Example 3 Activity of Kallikrein Inhibitor of the Blood Plasma All the preparations obtained from re-seeding on solid phase (more than 8 times) and liquid phase (3 times), exhibit a kallikrein inhibitor activity independently of antithrombin activity of hirudin. This determination uses a test of the recalcification time of the blood plasma.

  The kallikrein inhibitor activity is determined by measuring the recalcification extension time of the blood plasma: the extension of the duration of one period by two corresponds to two APC units (antiprocoagulant units) of the inhibitor. The mixture used comprises 0.2 ml of blood plasma, 0.1 ml of the bacterial extract preparation of the leeches (or 0.9% NaCl for the control) incubated 1.5 minutes at 37 ° C. and 0.1 ml. 0.25 M Ca C12 solution.

  Examples are 50 - 250 APC mg-I. Example 4: All bacterial extract preparations inhibit the activation of the contact phase of blood coagulation. The results were performed using dextran sulphate activated blood plasma in the presence of an S-2302 chromogenic substrate used according to the "end point" method: the reaction was stopped 10 minutes after substrate addition.

A405: 0.2 - 0.95

Example 5

  The caseinase and BAME-esterase activity of the blood extract is practically nil.

  On the other hand, the destabilase activity is demonstrated in all the bacterial extract preparations obtained after re-seeding on solid phase. This activity is demonstrated for both flushing waters and pulp precipitates after centrifugation of bacterial leech cells. Result after 16 hours of incubation at 37 C: 49 - 825 mm2 / mg.

  The cells obtained cultured in the liquid phase do not exhibit destabilase activity. The destabilase activity was analyzed on stabilized fibrin portions.

Example 6

  Non-specific lipolytic activity has been demonstrated both for cells from a solid support culture and a culture in a liquid medium. The substrates used are in particular paranitrophenylacetate, glycerol-3- (I-C14) -triolate and cholesterol (IC14) -oleate.

  Results with the following substrates: paranitrophenylacetate: 10-8 - 26 10-8 M mg-1 after 10 minutes of incubation glycerin-3- (I-C14) -thriolate: 2 10-8 8.6 10-8 M mg-I after 60 minutes of incubation cholesterin- (I-C14) -oleate 18 10-8 42 10-8 M mg -1 after 60 minutes of incubation.

Example 7

  The bacterial extract further comprises prostacyclin-like substances identified in both the liquid phase and the solid phase (78 gg mg 1). This location used a radioimmunoassay for 6-keto-prostaglandin F1a. The bacterial extract contains ten times more prostaglandin than the secretions of the salivary glands of medicinal leeches.

  The level of thromboformation is 10 to 15%, compared to 80-95% for the control.

  The prostaglandin content was calculated from the amount of 6-keto PGF1a using the set of [125 I] -6-keto-prostaglandin Fia reagents from the Amersham test system.

Claims (16)

claims   A method for obtaining an active bacterial extract of leeches comprising the following steps: (a) culture of leeches for a period of at least 2 months without food, (b) removal in the digestive tract of Aeromonas bacteria leeches .   (c) culturing Aeromonas hydrophila bacteria in a suitable medium, (d) separating the bacteria and the culture medium, (e) lysing the cells by sonication, (f) centrifugation, (g) recovering the supernatant, characterized in that the bacteria undergo at least one heat shock before the sonication step.   2. Production process according to claim 1, characterized in that the bacteria undergo at least two thermal shocks before the sonication step, each heat shock then being followed by a centrifugation step and a supernatant recovery step. .   3. Method of obtaining according to one of claims 1 or 2, characterized in that the shock (s) thermal (s) are made by temperature decreases at -80 C tracked or separated, in the case of several thermal shocks, by one or more defrosting steps.   4. Production method according to one of claims 1 to 3, characterized in that the culture medium is solid, and in this case the separation of bacteria is by gentle scraping of the solid medium and washing.   5. Method of obtaining according to one of claims 1 to 3 characterized in that the culture medium is liquid, and in this case the separation of bacteria is by centrifugation.   6. Method according to one of claims 1 to 5, characterized in that in step (c), is added a small amount of intestinal secretions or a total extract of leeches taken or obtained from leeches fasting since at less than 2 months.   7. Method of obtaining according to one of claims 1 to 6 characterized in that the leeches are subject to a fast period of 2 months.   8. Production method according to one of claims 1 to 7, characterized in that the leeches are Hirudo medicinalis leeches.   9. Aeromonas hydrophila bacterial extract obtainable by a process according to one of claims 1 to 8.   10. Bacterial extract according to claim 9, characterized in that it contains at least one substance chosen from dopamine, 5-hydroxytryptamine, acetylcholine, GABA, serotonin, substance P, vasoactive peptides, enkephalin, somatostatin, destabilase, kallikrein inhibitors, granulocyte neutral proteases, hyaluronidase, kininase, cholesterol esterase, bdeline, egline, antioxidants, and 6-ketoprostaglandin.   11. A pharmaceutical composition containing Aeromonas hydrophila bacterial extract according to claim 9 in combination with at least one pharmaceutically acceptable carrier.   Aeromonas hydrophila bacterial extract according to one of claims 9 or 10 as a medicament.   13. Use of a bacterial extract of Aeromonas hydrophila according to one of claims 9 or 10 for the preparation of a pharmaceutical composition anticoagulant, anti-inflammatory, anti-thrombolytic, immunostimulant, hypotensive, lipolytic, antihypoxia, and / or against edema.   14. Use of a bacterial extract Aeromonas hydrophila according to one of claims 9 or 10 as a dietary supplement to prevent and / or improve conditions related to cardiovascular disease.   15. Implantable medical prosthesis in which at least a portion of the prosthesis is covered with a covering comprising a bacterial extract Aeromonas hydrophila according to one of claims 9 or 10.   16. Implantable medical prosthesis according to claim 15, characterized in that it is a stent support.