Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs

Definitions

• the present invention relates to a process for obtaining a liquid comprising extracting from a mollusk the liquid located between the shell and the body of the mollusk, the liquid capable of being obtained by this process, its use in the pharmaceutical field, and in particular to improve skin healing, to regenerate tissues, or to promote osteogenesis or bone mineralization. It also relates to compositions, in particular pharmaceuticals, medical devices or food supplements containing it.
• mother-of-pearl has made it possible to offer effective solutions in specific cases.
• implants made from mother-of-pearl have been proposed for endosseous repairs.
• Powdered mother-of-pearl has also been suggested as a filling material where it then shows biodissolution properties; there is a gradual replacement with newly formed compact and spongy bone depending on the nature of the recipient bone.
• the use of mother-of-pearl as a biomaterial is unfortunately not always easy or possible in many cases, for example during large local repairs of degenerated tissue or loss of substances.
• the applicant has just developed a technique for obtaining the extrapalleal fluid located between the shell and the body of a mollusk by extraction of said fluid (or liquid).
• the fluid thus obtained can retain its self-organizing properties and that the latter can be used, even in a context external to the animal.
• this fluid is of great industrial interest because of its physical nature, since it can be in many forms, such as for example in the form of a liquid, a film, a coating, fibers or in the form of a porous material. Thus, it can easily be handled and used in different forms, while retaining its biological properties.
• the present invention therefore relates to a process for obtaining an extrapalleal fluid comprising recovering the extrapalleal fluid located between the interior of the shell and the body of a mollusk, in particular the mantle of said mollusk.
• the fluid obtained is thus pure and taken in its entirety, in particular unmodified and unprocessed, after recovery, by a process modifying its intrinsic qualities. It can then be formulated, to be used in particular in a pharmaceutical composition, a medical device or food supplements.
• the pharmaceutical composition according to the invention is in particular intended to improve the healing phenomena, to regenerate tissues, biological or bone, to promote osteogenesis or bone mineralization.
• the method for preparing a liquid therefore comprises a step of recovering the extrapalleal fluid located between the shell and the body of a mollusk, in particular the mantle of said mollusk, said fluid having been extracted from 'a mollusk.
• the fluid thus recovered corresponds to natural secretions of the mollusk which are found on the external surface of the mantle of the molluscs on the side of the shell.
• this liquid even once extracted and therefore outside of its natural context, could be used for its regenerative and mineralizing properties, and thus promote the production of the shell (organomineralized material) of a mollusc, and more broadly substitute for, or regenerate, secretions from any other tissue known to form an extracellular matrix.
• the liquid thus has the advantage of regenerating the tissues, biological or bone, in particular by stimulating the activity of the cells which form said tissues or by providing the basic materials useful for said regeneration, and by promoting osteogenesis or bone mineralization. .
• tissue regeneration is meant the reconstruction and renewal of any tissue that has suffered damage or alteration (or injured tissue).
• mineralization we mean the action (capacity) which consists in providing mineral elements or which favors the fixing of mineral elements on organic matter.
• mineral species crystals, atoms, ions, molecules containing “mineral” elements and / or trace elements, as opposed to organic elements.
• the regeneration of biological or bone tissue generally consists of reconstructing or renewing all or part of the altered tissue, in particular thanks to molecules of specific structure, to “signal” molecules and to organic and / or mineral nourishing elements present in the liquid obtained according to the invention. In addition, no intolerance was detected during the application of the liquid thus extracted and / or subsequently modified on or in a mammal.
• the step of removing or extracting the liquid is generally carried out as follows.
• the mollusc from which the liquid is extracted is generally a mono- or bi-valve mollusk, pearly or not, advantageously pearly.
• the molluscs are in particular oysters and more specifically oysters of the genus Pinctada and more particularly of the genus Pinctada species margaritifera or maxima.
• Oysters can also be chosen from those of the genus Ostrea, in particular of the species edulis, or of the genus Crassostrea, in particular of the species gigas. They may also be cephalopods, such as the nautilus, or gastropods, such as the periwinkle.
• the mollusc used can come from aquaculture or land farming.
• the mollusc can be sacrificed before, during or after the process according to the invention. It can be used dead or alive.
• the mollusk for example can be sacrificed before the process according to the invention, taking care to keep at least partially (preferably completely) the liquid to be extracted in the shell.
• the extraction process or step is a non-invasive method.
• the liquid located between the interior of the shell and the body of the mollusk, in particular the mantle of said mollusk corresponds to natural secretions of the mollusk which are found on the external surface of the mantle of the molluscs on the side of the shell. and therefore which are outside the body of the mollusk proper.
• the shell of the mollusc is preferably peeled off in whole or in part.
• the mollusk is bi-valve, at least one of the two valves is raised so as to be able to access the body of the mollusk, prior to extraction.
• the detachment (or separation) of the liquid from the shell can be carried out manually, generally using a suitable tool (such as: scraper or flexible spatula).
• a suitable tool such as: scraper or flexible spatula
• the detachment of the liquid from the shell is carried out chemically.
• an enzymatic reaction advantageously non-denaturing, and generally at room temperature, can also be implemented so as to separate, more finely, the liquid from the shell.
• this enzymatic reaction is carried out in the presence of at least one protease, such as for example trypsin.
• the reaction can then be stopped by an anti-protease or by simple dilution.
• the liquid is then withdrawn from the surface of the shell using an appropriate instrument, in particular using a syringe (in particular provided with a needle).
• the liquid withdrawn is advantageously sterilized or decontaminated by any method known per se, in particular by filtration, for example on a Millipore® filter (example: 0.20 ⁇ m). It can also be dehydrated, in particular by lyophilization. Liquid can also be frozen, preferably just after recovery or after filtration.
• the sampling can be carried out on living molluscs, for example in the case of large molluscs, such as pearly bivalves of the Pinctada family.
• This variant preferably consists in keeping the shell of the living bivalve open using an instrument, such as a wedge and / or forceps forceps (or retractor).
• the instrument is preferably chosen so as not to injure the animal, in particular it is chosen so as not to irreversibly damage its metabolism.
• the mantle is preferably lifted in whole or in part to separate it from at least one of the shells.
• the liquid is then taken from the surface of the shell using an appropriate instrument, such as in particular a scraper or flexible spatula.
• a scraper plastic
• the liquid is harvested using a syringe generally equipped with a needle, preferably with a diameter between 1 mm and 5 mm.
• any other material suitable for harvesting the liquid can be considered.
• the process for obtaining the fluid according to the invention may also comprise subsequent non-denaturing steps of the fluid thus recovered, such as steps of filtration, decontamination, sterilization, freezing / thawing, changes in viscosity, and / or various layouts.
• the liquid obtained according to the method described above can be used such that, in its pure state (taken in its entirety), in particular it is unmodified and unprocessed, after recovery, by a process modifying its intrinsic qualities (denaturing stages), in particular by a demineralization process.
• the liquid obtained can be formulated subsequently according to its subsequent use.
• the subsequent use of the liquid may require a suitable method of preparation.
• Experiments carried out with the liquid thus recovered show that it is very flexible to handle and that it adapts very easily to all kinds of constraints linked to its implementation. It can thus undergo several thawing and re-freezing without affecting its biological properties. It is the same for lyophilization.
• a person skilled in the art will take care not to modify the intrinsic qualities of the liquid thus extracted and will therefore avoid subjecting this liquid to any operation detrimental to its interest, such as a demineralization operation.
• the fluid may for example be in the form of a film or a coating.
• Any method known per se for forming a liquid or a film can be used. It can for example be spread on a smooth or porous surface or any other support and form a film (film-forming character of the fluid obtained).
• the film or coating thus formed retains the regenerating and healing character of the liquid. It can thus constitute an organic biological film on the surface, with a mineralizing power.
• the thickness of the film can vary to a large extent depending on its end use.
• a thick film can for example be obtained with the triple property: (i) sealing, (ii) self-supporting structure, (iii) intrinsic mineral contribution, while having the regenerating and healing character.
• This material formed by an intimate association between organic matter and mineral matter, forms a film on any surface and crystallizes polymorphically depending on the condition and texture of the surface.
• the film thus obtained can be used on its support or be removed from the support for later use.
• the liquid can also be used later in the form of fibers. Any method known per se for forming a liquid into fibers can be used.
• This liquid has the advantage of being spinnable over a wide range of viscosities. In particular, a very simple stretching technique, in particular using tweezers, makes it possible to stretch fibers from the liquid. It is possible in this way to use it in situ (locally) or in another way, as a porous, invasive filling material, to fill as needed a given volume.
• the invasive nature of this material allows in particular biological materials, such as cells, to penetrate there and exert their effects in association with the properties of the shaped liquid.
• the same material can be used to bridge two distant tissue banks.
• the same principle can be applied to very large volumes, in particular by coating fibers of another chemical nature (for example carbon fibers).
• the fibers thus coated have a coating on their surface comprising the constituent elements of the liquid according to the invention and can be used to provide, for example, a healing interface.
• the fluid can thus come in different forms, including liquid, films, coatings and fibers.
• it can also be in the form of a porous material, in particular by using, during its shaping, a porous support.
• the porous material may be a film, a coating or be in any other form, such as a spherical shape.
• the fluid according to the invention generally comprises a mixture of the constituent elements of living matter, in particular a mixture of proteins, proteoglycans, polysaccharides, lipids and mineral salts, this is more particularly the case for a mother-of-pearl mollusc, advantageously an oyster of the genus Pinctada (generally taken in its natural biotope).
• a mother-of-pearl mollusc advantageously an oyster of the genus Pinctada (generally taken in its natural biotope).
• the nature and quantity of these elements can vary to a large extent, particularly in function of the mollusc, its biotope, the season of the sample and the conditions of the sample.
• composition of this liquid preferably comprises at least the following compounds: proteins in an average amount of 0.5 mg / mL, proteoglycans in an average amount of 3 mg / mL, polysaccharides in an average amount of 1.5 mg / mL , lipids in an average amount of 1 mg / mL and mineral salts in an average amount of 7.5 mg / mL.
• a quantitative analysis of the trace elements present in a specific liquid (identical to that identified above) according to the invention was carried out, in particular by quantitatively measuring the trace elements present by analysis by neutron activation (ANA) and spectrometry of mass coupled to a plasma torch (ICP-MS).
• ANA neutron activation
• ICP-MS spectrometry of mass coupled to a plasma torch
• composition of this liquid preferably comprises the trace elements chosen from: S, Mg, La, Zn, Br, Ce, Fe, Mn, Cl, Cu, K, Sr, Na and Ca
• trace elements chosen from: S, Mg, La, Zn, Br, Ce, Fe, Mn, Cl, Cu, K, Sr, Na and Ca
• the amounts of these trace elements are respectively as follows: 0.02; 2; 0.28; 0.4; 1.78; 5.5; 13.6; 50; 296; 143; 582; 1000; 5420 (expressed in ⁇ g / g) and 38.8 (expressed in ⁇ g / 100g).
• a quantitative analysis of the amino acids present in free form or bound in a fluid according to the invention was carried out, in particular by quantitatively measuring the amino acids present by the amino acid analyzer Beckmann 6300 using the conventional method with ninhydrin. The result of this analysis is given in FIG. 1 (% by weight of amino acids relative to the total weight of amino acids).
• the present invention also relates to the fluid capable of being obtained by the process as described above.
• the liquid (or fluid) can be not only the liquid obtained directly after the recovery stage described above, but also that obtained after subsequent non-denaturing stages, such as stages of filtration, decontamination, sterilization, freezing / thawing, changes in viscosity, and / or various forms, such as in particular those identified above.
• the fluid according to the invention is used as a medicament.
• the present invention also relates to a composition, in particular pharmaceutical, characterized in that it comprises the fluid as defined above, in particular in combination with a pharmaceutically acceptable excipient.
• the liquid used as a medicament or in the composition detailed below may be that obtained directly after the recovery stage described above, but also that obtained after subsequent stages of filtration, decontamination, sterilization, freezing / thawing, changes in viscosity, and / or various forms, such as in particular those identified above.
• the administration of the pharmaceutical composition or of the medicament according to the invention can be carried out by topical, enteral or parenteral route.
• the pharmaceutical composition is packaged in a form suitable for topical application, that is to say applied in situ to the place where the tissue is injured.
• the pharmaceutical composition can be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or vesicles lipidic or polymeric allowing controlled release.
• the composition can be in the form of solutions or suspensions for infusion or for injection.
• the pharmaceutical composition is intended for local use on or in the injured tissue.
• the pharmaceutically acceptable carrier is in particular an excipient suitable for topical application.
• the pharmaceutical composition according to the invention is more particularly intended for the treatment of the tissues mentioned above, in particular for the treatment of the skin and bones of a mammal, in particular human. It can be in the form of ointments, creams, milks, ointments, powders, soaked pads, solutions, gels, sprays, lotions or suspensions. It can also be in the form of microspheres or nanospheres or lipid or polymeric vesicles or of polymeric patches and hydrogels allowing controlled release.
• This topical composition can be in anhydrous form, in aqueous form or in the form of an emulsion (water / oil, oil / water emulsion or multiple emulsion).
• the liquid according to the invention is used, preferably topically, at a concentration generally between 0.02% and 20% by weight, preferably between 0.25 and 10% by weight, and advantageously between 0.5 and 5%, relative to the total weight of the composition.
• the therapeutic composition according to the invention is characterized in that it contains, in association with inert excipients, a therapeutically effective amount of the liquid according to the invention, in particular for improving skin healing, for regenerating the tissues, or to promote osteogenesis or bone mineralization.
• Tissues include soft tissue or skeletal, including in particular the skeleton, bones, cartilage, teeth, or any other tissue, in particular the skin and its appendages.
• the composition according to the invention makes it possible to treat in particular bone, cartilaginous or dental disorders.
• the invention also resides in a method for improving skin healing, for regenerating tissues, in particular those identified above, or for promoting osteogenesis or bone mineralization, comprising administration to subjects in need of such treatment a therapeutically effective amount of the liquid as defined above.
• treatment designates preventive, curative, palliative treatment, as well as the management of patients (reduction of pain, improvement of the quality of life, slowing down of the progression of the disorder, trauma or illness), etc.
• the method or treatment can also be carried out in combination with other ingredients or treatments, such as in particular other active compounds to improve skin healing, to regenerate tissues, or to promote osteogenesis or bone mineralization.
• compositions or medicaments according to the invention can also comprise at least one other therapeutically active ingredient, for simultaneous, separate or spread over time use.
• compositions or medicaments according to the invention advantageously comprise one or more inert excipients or vehicles, that is to say pharmaceutically inactive and non-toxic. Mention may be made, for example, of saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art.
• the essays may contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, preservatives, etc.
• Agents or vehicles usable in formulations are in particular methylcellulose, Phydroxymethylcellulose, carboxymethylcellulose, cylclodextrins, polysorbate 80, mannitol, gelatin, lactose, vegetable or animal oils , acacia, etc.
• the compositions can be formulated in the form of an injectable suspension, gels, oils, tablets, suppositories, powders, capsules, capsules, etc., optionally by means of dosage forms or devices ensuring sustained and / or delayed release.
• at least one agent such as cellulose, carbonates or starches is used.
• compositions as described above can of course also contain pharmacodynamically active additives or a combination of these additives, and in particular: wetting agents, emollients, hydrating agents, such as glycerol, PEG 400 or else urea .
• compositions may also contain flavor enhancers, preservatives such as parahydroxybenzoic acid esters, stabilizers, moisture regulators, pH regulators, osmotic pressure modifiers , emulsifying agents, antioxidants, such as ⁇ -tocopherol, butylhydroxyanisole or butylhydroxytoluene.
• flavor enhancers such as parahydroxybenzoic acid esters, stabilizers, moisture regulators, pH regulators, osmotic pressure modifiers , emulsifying agents, antioxidants, such as ⁇ -tocopherol, butylhydroxyanisole or butylhydroxytoluene.
• the administration can be carried out by any method known to those skilled in the art, in particular by the oral, topical or injection route, typically by the intraperitoneal, intra-cerebral, intra-thecal, intravenous, intra-arterial or intramuscular route. Topical or oral administration is preferred. For long-term, non-topical treatment, the preferred route of administration will be sublingual, oral or transcutaneous.
• the compounds are generally packaged in the form of liquid suspensions, which can be injected using syringes or infusions, for example. It is understood that the flow rate and / or the dose injected, or in general the dose to be administered, can be adapted by a person skilled in the art according to the subject to be treated, the disorder, the mode of administration, etc. It is understood that repeated administrations can be carried out, optionally in combination with other active ingredients or any pharmaceutically acceptable vehicle (buffers, saline, isotonic solutions, in the presence of stabilizing agents, etc.).
• the invention relates to a device, more specifically suitable for subcutaneous or percutaneous injection, comprising the liquid as defined above and a physiologically acceptable excipient or support.
• This device can in particular be in the form of syringes or infusions.
• the medical device of the present invention may be intended to be implanted in or on the body of a mammal, the liquid as defined above may be located inside a membrane suitable for this use .
• the invention relates to a food supplement comprising the liquid as defined above.
• This food supplement is in particular intended to improve cutaneous healing, to regenerate tissues, or to favor osteogenesis or mineralization of bones or teeth.
• the invention can be used in mammals, in particular in humans. Other aspects and advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.
• Example 1 Recovery of the liquid from a sacrificed animal The mollusk is sacrificed so that the liquid remains in the shell. We take off the coat carefully; an enzymatic reaction is then used to completely detach the liquid from the shell. The reaction is then stopped by an anti-protease. The liquid is then taken from the surface of the shell. As soon as collected, the liquid can be frozen. It can be sterilized by filtration on a 0.20 ⁇ m millipore and can be freeze-dried while retaining its properties.
• An oyster of the species of Pinctada margaritifera is half-opened thanks to a retractor according to the practices well known to those skilled in the art in pearl farming.
• the excess water still present is discharged.
• the animal is arranged horizontally.
• the mantle is delicately separated from the lower shell using forceps, then the liquid is withdrawn using a syringe, for example.
• the liquid is: - filtered - lyophilized - a few weeks later, the liquid is reformed with sterile water - it is spread in the form of a thick film.
• the oyster is fixed and the coat is peeled off. In this way, it is possible to collect the liquid from the first valve. The animal is then turned over to operate on the other side. In this case, an amount of 50 to 100 ⁇ l per valve is obtained.
• the liquid collected from a population of around twenty animals is subjected to conditioning similar to that of Example 2.
• the liquid is: - filtered - lyophilized - a few weeks later, the liquid is reformed with sterile water - it is spread in the form of a thick film
• the liquid is reconstituted even after lyophilization, that it forms a film on a polypropylene support.
• This film solidifies by self-organization and self-healing.

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• Health & Medical Sciences (AREA)
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• Pharmacology & Pharmacy (AREA)
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