CA2344215A1 - Functionalized derivatives of hyaluronic acid and formation of hydrogels in situ using same - Google Patents

Abstract

Methods for chemical modification of hyaluronic acid, formation of amine or aldehyde functionalized hyaluronic acid, and the cross-linking thereof to form hydrogels are provided. Functionalized hyaluronic acid hydrogels of this invention can be polymerized in situ, are biodegradable, and can serve as a tissue adhesive, a tissue separator, a drug delivery system, a matrix for cell cultures, and a temporary scaffold for tissue regeneration.

Description

FUNCTIONALIZED DERIVATIVES OF HYALURONIC ACID AND FORMATION
OF HYDROGELS IN STfU USING SAME

This invention is directed to biomaterials for spatially and temporally controlled delivery of bioactive agents such as drugs, growth factors, cytokines or cells. In particular, this invention teaches versatile methods for chemical crosslinking of high molecular weight hyaluronic acid under physiological 10 conditions in situ, to form polymerizable biodegradable materials. The methods are based on the introduction of functional groups into hyaluronic acid (HA) via formation of an active ester at the carboxylate of the glucuronic acid moiety as an intermediate and subsequent substitution with a side chain containing a nucleophilic group on one end and a (protected) functional group on the other end. The 15 introduced functional groups allow for crosslinking of the HA derivatives.
Crosslinked hyaluronic acid hydrogels of this invention are useful in various surgical applications and as a temporary scaffold for tissue regeneration, e.g., in cartilage repair.
SUBSTITUTE SHEET (RULE 26~

BACKGROUND OF THE INVENTION
Repair of articular cartilage The failure of regenerating persistent hyaline cartilage by surgical procedures prompted investigators to attempt repair using biological strategies.
5 The biological repair of articular cartilage is, with a few exceptions, still at an experimental stage. Biological cartilage repair has been approached in two basic ways. First, autologous chondrocytes have been transplanted into a lesion to induce repair (Grande et al., J. Orthrop. Res. 7, 208-214 (1989); Brittberg et al., New En~l. J. Med. 331, 889-895 (1994); Shortkroffet al., Biomaterials 17, 147-154 10 (1996)). Chondrocytes may be obtained from a low-loaded area of a joint and proliferated in culture (see Grande; Brittberg; Shortkroff, supra), or mesenchymal stem cells may be harvested, e.g., from the iliac crest marrow, and induced to differentiate along the chondrocyte lineage using growth factors (Harada et al., Bone 9, 177-183 (1988); Wakitani et al., J. Bone Joint Sure. 76-A, 579-592 15 (1994)). The chondrocyte transplantation procedures currently attempted clinically, although promising, are hampered because technically they are very challenging, the cell preparation is very expensive, and the potential patient pool is limited by age, defect location, history of disease, etc. Cells have also been transplanted into cartilage defects in the form of perichondral grafts, e.g. obtained from costal 20 cartilage, but with limited success due to the limit in donor material and the complication of endochondral ossification of the graft site observed in longterm follow-up (Amiel et al., Connect. Tissue Res. 18, 27-39 ( I 988); O'Driscoll et al., J.
Bone Joint Sure. 70-A, 595-606 (I988); Homminga et al., Acta Orthrop. Scand.
326-329 (1989); Homminga et al., J. Bone Joint Sure. 72-B, 1003-100? (1990)).
A
25 second approach is aimed at the recruitment of mesenchymal stem cells from the surrounding connective tissue, e.g., synovium, using chemotactic and/or mitogenic factors (Hunziker and Rosenberg, J. Bone Joint SurQ. 78-A, 721-733 (1996); see also U.S. Patent No. 5,368,858). The availability of growth factors and cytokines in recombinant form and the lack of complicated cell transplantation make this 30 procedure a very attractive alternative. The shortcoming of both procedures is the SUBSTfTUZ'E $HEET (RULE 26) diffculty to stably anchor the repair inducing factors, whether tissue grafts, cells or growth factors, within the defect site. Also, outlining of the space that is to be repaired, e.g., by filling it with a matrix material, appears to be crucial to recreate a level cartilage surface (Hunziker and Rosenberg, supra). Thus far, the availability 5 of candidate matrix materials has been the limiting factor, and anchoring of materials seeded with chondrocytes and/or chondrogenic factors difficult, explaining the unsatisfactory results obtained with currently available materials such as polylactic acid and polyglycolic acid scaffolds (Freed et al., J. Biomed. Mat.
Res.
28, 891-899 (1994); Chu et al., J. Biomed. Mat. Res. 29, 1147-1154 (1995)), 10 calcium phosphate minerals (Nakahara et al., Clin. Orthrop. 276, 291-298 (1992)), fibrin sealants (Itay et al., Clin. Orthron. 220, 284-303 (1987)), and collagen gels (Wakitani et al., J. Bone 3oint Sure. 71-B, 74-80 (1989)). We have developed novel biodegradable materials based on hyaluronic acid which are optimized for the biological requirements posed on a repair material in a synovial joint and allow in 15 situ polymerization.
Biology of hyaluronic acid and its therapeutic use Hyaluronic acid (HA) is unique among glycosaminoglycans in that it is not covalently bound to a polypeptide. HA is also unique in having a relatively simple structure of repeating nonsulfated disaccharide units composed of D-20 glucuronic acid (GIcUA) and N acetyl-D-glucosamine (GIcNAc) (Scott et al., The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives, T.C. Laurent (ed.), Portland Press, London, (hereinafter "Hyaluronan and Its Derivatives"), pp. 7-15 (1998)). Its molecular mass is typically several million Daltons. HA is also referred to as hyaluronan or hyaluronate, and exists in several 25 salt forms (see formula I).
SUBSTITUTE SHEET (RULE 26) (I) OH
O O HO O HO
O O /
HO "O
OH NH H
O O OH

GIcUA GIcNAc GIcUA GIcNAc HA is an abundant component of cartilage and plays a key structural role in the organization of the cartilage extracellular matrix as an organizing structure for the assembly of aggrecan, the large cartilage proteoglycan (Laurent S and Fraser, FASEB J. 6, 2397-2404 {1992); Morgelin et al., Biophvs. Chem.
50, I 13-128 (1994)). The noncovalent interactions of aggrecan and link protein with HA lead to the assembly of a large number of aggrecan molecules along the HA-chain and mediate retention of aggrecan in the tissue. The highly negatively charged aggrecan/HA assemblies are largely responsible for the viscoelastic 10 properties of cartilage by immobilizing water molecules. A number of cell surface receptors for HA have been described and shown to play a critical role in the assembly of the pericellular matrix of chondrocytes and other cells, e.g., isoforms of CD44 and vertebrate homologues of Cdc37 (Knudson and Knudson, FASEB J. 7, 1233-1241 (1993); Grammatikakis et al., J. Biol. Chem. 270, 16198-16205 (1995)), 1 S or to be involved in receptor-mediated endocytosis and degradation of HA
to control HA levels in tissues and body fluids (Laurent and Fraser, supra;
Fraser et al., Hvaluronan and Its Derivatives, pp. 85-92 (1998)). Blocking of the interaction of these receptors with HA in prechondrogenic micromass cultures from embryonic limb bud mesoderm inhibits chondrogenesis, indicating that the 20 establishment and maintenance of a differentiated chondrocyte phenotype is at least in part dependent on HA and HA-receptor interactions (Maleski and Knudson, EXp.
Cell. Res. 225, 55-66 (1996)).
SUBSTITUTE SHEET (RULE 26) HA and its salts are currently being used in therapy for arthropathies by intraarticular injection (Stachnan et al., Ann. Rheum. Dis. 49, 949-952 ( 1990);
Adams, Hvaluronan and Its Derivatives, pp. 243-253 (1998)), in ophthalmic surgery for intraocular lens implantation Denlinger, Hvaluronan and Its Derivatives, pp.
5 235-242 (1998), to promote wound healing in various tissues (King et al., Sur a 109, 76-84 (1991)), or more recently, in derivatized and/or crosslinked form to manufacture thin films which are used for tissue separation (for review see Laurent and Fraser, supra; Weiss, Hyaluronan and Its Derivatives, pp. 255-266 (1998);
Larsen, Hyaluronan and Its Derivatives, pp. 267-281 (1998); Band, Hyaluronan and 10 Its Derivatives, pp. 33-42 (1998)). Extensive efforts have been made by various laboratories to produce derivatives of HA with unique properties for specific biomedical applications. Most of the developments have been focusing on the production of materials such as films or sponges for implantation and the substitution of HA with therapeutic agents for delayed release and/or prolonged 15 effect (for review see Band, supra; Prestwich et al., Hyaluronan and Its Derivatives, pp. 43-65 (1998); Gustafson, Hyaluronan and Its Derivatives, pp. 291-304 (1998)).
Strategies have included esterification ofHA (U.S. Patent Nos. 4,957,744 and 5,336,767), acrylation of HA (U. S. Patent No. 5,410,016), and cross-linking of HA
using divinyl sulfone (U.S. Patent No. 4,582,865) or glycidyl ether (U.S.
Patent No.
20 4,713,448). However, the modified HA molecules show altered physical characteristics such as decreased solubility in water and/or the chemical reaction strategies used are not designed for crosslinking of HA under physiological conditions (in an aqueous environment, at pH 6.5-8.0).
It is well known that polyaldehydes can be generated by oxidising 25 sugars using periodate (along, CRC Press, Inc., Boca Rayton, FL, pp. 27 (1993);
European Patent No. 9615888). Periodate treatment oxidizes the proximal hydroxyl groups (at C2 and C3 carbons of glucuronic acid moiety) to aldehydes thereby opening the sugar ring to form a linear chain (Scheme 1). While periodate oxidation allows for the formation of a large number of functional groups, the 30 disadvantage is the loss of the native backbone structure. Consequently, the SUBSTITUTE SHEET (RULE 26) generated derivative may not be recognized as HA by cells. In fact hydrogels formed by using periodate oxidized HA as a crosslinker, e.g., in combination with the HA-amines described herein, showed very limited tissue transformation and poor cellular infiltration in the rat ectopic bone formation model (Fig. 6).
This is in S sharp contrast to the HA-aldehyde derivatives described herein.
The introduction of free amino groups on HA, which could be used for further convenient coupling reactions under mild physiological conditions, has been a subject of great interest. Previous methods have produced a free amino group on high molecular weight HA by alkaline N-deacylation of its glucosamine 10 moiety (Curvall et al., Carbohvdr. Res. 41, 235-239 (1975); Dahl et al., Anal.
Biochem. 175, 397-407 (1988)). However, concomitant degradations ofHA via beta-elimination in the glucuronic acid moiety was observed under the harsh reaction conditions needed. This is of particular concern because low molecular weight HA fragments, in contrast to high molecular weight HA, have been shown 15 to be capable of provoking inflammatory responses (Noble et al., Hyaluronan and Its Derivatives, pp. 219-225 ( 1998)). An early report claimed that carbodiimide-catalyzed reaction of HA with gIycine methyl ester, a monofunctional amine, led to the formation of an amide linkage (Danishefsky and Siskovic, Carbohydr. Res.
16, 199-201 ( 1971 )). This however, has been proven by a number of studies not to be 20 the case (Kuo et al., Bioconju~ate Chem. 2, 232-241 (1991); Ogamo et al., Carbohydr. Res. 105, 69-85 ( 1982)). Under mildly acidic conditions the unstable intermediate O-acylisourea is readily formed, which in the absence of nucleophiles, rearranges by a cyclic electronic displacement to a stable N acylurea (Kurzer and Douraghi-Zedeh, Chem. Rev. 67, 107-152 (I967)). This O,N migration ofthe O-25 acylisourea also occurs when the nucleophile is a primary amine (Kuo et al., supra) and any amide formation that does occur is insignificant as reported by Ogamo et al., supra. Experiments where high molecular weight HA (Mr ~ 2 x 106 Da) was reacted with an excess of the fluorescent label 5-aminofluorescine in the presence of the carbodiimide EDC achieved only 0.86% of theoretical labelling. The 30 introduction of a terminal hydrazido group on HA with a variable spacer has SUBSTITUTE SHEET (RULE 26).

_7_ recently been achieved and has led to the ability to conduct further coupling and crosslinking reactions (Pouyani and Prestwich, Bioconjut~ate Chem. 5, 339-347 (1994), U.S. Patent Nos. 5,616,568, 5,652,347, and 5,502,081; Vercruysse et al., Bioconiueate Chem. 8, 686-694 (1997)) It is the objective of this invention to provide a method for more versatile modification of HA with various functional groups that allow for crosslinking of the HA derivatives under physiological conditions. It is another objective that the method of functionalization does not compromise the molecular weight or chemical identity (except of the target carboxyl group for coupling) of 10 HA. It is a further objective that the method of functionalization provides HA
molecules that are well tolerated in vivo and are biodegradable.
It is also the objective of this invention to identify HA derivatives and methodology for in situ polymerization thereof to provide a biodegradable scaffold for tissue regeneration. It is another objective that the HA materials can be I 5 polymerized in the presence of cells to serve as a vehicle for cell transplantation. It is a further objective to provide methodology for functionalization and cross-linking of HA that allows for variations in the biomechanical properties of the formed gels as well as in the sensitivity to cellular infiltration and degradation.
SUMMARY OF THE INVENTION
20 Biomaterials for spatially and temporally controlled delivery of bioactive agents such as drugs, growth factors, cytokines or cells, are a key factor for tissue repair. In particular, in .situ polymerizable biodegradable materials are needed for cartilage resurfacing that are designed to withstand the mechanical forces in a joint. We have developed a versatile method for chemical crosslinking of 25 high molecular weight hyaluronic acid under physiological conditions. The method is based on the introduction of functional groups into hyaluronic acid by formation of an active ester at the carboxylate of the glucuronic acid moiety and subsequent substitution with a side chain containing a nucleophilic group on one end and a (protected) functional group on the other end. We have formed hyaluronic acid SUBSTITUTE SHEET (RULE 26~

_g_ with amino or aldehyde functionality, and formed hydrogels with modified hyaluronic acid and bifunctional crosslinkers or mixtures of hyaluronic acid carrying different functionalities using active ester- or aldehyde-mediated reactions.
Physical and chemical properties of the hydrogels of this invention were evaluated using S biomechanical testing, and by assaying sensitivity towards degradation by glycosidases such as testicular hyaiuronidase. Biocompatibility was evaluated using cell culture assays and subcutaneous implantation of the hyaluronic acid materials in rats. This in vivo assay is also the established model for induction of ectopic bone formation by members of the transforming growth factor (3 family (TGF-~3), and 10 several crosslinked hyaluronic acid materials of this invention gave excellent ectopic bone formation in vivo when loaded with appropriate growth factor(s).
As set forth below in the detailed description of the invention, the compositions of the invention have many therapeutic uses. For example, compositions of the invention may be used to stem hemorrhage in general surgery, 15 reconstruct nerves and vessels in reconstructive, neuro- and plastic surgery, and to anchor skin, vascular, or cartilage transplants or grafts in orthopedic, vascular, and plastic surgery. Compositions of the invention are also useful as vehicles for the delivery of cells or bioactive molecules such as growth factors to stimulate focal repair. Local delivery of growth factors facilitates wound healing and tissue 20 regeneration in many situations, not only in promoting bone formation and stimulating cartilage repair in orthopedic procedures, but also, e.g., in treating pathological wound conditions such as chronic ulcers. These compositions may also serve as a scaffold to generate artificial tissues through proliferation of autologous cells in culture. On the other hand, the anti-adhesive property of some 25 compositions with respect to cells render such compositions particularly suitable to generate tissue separations and to prevent adhesions following surgery. The viscoelastic properties of HA make it particularly well suited for this purpose, and it is used clinically to achieve temporal pain relief by repeated intraarticular injections in arthropathies as a "joint lubricant", as a protective agent for eye irritations and in 30 opthalmic surgery, as a barrier to cells in facial and other reconstructions in plastic SUBSTITUTE SHEET (RULE 26) surgery and dentistry, in reconstructive surgery of tendons, in surgical procedures in the urogenital system, and in thoracic surgery. The injectable nature of the compositions of the invention also renders them suitable for tissue augmentation in plastic surgery, where the HA matrix serves primarily as an inert biocompatible filler S material (Balasz and Laurent, Hyaluronan and Its Derivatives, pp. 325-326 (1998)), e.g., for filling dermal creases or lip reconstruction.
HA hydrogels match several of the desired properties for a biodegradable material biocompatible with cells. The relatively simple repetitive structure of HA allows for specific modification and introduction of a large number 10 of functional groups, for crosslinking to generate hydrogels with excellent physical properties. HA hydrogels have also successfully been used as a delivery vehicle in chondrocyte transplantation studies (Robinson et al., Calcif. Tissue Int. 46, (1990)) and HA has proven its biocompatibility in various forms in clinical practice (for review see Laurent and Fraser, supra; Balazs and Laurent, supra).
15 The reaction mechanisms we have explored for in situ polymerization of HA derivatives are compatible with an aqueous environment and are non-toxic to cells. The aldehyde-mediated crosslinking strategies follow reactions occurring physiologically in crosslinking of fibrillar collagens and elastin.
NHS-esters provide an alternative for rapid formation of stable bonds under 20 physiological conditions, primarily by reaction with primary amines. The technology ofNHS-ester-mediated protein crosslinking has been developed for materials with applications in plastic surgery that require in situ polymerization (U.S. Patent No. 5,413,791).
BRIEF DESCRIPTION OF THE DRAWINGS
25 Fig. 1 shows the results of a ninhydrin test after reductive alkylation of HA and HA-aldehyde in the presence of putrescine. Reductive alkylation was carried out with an excess of putrescine in the presence of pyridine borane.
HA or derivatives thereof were purified by repeated ethanol precipitation prior to detection SUBSTITUTE SHEET (RULE 26) WO 00/16818 PC'T/EP99/06913 of free amino groups on the HA chain by using the ninhydrin test (Sheng et al., Anal. Biochem. 211, 242-249 (1993)).
Fig. 2 shows 'H NMR of native HA (Fig. 2A) and an HA-derivative with protected aldehyde functionality (Fig 2B) in D,O at 300 MHz. Peaks are 5 assigned as indicated on the structural formula.
Fig. 3 shows ~H NMR of HA-derivatives with amine functionality formed from putrescine (Fig. 3A), histidine (Fig. 3B), lysine (Fig. 3C), and adipic dihydrazide (Fig. 3D) in D,O at 300Mhz. Peaks are assigned as indicated on the structural formula.
10 Fig. 4 shows digestion of crosslinked HA hydrogels with hyaluronidase. In Fig. 4A, HA-hydrogels were formed by crosslinking l2mg/ml highly modified (~65-70%) HA-amine (adipic dihydrazido-HA) with 15 mg/ml (SPA); PEG. Gels were incubated with different concentrations of bovine testicular hyaluronidase (HYALase) for the indicated time and the degradation of the gels was 15 measured by the release of glucuronic acid into the supernatant using the carbazole method. (Bitter and Muir, Anal. Biochem. 4, 330-334 (1962)). In Fig. 4B, HA-hydrogels were formed by crosslinking 12 mg/ml optimally modified (~20-25%) HA-anine (adipic dihydrazido-HA) with 15 mg/ml (SPA)z-PEG (0); 12 mg/ml highly modified (~65-70%) adipic dihydrazido-HA with I 5 mg/ml (SPA)2-PEG (D);
20 12 mg/ml optimally modified (~20-25%) lysine methylester-HA with either 15 mg/ml (SPA)2-PEG (A) or 0.44 mg/ml glutaraldehyde (o), and 12 mg/ml optimally modified (~10=15%) diaminobutyl-HA with 15 mg/ml (SPA)2-PEG (o). Gels were incubated with different concentrations of bovine testicular hyaluronidase for the indicated time and the degradation of the gels was measured as in Fig. 4A
above.
25 Fig. 5 shows phase contrast images of cells cultured on different crosslinked HA hydrogels. Fig. SA: Dedifferentiated chondrocytes cultured on a hydrogel formed from highly modified (~65-70%) HA-amine (adipic dihydrazido-HA) crosslinked with S mg/ml (SPA):-PEG aggregate as a consequence of inability to adhere to substratum. Fig. SB: Cells cultured on a hydrogel made up by the 30 same HA-amine crosslinked with 0.25 mg/ml glutaraldehyde show a rounded SUBSTITUTE SHEET (RULE 26) morphology and no aggregation indicating that they are able to adhere to the substratum. Fig. SC: Cells cultured on a hydrogel formed from the HA-amine (adipic dihydrazido-HA) modified to a degree of ~20-25% and crosslinked with 2 mg/ml (SPA)= PEG adhere to the substratum, spread and subsequently infiltrate the 5 hydrogel. All images show cells 24h post seeding but morphology remains the same even after several days in culture.
Fig. 6 shows in vivo evaluation of HA hydrogels formed from different HA derivatives using aldehyde-mediated crosslinking. Subcutaneous implantation in rats of HA hydrogels consisting of (Fig. 6A) 12 mg/ml optimally 10 modified (~20-25%) HA-amine (adipic dihydrazido-HA) crosslinked with 0.25 mg/ml glutaraldehyde, (Fig. 6B) 7 mg/ml of the same HA-amine crosslinked with mg/ml HA-aldehyde (periodate oxidized), (Fig. 6C) 7 mg/ml of the same HA-amine crosslinked with 7 mg/ml HA-aldehyde (deprotected amino-dimethyl acetal-HA, 10-15% modified), or (Fig. 6D) 7 mg/ml of the same HA-amine crosslinked with 7 15 mg/ml HA-aldehyde (deprotected hydrazido-dimethyl acetal-HA, 40-45%
modified). The hydrogels also contained 1 mg/mI prefibrillized intact collagen type I, 200 pg/ml BMP-2 and 500 ng/ml IGF-1 to induce bone formation. Tissue specimens were harvested 10 days post implantation, fixed in formalin and processed for histology by paraffin embedding. Sections were stained with 20 Haematoxylin/Eosin. m, matrix material (note: matrix material shrinks during dehydration); s, skin (indicates orientation of implant).
Fig. 7 shows in viao evaluation of HA hydrogels crosslinked with different NHS-esters. Subcutaneous implantation in rats of HA hydrogels consisting of (Fig. 7A) 12 mg/ml highly modified (~65-70%) HA-amine (adipic 25 dihydrazido-HA) crosslinked with 15 mg/ml (SPA),-PEG, (Fig. 7B) 12 mg/ml optimally modified (~20-25%) HA-amine (adipic dihydrazido-HA) crosslinked with 15 mg/ml SPA:-PEG, or (Fig. 7C) 12 mg/ml of the same optimally modified HA-amine crosslinked with 3 mg/ml DTSSP (crosslinker concentrations are equal on a molar basis). The hydrogels also contained I mg/ml prefibrillized intact collagen 30 type I, 200 pg/ml BMP-2 and 50 ng/ml TGF-~i2 to induce bone formation.
Tissue SUBSTITUTE SHEET (RULE 26) specimens were harvested 10 days post implantation, fixed in formalin and processed for histology by paraffin embedding. Sections were stained with Haematoxylin/Eosin. m, matrix material (note: matrix material shrinks during dehydration); s, skin (indicates orientation of implant).
S Fig. 8 shows differential effect of growth factors incorporated into HA hydrogels on tissue transformation. Subcutaneous implantation in rats of the HA hydrogel formed from 12 mg/ml optimally modified (~20-25%) HA-amine (adipic dihydrazido-HA) crosslinked with 15 mg/ml (SPA)= PEG. The hydrogels also contained 1 mg/ml prefibrillized intact collagen type 1, and were supplemented 10 either with 200 pg/ml BMP-2 and 500 ng/ml IGF-1 (Fig. 8A), or 200 ~g/ml BMP-and 50 ng/ml TGF-~i2 (Fig. 8B). Tissue specimens were harvested 10 days post implantation, fixed in formalin and processed for histology by paraffin embedding.
Sections were stained with Haematoxylin/ Eosin.
BRIEF DESCRIPTION OF THE REACTION SCHEMES
15 Scheme I illustrates periodate oxidation of hyaluronic acid.
Scheme 2 illustrates coupling of amines to hyaluronic acid with EDC
via an active triazole ester intermediate.
Scheme 3 illustrates coupling of amines to hyaluronic acid with EDC
via an active N hydroxysuccinimide ester intermediate.
20 Scheme 4 illustrates crosslinking of amine functionalized hyaluronic acid with various bifianctional N-hydroxysuccinimide ester crosslinkers to form hydrogels. (1. (SPA):-PEG; 2. DTSSP).
Scheme S illustrates crosslinking of amine fianctionalized hyaluronic acid with glutaraldehyde to form hydrogels. In addition to the conventional 25 reaction of aldehydes with amines that results in the formation of a Schiff base, glutaraldehyde is also known to undergo polymerization by aldol condensation yielding polymers with a, ~3-unsaturated aldehydes at neutral or slightly alkaline pH
(Richards and Knowles, J. Mol. Biol. 37, 231-233 (1968)). Subsequent, SUBSTITUTE SHEET (RULE 26) nucleophilic addition of amines at the ethylenyl double bond creates a stable crosslink.
Scheme 6 illustrates formation of hydrogels with aldehyde functionalized hyaluronic acid. ( 1. amine functionalized HA; 2. bifunctional 5 amine).
Scheme 1 O

O
HO
H NH
O

-J n IOQ

O
w0 0 _ (a) NH
O'' \CH3 n SUBSTITUTE SHEET (RULE 26), Scheme 2 HA-COOH (I) R1-N=C=N-R2 (XII) carbodiimide pH 2-9 O R~
N
HA O ~ (X~) NH
Rz N (XIV) R N

O OH
N
HA O N'~ ~ N
(XV) Ra R3 pH 5.5-7.0 H2N-R (III) pKa X7.0 O
(IV) HA ~NHR
SUBSTITUTE SHEET (RULE 26) - IS -Scheme 3 HA-COOH (I) R1-N=C=N-R2 (XII) carbodiimide pH 2-9 O R~
,N
HA 0 (X~) NH

HO N (XVI) O

HA O J (XVII) O
pH 7.0-8.5 HZN-R (III) pKa >7.0 O
(IV) HA ~NHR
SUBSTITUTE SHEET (RULE 26) Scheme 4 O
HA~N-Ri-NHz H
Rz O O
O O Rz N-O~ ~-O-N
O-PEG-O
O O
O
Rz O
O O R
N-O--~ ~O-N z S-S
O O
O O O O
1. HA~H-R~-H-~O-PEG-O~H R~ H~HA
O O O O
2. HA~H-R~-H-~S S~--H-R~-H-~L-HA
SUBSTITUTE SHEET (RULE 26) Scheme 5 HA~N-R-NHz H
O O O CHO CHO O
H-C.-.(CHz)s-C-H , H-C-(CHz)z C=H-(CHz)z-C H-(CHz)s-C-H
n self condensation product O
O
HA~N-R-N=C-(CHz)3-C=N-R-N~HA Schiff base product H H H H
O CHO CHO O
II I I fl H-C-(CHz)z H- j H-(CHz)z-H CH-(CHz)3-C-H
HN ~ NH
Michael addition product R I
I NH
NH
O
O
HA
HA
SUBSTITUTE SHEET (RULE 26~

Scheme 6 O
HA~H-R~-CHO
O
1. HA~H-R2-NH2 2. H2N-R3-NH2 O O
1. HA-u-N-R~-C=N-R2-N~HA
H H H
O O
2. HA-~H-R~-H=N-R3-N=H-R~-H-~L-HA
DETAILED DESCRIPTION OF THE INVENTION
Using the methods of our invention, we generate an activated form of HA that differs minimally from native HA to conserve its unique physico-5 chemical properties. We also efFect a minimal change affecting only a relatively small number of dissaccharide units of native HA so that we do not alter its property to serve as a cell substratum.
We initially attempted to generate an aldehyde derivative of HA by reduction of the carboxyl groups of the glucuronic acid moieties into aldehydes 10 using 9-borabicyclo-3,3-nonane, a method that allows direct conversion of the carboxylic acid into the aldehyde (Cha et al., Bull Korean Chem. Soc. 9, 48-52 (1988), Cha et al., Org. Prey Proc. Int. 21, 451-477 (1989)):
1) HA-COOH (I) -~ HA-CHO (II) However, even though preliminary testing indicated the conversion 15 ofthe carboxyl groups into aldehydes to a degree of approximately S-10%
(Fig. 1), SUBSTITUTE SHEET (RULE 26) mixtures of concentrated, viscous HA-aidehyde solutions (~10 mg/ml) with 'small' polyamines such as putrescine, lysine, polylysine, histidine, or polyhistidine did not generate stable gels in a reasonable time frame to be suitable for in situ polymerization. It is important to note that the chemical properties of HA are 5 determined not merely by its functional groups per se but also by the accessibility of these functional groups of HA in an aqueous environment, which is related to the overall conformational structure and rheological properties of HA. HA behaves like a hydrogel in an aqueous media even in the absence of crosslinks because it forms a network stabilized by hydrogen bonds and van der Waals forces (Laurent and 10 Fraser, supra). To increase the accessibility of functional groups, we introduced a spacer between the functional group and the HA chain.
Introducing a functionalized side chain onto HA
We have subsequently developed methodology for introducing side chains into HA by carbodiimide-mediated coupling of primary or secondary amines 15 to the carboxyl group of the glucuronic acid moiety using an active ester intermediate. We have used this methodology to generate HA with different terminal functional groups for crosslinking including acetals, aldehydes, amines, and hydrazides. A wide range of functionalized amines are commercially available which allows us to introduce a wide variety of different functional groups useful for 20 crosslinking under physiological conditions using this methodology, including maleimides that react specifically with sulfhydryls or arylazides for photocrosslinking besides the amines and aldehydes described below.
Direct carbodiimide-mediated coupling of amines to the carboxyl group of HA in an aqueous environment, e.g., with EDC ( 1-ethyl-3-[3-25 dimethylaminopropylJ carbodiimide), does not yield the predicted product since the O-acyl isourea that is formed as a reactive intermediate rearranges rapidly to a stable N acyl urea (Kuo et al., supra). We demonstrate that by "rescuing" the active O-acyl isourea by formation of a more hydrolysis resistant and non-rearrangable active ester intermediate, the coupling of primary amines to HA is possible. A
wide SUBSTITUTE SHEET (RULE 26) variety of active carboxylic esters exist and could be formed for further reaction including NHS-esters, nitrophenol esters, triazole esters, sulfonic esters, etc., as long as the reagent for their preparation is soluble in H,O or in other polar solvents such as dimethylsulfoxide or dimethylformamide. HA is soluble in H=O or other 5 aprotic polar solvents in native form and when prepared as a sodium salt or when prepared as a tetrabutylammonium salt as described in U.S. Patent No.
4,957,744, respectively. We have formed active esters of HA with 1-hydroxybenzotriazole (HOBT) or N hydroxysulfo-succinimide using the H,O soluble carbodiimide EDC
for coupling. Nucleophilic addition to the ester formed from HOBT requires the 10 amine to be presented in unprotonated form at acidic pH (about S.5 to 7.0).
Only a limited number of amines including hydrazines and activated amines, e.g., ethylene diamine, have pKa values in a suitable range and are consequently unprotonated and reactive with the ester-intermediate formed with HOBT (Scheme 2). Simple primary amines, e.g., putrescine, which typically have pKa values > 9 do not form 15 significant amounts of adduct under acidic coupling conditions. The N
hydroxysulfosuccinimide-derived intermediate allows for the coupling reaction to be carried out at neutral pH (about 7.0 to 8.5) and consequently yields products by reaction with simple primary amines (Scheme 3).
Consequently, this methodology allows for the following reactions 20 to be carried out:
HA-COOH (I) + H=N-R (III) --~ HA-CO-NH-R (IV) HA-COOH (I) + R'-NH-R (V) ---r HA-CO-NR'-R (VI) wherein R and R' are alkyl, aryl, alkylaryl or arylalkyl side chains which may contain hetero atoms such as oxygen, nitrogen, and sulfur. The side chain may be 25 branched or unbranched, and be saturated or may contain one or more multiple bonds. The carbon atoms of the side chain may be continuous or may be separated by one or more functional groups such as an oxygen atom, a keto group, an amino group, and oxycarbonyl group, etc. The side chain may be substituted with aryl moieties or halogen atoms, or may in whole or in part be formed by ring structures SUBSTITUTE SHEET (RULE 26) such as cyclopentyl, cyclohexyl, cycloheptyl, etc. The side chain may have a terminal functional group for crosslinking such as aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, etc. The side chain may be a bioactive peptide, e.g., containing receptor binding sites or proteolytic cleavage sites.
5 Terminal functional groups of the side chain useful for crosslinking of HA under physiological conditions may be selected from the following list:
1. aldehydes, see examples (VII) HZN-R-CHO
2. amines, see Examples 10 (VIII) HZN-R-NH, 3. arylazides, e.g., 4-y-azidosalicylamido) butylamine (IX) HzN-R \ / N3 \ /
4. maleimides, e.g., 4-(N maleimidomethyl) cyclohexane-1-carboxylhydrazide 15 (X) O

O
5. sulfhydryls, e.g., S-methylsulfide cysteine (XI) HzN-R-SH
SUBSTITUTE SHEET (RULE 26) WO 00/16818 PCT/EP99l06913 6. peptides, e.g., H,N-APQQEA, comprising substrate sites for enzymatic crosslinking, e.g., by transglutaminases (Parameswaran et al., Proc.
Natl.
Acad. Sci. U.S.A. 87, 8472-8475 (/990); Hohenadl et al., J. Biol. Chem. 270, 23415-23420(1995)) 5 The carbodiimides useful in this reaction may be represented by the following formula:
(XII) R-N=C=N-R' wherein R and R' comprise side chains of variable structure as described above in detail. Carbodiimides which are soluble in an aqueous media are preferred.
10 The active ester may be of the following class and be formed by carbodiimide-mediated coupling of a compound for preparation of these active esters known to a person in the art:
1. triazole esters, e.g. 1-hydroxybenzotriazole (XIII) O
N
HA O N~ ~ N

'R R"
15 2. NHS-esters, e.g. N hydroxysulfosuccinimide SUBSTITUTE SHEET (RULE 26) - z3 -(xIV) O O
R
HA O N
O
3. nitrophenol esters, e.g, p-nitrophenol (XV) O R
HA O ~ ~ N02 Preparation of HA-aldehyde derivatives A side chain containing a protected aldehyde in the form of an acetal was prepared as follows. N (2,2-dimethoxyethyl)-4-(methoxycarbonyl) butanamide was obtained from aminoacet-aldehyde dimethyl acetal and mono-methyl succinate using EDC coupling. An amino group for the coupiing to HA was subsequently introduced by reacting the product with hydrazine, yielding the desired side chain 10 with the protected aldehyde, N (2,2-dimethoxyethyl)-4-(hydrazido)butanamide.
The side chain was coupled to HA using HOBT and EDC (Scheme 2). An acetal side chain with a simple primary amine, I -aminoethyl-dimethylacetal, was conjugated to HA using N hydroxysuccinimide and EDC (Scheme 3). The HA-derivatives were purified by ethanol precipitation. The nature of the HA-derivatives 15 was confirmed by 'H NMR (Fig. 2). The HA-acetal derivatives are easily activated to the reactive aldehydes by mild acid treatment. Other HA-aldehyde derivatives with variations in the length of the side chain have been prepared in a similar manner. See Examples 1-3.
SUBSTITUTE SHEET (RULE 26) Preparation of HA-amine derivatives Diaminoethane, lysine methyl ester, histidine, and adipic, succinic or suberic dihydrazide was coupled to HA using HOBT and EDC (up to 5 fold excess depending on the desired degree of modification) and adjusting the pH to ~6.5 by 5 repeated addition of O.1M HCl during the reaction (Scheme 2). HA-derivatives were also prepared in a similar manner using N hydroxy-sulfosuccinimide and primary amines containing unconjugated amino groups with a higher pKa (>9) such as 1,4-diaminobutane or 1,6 diaminohexane (Scheme 3). The HA derivatives were purified by repeated ethanol precipitation or by extensive dialysis, and the nature of 10 the HA derivatives was confirmed by 'H NMR (Fig. 3). The degree of modification was calculated from the NMR data and found to be as high as 70%.
Reaction conditions were subsequently adjusted such that a degree of modification of approximately 20% was achieved. Limiting the amount of carbodiimide proved to be most successful in controlling the degree of modification. A degree of 15 modification of 10-25% yielded efficient crosslinking but also a molecule that would still be recognized by glycosidases and by HA receptors as HA and thus allow for recognition and processing of the material by cells (see below). Similar HA
derivatives were synthesized using succinic, adipic or suberic dihydrazide or diaminoethane, -butane, or -hexane to study the ef~'ect of the length of the spacer 20 separating the introduced functional group from the HA-chain on the subsequent crosslinking. See Examples 4-8.
Crosslinked HA hydrogels The functionalized HA molecules can be crosslinked by reacting HA
derivatives with different functionalities or using homo- or heterobifunctional 25 crosslinkers which are available in large variety. The following basic reaction schemes are suitable for crosslinking of the described forms of modified HA
(see Examples 9-12):
1. aldehyde-mediated crosslinking SUBSTITUTE SHEET (RULE 26) R~ CHO + H2N-R2 ---~- R~ H=N-R2 2. active ester-mediated crosslinking O
R O

_ pH >7.0 ~~
R~ O--N + H2N R3 R~~N--R3 H
O
3. maleimide-mediated crosslinking O O
H
+ R--SH pH >G.5---5 ~ R-N
R-N ~ ' \S-R' O O
4. photo-crosslinking 265-275 nm R~ N3 + R2 ~'- R~ N-R2 (30060 nm with H
vitro group) 5. enzymatic crosslinking (transglutaminase) Rl-(CH),-CONHz + HzN-RZ --~ R,-(CH)z-CO-NH-RZ
Crosslinking of the HA-amine derivatives (Mr ~ 106) with bifunctional active esters, e.g., polyethyleneglycol-bis-succinimidyl-propionate [(SPA),-PEG] and reducible 3,3'-dithiobis(sulfo-succinimidyl-propionate) (DTSSP) (Scheme 4), or bifunctional aldehydes, e.g. glutaraldehyde (Scheme 5), generated 10 excellent hydrogels. Stable gels could be formed by crosslinking 5 to 25 mg/ml HA
SUBSTITUTE SHEET (RULE 26) derivative with >0.05 mM aldehyde or >0.2 mM active ester (numbers are reflecting functional group concentrations). Optimal gels were generated by crosslinking 1 Smg/ml HA derivative, modified to a degree of about 10-25%, with about 0.2mM
aldehyde or 0.6mM active ester. Similarly, crosslinking of the HA-aldehyde 5 derivatives (Mr 106) (optimally about 10-lSmg/ml) with bifirnctional amines (optimally about 0.2mM) yielded excellent gels (Scheme 6}. Conjugated amines such as dihydrazines or benzylamines are required for in situ polymerization of HA
in this case to resonance stabilize the unstable Schiff base product formed by reaction of an aldehyde with a primary amine (i.e., hydrazines yield a more stable 10 hydrazone linkage). Hydrogels were also formed from an equimolar mixture of HA-aldehyde derivatives and the different HA-amine derivatives (Scheme 6).
Optimal gels were formed with ~ 1 Smg/ml of the HA derivatives. At the optimal concentrations of HA and crosslinker, geiation occurred typically in about 30 sec.
to 5 min. which is suitable for in sitr~ polymerization. The crosslinked HA
15 hydrogels were sensitive to glycosidases, i.e., testicular hyaluronidase, indicating that they are biodegradable (Fig. 4).
A number of different tests including cell culture assays and animal experiments served to assess biocompatibility of the formulated biomaterials.
Embedding of chondrocytes into the polymerizing HA hydrogels showed that 20 neither aldehyde nor NHS-ester-mediated crosslinking was toxic to cells at the concentrations employed. Seeding of cells on top of prepolymerized HA
hydrogels showed a wide variety of cellular behaviours depending on the nature of the crosslinker and crosslinking density (Fig. 5). Highly crosslinked HA hydrogels were impenetrable to cells (Fig. S, A and B), while optimally crosslinked gels were 25 infiltrated (Fig. SC). Supplementation of the HA hydrogels with cell adhesion molecules such as fibronectin (in the range of 0.1 to 1 mg/ml) did induce adhesion and spreading of cells on the materials independent of the nature of the crosslinker and the crosslinking density, but did not change the results with regard to cell infiltration, demonstrating that the lack of infiltration is due to the high crosslinking 30 density and not the absence of cell-matrix interactions. See below and Fig.
7.
SUBSTITUTE SHEET (RULE 26) Subcutaneous implantation of biomaterials in rats is the established model for evaluation of biocompatibility of biomaterials (Laurencin et al., J. Biomed. Mat. Res. 24, 1463-1481 (1990)) and for induction of ectopic bone formation by members of the TGF-~i gene family, and bone morphogenetic proteins 5 (BMP) in particular (Wang et al., Proc. Natl. Acad. Sci. U.S.A. 87,2220-2224 (1990); Sampath et al., J. Biol. Chem. 267, 20352-20362 (1992)). Taking into consideration the cell culture results, we have formulated a number of HA
hydrogels for in vivo biocompatibility testing in this model. Implantation of prepolymerized HA hydrogel discs loaded with recombinant BMP-2 and IGF-1 or 10 TGF-~i2 subcutaneously in rats showed a mild fibrosis with a varying degree of cartilage and bone formation depending on the nature of the HA biomaterial (Figs. 6 and 7). The growth factors were mixed with the HA derivatives prior to gelling and the induction of bone formation suggests that neither reaction mechanism used for HA crossiinking (aldehyde or active ester-mediated reactions) significantly affected 15 the biological activity of the growth factors. Little inflammation was observed with active ester crosslinked HA-amine derivatives (Fig. 7) or with HA-amine derivatives crosslinked with various HA-aldehyde derivatives (Fig. 6B-6D) while a stimulation of foreign body giant cells was seen when the same HA-amine derivatives were crosslinked with glutaraldehyde (Fig. 6A). The degree of modification of RA
20 strongly affected the resorption and transformation rate of the biomaterials (Fig.
7A, 7B). Nevertheless, limited bone formation was seen even with a biomaterial formed from a highly modified (65-70%) HA-amine derivative (Fig. 7A). The absence of bone formation with a smaller bifunctional NHS-ester crosstinker indicates that the size of the generated crossbridge is crucial for resorption and 25 cellular infiltration (Fig. 7C). This is probably due to the difference in pore size of the material formed with crosslinkers of different sizes. The infiltration and transformation rate was similar with BMP-2/IGF-1 and BMP-2/TGF-X32 loaded biomaterials, indicating that the resorption rate is a material property.
However, at ten days post-implantation, the newly formed tissue was largely cartilage in the first 30 group and largely bone in the second group (Fig. 8), exemplifying the angiogenic SUBSTITUTE SHEET (RULE 26) effect of TGF-~i2 (Yang and Moses, J. Cell. Biol., 111, 731-741 (1990)). This demonstrates that the biological activity of the HA material can be modulated by inclusion of different bioactive factors. The lack of significant adverse erects and the demonstration of the desired biological activity of these novel HA
biomaterials 5 in vivo demonstrates their usefulness as a delivery vehicle for cells and growth factors in the field of tissue regeneration.
There are several approaches to the production of HA, including extraction from tissue and biosynthesis. Extraction from tissue typically uses fresh or frozen cocks' combs (U.S. Pat. No. 5,336,767), although other tissues including 10 the synovial fluid of joints (Kvam et al., Anal. Biochem. 211, 44-49 (1993)), human umbilical cord tissue, bovine vitreous humor, and bovine tracheae, have been used.
It is also possible to prepare HA by microbiological methods, such as by cultivating a microorganism belonging to the genus Streptococcus which is anhemolytic and capable of producing HA in a culture medium (U.S. Pat. Nos. 4,897,349;
15 4,801,539; 4,780,414; 4,517,295; 5,316,926). The HA raw material for preparing the compositions of the invention preferably consists of high molecular weight HA, more preferably of molecular weight greater than 0. 5 million daltons, and more preferably of molecular weight greater than one million daltons. The HA raw material for the compositions of examples of this invention described herein was 20 obtained from Genzyme Corp. (Cambridge, MA), and had a molecular weight greater than one million daltons. The size of the HA was unchanged after derivatization.
The compositions of the invention have many therapeutic uses. The fact that the compositions may be cured in a surgically practical time frame of one 25 to five minutes in situ with concurrent crosslinking to the tissue surfaces allows for employment as a tissue glue. Many situations in various surgical applications require such adhesives. For example, the compositions of the invention may be used to stem hemorrhage in general surgery, reconstruct nerves and vessels in reconstructive, neuro- and plastic surgery, and to anchor skin, vascular, or cartilage 30 transplants or grafts in orthopedic, vascular, and plastic surgery. Those of skill in SUBSTITUTE SHEET (RULE 26) the art may choose and design particular embodiments of the invention which are particularly suitable for a desired application, by adjusting several factors, including:
(1) the degree of functionalization of HA, which affects the crosslinking density of the material and interaction with cellular proteins, including receptors and 5 glycosidases; (2) the concentration of the crosslinker, which affects the crosslinking density of the material; (3) the size of the generated cross-bridge, which affects the pore size of the material; (4) the nature of the crosslinking mechanism, which determines polymerization time and the specificity of the reaction; and (5) the nature of the cross-bridge, which provides biological cues. See Figs. 4, 5, and 7 for 10 data concerning HA hydrogels with different crosslinking densities and pore sizes.
Generally, active ester- or photo-crosslinking are preferred to form materials for applications requiring fast gelation and strong bonding with tissue surfaces, such as tissue glues. Materials with anti-adhesive properties, which are useful to form tissue separations or for tissue augmentation, are formed from highly modified HA
15 derivatives with low molecular weight crosslinkers, which generates a dense material with very small pores, thereby minimizing cell adhesion and infiltration.
Conversely, biodegradable scaffolds for tissue repair are formed from HA with a limited degree of derivativization and high molecular weight crosslinkers, which generate a porous, biodegradable material. The crossbridge may even contain 20 biological cues, such as peptide sequences, which facilitate material degradation by, for example, proteolysis or cellular infiltration (e.g., the RGD sequence).
Compositions of this invention were designed to serve as a vehicle for the delivery of cells or bioactive molecules such as growth factors to stimulate focal repair. The crosslinked HA derivatives are porous hydrogels in which 25 biologically or therapeutically active compounds (e.g., growth factors, cytokines, drugs, and the like) can be physically or chemically incorporated. These compounds will then be subject to sustained release by chemical, enzymatic, and physical erosion of the hydrogel and/or the covalent linkage between the HA
chain and biologically active compound over a period of time. Local delivery of growth 30 factors with such a scaffold facilitates wound healing and tissue regeneration in SUBSTITUTE SHEET (RULE 26) many situations. For example, the compositions of the invention may be used not only to promote bone formation and stimulate cartilage repair in orthopedic procedures, as described more fully below, but also to treat pathological wound conditions such as chronic ulcers. They may also serve as a scaffold to generate 5 artificial tissues, e.g., cartilage (Hauselmann et al., Am. J. Physiol. 271, (1996)), through proliferation of autologous cells in culture. Similar procedures for generation of equivalents of other tissues or organs, including skin, liver, and others, in culture may be developed in the future and may be used in combination with the compositions of the invention.
10 Highly crosslinked materials have an anti-adhesive property with respect to cells, and such compositions may be used to generate tissue separations and to prevent adhesions following surgery. See Figs. SA and 7C, showing highly modified HA-amine, i.e., adipic dihydrazido HA, preferably crosslinked with low molecular weight bifunctional NHS-ester. The viscoelastic properties of HA
make 15 it particularly well suited for this purpose, and it is used clinically to achieve temporal pain relief by repeated intraarticular injections in arthropathies as a "joint lubricant", and as a protective agent for eye irritations and in ophthalmic surgery.
The technique of tissue separation is used in facial reconstruction in plastic surgery and dentistry. Prevention of the formation of adhesions is particularly relevant in 20 reconstructive surgery of tendons, in surgical procedures in the urogenital system, and in thoracic surgery. Many different HA-based materials are already in clinical use in these areas. (See products manufactured by Anika Therapeutics, Inc.
(Woburn, MA), Biomatrix, Inc. (Ridgefield, NJ), Genzyme Corp. (Cambridge, MA), and Fidia, S.p.A. (Abano Terme, Italy)). Those of skill in the art may choose 25 and design particular embodiments of the invention which are particularly suitable for a desired application by selecting distinct features as outlined above.
The injectable nature of the compositions of the invention also renders them suitable for tissue augmentation in plastic surgery, where the HA
matrix serves primarily as a biocompatible filler material, e.g., for filling dermal 30 creases or lip reconstruction. ADain, those of skill in the art may choose and design SUBSTITUTE SHEET (RULE 26) particular embodiments of the invention which are particularly suitable for a desired application, as outlined above.
The half life of pharmacological compounds, both synthetic and biological, has been shown to be drastically increased when delivered in a form 5 conjugated to HA (Larsen and Balazs, Adv. Dru Delivery Rev. 7, 279-293 (1991);
Drobnik, J., Drug Delivery Rev. 7, 295-308 (1991)). The functionalized forms of HA provided by this invention allow for easy substitution with pharmacologically active agents, such as anti-inflammatories, analgesics, steroids, cardiovascular agents, anti-tumor agents, immunosuppressants, sedatives, anti-bacterial, 10 anti-fungal, and anti-viral agents, etc., and may be used for sustained drug release over time, either locally in hydrogel form or systemically in free form.
In orthopedic surgery, the functionalized farms of HA of this invention have applications as a tissue glue or bioactive matrix material in the treatment of chondral and osteochondral fractures, osteochondritis dissecans, 1 S meniscal tears, as well as ruptured ligaments, tendons, or myotendineous junctions.
The HA materials of this invention may serve to facilitate anchorage of chondral or osteochondral transplants or grafts, or other biological or artificial implant materials, or to stimulate new bone or cartilage formation by serving as a scaffold for cells or as a delivery vehicle for growth factors. One general approach to 20 promote articular cartilage repair based on the compositions of the invention comprises using: (1) iu sitzr polymerized HA hydrogel as a matrix to fill the defect which is to be repaired and to provide a scaffold for repair cells, (2) an optional chemotactic agent to attract repair cells to the matrix and defect site, or alternatively, autologous chondrocytes or mesenchymal stem cells, (3) an optional 25 factor to promote cellular proliferation of repair cells in the matrix and defect site;
(4) sustained release of a transforming factor by the HA hydrogel over time to promote differentiation of the repair.
Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a SUBSTITHTE SHEET (RULE 26) stated integer or group of integers but not the exclusion of any other integer or group of integers.
In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and 5 are not to be construed as limiting the scope of the invention in any way.
Example 1 Preparation of N (2, 2-Dimethoxyethyl)-4-(methoxycarbonyl)butan-amide (1) - EDC (4.98g, 0.026mo1) was added to a mixture of aminoacetaldehyde dimethyl acetal (2. l8ml, 20mmol) and methyl monoester of succinic acid (2.648, 10 20mmo1) in 75m1 of dichloromethane, and the reaction mixture stirred for 24 h at room temperature. The solution was extracted successively with SOmI each of ice cold solutions of 0.75M sulfuric acid, 1M NaCI, saturated sodium bicarbonate, and 1M NaCI. The organic phase was collected and dried with sodium sulfate. The solvent was evaporated under reduced pressure yielding a syrup, which showed a 15 single spot on charring upon TLC in solvent A (Rt 0.75) and solvent B (Ri 0.24).
The apparent yield of 1 was 65%: 'H NMR in CDCI, b 5.70 (bs, 1H, NH), 4.34 (t, 1H, CH-(OCH,), 3.67 (s, 3H, COOCH,), 3.43-3.35 (s and t, 8H, CH,OC and CHCH,NH}, 2.38-2.26 (m, 4H, CH,CO).
Formation of Acyl-hydrazide (2) from 1 - Anhydrous hydrazine 20 (248111, 7.9mmol) was added to a solution of 1 (1.73g, 7.9mmol) in Sml of anhydrous methanol. The mixture was stirred at room temperature overnight and the solvent subsequently evaporated under reduced pressure yielding a solid residue.
The residue was dissolved in H,O (6m1) and extracted three times with an equal volume of dichloromethane. The aqueous solution was evaporated to dryness 25 under reduced pressure and then further dried overnight in vacuo. The crystalline solid (1.048, 82% yield) was homogeneous on TLC in solvent A (R, 0. IO) when visualized by charring. The 'H NMR spectrum indicated the loss of the ester methoxy group when compared to 1.
SUBSTITUTE SHEET (RULE 26) Preparation of Hydrazido-dimethyl acetal-HA (formula XIX) -Sodium hyaluronate (100mg, 0.25mmol) and N (2,2-dimethoxyethyl)-4-(hydrazido)butanamide (2) ( 1.646g, 7. Smmol) was dissolved in H,O (40m1, 2.Smg/ml HA). The pH was adjusted to 6.5 and HOBT (169mg, 1.25mmol) 5 predissolved in a 1:1 mixture of water and DMSO (lml) and EDC (240mg, 1.25mmo1) was added and the reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with I M NaOH and NaCI added to produce a 5% w/v solution. HA was precipitated by addition of three volume equivalents of ethanol.
The precipitate was redissolved in H:O at a concentration of approximately Smg/ml 10 and the precipitation repeated twice. The purified product was freeze dried and kept at 4°C under N,. See Fig. 2B for NMR data of the product.
(XIX) H
N
HA ~ - ~ OCH3 H H O
Example 2 Preparation of Aminoacetaldehyde-dimethyl acetal-HA (formula 15 XX) - Sodium hyaluronate (IOOmg, 0.25mmo1) and 2,2-dimethoxyethylamine (0.7888, 7.Smmo1) was dissolved in H=O (40m1, 2.Smg/ml HA). The pH was adjusted to 7.5 and NHS.SO,Na (268mg, 1.25mmo1) and EDC (240mg, 1.25mmol) was added and the reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with 1M NaOH and NaCI added to produce a 5% w/v 20 solution. HA was precipitated by addition of three volume equivalents of ethanol.
The precipitate was redissolved in H,O at a concentration of approximately Smg/ml and the precipitation repeated twice. The purified product was freeze dried and kept at 4°C under N,.
SUBSTITUTE SHEET (RULE 26) ( O

HA

Example 3 Deprotection of HA-acetals to_ form HA-aldehydes - The acetal modified HA (formula XXI) was dissolved in H:O to a concentration of 5-IOmg/ml 5 and IM HCl was added to give a final concentration of 0.025M. The solution was then allowed to stand at room temperature for 0.5 to 1.Oh. The solution was neutralized by the addition of 1M NaOH, yielding the deprotected HA-aldehyde (formula XXII).
HA-CO-R-CH(OCH,). (XXI) -r HA-CO-R-CHO (XXII) 10 Example 4 Preparation of I~iaminoethane-HA (formula XXIII) - Sodium hyaluronate (IOOmg, 0.25mmol) and 1,2-diaminoethane HCl (0.9988, 7.5mmol) was dissolved in H=O (40m1, 2.Smg/ml HA). The pH was adjusted to 6.5 and HOBT ( 169mg, 1.25mmol} predissolved in a I :1 mixture of water and DMSO
15 (lml) and EDC (240mg, 1.25mmo1) was added and the reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with 1M NaOH and NaCI
added to produce a 5% w/v solution. HA was precipitated by addition of three volume equivalents of ethanol. The precipitate was redissolved in H,O at a concentration of approximately Smg/ml and the precipitation repeated twice.
The 20 purified product was freeze dried and kept at 4°C under N,.
SUBSTITUTE SHEET (RULE 26) (XXIII) O

HA N
H
Example 5 Preparation ofL-Lysine methyl ester-HA (formula XXIV) - Sodium hyaluronate (i00mg, 0.25mmol) and L-lysine methyl ester dihydrochloride (1.7488, 5 7.Smmo1) was dissolved in H:O (40m1, 2.Smg/ml HA). The pH was adjusted to 6.5 and HOBT (169mg, 1.25mmo1) predissolved in a 1:1 mixture of water and DMSO
(lml) and EDC (240mg, 1.25mmol) was added and the reaction mixture was stirred overnight. The pH was subsequeritiy adjusted to 7.0 with 1M NaOH and NaCI
added to produce a S% w/v solution. HA was precipitated by addition of three 10 volume equivalents of ethanol. The precipitate was redissolved in H20 at a concentration of approximately Smg/ml and the precipitation repeated twice.
The purified product was freeze dried and kept at 4°C under N2. See Fig. 3C
for NMR
data of the product.
(XXIV) 15 Example 6 Preparation of L-Hi.stidine methyl ester HA (formula XXV) -Sodium hyaluronate ( 1 OOmg, 0.25mmol) and L-histidine methyl ester dihydrochloride ( 1.8158, 7. Smmol} was dissolved in H,O (40m1, 2. Smg/ml HA).
The pH was adjusted to 6.5 and HOBT(169mg, 1.25mmol) predissolved in a l:l 20 mixture of H:O and DMSO ( 1 ml) and EDC (240mg, 1.25mmo1) was added and the SUBSTITUTE SHEET (RULE 26) reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with 1M NaOH and NaCI added to produce a 5% w/v solution. HA was precipitated by addition of three volume equivalents of ethanol. The precipitate was redissolved in H,O at a concentration of approximately Smg/ml and the precipitation 5 repeated twice. The purified product was freeze dried and kept at 4°C
under N=.
See Fig. 3B for NMR data of the product.
( NH
HA N
H
Example 7 Preparation of Hydrazido-HA (formula XXVI) - Sodium 10 hyaluronate (100mg, 0.25mmol) and dihydrazide i.e. adipic dihydrazide (1.31g, 7.Smmo1) was dissolved in H,O (40m1, 2.Smg/ml HA). The pH was adjusted to 6.5 and HOBT (169mg, 1.25mmol) predissolved in a 1:1 mixture of water and DMSO
(lml) and EDC (240mg, 1.25mmol) was added and the reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with 1M NaOH and NaCI
15 added to produce a S% w/v solution. HA was precipitated by addition of three volume equivalents of ethanol. The precipitate was redissolved in H,O at a concentration of approximately Smg/ml and the precipitation repeated twice.
The purified product was freeze dried and kept at 4°C under N:. See Fig. 3D
for NMR
data of the product.
SUBSTITUTE SHEET (RULE 26) (XXVI) O

HA
Example 8 Preparation of Dian~inoalkyl-HA (formula XXVII) - Sodium hyaluronate (100mg, 0.25mmo1) and a diaminoalkane, i.e. 1,2-diaminobutane HCl 5 (1.208g, 7.Smmol) was dissolved in H20 (40m1, 2.Smg/ml HA). The pH was adjusted to 7.5 and NHS.SO,Na (268mg, 1.25mmol) and EDC (240mg, 1.25mmo1) was added and the reaction mixture was stirred overnight. The pH was subsequently adjusted to 7.0 with 1M NaOH and NaCI added to produce a 5% w/v solution. HA was precipitated by addition of three volume equivalents of ethanol.
10 The precipitate was redissolved in H:O at a concentration of approximately Smg/ml and the precipitation repeated twice. The purified product was freeze dried and kept at 4°C under N2. See Fig. 3A for NMR data of the product.
(XXVII) O

HA N
H
Example 9 15 Forn:anon of crosslinked HA hydrogels~ - The general procedure for forming crosslinked HA hydrogels is as follows: Modified HA is dissolved by agitation in H,O or phosphate buffered saline (pH 7.4-8.5) at a concentration of S-25mg/ml. The degree of modification of the HA derivative is derived from the integration ofthe'H NMR peaks. After complete dissolution, the HA derivative 20 solution is transferred to a 1 ml syringe. When reacting the HA derivatives with SUBSTITUTE SHEET (RULE 26) WO 00/16818 PCT/EP99/069t3 low molecular weight crosslinkers, a slight excess of the compound (about 1.1 molar equivalent of functional groups) is dissolved in a second 1 ml syringe in 1/10 of the HA derivative volume immediately prior to use. The syringes are connected while paying special attention to excluded air, the contents are rapidly mixed, 5 typically with 20 passages, and then extruded. When reacting HA derivative molecules with different functionalities, 0.5-1.0 equivalent of HA-aldehyde is mixed with equivalent of HA-hydrazine, depending on the degree of modification of HA
derivatives. At room temperature, gelation occurs within about 30 seconds to several minutes, depending on the formulation, and the gel properties do not 10 significantly change after approximately 5 minutes.
Example 10 Digestion of crosslinked HA hydrogels with hyaluronidase - The general procedure for digestion of crosslinked HA hydrogels is as follows: HA
hydrogels are formed in 1 ml syringes by crosslinking l2mg/ml HA-amine in 15 phosphate buffered saline with various crosslinkers as indicated in Fig. 4.
Gelling is allowed to occur for 1 hour at 37°C for the reaction to be complete, after which identical ~100p1 cylindrical gels are formed by cutting the syringes with a razor blade. The gels are incubated with different concentrations of bovine testicular hyaluronidase (Sigma) 50-SOOOU/mL in 400p1 of 30mM citric acid, 1 SOmM
20 Na=HPO" pH 6.3, I SOmM NaCI for the indicated time 0-48 hours. Degradation of the gels was determined from the release of glucuronic acid into the supernatant as measured using the carbazole method (Bitter and Muir, Supra).
Example 11 Crosslinked HA hydrogels as a matrix for cell culture -25 Chondrocytes were isolated from bovine nose cartilage according to established procedures (Hauselmann et al., Matrix 12, I 16-129 (1992; Kuttner et al., J.
Cell Biol. 93, 743-750 (1982)), cultured in Ham's F12 medium containing 5% fetal bovine serum and antibiotics, and dedifferentiated by monolayer culture on plastic.
SUBSTITUTE SHEET (RULE 26) For cytotoxicity studies, cells (2. Sx 10') were embedded into the HA
hydrogels by gently mixing the trypsinized cells ( about 50 to 100p.1) with the polymerizing HA
and crosslinker mixture (approximately 400p1 gel volume) prior to complete setting.
Agarose embedded cells served as a control. After adaptation to the culture 5 conditions (24h), cell proliferation and metabolic activity was assessed by pulse labeling with ['H]thynudine and [3'S]methionine. For cell infiltration studies, HA
hydrogels were polymerized in 24-well plates (~l5mm diameter and 3mm height) for 1 h at room temperature, and extensively rinsed with phosphate buffered saline.
Cell adhesion molecules or chemotactic factors, e.g. IGF-l, were added to the HA
10 solution prior to crosslinking when desired. After 24h, cells (2. Sx 10') were seeded on top of the HA-hydrogels and cultured as above. At different time points post seeding, gels were fixed in phosphate buffered 4% paraformaldehyde and processed for paraffin embedding. Cell infiltration was assessed by staining sections with Haematoxylin/Eosin. See Fig. 5.
15 Example 12 Subcutaneous implantation of HA hydrogels in rats - Rats (2-3 per test material) were anaesthetized with ketamine/xylazine, the ventral thorax and abdomen shaved, and prepared aseptically. A small vertical incision was made on either side of the xiphoid cartilage of the sternum and the skin undermined with a 20 blunt instrument to separate the skin from the underlying tissue. HA
hydrogels were polymerized in 3m1 syringes as described. For induction of chondro-osseous differentiation, lmg/ml prefibrillized intact collagen type I (Organogenesis, Canton, MA), 200 pl/ml recombinant BMP-2 (Genetics Institute, Cambridge, MA), and 500 ng/ml IGF-1 (Celtrix Pharmaceuticals, Santa Clara, CA) or SOng/ml TGF-~i2 25 (Celtrix Pharmaceuticals, Santa Clara, CA) were mixed with the HA solution prior to crosslinking. Collagen fibrils were prepared by slow polymerization (from dilute solutions of 2-3mg/ml) of acid-solubilized collagen in phosphate buffered saline and harvested by centrifugation following standard protocols (McPherson et aL, Collagen Rel. Res. 5, 119-135 ( 1985)). Gelling of the HA hydrogels was allowed SUBSTITUTE SHEET (RULE 26) to occur for 24h at room temperature for the reaction to be complete, after which identical ~3mm thick cylindrical gels were prepared by cutting the syringes with a razor blade. HA hydrogel discs were then placed in each pocket and the skin incisions closed with sutures. Ten days post operatively, the rats were euthanized 5 and the appearance of the implant sites, i. e. degree of inflammation, grossly examined and tissue specimens harvested and processed for histology by fixation in phosphate buffered formalin and paraffin embedding. Sections were stained with Haematoxylin/Eosin and with Safranin-O/fast green, and cell infiltration and transformation (cartilage and bone formation) induced by the biomaterial as well as 10 signs of fibrosis and inflammation in the surrounding tissue evaluated. See Figs.
6-8.
SUBSTITUTE SHEET (RULE 26)

Claims (39)

We claim:

1. A composition comprising a derivative of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such 5 that the substituted disaccharide subunit is of the formula wherein R' and R" is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl;
alkylaryl;
arylalkyl substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur; phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur;
phosphorous, halogen or metal ion; and said side chain functional groups being bound directly to each other or separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and said side chain terminating with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl.

2. The composition of claim 1, wherein at least 5% of said disaccharide subunits are substituted disaccharide subunits.

3. The composition of claim 1, wherein at most 95% of said disaccharide subunits are substituted disaccharide subunits.

4. The composition of claim 1, wherein at most 95% of said disaccharide subunits are substituted disaccharide subunits.

5. The composition of claim 1, wherein at least one of said terminal functional groups is selected from the group consisting of peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, and active ester, whereby said composition is amenable to crosslinking.

6. The composition of claim 1, wherein the molecular weight of said composition is at least 100,000 daltons

7. The composition of claim 1, wherein the molecular weight of said composition is at most 100,000 daltons.

8. The composition of claim 1, wherein the molecular weight of said composition is at least 1,000,000 daltons.

9. The composition of claim 1, wherein said composition is water soluble.

10. A composition comprising an activated ester of hyaluronic acid of the formula:

wherein R is selected from the group consisting of a substituted triazole, N-sulfosuccinimide, nitrophenol, partially halogenated phenol, perhalophenol, and pentafluorophenol.

11. A hydrogel of crosslinked HA derivatives, wherein said HA
derivatives are compositions according to claim 1.
Next, a set of claims to the hydrogels.

12. A hydrogel of crosslinked HA derivatives, wherein said HA
derivatives are selected from the group consisting of the compositions of claim 10.

13. The hydrogel of crosslinked HA derivatives of claim 11, wherein said hydrogel is biodegradable.

14. A method for making a derivative of hyaluronic acid, comprising the steps of:
a) forming an activated ester at a carboxylate of a glucuronic acid moiety of hyaluronic acid; and b) substituting at the carbonyl carbon of the activated ester formed in step (a), a side chain comprising a nucleophilic portion and a functional group portion.

15. The method of claim 14, wherein the nucleophilic portion is selected from the group consisting of ammonia, primary amine, secondary amine, hydroxyl, and sulfhydryl.

16. The method of claim 14, wherein the functional group portion is selected from the group consisting of active ester, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, and peptide.

17. The method of claim 14, wherein step (a) is performed with an active ester selected from the group consisting of a substituted triazole, N-sulfosuccinimide; nitrophenol, partially halogenated phenol, perhalophenol, pentalourophenol, HOBT, and NHS, by carbodiimide-mediated coupling.

18. The method of claim 14, comprising the additional step of (c) forming a cross-linked hydrogel from the hyaluronic acid derivative.

19. A method for forming a matrix for a temporary scaffold for tissue repair according to the method of claim 18, wherein the crosslinker is selected from the group consisting of polyvalent active ester, aldehyde, amine, arylazide, maleimide, and sulfhydryl.

20. A method for forming a matrix for a temporary scaffold for tissue repair according to the method of claim 18, wherein the HA derivative comprises a peptide substrate for transglutaminase, and wherein the HA
derivative is crosslinked using transglutaminase.

21. The method of claim 18, wherein step (c) is performed in the presence of cells.

22. The method of claim 18, wherein step (c) is performed in the presence of at least one member selected from the group consisting of growth factors, cytokines, drugs, and bioactive peptides.

23. The method of claim 22, wherein the bioactive peptide is RGD.

24. The method of claim 22, wherein the biactive peptide is a substrate for transglutaminase.

25. The method of claim 24, wherein the bioactive peptide is APQQEA.

26. The method of claim 24, wherein the growth factor is TGF-.beta. or BMP.

27. The method of claim 18, wherein step (c) is performed in situ in a patient in need of tissue repair.

28. A tissue adhesive comprising a hydrogel of claim 11, wherein the side chain is selected from the group consisting of activated ester, aldehyde, arylazide, and maleimide.

29. A tissue adhesive comprising an HA derivative of claim 10.

30. A tissue adhesive comprising a hydrogel of claim 11, wherein the crosslinked HA derivatives are formed using a cross-linker selected from the group consisting of polyvalent active ester, aldehyde, arylazide, and maleimide.

31. A tissue adhesive comprising a hydrogel of claim 11, wherein the cross-linked hydrogel is formed in the presence of at least one member selected from the group consisting of growth factors, cytokines, drugs, and bioactive peptides.

32. A tissue adhesive of claim 31, wherein the growth factor is TGF-.beta. or BMP-2.

33. A matrix for cell structures comprising a hydrogel of claim 11, wherein the crosslinked HA-derivatives are formed using a cross-linker selected from the group consisting of polyvalent active ester, aldehyde, amine, arylazide, maleimide, and sulfhydryl.

34. A matrix for cell structures comprising a hydrogel of claim 11, wherein the crosslinked hydrogel is formed in the presence of at least one member selected from the group consisting of growth factors, cytokines, drugs, and bioactive peptides.

35. A matrix for cell cultures according to claim 34, wherein the growth factor is TGF-.beta. or BMP-2.

36. A matrix for a scaffold comprising a hydrogel of claim 11, wherein the crosslinked HA-derivatives are formed using a cross-linker selected from the group consisting of polyvalent active ester, aldehyde, amine, arylazide, maleimide, and sulfhydryl.

37. A matrix for a scaffold comprising a hydrogel of claim 11, wherein the crosslinked hydrogel is formed in the presence of at least one member selected from the group consisting of growth factors, cytokines, drugs and bioactive peptides.

38. A matrix for a scaffold according to claim 37, wherein the growth factor is TGF-.beta. or BMP-2.

39. The matrix of claim 37, wherein the matrix further comprises cells.