WO2010099818A1 - Hydrogel de polysaccharide thermoréversible - Google Patents

Hydrogel de polysaccharide thermoréversible Download PDF

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Publication number
WO2010099818A1
WO2010099818A1 PCT/EP2009/052487 EP2009052487W WO2010099818A1 WO 2010099818 A1 WO2010099818 A1 WO 2010099818A1 EP 2009052487 W EP2009052487 W EP 2009052487W WO 2010099818 A1 WO2010099818 A1 WO 2010099818A1
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copolymer composition
functional group
reaction
hydrogel
group
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PCT/EP2009/052487
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English (en)
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Mauro Alini
David Olivier Eglin
Derek Mortisen
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Ao Technology Ag
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Publication of WO2010099818A1 publication Critical patent/WO2010099818A1/fr

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    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • 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
    • 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
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C08J3/246—Intercrosslinking of at least two polymers
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C08L33/26—Homopolymers or copolymers of acrylamide or methacrylamide

Definitions

  • the present invention describes an injectable, in situ gelling hydrogel based on a polysaccharide, preferably hyaluronic acid, and a thermoreversible polymer, preferably poly (N- isopropylacrylamide) , its method of production and its use in in vitro cell studies and medical applications.
  • a polysaccharide preferably hyaluronic acid
  • a thermoreversible polymer preferably poly (N- isopropylacrylamide)
  • Hydrogels have become an important material for biomedical applications due to their high water content, tissue-like elasticity, tunable diffusion characteristics, and their favorable tolerance by the body.
  • polysaccharides are a naturally derived hydrogel .
  • Polysaccharides are relatively complex carbohydrates made from monosaccharides joined by glycosidic bonds. In nature they have critical roles in cell signaling, extracellular matrix structure, lubricious fluids, and as energy sources. Due to their important roles in organ development and cell signaling, in addition to their tolerance and clearance in the body, polysaccharides have long been viewed as candidates for biomaterials.
  • polysaccharides chitosan, alginate, agarose, and gycosaminoglycans such chondroitin sulfate, heparin, and hyaluronic acid.
  • Hyaluronic acid is an endogenous linear heteropolysaccharide composed of D-glucuronic acid and D-N- acetylglucosamine, which are linked together via alternating ⁇ -1,4 and ⁇ -1,3 glycosidic bonds.
  • This polysaccharide can be composed of up to 25,000 disaccharide repeats, and range in size from 5,000 to 20,000,000 Da depending on the origin. Each repeat unit bears a carboxylic chemical group on the D- glucuronic acid motif. It is a chief component of the extracellular matrix in connective, epithelial, and neural tissues, and is known to play an important role in organ development, cell proliferation and migration. Additionally, HA contributes to the lubrication and maintenance of cartilage, where it is a major component of synovial fluid and forms a coating around chondrocytes .
  • HA hydroxyadiene sulfate
  • Pharmacia a commercial use of HA was as a material for eye surgery in the 1970' s by Pharmacia. Since then several other companies have developed HA based products for viscosupplementation, osteoarthritis, wound healing, tissue fillers, and cell scaffolds for tissue engineering. In many of these products, the HA is synthetically modified to alter its mechanical properties and slow its degradation when implanted in the body. The most common approach is to crosslink the linear polysaccharide to avoid rapid degradation and clearance from the implant site of unmodified polysaccharide. This may be accomplished by e.g. thiolating the carboxylic acid groups present on the HA molecule and subsequently crosslinking the gel by exposure to oxygen through ambient air exposure or by soaking in hydrogen peroxide (see Shu, X.
  • a further method includes direct methacrylation of HA, making it amenable to in situ crosslinking by exposure to a suitable free radical initiator (see Bencherif, S. A. et al Biomaterials 2008, 29(12), 1739-1749.) .
  • Yet another method includes use of a UV activated initiator to increase the control over polymerization timing, such that a monomer solution can be introduced to a site and subsequently cured by exposure to a UV source (see Masters, K. S. et al Biomaterials 2005, 26(15), 2517-2525) .
  • 'click chemistry' refers to a variety of chemical reactions that are characterized by their high yield, regioselectivity, large thermodynamic driving force, stability, and ability to proceed without the generation of offensive byproducts (see KoIb, H. C. et al Chem. Int. Ed Engl. 2001, 40(11), 2004-2021) . Importantly, these reactions can occur in a benign solvent, are insensitive to water and oxygen, and have a thermodynamic driving that is usually greater than 20 kcal.mol "1 .
  • HA has been functionalized with azide and alkyne groups, which are subsequently reacted together with a copper catalyst in order to crosslink the polysaccharide via formation of a triazole ring (WO 2008/031525, Di Meo et al, Macromol . Biosci . 2008, 8(7) , 670-681) .
  • HA has been similarly modified for the attachment of carborane rings intended for the cellular delivery of boron atoms in boron neutron-capture anti-cancer therapy (BNCT) (Crescenzi et al Biomacromolecules .
  • BNCT boron neutron-capture anti-cancer therapy
  • RAFT reversible addition-fragmentation chain transfer polymerization
  • PDI polydispersity
  • CTA trithiocarbonate based chain transfer agent
  • a polysaccharide modified with a thermoreversible polyamide e.g. poly (N-isopropylacrylamide) (pNIPAM)
  • pNIPAM poly (N-isopropylacrylamide)
  • pNIPAM poly (N-isopropylacrylamide)
  • FIG. 1 Exemplary grafting of an azide functionalized R group to the backbone of an alkyne functionalized HA polysaccharide.
  • R can equal a pNIPAM polymer and/or a bioactive agent.
  • FIG. 1 A multi-arm configuration of pNIPAM using a central linkage and multiple 'click' reactive sites can be used to increase the stiffness of the gel and decrease the degradation rate .
  • Figure 3 Schematic representation of thermally induced gelation of a polysaccharide using pNIPAM grafts via 'click chemistry' .
  • Figure 4. Schematic representation showing the diversity of groups that could occupy 'click chemistry' reactive sites on the polysaccharide backbone.
  • FIG. 6 Imaging of (A) azide-modified fluorescein mixed with water and non-functionalized HA; (B) fluorescein covalently grafted to a HA-pNIPAM-copolymer composition of the invention; and (C) HA-pNIPAM-copolymer composition of the invention without fluorescein as control .
  • the present invention provides a copolymer composition based on a hydrophilic polysaccharide and a thermosensitive polyamide that are crosslinked or grafted by a click chemistry reaction.
  • the copolymer composition of the invention is obtained by reacting an alkyne modified polysaccharide, preferably HA, with an azido-terminated pNIPAM. Upon solubilization in an aqueous solvent, the copolymer composition may form a thermosensitive hydrogel showing a liquid-to-gel phase transition with temperature change.
  • thermoreversible hydrogel which is biodegradable and biocompatible .
  • thermoreversible biodegradable hydrogel is in solution or liquid state at or around room temperature or lower and in a gelled state at or around physiological (or in vivo) temperature or higher.
  • thermosensitive biodegradable hydrogel having utility in many medical applications such as a bioactive agent or cell delivery system, in tissue engineering, cell culture systems, and the like.
  • the copolymer composition or hydrogel of the invention further comprises a bioactive agent, which is either dispersed in or covalently attached to the copolymer composition.
  • a bioactive agent which is either dispersed in or covalently attached to the copolymer composition.
  • the present invention is directed towards the use of a thermoreversible biodegradable hydrogel as a bioactive agent delivery system.
  • bioactive agent delivery system is easily administrable, e.g. can be injected parenterally.
  • Yet still another object of this invention is a method of preparation of a copolymer composition or hydrogel of the invention.
  • the invention relates to a kit comprising the copolymer composition of the invention, e.g. in a lyophilized form. Additional objects and advantages of this invention will become apparent from the following detailed description of the various embodiments making up this invention.
  • the present invention relates to copolymer compositions based on a polysaccharide and a thermoreversible polyamide, an injectable, in situ gelling hydrogel obtained thereof as well as their methods of production and their use in various medical applications, e.g. delivery systems and in vitro cell studies .
  • grafting density or “crosslinking density” is defined as the ratio of polysaccharide (units or mol%) that carry a functional group that has undergone a reaction with a complementary functional group to total polysaccharide (units or mol%) .
  • degree of crosslink or graft conversion means the quantity of pairs of functional groups converted into crosslinking or grafting bonds relative to the total quantity of pairs of functional groups initially present on the polysaccharide and thermoreversible polyamide, expressed as a percentage .
  • degree of conversion as used herein with respect to the polysaccharide means the quantity of carboxylate groups converted into functional groups suitable to undergo crosslinking or grafting via 'click chemistry' relative to the total quantity of carboxylate groups initially present on the polysaccharide, expressed as a percentage.
  • biodegradable refers to the ability of the compositions of the present invention to be degraded and broken down in vivo into non toxic substances, which are excreted.
  • biocompatible refers to the ability of the compositions of the present invention to be applied to tissues without eliciting adverse tissue responses, such as significant inflammation, fibrosis, and the like.
  • liquid-to-gel-phase transition in the present invention refers to a composition that is present as a flowable liquid below a lower critical solution temperature (LCST) and is changed into a gel above said critical temperature. Such a transition may occur reversibly.
  • LCST critical solution temperature
  • gelation temperature , “gelling temperature” or “LCST” may be used interchangeably to describe the temperature at which the liquid-to-gel-phase transition occurs. This temperature (or temperature range) depends on the nature of the composition.
  • hydrogel formed of a copolymer composition this includes factors such as molecular weight, ratios of the co-monomers, graft and/or crosslinking density of the copolymer composition, the concentration of the copolymer composition in the aqueous solution to form a liquid hydrogel, the absence or presence of one or more bioactive agents, additives, etc.
  • the terms “hydrogel in liquid state”, “liquid hydrogel” and “liquid copolymer solution” may be used interchangeably to describe the flowable liquid below the LCST.
  • hydrogel in gelled state stable hydrogel” or “robust hydrogel” may be used interchangeably to describe the gelled state above the LCST.
  • thermosensitive refers to a physical property of a composition in which the composition can undergo a physical change of a liquid state to a gel state (i.e. a liquid-to-gel- transititon) repeatedly when exposed to an increase in temperatures.
  • a gel state i.e. a liquid-to-gel- transititon
  • room temperature refers to a temperature range from about 18 0 C to 28 0 C, with a preferred ambient temperature of about 25 0 C.
  • in vivo temperature refers to a temperature range from about 28 0 C to 41 0 C, with a preferred in vivo temperature of about 35 0 C to 39 0 C.
  • physiological conditions refers to a temperature range of from 35 0 C to 39 0 C, with a preferred temperature of a human body of 37 0 C.
  • graft refers to two compounds (e.g. two polymers or a polymer and a bioactive agent, etc) that are covalently bound two each other via l click chemistry' type reactions.
  • crosslink refers to any covalent, ionic, or physical association of grafted polymers to link the polymer segments together.
  • chain transfer agent refers to a thiocarbonylthio compound containing at least one weak chemical bond by which monomers can be polymerized from in a controlled, living polymerization with end group specificity.
  • RAFT reversible addition-fragmentation chain transfer
  • carboxylic acid reactive group refers to any group that undergoes a reaction with carboxylic acids and includes halogen, hydroxy or alkoxy groups, amines, carboxylic acid derivatives, such as an acid chloride or an acid anhydride and the like.
  • alkyl used alone or in combination with other groups, e.g. alkoxy, refers to saturated, straight or branched-chain hydrocarbon radicals containing between one and ten carbon atoms including, but not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl and neopentyl .
  • alkyl is limited to 1-4 carbons.
  • halogen refers to fluorine, chlorine, bromine and iodine .
  • the present invention relates to a crosslinked or grafted copolymer composition suitable for in situ formation of a hydrogel in aqueous solution comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a "click chemistry' reaction, hereinafter also called copolymer composition of the invention.
  • click chemistry refers to a selection of chemical reactions characterized by their rapidity, regioselectivity, high yield, and high thermodynamic driving force. They are powerful and efficient chemical reactions that are modular and wide in scope, and have the benefit of being insensitive to solvents and pH conditions. Common features of 'click chemistry' reactions include i) stable linkages; ii) minimal cross-reactivity with other functional groups; iii) high conversions/yields that react to completion; iv) absence of appreciable amounts of side product (s); and v) proceed under relatively benign reaction conditions. Thus 'click chemistry' reactions provide stereoselective conversion with high to very high yields.
  • click reactions are driven by a high thermodynamic force (e.g. > 20 kcal.rn.ol ⁇ 1 ), which gives rise to highly modular and stereospecific reactions with high yields.
  • 'click chemistry' is not limited to a specific type of reaction but rather includes a range of different reactions, with different reaction mechanisms. These include, but are not limited to, a [1, 3] -dipolar cycloaddition (Huisgen) ; a [2,4] -cycloaddition (Diels-Alder) ; nucleophilic substitution reaction-ring opening reactions; carbonyl reactions of the non- aldol type; and addition to carbon-carbon multiple bonds.
  • Preferred reactions are pericyclic reactions, preferably a cycloaddition reaction, preferably a 1,3 -dipolar cycloaddition reaction or a Diels-Alder reaction.
  • the present invention relates towards a copolymer composition of the invention, wherein the 'click chemistry' reaction is a 1,3 -dipolar cycloaddition reaction between at least one first functional group present on a hydrophilic polysaccharide capable of participating in a 'click chemistry' reaction and at least one second complementary functional group present on a thermoreversible polyamide capable of participating in a 'click chemistry' reaction with the first functional group.
  • the first functional group is introduced by reacting a group RG-L-FG, wherein RG is a carboxylic acid reactive group, L is a linker and FG is the first functional group, with a carboxylic acid group present on the hydrophilic polysaccharide.
  • the linker may be selected from the group consisting of a covalent bond, straight or branched C(I- 8)alkyl groups and polyethers of the formula -(CH 2 -CH 2 -O) n - wherein n is 1 to 20, preferably 1 and 8.
  • 0.1 to 100%, more preferably 5 to 70%, more preferably 15 to 50% of the at least one carboxylic acid groups present on the hydrophilic polysaccharide are subjected to conjugation to a group RG-L-FG.
  • 0.1 to 100%, preferably 5 to 50 % of the functional group FG present on the hydrophilic polysaccharaide have been subjected to reaction with the complementary functional group present on the thermoreversible polyamide.
  • the functional groups involved in 'click chemistry' include e.g. a diene and/or a dienophile as used in the Diels Alder reaction, and an azide and/or alkyne group as used in a 1,3 cycloaddition .
  • the invention is directed towards copolymer compositions of the invention, wherein the at least one first functional group is an alkyne group and the at least one second functional group is a terminal azide group.
  • the invention is directed towards copolymer compositions of the invention, wherein the at least one first functional group is an azide group and the at least one second functional group is a terminal alkyne group .
  • the copolymer compositions of the invention have a grafting and/or crosslinking density between the polysaccharide and the thermoreversible polyamide of from 0.01 to 100 % preferably 5 to 50 % more preferably 10 to 30 %.
  • the present invention is directed towards a copolymer composition of the invention comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a Huisgen 1,3 -dipolar cycloaddition.
  • This reaction is well described in the literature (see e.g. WO 03/101972, incorporated herein by reference in its entirety) and includes the reaction between an azide and an alkyne group to form a 1, 2, 3-triazole ring (Huisgen et al . , Chem. Bex. 1967, 100, 2494-2507) . It may proceed in water alone or in the presence of a catalyst, such as a copper (I) catalyst, to enhance its efficiency, regioselectivity, and/or reaction rate.
  • a catalyst such as a copper (I) catalyst
  • polysaccharide includes any complex carbohydrate made of monosaccharides joined by glycosidic bonds. Typical monosaccharide units include arabinose, fructose, galactose, galactopyranosyl , galacturonic acid, guluronic acid, glucuronic acid, glucose, glucoside,N- acetylglucosamine, mannuronic acid, mannose, pyranosyl sulfate, rhamnose, or xylose.
  • Polysaccharides containing the foregoing units include cyclodextrins, starch, chitosan, trehalose, cellobiose, maltotriose, maltohexaose, chitohexaose, agarose, chitin 50, amylose, glucans, xylan, pectin, galactan, dextran, aminated dextran, cellulose, hydroxyalkylcelluloses, carboxyalkylcelluloses, fucoidan, sulfate polysaccharides, mucopolysaccharides, gelatin, zein, collagen, alginic acid, agar, carrageean, guar gum, gum arabic, gum ghatti, gum karaya, gum konjak, gum tamarind, gum tara, gum tragacanth, locust bean gum, pectins, xanthan gum, and the glycosaminoglycans such as HA
  • Preferred polysaccharides include chitosan and members of the glycosaminoglycan family, such as HA, heparin, chondroitin sulphate, most preferably HA.
  • the hydrophilic polysaccharide of choice has a molecular weight of between 1 kDa to 10 MDa, preferably between 10 kDa to 5 MDa. It is understood that the molecular weight of choice will depend on the intended function of the copolymer composition of the invention and hydrogel formed thereof (and thus by such factors as the desired copolymer composition degradation rate and the desired release rate of a bioactive agent optionally incorporated therein, i.e. the therapeutic condition to be treated) . For example, a polysaccharide with a higher molecular weight will exhibit increased stiffness of a hydrogel formed thereof and decreased clearance rate when implanted in vivo.
  • the present invention relates to a copolymer composition of the invention comprising a HA and a thermoreversible polyamide grafted or crosslinked via a 'click chemistry' reaction, such as the Huisgen 1,3-dipolar cycloaddition .
  • HA is a polymer based on disaccharide units (composed of D- glucuronic acid and D-N-acetylglucosamine linked together via alternating ⁇ -1,4 and ⁇ -1,3 glycosidic bonds) each having one carboxylate function, wherein the carboxylate function of the disaccharide unit of HA may be conjugated to a functional group suitable to undergo ' click chemistry' reactions, such as the Huisgen 1,3-dipolar cycloaddition.
  • a functional group suitable to undergo ' click chemistry' reactions such as the Huisgen 1,3-dipolar cycloaddition.
  • HA may be used to undergo a 1,3 -dipolar cycloaddition with a thermosensitive polyamide carrying the corresponding azide-
  • carboxylic acid group on the disaccharide D-glucuronic acid subunit of HA may be functionalized with propargylamine, whereby the percentage of carboxylic acid groups functionalized may be controlled by stoichiometric addition of propargylamine.
  • thermosensitive polyamide may include any polyamide capable of forming a hydration shell in an aqueous solution and capable to undergo significant changes in hydration in response to a change of temperature of said solution. More specifically, the polyamide may be selected from the group polyacrylamides , polyvinylamides, polyalkyloxazolines, polyvinylimidazoles, and their copolymers and blends. Specific examples for such polymers are poly (N- isopropylacrylamide) (pNIPAM) , poly (N-vinylcaprolactam) , poly
  • N-isopropyloxazoline poly (vinylimidazole) and poly (N- acryloyl-pyrrolidin) , most preferably poly (N- isopropylacrylamide) (pNIPAM) or a copolymer or derivative thereof .
  • the present invention relates to a copolymer composition of the invention comprising HA and a pNIPAM polymer grafted or crosslinked via a 'click chemistry' reaction, such as the Huisgen 1,3 -dipolar eye1oaddition .
  • an alkyne- (or azide-) functionalized HA may be used to undergo a 1,3 -dipolar cycloaddition with a pNIPAM polymer functionalized with the corresponding azide- (or alkyne-) group.
  • a pNIPAM polymer may be functionalized with a single azide group.
  • These azide- functionalized pNIPAM polymers may then be grafted onto an alkyne-functionalized polysaccharide backbone (such as HA) , where R is equal to the pNIPAM polymer ( Figure 1) .
  • thermoreversible synthetic polymers containing more than one group functionalized for 'click chemistry' can be introduced to covalently crosslink the polysaccharide.
  • this can be achieved by polymerization of NIPAM using a chain transfer agent that has been modified to include 2 or more azide groups.
  • the azide/alkyne crosslinking of the polysaccharide would form a weak gel, and further gelation of the material would occur via association of hydrophobic domains in the crosslinker. This would result in a stiffer hydrogel with slower degradation and slower drug release kinetics than a hydrogel formed from a polysaccharide grafted with pNIPAM containing only one azide group .
  • the crosslinker could be a pNIPAM polymer in a branched or multi-armed configuration, with multiple sites activated for coupling to the polysaccharide via * click chemistry' .
  • Figure 2 shows e.g. a four-arm pNIPAM configuration connected through a central linkage, whereby each "pNIPAM-arm" is linked through a 'click' reactive site to one or more polysaccharides.
  • a branched or multi-armed thermoreversible crosslinker of this sort may reduce the viscosity of the liquid copolymer solution at room temperature or produce a greater increase in stiffness during temperature induced formation of a hydrogel .
  • the molecular weight of the polyamide e.g.
  • the pNIPAM polymer is 1 to 200 kDa, preferably 5 to 100 kDa.
  • the molecular weight is chosen below 30 kDa, e.g. preferably from 5 to 30 kDa, to facilitate clearance from the body.
  • the molecular weight may be chosen higher than 30 kDa, e.g. from 30 to 100 kDa, to facilitate formation of a more robust hydrogel. It is understood that if a polyamide, e.g. a pNIPAM polymer comprising a degradable linkage is used, its molecular weight may be chosen higher than 3OkDa without impact on its clearance from the body.
  • the molecular weight of the copolymer compositions of the invention is 1 kDa - 1 GDa, more preferably 5 kDa to 500 MDa, most preferably between 1 MDa to 500 MDa.
  • the molecular weight of choice will depend on the intended function of the copolymer composition of the invention and hydrogel formed thereof (and thus by such factors as the desired copolymer composition degradation rate and the desired release rate of a bioactive agent optionally incorporated therein, i.e. the therapeutic condition to be treated) .
  • the molecular weight of the copolymer composition degradation products may be chosen sufficiently low, preferably below 30 kDa, to facilitate clearance from the body via the renal system.
  • the molecular weight of the copolymer compositions of the invention may be chosen higher, preferably higher than 30 kDa, to facilitate formation of a more robust hydrogel, e.g. a hydrogel with a higher modulus, or to slow the degradation of the material .
  • the copolymer composition of the invention is designed such that upon dissolution in an aqueous solvent it forms a hydrogel, which is in a flowing, deformable liquid state (i.e. a copolymer solution) at a lower temperature, such as room temperature (e.g. below the LCST), and will undergo a transition into a stable, robust, gelled state (i.e. a stable hydrogel) when exposed to temperatures at or around in vivo temperature (e.g. above the LCST) , such as mammalian body temperature of 37 0 C. Thermally induced gelation of the liquid copolymer solution, i.e.
  • FIG. 3 gives a schematic representation of a thermally induced gelation of a polysaccharide using pNIPAM grafts via 'click chemistry' : at temperatures below body temperature, e.g. ⁇ 37 0 C, the two pNIPAM polymers occupying each a click reactive site on the hydrophilic polysaccharide backbone are in an extended, hydrophilic state. Upon increasing temperature to body temperature or higher, e.g. ⁇ 37 0 C the two pNIPAM polymers are in a coiled, hydrophobic state. Association of the hydrophobic domains leads to gelation.
  • the invention is directed to a hydrogel which is characterized by showing a liquid-to-gel phase transition around body temperature, i.e around 18 0 C to
  • hydrogel of the invention is in a liquid state, and above said transition temperature the hydrogel is in a gelled state.
  • the hydrogel may be a solution based on 1 to 50 wt%, more preferably from 1 to 20 wt%, more preferably between 5 to 15 wt% of the copolymer composition of the invention dissolved in an aqueous solvent.
  • the aqueous solvent may be any of the commonly used aqueous solvents, which are all well known by those of ordinary skill in the art. This may be selected from the group consisting of water (including water for injection) , buffer solutions, acid solutions, basic solutions, salt solutions, saline solution, and glucose salt solution, for example sodium acetate, Tris, sodium phosphate, MOPS, PIPES, MES and potassium phosphate (e.g. in the range of 25 mM to 500 mM and in the pH range of 4.0 to 8.5) .
  • the ratios of hydrophilic polysaccharide to thermosensitive polyamide in the copolymer compositions of the invention are chosen such that they show retention of the desired water solubility and gelling properties of the hydrogel formed.
  • the copolymer compositions of the invention should show sufficient water solubility at temperatures below the LCST, and instant gelation under physiological conditions (e.g. pH 7.0 and 37°C) .
  • the hydrophilic polysaccharide is present in the copolymer compositions of the invention from about 2 % to 90 wt%, preferably from 10 to 50 wt% of the copolymer compositions of the invention.
  • the hydrophilic polysaccharide is present in hydrogels of the invention (i.e. the hydrated copolymer compositions of the invention) from about 1 % to 20 wt%, preferably from 2 to 10 wt% of the hydrogels of the invention.
  • the thermosensitive polyamide graft or crosslinks are present in the hydrogels of the invention from about 2 to 50 wt%, preferably 5 to 20 wt% of the hydrogels of the invention (or the hydrogels formed thereof) . It is understood that the exact ratios will also depend on the nature of the polyamide, i.e. linear vs. branched vs. star shape polyamide and degree of graft vs. crosslinking.
  • the concentration at which the copolymer compositions of the present-invention remain soluble below the LCST and form an instant gel at physiological conditions is typically up to about 60% by weight, with a preferred concentration range from about 1% to 50%, more preferred from about 2% to 10%. It is understood that those concentrations also depend on the exact nature of the copolymer composition.
  • copolymer compositions of the invention as well as the hydrogel formed thereof are bioresorbable, cytocompatible, nontoxic, and biocompatible and are therefore particularly useful in various medical applications.
  • the invention is directed towards the use of the copolymer compositions and hydrogels of the invention in in vivo drug delivery, in vitro cell culture, tissue filler, cosmetic, and tissue engineering applications.
  • copolymer compositions and hydrogels of the invention may be used as a bioactive agent delivery system for various bioactive agents ranging from small molecule compounds to macromolecular compounds to cells.
  • the present invention provides a bioactive agent delivery system comprising a hydrogel of the invention and at least one bioactive agent dissolved in an aqueous solvent.
  • Figure 4 illustrates schematically a hydrophilic polysaccharide backbone having two click reactive sites occupied by pNIPAM polymers and two click reactive sites occupied by a bioactive agent, drug depot or the like as well as one free click reactive site.
  • the copolymer compositions of the invention or the hydrogel of the invention may further comprise at least one bioactive agent .
  • bioactive agent is used throughout the specification to describe ay agent with biological activity to be incorporated into a copolymer composition of the invention.
  • the at least one bioactive agent may be natural, synthetic, semi-synthetic or derivatives thereof and may include both hydrophobic and hydrophilic, soluble and insoluble compounds. More specifically, the at least one bioactive agent may be any bioactive agent useful for the treatment and/or prevention and/or diagnosis of conditions in any therapeutic area known in mammals, such as animals and humans, particularly humans, which include, but are not limited to, infectious disease (anti-bacterial, anti-fungal and anti-viral activity, vaccines,), inflammatory disease (including arthritis, and hypertension) , neoplastic disease, diabetes, osteoporosis, pain management, general cardiovascular disease and lung disease (e.g.
  • asthma whemphysema
  • lung cancer chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • bronchitis influenza, pneumonia, tuberculosis, respiratory distress syndrome, cystic fibrosis, sudden infant death syndrome (SDKs) , respiratory synctial virus (RSV) , AIDS related lung diseases, sarcoidosis, sleep apnea, acute respiratory distress syndrome (ARDS) , bronchiectasis, bronchiolitis, bronchopulmonary dysplasia, coccidioidomycosis, hantavirus pulmonary syndrome, histoplasmosis, pertussis and pulmonary hypertension) , as well as in general conditions caused by growth of harmful or pathogenic organisms, including, but not limited to bacteria, yeast, viruses, protozoa or parasites, conditions to be treated by gene therapy or diagnosed by imaging means .
  • harmful or pathogenic organisms including, but not limited to bacteria, yeast, viruses, protoz
  • the at least one bioactive agent may be selected from a macromolecular compound or a small molecule compound, such as peptides, proteins, oligo- and poly-nucleotides, antibiotics, antimicrobials, growth factors, enzymes, antitumoral drugs, anti-inflammatory drugs, antiviral drugs, antifungal drugs, anesthetics, anti neoplastic drugs, antimitotic drugs, analgesics, narcotics, antithrombotic drugs, anticoagulants, haemostatic drugs.
  • a macromolecular compound or a small molecule compound such as peptides, proteins, oligo- and poly-nucleotides, antibiotics, antimicrobials, growth factors, enzymes, antitumoral drugs, anti-inflammatory drugs, antiviral drugs, antifungal drugs, anesthetics, anti neoplastic drugs, antimitotic drugs, analgesics, narcotics, antithrombotic drugs, anticoagulants, haemostatic drugs.
  • the at least one bioactive agent may be selected from the group consisting of (a) proteins or (poly) peptides, such as erythropoietin (EPO) , interferon-alpha, interferon-beta, interferon- gamma, growth hormone (human, pig, cow, etc.), growth hormone releasing factor, nerve growth factor (NGF) , granulocyte-colony stimulating factor (G-CSF) , granulocyte macrophage-colony stimulating factor (GM-CSF) , macrophage-colony stimulating factor (M-CSF) , blood clotting factor, insulin, oxytocin, vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet-derived growth factor (PDGF) , prolactin, luliberin, luteinizing hormone releasing hormone (LHRH) , LHRH agonists, LHRH antagonists, somatostatin, glucagon
  • Marimastat 2-Methoxyestradiol, Squalamine, Thalidomide,
  • the at least one bioactive agent may also be a (therapeutic) cell and may be selected from the group consisting of preosteoblast , chondrocyte, umbilical vein endothelial cell (UVEC) , osteoblast, adult stem cell, Schwann cell, oligodendrocyte, hepatocyte, mural cell (used in combination with UVEC), myoblast, insulin-secreting cell, endothelial cell, smooth muscle cell, fibroblast, [beta] -cell, endodermal cell, hepatic stem cell, juxraglomerular cell, skeletal muscle cell, keratinocyte, melanocyte, langerhans cell, merkel cell, dermal fibroblast, and preadipocyte .
  • preosteoblast chondrocyte, umbilical vein endothelial cell (UVEC) , osteoblast, adult stem cell, Schwann cell, oligodendrocyte, hepatocyte, mural cell (used in combination with UVEC), myoblast, insulin-
  • the at least one bioactive substance may be present in an amount of between about 0.01 to 50 wt%, preferably about 0.1 to 20 wt% based on the total amount of the copolymer composition or the hydrogel of the invention.
  • the bioactive agent may be dispersed within the copolymer composition or the hydrogel of the invention.
  • the bioactive agent may be conjugated to the copolymer composition or the hydrogel of the invention via a ⁇ click chemistry' reaction, e.g. by conjugation to functional groups on the polysaccharide that have not been coupled to a polyamide .
  • the ⁇ click chemistry' reaction is 1,3 -dipolar cycloaddition reaction between a first functional group on the hydrophilic polysaccharide and a third complementary functional group capable of participating in a 'click chemistry' reaction with the first functional group on the at least one bioactive agent.
  • the third complementary functional group is an azide group when the first functional group is an alkyne group.
  • the third functional group is an alkyne group when the first functional group is an azide group (see e.g. Figure 1, wherein the R group in may act as a bioactive agent) .
  • copolymer compositions of the invention or the hydrogel of the invention may further comprise at least one additive to modulate their properties (and thereby increasing the efficacy thereof), e.g. by modulating the water content and/or ionic charge thereof and/or by altering the solidity and gelling temperature thereof and/or by modulating the degradation rate and/or release rate thereof as well as by modulating the stability of one or more bioactive agents optionally incorporated therein.
  • the present invention provides a bioactive agent delivery system comprising a hydrogel of the invention and at least one bioactive agent and at least one additive dissolved in an aqueous solvent.
  • additives may include, but are not limited to, cationic polymers; anionic polymers; sugars; polyols, sugar-containing polyols and polymer-containing polyols, amino acids and sugar- containing amino acids, other bioavailable materials, sugar- containing ions, surfactants, organic solvents, preservatives, etc.
  • the at least one additive may be selected from the group consisting of: (a) cationic polymers having the molecular weight from 200 to 750,000), such as, poly-L-arginine, poly-L-lysine, poly (ethylene glycol), polyethylenimine , chitosan, protamin, and the like; (b) anionic polymers such as poly (N-vinyl-2 -pyrrolidone) , polyvinylacetate (PVA) , alginate, and the like; (c) bioavailable materials such as amiloride, procainamide, acetyl-beta- methylcholine, spermine, spermidine, lysozyme, fibroin, albumin, collagen, growth factors such as transforming growth factor-beta (TGF-beta) , fibroblast growth factor (bFGF) , vascular endothelial growth factor (VEGF) , and the like, bone morphogenetic proteins (BMPs) , deposition
  • the at least one additive may be physically incorporated in the copolymer composition or the hydrogel of the invention, e.g. by dispersion, and may be present from about 0.001 to 30 wt%, preferably about 0.1 to 10 wt% based on the total weight of the copolymer composition or the hydrogel of the invention.
  • the invention is directed to a crosslinked or grafted copolymer composition or a hydrogel of the invention comprising HA and pNIPAM grafted or crosslinked via a * click chemistry' reaction between at least one alkyne group present on the HA and at least one terminal azide group present on the pNIPAM optionally further comprising at least one bioactive agent, wherein the bioactive agent is either dispersed within the copolymer composition or the hydrogel or conjugated to the copolymer composition or the hydrogel via a "click chemistry' reaction between at least one alkyne group present on HA and an azide group present on the bioactive agent and optionally further comprising at least one additive .
  • the invention is directed to a method of preparing a copolymer composition of the invention comprising grafting or crosslinking a hydrophilic polysaccharide and a thermoreversible polyamide via a x click chemistry' reaction as defined hereinabove.
  • the 'click chemistry reaction' is a pericyclic reaction, preferably a cycloaddition reaction, preferably a 1,3 -dipolar cycloaddition reaction or a Diels- Alder reaction.
  • a preferred method of the invention comprises the steps of:
  • thermoreversible polyamide having at least one second complementary functional group capable of participating in a 'click chemistry' reaction with the first functional group ;
  • Further steps may include physically dispersing or covalently linking at least one bioactive agent in the copolymer composition obtained in step d) . More specifically the method may comprise the steps of:
  • the methods of the invention may further comprise the steps of dispersing at least one additive in the copolymer compositions of the invention (or the hydrogels obtained therefrom) .
  • the invention also provides a method for preparing a hydrogel comprising dissolving a copolymer composition according to the invention in an aqueous solvent as defined hereinabove .
  • the at least one bioactive agent and optionally the at least one additive may be present in the copolymer composition prior to dissolution in an aqueous solvent to form the hydrogel .
  • the at least one bioactive agent and optionally the at least one additive may be after dissolution of the copolymer composition.
  • a bioactive agent and optionally an additive are added to a copolymer composition of the invention.
  • the obtained bioactive agent composition may be used immediately by dissolution in an aqueous solution to form a hydrogel, i.e. a bioactive agent delivery system.
  • the obtained bioactive agent composition may be stored, e.g. in a lyophilized state, until further use is required.
  • a bioactive agent and/or an additive are added to a hydrogel of the invention to form a bioactive agent delivery system which may be used immediately.
  • the invention is directed towards further uses of the copolymer compositions and hydrogels of the invention, such as in vitro cell culture, tissue filler, cosmetic, and tissue engineering applications.
  • bioactive agents grafted onto a copolymer composition of the invention may be used to investigate the impact of said bioactive agents on cells cultured in vitro.
  • the copolymer compositions of the invention may be used as a cell culture device.
  • Cells may be suspended in the liquid copolymer solution at room temperature and then warmed to create a robust, stable hydrogel for three dimensional cell culture. Retrieval of the cells may be achieved by simply cooling the hydrogel such that it returns to its liquid copolymer solution state .
  • hydrogel or bioactive agent delivery system of the invention may be administered to a subject through various routes depending on its final use.
  • routes of administration may include oral administration, buccal administration, mucosal administration, nasal administration, intraperitoneal administration, hypodermic injection, muscular injection, percutaneous administration, and intratumoral administration, whereby a local administration such as hypodermic injection, muscular injection, or percutaneous administration is preferred.
  • a bioactive agent delivery system may be injected as an aqueous solution into a living body (at a temperature below the LCST) .
  • the bioactive agent delivery system forms a bioactive agent-containing depot in a gel state at in vivo temperature. Release (or diffusion) of the bioactive agent occurs upon degradation of the copolymer composition or via simple diffusion from the gelled hydrogel depot. In case of a chemically incorporated bioactive agent (i.e. by covalent linkage) the release or diffusion also depends on the degradation of the chemical linkage to the polysaccharide backbone .
  • the copolymer composition Prior to its use as a bioactive agent delivery system, the copolymer composition may be stored in lyophilized form until further use.
  • the invention provides a single or multi compartment kit comprising a copolymer composition according to the invention in a sterile, lyophilized form.
  • the kit may comprise in separate compartments a defined amount of aqueous solvent for reconstitution of the copolymer composition and/or a defined amount of a bioactive agent and/or a defined amount of an additive.
  • Example 1 Synthesis of a thermoreversible hydrogel using HA grafted with pNIPAM
  • HA modified with alkyne groups (a) HA modified with alkyne groups (HA-PA) .
  • the solution was then transferred to a 50 ml conical tube, frozen at -80 0 C, and lyophilized.
  • the degree of substitution was determined by reacting a portion of the HA-PA with excess sodium azide using copper sulfate (CuSO4) as the catalyst and ascorbic acid as the reducing agent . After dialysis and lyophilization, the material was dissolved in deuterium oxide
  • the degree of substitution was determined by comparing the integration of the triazole proton peak ( ⁇ 7.88 ppm) to the integration of the acetyl proton peak ( ⁇ 2.00 ppm) .
  • the degree of substitution varied between 6-28%, depending on the amount of propargylamine, EDC, and NHS used during the reaction.
  • N3- pNIPAM N-isopropylacrylamide
  • N3-pNIPAM was prepared according to procedures already established in the field.
  • a chain transfer agent (CTA) consisting of S-1-dodecyl-S' - ( ⁇ , ⁇ ' -dimethyl- ⁇ " -acetic acid) trithiocarbonate was first prepared according to Lai JT et al , Macromolecules 2002, 35, 6754-6756. This CTA was then modified with an azido group according to Gondi et al (Macromolecules 2007, 40, 474-481) .
  • NIPAM N-isopropylacrylamide
  • the grafting of N3-pNIPAM to HA-PA was performed by dissolving HA-PA in distilled water at 0.5% w/v.
  • a catalyst solution was prepared with 0.3731 g ascorbic acid sodium salt, 0.0470 g CuSO4-5H20, and 2 ml of distilled water. After thorough mixing of both solutions, 200 ⁇ l of the catalyst solution was added to the HA-PA and N3 -pNIPAM solution.
  • Example 2 Thermoresponsive Copolymer comprising covalently grafted fluorescein.
  • a 0.5% w/v solution of HA-PA was prepared as in Example 1.
  • Azido modified fluorescein (0.0047 g) was dissolved in 125 ⁇ l of dimethylsulfoxide (DMSO) .
  • DMSO dimethylsulfoxide
  • To 3 ml of the 0.5% HA-PA solution was added 50 ⁇ l of azido fluorescein and 0.45 g of N3 -pNIPAM with a Mn of 29 kDa. Based on these weights, the fluorescein and pNIPAM chains would occupy 10 and 8% of the disaccharide subunits, respectively.
  • the solution was mixed and a catalyst solution consisting of 200 ⁇ l water, 0.0047 g CuSO4 , and 0.0373 g ascorbic acid sodium salt was added.
  • thermoresponsive copolymer may be used for imaging purposes by itself or in the presence of other bioactive agents coupled to any of the additional active sites on the HA.

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Abstract

La présente invention porte sur une composition de copolymère réticulé ou greffé appropriée à la formation in situ d'un hydrogel en solution aqueuse comprenant un polysaccharide hydrophile et un polyamide thermoréversible greffé ou réticulé par réaction de « chimie click », sur son procédé de production et sur son utilisation dans des études de cellule in vitro et des applications médicales.
PCT/EP2009/052487 2009-03-03 2009-03-03 Hydrogel de polysaccharide thermoréversible WO2010099818A1 (fr)

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