CA2179105A1 - Antioxidant grafted polysaccharides and their uses - Google Patents

Abstract

Grafted polysaccharide compositions comprising polysaccharides grafted with antioxidants on at least one hydroxyl group of the polysaccharide. The use of antioxidant grafted polysaccharides or antioxidant grafted crosslinked polysaccharides as a treatment for arthritis, as a drug delivery vehicle, to reduce the incidence of post-operative adhesion formation, to promote the healing of chronic wounds and ulcers, and as a component of cosmetic formulations.

Description

This invention relate< to polysaccharides grafted with antioxidants, and preferably hyaluronic acid or crosslinked hyaluronic acid grafted with hindered phQnols.
The ~ynovial fluid found in ~mm-lian joint~ functions as a lubricant and ~hock ab~orber. The most important component of the ~ynovial fluid is sodium hyaluronate, which makes the greatest contribution to the mechanical propQrties of the fluid. Hyaluronic acid is a naturally occurring high molecular weight glycosaminoglycan having a repeating di~accharide unit of 2-amino-2-deoxy-3-0-(~-D-glucopyranosyluronic acid)-D-glucoso. The di~accharide~ are joined to form an unbranchQd, uncro~Qlinked poly~accharide chain by ~1-4 glucosidic bond~. In addition to its presence in ~ynovial fluids, hyaluronic acid occurs in cell coats, pericellular gels, the extracellular matrix substance of connective tissues of vertebrates, the vitreous humor of the eye, human umbilical cord tis~ue, rooster combs and in some bacteria.
During inflammation of a joint caused, for example, by osteo- or rheumatoid arthritis both the molecular weight of hyaluronic acid and its concentration are reduced. Thiq lowering of molecular weight decreases the ability of ~ynovial fluid to act as a shock absorber, and thuQ the fluid does not provide adequate protection for the cartilage of the joint. Moreover, the lowering of molecular weight al~o reduces viscosity and thus promotes leakage from the joint. In the case of advanced arthritis the cartilage i5 eroded away, leading to pain when the joint is in motion (see for example, "The Merck Manual of Diagnosis and Therapy-16th Edition", p 1338-42).
One of the cause~ for the lowering of molecular weight and for the high rate of 108~ of sodium hyaluronate from the synovial cavity, is the degradation of the molecule by hydroxyl radicals. Hydroxyl radicals come from two sources.
The primary source i~ white blood cells which enter the joints when they are inflamed, and release xanthine peroxidase and other enzymes to form superoxide anion, hydrogen peroxide and hypochlorite, which upon breakdown form hydroxyl radical. Another source for hydroxyl radical i~ the reduction of oxygen by reducing agents in the presence of iron. A common reducing agent in the body is a~corbic acid. Oxygen i reducQd by iron(II) to form ~uperoxide anion, which then rQacts with iron ~III) to form hydrogen peroxide. Hydrogen peroxide is reduced to hydroxyl radical.
The u~e of sodium hyaluronate of relatively moderate molecular weight a~ a supplQmQntal ~ynovial fluid in the leg joint of race horse~ has been reported (Balazs et al., J.
Equine Vet. Sci., p. 217-228, 1985). However, synovial fluid in the joints of humans contains substantially higher molecular weight sodium hyaluronate than that of horse~.
Solutions of ~odium hyaluronate also have been tested as ~upplemental synovial fluid for human osteoarthritic jointJ by injection into the joint~. Treatment of arthritis by injection of sodium hyaluronate has been disclosed by Weiss et al., Semin. Arthritis Rheum., 11, p. 143, (1981);
Na~i~i et al., J. Clin. Pharmcol. Therapy Toxicology, 20, p.
501, (1982); Grecomoro et al., Pharmatherapeutica, 5, p.
137, (1987) and Briganiti Qt al., Clinical Trials Journal, 24, p. 333, ~1987). However, it ha~ been reported that intra-articular injQctions of sodium hyaluronate ~olutions do not perform measurably differently from placebos ~Dahlberg et al., in "Arthriti~ 6 Rheumatism" 37, p.521, 1994). Brown et al. in Exp. Physiol. 76, p.l25, (1991), reported that tho half-life of hyaluronic acid injected in a joint i~ only about 13 hour~ h~rg, vide supra, has di~closed that a 13 hour half-life i~ ~hort for therapeutic value. It i~ believed that the short half-life of injected hyaluronic acid is due in part to degradation by hydroxyl radicals ~J.M. McCord, Science, 185, p.529, 1974).
Thi~ invention relates to novel ways of increasing the re~istance of poly~accharide~, in particular hyaluronic acid and sodium hyaluronate, to hydroxyl radical~ by grafting them with antioxidants.
Thi~ invention pertains to grafted polysaccharide compositions comprising polysaccharide grafted with antioxidant on at least one hydroxyl group of the polysaccharide.
In a preferred e~hoAi~ent the polysaccharide comprises polysaccharide containing acidic group~. In the most preferrod embodiment, the polysaccharide comprise~
hyaluronic acid or a ~alt of hyaluronic aeid; the antioxidant comprise~ hindered phenol; and the grafted co~rosition ha~ substantially greater resistance to hydroxyl radical~ than doe~ un-grafted hyaluronic acid or its sodium salt.
In another embo~i~ent the invention pertains to a method for grafting a polysaccharide which comprises reacting the polysaccharide with a hydroxyl-reactive derivative of an antioxidant.

In another ~ho~iment the invention also pertains to pharmaceutical compositions for treating inflammation of m~m~lian joints; e.g., arthritis, for preventing post-operative adhesion fonmation and for promoting the healing of chronic wounds, comprising as the active component the antioxidant grafted polysaccharide of thi~ invention.
In a further ~mbo~imsnt the invention pertains to drug delivery sy9tems comprising antioxidant grafted polysaccharide which ha~ been crosslinked.
The invention also pertains to cosmetic compositions comprising the grafted polysaccharide of this invention.
In yet another embodiment the invention pertains to method~ for treating inflammation of mammalian joint~, e.g.
arthritis, reducing the incidence of post-operative adhe~ion formation and promoting the healing of chronic wounds and ulcers comprising injecting or applying an effective amount of a phanmaceutical composition comprising a~ the active component antioxidant grafted polysaccharide of this invention.
The grafted polysaccharide compositions of this invention comprise poly~accharide grafted with antioxidant on at lea~t on~ hydroxyl group of the polysaccharide.
The compo~itions of thi~ invention compri~e material having a formula ~elected from the group consisting of:

s ~(o~

~ a~R ~R ~

,~
R~--~H ~(O~

wherein R represents the backbone of poly~accharide or cro~linked polysaccharide containing acidic groups or salts thereof, -(0)- is the residue of a polysaccharide hydroxyl group, ~ is hydrogen, Cl-C20 alkyl, phenyl or substituted phenyl, R2is Cl-C20 alkyl, phenyl or substituted phenyl, and Ar is aryl or sub~tituted aryl.
Preferably the compo ition~ of thi~ invention comprise material having a fonmula ~elected from the group consisting of:

r II

~ O ~ ~--~R 10~

III IV

o~3 v wherein R represents the backbone of polysaccharide or cros~linked poly accharide containing acidic groups or salt~
thereof, -(O)- is the residue of a polysaccharide hydroxyl group; ~ is hydrogen, C~-C20 alkyl, phenyl or subqtituted phenyl; and ~ i~ Cl-C20 alkyl, phenyl or substituted phenyl.
In formulas I-V, preferably ~ i~ ~elected from the group con~isting of hydrogen, methyl, ethyl, i-propyl and t-butyl, and ~ i~ ~elected from the group consiating of methyl, ethyl, i-propyl and t-butyl.
More preferably the com.~po~ition~ comprise material of formulas I, II and rv wherein ~ and ~ are t-butyl, and wherein the polysaccharide comprises hyaluronic acid.
The polysaccharide~ for use in thi~ invention may include, for example, m^~bsrQ selected from the group con~isting of gum arabic, gum karaya, gum tragacanth, locust bean gum, guar gum, p~yllium gum, starch, pectin, agar, alginic acid, furcellaran, dextran, xanthan, carboxymethyl cellulosQ, methyl cellulose, hydroxyethylcarboxymethyl cellulo~e, carboxymethyl starch, cationic ~tarch, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan ~ulfate, heparan sulfate, heparin, polygalacturonic acid, polymannuronic acid, polyglucuronic acid and carrageenan.
Preferably the polysaccharides of this invention comprise polysaccharide containing acidic group~ or salts of th~ acidic groups. The preferred acidic groups comprise at least one me~h4r qelected from the group con~isting of carboxyl, ~ulfate, sulfite and pho~phate. The most preferred acidic group is the carboxyl group.
Poly~accharide~ containing acidic group may include, for example, m~mhsrs selQcted from the group consisting of hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, heparin, carboxymethyl cellulose, hydroxyethylcarboxymethyl cellulosQ, car~oxymethyl starch, pectin, xanthan, alginic acid, polygalacturonic acid, polymannuronic acid, polyglucuronic acid and carrageenan. The most preferred is hyaluronic acid .
The preferred salts in accordance with this invention comprisQ salt~ of an alkali or alkaline earth metal, aluminum or ammonium. The most preferred ~alt i~ a salt of ~odium.
The grafted polysaccharides of this invention may also be crosslinked. Crosslinked polysaccharides may be prepared by any methods di~closed in the art. Sakurai et al. in U.S.
Patent No. 4,716,224, disclo~e crosslinked hyaluronic acid or salts thereof prepared by crosslinking hyaluronic acid or its salts with a polyfunctional epoxide. In U.S. Patent No.
4,863,907 Sakurai et al. disclosQ cro~slinked ~5 glycosaminoglycan or salts thereof, prepared by crosslinking a glycosaminoglycan or a salt thereof with a polyfunctional epoxy compound. Huang et al., in European Patent Application No. 0 507 604 A2, disclose ionically crosslinked carboxyl-containing polysaccharides where the crosslinking agent i~ a compound possessing a trivalent cation. Malson et al., in U.S. Patent No. 4,716,154 disclose crosslinking hyaluronic acid with bi- or polyfunctional Qpoxides or their corre~ponding halohydrins, epihalohydrins or halides, and divinyl sulfone. Mal~on et al., in U.S. Patent No.
4,772,419 also disclose crosslinking hyaluronic acid with polyfunctional epoxides. In U.S. Patent No. 4,957,744 della Valle et al. disclosQ cross1inked QstQrs of hyaluronic acid preparQd by esterifying the carboxyl groups of hyaluronic acid with polyhydric alcoholQ. Balazs et al., in U.S.
Patent Nos. 4,582,865, 4,605,691 and 4,636,524, disclosQ
crosslinking of hyaluronic acid and its salts, and of other polysaccharides, by reaction with divinyl~ulfone. In U.S.
Patent Nos. 5,128,326 and 4,582,865, Balazs et al. disclose crosslinking hyaluronic acid with formaldehydQ, epoxidQs, polyaziridyl compounds and divinyl sulfonQ. In U.S. Patent No. 4,713,448 Balaz~ et al. di~clo-~e chemically modifying hyaluronic acid by reaction with aldQhydes such a~
formaldehyde, glutaraldehyde and glyoxal, and teach the pos~ibility that crosslinking has occurred. In U.S. Patent No. 5,356,883 Kuo et al. disclosQ crosslinking hyaluronic acid by reaction with biscarbodiimides.
A preferred method for crosslinking polysaccharides i8 by their reaction with carboxylic acid di- or poly-anhydrides. A preferrred cro-~linked composition comprises material of above formula I wherQ Rl and ~ are t-butyl, and the polysaccharide i~ hyaluronic acid or its ~odium salt crosslinkQd by reaction with pyromellitic dianhydride.
In the grafted compositions, the grafting level is most readily expressed in term~ of the number of equivalents of antioxidant that are present per equivalent of poly~accharide repeating unit. For the purpose of the invention the minimum level of grafting could be as low as about 1 equivalent of antioxidant per 1000 equivalents of poly~accharide repeating unit~. A preferred minimum level i~ about 1 equivalent per 700, and the most preferred minimum level about 1 equivalent per 600 polysaccharide repeating unit~.
The maximum level of grafting could be a~ high as about 1 equivalent per 10 equivalents of poly~accharide repeating units. A preferred ~ximllm level is about 1 equivalent per 100, and the most preferred m~Ximum level about 1 equivalent per 400 polysaccharide repeating units.
It ha~ been found that when the antioxidant grafted composition in accordance with thi~ invention is derived from hyaluronic acid or it~ ~alt~, it ha~ ~ubstantially greater resistance to degradation caused by hydroxyl radical than does un-grafted hyaluronic acid or it~ salt~.
In test-~ for degradation by hydroxyl radical, the hydroxyl radicalA wero generated by reaction of ferric chloride with a~corbic acid a~ de~cribed by Wong et al.
Inorganic ~iochemistry, 14, p. 127 (1981). In a typical test the viscosity half-life was 0.9 hours for hyaluronic acid. When the ~ame test was carried out under the same conditions on grafted compositions, both cro-~linked and non-crosslinked, of this invention having formula I wherein R, tho polysaccharide, wa~ hyaluronic acid, and R1 and R2 wore t-butyl, the viscosity half lives ranged from 3 to 48 hour~, indicating the sub~tantially greater resi~tance of the grafted compositions to degradation by hydroxyl radical.
The antioxidant grafted compositions are prepared by reaction of polysaccharide~ with hydroxyl-reactive antioxidant derivative~. The term "hydroxyl-reactive antioxidant derivative~ is intended to mean an antioxidant containing a functional group capable of reacting with hydroxyl groups contained in a polysaccharide.
The preferred hydroxyl-reactive antioxidants for preparing the compositions of this invention are selected 5from the group consisting of:

10B-F~F~ B~OP(Of~ tl 11~ l lNP~
VI ' VII ' VIII

B ~ J~r Nll J~r X ~ l~r Nll XII

wherein ~ is hydrogen, Cl-C20 alkyl, phenyl or substituted phQnyl; R2 is C~-C20 alkyl, phenyl or substituted phenyl; A
is -C(O)X, -C(O) ~2C~20CH2CH2Y, -C~O)NHCH2CH2CH2Y, -OCH2CH20CH2CH2Y or -CH2Yi B i~ -C(O)X, -CH2Y, or X; X is halogen, l-imidazole, phenoxy, nitrophenoxy, p-toluQnesulfonatQ, mQthanesulfonate or alkyl or aryl carboxylate; Y is halogen, p-toluenesulfonate and methanesulfonate.

The preferred hydroxyl-reactive antioxidants comprise hydroxyl-reactive hindered phenols of structure XII where R
is hydrogen, methyl, ethyl, i-propyl or t-butyl, and ~ is methyl, ethyl, i-propyl or t-butyl. The term "hindered phenol" refer~ to phenols having at lea~t one position ortho to the hydroxyl group occupied by phenyl, substitutQd phenyl or Cl-C20 alkyl substituent.
Preferred hydroxyl-reactive hindered phenols of structure XII comprise those wherein Rl and ~ are t-butyl, A is -C(O)X, -CH2Y or -C(O)OCH2CH20CH2CH2Y, where X iQ
chlorine or l-imidazole and Y is bromine. Specifically, preferred hydroxyl-reactive hindered phenols are 3,5-di-t-butyl-4-hydroxybenzoyl chloride, 2,6-di-t-butyl-4-bromomethylphenol, 3,5-di-t-butyl-4-hydroxybenzoyl 2-~2-(chloroethoxy)ethoxy]ethyl ester and 3,5-di-t-butyl-4-hydroxybenzoyl-l-imidazole.
Because hydroxyl-reactive antioxidant derivatives may readily react with water, it is preferred that their reaction with polysaccharide b~ carried out in a dry, polar, aprotic solvent. Preferred solvents are N-methyl pyrrolidinone, N-ethyl pyrrolidinone, N-cyclohexyl pyrrolidinone, 4-methyl morpholine N-oxide, dimethyl formamide, sulfolane and dimethyl sulfoxide.
Salts of acidic polysaccharides may not be soluble in the preferred solvents. In particular, the sodium salt of hyaluronic acid i~ not soluble in the preferred solvents, and so it is generally convenient for the purpose of this ~ me~t to convert the sodium salt to a tetraalkyl ammonium salt to increase solubility. After the grafting reaction of tetraalkyl ammonium salt and hydroxyl-reactive 21 7~1 05 antioxidant derivative, the product can be converted back to the sodium form by ion exchange.
If it is desired to prepare an antioxidant grafted crosslinked composition, the crosslinking reaction can bQ
carried out either before or after the grafting reaction.
It may al~o be carried out simultaneously with the grafting reaction in those casQs where the solvents used for crosslinking are the ~ame as those used for the grafting reaction.
In the grafting reaction, the ratio of hydroxyl-reactive antioxidant derivative to polysaccharide i~ most readily Qxpressed in terms of the moles of antioxidant derivative utilized per equivalent of repeating unit in the polysaccharidQ. For the purposQ of the invention, the minimum levQl of hydroxyl-reactive antioxidant derivative could be a~ low a-~ about 1 equivalent per 1000 equivalent~
of polysaccharide repeating units. A preferred minimum level is about 1 equivalent per 700, and the most preferred minimum level about 1 equivalent per 600 polysaccharide repeating unit~.
The m~Yimll~ level of hydroxyl-reactive antioxidant derivative could b~ a~ high a~ about 1 equivalent per 10 equivalentQ of polysaccharide repeating units. A preferred m~xjml-m level is about 1 equivalent per 100, and the most preferrQd m~xi~u~ level about 1 equivalent per 400 polysaccharide repeating units.
As noted above, in other emboAiment-~ the invention pertain~ to compositions for treating inflamed m~mm~l ian joint~, e.g., arthritis, for preventing post-operative adhesions and for promoting the healing of chronic wounds and ulcers. The active component of these compositions comprisQ~ the antioxidant grafted polysaccharides of this invention. The preferred antioxidant grafted polysaccharidQs are hindered-phenol grafted hyaluronic acid or hindered-phenol grafted cros~linked hyaluronic acid, or S pharmaceutically acceptable salts thereof.
Pharmaceutically acceptable salts include salts of an alkali or alkaline earth metal, aluminum or ammonium. The preferred phanmaceutically acceptable salt is sodium.
The antioxidant grafted polysaccharide compositions of thiq invention can be used to prevent po~t-operative adhesions in any animal that i~ susceptible to unwanted adhesion fonmation following surgery. The compositions are used to prevent adhesions from developing in ~-m~?l8, preferably human being~. They are useful in all types of surgery where it is desired to inhibit the fonmation of post-surgical adhesions, e.g., ~h~ominal surgery, gynecological surgQry, thoracic surgery, orthopedic surgery, neurological surgery and ophthalmological surgery. The preferred compositions for this use are hindered-phenol grafted hyaluronic acid or hindered-phenol grafted crosslinkQd hyaluronic acid, or pharmaceutically acceptable ~alt~ thereof.
Adhesion preventatives may be administered to the site of surgical trauma by any convenient mode such as, for example, by lavage, by coating directly on the site in a gel, cream, film or foam, or by any other convenient method.
The admini~tration of adhesion preventatives can occur at any time before significant wound healing has occurred. It i8 preferred to administer it at the conclusion of surgery, just prior to closing of the wound. However, in some cases it may be desirable to administer the preventative 217~105 continually over a period of time. An effQctive amount of adhesion preventative i~ an amount necessary to affeet a reduction in the incidence of post-operative surgical adhesion~. Preferably, the amount should bo enough to coat the entire area exposed to the surgical trauma, and if de~ired an additional amount sufficient to coat body tissue adjacent to the area of trauma.
The antioxidant grafted poly~aeeharide eompositions of this invention ean also be u~ed to promote the healing of ehronie wounds, e.g. burn~, and ulcers, e.g. diabetes foot ulcer~ in ~mm~ls, in partieular, human being~. The preferred composition~ for this use are hindered-phenol grafted hyaluronic acid or hindered-phenol grafted cro~slinked hyaluronic aeid, or phanmaeeutieally aeeeptable saltq thereof. Hyaluronie aeid retain~ moisturQ and also ha~ angiogene~is eharaeteristies that make it useful for this applieation. When utilized for wound healing the eompo~ition~ may be used alone in aqueouq solution, preferably physiologieal saline solution, or the solutions may be eombined with wound healing drug~ and other water soluble polymer~. They may be administered to the sitQ of the wound or uleer by any eonvenient mode sueh as, for example, by lavage, by eoating direetly on the site in a gel, eream, film or foam, by impregnation in a bandage or wound dres-~ing that is applied to wound or ulcer, or by any other convenient method. An effective amount to promote healing i~ enough to coat the entire area of the wound or ulcer and if desired an additional amount sufficient to coat body tissue adjacent to the wound or ulcer. A typical antioxidant grafted polysaccharide may contain as other ingredients water-soluble polymers, antibiotics, immunosuppressants and pain reducers.
In the application of grafted hyaluronic acid or crosslinked grafted hyaluronic acid of this invention to the treatment of inflamed joint-~, e.g., arthritis, in mammals, in particular human bQings, the hyaluronic acid derivative is usually dis~olved in phy~iological saline to a sufficient viscosity to pas~ through an injection needle. The maxi mllm viscosity is about 50,000 cps, preferably about 30,000 cps.
The minimum viscosity is about 5,000 cps. The treatment solution is then injected into the diseased joint.
A typical knee joint synovial fluid supplementation injection procedure is similar to one described by Miller et al. in J. Bone and Joint Surgery, 40, p.636 ~1985). In the procedure a sterile solution, 2.5 ml, of the sodium salt of antioxidant grafted hyaluronic acid (concentration of grafted hyaluronic acid: 10 mg/ml) in buffered saline (sodium chloride 8.5 mg/ml, dibasic sodium phosphate 0.537 mg/ml, sodium dihydrogen phosphate 0.016 mg/ml) is slowly drawn into a syringe to ensure the absence of air pockets.
The knee is then prepared for injection by cleaning with soap, wiping with cetyl trimethylammonium bromide and painting with tincture of iodine. The solution is injected into a synovium cavity through a premarked triangular arc at the lateral side of the joint bound by the tibial plateau, the edge of the ligamentum patellae, and the curve of the lateral femoral condyle. Local anae~thesia may be used prior to injection. In certain cases knee aspiration with the buffered saline solution may be needed prior to the synovial fluid supplementation injection. Such a procedure is de~cribed by Dahlberg et al. in Arthritis & Rheumatism, 37, 1994, page 521.
The injectable Qolution may contain materials in addition to the grafted composition. These include water soluble polymers such as chondroitin sulfate, dermatan sulfate, and/or a phospholipid to improve the lubricity of the solution. AnesthQtics, anti-inflammatory rQagents, antibiotics, antibacterial~, cytotoxin~ and sugars may be added also.
Antioxidant grafted polysaccharides of this invention particularly in the crosslinked form, may be used as a drug delivery system. The preferred compositions for this use are hindered-phenol grafted hyaluronic acid or hindered-phenol grafted erosQlinked hyaluronic aeid, or phanmaceutieally acceptable ~alts thereof. Crosslinked hyaluronic aeid forms a molecular eage in whieh moleeules with pharmacological aetivity can be dispersed. The substances contained in the cage are delivered into the environment by diffusion. The drug molecule, or mixture of drug moleeules, may be covalently or non-covalently bonded to the hyaluronie aeid. The eovalent bonding can be via attachment to the earboxylic aeid or hydroxyl groups of the hyaluronie aeid moieties. The gel~, films, threads, partielQs or sponges of hyaluronie aeid-based eomposition may be plaeed, sprayed, ingested, injeeted or implanted at the loeation where the contained pharmaeologieal substance i~ needed. These sub~tancQs may be therapeutie drugs (sueh as anesthetics, analgesics, anti-inflammatories, diuretics, antagonists, antibiotics, hormones, antirheumatics, adrenergie agonist~, cytostaties, antihypertensives or immunosuppressant agents), growth factor~, enzymes or cellular anti-adhesion compounds.
Antioxidant grafted polysaccharides of thi~ invention can also function aJ components of cosmetic~ for topical S use~. The preferred composition~ for this U8Q are hindered-phenol grafted hyaluronic acid or hindered-phenol grafted crosalinked hyaluronic acid, or pharmaceutically acceptable salts thereof. Because hyaluronic acid has been shown to hold moisture under low relative humidity conditions and yield a pleasant and smooth feeling at high relative humiditie~, it has been used as a moisturizer in cosmetic formulations. The composition~ of this invention will provide ~imilar effects. Mixtures of the grafted hyaluronic acid composition~ with other low cost water-soluble polymer~
lS such as carboxymethyl cellulose, pectin, alginate, soy protein, casein and gelatin may also be employed.
Natural extracts of plant sources, ~uch as cactus aloe vera, mesquite, matricaria ch~momilla, tumeric, carrot, jojoba, rose and other~, may be blended into a co~metic fonmulation containing grafted hyaluronic acid. Alpha hydroxy acids ~uch as lactic and hydroxyethanoic may be added to the formulation to improve the pla~ticity of the ~kin.
A typical anti-aging cosmetic composition is:

2-hydroxyethanoic acid, 7%, propylene glycol, lS%, hindered-phenol grafted hyaluronic acid solution (1 g/100 ml), 1%, water, 60% and ethyl alcohol, 17%, where all percentages are by weight.
A formulation for facial soft gel is: aqueou~ slurry of carboxymethyl cellulose (3 g/100 ml), 2S%, aqueous solution of triethanolamine (10 g/100 ml), 11%, Methyl Gluceth-10, 5%, hindered-phenol grafted hyaluronic acid aqueous solution (1 g/100 ml), 1%, perfume and preservatives, 1%, water, 57%, where all percentage~ are be weight.
A typical essential skin moisturizer composition i8 hydroxyethyl celluloqe, 0.5%, Methyl Gluceth-10, 2%, glycerin, 2%, hindered-phenol grafted hyaluronic acid aqueous ~olution (1 g/100 ml), 1%, water, 94%, preservatives and perfume, 0.5%, where all percentage~ are by weight.
The invention is illustrated by the following examples, which are provided for the purpo~e of repreqentation, and are not to be construed a~ limiting the scope of the invention. All parts and percentageq in the examples are by weight unless otherwise qpecified.
Example 1 Synthesis of 3,5-di-t-butyl-4-hydroxybenzoyl chloride A mixture of 3,5-di-t-butyl-4-hydroxybenzoic acid (4 gram ), 20 ml of hexane and 20 ml of thionyl chloride was boiled for 4.5 hours. The solvent and exces~ thionyl chloride were removed by distillation, leaving a solid which was used without further purification. To confirm the identity of the acid chloride, a qmall ~ample wa~ quenched with excess anhydrous ethanol and triethylamine. The quenched sample a~ extracted with water and methylene chloride. After the solvent was evaporated, an oil wa~
obtained. The lH NMR spectrum (CDC13, ppm from TMS) confirmed that the oil was the corre_ponding eqter: 7.9 ppm (8, Ar, 2), 5.25 ppm (8, OH, 1), 4.3 ppm (q, CH3, 18) and 1.44 ppm (t, CH3, 3). Thi~ re_ult confinms that 3,5-di-t-butyl-4-hydroxybenzoyl chloride haq been formed.

217qlO5 Example 2 Synthesis of 2,6-di-t-butyl-4-bLo...o...ethyl phenol A mixture of 1 g of 2,6-di-t-butyl-4-methyl phenol, 0.9 g of N-bromosuccinimide an~ 40 ml of carbon tetraehloride was boiled for 1.5 hours. Suceinimide was removed by filtration, and the resulting organic ~olution was evaporated to give a viscous liquid with the following lH NMR spectrum (CDCl3, ppm from TMS): 7.05 ppm ~8, ArH, 2), 5,15 ppm (8, OH, 1), 4.35 ppm (~, CH2~r, 2) and 1.3 ppm (8, CH3, 18).

Example 3 Synthesis of 3,5-di-t-butyl-4-hydroxybenzoyl-2-[2-(2-chloroethoxy)ethoxy] ethyl ester In a 250 ml flask equipped with a Dean-Stark trap, a mixture of 5 g of 3,5-di-t-butyl-4-hydroxybenzoic acid, 3.7 g of 2-[2-(2-chloroethoxy)ethoxy] ethanol, 0.1 g of p-toluenesulfonic acid and 60 ml of toluene was heated at boiling for 4 days. The resulting mixture was extracted with water and methylene chloride, and then the methylene chloride ~olution was dried over MgSO~. Evaporation of the ~olvent gave 6.97 g of the ester with the following lH NMR
spectrum (CDCl3 ppm from TMS): 7.9 ppm (q, ArH, 2), 5.72 ppm (8, OH, 1), 5.45 ppm (m, CH2, 2), 3.86 ppm (m, CH2, 2), 3.7 ppm (m, CH2, 6), 3.6 ppm (m, CH2, 2) and 2.45 ppm (8, CH3, 18).

Example 4 Synthe i~ of 3,5-di-t-butyl-4-hydroxybenzoyl-1-imidazole A mixture of 1 g of 3,5-di-t-butyl-4-hydroxybenzoic acid, 0.7 g of 1,1'-dicarbonyl diimidazole and 50 ml of methylene chloride was stirrQd togQther at room temperature for 17 hour~. After the solvent was removed, the product wa~ recrystallized in toluene and hexane to yield a hygroscopic ~olid. A small sample of the solid was mixed with 0.2 g of ethanol and 2 ml of methylene chloride and allowed to stand for 10 hours. After the solvQnt was removed, only the corresponding ethyl ester was identified by lH NMR as in Example 1. Thi~ result indicated that 3,5-di-t-butyl-4-hydroxybenzoyl-1-imidazole had been fonmed.
Example 5 Preparation of the methyltricaprylammonium salt of hyaluronic acid To a solution of 10 g of sodium hyaluronate (fermentation product, Chi~so Corporation, Chiba, Japan) in 1000 ml of water wa~ added a solution of S0 g. of methyltricaprylammonium chloride (Aliquat 336, Aldrich Chemical, Milwaukee, Wiscon~in) in 50 ml of acetone. The mixture wa~ ~tirred overnight, and then the rubbery precipitate was filtered, washed with water and acetone, and then dried in vacuo overnight. It was again soaked in 500 ml of acetone for 7 hours and dried in vacuo overnight to yield 46.9 g of rubbery material.

Example 6 Grafting 3,5-di-t-butyl-4-hydroxybenzoyl chloride to hyaluronic acid To a solution of 10 g of the methyltricaprylammonium salt of hyaluronic acid (prepared by the method of Example 5) in 1000 ml of dry N-methylpyrrolidinone (NMP), 0.4 g of 3,5-di-t-butyl-4-hydroxybenzoyl chloride (Example 1) was added. The mixturQ was tumbled for 3 hour~ and then stored at 10C for 16 hours. For ion exchange back to the sodium ~alt, an aqueou~ ~olution of NaCl (15 g in 250 ml of water) was then added to the reaction mixture. After the resulting ~olution had been stirred for 1 hour, 3 g of ~odium bicarbonate and 200 ml of acetone were added to precipitate the product. The polymer was filtered and washQd five time~
with acetone/water (ratio: 4/1, 200 ml) and then with 100 ml of acetone. The product wa~ further purified by redissolving it in 1 1 of water and then precipitating it into a large volume of methanol. Four grams of product was obtained.

Example 7 Grafting 3,5-di-t-butyl-4-hydroxybenzoyl-1-imidazole to hyaluronic acid To a ~olution of 2 g of the methyltricaprylammonium ~alt of hyaluronic acid (prepared by the method of Example 5) in 280 ml of NMP wa~ added 0.25 g of 3,5-di-t-butyl-4-hydroxybenzoyl-l-imidazole (Example 4). The mixture waq maintained at 4SC for 20 hours, and then it was ion excha~ged back to the sodium salt fonm a~ de~cribed in Example 6. The product weighed 0.92 g.

Example 8 Grafting 3,5-di-t-butyl-4-hydroxybenzoyl chloride to hyaluronic acid cro~qlinked with th~ diglycidyl ether of bi~phenol A
The crosslinking reaction was carried out fir~t, by mixing 2 g of the methyltricaprylammonium salt of hyaluronic acid (prepared by the method of Example 5), 0.2 g of 217qlOS

diglycidyl ether of bisphenol A, and 280 ml of NMP, and maintaining the mixture at 45C for 24 hours. To this mixture wa-~ then added 0.2 g of 3,5-di-t-butyl-4-hydroxybenzoyl chloride, and then the entire reaction mixture wa~ maintained at room temperature for 17 hours.
The product wa~ converted to the ~odium ~alt and worked up aJ described for Example 6.

Example 9 Grafting 3,5-di-t-butyl-4-hydroxybenzoyl chloride to hyaluronic acid crosslinked with 1,2,4,5-benzenetetracarboxylic acid dianhydride To a solution of 5 g of the methyltricaprylammonium ~alt of hyaluronic acid ~prepared by the method of Example 5) in 700 ml of NMP, there was added 0.5 g of 1,2,4,5-benzenetetracarboxylic acid dianhydride. The mixture was maintained at 10C for 7 days. Triethyl amine ~1 ml) and 3,5-di-t-butyl-4-hydroxybenzoyl chloride (0.5 g) were then added to the reaction mixture. After 16 hour~, the product wa~ ion sYch~nged with a ~olution of NaCl (0.5 g) and NaHC03 (O.S g) in S0 ml of water, and then precipitated by addition of 400 ml of acetone. The product was filtered and washed with 4/1 acetone/watQr and then pure acetone. After drying 2.9 g of polymer was obtained.
.25 Example 10 Grafting of 3,5-di-t-butyl-4-hydroxybenzoyl-2-[2-(2-chloroethoxy)ethoxy] ethyl ester to hyaluronic acid A mixture of 2 g of methyltricaprylammonium salt of hyaluronic acid (prepared by the method of Example 5) and 0.25 g of 3,S-di-t-butyl-4-hydroxybenzoyl-2-[2-(2-chloroethoxy)ethoxy] ethyl e~ter in 280 ml of MMP wa~
maintained at 45C for 20 hours. The mixture was treated with a solution of 0.1 g NaCl and 0.1 g of NaHC03 in 20 ml of water and worked up as in Example 9. After drying 1.0 g of polymer wa~ obtained.

Example 11 Resistance of grafted hyaluronic acid to degradation by hydroxyl radical~
Hydroxyl radicals were generated by the reaction of ferrie ion with aseorbie aeid a~ deseribed by Wong et al.
Inorganie Biochemistry, 14, p. 127 (1981).
To a 100 ml aqueou~ solution of sodium ~alt of hindered-phenol grafted hyaluronate 0.3 to 0.6% wt/vol as indicated, buffered at pH 7.4) 0.25 ml of a ferric ehloride solution ~1.1 g in 20 ml of water) and 0.25 ml of an aseorbie aeid ~olution (0.64 g in 20 ml of water) were added. The mixture wa~ shaken for about 10 seeonds, and then the first vi~eosity ~Brookfield) wa~ measured after 10 minutes. The vi~eosity at this point was eonsidered to be 100%. The degradation reqi~tanee of the sample wa~ assessed by mea~uring the time required to reduee the viseosity to 50% of the 10 minute value (viseo~ity half-life). The eontrol eon~i~ted of sodium hyaluronate. The result~ are in Table 1.

Tabl e F-rric Chlorldo/~cor~ic Acld T -t of Hlndbr~d-Ph-nol Or~ft-d ~y-luron c Acid ~mpl- Vi~co~ity Conc ntr~t~on, ~lf-Product of FY~mpl- ~ Rt /Vol Li--, hr~
Control 0 4 0 9 6 0 ~ 3 8 0 3~ 5 9 0 6 ~8

Claims (21)

1. A grafted polysaccharide composition comprising polysaccharide grafted with antioxidant on at least one hydroxyl group of the polysaccharide.

2. The composition of claim 1 wherein the antioxidant comprises a hindered phenol.

3. The composition of claims 1 or 2 wherein the polysaccharide containing acidic groups or salts thereof.

4. The composition of claim 3 wherein the acidic groups comprise at least one member selected from the group consisting of carboxyl, sulfate, sulfite and phosphate .

5. The composition of any of claims 3 or 4 wherein the salts comprise salts of an alkali or alkaline earth metal, aluminum or ammonium.

6. The composition of any of the preceding claims wherein grafted antioxidant is present at a level of from 1 equivalent per 1000 repeating units of polysaccharide to 1 equivalent per 10 repeating units of polysaccharide.

7. The composition of any of the preceding claims wherein grafted antioxidant is present at a level of from 1 equivalent per 700 repeating units of polysaccharide to 1 equivalent per 100 repeating units of polysaccharide.

8. The composition of any of the preceding claims wherein grafted antioxidant is present at a level of from 1 equivalent per 600 repeating units of polysaccharide to 1 equivalent per 400 repeating units of polysaccharide.

9. The composition of any of the preceding claims wherein the srafted polysaccharide comprises at least one member selected from the group consisting of:

, , , , , , , , , , , , and wherein R repreqents the backbone of polysaccharide or crosslinked polysaccharide containing acidic groups or salts thereof, -(O)- is the residue of a polysaccharide hydroxyl group, R1 is hydrogen, C1-C20 alkyl, phenyl or substituted phenyl, R is C1-C20 alkyl, phenyl or substituted phenyl, and Ar is aryl or substituted aryl.

10. The composition of any of the preceding claims wherein the grafted polysaccharide composition is crosslinked.

11. The composition of any of the preceding claims wherein the grafted polysaccharide composition is crosslinked by reaction with carboxylic acid di- or poly-anhydride.

12. The composition of any of the preceding claims wherein the polysaccharide comprises at least one member selected from the group consisting of hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, heparin, carboxymethyl cellulose, hydroxyethylcarboxymethyl cellulose, carboxymethyl starch, pectin, xanthan, algnic acid, polygalacturonic acid, polymannuronic acid, polyglucuronic acid and carrageenan.

13. The composition of any of the preceding claims wherein the grafted polysaccharide comprisec grafted hyaluronic acid.

14. The composition of claim 13 wherein the grafted hyaluronic acid has substantially qreater resistance to hydroxyl radicals than does un-grafted hyaluronic acid.

15. A method for treating mammalian arthritis comprising injecting into an affected joint an effective amount of a composition wherein the active component is the composition of claims 13 or 14.

16. A method for preparing polysaccharide grafted with antioxidant on at least one hydroxyl group of the polysaccharide, comprising reacting said polysaccharide with at least one hydroxyl-reactive antioxidant derivative selected from the group consisting of:

, , , VI VII VIII

, , and IX X XI

XII

wherein R1 is hydrogen, C1-C20 alkyl, phenyl or substituted phenyl; R2 is C1-C20 alkyl, phenyl or substituted phenyl; A
is -C(O)X, -C(O)OCH2CH2OCH2CH2Y, -C(O)NHCH2CH2CH2Y, -OCX2CH2OCH2CH2Y or -CH2Y; B is -C(O)X, -CH2Y, or X; X is halogen, 1-imidazole, phenoxy, nitrophenoxy, p-toluenesulfonate, methanesulfonate, alkyl carboxylate or aryl carboxylate; Y is halogen, p-toluenesulfonate or methanesulfonate.

17. The method of claim 16 wherein the polysaccharide contains acidic groups or salt thereof.

18. The method of claims 16 or 17 wherein the polycaccharide comprises at least one member seiected from the group consisting of hyaluronic acid, chondroitin qulfate, keratan sulfate, dermatan sulfate, heparan sulfate, heparin, carboxymethyl cellulose, hydroxyethylcarboxymethyl cellulose, carboxymethyl starch, pectin, xanthan, alginic acid, polygalacturonic acid, polymannuronic acid, polyglucuronic acid and carrageenan.

19. The method of any of claims 16, 17 or 18 wherein the grafted polysaccharide is crosslinked.

20. The method of any of claims 16 to 19 wherein the polysaccharide is hyaluronic acid or a salt of hyaluronic acid.

21. The method of any of claims 16 to 20 wherein the hydroxyl-reactive antioxidant derlvative is used at a level of from 1 equivalent per 1000 repeating units of polysaccharide to 1 equivalent per 10 repeating units of polysaccharide.