Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1992-02-11
2001-01-16
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S055200, C536S055300, C536S056000, C536S102000, C514S053000, C514S054000, C514S056000, C514S057000, C514S060000, C514S062000
Reexamination Certificate
active
06174999
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to biocompatible films and gels formed from chemically modified polyanionic polysaccharides.
Hyaluronic acid (“HA”) is a naturally occurring mucopolysaccharide found, for example, in synovial fluid, in vitreous humor, in blood vessel walls and umbilical cord, and in other connective tissues. The polysaccharide consists of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues joined by alternating &bgr; 1-3 glucuronidic and &bgr; 1-4 glucosaminidic bonds, so that the repeating unit is -(1→4)-&bgr;-D-GlcA-(1→3)-&bgr;-D-GlcNAc-. In water, hyaluronic acid dissolves to form a highly viscous fluid. The molecular weight of hyaluronic acid isolated from natural sources generally falls within the range of 5×10
4
up to 1×10
7
daltons.
As used herein the term “HA” means hyaluronic acid and any of its hyaluronate salts, including, for example, sodium hyaluronate (the sodium salt), potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
HA, in chemically modified (“derivatized”) form, is useful as a surgical aid, to prevent adhesions or accretions of body tissues during the post-operation period. The derivatized HA gel or film is injected or inserted into the locus between the tissues that are to be kept separate to inhibit their mutual adhesion. To be effective the gel must remain in place and prevent tissue contact for a long enough time so that when the gel finally disperses and the tissues do come into contact, they will no longer have a tendency to adhere.
Chemically modified HA can also be useful for controlled release drug delivery. Balazs et al., 1986, U.S. Pat. No. 4,582,865, states that “cross-linked gels of HA can slow down the release of a low molecular weight substance dispersed therein but not covalently attached to the gel macromolecular matrix.” R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et al.,
Controlled Release Delivery Systems,
Marcel Dekker, Inc., New York, describes sustained release of chloramphenicol covalently attached to hyaluronic acid via ester linkage, either directly or in an ester complex including an alanine bridge as an intermediate linking group.
I. Danishefsky et al., 1971, Carbohydrate Res., Vol. 16, pages 199-205, describes modifying a mucopolysaccharide by converting the carboxyl groups of the mucopolysaccharide into substituted amides by reacting the mucopolysaccharide with an amino acid ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”) in aqueous solution. They reacted glycine methyl ester with a variety of polysaccharides, including HA. The resulting products are water soluble; that is, they rapidly disperse in water or in an aqueous environment such as is encountered between body tissues.
Proposals for rendering HA compositions less water soluble include cross-linking the HA. R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et al.,
Controlled Release Delivery Systems,
Marcel Dekker, Inc., New York, describe modifying HA by attaching cysteine residues to the HA via amide bonds and then cross-linking the cysteine-modified HA by forming disulfide bonds between the attached cysteine residues. The cysteine-modified HA was itself water soluble and became water insoluble only upon cross-linking by oxidation to the disulfide form.
De Belder et al., PCT Publication No. WO 86/00912, describe a slowly-degradable gel, for preventing tissue adhesions following surgery, prepared by cross-Linking a carboxyl-containing polysaccharide with a bi- or polyfunctional epoxide. Other reactive bi- or polyfunctional reagents that have been proposed for preparing cross-linked gels of HA having reduced water solubility include: 1,2,3,4-diepoxybutane in alkaline medium at 50° C. (T. C. Laurent et al., 1964, Acta Chem. Scand., vol. 18, page 274); divinyl sulfone in alkaline medium (E. A. Balasz et al., U.S. Pat. No. 4,582,865, (1986); and a variety of other reagents including formaldehyde, dimethylolurea, dimethylolethylene urea, ethylene oxide, a polyaziridine, and a polyisocyanate (E. A. Balasz et al., U.K. Patent Appl. No. 84 20 560 (1984). T. M{umlaut over (a)}lson et al., 1986, PCT Publication No. WO 86/00079, describe preparing cross-linked gels of HA for use as a vitreous humor substitute by reacting HA with a bi- or polyfunctional cross-linking reagent such as a di- or polyfunctional epoxide. T. M{umlaut over (a)}lson et al., 1986, EPO 0 193 510, describe preparing a shaped article by vacuum-drying or compressing a cross-linked HA gel.
SUMMARY OF THE INVENTION
In one aspect, the invention features a method for making a water insoluble biocompatible composition, the method including combining, in an aqueous mixture, a polyanionic polysaccharide, an activating agent, and a nucleophile, under conditions sufficient to form the composition.
In preferred embodiments of this aspect of the invention, the activating agent which are used include benzotriazole-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate, O-benzotriazole-1-yl-N,N,N′,N′tetramethlyluronium hexafluorophosphate, bromotris(dimethylamino)phosphonium hexafluorophosphate, or the corresponding halide salts thereof.
The preferred concentration of polyanionic polysaccharide in the reaction is 0.0002-0.1M, more preferably 0.0005-0.02M. The preferred pH for carrying out the reaction is 3.5 to 8.0. The preferred reagent stoichiometry is at least 0.1 molar equivalents of activating agent per molar equivalent of polyanionic polysaccharide.
Another aspect of the invention features; a method for making a water insoluble biocompatible composition, the method including combining, in an aqueous mixture, a polyanionic polysaccharide, an activating agent, a modifying compound, and a nucleophile, under conditions sufficient to form the composition.
In preferred embodiments of this aspect of the invention, modifying compounds include, 1-hydroxybenzotriazole hydrate, 1-hydroxybenzotriazole monohydrate, N-hydroxysulfosuccinimide, N-hydroxysuccinimide, 4-nitrophenol, 2-nitrophenol, 4-nitrothiophenol, 2-nitrothiophenol, pentachlorophenol, pentafluorophenol, imidazole, tetrazole, 4-dimethylaminopyridine or other related compounds. The activating agent is preferably a diimide, more preferably a carbodiimide, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide.
Also in preferred embodiments of the second aspect of the invention, the preferred polyanionic polysaccharide is present in the reaction at a concentration of 0.0002-0.1M, more preferably 0.005-0.02M. The preferred pH for carrying out the reaction is 3.5 to 8.0. The preferred reagent stoichiometry is at least 0.1 molar equivalents of activating agent per molar equivalent of polyanionic polysaccharide, and at least
1
molar equivalent of modifying compound per molar equivalent of activating agent. Preferred polyanionic polysaccharides for use in the methods of the invention include hyaluronic acid (HA), carboxymethyl cellulose (CMC), carboxymethyl amylose (CMA), chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate; HA, CMC, and CMA are particularly preferred. It is also well understood that two or more polyanionic polysaccharides may be employed in the methods of the invention.
Also in both aspects of the invention, preferred nucleophilic compounds which are capable of reacting with the activated polyanionic polysaccharide include amino acid amides (preferably leucinamide hydrochloride), monofunctional amines (preferably 3-amino-1-propanol), amino acid esters (preferably a methyl ester or a butyl ester, including t-butyl ester), amino alcohols, amino thiols, amino phenols, amino cathechols, amino acids, salts of amino acids, peptides, proteins and other ambident nucleophilic compounds in which only one electron rich moiety reacts as a nucleophile with the activated polyanionic polysaccharide.
The term “aqueous mixture”, as used herein, generally refers to a solution composed primaril
Miller Robert J.
Xu Xuejian
Geist Gary
Genzyme Corporation
White Everett
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