EP2294126A1 - Injectable hydrogel forming chitosan mixtures - Google Patents

Injectable hydrogel forming chitosan mixtures

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Publication number
EP2294126A1
EP2294126A1 EP09762176A EP09762176A EP2294126A1 EP 2294126 A1 EP2294126 A1 EP 2294126A1 EP 09762176 A EP09762176 A EP 09762176A EP 09762176 A EP09762176 A EP 09762176A EP 2294126 A1 EP2294126 A1 EP 2294126A1
Authority
EP
European Patent Office
Prior art keywords
chitosan
composition
kda
highly
range
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.)
Withdrawn
Application number
EP09762176A
Other languages
German (de)
English (en)
French (fr)
Inventor
Noah Ben-Shalom
Zvi Nevo
Avraham Patchornik
Dror Robinson
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.)
Chi2Gel Ltd
Original Assignee
Chi2Gel Ltd
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
Priority claimed from US12/155,916 external-priority patent/US8153612B2/en
Application filed by Chi2Gel Ltd filed Critical Chi2Gel Ltd
Publication of EP2294126A1 publication Critical patent/EP2294126A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/042—Gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/736—Chitin; Chitosan; Derivatives thereof
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • 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
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/91—Injection
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the present invention relates to combinations of chitosans that form hydrogels in a pH-dependent and thermo-sensitive manner, aqueous solutions used to form such hydrogels, and methods of use thereof.
  • Hydrogels are highly hydrated, macromolecular networks, dispersed in water or other biological fluids. Hydrogels that are thermo-sensitive (or thermosetting) exhibit increased viscosity upon increases in temperature. Such hydrogels have been shown to have favorable application properties and longer survival periods at the site of application as compared to non-thermosensitive hydrogels, and are therefore advantageous as vehicles for slow-release drug delivery systems.
  • Chitosan occurs as a family of polymers, obtained by partial to substantial N- deacetylation of chitin, the latter being a linear polysaccharide of N-acetyl-D- glucosamine (GIcNAc) units in ⁇ -1,4 linkage. Chitin is present in abundance in invertebrates, particularly in the exoskeletons of crustaceans (e.g. shrimps, crabs, lobsters).
  • Commercial deacetylation processes used for chitosan production generally involve treatment of shellfish food processing waste products with concentrated alkaline solutions, usually sodium hydroxide.
  • chitosan may be a heteropolymer of D-glucosamine (GIcN) and GIcNAc units, or it may be a homopolymer formed exclusively of GIcN units.
  • GIcN D-glucosamine
  • Various techniques are available for varying the extent of deacetylation, and for re-acetylating the polymer in order to obtain chitosan having a desired degree of acetylation.
  • the deacetylation process results in free amino groups along the polymer chain, which render the polymer soluble in selected aqueous acid systems.
  • the degree of solubility of chitosan with a given degree of deacetylation depends on multiple parameters, including polymer molecular weight, temperature, and concentration and nature of the acid solvent.
  • one parameter used to characterize chitosan is the proportion of GIcNAc units to GIcN units, expressed as the degree of acetylation (DA), or as the reciprocal degree of deacetylation (DD or DDA).
  • the structure of chitosan may be further characterized by the mode of distribution of the GIcNAc and GIcN units within the polymer chain, wherein for example, a non-homogeneously deacetylated chitosan has deacetylated units occurring in blocks of variable size and/or location along the chain, whereas a homogeneously deacetylated chitosan has a randomly distribution of deacetylated units along the polymer chain.
  • the molecular weight is an important parameter, which together with the degree, and distribution mode of acetylation, determine properties of chitosan, such as solubility, biodegradability and viscosity.
  • Chitosan is bioadhesive, biocompatible, non-toxic, and non-immunogenic, allowing its use in the medical, pharmaceutical, cosmetic and tissue construction fields.
  • the use of chitosan for topical ocular application, intraocular injection and and transplantation in the vicinity of the retina has been disclosed (Felt et al., 1999; Patashnik et al., 1997; Song et al., 2001).
  • uniform submicron chitosan fibers prepared by electro-wet-spinning technology, and their use in development of artificial muscles, biosensors, and artificial organ components has been disclosed (Lee et al.
  • chitosan is specifically recognized and cleaved by certain enzymes, e.g. lysozyme, it is therefore bioerodable and biodegradable (Muzzarelli, 1997; Koga, 1998).
  • hydrogels comprising cross-linked chitosan and/or chitosan in combination with additional polymers are known, including for example, those comprising: chitosan chlorides or chitosan glutamates cross-linked with genipin (Mwale et al., 2005), reportedly useful as scaffolds for the encapsulation of invertebral disc cells (Roughley et al., 2006); chitosan-graft-poly(N-isopropylacrylamide), reportedly useful for entrapping chondrocytes and meniscus cells (Chen et al., 2006); a chitosan/poly(acrylic acid) pH-sensitive hydrogel (Shi et al., 2004); chitosan, ⁇ -glycerol phosphate and hydroxyethyl cellulose (Li et al., 2002); a chitosan/polyvinyl pyrrolidone pH-sensitive hydrogel (Risbud a
  • U.S. Patent No. 6,344,488 discloses a polysaccharide based gel solution comprising 0.1 to 5.0% by weight of chitosan; and 1.0 to 20% by weight of a monophosphate dibasic salt of polyol or sugar; wherein said gel solution is a solution at pH between 6.5 and 7.4 at a temperature below 20 0 C, and forms a gel within a temperature range from 20 to 70 0 C.
  • the thermosensitive chitosan hydrogel may be prepared by neutralizing a chitosan having a deacetylation degree of about 80% with ⁇ -glycerophosphate.
  • Thermosensitive chitosan hydrogels containing ⁇ -glycerophosphate include those referred to under the trade names BST-CarGelTM, intended for filling cartilage defects and cartilage repair; BST-DermOnTM, intended for wound healing; and BST-InPodTM, intended for treatment of heel pain.
  • ⁇ -glycerophosphate is a negatively charged molecular entity that can react with positively charged drugs, leading to their precipitation, or to the disturbance of their release from the hydrogel. Therefore, the presence of ⁇ -glycerophosphate decreases the range of drugs with which chitosan/ ⁇ - glycerophosphate hydrogels can be used.
  • the modulation of the properties of the hydrogel depends on the concentration of glycerophosphate, and is therefore limited by the solubility of ⁇ -glycerophosphate.
  • a high concentration of ⁇ - glycerophosphate is required for a low gelation time, and for avoiding the rapid elimination of the hydrogel after its administration.
  • a high concentration of ⁇ - glycerophosphate also decreases the viscosity of the hydrogel. Accordingly, these hydrogels lack the desirable combination of a low gelation time together with high viscosity.
  • the high concentration of ⁇ -glycerophosphate may induce precipitation of the hydrogel at the administration site.
  • chitosan/ ⁇ - glycerophosphate gels are hampered by turbidity, thus rendering them inappropriate for particular applications such as ocular or topical administration.
  • turbidity of chitosan/ ⁇ -glycerophosphate hydrogels is modulated by the degree of deacetylation of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the glucose amine monomers.
  • WO 2005/097871 discloses a pseudo-thermosetting neutralized ' chitosan composition, which comprises 0.1 to 2.0 wt/v %, preferably 0.5 to 1 wt/v %, based on the total composition, of a homogeneously reacetylated chitosan derived from a chitosan having a deacetylation degree of 80 to 90%, having a molecular weight of not smaller than 200 kDa, preferably not smaller than 600 kDa, and a deacetylation degree of 30 to 60%, preferably 45 to 55%, neutralized with an hydroxylated base, such as NaOH, wherein said composition forms a phosphate-free transparent hydrogel at a temperature higher than 5 0 C.
  • a pseudo-thermosetting neutralized ' chitosan composition which comprises 0.1 to 2.0 wt/v %, preferably 0.5 to 1 wt/v %, based on the total composition, of a homogene
  • the homogeneous distribution of the acetylated and deacetylated monomers of chitosan is an essential criterion for obtaining the subject hydrogels. It is furthermore disclosed that the consistency of the subject hydrogels may be improved by the addition of a diol, in particular 1,3-propandiol.
  • U.S. Patent No. 4,738,850 discloses a controlled release formulation comprising a drug selected from angiotensin converting enzyme inhibitors and ascorbic acid, in combination with chitosan, wherein a gel-like complex is formed of the drug and the chitosan in environments ranging from neutral to acidic.
  • the chitosan is 80 to 90% deacetylated and is present in the formulation at a concentration from about 5 to about 70% by weight.
  • U.S. Patent No. 6,140,089 discloses an encapsulation device comprising viable cells dispersed in a three-dimensional particulate, essentially non-cross-linked, chitosan core matrix encapsulated in a thermoplastic semipermeable membrane, wherein the chitosan core matrix is formed by precipitation of a chitosan solution containing said cells.
  • chitosan precipitation can be achieved by any method which removes or masks the charge of a sufficient number of the free amino groups, such as by placing chitosan-containing capsules in a buffered solution containing monovalent ions, or by adjusting the pH of a chitosan solution.
  • U.S. Patent No. 6,486,140 discloses use of a combination of an agent comprising chitosan and heparin immobilized to the chitosan for prevention of undesirable tissue adhesion.
  • the chitosan has a degree of N-acetylation of no more than about 90%, and the agent may be applied in the form of inter alia a solution or a gel.
  • U.S. Patent Application Publication No. 2005/0042265 discloses a composition comprising a chitosan hydrogel, having a degree of acetylation no greater than about 40%, and preferably between about 2 and about 6%, for use in repair and cicatrization of cutaneous lesions of chronic or acute wounds.
  • U.S. Patent No. 6,521,243 discloses an ionic chitosan iodine complex in the form of solutions and hydrogels, comprising chitosan or a derivative thereof; an aqueous vehicle; elemental iodine; and an iodide source.
  • the chitosan has an average molecular weight between 10 to 1000 kDa, preferably 100 to 800 kDa, and most preferably 250 to 750 kDa, and a degree of deacetylation of 40% to 95%, preferably 60% to 90%.
  • U.S. Patent No. 6,858,222 discloses a drug releasing fiber, comprising segments of chitosan having different degrees of deacetylation, and a method of production thereof. This disclosure does not specify any particular chitosan degree of deacetylation.
  • U.S. Patent No. 6,329,337 discloses an adhesive for biological tissue comprising a recombinant human serum albumin as a glue agent, and a bifunctional or multifunctional aldehyde as a cross-linking agent.
  • the glue agent may contain an additional component inter alia a partially-acetylated chitosan, such as a 50% acetylated chitosan, and further, an aqueous solution of the glue agent may be lyophilized, and, prior to use, reconstituted with water or saline for injection.
  • an additional component inter alia a partially-acetylated chitosan, such as a 50% acetylated chitosan
  • an aqueous solution of the glue agent may be lyophilized, and, prior to use, reconstituted with water or saline for injection.
  • WO 03/011912 teaches a process of preparing chitosan comprising subjecting particulate chitin to low temperature swelling with an aqueous solution, e.g. IO N alkali, for a period of at least 36 hours, followed by reacting the swollen particulate chitosan with an alkaline solution at an elevated temperature so as to cause deacetylation.
  • an aqueous solution e.g. IO N alkali
  • the swelling is carried out at a temperature of up to 30 0 C
  • the deacetylation is carried out at a temperature that is at least 5 °C and preferably at least 25 °C higher than the swelling stage, and acetylation is effected to give a chitosan product having a degree of acetylation of 0.2 to 0.7, especially 0.45 to 0.6.
  • the disclosed method further comprises a washing step following deacetylation, and the product may be optionally further modified e.g. by gel or solution formation.
  • the chitosan product is fully water soluble i.e. more than 97% by weight may be dissolved in a dilute acid solution.
  • WO 2004/069230 discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a release sustaining or mucoadhesive agent, and a physiologically active agent, wherein the release sustaining or mucoadhesive agent comprises at least two chitosans having different degrees of acetylation (F A ), wherein at least one chitosan has an FA value in the range from 0.25 to 0.80 (e.g., 0.30 to 0.60 or 0.33 to 0.55).
  • F A degrees of acetylation
  • the compositions are in a form suitable for administration into the gastrointestinal tract, e.g. orally or rectally, and may include gels.
  • WO 2004/068971 teaches foodstuffs comprising a nutritional food substance and a chitosan having an acetylation degree (F A ) of from 0.25 to 0.80, or a mixture of chitosans.
  • F A acetylation degree
  • WO 2004/069230 and WO 2004/068971 do not teach injectabgle solutions or ge forming solutions. These publications fail to teach chitosan hydrogels of defined viscosity, and further fail to describe preparation of hydrogels from mixtures of chitosans. In fact, the compositions and products exemplified in WO 2004/069230 and WO 2004/068971 comprise either only highly deacetylated chitosans which are not capable of forming hydrogels under physiological conditions since they will precipitate at pH values in the neutral range, or only highly acetylated chitosans which remain soluble at neutral pH and at 37 °C.
  • WO 2008/072230 published after the priority date of the present invention, discloses a chitosan composition comprising at least one type of chitosan having a degree of acetylation in the range of from about 30% to about 60%, and at least one type of chitosan having a degree of acetylation of at least 70%, wherein the composition undergoes pH- and temperature-dependent gelation to form a hydrogel.
  • the molecular weight of the each of the highly deacetylated and the highly acetylated chitosans is in the range of 10 kDa to about 4000 kDa, wherein the highly deacetylated chitosan preferably has a molecular weight of greater than about 200 kDa and the highly acetylated chitosan preferably has a molecular weight of greater than about 60 kDa.
  • hydrogel forming chitosan compositions that combine the properties of viscosity, mechanical integrity, favorable rates of gel formation and degradation at physiological conditions, and predictable drug release profiles, which can be utilized for development of slow release drug formulations and for scaffolds and implants for regenerative medicine.
  • the present invention provides a pH- and temperature-dependent hydrogel forming chitosan composition, gels comprising mixtures of chitosans and uses thereof.
  • the present invention is based, in part, on the finding that a composition comprising two different types of chitosans, which differ with respect to their degree of acetylation/deacetylation and having specific molecular weight ranges, forms a hydrogel at physiological conditions of pH and temperature, yet remains in a liquid state at pH values below the neutral range and/or at low temperatures, e.g. under refrigeration.
  • the composition comprises: a first type of chitosan having a degree of acetylation in the range of about 20% or less (also referred to interchangeably herein as “highly deacetylated chitosan” or “type 1 chitosan”); and a second type of chitosan having a degree of acetylation in the range from about 40% to about 60% (also referred to interchangeably herein as “highly acetylated chitosan” or “type 2 chitosan”).
  • the degree of deacetylation of each of the first and the second type of chitosan are respectively in the range from about 80% to about 100%, and in the range from about 40% to about 60%.
  • the chitosan hydrogels of the present invention are advantageous over prior art chitosan gel-forming compositions comprising a single type of chitosan.
  • the use of a combination of chitosans as described herein confers improved physical, chemical and pharmacokinetic properties of the resultant hydrogel, as compared to gels or hydrogels comprising a single type of chitosan.
  • the present invention utilizes the ability of highly acetylated chitosan to interact with highly deacetylated chitosan and thus avoid precipitation of the highly deacetylated chitosan, which would normally occur under the same pH conditions, in the absence of the highly acetylated chitosan. More specifically, highly deacetylated chitosans precipitate in aqueous solutions at pH values higher than about 6.5 due to protonization of the free amine groups, and are therefore unsuitable on their own for injection into human subjects for hydrogel formation.
  • highly deacetylated chitosans are advantageous for hydrogel formation as they confer mechanical strength and rigidity, and exhibit relatively long term stability since they are poor substrates for lysozyme and are therefore slowly degraded.
  • Highly acetylated chitosans can be used to complement the properties of highly deacetylated chitosans, since the former remain soluble at physiological pH, contribute elasticity and softness to hydrogels comprising them, and are more rapidly degraded by lysozyme.
  • the disclosed hydrogel forming composition can be utilized in various medical applications, in particular, slow release drug formulations, viscoelastic treatment of degenerative conditions such as osteoarthritis, and implants and scaffolds for tissue regeneration.
  • the present invention provides a chitosan composition
  • a chitosan composition comprising a highly acetylated chitosan having a degree of acetylation in the range of from about 40% to about 60%, and a highly deacetylated chitosan having a degree of acetylation of no greater than about 20%, wherein at 25 °C and at pH in the neutral range the composition has a viscosity of at least about 100 mPa-s at a shear rate of 50 rpm. According to particular embodiments, at 25 °C and at pH in the neutral range, the composition has a viscosity of no greater than about 70 mPa-s at a shear rate of 200 rpm.
  • Neutral pH as used herein refers to pH 7.0 ⁇ 0.2.
  • the composition has a viscosity of at least about 400 rnPa-s at a shear rate of 20 rpm, and a viscosity of no greater than about 200 mPa-s at a shear rate of 100 rpm.
  • the composition has a viscosity of at least 2 fold, preferably at least 3 fold that of the same composition at pH 6.0, wherein the viscosity is determined at a shear rate of 20 rpm.
  • the composition is in the form of a liquid.
  • the composition is in the form of a liquid.
  • the invention further provides a chitosan composition in the form of a hydrogel, the composition comprising a highly acetylated chitosan having a degree of acetylation in the range of from about 40% to about 60%, and a highly deacetylated chitosan having a degree of acetylation of no greater than about 20%, wherein at 25 0 C and at pH 7.0 ⁇ 0.2 the composition has a viscosity of at least 100 mPa-s at a shear rate of 20 rpm.
  • the composition has a viscosity of at least about 100 mPa-s at a shear rate of 50 rpm. According to particular embodiments, at 25 °C and at pH 7.0 ⁇ 0.2, the composition has a viscosity of at least about 200 rnPa-s at a shear rate of 50 rpm. According to particular embodiments, at 25 0 C and at pH 7.0 ⁇ 0.2, the composition has a viscosity of at least about 400 mPa-s at a shear rate of 20 rpm.
  • the invention further provides a chitosan composition in the form of an injectable solution, the composition comprising a highly acetylated chitosan having a degree of acetylation in the range of from about 40% to about 60%, and a highly deacetylated chitosan having a degree of acetylation of no greater than about 20%, wherein at 25 °C and at pH below the neutral range the composition has a viscosity of no greater than about 150 mPa-s at a shear rate of 50 rpm.
  • the composition has a viscosity of no greater than about 100 mPa-s at a shear rate of 50 rpm.
  • the highly acetylated chitosan has a degree of acetylation in the range of from about 45% to about 55%, and the highly deacetylated chitosan has a degree of acetylation of about 15% or less.
  • the highly acetylated chitosan is a homogenously reacetylated chitosan.
  • the highly deacetylated chitosan is a non-homogenously deacetylated chitosan.
  • the highly deacetylated chitosan has a molecular weight of at least about 100 kDa, and the highly acetylated chitosan has a molecular weight of at least about 200 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 100 kDa to about 2000 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 2000 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 100 kDa to about 700 kDa, such as for example, about 100 kDa to about 400 kDa, or about 400 kDa to about 700 kDa; and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 400 kDa to about 700 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • the highly deacetylated chitosan has a molecular weight selected from the group consisting of about 100 kDa; about 400 kDa, and about 650 kDa; and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • the highly deacetylated chitosan and the highly acetylated chitosan are each present at a concentration of about 0.2% to about 3% w/v of the total composition. According to another particular embodiment, the highly deacetylated chitosan and the highly acetylated chitosan are each present at a concentration of about 0.5 % to about 2% w/v of the total composition. According to another particular embodiment, the highly deacetylated chitosan and the highly acetylated chitosan are each present at a concentration of about 1% to about 1.2% w/v of the total composition.
  • the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is selected from the group consisting of 1:1; 1:2; 1:3 and 1 :4.
  • each of the highly deacetylated chitosan and the highly acetylated chitosan are present at a concentration of about 1% to about 1.2 % w/v of the total composition, and the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is 1 : 1.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 400 kDa to about 700 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa, wherein each of the highly deacetylated chitosan and the highly acetylated chitosan are present at a concentration of from about 1% to about 1.2% w/v of the composition; and wherein the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is 1 : 1.
  • the highly deacetylated chitosan has a molecular weight of about 2000 kDa and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa, wherein the concentration of the highly deacetylated chitosan is 0.5% w/v of the total composition, and wherein the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is selected from the group consisting of 1 :2; 1 :3 and 1 :4.
  • the hydrogel has a degradation profile in which no more than about 50% of the hydrogel is degraded by 4 days following its formation.
  • the composition further comprises a lysozyme inhibitor, wherein the lysozyme inhibitor is selected from the group consisting of a protein and a saccharide.
  • the saccharide is tri-N-acetyl-glucosamine.
  • the lysozyme inhibitor is bound to the highly acetylated chitosan.
  • the composition comprises tri-N-acetyl-glucosamine bound to the highly acetylated chitosan.
  • the chitosan composition further comprises at least one negatively charged substance selected from a polysaccharide, a phospholipid and combinations thereof.
  • the negatively charged polysaccharide is selected from the group consisting of an animal- derived polysaccharide, a plant-derived polysaccharide, a glycosaminoglycan, and combinations thereof.
  • the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, dermatan sulfate, dextran sulfate, heparan sulfate, heparin, hyaluronic acid and keratan sulfate.
  • the phospholipid is phosphatidylcholine.
  • the chitosan composition further comprising hyaluronic acid and phosphatidylcholine is for use in synovial fluid replacement and/or in viscoelastic treatment, for example in the treatment of osteoarthritis.
  • the composition comprises microspheres of chitosan encapsulating a drug and/or electrospun chitosan fibers embedded in the gel.
  • the composition further comprises at least one of a drug, a polypeptide and a cell (such as an animal cell or a plant cell).
  • the composition further comprises an emulsif ⁇ er.
  • the chitosans and the emulsifier are in the form of nanoparticles.
  • the nanoparticles are encapsulated in the hydrogel.
  • the composition is formulated for administration by a route selected from direct instillation, injection and endoscopic administration.
  • an implantable device comprises the composition in the form of a hydrogel.
  • Such an implantable device may be for a use selected from tissue repair, tissue reconstruction, tissue construction, and tissue replacement.
  • an anti-adhesion device comprises the composition of the invention, which may be used in applications such as cardiothoracic surgery and abdominal surgery.
  • a drug delivery device or system comprises the composition of the invention.
  • the drug delivery device or system may be for slow release of an embedded medication.
  • drugs for use in this system include proteins and non-protein agents such as, for example, ACE- inhibitors, anti-inflammatory drugs, opthalmological drugs and urological drugs.
  • a cosmetic agent delivery device comprises the composition of the invention.
  • An example of a suitable cosmetic agent is an anti-wrinkle agent
  • the drug delivery device or system, or the cosmetic agent delivery device may also optionally comprise one or more of a mineral, a vitamin, a food additive or natural extract such as a plant derived extract.
  • the hydrogel itself, optionally with an active ingredient, may be used as a food additive.
  • any of the drug, the cosmetic agent, the mineral, the vitamin, the food additive or the natural extract are in the form of nanoparticles, wherein the nanoparticles are encapsulated in the hydrogel of the invention.
  • a three-dimensional gel construct comprises the hydrogel of the invention, wherein the hydrogel is a support for cells.
  • the cells are endogenous cells or exogenous cells, and the construct optionally further comprises exogenous growth factors.
  • a cell-loaded artificial matrix comprises the hydrogel of the invention.
  • the cells are selected from the group consisting of chondrocytes, fibrochondrocytes, ligament fibroblasts, skin fibroblasts, tenocytes, myofibroblasts, mesenchymal stem cells and keratinocytes.
  • composition of the invention for the preparation of a medicament for treating heel pain.
  • a use of the composition of the invention for the preparation of a medicament for wound healing there is provided a use of the composition of the invention for the preparation of a medicament for disrupting biofilm.
  • a use of the composition of the invention for the preparation of a medicament for preventing or treating surgical adhesions there is provided a use of the composition of the invention for the preparation of a medicament for treating rotator cuff damage, including rotator cuff tears, wherein the medicament optionally further comprises autologous cells.
  • composition of the invention for the preparation of a medicament for treating osteoarthritis, wherein the composition further optionally includes hyaluronic acid and phosphatidylcholine as a synovial fluid replacement, as described herein.
  • the chitosan hydrogel may be used as a lubricating agent for treatment of conditions such as vaginal atrophy, dry eyes, conjunctivitis sicca, dry nose following upper respiratory infections, as well as a general soothing agent for various abrasions.
  • the invention provides a chitosan composition
  • a chitosan composition comprising a highly deacetylated chitosan having a molecular weight in the range of from about 100 to about 4000 kDa, such as about 100 kDa to about 2000 kDa, and having a degree of acetylation of no greater than about 20%, and a saccharide oligomer having a molecular weight in the range of from about 200 to about 20000 Da, wherein the composition is in a form of an aqueous solution.
  • the saccharide oligomer is selected from the group consisting of a chitosan oligomer; a D-glucosamine oligomer and an N-acetyl-D- glucosamine oligomer.
  • the chitosan oligomer is selected from the group consisting of a highly deacetylated chitosan oligomer having a degree of acetylation of no greater than about 20%, and a highly acetylated chitosan oligomer having a degree of acetylation of from about 40% to about 60%.
  • the highly acetylated chitosan oligomer has a degree of acetylation in the range of from about 45% to about 55%. According to particular embodiments, the highly deacetylated chitosan oligomer has a degree of acetylation of about 15% or less.
  • the N-acetyl-D-glucosamine oligomer has up to about 7 units. According to particular embodiments, the D-glucosamine oligomer has between 3 and about 100 units. In a particular embodiment, the D-glucosamine oligomer has between 3 and about 50 units.
  • the ratio of the saccharide oligomer and the highly deacetylated chitosan having a molecular weight in the range of from about 100 kDa to about 2000 kDa is greater than 1:1.
  • the ratio ranges from 2:1 to 20:1.
  • the composition has a viscosity of no greater than 150 rnPa-s at a shear rate of 50 rpm.
  • the composition has a viscosity of no greater than 100 mPa-s at a shear rate of 50 rpm.
  • the composition has a viscosity of no greater than 70 mPa-s at a shear rate of 200 rpm.
  • the present invention provides a method for the production of a stable hydrogel, wherein the method comprises dissolving in an acidic aqueous solution at least one highly acetylated chitosan having a degree of acetylation in the range of from about 40 to about 60%, and at least one highly deacetylated chitosan having a degree of acetylation of no greater than about 20%, so as to form a composite solution; adjusting the pH of the composite solution to a value of 6.5 to 7.2; and increasing the temperature of the composite solution to 37 °C while raising the pH to a value of 7.0 to 7.6; so as to produce a hydrogel which at 25 °C has a viscosity of at least 100 mPa-s at a shear rate of 50 rpm.
  • FIG. 1 illustrates the formation of a hydrogel according to some embodiments of the present invention from a liquid composition comprising two different types of chitosan.
  • FIG. 2 illustrates degradation over time of different hydrogel compositions formed from a combination of type 1 and type 2 chitosans.
  • FIG 2A shows degradation of compositions comprising a type 2 chitosan (MW 220 kDa; DA 50) in combination with a type 1 chitosan (MW 65 kDa; DA 15) in a ratio of 1:1 (diamond symbols); 1:2
  • FIG. 2B shows degradation over time of hydrogel compositions formed from a combination of a type 2 chitosan (MW 220 kDa; DA 50) and a type 1 chitosan of molecular weight of either 65 kDa and DA 15 (triangle symbols) or 100 kDa and DA 9 (square symbols), at a ratio of 1 : 1.
  • FIG. 3 illustrates release of hemoglobin from a hydrogel composition formed from a combination of a type 1 chitosan (MW 65 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50), as measured by the amount of protein ( ⁇ g/ml) in eluent.
  • the systems represented by diamond, square, triangle, star and asterisk symbols correspond to repetitions of the experiment, and the circle symbols represent the mean of the experiments.
  • FIG. 4 illustrates release of bovine serum albumin (BSA) from a hydrogel composition formed from a combination of a type 1 chitosan (MW 65 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50), as measured by optical density (OD) of the eluent.
  • BSA bovine serum albumin
  • the systems represented by diamond, square, triangle, star and asterisk symbols correspond to repetitions of the experiment, and the circle symbols represent the mean of the experiments.
  • FIG. 5 presents a bar chart illustrating release of BSA from a hydrogel formed from a combination of type 1 chitosan (MW 65 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50).
  • FIG. 6 illustrates the degradation over time of hydrogel compositions formed from a combination of a type 1 chitosan (MW 65 kDa; DA 50) and a type 2 chitosan (MW 220 kDa; DA 50), either including (square symbols) or lacking BSA (diamond symbols), expressed as a percentage of the weight of the gel remaining undegraded.
  • FIG. 7 illustrates the integration of the release profile of BSA (square symbols) with the degradation profile (triangle symbols) of a hydrogel composition formed from a combination of a type 1 chitosan (MW 65 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50) and BSA.
  • FIG. 7 illustrates the integration of the release profile of BSA (square symbols) with the degradation profile (triangle symbols) of a hydrogel composition formed from a combination of a type 1 chitosan (MW 65 kDa; DA 15) and a type 2
  • FIG. 8 shows histopathology of wound bed biopsies taken from rats, either treated with a hydrogel composition formed from a combination of a type 1 chitosan (MW 660 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50) (Fig. 8A), or untreated (Fig. 8B).
  • a hydrogel composition formed from a combination of a type 1 chitosan (MW 660 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50) (Fig. 8A), or untreated (Fig. 8B).
  • FIG. 9 shows a graph of the results of treating inflicted wounds in diabetic rats with a hydrogel composition formed from a combination of a type 1 chitosan (MW 660 kDa; DA 15) and a type 2 chitosan (MW 220 kDa; DA 50) and acetylglucosamine oligomers (Gel), expressed as the surface area of wound bed biopsies on Day 7 following biopsy.
  • the wound was either covered by a plaster bandage (Control + Cover) or left uncovered (Control no Cover).
  • FIG. 10 shows the results of in vivo experiments performed on rats for rotator cuff damage.
  • FIG. 10A Histological slices from rats treated with chitosan hydrogel containing bone marrow cells following suturing (Fig. 10A) show migration of inflammatory cells and repair in the defect area, whereas slices from rats that were untreated following suturing (Fig. 10B) show empty defect areas with no signs of repair.
  • FIG. 11 presents a schematic illustration of measuring a friction coefficient.
  • FIG. 12 shows the static friction coefficient measured between two layers of normal cartilage to which were applied: saline; a hydrogel composition formed from a combination of a highly deacetylated chitosan (MW 660 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) (Combination); the same chitosan combination further containing chondroitin sulfate (+CS); the same chitosan combination further containing chondroitin sulfate and phosphatidyl choline (+CS/PC); or hyaluronic acid (HA).
  • a hydrogel composition formed from a combination of a highly deacetylated chitosan (MW 660 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) (Combination); the same chitosan combination further containing chondroitin
  • FIG.13 shows the viscosity of various chitosan compositions as a function of pH when measured at 25 0 C.
  • Compositions contained a highly acetylated chitosan (MW 220 kDa DA 50) and a highly deacetylated chitosan (MW 420 kDa DA 9), either each on its own or in combination.
  • FIG.14 shows the viscosity of compositions comprising two types of chitosans as a function of pH when measured at 25° C.
  • Compositions contained a combination of a highly deacetylated chitosan (MW 65 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1, or a combination of a highly deacetylated chitosan (MW 100 kDa; DA 9) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1.
  • a highly deacetylated chitosan MW 65 kDa; DA 15
  • a highly acetylated chitosan MW 220 kDa; DA 50
  • FIG.15 shows the viscosity of compositions comprising two types of chitosans at different pH values when measured at 25 °C and at varying shear rates.
  • Compositions contained a combination of a highly deacetylated chitosan (MW 100 kDa; DA 9) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1 at various pH values
  • Figure 15A or a combination of a highly deacetylated chitosan (MW 65 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1 at various pH values (Figure 15B).
  • a highly deacetylated chitosan MW 65 kDa; DA 15
  • a highly acetylated chitosan MW 220 kDa; DA 50
  • FIG.16 shows the viscosity of compositions comprising two types of chitosans at different pH values when measured at 1 0 C and at varying shear rates.
  • Compositions contained a combination of a highly deacetylated chitosan (MW 100 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) (SKl 0:50) at a ratio of 1:1 at various pH values; or a combination of a highly deacetylated chitosan (MW 65 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) (FVL10:50) at a ratio of 1:1 at various pH values.
  • a highly deacetylated chitosan MW 100 kDa; DA 15
  • a highly acetylated chitosan MW 220 kDa;
  • FIG.17 shows the viscosity of compositions comprising a combination of a highly deacetylated chitosan (MW 100 kDa; DA 9) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1 when measured at varying shear rates, either at 1 °C ( Figure 17A); or at 25 0 C ( Figure 17B), both at various pH values.
  • a highly deacetylated chitosan MW 100 kDa; DA 9
  • a highly acetylated chitosan MW 220 kDa; DA 50
  • FIG.18 shows the viscosity of compositions comprising a combination of a highly deacetylated chitosan (MW 65 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1:1 when measured at varying shear rates and at 1 °C (Figure 18A); or at 25 0 C ( Figure 18B), both at various pH values.
  • a highly deacetylated chitosan MW 65 kDa; DA 15
  • a highly acetylated chitosan MW 220 kDa; DA 50
  • FIG.19 shows the viscosity of a composition comprising two types of chitosans at different pH values when measured at 25° C at varying shear rates.
  • the composition contained a combination of a highly deacetylated chitosan (MW 660 kDa; DA 15) and a highly acetylated chitosan (MW 220 kDa; DA 50) at a ratio of 1 : 1 at various pH values.
  • the pH-dependent and thermosensitive chitosan hydrogels of the present invention are advantageous over prior art chitosan gel-forming compositions comprising a single type of chitosan.
  • the use of a combination of chitosans " as described herein confers improved physical, chemical and pharmacokinetic properties of the resultant hydrogel, as compared to gels or hydrogels comprising a single type of chitosan.
  • a composition "comprising a single type of chitosan” refers to compositions in which either a highly deacetylated chitosan or a highly acetylated chitosan is the sole chitosan present in the composition.
  • hydrogels which contain only a highly acetylated chitosan are mechanically much weaker than the hydrogels of the present invention, possibly due to the different interactions responsible for gel formation.
  • gels formed solely from highly acetylated chitosans generally require a high chitosan concentration to effect gel formation, and/or require an additional agent, such as 1,3 -propanediol for improving the viscoelastic behavior of the gel, as disclosed for example in WO 2005/097871.
  • compositions which contain only a highly acetylated chitosan will form gels only at low temperatures, which negates their use for in vivo applications.
  • gels formed solely from highly acetylated chitosans are subject to rapid enzymatic degradation by serum enzymes such as lysozyme, for which they are favorable substrates. Accordingly, such gels have relatively shorter half-life in vivo in comparison to the hydrogels of the invention, and accordingly, the former are not suitable for preparation of slow release drug formations, implants and devices which require prolonged resilience and stability.
  • the term "pseudo-thermosetting” in connection with the composition of the present invention means that temperature does not induce the gelation of the composition but acts rather as a catalyst which dramatically shortens the gelation time when risen.
  • the term “hydrogel” refers to a three-dimensional macromolecular network having a degree of hydration of at least 90%, and which exhibits substantially no flow when in the steady-state.
  • the chitosan hyrogels of the present invention generally attain a steady state at pH values greater than about 6.5 and at temperatures greater than about 4 °C.
  • a chitosan hydrogel according to the invention does not encompass a chitosan gel formed from a single type of chitosan such as a highly deacetylated chitosan in the swollen state.
  • viscosity refers to a measure of the resistance of a fluid which is being deformed by either shear stress or extensional stress.
  • Newtonian fluids exhibit constant viscosity over a wide range of shear rates i.e. they are independent of the shear rate, whereas non-Newtonian fluids (such as polymeric gels) exhibit varying viscosity in response to different shear rates.
  • Non-Newtonian fluids exhibit a variety of different correlations between shear stress and shear rate. Generally, the viscosity of non-Newtonian fluids decreases at high shear rates (a phenomenon known as shear thinning), and the viscosity increases at low shear rates.
  • the SI physical unit of dynamic viscosity is the pascal-second (Pa- s), which is identical to kg-m ⁇ -s "1 .
  • the millipascal (mPa-s) is 0.01 Pa s.
  • Viscosity of a non-Newtoninan fluid may be measured with a rheometer, which imposes a specific stress field or deformation to the fluid, and monitors the resultant deformation or stress.
  • non-homogeneous deacetylation in reference to chitosan means that the deacetylated units of D-glucosamine occur in blocks of variable size and/or distribution.
  • homoogeneous deacetylation in reference to chitosan means that the deacetylated units of D-glucosamine are randomly distributed along the polymer.
  • homogeneous reacetylation in reference to chitosan means that deacetylated chitosan is reacetylated in a manner in which the N-acetylD- glucoasamine units and the D-glucosamine units are randomly distributed along the polymer.
  • neutral pH As used herein, the terms “neutral pH”, “pH in the neutral range”, “neutralized”, “physiological pH” and the like mean a pH of 7.0 ⁇ 0.2.
  • saccharide oligomer means a saccharide having a degree of polymerization of 3 to about 100 monomelic units.
  • the oligomer may be formed from a single type of saccharide unit, for example only D-glucosamine units, or it may contain different types of saccharide units, for example D-glucosamine units and N-acetyl-D-glucosamine units, as in a chitosan oliogmer.
  • compositions, methods or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • Chitosans which are deacetylated to a degree of deacetylation (also referred to herein as DD or DDA) of about 70-100% (i.e. degree of acetylation, DA, of up to about 30%), such as commercially available chitosan, are termed herein type 1 chitosans or chitosans type 1. These chitosans are insoluble at physiological pH, and are poorly recognized by Iyso2yme.
  • Such chitosans when utilized in in vivo applications, are typically characterized by relatively slow biodegradation, which, depending on the degree of deacetylation, can last from a few days to a few months. Gels formed by chitosans of this type have a low degree of acetylation, such that the free amine groups participate in dense hydrogen bonds with many hydrophobic interactions.
  • the degradation rate of chitosans has been shown to be a function of the degree of deacetylation. Degradation of chitosan has an influence on cell proliferation and remodeling.
  • chitosans Highly homogeneously deacetylated or reacetylated chitosans (having a degree of acetylation of from about 30% to about 60%) are termed herein type 2 chitosans or chitosans type 2.
  • Such chitosans are readily digested/degraded by lysozyme, thereby enabling, for example, controlled drug release of a drug encapsulated therein.
  • the degree of deacetylation of chitosan is lower than 30%, the chitosan becomes a polymer close to chitin that is insoluble in acidic conditions and therefore not suitable for use in embodiments of the present invention.
  • a degree of deacetylation greater than 70% precipitation of chitosan occurs.
  • the degree of ' deacetylation of chitosan may be determined by a spectrophotometric method such as described, for example, in the literature by R.A. Muzarelli and R. Richetti [Carbohydr. Polym. 5, 461-472, 1985 or R.A. Muzarelli and R. Richetti in "Chitin in Nature and Technology", Plenum Press 385-388, 1986].
  • chitosan is solubilized in 1% acetic acid and the DD is determined by measuring its content of N-acetyl-glucosamine by UV at 200, 201, 202, 203 and 204 nm using N-acetyl-D-glucosamine solutions as standards.
  • the present invention relates to a polysaccharide chitosan composition
  • a polysaccharide chitosan composition comprising a combination of at least one highly acetylated chitosan (type 2) having a degree of acetylation in the range of from about 40% to about
  • the highly acetylated type 2 chitosans can interact through electrostatic, hydrogen and hydrophobic interactions with the highly deacetylated chitosans type 1.
  • the extent of interaction increases with increasing pH.
  • a composition comprising solutions of both types of chitosan can form a stable gel at physiological pH, without the need for glycerophosphate. The obtained composition is therefore devoid of glycerophosphate.
  • the chitosan composition described is either in the form of an aqueous solution, or a hydrogel.
  • the transition from the liquid state to a hydrogel occurs at pH conditions above about 6.5.
  • the hydrogel is stable at physiological conditions i.e. pH in the neutral range and 37 °C.
  • composition described herein can form a gel at room temperature or at lower temperatures (e.g., 4 0 C). Nonetheless, the gel formation at such conditions is slow and may last from a few days to a few months, thus enabling to store and transfer the composition as an aqueous solution.
  • Type 1 chitosans in the unprotected state precipitate at a pH of about 6.5, which is less than physiological pH.
  • Interaction of the highly hydrophobic, homogenously acetylated chitosan type 2 with chitosan type 1 prevents this precipitation of the non- homogenously acetylated type 1 chitosan, by formation of hydrogen and hydrophobic bonds, allowing the formation of a stable semi-solid hydrogel at pH 7.0 in the neutral range.
  • the secondary bonds which are formed allow the encapsulation of the non- homogenous chitosan chains and maintain its solubility at pH greater than its pKa value.
  • secondary chain interactions are the main molecular forces involved in gel formation (Chenite et al. 3 2000; Berger et al., 2005).
  • Type 1 chitosans mainly contribute to the stability, strength and rigidity of the gel, and provide slow degradation, while type 2 chitosans contribute to the softness, elasticity and fast solubilization of the gel.
  • the degradation profiles of compositions comprising type 1 and type 2 chitosans are discussed further in Example 2 below, and are shown in Figure 2.
  • the present inventors have shown that a hydrogel formed from a type 1 chitosan having a molecular weight of about 100 kDa and a type 2 chitosan having a molecular weight in the range of 200 to 250 kDa provides a substantially linear degradation rate, wherein at least 50% of the hydrogel remains undegraded by 4 days following gel formation (Figure 2B).
  • gels formed from the same type 2 chitosan and a type 1 chitosan of lower molecular weight display less desirable two-phase degradation patterns ( Figure 2A).
  • the type 2 chitosan can be regarded as a "protector” or “coating”, which provides a shell around the type 1 chitosan and thus prevents its precipitation.
  • the type 2 chitosan is recognized by lysozyme.
  • This feature enables control of the degradation rate of a hydrogel formed from the composition described herein.
  • binding a lysozyme inhibitor which may be a protein or a saccharide
  • a suitable example of a lysozyme inhibitor is the saccharide tri-N- acetylglucosamine.
  • the physical and chemical properties of the hydrogel formed from the described composition are altered by raising or lowering the molecular weight of the chitosans and/or their degree of acetylation, and by the natural acetylation diversity of chitosans from different sources.
  • the properties of the gel can further be controlled by selection of the type of reacetylation (i.e. homogenous or non-homogenous), or by mapping the patterns of distribution of the deacetylated/acetylated sites.
  • the highly acetylated chitosan is homogenously reacetylated. Further preferably, the highly deacetylated chitosan is non-homogenously deacetylated.
  • chitosan is industrially prepared by deacetylation of dry chitin flakes (Muzzarelli, 1986). Deacetylation preferentially occurs in the amorphous zones of the chitin molecules at the surface of the flakes, resulting in non-homogeneous monomers with variable block size of deacetylated unit distribution (Aiba, 1991).
  • reacetylated chitosan under homogeneous conditions adopts a random distribution of deacetylated monomers, which induces a decrease of the crystallinity of chitosan and in turn increases its solubility (Aiba, 1991, 1994; Ogawa and Yui, 1993 ; Milot et. al., 1998).
  • Homogeneous reacetylation of chitosan has the effect of increasing the number of hydrophobic sites by replacing amine groups with acetyl groups, and also reduces the crystalline structure that makes chitosan tend to fold, cumulating in increased solubility of the chitosan. Reacetylation prevents refolding of the polymer, maintaining the straight chain, and thus preventing the pH-related decrease in solubility.
  • any commercial chitosan of pharmaceutical grade and sufficient molecular weight may be used in the preparation of reacetyated chitosan. Methods for homogeneous reacetylation are disclosed for example in WO 2005/097871 and WO 03/011912.
  • the product may be characterized with respect to the degree of deactylation, for example as measured by a UV method (Muzarelli et al. in "Chitin in Nature and Technology", Plenum Press, New York, 385- 388, (1986)), and with respect to the molecular weight, for example as determined by size exclusion chromatography (Felt et al. Int. J. Pharm. 180:185-193 (1999)).
  • the chitosan composition disclosed herein has a viscosity of at least 100 mPa-s at a shear rate of 50 rpm when the composition is at 25 0 C and at pH in the neutral range.
  • the viscosity is not greater than 70 mPa-s, when the composition is at the same temperature and pH conditions.
  • the composition at 25 0 C and at pH 7.0 ⁇ 0.2 the composition has a viscosity of at least 100 mPa-s at a shear rate of 20 rpm.
  • the composition at 25 °C and at pH in the neutral range, has a viscosity of at least 400 mPa-s at a shear rate of 20 rpm.
  • the composition has a viscosity of no greater than 200 mPa-s at a shear rate of 100 rpm.
  • the hydrogel composition has a viscosity of at least 100 mPa-s at a shear rate of 20 rpm.
  • the hydrogel composition has a viscosity of at least 100 mPa-s, or at least 200 mPa-s, at a shear rate of 50 rpm.
  • the composition has a viscosity that is at least 2 fold greater than that of the same composition at pH 6.0, when the viscosity is determined at a shear rate of 20 rpm.
  • the composition has a viscosity that is at least 3 fold greater than that of the same composition at pH 6.0, when the viscosity is determined at a shear rate of 20 rpm.
  • the liquid composition has a viscosity of no greater than about 150 mPa-s, at a shear rate of 50 rpm.
  • the liquid composition has a viscosity of no greater than about 100 rnPa-s, at a shear rate of 50 rpm.
  • the compositions remain in substantially a liquid state, indicating their suitability for low pH and/or refrigeration storage in the liquid state prior to administration, for example by injection for in vivo gel formation.
  • Increasing the molecular weight of the chitosan increases its viscosity, such that the polymer is highly hydrated and highly hydrophobic. This is demonstrated for example in Example 9 and Figure 19 which disclose that the viscosity of the composition FM80- 50 1 : 1 comprising a type 1 chitosan of molecular weight 660 kDa is substantially higher than that of the composition SKl 0:50 1:1, the latter of which comprises a type 1 chitosan of molecular weight of only 100 kDa.
  • these compositions exhibit viscosities of about 420 and about 110 mPa-s, respectively. Accordingly, the invention enables formation of hydrogels of varying strength and water retention. This enables one of ordinary skill in the art to produce a hydrogel of selected degradation rate, drug release, and mechanical strength.
  • each of the highly acetylated and highly deacetylated chitosans has a molecular weight of greater than about 100 kDa.
  • the highly deacetylated chitosan may have a molecular weight of at least about 100 kDa, and the highly acetylated chitosan may have a molecular weight of at least about 200 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 100 kDa to about 2000 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 2000 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 100 kDa to about 700 kDa, such as for example, about 100 kDa to about 400 kDa, or about 400 kDa to about 700 kDa; and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 400 kDa to about 700 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • the highly deacetylated chitosan has a molecular weight selected from the group consisting of about 100 kDa; about 400 kDa, and about 650 kDa; and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa.
  • Molecular weight of chitosan may be determined by size exclusion chromatography as reported for example by O. Felt, P. Purrer, J. M. Mayer, B. Plazonnet, P. Burri and R. Gurny in Int. J. Pharm. 180, 185- 193 (1999).
  • the upper limit of MW is determined by the required ease of administration, which depends on the chosen application.
  • the degree of acetylation results in increased hydrophobicity in the range of 0 to 30% DA, but at higher values, such as 40 to 60% DA, the polymer become more soluble as the DA is increased. Furthermore, increasing the number of N-acetyl glucosamine groups increases the rate of degradation in the body, due to increased recognition sites for lysozyme. Hence, the rate of release of drug from the hydrogel can be controlled by varying the degree of chitosan acetylation.
  • Variations in the molecular weight, degree of deacetylation and the distribution of the acetylated sites, concentration and ratio of the two or more chitosans affect the conditions (pH, temperature etc.) under which gel formation occurs; solubility; biodegradability; degree of reactivity with proteins, active pharmaceutical ingredients or other chemicals; hydrophobicity/hydrophilicity; degree of hydration; as well as biological and biocompatibility properties of the gel, such as effect on cell growth, proliferation and survival, ability of chitosans to function as inflammatory or antiinflammatory mediators, and the effect of chitosans on acceleration or deceleration of wound healing.
  • type 1 chitosans of higher molecular weight have higher hydrophobicity and higher viscosity, resulting in a stronger gel due to higher inter- molecular interactions.
  • Type 1 chitosans of higher DDA have a lower rate of degradation.
  • Type 1 chitosans having higher crystallinity have a lower degradation rate due to the fact that the crystalline form is non-soluble.
  • each of the highly acetylated and the highly deacetylated chitosans is independently present at a concentration of about 0.2% to 3% w/v of the total composition.
  • each of highly acetylated and the highly deacetylated chitosans is independently present at a concentration of about 0.5% to 2% w/v of the total composition.
  • each of highly acetylated and the highly deacetylated chitosans is independently present at a concentration of about 1% to 1.2% w/v of the total composition.
  • the ratio between the highly acetylated and the highly deacetylated chitosans is 1:1, such that the above concentrations are for each of the highly acetylated and the highly deacetylated chitosans.
  • the ratio can be 2:1, 3:1 and even 4:1. Also contemplated are ratios such as 1.1:1, 1.2:1, 1.5:1 1.8:1, and any other ratio in the range of from 1:1 to 4:1.
  • each of the highly deacetylated chitosan and the highly acetylated chitosan are present at a concentration of about 1% to about 1.2% w/v of the total composition, and the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is 1 : 1.
  • the highly deacetylated chitosan has a molecular weight in the range of from about 400 kDa to about 700 kDa, and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa, wherein each of the highly deacetylated chitosan and the highly acetylated chitosan are present at a concentration of from about 1% to about 1.2% w/v of the composition; and wherein the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is 1:1.
  • the highly deacetylated chitosan has a molecular weight of about 2000 kDa and the highly acetylated chitosan has a molecular weight in the range of from about 200 kDa to about 250 kDa, wherein the concentration of the highly deacetylated chitosan is 0.5% w/v of the composition, and wherein the ratio of the highly deacetylated chitosan and the highly acetylated chitosan in the composition is selected from the group consisting of 1 :2; 1 :3 and 1 :4.
  • composition described herein offers greater possibility of controlling the properties of the formed hydrogel, including, for example, the hydrogel strength, rate of degradation, and release rate, as compared for example to previously disclosed chitosan/ ⁇ -glycerophosphate hydrogels.
  • the hydrogel of the present invention may further comprise a third chitosan, selected from either type 1 or type 2, having a different molecular weight or degree of deacetylation, thus extending control over the resultant hydrogel.
  • the polysaccharide hydrogel according to the present invention may optionally further comprise a negatively charged substance, such as a negatively charged polysaccharide or a negatively charged -phospholipid.
  • Negatively charged polysaccharides include various animal-derived polysaccharides, plant-derived polysaccharides, glycosaminoglycans and combinations thereof.
  • Glycosaminoglycans include for example, chondroitin sulfate, dermatan sulfate, dextran sulfate, heparan sulfate, heparin, hyaluronic acid, keratan sulfate and combinations thereof.
  • an exemplary suitable phospholipid is phosphatidylcholine.
  • the chitosan composition further comprises hyaluronic acid and phosphatidylcholine.
  • Such a composition is highly beneficial for use as a synovial fluid replacement in osteoarthritis treatment, as it lowers the friction between cartilage surfaces, as disclosed in Example 6.
  • the chitosan composition described herein further comprises both a glycosaminoglycan and a phospholipid.
  • compositions and mixtures based on these two types of chitosans may be used to provide hydrogels with suitable properties for a wide range of applications.
  • Exemplary applications include, but are not limited to drug delivery systems e.g. for slow release of agents or medications, scaffolding of various consistencies, including gels for supporting cell growth or bone structural support; cartilage repair; tissue reconstruction; in wound-dressings, promoting scar free healing and macrophage activation; for production of artificial skin; as an artificial kidney membrane; for bone filling; and heel pain relief and as synovial fluid replacement compositions.
  • the hydrogel may be formed in situ (in vivo) sub-cutaneously, intra-peritoneally, intra-muscularly or within biological connective tissues, bone defects, fractures, articular cavities, body conduits or cavities, eye cul-de-sac, or solid tumors.
  • the polysaccharide solution may be introduced within an animal or human body by injection or endoscopic administration.
  • Drugs, polypeptides, living microorganisms, animal or human cells may be incorporated within the polysaccharide solution prior to gelation.
  • a hydrogel encompasses a semi-solid gel formed from the chitosan aqueous solutions described herein, upon subjecting these solutions to the physiological conditions described herein.
  • the hydrogel is preferably formed in vivo, upon administration of the chitosan composition, but can alternatively be formed ex vivo prior to its utilization, for example as an implant.
  • the gel may be incorporated as a whole, or as a component, into implantable devices or implants for repair, reconstruction and/or replacement of tissues and/or organs, either in animals or humans.
  • the gel may be used as a whole, or as a component of, implantable, transdermal or dermatological drug delivery systems.
  • the gel may be used as a whole, or as a component of, ophthalmological implants or drug delivery systems.
  • the gel may be used for producing cells-loaded artificial matrices that are applied to the engineering and culture of bioengineered hybrid materials and tissue equivalents.
  • the loaded cells may be selected from the group consisting of chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells, keratinocytes (skin), and neurons, as well as adipocytes or bone marrow cells.
  • chondrocytes articular cartilage
  • fibrochondrocytes meniscus
  • ligament fibroblasts ligament fibroblasts
  • skin fibroblasts skin fibroblasts
  • tendons tenocytes
  • myofibroblasts muscle
  • mesenchymal stem cells keratinocytes (skin)
  • neurons as well as adipocytes or bone marrow cells.
  • cells from any tissue which are capable of proliferation may optionally be embedded in such a construct.
  • Biofilm is composed of at least 80 percent extracellular macromolecules that are usually positively charged, similar to chitosan.
  • Chitosan may optionally be used as a biofilm disruptor thus helping wound hygiene and limiting the inhibitory effect of biofilm on destruction of bacteria.
  • Chitosan gel mixed with lactoferrin may optionally act as a slow release reservoir to destroy biofilm in any chronic wound or a wound that may become chronic.
  • Chitosan gel mixed with xylitol may optionally also be a specific biofilm disruptor.
  • loaded polysaccharide gel as injectable or implantable gel biomaterials which act as supports, carriers, reconstructive devices or substitutes for the formation in situ of bone-like, fibrocartilage-like or cartilage-like tissues at a physiological location of an animal or a human.
  • chitosan gels according to the present invention may be useful as a sustained delivery drug-system for treatment of the eye.
  • Results based on the ocular irritation test of chitosan compounds have indicated that chitosan preparations are suitable for use as ophthalmic gels based on their excellent tolerance 1 (Molinaro et. al., 2002).
  • a slow release drug delivery hydrogel system comprising highly acetylated type 1 chitosans and highly deacetylated type 2 chitosans.
  • any of the drug delivery systems of the present invention may be used for delivery of a wide variety of drugs, including, but not limited to, analgesics, anesthetics, antiacne agents, antiaging agents, antibacterials, antibiotics, antiburn agents, antidepressants, antidermatitis agents, antiedemics, antihistamines, antihelminths, antihyperkeratolyte agents, antiinflammatory agents, antiirritants, antilipemics, antimicrobials, antimycotics, antioxidants, antipruritics, antipsoriatic agents, antirosacea agents antiseborrheic agents, antiseptics, antiswelling agents, antiviral agents, antiyeast agents, cardiovascular agents, chemotherapeutic agents, corticosteroids, fungicides, hormones, hydroxyacids, keratolytic agents, lactams, mitocides, non-steroidal anti- inflammatory agents, pediculicides, progestins, sanatives
  • the present invention provides a method for the production of a stable hydrogel, wherein the method comprises dissolving in an acidic aqueous solution at least one highly acetylated chitosan having a degree of acetylation in the range of from about 40 to about 60%, and at least one highly deacetylated chitosan having a degree of acetylation of no greater than about 20%, so as to form a composite solution; adjusting the pH of the composite solution to a value of 6.5 to 7.2; and increasing the temperature of the composite solution to 37 °C while raising the pH to a value of 7.0 to 7.6; so as to produce a hydrogel which at 25 °C has a viscosity of at least 100 mPa-s at a shear rate of 50 rpm.
  • dissolving of the highly acetylated chitosan and the highly deacetylated chitosan is performed simultaneously in the same vessel.
  • dissolving the highly acetylated chitosan and the highly deacetylated chitosan is performed in separate vessels to form two solutions.
  • the process further comprises mixing these two solutions to form the composite solution.
  • a pH-dependent and temperature-dependent hydrogel formed by the process described herein.
  • the present inventors have further found that a mixture of a highly deacetylated chitosan and various saccharide oligomers including chitosan oligomers, also forms a hydrogel having the desired properties, as described hereinabove.
  • a chitosan composition comprising at least one highly deacetylated chitosan having a molecular weight in the range of from about 100 to about 4000 kDa, such as from about 100 kDa to about 2000 kDa (a chitosan polymer) and a degree of acetylation of no greater than about 20%, and at least one chitosan oligomer having a molecular weight in the range of from about 200 to about 20000 Da, the composition being in a form of an aqueous solution.
  • the chitosan oligomer may be a highly deacetylated chitosan oligomer having a degree of acetylation of no greater than about 20%, or alternately, it may be a highly acetylated chitosan oligomer having a degree of acetylation of in the range from 40% to 60%, for example, about 45% to about 55%.
  • the highly deacetylated chitosan oligomer has a degree of acetylation of about 15% or less.
  • the saccharide oligomer is a D-glucosamine oligomer having between 3 and about 100 units.
  • the D- glucosamine oligomer has between 3 and about 50 units.
  • the saccharide oligomer is an N-acetyl-D-glucosamine oligomer having up to about 7 units.
  • the ratio between the saccharide oligomer and the highly deacetylated chitosan polymer is greater than 1:1, and can be in the range of from 2:1 to 20:1, depending, inter alia, on the MW of the highly acetylated chitosan and the chitosan oligomers.
  • the concentration of the highly acetylated chitosan polymer can be, for example, 1%, 2%, 4%, 10% and any concentration is the range of 1-20% (w/w).
  • the concentration of the oligomers is selected according to the desired ratio.
  • the saccharide chitosan oligomers are water-soluble at a pH of 6.5 and higher and thus can also serve as "protectors" of the highly deacetylated chitosan polymer, in a manner analogous to that effected by a highly acetylated chitosan polymer, as discussed herein.
  • the composition has a viscosity of at least 100 mPa-s at a shear rate of 50 rpm. In a particular embodiment, at 25 °C and at pH in the neutral range the composition has a viscosity of no greater than 70 mPa-s at a shear rate of 200 rpm.
  • Hydrogels comprising a combination of a chitosan polymer and a chitosan oligomer can be utilized in any of the applications described herein and may be prepared as described in Example 8 herein.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Chitosan with a degree of acetylation of 15% and molecular weight of 65 kDa (Koyo, Japan) was dissolved by mixing with 0.9 % HCl for 24 hours, forming a type 1 chitosan solution having a chitosan concentration of 3 % (w/v).
  • the type 1 and type 2 chitosan solutions were mixed according to the following ratios of type- 1 to type-2: 1:1, 1 :2 and 1:3, titrated to pH 6.8 and left for 24 hours at 4 0 C, followed by further titration to pH 7.2 at 4 0 C with sodium hydroxide.
  • the resulting composition was liquid at room temperature.
  • the liquid solution formed a stable semisolid hydrogel, as illustrated in Figure 1.
  • Pseudo-thermosetting hydrogel (3% w/v) was prepared using homogenously acetylated type 2 chitosan and non-homogenously deacetylated type 1 chitosan, as described in Example 1 above (hydrogel A). Hydrogel A was prepared at ratios of 1:1, 1:2 and 1:3. A different hydrogel (hydrogel B) was prepared using the same type 2 chitosan as used for hydrogel A, and a type 1 non-homogenous chitosan of molecular weight 100 kDa.
  • the gel was then frozen, lyophilized and weighed. Weight degradation was calculated from the change in weight of the samples, as a function of time interval.
  • hydrogel A The degradation of hydrogel A by serum enzymes is shown in Figure 2 A.
  • Two distinct types of degradation kinetics are exhibited by each of the compositions shown in Figure 2 A: an initial fast phase that terminates within 3-6 days and a slower one that exhibits only partial degradation after 14 days.
  • the rate of degradation of the gel is more rapid in compositions having a higher ratio of type 2 chitosan to type 1 chitosan i.e. a 1:1 composition degrades more rapidly than a 1:3 composition.
  • the fast phase reflects degradation of type 2 chitosan, which is highly soluble and readily recognized by serum enzymes.
  • the slow kinetic phase is related to chitosan type 1 chitosan, which is not readily recognized and digested by serum enzymes.
  • Controlling the reacetylation of glucosamine polymer is a very important tool for manipulating the extent of recognition of the chitosan by lysozyme and consequently for manipulating the rate of hydrogel degradation.
  • the main factor that controls the activity of the enzyme is the percentage of N-acetyl glucosamine (GIcNAc) in the polymer (Ran et al., 2005). For this reason decreasing the reacetylation degree from 50% to 35% in chitosan type 2 should allow the rate of degradation to be significantly decreased, resulting in a much shallower slope. On the other hand, increasing the degree of acetylation of type 1 chitosan should result in faster degradation of the polymer.
  • thermosetting hydrogel presented herein as a slow release vehicle, hemoglobin and bovine serum albumin
  • BSA bovine serum
  • the data shown in Figure 7 relate to a single degree of acetylation of type 1 chitosan and a single molecular weight, which resulted in a protein release profile having a rate of release which decreased each day.
  • appropriate selection of additional variables such as degree of reacetylation and molecular weights of the two types of chitosans allows the characteristics of the gel to be determined, and enables affinity of the protein drug for the chitosan structure to be improved.
  • Such specific combinations would be expected to provide a fixed rate of release of a specific drug, reflecting a combined diffusion and matrix degradation rate.
  • Psammomys obesus strain rats which are known to develop diabetic symptoms when raised in captivity on a high fat diet, were used as a model of type II Diabetes mellitus. These animals are considered to be an excellent model for simulating chronic skin ulcers of diabetics, and study of skin wound healing, due to their tendency to develop profound infections, gangrene and sepsis, leading to morbidity and even mortality.
  • a chitosan-based hydrogel was used as a biological dressing, avoiding the need for bandaging or suturing, and providing a direct coating of the wound bed.
  • the composition of the chitosan mixture includes: a highly deacetylated chitosan (660 kDa, DA 15), a highly acetylated chitosan (220 kDa, DA 50) and a mixture of N-acetyl-D-glucosamine oligomers from 1-7 units (Koyo, Osaka, Japan).
  • the formulation includes the three components in a ratio of 1 :0.8:0.2, respectively.
  • mice Thirteen animals were found to have developed diabetes following administration of a high fat diet, starting from 4 to 6 weeks prior to day zero.
  • the injuries of seven diabetic animals were treated by administration of the chitosan based-gel of Example 1 to the wound area, while a further six animals were left untreated.
  • the gel was reapplied to the wound area of the treatment group every day.
  • a conventional plaster bandage was applied over the gel in the treatment group.
  • a plaster bandage was applied over the wound, whereas in the other half the wound area was left uncovered.
  • Rotator cuff tears are a common source of shoulder pain.
  • the incidence of rotator cuff damage increases with age and is most frequently caused by degeneration of the tendon, rather than injury from sports or trauma.
  • Treatment recommendations vary from rehabilitation to surgical repair of the torn tendon(s). The best method of treatment is different for every patient and indeed many patients do not achieve satisfactory repair of their injuries.
  • the present invention overcomes these drawbacks of the background art by providing an injectable product allowing delivery of autologous cells into rotator cuff tears under ultrasonographic control.
  • the injectable product allows the incorporation of bone marrow cells as well, for example for tissue healing.
  • the procedure is performed as an outpatient procedure or an ambulatory procedure requiring local anesthesia.
  • a chitosan mixture was designed so as to serve as a reservoir of negatively charged substances such as proteoglycans (e.g. chondroitin sulfate), hyaluronate, and/or phospholipids (e.g., phosphatidyl choline).
  • proteoglycans e.g. chondroitin sulfate
  • hyaluronate e.g., phospholipids
  • phospholipids e.g., phosphatidyl choline
  • the static coefficient of friction was measured in vitro between two flat cartilage surfaces, to which various test compositions were applied. Following application of the compositions, the cartilage surfaces were placed one on top of the other, and one edge was slowly lifted manually.
  • Figure 12 presents the static friction coefficient between two layers of normal cartilage, as measured for: a control system with no applied hydrogel (denoted as
  • Saline a combination chitosan hydrogel containing a chitosan type 1 (MW of 660 kDa; DA 15), and a chitosan type 2 (MW of 220 kDa; DA50) (denoted as
  • Combination the aforementioned combination chitosan hydrogel and further comprising 0.1 % chondroitin sulfate (denoted as +CS), the aforementioned combination chitosan hydrogel further comprising 0.1% chondroitin sulfate and 0.1% phosphatidyl choline (denoted as +CS/PC), and a 1% solution of hyaluronic acid (denoted as HA).
  • a chitosan polymer (or oligomer) is defined by its molecular weight, its degree of deacetylation, its crystallinity and the mode of distribution of its acetyl groups.
  • the solubility of deacetylated chitosan in aqueous solutions is limited.
  • a chitosan having a MW above 200 kDa provides solutions having concentrations less than about 10% (w/v), and as the molecular weight increase, the maximal solution concentration decreases.
  • HCl 0.15N is a concentration which when fully titrated becomes the physiological NaCl concentration. Higher concentrations of HCl (or other acids such as acetic acid) allows higher concentrations of chitosan solutions.
  • Type 1 chitosan precipitates from solution when at a pH above 6.5.
  • Type 1 chitosan solutions at physiological environments therefore do not exist, and most of the currently practiced applications involving implementation of chitosan utilize various types of solid chitosan.
  • Chitosans homogenously deacetylated to 50% or homogenously reacetylated to 30-60 % are referred to as type 2 chitosans.
  • Such a chitosan has a superior solubility in aqueous solutions, as compared to highly deacetylated chitosans (type 1) and typically remains soluble at neutral and physiological pH, depending on its concentration.
  • Hvdrogel Formation using a mixture of chitosan polymers When mixing type 1 chitosan and type 2 chitosan at a physiological pH and under certain conditions, no precipitation of the polymers is observed and instead, the mixture forms a hydrogel. Hydrogel formation involves "coating” (or “protection") of the type 1 chitosan by the type 2 chitosan , and is effected by the affinity between the two chitosan types, which leads to interactions therebetween (e.g., hydrogen bonds, hydrophobic interactions and/or Van der Waals interactions).
  • the gelation process may be as short as several minutes or as long as many days and is demonstrated by a gradual yet continuous increase in the viscosity of the system.
  • hydrogel formation is determin by turning a glass tube that contains the initial solution on its side and assessing whether the solution flows, or alternately whether a semi-solid gel is formed which remains stuck to the bottom of the glass tube. Gel formation depends on the type, shape and parameters (e.g., diameter) of tube, as well as the assay time frame.
  • hydrogel formation was defined as follows: a one ml solution was placed in a 14 mm (in diameter) round-bottomed glass tube and was incubated at 37 °C overnight. Thereafter, the glass tube was turned into a horizontal position and the presence or absence of liquid flow was determined. Absence of liquid flow indicated that a hydrogel was formed. The hydrogel contains the whole amount of water and remained rigid in a semi solid state. Presence of flow but in a "well distinguished" structure also indicated that a hydrogel was formed. The presence of liquid flow and/or the formation of two separate phases, solid and liquid, indicated that a hydrogel was not formed.
  • Mixture formation Using the above-described stock solutions, mixtures having a defined final concentration (w/v) of each chitosan and a defined ratio thereof, were prepared.
  • An exemplary mixture is FMTM80:DACTM50 1.2:1.2, in which each of the chitosans are at a final concentrations of 1.2% (w/v), and the ratio therebetween is 1 : 1.
  • the pH range in which a gel is formed at 4 0 C is 7.4-7.7. At higher pH values, precipitation is observed.
  • pH values at 4 °C correlate to pH values lower by 0.5 units at 25 0 C.
  • pH 7.4 at 4 0 C is found to be pH 6.9 at 25 °C.
  • the type 1 chitosan is considered the backbone of the hydrogel.
  • hydrogel formation depends on the final concentration of type 1 chitosan.
  • the MW of the type 1 chitosan is increased, its final concentration in the composition should be decreased, and vice versa, i.e. as the MW decreases its final concentration in the composition should be increased.
  • type 1 chitosan Use of type 1 chitosans of high MW, decreases the pH working range i.e. the range that allows hydrogel formation. For example, a composition comprising a type 1 chitosan of MW 660 kDa forms a hydrogel in the pH range 7.4-7.7, while a composition comprising a type 1 chitosan of MW 2,000 kDa forms a hydrogel in the pH range 7.0-7.3. Furthermore, as the MW of the type 1 chitosan is increased, the relative concentration of the type 2 chitosan required is increased. Furthermore, as the concentration of the type 1 chitosan is decreased (e.g., to 0.5 %), the pH range at which hydrogel formation occurs shifts down to 7.2-7.4 (at 4
  • the concentration of that chitosan required for hydrogel formation is increased.
  • Exemplary concentrations required for type 1 chitosans of MW of 200 kDa or less are in the range 1.2-1.5% (w/v) or higher. Under such conditions pH values for hydrogel formation may shift to 7.5-7.8 (at 4 °C).
  • Concentration of type 2 chitosan A minimal relative concentration of the type 1 chitosan and the type 2 chitosan is required for hydrogel formation (e.g., a 1:1 ratio).
  • type 2 chitosan Molecular Weight of type 2 chitosan.
  • Using a type 2 chitosan of MW higher than 220 kDa enables use of a reduced relative concentration of type 2 chitosan in the mixture.
  • a type 2 chitosan of higher MW results in high protection and improved stability of the chitosan type 1 (over a wide range of MW of type 1 chitosan). At such conditions, the pH range for hydrogel formation may increase.
  • the pH for hydrogel formation should be about 7.8, whereas using a type 2 chitosan of MW 2000 kDa and a type 1 chitosan of MW 2000 kDa, the pH for hydrogel formation should be about 7.6.
  • Temperature The temperature affects the rate of hydrogel formation linearly. Thus, at 37 0 C, the hydrogel will form faster than at room temperature or at 4 °C.
  • Relative ratio of type 1 chitosan and type 2 chitosan depends on the MW of each chitosan. For example, as the MW of type 1 chitosan increases (e.g., to 2000 kDa), its required concentration can be reduced possibly to about 0.5%. However, it is assumed that the ratio between type 2 and type 1 would increase to, for example, 2:1, 3:1 and even 4:1.
  • the minimal concentration thereof required for hydrogel formation decreases to e.g., 0.5% (instead of 1%), such that when high MW type 1 chitosan is used, the ratio would be about 1:1.
  • Chitosan hydrogels formed from highly acetylated and highly deacetylated chitosan oligomers and a highly deacetylated chitosan polymer
  • Oligomers of highly deacetylated chitosan are soluble at pH higher than 6.5, in contrast to highly deacetylated chitosan polymers having a similar degree of deacetylation.
  • a hydrogel from a mixture of highly deacetylated chitosan polymers (e.g., MW of 200-2000 kDa), in combination with chitosan oligomers (e.g., MW of 200-20000 Da) was tested.
  • Solutions of a type 1 chitosan polymer (MW 660) were each mixed with a solution of a highly deacetylated oligomer (MW 200-1500 Da) for assessing hydrogel formation.
  • the highly deacetylated oligomer was present in the solutions tested at final concentrations of 1%, 2%, 4% and 10%, and the highly deacetylated polymer was present at a final concentration of 1%.
  • the tested ratios (oligomer to polymer) were 1:1; 2:1; 4:1; and 10:1.
  • Example 7 The principles discussed in Example 7 were validated in viscosity studies of various hydrogels formed from chitosan polymers. Materials and Methods
  • Combination chitosan solutions were prepared in either of two ways.
  • individual stock solutions of a type 1 and a type 2 chitosan were prepared at the desired concentrations, as described in Example 7.
  • Slow drop wise titration with NaOH at either 1 °C or 25 0 C was carried out until the desired pH was achieved.
  • pre- weighed amounts of type 1 and a type 2 chitosans in powder form were combined in an acid solution e.g. HCl (0.15N) or acetic acid (0.25N). Following solubilization the solutions were slowly titrated with NaOH at either 1 0 C or 25 0 C until the desired pH was achieved.
  • Figure 13 illustrates results obtained with compositions containing DACTM50 on its own at a concentration of 1% (diamond symbols) or 2% (square symbols); or FMTM80S on its own at a concentration of 1% (triangle symbols); or FMTM80S on its own at a concentration of 1% (asterisk symbols); or a combination of FMTM80 and DAC -,TM M 50 at a ratio of 1 : 1 (oval symbols).
  • 1% solutions of individual type 1 chitosans (FMTM80S or FMTM80) have low viscosity at 25 0 C in the tested pH range from about 4 to 6.5.
  • the combination solution FM80:50 1:1 transforms from a liquid to a semi-solid gel at a pH of about 6.5 at 25 0 C, as indicated in Figure 13.
  • the composition starts to display non-Newtonian behavior, and a further increase in pH significantly increases its viscosity i.e. to about 175 mPa-s at pH 6.9.
  • a 1:1 mixture of a 15% acetylated (i.e. highly deacetylated) chitosan of molecular weight 660 kDa and a 50% acetylated (i.e. highly acetylated) chitosan of molecular weight 220 kDa is suitable for hydrogel formation at physiological conditions.
  • Figure 14 illustrates results obtained with compositions containing a combination of the highly deacetylated chitosan FVLTM and the highly acetylated chitosan DACTM50 at a ratio of 1 :1 (diamond symbols), or a combination of the highly deacetylated chitosan SKTM10 and DACTM50 at a ratio of 1:1 (square symbols).
  • Figure 14 shows that each of the mixtures FVL:50 1:1 and SK10:50 1:1 display a significant increase in viscosity at pH values approaching neutrality, leading to the formation of a semi-solid hydrogel.
  • Figure 15A indicates that the viscosity of SK10:50 1:1 increases dramatically upon a decrease in shear rate, when the pH value of the solution is in the neutral range i.e. 6.87 and 7, whereas the same compositions at lower pH values do not exhibit increased viscosity at decreasing shear rates. That is, SK10:50 1:1 exhibits liquid Newtonian behavior at pH values below the neutral range, and exhibits non-Newtonian behavior at pH values in the neutral range.
  • FVL:50 1:1 displays only a minor increase in viscosity upon a decrease in shear rate at neutral pH and remains in the liquid state (Fig. 15B), reaching a peak viscosity of about 35 mPas, as compared to 105 mPa-s in SK10:50 1:l.
  • Figure 16 shows that at low temperature i.e. 1 0 C, the pH-induced increase in viscosity in compositions of the invention is diminished.
  • chitosan compositions according to the invention may be stored at low temperatures in order to preserve them in the liquid or relatively non-gelled state.
  • FVL:50 1:1 exhibits a greater viscosity at 1 °C as compared to that at 25 °C (compare Figure 15B to Figure 16), suggesting the unsuitability of this composition for hydrogel formation in vivo, due to its tendency to have decreased viscosity upon an increase in temperature to that of room temperature and above.
  • Figure 17 shows results obtained with compositions comprising a combination of SKTM10 and DACTM50 at a ratio of 1:1 when measured at varying shear rates and at 1 °C
  • Figure 17A the compositions having pH values of 5.35 (vertical bar symbols); 5.92 (circle symbols); 6.22 (asterisk symbols); 6.45 (x symbols); 6.81 (triangle symbols); 6.95 (square symbols) and 7.01 (diamond symbols); or at 25 °C
  • Figure 17B the compositions having pH values of 5.13 (x symbols); 5.67 (empty square symbols); 6 (empty triangle symbols); 6.24 (empty diamond symbols); 6.51 (asterisk symbols); 6.65 (filled circle symbols); 6.75 (empty circle symbols); 6.87 (filled diamond symbols) and 7 (filled square symbols).
  • SKl 0:50 1:1 exhibits Newtonian behavior over a range of pH values from 5.35 to about 6.8, indicated by the observation that the viscosity remains relatively unchanged irrespective of the shear force i.e. there is no appreciable difference in viscosity between a shear force of 200 versus that of 50 rpm.
  • the composition exhibits increased viscosity upon decreased shear force, indicative of non-Newtonian behavior. This increase is particularly evident at 25 °C, wherein at pH 7 the viscosity increases from 65 to 110 rnPa-s upon a decrease in shear force from 200 to 50 rpm ( Figure 17, panel B).
  • the composition continues to increase in viscosity and undergoes hydrogel formation over a period of time.
  • the viscosity increases only from about 55 to about 65 mPa-s ( Figure 17, panel A).
  • the compositions having pH of 6.8 and less exhibit lower viscosity at 25 0 C, as compared to the same compositions at 1 0 C, suggesting that the combined conditions of physiological pH and temperature are optimal for hydrogel formation.
  • Figure 18 shows that a combination chitosan composition comprising a highly deacetylated chitosan of molecular weight of less than about 100 kDa is not suitable for gel formation under physiological conditions.
  • the results in this figure were obtained with a combination of FVLTM and DACTM50 at a ratio of 1:1 when measured at varying shear rates and at 1 0 C (Figure 18A), the compositions having pH values of 5.85 (circle symbols); 6.2 (asterisk symbols); 6.44 (x symbols); 6.76 (triangle symbols); 6.94 (square symbols) and 7.00 (diamond symbols); or at 25 0 C (Figure 18B), the compositions having pH values of 5.45 (open diamond symbols); 5.7 (open square symbols); 6.04 (open triangle symbols); 6.3 (x symbols); 6.56 (asterisk symbols); 6.68 (circle symbols); 6.82 (closed triangle symbols); 6.88 (closed diamond symbols) and 7.03 (closed square symbols).
  • FVL:50 1:1 exhibits an increase in viscosity as the pH approaches neutrality but does not undergo gel formation at either 1 0 C ( Figure 18A) or 25 °C ( Figure 18B).
  • this composition exhibits a higher viscosity at 1 °C (neutral pH, low shear rate) as compared to that at 25 °C.
  • molecular weight of the highly deacetylated chitosan is an important parameter for determining gel formation in the compositions of the invention.
  • Figure 19 shows that increasing the molecular weight of the highly deacetylated chitosan improves the viscosity thereof, as shown with a composition containing a combination of FMTM80 and DACTM50 at a ratio of 1:1 at pH values of 4.81 (diamond symbols); 5.93 (square symbols); 6.24 (triangle symbols); 6.43 (x symbols); 6.64 (asterisk symbols) and 6.91 (circle symbols).
  • a composition containing a combination of FMTM80 and DACTM50 at a ratio of 1:1 at pH values of 4.81 (diamond symbols); 5.93 (square symbols); 6.24 (triangle symbols); 6.43 (x symbols); 6.64 (asterisk symbols) and 6.91 (circle symbols).
  • hydrogel formation of FM80-50 1 :1 is initiated at a lower pH (between about 6.4 to about 6.6) as compared to that for SKl 0:50 1:1 (about 6.8 to about 6.9) (see Figure 17B.
  • the viscosity achieved at neutral pH is significantly higher in the composition containing type 1 chitosan of higher molecular weight i.e. 420 mPa-s for FM80-50 1:1 versus about 110 mPa-s for SKl 0:50 1:1.
  • the molecular weight of the type 1 chitosan can be varied for different applications, while still achieving hydrogel formation. For example, a more viscous hydrogel may be desirable for an implant requiring prolonged durability in vivo, whereas a hydrogel of lower viscosity may be sufficient to achieve the desired release properties of a slow release drug formulation.
  • Muzzarelli 1986: In: Muzzarelli, R.A.A., Jeuniaux, C, Gooday, G. W. (Eds.), The

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