[go: up one dir, main page]

WO2011060553A1 - Formulation et procédé de préparation rapide de solutions de chitosane isotoniques et cytocompatibles sans provocation de la précipitation du chitosane - Google Patents

Formulation et procédé de préparation rapide de solutions de chitosane isotoniques et cytocompatibles sans provocation de la précipitation du chitosane Download PDF

Info

Publication number
WO2011060553A1
WO2011060553A1 PCT/CA2010/001856 CA2010001856W WO2011060553A1 WO 2011060553 A1 WO2011060553 A1 WO 2011060553A1 CA 2010001856 W CA2010001856 W CA 2010001856W WO 2011060553 A1 WO2011060553 A1 WO 2011060553A1
Authority
WO
WIPO (PCT)
Prior art keywords
cytocompatible
chitosan
salt
polymer composition
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CA2010/001856
Other languages
English (en)
Inventor
Caroline Hoemann
Jun Sun
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ecole Polytechnique de Montreal
Original Assignee
Ecole Polytechnique de Montreal
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ecole Polytechnique de Montreal filed Critical Ecole Polytechnique de Montreal
Publication of WO2011060553A1 publication Critical patent/WO2011060553A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • 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
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices

Definitions

  • the present disclosure relates to a two-part cytocompatible composition for use in repairing tissue of a patient comprising an admixture of a first-part liquid polymer solution with a second-part liquid salt solution, the composition being free of polymer precipitation at room temperature and the composition being admixed just prior to administration or use in the patient.
  • a wound-stimulatory implant consisting of an autologous, in situ solidifying scaffold-stabilized blood clot.
  • the scaffold- stabilized clot is generated by mixing a cytocompatible polymer solution such as glycerol phosphate (GP)-buffered chitosan with unclotted whole blood (International application publication No. WO 02/000272, the content of which is enclosed herewith by reference).
  • GP glycerol phosphate
  • thermogelling chitosan- GP solutions tended to gel or precipitate with extended storage at room temperature.
  • Lowering the pH by reducing the level of disodium GP resulted in formulations that showed a slower time to gel during room temperature storage but these pH 6.0 chitosan solutions were hypotonic ( ⁇ 70 mOsm) and not cytocompatible, as evidenced herein below:
  • the initial chitosan-GP formulation that improved cartilage repair in animal models consisted in 1.5% to 1.7% chitosan, 70 mM HCI and 135 mM disodium ⁇ -glycerol phosphate, pH 6.7 to 6.8 (390 to 520 mOsm) (Hoemann et al., 2007, Osteoarthritis and Cartilage, 15: 78-89; Hoemann et al., 2005, Journal of Bone and Joint Surgery-American Volume, 87A(12): 2671-2686; Chevrier et al, 2009, Osteoarthritis and Cartilage, 15: 316-327).
  • the chitosan powder was dissolved in 77 mM HCI to achieve 90% to 100% chitosan protonation (pH 4.5 to 5.0), autoclave sterilized, and then combined drop-wise at 4°C with 1/10 th volume of filter-sterile 1.35 M disodium ⁇ - glycerol phosphate pH 9.3.
  • Chitosan-GP can also serve as a delivery vehicle for biologically active therapeutics (Chenite et al., 2000, Biomaterials, 21 : 2155-2161 ; International application publication No. WO 02/000272). Therefore, for additional practical reasons, it would be desirable and advantageous to be provided with a method and formulation that favors non-covalent coupling of specific biological factors with chitosan, to immobilize the factor at the site of implantation. However it is currently unknown how to couple proteins non- covalently with chitosan particles
  • a two-part cytocompatible system for providing a cytocompatible polymer composition for use in repairing tissue(s) of a subject in need thereof.
  • the system comprises at least two containers, the first one comprising a first-part liquid solution of a polymer and the second one comprising a second-part liquid solution of a salt.
  • This system enables the generation, just prior to administration, of a cytocompatible polymer composition that is substancially homogeneous and/or substantially free of polymer precipitation at room temperature.
  • the present application provides a two-part cytocompatible system for preparing a cytocompatible polymer composition for use in repairing a tissue of a subject.
  • the system comprises a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container.
  • the first part and said second part are to be combined, prior to said use, to provide said cytocompatible polymer composition and wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the polymer is a modified or natural polysaccharide.
  • the polymer is dissolved in a mineral acid or an organic acid to prepare the first part liquid solution, such as, for example, the acid is hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid and/or hydrobromic acid.
  • the polysaccharide is chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, heparin and/or heparin sulfate.
  • the salt is an organic salt or an inorganic salt, such salts including, but not limited to sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt (for example NaCI, KCI, CsCI, CaCI 2 , CsF, KCI0 4 , NaN0 3 and/or CaS0 4 ).
  • the organic salt is glycerol-phosphate.
  • the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower about 7.8.
  • the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than about 6.8.
  • the polymer is chitosan.
  • the chitosan has a degree of deacetylation equal to or higher than about 20% and equal to or lower than about 100% deacetylated.
  • the chitosan has an average molecular weight equal to or higher than about 1 kDa and equal to or lower than about 10 MDa.
  • the chitosan has an average molecular weight equal to or higher than about 10 kDa and equal to or lower than about 150 kDa.
  • the concentration of chitosan in the first part liquid solution is equal to or higher than about 1.5% and equal to or lower than about 2.2% w/v.
  • the system further comprises a third part blood component.
  • the third part blood component is whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood and/or placenta blood.
  • the third part blood component is plasma, erythrocytes, leukocytes, monocytes, platelets, fibrinogen, stem cells and/or thrombin.
  • the third part blood component comprises a platelet-rich plasma substantially free of erythrocytes.
  • the mixing ratio of (i) the third part blood component and (ii) the first part liquid solution of polymer solution combined with the second part liquid solution of the salt is equal to or higher than about 1 :3 and equal to or lower than about 1 :6.
  • the cytocompatible polymer composition is capable of activating complement through platelet activation and/or capable of stimulating release of C5a peptide in whole blood or blood fractions containing platelets.
  • the concentration of glycerol-phosphate in the second part liquid solution is equal to or higher than about 400 and equal to or lower than about 1000 mM.
  • the system further comprises a buffer to achieve near-neutral pH and isotonicity of the cytocompatible polymer solution.
  • the first and second biological containers are independently selected from a vial or a syringe.
  • the present application provides a cytocompatible polymer composition for use in repairing a tissue of a subject, wherein said cytocompatible polymer.
  • the composition is prepared by admixing a first part liquid solution of a polymer with a second art liquid solution of a salt prior to said use.
  • the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the polymer type and method of preparing the solution
  • the acid type, concentration
  • the salt type, concentration
  • the cytocompatible polymer composition characterisitics pH, biological properties
  • the chitosan degree of deacetylation, average molecular weight, concentration in first part liquid solution
  • the third part blood component type
  • mixing ratio the buffer and the first and second biological containers
  • the present application provides the use a cytocompatible polymer composition as defined herein or prepared from a two- part composition as defined herein for repairing a tissue in a subject.
  • the tissue is cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors and/or ulcers.
  • the cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, bone remodeling and/or osteochondral repair.
  • the present application provides the use of a cytocompatible polymer composition (i) prepared with a two-part system as defined herein or (ii) defined herein for delivering a therapeutic substance to a subject.
  • the therapeutic substance is a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a cytokine, a complement factor, and/or an angiogenic factor.
  • the present application provides a method for repairing and/or regenerating a tissue of a subject in need thereof.
  • the method comprises administering a cytocompatible polymer composition (i) prepared with a two-part system as defined herein or (ii) as defined herein into said tissue in need of repair and/or regeneration.
  • the cytocompatible composition is capable of adhering to the tissue to be repaired and/or regenerated so as to repair and/or regenerate the tissue.
  • the tissue is cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors and/or ulcers.
  • the cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, bone remodeling and/or osteochondral repair.
  • the present application provides a method for delivering a therapeutic substance in a subject in need thereof.
  • the method comprises administering a cytocompatible polymer composition admixed with the therapeutic substance into said subject, wherein said cytocompatible polymer composition (i) is prepared with a two-part system as defined herein or (ii) is defined herein.
  • the therapeutic substance is a polysaccharide, a polypeptide, a drug, a liposome, a DNA, a DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a complement factor, a cytokine and/or an angiogenic factor.
  • the present application provides a kit for preparing a cytocomptabile polymer composition for use in repairing and/or regenerating a tissue of a subject.
  • the kit comprises i) a first container comprising a liquid polymer solution and ii) a second container comprising a liquid salt solution.
  • the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the polymer is dissolved in an acid solution to provide the polymer solution, such as, for example, hydrochloric acid, acetic acid, lactic acid, citric acid and/or glycolic acid solution.
  • the kit can further comprise a concentrated buffer solution so as to adjust the cytocompatbile polymer solution to a cytocompatible pH and isotonicity.
  • the polymer is a modified or natural polysaccharide including, but not limited to chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, heparin, and/or heparin sulfate.
  • the salt in said liquid salt solution is an organic or inorganic salt, such as, for example, sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt and/or carboxylate salt (e.g. NaCI, KCI, CsCI, CaCI 2 , CsF, KCI0 4 , NaN0 3 and/or CaS0 4 ).
  • the salt is an organic salt, for example, glycerol-phosphate.
  • the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than about 7.8.
  • the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than 6.8.
  • the polymer is chitosan.
  • chitosan has a degree of deacetylation equal to or higher than about 20% and equal to or lower than about 100%.
  • the chitosan has an average molecular weight equal to or higher than about 1 kDa and equal to or lower than about 10 MDa.
  • the chitosan has an average molecular weight equal or higher than about 10 kDa and equal to or lower than about 150 kDa.
  • the concentration of in the polymer solution equal or higher than about 1.3% and equal or lower than about 2.5% w/v.
  • concentration of glycerol-phosphate in the salt solution is equal or higher than about 600 and equal or lower than 1000 mM.
  • the cytocompatible polymer composition has a pH equal to or higher than about 6.9 and equal to or lower about 7.4.
  • the kit further comprises instructions for use of said kit to repair and/or regenerate the tissue of the subject.
  • the first and second biological containers are independently selected from a vial and a syringe.
  • the present application provides a method for preparing a cytocompatible polymer solution for use in repairing a tissue of a subject.
  • the method comprises combining a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container.
  • first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the cytocompatible polymer composition is administered rapidly to the subject in need thereof.
  • the method can also comprise the combination of other solution such as a blood component or a buffer.
  • the polymer type and method of preparing the solution
  • the acid type, concentration
  • the salt type, concentration
  • the cytocompatible polymer composition characterisitics pH, biological properties
  • the chitosan degree of deacetylation, average molecular weight, concentration in first part liquid solution
  • the third part blood component type
  • mixing ratio the buffer and the first and second biological containers
  • the present application provides a method for repairing a tissue of a subject.
  • the method comprises combining a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container and administering the resulting composition to the subject.
  • said first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the cytocompatible polymer composition is administered rapidly to the subject in need thereof.
  • the method can also comprise the combination of other solution such as a blood component or a buffer.
  • the polymer type and method of preparing the solution
  • the acid type, concentration
  • the salt type, concentration
  • the cytocompatible polymer composition characterisitics pH, biological properties
  • the chitosan degree of deacetylation, average molecular weight, concentration in first part liquid solution
  • the third part blood component type
  • mixing ratio the buffer and the first and second biological containers
  • the present application provides a method for administering a therapeutic substance to a subject.
  • the method comprises combining a first part liquid solution of a polymer in a first container, a second part liquid solution of a salt in a second container and the therapeutic substance and administering the resulting composition to the subject.
  • said first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.
  • the cytocompatible polymer composition is administered rapidly to the subject in need thereof.
  • the method can also comprise the combination of other solution such as a blood component or a buffer.
  • the polymer type and method of preparing the solution
  • the acid type, concentration
  • the salt type, concentration
  • the cytocompatible polymer composition characterisitics pH, biological properties
  • the chitosan degree of deacetylation, average molecular weight, concentration in first part liquid solution
  • the third part blood component type
  • mixing ratio the buffer and the first and second biological containers
  • a two-part cytocompatible composition for use in repairing tissue of a patient prepared by admixing just before use a first-part liquid polymer solution with a second-part liquid salt solution, said composition being free of polymer precipitation at room temperature.
  • the polymer is a modified or natural polysaccharide, such as polysaccharide selected from the group consisting of chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, decorin, heparin, and heparin sulfate.
  • polysaccharide selected from the group consisting of chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, decorin, heparin, and heparin sulfate.
  • the acid comprises a mineral acid or an organic acid, such as, but not restricted to, hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid.
  • a mineral acid or an organic acid such as, but not restricted to, hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid.
  • the salt can be an inorganic or organic salt, such as sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt; at least one of NaCI, KCI, CsCI, CaCI 2 , CsF, KCIO4, NaN03 or CaS0 4 ; or disodium sodium glycerol-phosphate.
  • the final composition has a pH between 5.8 and 7.8, more preferably between 5.8 and 6.8.
  • the chitosan solution can have a pH 4.5 to 5.6 and the salt solution either with no fixed pH (NaCI) or pH range 6.4 to 7.4 (GP-HCI).
  • the chitosan can be 20% to 100% deacetylated with an average molecular weight ranging from 1 kDa to 10 MDa, or preferably having an average molecular weight ranging from 10 kDa to 150 kDa, and in a concentration between 1.5% to 2.2% w/v.
  • chitosan- HCI preparations with 70% to 90% protonation could generate a room temperature-stable chitosan solution.
  • the HCI concentration does not exceed 79 mM of the re-constituted 1.6% w/v chitosan- HCI and glycerol phosphate solution.
  • a 2-part chitosan/blood system is feasible when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of ⁇ -GP in 50 mM or 100 mM HCI is injected directly into the vial and shaken immediately before use.
  • Another embodiment is when 1.2 ml chitosan-HCI can be packaged in a mixing vial, and 0.3 ml of 0.5M ⁇ -GP in 50 mM HCI is added directly into the vial immediately before use.
  • the chitosan will be in non-aggregated form when prepared the day of use, and will have the same formulation as the chitosan-GP solution that generated excellent repair results.
  • Another embodiment is when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of 750 mM NaCI is used to generate an isotonic and slightly acidic (pH 5.8 to pH 6.1) chitosan solution.
  • the composition further comprises a blood component which can be selected from the group consisting of whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood and placenta blood; and further comprises plasma or platelet rich plasma free of erythrocytes.
  • a blood component which can be selected from the group consisting of whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood and placenta blood; and further comprises plasma or platelet rich plasma free of erythrocytes.
  • the blood component can also be selected from the group consisting of plasma erythrocytes, lymphocytes, polymorphonuclear cells, monocytes, platelets, fibrinogen, stem cells and thrombin; more preferably is plasma, erythrocytes, polymorphonuclear cells, monocytes, or platelets.
  • the glycerol-phosphate or salt component is generated as a stock solution and mixed with the liquid polymer solution such that the final salt concentration is cytocompatible between 250 to 520 milliOsmolar.
  • the mixing ratio of blood component and admixture of the liquid polymer-salt solution is in between and including 1 :3 to 1 :12 v/v admixture:blood.
  • composition permits propagation of the clotting cascade, and can activate complement through platelet activation, stimulating release of C5a peptide.
  • the composition also comprises admixing a buffer to achieve neutral pH and isotonicity, the chitosan being dissolved in an acid solution, such as hydrochloric acid, acetic acid, lactic acid or glycolic acid.
  • a buffer to achieve neutral pH and isotonicity
  • the chitosan being dissolved in an acid solution, such as hydrochloric acid, acetic acid, lactic acid or glycolic acid.
  • the tissue can be selected from the group consisting of cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, infarcted cardiac tissue, ischemic tissue, resected tumors and ulcers.
  • composition can also stimulate subchondral angiogenesis, bone remodeling, osteoclast formation, or osteochondral repair.
  • a method for repair and/or regeneration of a tissue of a patient comprising administering a composition as defined herein into the tissue in need of repair and/or regeneration, wherein composition when placed at the site in need of repair will adhere to the site in need of repair to effect repair and/or regeneration of the tissue.
  • Also provided is a method for delivering a therapeutic substance in a patient comprising administering a two-part composition as defined herein.
  • a method of preparing a cytocompatible composition for use in repairing tissue of a patient comprising dissolving a polymer in order to obtain a polymer solution free of precipitate, mixing a liquid salt solution to the polymer solution obtaining the cytocompatible composition free of polymer precipitate at room temperature.
  • the composition can be also be admixed at 4°C, preferably in a temperature range between 4°C and 37°C.
  • the mixing of the liquid salt solution to the polymer solution obtaining the cytocompatible composition free of polymer precipitate at room temperature can be done 0.5 minutes to 1 day prior to mixing the composition with a therapeutic substance or blood component.
  • kits for repairing and/or regenerating a tissue of a patient comprising i) a soluble acidic polymer solution and ii) a liquid salt solution.
  • the kit can also comprise instruction(s) for use.
  • the liquid polymer solution and the liquid salt solution are packaged prior two admixture in two separate biological containers. It is encompassed herein any biological container allowing storage of the polymer solution and the liquid salt solution prior to admixing them together, such as for example but not limited to, a vial or a syringe.
  • anionic biologically active factors can be added to the polysaccharide solution and remain tethered to the polysaccharide, and immobilized within the implant following mixture with blood or blood factors.
  • the present description provides for the preparation of a chitosan composition via the mixing of two compositions, where the first composition, optionally referred to as the mixing composition, is prepared from or contains water, chitosan and hydrochloric acid, while the second composition, optionally referred to as the additive composition, is prepared from or contains water, glycerolphosphate salt, and hydrochloric acid. It is a feature of this aspect of the disclosure that each of these two precursor compositions contains hydrochloric acid.
  • the additive composition may comprise, or consist entirely of, water, hydrochloric acid at a concentration of 25 to 75 mM, preferably 35 to 65 mM, and more preferably 40 to 60 mM, and glycerolphosphate salt at a concentration of 250 to 750 mM, preferably 350 to 650 mM, and still more preferably 400 to 600mM.
  • the mixing composition may comprise, or consist entirely of, water, chitosan at a %wt/wt concentration in the range of 1.8 to 2.3, preferably 1.9 to 2.2, and more preferably about 2.05, while the hydrochloric acid concentration in term of millimolar, is in the range of 60 to 80 mM, preferably 65 to 75 mM, and more preferably 69-70 mM.
  • the additive composition may comprise, or consist essentially entirely of, water, hydrochloric acid and glycerolphosphate salt such that the pH of the additive composition is within the range of 6.7 to 7.3, preferably 6.9 to 7.1 , and most preferably around 7.0, while the mixing composition comprises, or consists essentially entirely of, chitosan and hydrochloric acid with a pH in the range of 5.5 to 6.2, preferably 5.7 to 6.0 and most preferably around pH 5.9.
  • the mixing composition comprises chitosan at a %wt/wt concentration in the range of 1.8 to 2.3, preferably 1.9 to 2.2, and more preferably about 2.05, while the pH of the mixing composition is in the range of about 5.5 to 6.2, preferably 5.7 to 6.0 and most preferably 5.9.
  • the additive composition comprises glycerol phosphate salt at a %wt/wt concentration in the range of 8 to 12, preferably 9 to 11 , and more preferably around 10.2, while the pH of the mixing composition is in the range of 6.7 to 7.3, preferably 6.9 to 7.1 , and more preferably around 7.0, where the pH of the additive composition is adjusted by use of hydrochloric acid.
  • the mixing composition has a dynamic viscosity as measured in mPa-s, in the range of 500 to 1500, preferably 1350 to 1450.
  • the chitosan concentration is preferably 1.4 to 1.8 wt%, more preferably 1.5 to 1.7 wt%, and still more preferably around 1.6wt%;
  • the glycerol phosphate salt concentration is preferably 1.8 to 2.2 wt%, more preferably 1.9 to 2.1 , and more preferably about 2.0wt%;
  • the pH of the combination is preferably 6.4 to 7.0, more preferably 6.6 to 6.8 and still more preferably about 6.7.
  • the osmolarity of the resulting combination, as determined mOsm/kg, is preferably within the range of 200 to 400, more preferably 220 to 380, and still more preferably 250 to 350, and yet more preferably 280 to 320.
  • each of the mixing and additive compositions is sterile.
  • the glycerol phosphate salt is selected from glycerol-2-phosphate salts, syn-glycerol-3- phosphate salts, and L-glycerol-3-phosphate salts, where a preferred glycerol phosphate salt is a salt of beta-glycerolphosphate (BGP), where suitable counterions are the disodium or dipotassium salts of the glycerol phosphate.
  • BGP beta-glycerolphosphate
  • the chitosan used in this aspect of the disclosure preferably has a degree of deacetylation in the range of 70 to 85%, more preferably 78 to 84%, and still more preferably around 81 %.
  • the resulting chitosan composition is preferably mixed with blood or component(s) thereof, in a volumetric ratio of about 3 parts blood to 1 part chitosan solution.
  • a kit comprising mixing and additive containers, the mixing container comprising chitosan at a concentration of 1.9 to 2.2 wt% and a pH of 5.7 to 6.0 adjusted with hydrochloric acid, while the additive container comprises beta- glycerol phosphate at a concentration of 8 to 12 wt% and a pH of 6.9 to 7.1 adjusted with hydrochloric acid.
  • the container can be for example a vial and/or a syringe.
  • Fig. 1 corresponds to a graphic representation of the pH of a 1 % w/v chitosan solution in relation with the protonation level.
  • Fig. 2 corresponds to a graphic representation of the correlation between HCI concentration needed to obtain a particular protonation level for a given chitosan solution (82.6% DDA) at a given w/v percent concentration.
  • Fig. 3 corresponds to the effect of increasing HCI in 1 volume of 500 mM ⁇ -GP admixed into 4 volumes of 2% w/v chitosan-HCI pH 5.6, on the ⁇ -GP solution (diamond symbol) and on the final pH of the isotonic chitosan-HCI- ⁇ - GP solution (square symbol), and the formation of visible precipitate in the vial after adding the ⁇ -GP solution.
  • Fig. 4 illustrates the effect of substituting NaCI for ⁇ -GP on an isotonic chitosan solution pH.
  • FIG. 5 illustrates the coagulation of mixtures of whole human blood and rapidly reconstituted chitosan-HCI pH 5.5 and ⁇ -GP pH 7.2, and C5a generation during coagulation of whole blood and chitosan-GP/blood mixtures as a function of time.
  • C5a appeared in parallel with platelet degranulation and thrombin activation in coagulating chitosan-GP/blood (A, C, E) and whole blood (B, D, F).
  • Panels A and B show the time-dependent increase in clot tensile strength (Amplitude (A), mm, black trace) and thrombin generation (Thrombin/Anti-Thrombin (TAT) levels, dashed bars) for a representative donor (out of 4 donors).
  • Panels C and D show time-dependent increase of serum C5a fragment detected by goat anti-C5a polyclonal antiserum, and a positive control zymosan-activated serum (ZAS) incubated 60 min.
  • Panels E and F show time- dependent increase of serum platelet factor 4 (PF4) as a marker of platelet activation, and a positive control chitosan-GP/blood incubated 75 min with thrombin (10 U/ml) (lla).
  • PF4 serum platelet factor 4
  • FIG. 6 illustrates an assay for C5a generation in serum and citrated plasma.
  • Panel A shows serum exposed to either 4 mg/ml zymosan particles dispersed in NaCI (ZAS-NaCI), isotonic glycerol phosphate buffer (GP), or 4 mg/ml 80% DDA chitosan in isotonic GP buffer (Chi80-GP).
  • Panels B and C C5a fragments in citrated plasma exposed to various test activators as indicated. Symbols: the black circle indicates 16 kDa C5a constitutively present in plasma and serum. The arrow indicates C5a produced by complement activation.
  • Fig. 7 illustrates an assay using citrated plasma for C3 binding to insoluble zymosan or chitosan particles.
  • Zymosan-NaCI (ZAP) and 80% DDA chitosan-GP (Chi80-GP) were exposed to citrated plasma for 60 min at 37°C, then the insoluble pellets were rinsed in EDTA buffer, extracted with 8 M urea, and urea extracts loaded on a nonreducing gel (Panel A), or a reducing gel (Panel B). Migration is shown relative to 0.2-5 ⁇ human plasma (P) that was incubated for 1 h at 37°C.
  • FIG. 8 illustrates an assay using citrated plasma to measure C5a/C3a generation and C5/C3 binding to insoluble zymosan or chitosan particles.
  • Fig. 9 illustrates native C5 and C3 bound to chitosan under complement-inhibiting conditions.
  • Zymosan or 95% DDA chitosan-NaCI (Chi95) were incubated at 4 mg/mL with pooled serum (-), serum with 10 mM EDTA (E), or methylamine-treated plasma (M) for 1 h at 37°C.
  • Resulting Supernatants were analyzed by Western blot for C5a (Panel A, nonreducing conditions) and 8M urea Pellet extracts from same samples were analyzed for C5 with an anti- C5a antibody (Panel B, nonreducing conditions), and C3 with an anti-C3b antibody (Panel C, reducing conditions).
  • Panel D shows Western blot of panel C that was stripped and reprobed with anti-C3a antibody. The boxes indicate the protein detected, and antisera used for immunodetection.
  • Fig. 10 illustrates the binding of pure C3 to chitosan verified by surface plasmon resonance (SPR) biosensing.
  • the top trace shows protein accumulation on biotinylated chitosan coupled to streptavidin (SA) sensorchip and the bottom negative control trace shows bare SA sensorchip exposed simultaneously to the same conditions.
  • Serial injections were as follows: buffer (1-4), pure C3 protein at 1 , 10, 20, and 100 pg/ml concentrations (5-8, respectively), anti-C3a antibody at 0.3 pg/ml (9), and anti-C3b antibody at 0.05 and 0.13 pg/ml (10, 11).
  • buffer 1-4
  • pure C3 protein at 1 , 10, 20, and 100 pg/ml concentrations (5-8, respectively)
  • anti-C3a antibody at 0.3 pg/ml (9)
  • anti-C3b antibody at 0.05 and 0.13 pg/ml (10, 11).
  • FIG. 11 illustrates an assay for association of factor B, factor B cleavage products, and antithrombin (AT) in citrated plasma to chitosan and zymosan particles.
  • Zymosan and chitosan particles were incubated in plasma for 1 h incubation at 37°C, the pellets extracted with 8M urea, and pellet extracts analyzed by Western blot under nonreducing conditions using anti-factor B polyclonal antiserum (Panel A) and monoclonal anti-human AT (Panel B).
  • Panel C shows a nonreducing Western blot analysis of factor B, using urea pellet extracts (lanes 1-4) and supernatants (lanes 5-7) from the same set of samples as those analyzed in Panel A. Appropriate quantities of purified protein standard (factor B, AT) and plasma were loaded onto each gel as a control.
  • Fig. 12 corresponds to a schematic representation of the vial/syringe used to mix the polymer and salt solution.
  • Fig. 13 corresponds in (A) to a photographic representation showing that the implant described herein was delivered to an acute rabbit femoral trochlear cartilage defect with 4,1 mm diameter microdrill holes pre-treated with 3 ⁇ _ purified human thrombin.
  • the contralateral drilled cartilage defect was treated with 3 ⁇ _ of purified human thrombin.
  • the implant-treated defects had significantly greater subchondral bone repair (compare A1 and B1) and significantly greater basal integration (compare C1 and D1 where open arrows indicate detached repair tissue) compared to the control defects.
  • the repair period was 6.5 months.
  • Treated repair tissue (see Fig. 14C) contained more glycosaminoglycan than controls throughout the defect (see Fig. 14B).
  • Fig. 15 corresponds to a graphic representation of the liquid mixture of chitosan-GP reconstituted in the clinic during the operative procedure. Blood was added and mixed with the solution, then the implant was solidified in situ, to treat microfracture lesions of human patients. In other patients, lesions were treated with microfracture-only. Repair tissue biopsies were obtained 13 months post-treatment in 21 patients from the estimated geometric center of the initial lesion (13 treated, 9 Microfracture-only). The cartilage repair tissue in biopsies from implant-treated defects was significantly thicker (A) and had a significantly higher over-all histology score (B) for quality of repair as judged by 3 independent blinded readers, for 14 different repair tissue features.
  • A over-all histology score
  • Fig. 16 corresponds to photographic representations of average control biopsies (A) and average treated biopsies (B) showing that the cartilage repair tissue in biopsies from implant-treated defects had a significantly higher quality of repair.
  • the resulting solution can be further combined with whole blood or blood derivatives to generate mixtures capable of in s/iiv-so I id if i cati o n over wounds or surgical defects, or extracorporeal devices.
  • the resulting solid implants stimulate the repair and regeneration of articular cartilage, joint tissues and other tissues including but not limited to meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, ulcers, aorta, and cardiac tissue.
  • the advantage of using a system with two distinct containers for the polymer solution and the salt solution is that the resulting polymer composition can be generated shortly prior to its administration to the subject. Because the polymer composition is produced shortly before it is administered, the polymer in the composition is substantially dissolved and/or in an unprecipitated form. As used herein, a "polymer is substantially dissolved", when the majority of the polymer in the solution are dissolved in the solution.
  • a "polymer is substantially in an unprecipitated form" when the majority of the polymer in the solution does not form a precipitate with other components of the solution (for example, other polymers).
  • the final product is thus considered to be more homogeneous and easier to handle than polymer compositions that have been generated and stored prior to use.
  • the system proposed for generating a room temperature-stable 2- part formulation consist in (1) a polymer composition (such as an acid chitosan solution) and (2) a compatible salt solution or buffer of appropriate pH that can be rapidly reconstituted to form a 1-part polymer solution (such as an isotonic chitosan solution) that is free of polymer precipitates.
  • a polymer composition such as an acid chitosan solution
  • a compatible salt solution or buffer of appropriate pH that can be rapidly reconstituted to form a 1-part polymer solution (such as an isotonic chitosan solution) that is free of polymer precipitates.
  • the resulting cytocompatible polymer solution can be further combined with or without bioactive factors, and with whole blood or blood fractions, to allow propagation of the enzymatic clotting cascade, coagulation, and the formation of a cytocompatible implant containing chitosan polymer, polymerized fibrin, and blood elements.
  • liquid chitosan in dilute HCI around 70 mM
  • pH 4.5 - 5.6 is prepared (see Fig. 12).
  • Viscosity of this chitosan-HCI solution may slowly decline over time due to spontaneous hydrolysis of the chitosan chains.
  • the solution could be frozen, or heated, with no fear of gellation or precipitation.
  • a syringe containing 5x to 10x solution ⁇ -GP/NaCI, for asceptic injection into the chitosan vial, is prepared. Passive diffusion can be allowed, or the solution may be vortex mixed or shaken (i.e. for 10 seconds) to reconstitute a chitosan-salt solution.
  • the correct ratio of chitosan and ⁇ -GP or NaCI is determined by the final solution transparency (lack of chitosan precipitation), osmolality (needs to be close to isotonic) and pH (isotonic NaCI brings chitosan to pH 5.8-6.1 and GP can bring the pH to pH 6.4 to 6.8).
  • a vented injection device could be use in order to relieve any pressure introduced by the injected volume of blood or ⁇ -GP/NaCI.
  • Chitosan-GP product performance and clinical ease-of-use is now improved by the method and formulation described herein that permit room temperature storage and rapid reconstitution to generate a formulation similar to those formulations showing efficacy in cartilage repair studies.
  • the rapidly reconstituted formulation should generate a chitosan solution that is isotonic, near-neutral pH and free of precipitate.
  • the following disclosure demonstrates the disclose method and formulation generate room temperature-stable formulations that can be rapidly combined to form a cytocompatible and sterile solution of chitosan-glycerol phosphate or chitosan-sodium chloride.
  • the in vivo efficacy of the composition described herein is demonstrated in rabbits and in humans (see Fig. 13-16).
  • the formulation and method described permits immobilization of anionic bioactive factors such as complement C3 and C5 in the chitosan- salt blood implant, and the release of cationic chemotactic factors C3a and C5a via specific non-covalent tethering of anionic proteins to the chitosan scaffold before and after liquid-solid phase transition (see Table 1).
  • Prothrombin P00734; C2, P06681 ; C4, P0C0L4; D, P00746; I, P05156; FVII, P08709; PF4, P02776.
  • Chitosan is a polycationic and biocompatible polysaccharide that is thrombogenic, chemotactic for neutrophils, and stimulates angiogenesis and wound repair (Hoemann et al., 2005, Journal of Bone and Joint Surgery- American Volume, 87A(12): 2671-2686) through mechanisms that remain unclear.
  • Chitosan is produced by chemical deacetylation of chitin, resulting in a polymer composed of ⁇ (1 ⁇ 4) O-linked -glucosamine (Glc) and N-acetyl-p-D- glucosamine (GlcNA) with variable degree of deacetylation (DDA), for example, 90% DDA chitosan has 90% Glc and 10% GlcNA monomer. Chitin and chitosan were previously shown to deplete or adsorb complement C3 and C5 proteins from serum and plasma, suggesting that chitosan may activate complement.
  • Glc O-linked -glucosamine
  • GlcNA N-acetyl-p-D- glucosamine
  • DDA variable degree of deacetylation
  • Complement activation is marked by the formation of a C3 convertase complex on the surface of target cells or activating surfaces.
  • C3 convertase generates a local amplification of the complement cascade and C5 cleavage, culminating in the release a 70 amino acid cationic C5a anaphylatoxin peptide fragment.
  • C5a provides a potent stimulus for neutrophil chemotaxis
  • C3b fragments adsorbed onto biomaterial surfaces serve as opsonins or coatings that mediate recognition and clearance by phagocytes (reviewed in Law and Dodds, 1997, Protein Sci, 6: 263-274).
  • Activation of complement could potentially explain how neutrophils become attracted to chitosan particles, yet evidence of C5a generation by chitosan was lacking.
  • acetylated chitosans may still bear resemblance to chitin, a yeast cell wall molecular pattern, highly deacetylated chitosans, (i.e., 90% Glc or higher) have little chemical similarity with chitin, and would not necessarily be predicted to interact with the complement system in the same manner.
  • Complement activation is propagated through a cascade of plasma serine proteases, and represents the liquid phase of the innate immune reaction to trauma and foreign molecular patterns.
  • Human complement C3 and C5 are ⁇ 190 kDa proteins that circulate as heterodimers of 115 kDa alpha and 75 kDa beta chains linked by a disulfide bond. Cleavage of the small peptide C3a from C3 generates a metastable C3b fragment with a reactive internal thioester bond that is able to form covalent linkages with activating surfaces.
  • Solid-phase C3b associates with the proenzyme factor B to form a C3bB complex that is cleaved once in the factor B subunit by factor D; the Ba cleavage product dissociates leaving an active C3bBb solid-phase convertase for C3 and C5 that locally amplifies the complement cascade.
  • C3b is rapidly cleaved in the alpha chain to form iC3b, by plasma protease I and its cofactor H, and cell-derived serine proteases can further degrade the iC3b alpha chain to produce C3c and C3dg.
  • C3b and C3b break-down products remain associated with the activating surface as opsonins, complement activation can therefore be assayed by the appearance of fluid- phase C5a, and solid-phase associated C3b and break-down products.
  • Zymosan a yeast ghost particle, activated complement in serum and citrated plasma, but not in EDTA-serum or methylamine plasma, to generate fluid-phase C5a, while C3b formed covalent cross-links with zymosan- associated proteins and became rapidly cleaved to iC3b, with factor Bb stably associated.
  • Chitosan forms a noncovalent and tight association with AT in a charge- and DDA-dependent manner (see Fig.8). Furthermore, pure C3 bound to chitosan biosensor surfaces in a manner that selectively exposed the cationic C3a epitope. Altogether, these collective data allow generating a new and predictive model of chitosan-blood protein interaction, whereby anionic proteins with a pi lower than the pK a of chitosan can deposit onto positively charged chitosan nonspecifically, and without requiring a reactive internal thioester bond (Fig. 9B and C; lane Chi95-M).
  • thrombin is a C5 convertase
  • the data indicates that it is a rather weak convertase in human plasma compared to zymosan, and suggest that platelet activation (Fig. 5) which is triggered by thrombin, is a more significant driver of C5a generation in normal whole blood.
  • C5a release from blood clots and from chitosan- GP/blood clots could be related to the increased neutrophil chemotaxis seen towards chitosan-GP/blood implants during guided cartilage repair (Chevrier et al., 2007, Osteoarthritis Cartilage, 15: 316-327).
  • Transient neutrophil chemotaxis toward chitosan-GP/blood implants was followed by angiogenesis, bone remodeling, and a more structurally improved cartilage repair tissue, compared to blood clots induced by surgical intervention alone (Hoemann et al., 2005, Bone Joint Surg-AM, 87: 2671-2686). These data suggest that transient C5a release could be a therapeutic initiating event in wound repair.
  • Chitosan is a nonactivating biomaterial that is positively charged, and binds to anionic plasma and serum proteins C3, C5, factor B, Ba fragment, and AT without leading to complement activation or release of C5a anaphylatoxin.
  • C5a fragment appeared in coagulating whole blood serum in parallel with platelet degranulation, with or without chitosan.
  • repair when applied to cartilage and other tissues is intended to mean without limitation repair, regeneration, reconstruction, reconstitution or bulking of cartilage or tissues.
  • blood is intended to mean whole blood, processed blood, venous blood, arterial blood, blood from bone-marrow, umbilical cord blood and placenta blood. It may be enriched in platelets.
  • blood component is intended to mean erythrocytes, leukocytes, monocytes, platelets, fibrinogen, and thrombin. It may further comprise platelet rich plasma free of erythrocytes. In another embodiment, blood component is intended to mean any component of the blood retaining clotting properties.
  • biocompatible polymer is intended to mean a polymer that can be contacted with a tissue, without altering the tissue viability and that is tolerated or accepted by the tissue or the organism.
  • the term "patient” is intended to mean a human or an animal.
  • solidification or "presolidification” is intended to mean the loss of the liquid state to the benefit of the solid state.
  • thermogelling is intended to mean the characteristic of a polymer which becomes non-liquid at a certain temperature over a certain period of time.
  • clotting is intended to mean a type of solidification involving formation of a blood clot or plasma clot.
  • cytocompatible is intended to define the property of a composition or solution to not be toxic to the living cells.
  • therapeutic substance is intended to refer to the property of any substance to have beneficial or therapeutic effect on the patient administered with the substance.
  • therapeutic substance can be, but not limited to, a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA- polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a cytokine and an angiogenic factor.
  • the optimal chitosan-GP solution should be close to isotonic and the solution pH should be between 5.8 to 6.8. Therefore it is necessary to determine appropriate level of acid needed in the composition to attain this pH range.
  • chitosan 83%DDA was dissolved at 1% wt/vol in 10 ml total volume with varying levels of HCI to give 70%, 80%, 90%, and 100% protonation.
  • LOD Loss on Drying
  • COA Certificate of Analysis
  • Fig. 1 the pH is maintained close to pH 5.5 when the protonation is below 90%, and drops abruptly when protonation exceeds 90%.
  • the HCI content could potentially influence the chitosan polymer conformation in solution, and the interaction of chitosan with blood components and cells. It is clear that chitosan solutions maintained around pH 5.5 could be room temperature stable. It is thus demonstrated herein that chitosan-HCI preparations with 70% to 90% protonation could generate a room temperature- stable chitosan solution.
  • Precipitation may happen within a few seconds.
  • a 2-part chitosan/blood system is feasible when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of ⁇ -GP in 50 mM or 100 mM HCI is injected directly into the vial and shaken immediately before use.
  • Another embodiment is when 1.2 ml chitosan-HCI can be packaged in a mixing vial, and 0.3 ml of 0.5M ⁇ -GP in 50 mM HCI is added directly into the vial immediately before use.
  • the chitosan will be in non-aggregated form when prepared the day of use, and will have the same formulation as the chitosan-GP solution that generated excellent repair results.
  • Another embodiment is when 1 ml chitosan- HCI is packaged in a mixing vial, and 0.11 ml of 750 mM NaCI is used to generate an isotonic and slightly acidic (pH 5.8) chitosan solution (see Figs. 3 and 4).
  • Chitosan was dissolved at 2.05% w/w, pH 5.6 in dilute HCI, and autoclave-sterilized to produce liquid solutions with dynamic viscosities of 1422 mPa s (80.2% DDA), 1849 mPa s (80.6% DDA), 2964 mPa s (94.6% DDA) at 25°C and stored as sterile aliquots at room temperature for up to 6 months or frozen at -80°C.
  • liquid chitosan-HCI was combined at a 4:1 v/v ratio with sterile ddH20, filter-sterile 500 mM disodium ⁇ -glycerol phosphate/ 50 mM HCI pH 7.2 (GP) or autoclave-sterile 750 mM NaCI to generate solutions with 1.6% w/v chitosan at pH 5.6 (chitosan-HCI, hypotonic), pH 6.6 (chitosan-GP, isotonic) or pH 6.1 (chitosan-NaCI, isotonic).
  • the disodium glycerol phosphate (GP) levels were adjusted to be isotonic and cytocompatible.
  • Clot tensile strength was evaluated with 4 Thromboelastograph® (TEG) instruments (5000 series TEG analyzer Software Version 3, Haemoscope, Niles IL) which permits the simultaneous analysis of 8 samples.
  • TEG Thromboelastograph®
  • Unmodified whole blood was homogenously mixed with chitosan-GP at a 3:1 ratio blood:chitosan-GP, deposited in plastic sample TEG cups, and allowed to coagulate for up to 75 min at 37°C. Samples were removed at specific intervals, the blood volume equivalent diluted 10-fold in ice- cold quench buffer (20 mM HEPES, 50 mM EDTA, 10 mM benzamidine, 150 mM NaCI, pH 7.4 with 100 ⁇ PMSF and 33 ⁇ FPR-ck), vortexed, and the serum cleared by centrifugation and stored at -80°C, as previously described. Whole unmodified blood was analyzed in parallel as a control.
  • Thromboelastography was used to monitor coagulation and the development of clot tensile strength and thrombin generation was monitored in serum by thrombin-antithrombin (TAT) levels as previously described (Marchand et al., 2009, Osteoarthristis Cartilage, 17: 950-957).
  • TAT thrombin-antithrombin
  • Fig. 5A-D whole blood and chitosan-GP/blood mixtures
  • PF4 platelet factor 4
  • Fig. 5E,F a marker of platelet degranulation
  • C5a, TAT, and PF4 levels began to decay in whole blood serum but not in chitosan-GP/blood clot serum after 75 min of coagulation (Fig. 5).
  • chitosan-GP/blood mixtures minor complement and platelet activation was seen before the onset of thrombin generation in experiments using four different blood donors. These data show that complement was activated both in whole blood, and chitosan-GP/blood mixtures, in parallel with platelet activation.
  • zymosan is a yeast ghost structure that is composed of chitin, the GlcNA parent molecule of chitosan, along with mannan polysaccharide, and residual yeast proteins and lipids.
  • Zymosan-activate serum contained abundant C5a fragments that migrated around 13 kDa, slightly faster than the 16.1 kDa marker (Fig. 6A).
  • the nonglycosylated recombinant C5a (rC5a) peptide standard migrated faster in the gel than the glycosylated 13 kDa zymosan-activated C5a.
  • citrated plasma in high sensitivity Western blots only contained the 16 kDa fragment (Fig. 6A, ECL+).
  • serum exposed to GP or chitosan-GP contained the 16 kDa and not the 3 kDa C5a fragments (Fig. 6A, ECL+).
  • chitosan-GP 80% or 95% DDA
  • chitosan-NaCI chitosan with LPS
  • solid chitosan particles all failed to generate detectable levels of the 13 kDa C5a fragment in plasma (Fig. 6B and C; Chi80, Chi95).
  • Purified thrombin also failed to generate C5a fragments in citrated human plasma (Fig. 6C; lla), even though a fibrin clot was produced.
  • C3 binds to chitosan noncovalently while C3b and iC3b become cross- linked to zymosan-associated proteins
  • zymosan-associated C3 proteins migrated at 190 kDa and as cross-linked higher molecular products (Fig. 7A; ZAP), and in reducing conditions C3 migrated as a strong ⁇ 42 kDa fragment along with a faint C3b band (Fig. 7B; ZAP).
  • the 42 kDa band could arise from iC3b or C3c, although the absence of a faster-migrating 35 kDa C3c product in the nonreducing gel, due to cleavage of the 40 kDa C3dg fragment (Fig. 7C), indicated that the 42 kDa fragment was a subfragment of iC3b.
  • C3 proteins were also eluted from the chitosan pellet.
  • C3 proteins migrated as intact C3 and discrete high molecular weight products (Chi80-GP; Fig. 7A).
  • C3 protein migrated as an intact C3 alpha chain (-115 kDa), and discrete high molecular weight products above 190 kDa, that comigrated with cross-linked C3 proteins present in plasma (C3-X; Fig. 7B).
  • C3 and C5 bind to chitosans with 80% DDA and 95% DDA as intact proteins.
  • Plasma C3a was much more F4 abundant than C5a, consistent with previous assays of C5a and C3a in human plasma and serum (Fig. 8C; Plasma).
  • Zymosan generated slightly more C3a in the fluid phase (Fig. 8C; ZAP).
  • iC3b was the main product eluted, which was detected by anti-C3b antisera and not anti-C3a antisera (Fig. 8D vs. E, respectively; ZAP).
  • Chitosan also captured 2 higher molecular weight C3 proteins from plasma that migrated above the intact 115 kDa C3 alpha chain. These proteins were not activation-fragments of C3, because they were recognized by both anti-C3b and anti-C3a antisera (8D, E). These species comigrated with or close to 2 cross- linked proteins in the purified C3 standard (C3-X; Fig. 8D and E). Although the nature of these 2 proteins is not completely clear, the apparent molecular mass and retention of C3a and C3b epitopes suggested that one of the species represented an intact 230 kDa alpha chain dimer (C3-X; Figs. 7B and 8D and E).
  • Complement C3 and C5 still bind to chitosan in EDTA and methylamine inhibiting conditions.
  • B-chitosan was dissolved in acetic acid pH 6.1 , 0,2 pm filtered, and further diluted to 50 pg/ml in Coupling Buffer (50 mM HEPES, 150 mM NaCI pH 6.1).
  • Coupling Buffer 50 mM HEPES, 150 mM NaCI pH 6.1.
  • Running buffer (10 mM HEPES, pH 7.4, 150 mM NaCI, 3.0 mM EDTA, 0.005% TweenTM 20) was used to equilibrate the chip and to dilute C3 and anti-C3 antibodies before injection.
  • C3 protein was serially injected (180 s, flow rate 5 ⁇ /min) of buffer (4 times), at 1 , 10, 20, and 100 pg/rnl concentration followed by anti-C3a antibody (1/200 dilutions of a 60 pg/ml stock) and injections of anti- C3b antibody (1/500 and 1/200 dilution of a 25 pg/ml stock with 50% v/v glycerol).
  • Control surface corresponded to bare streptavidin-coated sensorchip flowcell for parallel monitoring of nonspecific protein adsorption.
  • Factor B is cleaved during complement activation by factor D to form factor Bb, the enzymatically active subunit of the C3/C5 convertase complex.
  • zymosan particles were incubated for 1 h incubation at 37°C in citrated plasma, rinsed with EDTA buffer and extracted with 8M urea, solid-phase Bb was abundantly present in the pellet extract (ZAP; Fig. 1 A and C).
  • chitosan pellet F7 extracts under identical conditions contained intact factor B and no fragment Bb (Chi95; Fig. 11A and C).
  • Factor B associated more tightly with 95% DDA than 80% DDA chitosan (Fig. 1 1A), as was the case for C5 and C3 (Fig. 8B, D and E respectively).
  • fragment Ba was eluted from chitosan particles after incubation in plasma (Fig. 1 1A).
  • Further analysis of the chitosan treated plasma supernatants revealed that chitosan adsorbed out low-level Ba fragment (and not Bb) already present in plasma (Fig. 11 C).
  • Antithrombin (AT) an anionic serum protease inhibitor, also adsorbed to chitosan particles, with stronger affinity for 95% DDA compared to 80% DDA chitosan (Fig. 11 B).
  • the implant-treated defects had significantly greater subchondral bone repair (compare A1 and B2 in Fig. 13A; and right panel in Fig. 13B) and significantly greater basal integration (compare C1 and D1 in Fig. 13A; and left panel in Fig. 13B) compared to the control defects.
  • the repair period was 6.5 months.
  • Treated repair tissue (Fig. 14C) contained more glycosaminoglycan than controls (Fig. 14B) throughout the defect .
  • a liquid mixture containing 1200 pL 2.05% w/w Chitosan solution (pH 5 to 6)), 300 pL 10.05% w/w beta-Glycerol Phosphate solution pH 7.2, and 4.5 mL fresh human unclotted whole blood of chitosan-GP was reconstituted in the clinic during the operative procedure.
  • the 2-part formulation described herein was used as a two vial product comprised of a mixing vial containing the chitosan solution and an additive vial containing the beta-glycerol phosphate buffer. Blood was added and mixed with the solution, then the implant was solidified in situ, to treat microfracture lesions of human patients. In other patients, lesions were treated with microfracture-only.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Materials For Medical Uses (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne un système cytocompatible en deux parties pour la préparation d'une composition cytocompatible destinée à une utilisation dans la réparation tissulaire d'un sujet. Le système comprend le mélange d'une solution liquide en première partie d'un polysaccharide, tel que le chitosane et une solution liquide en seconde partie d'un sel, tel que le phosphate de glycérol. Lorsque les solutions de chitosane et de phosphate de glycérol sont combinées, il est produit une composition cytocompatible qui est dépourvue de précipitation de polymère à température ambiante. La composition active également la formation de complexes non covalents entre le chitosane et des protéines anioniques thérapeutiques avec un pl < 6 78.
PCT/CA2010/001856 2009-11-19 2010-11-19 Formulation et procédé de préparation rapide de solutions de chitosane isotoniques et cytocompatibles sans provocation de la précipitation du chitosane Ceased WO2011060553A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26280809P 2009-11-19 2009-11-19
US61/262,808 2009-11-19

Publications (1)

Publication Number Publication Date
WO2011060553A1 true WO2011060553A1 (fr) 2011-05-26

Family

ID=44059164

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2010/001856 Ceased WO2011060553A1 (fr) 2009-11-19 2010-11-19 Formulation et procédé de préparation rapide de solutions de chitosane isotoniques et cytocompatibles sans provocation de la précipitation du chitosane

Country Status (1)

Country Link
WO (1) WO2011060553A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017009346A1 (fr) 2015-07-13 2017-01-19 Kiomed Pharma Chitosane pour mélange avec un fluide coagulable
US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999007416A1 (fr) * 1997-08-04 1999-02-18 Bio-Syntech Ltd. FORMATION, TRIBUTAIRE DU pH, ET A TEMPERATURE CONTROLEE, DE GELS DE POLYSACCHARIDE IONIQUE
US20070037737A1 (en) * 2000-06-29 2007-02-15 Hoemann Caroline D Composition and method for the repair and regeneration of cartilage and other tissues
CA2672936A1 (fr) * 2006-11-30 2008-06-05 Bio Syntech Canada Inc. Methode de solidification in situ de compositions sang-polymere pour medecine regenerative et applications de reparation du cartilage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999007416A1 (fr) * 1997-08-04 1999-02-18 Bio-Syntech Ltd. FORMATION, TRIBUTAIRE DU pH, ET A TEMPERATURE CONTROLEE, DE GELS DE POLYSACCHARIDE IONIQUE
US20070037737A1 (en) * 2000-06-29 2007-02-15 Hoemann Caroline D Composition and method for the repair and regeneration of cartilage and other tissues
CA2672936A1 (fr) * 2006-11-30 2008-06-05 Bio Syntech Canada Inc. Methode de solidification in situ de compositions sang-polymere pour medecine regenerative et applications de reparation du cartilage

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AHMADI, R. ET AL.: "Biocompatibility and gelation of chitosan-glvcerol phosphate hydrogels", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PARTA, vol. 86, 2008, pages 824 - 832, XP055159287, DOI: doi:10.1002/jbm.a.31676 *
CHO, M.H. ET AL.: "Chitosan Gel as an In Situ-Forming Scaffold for Rat Bone Marrow Mesenchymal Stem Cells In Vivo", TISSUE ENGINEERING: PART A, vol. 14, 2008, pages 1099 - 1108 *
ILIESCU, M. ET AL.: "Ultrastructure of Hybrid Chitosan-Glycerol Phosphate Blood Clots by Environmental Scanning Electron Microscopy", MICROSCOPY RESEARCH AND TECHNIQUE, vol. 71, 2008, pages 236 - 247 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
US11529420B2 (en) 2013-12-09 2022-12-20 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
WO2017009346A1 (fr) 2015-07-13 2017-01-19 Kiomed Pharma Chitosane pour mélange avec un fluide coagulable

Similar Documents

Publication Publication Date Title
JP2017538751A (ja) 流動性止血組成物
Barrowcliffe History of heparin
CN103687631B (zh) 促凝血肽及其衍生物以及它们的用途
US20100178355A1 (en) Method for in situ solidification of blood-polymer compositions for regenerative medicine and cartilage repair applications
US20070110813A1 (en) Polycation-polyanion complexes, compositions and methods of use thereof
Cheung et al. Analysis of the complement C3 fragments associated with hemodialysis membranes
EP2501392B1 (fr) Formulations de chitosane physiologiques solubles combinées à du plasma riche en plaquettes (prp) pour la réparation de tissus
ES2779228T3 (es) Uso de oligómeros de alginato como anticoagulantes sanguíneos
JP2009535341A (ja) 血液凝固を軽減するためのフコイダン
JP6894993B2 (ja) 血漿タンパク質にコンジュゲートしたヘパリンを含む治療用ａｐａｃ分子
Jaques et al. Pharmacodynamics and clinical effectiveness of heparin
WO2011038047A1 (fr) Méthodes de traitement par une composition d&#39;héparine de bas poids moléculaire
WO2011060553A1 (fr) Formulation et procédé de préparation rapide de solutions de chitosane isotoniques et cytocompatibles sans provocation de la précipitation du chitosane
EP2501754B1 (fr) Nouvelle formulation de mélanges sang/solution salée minérale-chitosane physiologique pour réparation de tissu
CN104302296B (zh) 用于治疗血友病a和/或冯维勒布兰德病的糖组合物
CN110381958A (zh) 中等分子量肝素
US20250099540A1 (en) Methods and pharmaceutical composition for treating thrombotic disorders
Szczubialka et al. Heparin-a key drug in the treatment of the circulatory degenerative diseases: controlling its action with polymers
Cortese et al. Idarucizumab: what should we know?
WO2011060554A1 (fr) Composition pré-solidifiée et procédé de mise en place in situ d&#39;implants en chitosane présentant une large gamme de masses moléculaires et comportant ou pas des médicaments pour des applications de médecine régénératrice et de réparation des cartilages
Buchanan et al. Anticoagulant and antithrombin effects of intimatan, a heparin cofactor II agonist
JP2018524067A (ja) アンチトロンビン−ヘパリン組成物を利用する医療機器、システムおよび方法
Gonzalez The Development of an Anti-thrombotic and Anti-inflammatory Coating for Medical Devices
THABETHE THE HEPARINS
Diaz Engineering Polymeric Platforms: The Design of Novel Diagnostic Systems & Drug Delivery Devices via Surface Modification

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10831009

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10831009

Country of ref document: EP

Kind code of ref document: A1