Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
  • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
  • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
  • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/41—Porphyrin- or corrin-ring-containing peptides
  • A61K38/42—Haemoglobins; Myoglobins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/60—Materials for use in artificial skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders

Definitions

• the object of the present invention is to provide globin preparations useful for administration to humans.
• These preparations can be, in particular, in the form of injectable pastes or solid implantable materials, or implants.
• human collagen which would be preferable to animal collagen, is possible from human skin tissue. But it is made very difficult because the removal of human tissue from corpses poses considerable ethical problems and requires costly tests to eliminate the risks of transmission of infectious, viral or other diseases. Preparing human collagen from placentas is expensive, complex and difficult to organize. The preparation of human collagen by modern methods of genetic recombination or cell cultures is also very expensive, which will certainly hinder the commercial development of this product.
• Globin is the constituent protein of hemoglobin which itself contains 4 peptide chains (2 ⁇ chains and 2 ⁇ chains) each associated with a heme.
• the heme consists of a tetrapyrole structure containing 1 positively charged iron atom.
• There are 4 heme molecule, responsible for the red coloring has hemoglobin.
• the processes for preparing globin have been known for a very long time and have been developed for the purpose of food application or for the preparation of injectable pharmaceutical solutions. Unlike hemoglobin which is perfectly soluble at physiological pH, globin is remarkably insoluble under the same conditions. The insoluble nature of globin under physiological conditions has hindered the development of its pharmaceutical applications until now.
• the subject of the invention is a pasty or solid preparation of globin which is insoluble at physiological pH, biocompatible, sterile and, preferably, biodegradable, in particular in the form of an injectable paste, of materials solids, for example granules, films, or insoluble implants.
• the present invention proposes to preserve the insoluble, natural character at neutral pH, of globin, for example by harvesting by centrifugation a globin protein precipitate formed by suspension of this precipitate in a pharmaceutically acceptable vehicle, for example a physiological aqueous solution of PBS type, containing 9g / l NaCl and buffered at neutral pH between 6.8 and 7.5.
• a pharmaceutically acceptable vehicle for example a physiological aqueous solution of PBS type, containing 9g / l NaCl and buffered at neutral pH between 6.8 and 7.5.
• the paste thus formed is injectable after homogenization using a fine needle.
• This paste can be prepared in the presence of viscous and lubricating agents such as solutions of triglycerides, polyethylene glycol, especially sodium hyaluronate, hyaluronic acid or other polysaccharides or mucopolysaccharides or oxidized cellulose.
• Such an additive facilitates the passage of the pasty precipitate through the finest needles (diameter 30 g) and its injection as an intradermal implant.
• the originality and the interest of this product resides in the fact that it is a protein paste, perfectly biocompatible with the surrounding tissues into which it is injected.
• This protein has not undergone any alteration or chemical modification, it is naturally insoluble as soon as it is in a physiological environment.
• a protein paste of human globin can be used in humans to fill cavities, wrinkles, skin scars or increase the volume of certain tissues (urinary, digestive sphincters, vocal cords, etc.) - This paste can be used to fill defects in the bone or cartilage and to facilitate healing.
• Insoluble globin particles can also be used in the culture of animal or human cells.
• the progressive degradation of the globin support, in contact with the cells which digest it progressively, can additionally provide a means of cell nutrition complementary or alternative to the liquid culture media used up to now.
• Homologous human globin is preferable and makes it possible to avoid any immunological reaction of the patient to be treated, during or after the injection.
• This product therefore represents an important advantage compared to collagen which up to now has been prepared from animal skins (calf, pork, etc.) and which requires a certain number of precautions and conditions to avoid immunological reactions in the patients.
• globin remains soluble at acidic or basic pHs and under these conditions can be sterile filtered through porous membranes.
• such solutions can be treated as protein solutions and make it possible to produce products such as: sponges, films, granules, using or combining the techniques of drying, lyophilization, crosslinking , precipitation.
• Globin is easy to purify from red blood cells from animal or human blood. Human red blood cells are available in sufficient quantities to from expired donations remaining in stock in blood transfusion centers and for which all the preliminary health tests were carried out at the time of collection.
• the preparation of insoluble, injectable globin or other biomaterials based on globin therefore represents new biomedical applications making it possible to efficiently valorize unused blood or expired blood donations and to avoid or reduce their destruction.
• the implementation of the invention is also possible from a blood sample from the patient to be treated of approximately 5 to 100 ml, and its transformation into autologous globin with the same methods as for large volumes, then the injection of the paste obtained for the correction of wrinkles from the same patient or other applications such as the healing of chronic wounds.
• the number of syringes prepared from a patient sample can be large and allow the patient to be treated for several years.
• the human placenta which is expelled after delivery contains blood which is generally destroyed by incineration, but which can also be used for the invention.
• Donor's blood bags are officially controlled by blood transfusion organizations, thanks to biochemical, bacteriological, serological examinations and screening tests for different viruses and other infectious agents.
• placental blood it would obviously be necessary to carry out the same examinations on samples of blood from the umbilical cord or from the mother before being able to collect, store and extract the blood from this raw material.
• the tests to be performed can be simplified. Carrying out the invention first of all requires collecting and purifying the red blood cells in these samples of blood, or blood liquids, by simple operations which are already known.
• the red blood cells are recovered by centrifugation at low speed.
• the plasma supernatant is separated and replaced by a physiological saline liquid of PBS type; containing 9g / l of NaCl and buffered to neutral pH.
• the pellet of purified red blood cells is added with 1 or 2 volumes of distilled water to produce an osmotic shock which causes the lysis of the membranes of the red blood cells and releases the hemoglobin in concentrated and purified solution.
• a high speed centrifugation step (10 to 20,000 rpm) eliminates the membrane and cell debris in the pellet.
• a final stage of filtration of the supernatant on a membrane with a porosity of 0.2 micron makes it possible to prepare a purified and sterile hemoglobin solution, devoid of particles and derivatives of tissue, cell or membrane origin.
• Heme-Globin cleavage at acid pH was described in the presence of alcohol by SCHULZ in 1898.
• ANSON and MIRSKY in 1930 then ROSSI-FANELLI et al. in 1958 use acetone in the presence of acid at 0 ° C.
• TEALE (1957) prefers the use of methyl ethyl ketone in place of acetone.
• AUTIO et al. (1984) separate the globin at acid pH thanks to the absorption and precipitation of heme with soluble carboxymethylcellulose.
• the globin thus prepared is soluble at acidic or alkaline pH but becomes insoluble as soon as the pH of the aqueous solution is neutralized between pH 6 and 8.
• Solubilization tests at neutral pH were carried out by STRUMIA et al. in 1951 and 1952 by a prolonged alkaline treatment which leads to a partial deamidation of g-Lobine at the level of the asparagine and glutamine residues transformed respectively as aspartic acid and glutamic acid (VARS 1952).
• Other solubilization tests were carried out by VOLCKMANN in 1988 by succinylation.
• the verification of the interest of the invention can be easily carried out from a preparation of rabbit globin.
• the physiological precipitated globin paste thus prepared can be injected subcutaneously in different places on the back or the wall of the rabbit. It is easy to verify safety by the absence of erythema locally. The persistence of the product under the skin can be observed by palpation over time. The absence of antigenic power of the product can be verified by immunization of rabbits with or without Freund's adjuvant by subcutaneous and intramuscular route. Blood samples taken after immunization makes it possible to verify the absence of anti-globin or anti-hemoglobin antibodies by the usual control tests.
• the blood is recovered in the presence of heparin or in the presence of sodium citrate to avoid its clotting. 210 ml of blood are thus obtained which are centrifuged for 30 minutes at 2500 rpm. The supernatant containing the plasma is removed with a pipette and the pellet is washed 5 times with 3 volumes of PBS buffer, containing 9 g / 1 NaCl and buffered to pH 7.2. The final pellet, washed, is added, with stirring, an equal volume of distilled water to lyse the red blood cells. The final suspension is centrifuged at 12,000 rpm to remove cell and membrane debris. The supernatant is filtered through a cellulose acetate membrane with a porosity of 0.22 microns. 82 ml are obtained containing 97 g / l of hemoglobin.
• Hemoglobin is transformed into globin according to the technique described by TAYOT and VERON (1983). This hemoglobin solution is poured with stirring into 275 ml of 96% ethanol containing 1 ml of concentrated HCL. The pH is adjusted to 3. The final concentration is 74% ethanol and 22 g / l of hemoglobin at acidic pH. 3 g of L4S activated carbon of the CECA brand are added with vigorous stirring for 15 hours at 4 ° C.
• the suspension is centrifuged at 15,000 rpm for 30 minutes to remove the char in the form of a pellet.
• the supernatant containing the discolored acid globin is filtered through a series of porous membranes until the porosity is the lower (0.2 micron) to remove fine particles of carbon.
• the filtrate is diluted with an equal volume of distilled water, the pH is adjusted to 7.4 by adding NaOH and the globin precipitates massively. After 15 hours, the globin precipitate is recovered by centrifugation, then washed twice with 3 volumes of PBS physiological solution, containing 9 g / l NaCl and buffered to pH 7.4. 58.2 g of globin precipitate at pH 7.4 are collected.
• the precipitate is homogenized by successive transfers between 2 syringes of volume 5 ml connected by a connector with an internal diameter of 1 to 0.2 mm, by successively pressing the plunger of each syringe to pass the precipitate into the other syringe.
• the homogenized precipitate is distributed into 1 ml syringes. It is possible to expel the globin paste precipitated from the syringe, through fine needles of 24 or 27g diameter.
• the concentration of globin in the dough can be adjusted to values between 30 and 150 mg / g.
• 210 ml are obtained containing 52 g / l of hemoglobin which are stored at 4 ° C.
• An equal volume of 210 ml of 0.1 N HCl at 4 ° C. is added, then the whole is poured into 4 1 of acetone, containing 40 ml of ICl HCl.
• the suspension is stirred vigorously and left to stand for 1 hour at room temperature, under a chemical hood.
• the heme dissolved in acetone is removed by filtration on a porous fabric and the globin precipitate is recovered, washed with acid acetone and dried under a stream of air.
• various mineral acids sulfuric, phosphoric, etc.
• carboxylic such as acetic acid, oxalic acid, or citric acid, for example, can be used in place of hydrochloric acid to acidify hemoglobin solution before discoloration.
• Another variant of this process consists in precipitating the acidic hemoglobin solution before it becomes discolored.
• the precipitation can be carried out by adding NaCl at a concentration of 40 to 60 g / l.
• the precipitate of acid hemoglobin is then discolored by suspension in a sufficient volume of ethanol and / or acetone.
• the pigment passes into solution in ethanol or / and acetone; the globin remains in precipitated form and can be collected by filtration on a porous fabric. Thanks to the elimination of any aqueous phase, this variant makes it possible to reduce the necessary volume of ethanol and / or acetone by a factor at least equal to 5.
• the globin is returned to an aqueous solution at a pH of between 2 and 3.
• the aqueous solution of acidic globin is sterile filtered through a membrane with a porosity of 0.2 micron, then precipitated by neutralizing the pH by adding NaOH to pH 7.4.
• Syringes of globin paste, precipitated at neutral pH can be prepared as in the previous example.
• the neutralization of the acidic globin solution can be carried out by adding sodium hyaluronate at pH alkaline. In this case, an insoluble globin paste complexed and impregnated with hyaluronate is formed, providing a lubricating function which improves the injectability through very fine needles (diameter 30g).
• Example 1 The process of Example 1 is carried out from a controlled and expired blood pellet obtained from a blood transfusion center. Syringes containing a paste of human globin, precipitated, biocompatible and implantable by injection, are obtained.
• Example 1 or 2 The process of Example 1 or 2 is carried out until the globin precipitate is obtained at pH 7.4, before washing. This precipitate is dissolved, again, in 3 volumes of 0.1 M NaOH soda at 1M at 20 ° C for 1 hour, with stirring.
• the solution is then neutralized by adding an equal volume of HCl of the same molarity and the pH of the suspension is adjusted between 7 and 7.4.
• the globin precipitate is then collected by centrifugation, then washed in PBS physiological solution as in the previous examples.
• the precipitated globin paste which can be supplemented with hyaluronic acid, or other viscous products and biocompatible lubricants: triglycerides, polyethylene glycol, oxidized cellulose, chitosan, etc. is distributed into syringes and the injectability of the product obtained is checked again using very fine needles for intradermal use.
• This alkaline treatment of globin makes it possible to improve the guarantees of health safety of the product without modifying in a way significant the insoluble nature of globin at neutral pH.
• Example 5 Preparation of a globin paste precipitated and crosslinked with glutaraldehyde. This treatment is possible to increase the resorption time of the implant. The final globin precipitate is suspended at 2% in PBS. Glutaraldehyde is added with stirring at a concentration of 1 mg / g of precipitate. After incubation for 1 hour at 20 ° C, the globin precipitate is washed and put into syringe as in the previous examples.
• crosslinking agents such as dialdehydes or polyaldehydes can be used, in particular polysaccharides oxidized by the periodic acid such as oxidized dextran, oxidized starch, or oxidized hyaluronic acid.
• Another method consists in distributing an acidic solution of soluble globin, sterile filtered in a first syringe and a second alkaline solution, sterile filtered in a second syringe.
• the pH of each syringe is adjusted so that their subsequent mixing is at neutral pH.
• the connection of these two syringes thanks to a sterile connector, makes it possible to produce a sterile, homogeneous mixture, of neutral pH, by successive transfers from one syringe to the other.
• a sterile precipitate is obtained by spontaneous precipitation of globin.
• the suspension obtained can be concentrated by extrusion through fine needles which allow only the aqueous phase to pass.
• Sterilization of the syringes prepared according to any one of the preceding examples can be carried out by irradiation at a dose of between 5 and 30 kilogray.
• the various preparations of globin are insoluble before and after their sterilization by irradiation. In both cases, the globin insoluble at neutral pH becomes soluble if acidification is carried out at pH 3 with any acidic aqueous solution.
• Example ⁇ Production of an insoluble gel or film from unmodified soluble globin.
• a solution of soluble globin is produced by dissolving the acetone globin powder at pH 3, at a concentration of 20 to 120 mg / ml in aqueous solution. This solution is sterile filtered on a porosity membrane 0.2 ⁇ , then adjusted to pH 5 by addition of sterile sodium hydroxide NaOH 1 N with stirring.
• oxidized starch at pH 3.5, or another cross-linking aldehyde, or polyaldehyde, containing at least 5 carbon atoms per molecule, at a concentration of 0.5% with stirring for 5 min.
• the sterile mixture is poured onto a flat surface to obtain a thickness of 1 to 3 mm of liquid, at a temperature of 20 to 37 ° C, under laminar flow.
• the liquid product gels gradually thanks to the crosslinking of the globin chains induced by the oxidized starch, then dehydrates under the current of air, if it is desired to obtain a film.
• the final film of thickness between 20 and 200 ⁇ , depending on the initial concentration of material, can be sterilized by beta or gamma irradiation or with ethylene oxide.
• Well known filming agents can be added such as collagen, gelatin, hyaluronic acid, oxidized cellulose or other polysaccharides or mucopolysaccharides, polyethylene glycol, glycerol etc. Such an additive makes it possible to give flexibility and / or resistance to the film.
• Such a film can be used alone to protect a skin or surgical wound, promote healing, or can be combined with various prostheses (vascular prostheses, reinforcing mesh, porous matrices) to make them waterproof, or improve their biocompatibility, or confer anti-adhesion properties or an adhesive character, or accelerate the cell colonization of these prostheses, according to known techniques and already used with other products.
• various prostheses vascular prostheses, reinforcing mesh, porous matrices
• a film from globin soluble at pH 5 as previously, but without introducing a crosslinking agent.
• the final crosslinking of the dried film is carried out by the final irradiation which creates covalent bonds between the globin chains.
• Such a film can then be glued to the tissues using biologically compatible adhesives, reactive with the amino groups of globin.
• the polyaldehydes obtained by periodic oxidation of polysaccharides can be used.
• oxidized dextran or oxidized starch are preferred.
• Example 9 Biomedical applications of insoluble globin particles as a support for cell cultures.
• the globin precipitate paste at neutral pH, sterile, obtained as in any one of the preceding examples, is incubated for example with DMEM medium for cell cultures, at a temperature in the region of 37 ° C.
• a suspension is obtained in which the cells are introduced at a density of 10,000 to 100,000 cells / ml. After stirring for 30 minutes and decanting for 1 hour to 15 hours, the cells attach to the globin particles and multiply on their surface during the duration of the culture, which can be 3 to 12 days.
• the cell culture medium is chosen according to the cell type according to the knowledge currently published.
• the cell suspension fixed to the globin particles can be concentrated by spontaneous decantation.
• the cell paste obtained can be put into syringes and injected as a biocompatible cellularized implant for various therapeutic applications known today.
• the culture of skin fibroblasts can allow the preparation of implants cellularized for skin healing or connective tissue filling applications.
• the culture of chondrocytes can allow the preparation of cellular implants for filling and healing applications of superficial cartilage wounds.
• the culture of osteoblasts can allow the preparation of cellular implants for filling and healing applications of fractures or losses of bone substance.
• stem cells in particular of embryonic origin, or of umbilical cord blood, or bone marrow, or isolated from different adult tissues, can be cultured on these globin particles and fulfill the desired functions after injection. or implantation of the cellularized paste.
• the preparations according to the invention, and in particular the insoluble globin syringes prepared according to any one of Examples 1 to 7 can be used in the following nonlimiting applications: Filling of wrinkles and skin defects - Filling of connective or sphincter tissues for applications in urology: vesicoureteral reflux in children, stress incontinence in women; in ENT: volume correction of the vocal cords. Hemostatic plug for percutaneous arterial wounds.
• Skin healing by using globin paste alone or in combination with other healing products or growth factors.
• Healing of cartilage or bone by using globin alone or in combination with other healing products: calcium phosphate, calcium carbonate, hydroxyapatite, growth factors like BMP.
• the invention also relates to methods of treating the human or animal body comprising at least one step of administering a therapeutically effective amount of an injectable or implantable preparation according to the invention to a patient who has the need thereof.
• These methods include in particular the administrations corresponding to the abovementioned applications, by the parenteral or surgical injection or implantation routes, or by the cutaneous route.
• I-Method for preparation from human erythrocytes I-Method for preparation from human erythrocytes.

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