Patent 6,548,081

The present invention relates to a composite and a method for reducing post-operative adhesion of tissues. The composite includes a biocompatible, biodegradable support, and a water-insoluble hyaluronic acid derivative at the support. The hyaluronic acid derivative includes an N-acylurea that results from cross-linking by the reaction of hyaluronic acid with a multifunctional carbodiimide. Optionally, a monocarbodiimide also may be employed. A pharmaceutically-active molecule may be added to the N-acylurea derivative of hyaluronic acid. Although the composite includes material that prevents adhesion between tissues, in order to reduce the need for suturing when the composite is being used during a surgical procedure, a material that enhances adhesion of the composite to tissues may be applied to a surface of the composite. A method of forming the composite for reducing post-operative adhesion of tissues, including the step of applying an N-acylurea derivative of hyaluronic acid resulting from cross-linking with a multifunctional carbodiimide, to a biocompatible, biodegradable support; a method of preparing a drug delivery vehicle that includes a pharmaceutically-active molecule with the N-acylurea derivative of hyaluronic acid resulting from cross-linking with a multifunctional carbodiimide; and a method of reducing post-operative adhesion of tissues are disclosed.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the composite and method of the invention will now be more particularly described with reference to the accompanying FIGURE, and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following.

Provided below are definitions of some of the terms used in the description. A "water-insoluble" gel, film or sponge of the invention, as that phrase and like terms are used herein, is one which is heterogeneous when suspended in a sufficient amount of water at room temperature.

A "biocompatible" substance, as that term is used herein, is one that has no medically unacceptable toxic or injurious effects on biological function.

A "biodegradable" substance, as that term is used herein, is one that is capable of being decomposed by natural biological processes.

A "nucleophile," as that term is used herein, is any molecule possessing an electron rich functional group (such as a primary amine).

A "polyanionic polysaccharide," as that term is used herein, is a polysaccharide other than HA containing more than one negatively charged group, e.g., a carboxyl group.

A "cross-linking agent," as that phrase is used herein, is a molecule containing two or more functional groups that can react with HA or a derivative thereof.

A "film," as that term is used herein, means a substance formed by compressing a gel or by allowing or causing a gel to dehydrate. A gel of the invention may be formed into such a film.

A "sponge," as that term is used herein, means a substance formed by freeze-drying a gel. A gel of the invention may be formed into such a sponge.

"Room temperature," as that phrase is used herein, includes temperatures in the range of from about 20^oC. to about 25^oC.

As used herein, the term "HA" means hyaluronic acid and any of its hyaluronate salts, including, but not limited to, sodium hyaluronate (the sodium salt), potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.

"Derivatized hyaluronic acid," as the term is used herein, means hyaluronic acid that has been derivatized with a carbodiimide, such as a monocarbodiimide or a multifunctional carbodiimide, or that has been derivatized with a mixture of a multifunctional carbodiimide and a monocarbodiimide. Preferably the derivatization is carried out in the absence of a polyanionic polysaccharide other than the hyaluronic acid. In a specific embodiment, the derivatized hyaluronic acid is water-insoluble.

An embodiment of a composite of the invention includes at least two components: a biocompatible, biodegradable support and a derivative of HA that includes an N-acylurea resulting from cross-linking with a multifunctional carbodiimide, such as a biscarbodiimide.

Examples of the physical form of a suitable support include: a biocompatible, biodegradable matrix, sponge, film, mesh, and a composite of particles which may be in the form of beads. The beads may be bound together by a bioabsorbable material. The biodegradable support may be formed from a material which is porous, and the pore sizes may be large enough so that when a layer of the hyaluronic acid (HA) derivative is spread on the support, the molecules of the HA derivative can partially or fully penetrate into the pores of the support to make an anchor. Examples of compositions to be used as a suitable support include: cross-linked alginates, gelatin, collagen, cross-linked collagen, collagen derivatives, such as, succinylated collagen or methylated collagen, cross-linked hyaluronic acid, chitosan, chitosan derivatives, such as, methylpyrrolidone-chitosan, cellulose and cellulose derivatives such as cellulose acetate or carboxymethyl cellulose, dextran derivatives such carboxymethyl dextran, starch and derivatives of starch such as hydroxyethyl starch, other glycosaminoglycans and their derivatives, other polyanionic polysaccharides or their derivatives, polylactic acid (PLA), polyglycolic acid (PGA), a copolymer of a polylactic acid and a polyglycolic acid (PLGA), lactides, glycolides, and other polyesters, polyoxanones and polyoxalates, copolymer of poly(bis(p-carboxyphenoxy)propane)anhydride (PCPP) and sebacic acid, poly(1-glutamic acid), poly(d-glutamic acid), polyacrylic acid, poly(d1-glutamic acid), poly(1-aspartic acid), poly(d-aspartic acid), poly(d1-aspartic acid), polyethylene glycol, copolymers of the above listed polyamino acids with polyethylene glycol, polypeptides, such as, collagen-like, silk-like, and silk-elastin-like proteins, polycaprolactone, poly(alkylene succinates), poly(hydroxy butyrate) (PHB), poly(butylene diglycolate), nylon-2/nylon-6-copolyamides, polydihydropyrans, polyphosphazenes, poly(ortho ester), poly(cyano acrylates), polyvinylpyrrolidone, polyvinylalcohol, poly casein, keratin, myosin, and fibrin. A sample of highly cross-linked HA may form a support for a sample of modified HA which is not highly cross-linked.

In general, the modified HA derivative is prepared by reacting hyaluronic acid, or a salt thereof, with a multifunctional carbodiimide, preferably a biscarbodiimide, in the absence of a nucleophile or a polyanionic polysaccharide other than HA, to form an N-acylurea resulting from cross-linking with the multifunctional carbodiimide. Additionally, a monocarbodiimide may be employed in combination with a multifunctional carbodiimide, the monocarbodiimide having the formula:

R¹ --N=C=N--R²

wherein R¹ and R² may include hydrocarbyl, substituted-hydrocarbyl, alkoxy, aryloxy, alkaryloxy. Examples of suitable monocarbodiimides include: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC); 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC); 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide methiodide (EAC); 1,3-dicyclohexylcarbodiimide (DCC); and 1-benzyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (BDC).

Use of a polycarbodiimide to prepare the modified HA derivative causes cross-linking of the hyaluronic acid. For example, use of a biscarbodiimide reactant to prepare the water insoluble gel of the invention results in a cross-linking between COOH groups present on the repeating disaccharide unit of hyaluronic acid, since the biscarbodiimide is difunctional. The COOH group may be present in the same polymer chain, resulting in an intramolecular cross-linked product, or present on two different polymer chains, resulting in an intermolecular cross-linked product.

Examples of suitable biscarbodiimides may be represented by those difunctional compounds having the formula: R¹ --N=C=N--R² --N=C=N--R³, wherein R¹, R² and R³ may include hydrocarbyl, substituted-hydrocarbyl, alkoxy, aryloxy, alkaryloxy and the like.

The term "hydrocarbyl" as used herein means the monovalent moiety obtained upon removal of a hydrogen atom from a parent hydrocarbon. Representatives of hydrocarbyls are alkyls of 1 to 25 carbon atoms, inclusive, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonodecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl and the isomeric forms thereof; aryl of 6 to 25 carbon atoms, inclusive, such as phenyl, tolyl, xylyl, naphthyl, biphenylyl, triphenylyl, and the like; aryalkyl of 7 to 25 carbon atoms, inclusive, such as benzyl, phenylethyl, phenylpropyl, phenylbutyl, phenylhexyl, naphthylalkyl and the like; cycloalkyl of 3 to 8 carbon atoms, inclusive, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like; alkenyl of 2 of 25 carbon atoms, inclusive, such as vinyl, allyl, butenyl, pentenyl, hexenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, tricosenyl, tetracosenyl, pentacosenyl, and isomeric forms thereof. Preferably, hydrocarbyl has 6 to 14 carbon atoms, inclusive.

The term "substituted hydrocarbyl" as used herein means the hydrocarbyl moiety as previously defined wherein one or more hydrogen atoms have been replaced with a chemical group which does not adversely affect the desired preparation of the product derivative. Representative of such groups are amino-, phosphino-, quaternary nitrogen (ammonium), quarternary phosphorus (phosphonium), hydroxyl, amide, alkoxy, mercapto, nitro, alkyl, halo, sulfone, sulfoxide, phosphate, phosphite, carboxylate, carbamate groups and the like. Preferred groups are amino, amide, ester, and ammonium groups.

Preferred N-acylureas and O-acylisoureas of the invention are those in which R¹ and/or R² are hydrocarbyl substituted with an amino group. The term "alkoxy" as used herein means a monovalent group of the formula:

--O-alkyl

wherein the alkyl preferably has 4 to 12 carbon atoms, inclusive. The term "aryloxy" as used herein means the monovalent group of the formula:

--O-aryl

wherein the aryl preferably has 6 to 10 carbon atoms, inclusive and may be substituted as described above. The term "alkylaryloxy" as used herein means the monovalent group of formula:

--O-alkylenearyl

such as oxybenzyl and the like.

Preferred biscarbodiimides include p-phenylene bis(ethylcarbodiimide) and 1,6-hexamethylene bis(ethylcarbodiimide). Other examples of suitable biscarbodiimides include 1,8-octamethylene bis(ethylcarbodiimide); 1,10 decamethylene bis(ethylcarbodiimide); 1,12 dodecamethylene bis(ethylcarbodiimide); PEG-bis(propyl(ethylcarbodiimide)); 2,2'-dithioethyl bis(ethylcarbodiimide); 1,1'-dithio-p-phenylene bis(ethylcarbodiimide); and 1,1'-dithio-m-phenylene bis(ethylcarbodiimide).

The reaction of HA with a biscarbodiimide rather than a monocarbodiimide does not change the mechanism of reaction, but causes the product to be cross-linked.

Those skilled in the art will know, or will be able to ascertain with no more than routine experimentation, methods of preparing suitable biscarbodiimides. See, for example, the methods described in U.S. Pat. Nos. 6,013,679; 2,946,819; 3,231,610; 3,502,722; 3,644,456; 3,972,933; 4,014,935; 4,066,629; 4,085,140; 4,096,334; and 4,137,386, all of which are incorporated herein by reference in their entirety.

In one embodiment, the reaction is carried out under conditions such that the resulting modified hyaluronic acid product is at least about 1% cross-linked. The conditions necessary to achieve at least about 1% cross-linking, are first described in general, and further and specifically described in the Examples. In another embodiment, at least about 2% of the carboxyl functionalities of the modified hyaluronic acid are derivatized. Generally, at least about 25% of the derivatized functionalities are O-acylisoureas or N-acylureas. Examples of sets of conditions that achieve at least about 2% derivatization are specifically described in the Exemplification section.

The steps required to make a biocompatible HA derivative of this invention include providing a sample of HA or a salt thereof, such as sodium hyaluronate. HA from any of a variety of sources, including HA extracted from animal tissues or harvested as a product of bacterial fermentation, can be used as a starting material. Alternatively, the HA used to make the composites of this invention can be produced in commercial quantities by bioprocess technology, as described, for example, in Nimrod et al., PCT Publication No. WO 86/04355. The sample of HA or its salt is dissolved in water to make an aqueous solution. Preferably, the concentration of HA in this first aqueous solution is in the range of between about 0.1% and 5% weight/weight ("w/w"), that is, 1 mg/ml solution to 50 mg/ml solution. More preferably, the reactions are carried out with a range of about between about 0.4% and 0.6%, or 4 to 6 mg of hyaluronic acid per milliliter. The precise concentration used will vary depending on the molecular weight of the HA. At significantly lower concentrations, the reactions are slower and less effective. At significantly higher HA concentrations, the end product may be difficult to handle due to the increase in viscosity. One skilled in the art will be able to determine, with no more than routine experimentation, an acceptable concentration of HA to be used for a particular embodiment. Examples of acceptable concentrations of HA are described in U.S. Pat. No. 5,356,883, to Kuo et al., the teachings of which are incorporated herein by reference in their entirety.

The pH of the HA solution is then adjusted by the addition of a suitable acid, so that the aqueous HA solution is acidic, preferably having a pH of about between 4.0 and 6.0, more preferably between about pH 4.75 and about pH 5.5.

Once the pH of the aqueous HA solution has been adjusted, the carbodiimide can be added. Generally an excess of the stoichometric proportion of carbodiimide is advantageous to promote the desired reaction. Preferably the molar equivalent ratio of the carbodiimide to HA is equal to or greater than about 5%.

Preferably, the carbodiimide is dissolved in an appropriate water-mixable solvent and added drop-wise. As the carbodiimide and the HA are mixed, the pH of the solution generally will increase. Films and gels with various desired physical properties can be obtained by simply allowing the pH to rise as the reaction proceeds. However, the reaction is monitored by a pH meter, and HCl may be added to maintain the pH of the reaction mixture between about 4.75 and about 5.50. The reaction is allowed to proceed at room temperature for about two hours.

The details of the reaction are described in U.S. Pat. Nos. 6,013,679, and 5,356,883, the contents of which are incorporated herein by reference in their entireties.

The reaction conditions for HA cross-linking with a biscarbodiimide are similar to those used for HA-monocarbodiimide coupling reactions. Advantageously, the cross-linking reactions are carried out with (1) an increase of the HA concentration in the reaction mixture, and (2) a decrease of the biscarbodiimide concentration in the addition solution. This creates a condition favorable to intermolecular cross-linking versus intramolecular cross-linking.

The reactions described above may be directed to favor the formation of the N-acylurea derivatives by increasing the pH with aqueous base.

The progress of the reaction described above may be followed by monitoring the pH. When the pH is stabilized, the reaction is substantially complete. At the conclusion of the reaction, the desired hyaluronic acid derivative may be separated from the reaction mixture by conventional methods of precipitation, washing and re-precipitation. The completeness of the reaction, the nature of the products and the extent of chemical modification can be determined by proton NMR.

If a colored product is desired, a solution of a biocompatible dye or stain, e.g., Coomassie.TM. Brilliant Blue R-250, can be admixed to the reaction mixture. The resulting product will have a blue color which makes the gel, film or sponge easy to see when it is handled during surgery and when it is in place.

When the reaction is complete, sodium chloride is added to the reaction mixture to adjust the sodium chloride concentration to 1M. Ethanol equal to three volumes of the reaction mixture is added to form a white, stringy precipitate of chemically-modified, HA derivative. The precipitate is separated from the solution, washed, and dried by vacuum.

To make a water-insoluble gel of the HA derivative, the precipitate is re-suspended in water and stirred in a cold room. The gel of the HA derivative is a hydrogel. The term "hydrogel" is defined herein to mean a macromolecular network swollen in water or biological fluids. The degree of gelation is dependent on the degree of cross-linking achieved.

To make a sponge, the precipitate is then re-suspended in water, poured into a mold having a desired shape, and, preferably, dried, such as by air-drying, freeze-drying or heat-drying. A film may be prepared by further drying the gel. Alternatively, a film can be formed by compressing a gel under conditions that permit water to escape, as, for example, by compressing the gel between two surfaces, at least one of which is porous. See, for example, Malson et al., U.S. Pat. No. 4,772,419, the teachings of which are incorporated herein by reference in their entirety.

There are several ways in which the gel, film or sponge can be immobilized on the support to make the composite device of this invention. For example, a layer of derivatized, cross-linked hyaluronic acid may be applied, either by soaking or dipping or spraying or spreading or by any other method of application, to at least one surface of a support to form a composite. A suitable support may be a matrix, sponge, film, or particles such as beads, which may be porous. If the surface of the support is porous, the HA derivative will soak into the pores on the support surface. For example, porous beads may be soaked in the hyaluronic acid derivative for a sufficient period of time to allow the hyaluronic acid derivative to be absorbed and adsorbed by the pores of the beads. The composite is then dried under conditions that permit the escape of water from the composite.

The dried beads may be suspended in a solution of a solvent and a second biopolymer, for example, polylactic--polyglycolic acid--copolymer (PLGA). The suspension may then be poured into a mold, and the solvent allowed to evaporate. The composite thus formed will have the beads of derivatized HA and first biopolymer entrapped in a fine film of a second biopolymer.

Dried beads may be compressed to form a sheet. The product is thus a composite sponge or film in which derivatized HA has formed a uniform layer in contact with at least a portion of the surface of the support. If the surface of the support is porous, a portion of the layer of derivatized HA is embedded and anchored into the surface of the support.

In another embodiment, a composite sponge or film having hyaluronic acid derivative on both sides of the support is prepared by pouring the water-insoluble gel of derivatized HA prepared according to the procedure described above, into a first mold having the desired shape and depth, and spreading the gel in the first mold to form a first gel layer of even thickness. A suitable support may be a matrix, sponge, film, or particles such as beads made from another biocompatible material, for example collagen or gelatin. The support is spread on top of the evenly-spread first gel layer of derivatized HA. A second mold of the same size, shape and depth, is placed on the top of the support. Gel is poured into the second mold, and spread to form a second gel layer of even thickness in the second mold. In this manner, the polymer used as supporting matrix is sandwiched between the two layers of derivatized HA gel which are molded to the support. The composite is freeze-dried. The freeze-dried composite is compressed and cut into specimens of the desired shape and size.

To make a composite having derivatized HA gel on one side of the composite and a different material on another side, the second mold can be filled with a different kind of gel. The composite that is formed will have derivatized HA on one side and the different material on another side. This embodiment of the invention provides a composite that could have a coating on one side that enhances adherence of the composite to wet tissue, and a coating on another side that prevents adhesion between tissues. Materials that are suitable for enhancing adherence of the composite to tissue include fibrin, collagen, cross-linked collagen, and collagen derivatives, and any other polymers containing the peptide sequence, arginine (R), glycine (G), and aspartic acid (D). In surgical sites where suturing is not possible, and keeping the composite in place is difficult, this composite may stay in place just by adhering to the tissue. Suturing may not be required.

These composites provide a sustained source of derivatized HA at the surgical site as the composite biodegrades and is absorbed by the tissues. The rate of biodegradation, and thus the rate of release of derivatized HA can be controlled, in part, by the degree of cross-linking of HA, and the quantity of the cross-linked HA loaded on the support. The residence time of unmodified HA in the human body is generally less than a week. However, when HA is derivatized, the residence time is appreciably increased. In general, an increase in the degree of cross-linking results in an increase in the time of residence. By controlling the degree of cross-linking, a cross-linked HA of desired residence time can be synthesized. Furthermore, coating the derivatized HA on a porous, biodegradable support enables the derivatized HA to penetrate into the pores of the support. When the derivatized HA present on the surface of the support is degraded, there is still some derivatized HA present in the pores of the support. Following the bio-degradation of the derivatized HA present on the surface of the composite, the biodegradable matrix also degrades. During the degradation of the matrix, the derivatized HA trapped in the pores of the matrix is released and thus provides a sustained supply of derivatized HA. After the resorption and degradation of the composite, the healed tissues come into contact with each other and resume their normal function. The derivatized HA selected for a particular use may have a biodegradation rate which is faster than the biodegradation rate of the support. The support, in fact, can be itself made of a sample of cross-linked HA having a slower rate of biodegradation than that of the derivatized HA loaded on the support.

The rate at which the gel, film or sponge degrades and diffuses also depends on the insolubility, the density, and the degree of cross-linking of the modified HA in the composite. Just as gels, films and sponges which have a high degree of cross-linking are slow to degrade, modified HA which is more insoluble, or which has a higher degree of cross-linking, will degrade at a slower rate. Preferably, the density of modified HA in the film or sponge will be in the range of from about 0.1 mg/cm² to about 10 mg/cm². Those skilled in the art will know, or will be able to ascertain with no more than routine experimentation, the appropriate combination of insolubility, density and cross-linking that will yield a gel, film or sponge having the desired rate of degradation for a given situation.

The composite structure can be used as a surgical aid to separate healing tissues or to prevent post-operative adhesion. Introduced to the tissues, either during surgery or post-operatively, the gel, film, or sponge made of derivatized HA according to the procedure described above, gets re-hydrated and forms a swollen gel. This swollen composite, placed between or among the tissues, keeps the healing tissues separated from each other and maintains the said separation during the healing process. The support matrix helps the composite stay in place. Preferably, the composite structure should remain in place for a long enough period so that when it has entirely degraded and dispersed and the tissues do come into contact, the tissues will no longer have a tendency to adhere. The length of the post-operative period during which a contact-inhibiting effect between tissues should be maintained varies according to the type of surgery involved.

Examples of surgical procedures in which the biocompatible gels, films and sponges of this invention may be used include, but are not limited to, cardiosurgery, operations performed in abdominal regions where it is important to prevent adhesions of the intestine or the mesentery; operations performed in the urogenital regions where it is important to ward off adverse effects on the ureter and bladder, and on the functioning of the oviduct and uterus; and nerve surgery operations where it is important to minimize the development of granulation tissue. In surgery involving tendons, there is generally a tendency towards adhesion between the tendon and the surrounding sheath or other surrounding tissue during the immobilization period following the operation.

In ophthalmological surgery, a biodegradable implant could be applied in the angle of the anterior chamber of the eye for the purpose of preventing the development of synechiae between the cornea and the iris; this applies especially in cases of reconstructions after severe damaging events. Moreover, degradable or permanent implants are often desirable for preventing adhesion after glaucoma surgery and strabismus surgery.

In yet another embodiment, this invention is directed to drug delivery systems. Free functional groups in the acylurea side-arm (e.g., amines, amides, and esters) can be further reacted to bond with reactive therapeutic drug molecules, under conventional and known reaction conditions, to obtain vehicles for delivery of therapeutic drugs. For example, hydrophobic and/or cationic "side-arms" may be attached to the HA polymer, to prepare useful polymer carriers for therapeutic drugs. In carrying out the preparation of the derivatized HA of the invention, a sufficient proportion of the carbodiimide is reacted with the HA, or salt thereof, to obtain a polymer chain having recurring polymer chain units, interrupted by at least one disaccharide unit per HA molecule having a pendant acylurea side-arm. See, for example, U.S. Pat. No. 5,356,883, to Kuo et al., the teachings of which are incorporated herein by reference in their entirety. The biscarbodiimide cross-linked hyaluronic acid possesses new drug-binding regions which do not interfere with biocompatibility.

A controlled-release drug delivery vehicle can be formed from a pharmaceutically-active substance, such as a therapeutic drug, which covalently bonds to, or noncovalently interacts with, the modified HA polymer of the invention. The non-covalent interactions include ionic, hydrophobic, and hydrophilic interactions in which the drug is dispersed within the gel, film or sponge. As used herein, the term "dispersed" shall refer to ionic, hydrophobic, and hydrophilic interactions between the drug and the modified HA. For example, by selection of appropriate carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), a cationic moiety can be immobilized on HA polymer chains. This cationic site may serve as a noncovalent, ionic binding site for anionic substances such as non-steroidal anti-inflammatory drugs (e.g. naprosyn). In both cases, the modified HA functions as a vehicle which provides the controlled release of a drug from the system. In both cases, the drug delivery system is then injected or implanted at the locus where delivery is desired.

Any substance which has biological or pharmaceutical activity and which is normally considered to be a drug can be used as the drug component in the delivery systems of the invention. The choice of the substance will depend upon the specific use of the drug delivery system. Suitable pharmaceutically-active substances include growth factors, enzymes, therapeutic drugs, biopolymers, and biologically compatible synthetic polymers.

A "therapeutic drug," as that term is used herein, includes, for example: compounds and compositions recognized in the official United States Pharmacopoeia, the official Homeopathic Pharmacopoeia of the United States, or the official National Formulary, or any supplement of any of them; compounds and compositions intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals; and compounds and compositions (other than food) intended to affect the structure or any function of the body of man or other animals.

Examples of classes of therapeutic drugs include steroidal and non-steroidal anti-inflammatory drugs, hormones and any synthetic analogues and pharmaceutically-active fragments thereof.

Therapeutic drugs which are suitable for use in the delivery system of the invention may be fat soluble, water-soluble, anionic or cationic, as long as they can interact with a group on the carbodiimide residue to form either covalent or ionic bonds or hydrophobic or hydrophilic interactions, including those described below.

A hydrophobic interaction between the drug and the modified HA can occur when, by appropriate selection of the carbodiimide, the hydrophilic HA is controllably converted in character to include a hydrophobic entity which is receptive to further interaction with a therapeutic drug having a hydrophobic moiety. Suitable drugs include fatty acid derivatives, steroids (e.g., dexamethasone) and their analogs, and other drugs with hydrophobicity.

The delivery system of the invention is particularly well-suited for administering growth factors (e.g., interleukins, prostaglandins, thromboxanes, leukotrienes and cytokines), steroidal and non-steroidal contraceptive agents, antibiotics (e.g., penicillin, streptomycin and linocomycin), analgesics, sedatives, barbiturates, aminoalkylbenzenes, catecholamines, narcotics, narcotic antagonists, anti-neoplastic agents and anticoagulants (e.g., heparin and heparin sulfate).

The drug concentration can be varied over very broad limits and preferably should be chosen depending on the solubility of the drug, its pharmaceutical activity, and the effect desired.

Those skilled in the art will appreciate that the functional carboxylic acid group of unmodified HA is sheltered by the molecule conformation, making it slow to react, it at all. The modified HA of this invention, however, is an HA acylurea which possesses at least one "side-arm" or "spacer" projecting outwardly from the polymer chain. This outwardly projecting side-arm includes one or more reactive sites, depending on the carbodiimide employed. The reactive site(s) include, at the least, a primary or secondary amino, amide, imino or ammonium group. The availability of a free amino group provides a reaction site free of steric hindrance associated with the polymer chain. This reaction site may then be used to couple the water-insoluble compositions of the invention to a therapeutic drug. For example, a primary amine-functionalized HA can be used as a tether for drug coupling. A carboxylate-containing anti-inflammatory drug, such as Ibuprofen (2-methyl-4-(2-methyl-propyl)benzeneacetic acid), can be converted to the corresponding N-hydroxysuccinimide (NHS) active esters, which can react with the primary amine under physiological conditions.

Peptides can also be linked to the amine tether of an amine-functionalized HA. A thiol-cleavable cross-linker such as dithiobis(succinimidyl)-propionate (DSP) is first used to cross-link the amine tethers of modified HA. Then, the sulfhydryl groups produced through the reduction of the disulfide bonds can react with the e-amino group of lysine of the peptides through the heterobifunctional cross-linker N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP).

Alternatively, therapeutic drugs containing reactive functional groups (e.g., hydroxyl, carboxyl, amino) can be covalently bonded to the carbodiimide prior to reaction with HA to form hydrolyzable bonds. Then, the carbodiimides which contain drugs can be attached to HA via the acylurea linkage. For example, a carboxyl-containing therapeutic drug can react with a carbodiimide precursor, e.g., a thiourea, bearing a pendant amine or hydroxyl group to tether the drug through an enzymatically labile amide or ester linkage. The resulting thiourea may then be converted to the corresponding carbodiimide. Upon reaction of the carbodiimide with the hyaluronic acid, a drug delivery system is obtained without further reactions.

There are generally two chemical interactions in the drug delivery systems of the invention. The first is a very stable acylurea linkage between the modified HA molecule and the carbodiimide moiety. The second is a less stable linkage or interaction between the carbodiimide moiety and the therapeutic drug moiety. The less stable linkage is readily broken, releasing the therapeutic drug at the site of administration. The more stable chemical bond between the HA molecule and the carbodiimide moiety will ensure the release of the therapeutic drug without the release of the carbodiimide residue, which might affect the therapeutic action of the drug.

Delivery is also related to the degradation of the gel, film or sponge as a result numerous metabolic processes taking place in vivo. The degradation process is usually slower than diffusion, which provides the delivery of a drug via delivery systems in which the drug non-covalently interacts with the derivatized HA of the gel, film or sponge vehicle. By choosing the concentration of derivatized HA, one can control the rate of degradation or diffusion and, thus, the rate of drug delivery.

The modification of the HA by reaction with a carbodiimide does not adversely degrade the polymer. At a low degree of chemical modification, the properties of viscoelasticity may be retained to produce a soluble product. One skilled in the art will know, or will be able to ascertain with no more than routine experimentation, the degree of chemical modification necessary to yield an insoluble gel.

The drug delivery products of the invention can be administered to a mammal, including humans, in pharmaceutically-acceptable dosage forms, with or without the use of pharmaceutically-acceptable carriers. Dosage forms include, but are not limited to, intravenous, intra-articular, sub-cutaneous, oral and topical administration forms.

In mammals, the majority of free HA in the body is taken up in the lymphatic system, this is especially true for the higher molecular weight HA. The HA circulating in the human body has a medium molecular weight in the range of 1.4x10⁵ to 2.7x10⁵ and is taken up by liver endothelial cells. HA with a molecular weight less than 2.5x10⁴ is within the filtration limit of human kidneys and is excreted in urine. Accordingly, one advantageous use of the composite for the treatment of certain neoplastic disease is an intravenous administration of an acylurea-cytotoxin combination, releasing the therapeutic drug at the site of neoplastic incursions. Also, by the selection of HA having an appropriate molecular weight, the kidneys can be targeted for drug administration.

Claim 1 of 89 Claims

What is claimed is:

1. A composite for reducing post-operative adhesion of tissues, comprising:

a) a biocompatible, biodegradable support; and

b) a hyaluronic acid derivative at the support, said hyaluronic acid derivative comprising an N-acylurea that results from reaction of hyaluronic acid with a multifunctional carbodiimide.

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