CA2081340A1 - Composition and method for topical treatment of damaged or diseased tissue - Google Patents

Abstract

COMPOSITION AND USE THEREOF FOR TOPICAL TREATMENT OF DAMAGED OR
DISEASED TISSUE
ABSTRACT
In accordance with the present invention, a composition and use thereof is provided for topically treating damaged or diseased tissue. The present invention comprises an aqueous gel or a pharmaceutically acceptable topical carrier containing an effective amount of an acid mucopolysaccharide. The composition containing the aqueous gel is liquid at room temperature and a gel at body temperature, thereby providing a composition that can be easily applied to the damaged or diseased tissue. After topical application to the tissue, the composition according to the present invention provides a covering over the tissue that protects the tissue and accelerates healing.

Description

COMPOSITION AND USE THEREOF POR TOPICAL TREATMENT OF DAMA~ED OR
DISEASED TISSUE
Technical Field The present invention relates to a composition and method of use thereof for the topical treatment of damaged or diseased tissue. More particularly, the present invention is a composition and method for effectively treating damaged or diseased skin and tissue and promoting the healing of the damaged or diseased skin and tissue.
Background of the Invention As used herein, the term "topical treatment" means the treatment of the surface of the body of an animal or human or an internal surface of the human or animal that has been externalized either during surgery or by traumatic injury. The term "damaged tissue" means tissue damaged by surgery, ischemia, burns, toxins, trauma or other noxious insult. The term "diseased tissue" means tissue that is infected with a microorganism such as a yeast, bacteria or virus or a combination of organisms. The infection can be independent of or as a result of damage to the tissue. The term "burns" means tissue injuries caused by thermal, electrical, ionizing or solar radiation or chemical agents.
Tissue damage caused by either surgery, ischemia, burns, toxins, trauma or other noxious insults illicits similar physiological responses. Injury to the skin, resulting in destruction of the epidermis and on occasion dermal layers of the organ, exposes the underlying tissue to the external environment and causes blood loss, plasma loss and an increased risk of infection. Tissue damage caused by a variety of topical insults results in similar physiological responses and requires the same care and precautions.
Burns are tissue injuries caused by thermal, electrical, ionizing or solar radiation, or chemical agents. The common mechanism of burns is denaturation of protein, resulting in cell injury or death. The effect is in accord wo 91/16058 Pcr/ US9 1/02912

2 2081340 with the Iype, duration, and intensity of action of the particular agent causing~he burn. Because such agents impinge on the organism from its environment, tissues in direct contact with this environment, e.g. skin, are damaged most quickly.
s In electrical burns, because most of the resistance to electric current is at the point of the contact of the skin with the conductor, these burns usnally involve skin and subjacent tdssues. These burns may be of almost any size and depth. Resultant slough is usually greater than the original regions would indicate.
Chemical burns may be due to strong acids and akalies, phenols, cresol, mustard gas, lewisite, phosphorus and the like. All produce areas of necrosis which may extend slowly for several hours. Radiation burns may result from ionizing (radioactdvity) or, more cornmonly, from solar radiation.
Bums of this type result in skin changes ranging from mild erythema to more IS pronounced swelling with blistering or ulceradon.
The first local evidence of a burn is dilation of capillaries and small vessels with increased capillary permeabiliy. The resultant plasma loss under the epidermis produces edema. Later, evidence of cellular injury can be seen histologically as swollen or pyknotic nuclei with coagulation of cytoplasm.In addition, collagen fibriles lose their distincmess.
In spontaneous healing, dead tissue is sloughed off as new cpithelium begins to cover the injured area. In superficial burns, regeneration occurs rapidly from uninjured cpidermal elements, hair, follicles, and sweat glands; little scarring results unless infection occurs. With deep burns (destruction of the epidermis and much of the derrnis), reepithelization starts from the edges of the wound or from the remains of scattered integumentary organ. This process is slow, and granulation tissue forms in excess amounts before being covered by the epithelium. Unlcss treated as soon as possible by skin grafting, such wounds gene}ally contract, and disfiguring or disabling scars result.
Systemic cffects of burns may quickly endanger the patient's life. Primary or neurogenic shock consists of sudden collapse from generalized vasodilation, presumably on a reflex basis from pain, fright, and anxiety; it israrely fatal. Secondary shock develops insidiously, or rapidly if the burn is extensive. Increased capillary permeabilily resulting from damage to vessel walls allows large amounts of fluid to exude into the wound area and from the . . .

WO 9 1 / 1 6058PC~ )S9 1 /029 1 2 ~U813~0 burn surface. This fluid consists of water, plasma, crystalloides, and about two-thirds of the plasma protein. The fluid loss is at the expense of the plasma.
When the epidermis is broken, evasion of bacteria may occur.
Dead tissue, warmth, and moisture provide ideal conditions for bacterial growth. The type of pathogen depends partly on the location of the burn;
nasalpharyngeal inhabitants such as streptococci and staphylococci predominate in upper body burns, while cold form bacteria and clostridia are often importantin lower body burns. What is needed is a method of covering the burn and protecting the bwned area from the environment.
Burns are usually classified into three types. In first degree buros, damage is limited to the outer layer of the epidermis with er~thema, increased warmth, tenderness? and pain. Edema, but not vesiculation, usuall~
occurs. In second degree burns, damage extends through the epiderrnis and involves the dermis, but not sufficiently to interfere with rapid regeneration of the epithelium. Vesicles, blebes, or bullae form. In third degree or full thickness burns, dcstruction of both the epidermis and dermis is seen.
Vesiculation is often absent and severe pain is unusual after the acute initial pain, because of the destruction of the nerve endings. The surface may be charred, coagulated, or white and lifeless (as from skulls), and sometimes is insensitive to pin prick. A burn may show varying degrees of damage in different areas. Frequently, it is difficult to distinguish between second and third degree burns until areas of third degree depth demarcate.
Conventional therapy for all burns includes relief of pain, strict asepsis, and carc of the wound, prevention or relief of shock, control of infection, correction of enema, and maintenanoe of nutrition. Local treatment ofchemical burns differs according to the causative agent. Following treatment, these wounds should be cared for similarly to thermal burns of comparable size and extent.
Conventional treatment for first and second degree burns involves clcaning the burned area gently with cold or warm water and hexachlorophene or green soap solution. The burned areas are then rinsed with sterile isotonic saline. All dirt, grease, and other contaminants on broken epidermis should be removed. Unbroken blebes are usually left intac~. For open or exposure treatment, the wounds are allowed to dry. For the closed type of treatment, sterile fine mesh absorbent gauze is applied. For burned limbs, a pressure dressing is oflen used consisting of additional layers of sterile .
~ i wo 91/16058 Pc-r/US91/02912 4 20813~0 absorbent gau~e covered by abdominal pads of mechanics' waste, all enclosed by f~ly applied absorbent gauze or by elastic bandages. If the burns are uncomplicated by signs of infecdon, the pressure dressings are left in place until the burn has healed. The burn will heal better if immobilized and elevated because these measures decrease the flow of Iymph and limit the spread of infection. ' For third degree burns, which are characterized by deep destruction of the skin so that'the reepithelization must take place largely or entirely from the edges of the wound, conventional treatment is identical with the previous outline. However, to avoid slow healing and disfiguring scars, burns of more than a few square centimeters require skin grafting. In small - areas of third degree depth, this can be done within a few days of the injury b~
cxcision of the burned area. This is followed by application of s'plit thicknessgrafts.
For burns involving greater than 15% of the body surface area, the fluid requirements of the patdent need to be irnmediately addressed. The surface wounds of patients with extensive third degree burns are usually treatedwith an 0.5% solution of silver nitrate or mafenide cream.
Some physicians, however, prefer open treatment. Its advantages are simplicity, better control of hyperpyrexia, and easier ambulation.
Special hospital ventilation is r~quired to prevent bacterial contamination. This therapy is less suitable for burns of the hand and foot than for circumferentialburns of the trunk. Open treatment does not rduce pain or loss of exudate. It probab1y should be regarded at present as a complimentary form of local therapy well suited to the treatment of burns of the face, neck, side of the trunk, or proximal part of the extremity. In general, after cleansing and debridement of the burned area, the patient is placed on surgically clean or sterile sheets in a room of proper temperature and humidity with a cradle over the bed. In 48 to 72 hours, the protein-rich exudate forms a crust over the burned areas. Fluid, antibody, electrolyte and colloid therapy, grafting, and other forms of treatment are conducted as in closed therapy.
The treatment of burns using polyoxyethylene-polyoxypropylene block copolymers is described in U.S. Paten~ No.
4,879,109. This patent describes use of the block copolymers either alone or in combination with othcr compounds, to increase the flow of biologic fluids to damaged tissue. To be effective, the composition must be introduced in~o the ~ , . .

circulatory system because the copolymer works by interfering with the pathologic hydrophobic interactions in blood and other biological fluids.
The treatment of burns using an aqueous gel with silver ions therein is described in U.S. Patent No. 3,689,575. This patent describes a polyoxyethylene-polyoxypropylene block copolymer which has the unique property of being liquid at low temperatures and forming a gel at higher temperatures. Silver ions are added to the gel and the composition is used to treat burns. The composition described in the '575 patent utilizes conventional silver ion treatment of burns by dissolving the silver ions in a copolymer matrix.
The preparation of an aqueous gel is described in U.S. Patent Nos. 3,867,533; 3,748,276 and 3,740,421, all of which may be referred to for further information. U.S. Patent No. 3,867,533 describes the addition of numerous water-insoluble pharmaceutically or cosmetically active organic ingredients. U.S. Patent No. 3,748,276 describes the addition of conventional antimicrobial agents to the gel. However, none of the aforementioned patents disclose the admixture of acid mucopolysaccharides with the copolymer for the purpose of preparing a therapeutically effective topical composition.
The treatment of soft tissue wounds using an aqueous mixture of fibrillar collagen, heparin and undegranulated platelets or platelet releasate is described in U.S. Patent No. 4,760,131. This patent indicates that collagen/glycosaminoglycan suspensions have only a minimal effect on the rate or extent of wound healing. For the composition to be effective in treating soft tissue wounds, a platelet fraction must be added.
In several articles by Saliba, et al, humans with burns were treated with a combination of intravenous, subcutaneous and topical application of large doses of heparin. Because the heparin was dissolved in an aqueou~ liquid, the liquid had to be applied constantly either by spraying through a needle onto the burn area or by dripping the solution onto the burn. Blisters had to be ruptured and the heparin injected into the blister by needle. In addition, the reports disclose that the topical treatment must be accompanied by the administration of large doses of intravenous heparin for several days. The effectiveness of the heparin therapy is limited in that the large doses required for therapeutic effect result in compromise of the blood coagulation system and an increased risk for hemorrhage. (See Saliba, M.J., ~Heparin Efficacy in Burns: II Human thermal burn treatment with large doses of topical and parenteral "~.

wo 91/16058 Pcr/US9l/02912 2~8134~

heparin" Clin. Aviat. and Aero. Med., 41 (I 1), pgs. 1302-1306 (1970); Saliba, M.l., e~ al., Large burns in humans, treatrnent with heparin," JAMA, 225(3), pgs. 261-269 (1973)) Articles by Rudolph and Tauschel disclose that heparin, in combinadon with allantoin and dexpanthenol, is effecdve in reducing the anti-inflammatory reactions of experimentally induced erythemas. The authors state that comparative testing of a heparin cream without allantoin and dexpanthenol had shown that heparin in cream does not differ fundamentally in effect from the cream base without heparin. (See Tauschel, H.D., et al., "Investigations of the Percutaneous Activity of a Combination of Heparin, Allantoin and Dexpanthenol in a Specific Ointment Base. Anti-lnflammatory Effect of UV
Erythema in the Guinea Pig," Anneim.-Forxch.lDrug Res. 32 (Il)(9), pgs.
1096-1100 (1982) and Rudolph, C., et al., "Investigations into the Percutaneous Acdvity of a Combinadon of Heparin, Allantoin, Dexpanthenol in a Specific Ointment Base. Antiallergic/Anti-lnflammatory Effect in the PCA
Test in Rats," Arzeim.-Forsch.lDrug Res. 34 (II)12), pgs. 176~8 (1984)) In a report by Raake, (Raake, W., "Comparison of the Anti-inflammatory Effect of Mucopolysaccharide Ointments with Ointments - Containing Heparin in the UV Erythema Tcst," Arzenim.-ForschlDrug P~es. 3~
(I)(4), pgs. 449-451 (1984)) the and-inflammatory effects of heparin are invesdgated. The Raake reference did not disclose any studies wherein heparin was used to treat wounds nor did the reference disclose the use of heparin with poloxamers.
What is needed is a treatmcnt for burns and other topical injuries that will cover thc damaged area and protcct the damaged area from the cnvironment. The treatment should inhibit inflammatory processes, provide relicf f~om pain and accelerate the healing process without compromise of endogcnous physiologic tnechanisms such as blood coaguladon. In addition, the treatment should be easily applied and should be effective over long periodsof time.

Summary of the In-~ention The present invendon provides a composidon and method for the topical treatment of damaged tissue. The composition of the presen~
invention comprises a solutdon of polyoxyethylene-polyoxypropylene bloc~;
copolymer and an effective concentration of an acid mucopolysaccharide.
..

wo 91~16058 PCr~ussl/02912 The polyoxyethylene-polyoxypropylene block copolymer has the following general formula:
HO(C2H40)b(c3H6o)a(c2H4o)b wherein a is an integer such that the hydrophobe represented by C3H6O has a molecular weight of at least 2250 daltons and b is an integer such - that the hydrophile portion represented by C2H4O constitutes from about 10 to 90 pèrcent by weight of the copolymer, with a preferable range of hydrophile portion of between 50% and 90%.
Aceording to the present invention, the copolymer is and an acid mucopolysacchaTide are dissolved in an aqueous solution. It is preferable that the acid mucopolysaccharide have little or no anticoagulant activity or to be ofsufficient molecular size or charge so as not to be absorbed in the ciruculation to avoid the toxicity exhibited by many acid mucopolysaccharides. The composition is then topically applied to damaged tissue. This allows is manifestation of the therapeutic effects of the acid mucopolysaccharide at concentrations producing minimal or no compromise to the blood coagulation system. Because the preferred copolymer is a liquid at room temperature or below, and is a gel at body temperature, the aqueous gel composition forrns a protective gel over the damaged tissue.
The solution which comprises the presen~ invention unexpectedly accelerates healing of the damaged or diseased tissue while at the same tirne keeping the darnaged tissue moist and proteeted from contamination by mieroorganisms. lt is believed that the copolymer forrns a matrix which slows the diffusion of mueopolysaccharide to the damaged or diseased tissue.
Thus, the gelled eopolymer with the acid mucopolysaceharide admixed therein provites a eontinuous and eonstant bathing of the area with the mueopolysaccharide over a long period of time. Beeause the copolymer gel is an aqueous gel, there is also the added advantage of the gel keeping the diseased or damaged dssue moist without the ineonvenience of having to bandage the dssue.
Other pharmaceutieally active agents ean be added to the eopolymer/aeid mucopolysaecharide admixture as deemed neeessary. For example, conventional antimicrobial agents could be added to the eopolymer/glycosaminoglycan admixture ~o protect the darnaged tissue from infection. Growth factors, such as human growth hormone, tissue derived growth factor, epidermal growth factor, platelet derived growth factor~

wo gl/16058 PcT/~;s9l/o2sl2 fibroblast growth factor, and/or nerve growth factor may be added to the copolymer/acid mucopolysaccharide admixture to promote the regrowlh of healthy tissue. This would be particularly important in treating burns.
The composiion of the present invention further comprises a S pharmaceutically acceptable topical carrier admixed with an acid mucopolysaccharide. The pharmaceutically acceptable topical carrier may be any sui~able, commercially available ointment, cerate or salve. Other pharmaceutically active agents can be added to the carrier. The composition is adrninistered topicaDy to damaged or diseased tissue.
Accordingly, it is an object of the presem invenion to provide a composition and method that is effective in topically treating dama~ed or diseased tissue.
It is another object of the present invention to provide a composition and method that is effective in treating burns.
lS It is another ob~ect of the present invention to provide a composiion and method that is effecive in treaing dssue damaged by traurna.
It is yet another object of the present invention to provide a composition and method that accelerates the healing process.
It is another object of the present invention to provide a eomposition and method that forms an aqueous gel over the damaged or diseased tissue thereby keeping the damaged or diseased issue moist.
It is another object of the present invention to provide a eomposition and method that eontains an anesthetie and will inhibit pain when applied to the damaged or diseased tissue.
It is another object of the present invention to provide a composidon and method that will eover damaged or diseased tissue with a protcctivc laycr that is transparcnt thereby allowing observaion of the damaged arca during the hcaling proccss.
These and other objects, features and advantages of the present . 30 invention will beeome apparent after a review of the following detailed deseripdon of the diselosed embodiments and the appended daims.
Detailed Description of the Disclosed Embodiments In aeeordance with the present invenion, an aqueous gel composition is provided that is effective in topically treating darnaged or diseased tissue. The aqueous gel eomposition of the present invention comprises an adrnixture of an aeid mueopolysaecharide and a polyoxyethylene- -.~ ~

wo 91/16058 Pcr/us91/02912 20~l3~a polyoxypropylene block copolymer that has the physical propeny of being a - liquid at room temperature or below and a gel at body temperature.
The polyoxyethylene-polyoxypropylene block copolymer has the following general formula:
HO(C2H40)b(c3H6o)a(c2H4o)b wherein a is an integer such that the hydrophobe represented by C3H6O has a molecular weight of at least 2250 daltons and b is an integer such that the hydrophile portion represented by C2H4O constitutes from about 50 to 90 percent by weight of the copolymer.
The polyoxyethylene-polyoxypropylene block copolymer preferably has a hydrophobe represented by C3H6O with a molecular weight between approximately 2250 and 6000 daltons with the most preferable molecular weight of the C3H6O of approximately 4000 daltons.
The surface acdve copolymer blocks are forme~ by condensation of ethylene oxide and propylene oxide at elevated temperature and pressure in the presence of a basic catalyst. There is some stadstical variation in the number of monomer units which combine to form a polymer chain in each copolymer.
The molecular weights given are approximations of the average weight of copolymer molecule in each preparation. It is to be understood that the blocks of propylene oxide and cthylene oxide do not have to be pure. Small amounts of other materials can be admixed so long as the overall physical chemical properties are not substantially changed. A more detailed discussion of the preparation of thcse products is found in U.S. Patent No. 2,674,619, which is incarporated herein by reference.
Preparation of the copolymer portion of the present invention is dcscribcd in U. S. Patent Nos. 3,92S,241 and 3,867,533, both of which are inco~porated herein by refence. Dlustrative block copolymers of the following general formula:
HO(C2H40)b(c3H6o)a(c2H4o)b which may be employed in the preparation of the gels of the prescnt invention are presented in Tablc 1.

f ,~, ~
r~ ' !

20~3~0 Table I
Mol. Wt of Wt percent ofApprox. total hydrophobe~ hydrophile~mol. wt.
_base (average)___ (A~erage) of copol,vmer Hydrophobe base is the (C3H6O)a por~ion of the copolymer mole cule.
Hydrophile base is the (C2H40)b por~ion of the copolymer molecule.
s Not all of the block copolymers of the formula:
HO(c2H40)b(c3H6o)a(c2H4o)b may be employed in thc present invention. Because of the nature of aqueous solutions of thcse block copolyrners, three variables affect the formation of the gels. Thereforer it is necessaly to recognize certain minima for the three variables. These variables are:
(1) the weight pacent.concentration of block copolymers in the gel;
(2) the molecular weight of the hydrophobe portion (C3H6O)a; and

(3) the weight percent of the hydrophile portion (C2H4O)b of the copolymcr.
Thcse minima define a minimum weight percent concentration of the block copolymer with a specific hydrophobe having a minimum weighl percent of cthylene oxide that is necessary to form a gel. Thus, at the minimurnconcentration with a specific molecular weight hydrophobe, a minimurn weight percent of ethylcne oxide is required before a specific block copolymer will form a gel in an aqueous solution. Examples of minimum weight percent conccntrations with specific molecular weight hydrophobes are set out in Table Il.

wo 91/16058 PCr/USsl/02912 Il 2081340 Table II
Moh wt of I Min. wt percent I Min. weight percent I Total mol. wt of hydrophobe base concentration to ethylene oxide block copolymer form a rel reauired 2250 _ ~ 50 40~0 20 70 13500 At least a 40 percent weight concentration of the block S copolymer having a hydrophobe of at least 2,250 molecular weight with at least about 50 weight percent of ethylene oxide condensed therewith will be necessary to form a gel in an aqueous solution. In all cases, the block copolymers above the minima indicated in Table I will form gels in aqueous solutions up to 90 weight percent concentration and higher. Above 90 weight percent concentration, however, the gels tend to become indistinguishable from the starting block copolymer itself. It is to be understood that the molecular weight of the hydrophobe may be other than those illustrated in Table 1. Thus, for example, if a hydrophobe of about 2500 molecular weight is used, it is recognized that a gel may be formed from the block copolymer at a concentration of 40 weight percent in an aqueous solution where about 45 weight percent of ethylene oxide is present in the block copolymer.
From the information presented in Tables I and II, it can be seen that the following provisions must be maintained to prepare gel compositions in accordance with the present invention:
1. When a in the following formula HO(c2H40)b(c3H6o)a(c2H4o)b is an integer such that the average molecular weight of the hydrophobe is about 2250 daltons, then the ethylene oxide content is from 50 to 90 weight percent of the copolymer, the total average molecular weight of the copolymer is from 4600 daltons to 10,750 daltons and the gel composition comprises from 40 to 50 weight percent of the copolymer.
2. When a in the following foqmula HO(C2H4 O)b(C3H6)a (C2H40)b . , . .

wo 91/~6058 Pcr/~lssl~02912 12 2~8l3~o is an integer such that the average molecular weight of the hydrophobe is abou~ 2750 daltons, then the ethylene oxide content is from 45 to 90 weight percent of the copolymer, the total average molecular weight of the copolymer is from 4910 daltons to 13500 daltons and the gel composition comprises from 40 to S0 weight percent of the copolymer.
3. When a in the following formula:
HO(c2H40)b(c3H6o)a(c2H4o)b is an integer such that the average molecular weight of the hydrophobe is about 3250 ~altons, then the ethylene oxide conten~ is from 3S to 90 weight percent of the copolymer, the total average molecular weight of the copolymer is from 4910 daltons to 15,510 daltons, and the gel composition comprises from 30 to 50 weight percent of the copolymer,

4. When a in the following formula-HO(C2H40)b(c3H6o)a(c2H4o)b is an integer such that the average molecular weight of the hydrophobe is about 4000 daltons, then the ethylene oxide content is from 35 to 90 weight percent of the copolymer, the total average molecular weight of the copolymer is from 6150 daltons to 20,000 daltons and the gel composition comprises from 30 to S0 weight percent of the copolymer, with the further proviso that when a in the formula is an integer such that the average molecularweight of the hydrophobe is about 4000 daltons, the ethylene oxide content is from 70 to 90 weight percent, the total average rnolecular weight of the block polymer is from 16,000 daltons to 20,000 daltons and thc gel composition comprises from IS to S0 weight percent of the copolymer.
The preferred polyoxyethylene-polyoxypropylene copolymer is poloxamer 407 which has a chcmical formula of ~-hydro- omega- hydroxy-poly(oxyethylene) 101 - poly(oxypropylene)s6 - poly(oxyethylene) 101 - The poloxamer 407 can also be represented by the following folmula:
HO(C2H4)b~C3H 6)a(C2H4)b wherein the molecular weight of the hydrophobe (C3H6O) is approximately 4000 daltons and the total molecular weight of the compound is approximately 13,S00 daltons.
Many different acid mucopolysaccharides are effective in the present invention. The acid mucopolysaccharides, also known as 35 ~ glycosaminoglycans, that are admixed or dissolved with the copolvmer wo 91/16058 Pcr/us9l/o29l2 generally consist of recurring disaccharide units, each of which contains a derivative of an arninohexose, usually D-glucosamine or D-galactosamine. At least one of the two sugars in the recurring disaecharide unit of acid mucopolysaccharides contains an acidic group having a negative charge at pH
7, the negative group being either a carboxylate or sulfate group. An exarnple of an acidie hexose is D-glucuronate, derived from D-glucose by oxidation of the ~ carbon atom to a carboxylate group.
Acid mucopolysaccharides are thus heteropolysaccharides because they consist of at least two kinds of monosaccharides in alternating sequence which vary in such characleristics as chain length and charge density.
The prefix muco- refers to the fact that these polysaccharides were first isolated from mucin, the slippery, lubricating proteoglycan of mucous secretions The acid mucopolysaccharide hyaluronic acid derived from the intercellular cement of animal tissues contains many alternating units of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid forms highly viseous, jellylike solutions. Hyaluronic acid is often found combined with other mucopolysaccharides.
Chondroitin, a major polysaccharide of cartilage proteoglycans, eontains altemating units of D-glueuronic acid and N-acetyl-D-galactosamine.
Chondroitin may be regarded as the parent material for other widely distributed polysaeeharides, such as chondroitin sulfate A and chondroitin sulfate C, which differ only in the posidon in which sulfate is esterified to the galactosarnine rnoiety.
Another irnporlant mucopolysaecharide, long termed ehondroitin sulfates is dcrmatan sulfate (c~-L-iduronosyl-(1-3)-B-D-N-acytylgalactosannine 4-sulfate). Dermatan eonsists of repeadng disaeeharide units of iduronosyl and acctylgalactosamine. It can bc isolated from various tissues ineluding blood vessd walls and skin. Dermatan sulfate catalyses the Ih~mbin-heparin cofactor II interaetion. Despite the andthrombode effeets of derrnatan sulfate, derrnatancornpounds gcncrally arc weak anticoagulants.
The aeid polysaeeharide heparin is generated by eertain types of eells that are espeeially abundant in the lining of arterial blood vessels.
Heparin, like other mueopolysaeeharides, is an anionic earbohydrate chain synthesized as part of a proteoglyean. The basic building blocks of heparin are alternating units of uronie aeid and N-aeetylglueosarnine with the predominan repeat disaccharide unit being L-iduronic acid and 2-amino-2~eoxy-D-glucose.

Wo 91/16058 PCI/US91/02912 14 2~1340 The L-iduronic acid arises from the epimerization of D-glucuronic acid at position 5 during its biosynthesis and, in general, increases in the L-iduronic acid residues parallels the increase in sulfation of the heparin. If the epimerization and the sulfation reactions went to completion, there would be 3 sulfate per disaccharide consisting of an N-sulfate and a 6-O-sulfate on the aminosugar and a 2-O-sulfate on the L-iduronic acid. There are approximately 2.2 sulfate residues per disaccharide in standard porcine mucosal heparin, suggesting that there is a significant number of unmodified glucuronic acid residues in heparin. Incomplete conversion of glucuronic acid to iduronic acid is essential for the antieoagulant function of heparin.
Heparin exhibits strong anticoagulant activity. Low molecular weight heparins have a mean molecular weight of less than approximately 6000 daltons show much stronger antieoagulant aetivity than higher molecular weight heparins. The preferred aeid mueopolysaccharides that are used in the present invention have little or no anticoagulant activity.
Heparans are another group of aeid mucopolysaeeharides.
Chemieally, heparans have the same earbohydrate baekbone as heparin but differ in their sulfate eontent and distribution of eharged groups. Hepatans andother aeid mueopolysaeeharides exhibit signifieantly less anticoagulant ptoperties eompated to heparin.
It has been found that strongly anionic acid mueopolysaeehatides are generally preferred when praetieing the present invention. "Strongly anionie" means a molecule with a high negative eharge density. The sttongly anionie aeid mueopolysaeeharides have a greater therapeutie effeet when compared to weakly anionie acid mueopolysaceharides.
Strongly anionie aeid mueopolysaeeharides ean be prepared by any method eapable of distinguishing moleeules with a higher negative eharge density from those of lowet eharge density. Eleetrophoresis or ion exehange ehromatography ate two sueh methods and ate well known by those of ordinary skill in the art. Another method would simply utilize the anionic mueopolysaceharide as the free base rather than the sodium, ealeium or lithium salt as they are eotnmonly rnanufaetuted.
The size of the aeid mucopolysaechande ean vary greatl~n For example, the molecular weight of the mueopolysaeeharide that is used in the topical treatrnent of the present invenion can be f~r between 3000 daltons to 3,000,000 daltons. The preferable average molecular weight of the acid wo 91~16058 PCI/US91/02912 ,5 20813~0 mucopolysaccharides is between approximately S000 and S0,000 daltons. The most preferable molecular weight of the acid mucopolysaccharides is between approximately 10,000 and 20,000 daltons. It is to be understood that the molecular weight of the acid mucopolysaccharide is not a critical factor in the S present invention, and that any one preparation of acid mucopolysaccharide will contain a population of molecules that can vary greatly in size. This variance in size of molecules does not necessarily reduce the effectiveness of the present invention. The present invention utilizes acid mucopolysaccharides of higher molecular weight to minirnize systermc absorption and restrict the biologic actions to the locally applied area. (See Emanuele, et al., "The Effect of Molecular Weight on the Bioavailability of Heparin", Thrombosis Research, Vol. 4B, pgs. 591-596, (1987) which is incorporated herein by reference) Thus, an acid mucopolysaccharide can be used in the present invention where the acid mucopolysaccharide is of sufficient molecular size such that it is not lS absorbed systemically.
The most preferred acid mucopolysaccharide for use in the present invention is dermatan sulfate. The average molecular weight of dermatan sulfate is estimated to be approximately 30,000 daltons depending on its sourcc and method of preparation. The concentration of dermatan sulfate in the composition of the present invention is preferably between approxirnately 10mg per ml and 100 mg per ml. The more preferred concentration of dermatan sulfate is between 25 mg per ml and 75 mg per ml with a most preferred concentradon of dermatan su1fate of approximatdy SOmg per ml.
Another preferred acid mucopolysaccharide for use in the present invention has a high molecular weight. The preferred average molecular weight of the heparin is approximately lS,000 daltons. The concentration of heparin in the composidon of the present invendon is preferably between approximately 10 mg per ml and lO0 mg per ml. The more preferred concentradon of heparin is between 20 mg per ml and S0 mg per ml with a most prefcrred concentraion of heparin of approximately 25 mg per ml. Each mg of heparin is expected to contain approximately lO0 units of acdvity.
Some of the commonly known acid mucopolysaccharides that can be used with the present invention include, but are not limited to, heparin,heparan sulfate, heparinoids, dermatan sulfate, pentosan polvsulfate.
chondroitin sulfate and hyaluronic acid.
=

. , .

16 20~134a In accordance with the present invention, the acid mucopolysaccharides may also be admixed with pharmaceutically acceptable topical carriers. The carrier may comprise any commercially available base including but not limited to waxes, petrolatum, and glycerides. More S specifically, the acid mucopolysaccharide may be admixed to com~nercially available salves, ointments or cerates containing a variety of pharmaceutically active agents.
The compositions prepared in accordance with the presem invention comprise at least the following ingredients: (1) a pharrnaceutically effective concentration of a acid mucopolysaccharide and (2) an aqueous gel eomprising, based on 100 parts by weight, (a) from 15 to 50 parts, preferably from 15 to 25 parts, of a polyoxyethylene-polyoxypropylene block copolymer and (b) from S0 to 85 parts, preferably from about 75 to 85 parts, of water.
The method of the present invention comprises the steps of topically administering to a human or animal with damaged or diseased tissue a eomposition comprising an admixture of a polyoxyethylene-polyoxypropylene block copolymer that has the physical property of being a liquid at room temperature or below and a gel at body temperature, and an acid mucopolysaccharide. The composition is deseribed hereinabove.
The composiion of the present invention can be administered by dabbing, dripping, pouring, spraying or painting the eomposition onto the damaged or diseased tissue. In addition, the eomposition of the present invention comprising the bloek eopolymer and aeid mucopolysaccharide can be administered by inser~ng the damaged or diseased tissue into a solution of the eomposition thereby allowing the eomposition to gel onto the tissue.
Preferably, the eomposition of the present invention should be adrninistered so that a gd layer forms over the damaged or diseased tissue thereby allowing the damaged or diseased tissue to be protected from the environment.
In addition, beeause the aeid mucopolysaceharide is dispersed ~roughout the gel, the aeid mueopolysaeeharide will be in eonstant eon; ict withthe damaged or diseased tissue thereby aecderating the healing of the tissue. Itis lo be understood that the topical administration of the composition of the present invention includes administration to the surface of the body as well as administration to tissue that is exposed through surgery or traurna.
The composition and method of the present invention is particularly effective in treating bums. These burns can be chemical burns.

'0 91/16058 PCT/US91/02912 thermal burns, electrical burns or radioactive burns. The present invention is also effective in treating other types of tissue damage such as traurnatic darnage, including bu~ not limited to, compound fractures, euts, abrasions or damage due to infection with bacteria, fungi or other rnicroorganisms.
S It may be advantageous to add additional pharrnaceutically active agents to the composition of the present invention depending upon the type of injury that is being treated. For example, if one is treating a serious burn according to the present invention, antimicrobial agents may be added to the composidon of the present invention to retard infeetion. The antimicrobial agentmay be an antibiotic, antifungal agent or a mixture thereof. Representative antimicrobial agents that ean be used in praetieing the present invention include, but are not limited to, penicillins, cephalosporins, bacitracins, aminoglycosides and polypeptide antibiotics and the baeteriastatie eompounds including, but not limited to, chloramphenicol, tetracyclines, macrolides, sulfonamides and lS aminosalicylic aeid. Antifungal agents that can be used in praeticing the present invention include, but are not limited to, nystatin, amphotericin B and griseofuvin.
In addition, silver ions ean be used in practicing the present invention. The silver salts that ean be employed in the preparation of the gels of the present invention are those silver salts which will preferably dissolve in water at a minimum eoneentration of 0.1% by weight. Representative silver salts include silver nitrate, silver aeetate, silver sulfate, and silver lactate. The arnount of silver salt that will produce a benefieial effeet is between about 0.1 and 1.0% by wdght of silver salt based on the weight of the water in the gel.
Growth faetors such as human growth hormone, dssue derived growth factor, epidermal growth faetor, platelet derived growth factor, fibroblast growth factor, and/or nerve growth factor may optionally be added to the eomposidon of the present invention, either singly or in eombination, to enhanee the growth and development of the damaged or diseased tissue. In addition, anti-inflammatory agents may be added to reduee inflammation in the damaged or diseased tissue.
The eomposition of the present invention ean optionally eontain anesthetics to alleviate pain when applied to the damaged or diseased tissue.
Representative anesthetics that can be employed in the present invention 3S include, but are not limited to, lidoeaine and proeaine.

. - .

WO 9 1 / 1 6058 PCr/l,'S9 1 /0291 2 18 2~1340 This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the descripion herein, may suggest themselves to those skilled in the art without departing from the spirit of the presenl invenion and/or the scope of the appended claims.
. .
Example I
A topically-applicable gel composition that is effective for treatihg damaged or diseased tissue is prepared using the following ingredients:
Parts In~redients 10% ................. Copolymer 2%................... Derrnatan Sutfate 78% ................. Water The copolymer in this example has the following general formula:
HO~C2H4O)b(c3H6o)a(c2H4o)b wherein the molecular weight of the hydrophobe (C3H6O) is approximately 4000 daltons and the total molecular weight of the compound is approximately 13,S00 daltons.

Example Il The composition of Example I is administered to a patient with a second degree burn on his arm. The composiion is placed in a syringe and is cooled to approximately 20C. The composition is slowly apptied to the surface of the burn, atlowing the liquid composition to gel on the burn. Enough of the composition is added to form a get that is approximately 0.2 cm in depth.

Example III
A topically-applicable gel composition that is effeci~e for treating dama~ed or diseased tissue is prepared using the following inr~edients:5 WO 91/16058 PCI/~_:S91/02912 Parts Ingredients 205~................ Copolymer 4% ................. Heparin 0 . 5 % ............ Gentamicin sulfate 7 5 . 5 % .......... Water The copolymer in this example has the following general formula:
HO(C2H40)b(c3H6o)a(c2H4o)b wherein the molecular weight of the hydrophobe (C3H6O) is approximatel~
4000 daltons and the total molecular weight of the compound is approximately 13,500 daltons.

Example IV
The composition of Example III is adrninistered to a patient with a first degree bum on his leg. The composition is placed in a syringe and is cooled to appr~ximately 20C. The composition is slowly applied to the surface of the burn, allowing the liquid composition to gel on the burn. Enou~h of the composiion is added to form a gel that is approximately 0.2 cm in depIh.
Example V
- A topically-applicable gel<composition that is effective for treating damaged or diseased tissue is prepared using the following ingredients:
Parts Ingredients 20%................. Copolymer 1% ........ ,....... De~natan sulfate 0.5% ...... ;....... Gentamicin sulfa~e 78.59~.............. Water The copolymer in this example has the following general fortnula:
HO(C2H40)b(c3H6o)a(c2H4o)b wherein the molecular weight of the hydrophobe (C3H6O) is approxima~el!
4000 daltons and the total molecular weight of the compound is approximately 13,500 daltons.

wo 91/16058 Pcr/~s9l/o2sl2 20 2~1340 Example VI
The composition of Example V is administered to a patient with a third degree burn on his torso. The composition is placed in a syringe and is cooled to approximately 20C. The composition is slowly applied by spraying S the composition onto the surface of the burn, allowing the liquid composition IO
gel on the burn. Enough of the composition is added to form a gel that is approximately 0.2 cm in depth.

Example VII
A topically-applicable gel composition that is effective for treating damaged or diseased tissue is prepared using the following ingredients:
Parts In~redients 20%.................. Copolymer 1% mg~ml............ Heparin 1% by weight........ Lidocaine 78%.................. Water The copolymer in this example has the following gelieral formula:
HO(c2H40)b(c3H6o)a(c2H4o)b wherein the molecular weight of the hydrophobe (C3H6O) is approximately 4000 daltons and the total molecular weight of the compound is approximately 13,500 daltons.
Exsmple VIII
The composition of Example VII is administered to a patient with a second degrec sunburn on his back. The composition is placed in a syringe at room lemperature. The composition is slowly applied by dripping thc composition onto thc surface of the burn, allowing the liquid composition togel on the burn. Enough of the composition is added to form a gel that is approximately 0.1 cm in depth.

Exsmple IX
A topically-applicable gel composition which is effective in treating burns is prepared from the following ingredients:

.' ..` :

wo 91/16058 Pcr/ussl/o29l2 2l 20813~0 Parts In~redients 20%................. Copolymer S%.................. ~ermatan 75%................. Saline s The copolymer (poloxamer 407, BASF Corporation, Parsippany, NJ)) in this example has the following general formula:
HO(C2H4 O) b(C3 H 6)a (C2 H4 )b wherein the molecular weight of the hydrophobe (C3H6O) is approximately l O 4000 daltons and the total molecular weight of the compound is approximately 13,500 daltons.
The effects of the topically-applicable gel compositiori in the treatment of thennally injured tissue were studied in a guinea pig model of split thickness burns. Deeply anesthetised, hairless guinea pigs were subjected to thermal tissue injury by placing a 5 cm2, 80g metal probe theated to 80C) on their back for exactly 5 seconds. Thirty minutes post burn, animals were either left untreated (control), treated with the 20% gd of poloxamer 407 without dermatan or treated with 20% poloxamer 407 and 5% dermatan sulfate (Sciendfic Protein Labs, Waunakee, WS). Treated animals received 0.5 ml applications of the appropriate test article at 0.5, l, 2, 3, 4, 5, 6, 7, 8, 24, 28, 32 and 48 hours post burn. All animals were assessed for wound area, skin thickness and erythema at various intervals post burn. Histologic sections demonstrated that r~duction in skin thickness correlated with a reduction in ne~collagen forrnation (scar) in the superficial dermis.
The effect of the topically-applicable gel composition on wound contracture is shown in Table III. Burn wound measurements are made by determining the distance between points tattooed at the wound periphery immediately post burn. The data clearly shows that the combination of heparin and copolymer had a significant effect on wound contracture at 24 hours and 7' hours.

. . .

wo 91/16058 Pcr/~;S91/02912 22 2~813~0 Table III
Area of Lesion Time l Untreated ¦Poloxamer ¦Poloxamer +
l lAlone lDermatan 30Min 5.56:~0.36 6.19+0.15 6.65to.18 24 hours 5.04:~û.31 4.7S+0.2; 3.91+0.1~
72 hours 4.32~û.45 3.53+0.50 2.73+0.~ .

The effects of the topically-applicable gel composition on skin thickness in the burn model are shown in Table IV. The poloxamer/derrnatan treated animals had a less skin thick;ness when compared to either poloxamer only trealed animals or control animals. This was especially true at one d~
post burn.
Table IV
Skin Thickness Time l Untreated lPoloxamer lPoloxamer lAlone Dermatan Dav I 7.43_0.32 i7.30+0.525.70:~0.31 Dav 2 4.30:tO.86 1 4.57+0.37 4.67+0.1 ~
Dav3 5 57-0.97 ¦4.50_0.25 3.80+0.10 Dav 14 4.57_0.27 l3.78+0.15 3.30_0.3~

The effects of the topically-applicable gel composition on crythema in the burn model are shown in Table V. Erythema was scored as 0 for little or no erythema to 3 for maximum erythema. As can be seen in Table V, thc animals that were treated with the poloxamer/dermatan combination had less erythema than did the animals that were treated with poloxarner onl~ or wcre left untreated.
Table V
Erythema (0 to 3) ~me IJntreatedPoloxamer Poloxamer Alone Dermatan ~...... ~ ._ ~u Mm 3~0 3_0 3+0 24 hours 3~û 0.9+0~ 1 û.7+0. ' 72 hours 2+0 û.7+0.7 0~0 I wee~ 0+0 0+() ()~0 ~ o 9 1 ~ 16058 pcrJ~ S9 1 /029 1 2 23 20~13~0 It should be understood, of course, that the foregoing relates only to a preferred embodiment of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims.

Claims (40)

WHAT IS CLAIMED IS:

1. A composition for topically treating damaged or diseased tissue comprising a solution of:
a. a polyoxyethylene-polyoxypropylene block copolymer having the following formula:

HO(C2H4O)b(C3H6O)a(C2H4O)bH
wherein a is an integer such that the hydrophobe represented by C3H6O has a molecular weight of between 2250 and 6000 daltons and b is an integer such that the hydrophile portion represented by C2H4O constitutes from about 50 to 90.
percent by weight of the copolymer, and b. an effective amount of dermatan sulfate.

2. The composition of Claim 1, wherein the hydrophobe represented by C3H6O has an average molecular weight of between approximately 2750 and 4000 daltons.

3. The composition of Claim 2, wherein the hydrophobe represented by C3H6O has an average molecular weight of approximately 4000 daltons.

4. The composition of Claim 1, further comprising an antimicrobial compound.

5. The composition of Claim 4, wherein the antimicrobial compound is an antibacterial compound.

6. The composition of Claim 4, wherein the antimicrobial compound is an antifungal compound.

7. The composition of Claim 1, further comprising an anti-inflammatory agent.

8. The composition of Claim 1, further comprising a growth factor.

9. The composition of Claim 1, further comprising an anesthetic.

10. The use of a composition for treating damaged tissue in a human or animal when of topically administered in an aqueous solution to the human or animal with diseased or damaged tissue, the aqueous solution containing:
a. a polyoxyethylene-polyoxypropylene block copolymer having the following formula:
HO(C2H4O)b(C3H6O)a(C2H4O)bH
wherein a is an integer such that the hydrophobe represented by C3H6O has a molecular weight of between 2250 and 6000 daltons and b is an integer such that the hydrophile portion represented by C2H4O constitutes from about 50 to 90 percent by weight of the copolymer, and b. an effective amount of an acid mucopolysaccharide.

11. The use according to Claim 10, wherein the acid mucopolysaccharide is selected from the group consisting of heparin, heparan sulfate, heparinoids, dermatan sulfate, pentosan polysulfate, chondroitan sulfate and hyaluronic acid.

12. The use according to Claim 11, wherein the acid mucopolysaccharide is heparin.

13. The use according to Claim 11, wherein the acid mucopolysaccharide is dermatan sulfate.

14. The use according to Claim 10, wherein the acid mucopolysaccharide has minimal anticoagulant activity.

15. The use according to Claim 10, wherein the hydrophobe represented by C3H6O has an average molecular weight of between approximately 2750 and 4000 daltons.

16. The use according to Claim 15 wherein the hydrophobe represented by C3H6O has an average molecular weight of approximately, 4000 daltons.

17. The use according to Claim 10, wherein the composition further comprises an antimicrobial compound.

18. The use according to Claim 17, wherein the antimicrobial compound is an antibacterial compound.

19. The use according to Claim 17, wherein the antimicrobial compound is an antifungal compound.

20. The use according to Claim 10, wherein the composition further comprises an anti-inflammatory agent.

21. The use according to Claim 10, wherein the composition further comprises an anesthetic.

22. The use of a composition for treating burns in a human or animal when topically administered to the human or animal with the burn, the composition comprising:
a. a polyoxyethylene-polyoxypropylene block copolymer having the following formula:
HO(C2H4O)b(C3H6O)a(C2H4O)bH
wherein a is an integer such that the hydrophobe represented by C3H6O has a molecular weight of between 2250 and 6000 daltons and b is an integer such that the hydrophile portion represented by C2H4O constitutes from about 50 to 90 percent by weight of the copolymer; and b. an effective amount of an acid mucopolysaccharide.

23. The use according to Claim 22, wherein the acid mucopolysaccharide is selected from the group consisting of heparin, heparin sulfate, heparinoids, low molecular weight heparin, dermatan sulfate, pentosan polysulfate, chondroitan sulfate and hyaluronic acid.

24. The use according to Claim 23, wherein the acid mucopolysaccharide is heparin.

25. The use according to Claim 23, wherein the acid mucopolysaccharide is dermatan sulfate.

26. The use according to Claim 22, wherein the acid mucopolysaccharide has minimal anticoagulant activity.

27. The use according to Claim 22, wherein the hydrophobe represented by C3H6O has an average molecular weight of between approximately 2750 and 4000 daltons.

28. The use according to Claim 27, wherein the hydrophobe represented by C3H6O has an average molecular weight of approximately 4000 daltons.

29. The use according to Claim 22, wherein the composition further comprises an antimicrobial compound.

30. The use according to Claim 29, wherein the antimicrobial compound is an antibacterial compound.

31. The use according to Claim 29, wherein the antimicrobial compound is an antifungal compound.

32. The use according to Claim 22, wherein the composition further comprises an anti-inflammatory agent.

33. The use according to Claim 22, wherein the composition further comprises an anesthetic.

34. The use of a composition for treating damaged tissue in a human or animal when topically administered to the human or animal with the diseased or damaged tissue, the composition comprising dermatan sulfate and a pharmaceutically acceptable topical carrier.

35. The use according to Claim 34 wherein the composition further comprises an antibacterial compound.

36. The use according to Claim 34 wherein the composition further comprises an antifungal compound.

37. The use according to Claim 34 wherein the composition further comprises an anti-inflammatory compound.

38. The use according to Claim 34 wherein the concentration of dermatan sulfate in the composition is between approximately 10 milligrams per milliliter and approximately 100 milligrams per milliliter.

39. The use according to Claim 34 wherein the concentration of dermatan sulfate in the composition is between approximately 25 milligrams per milliliter and approximately 75 milligrams per milliliter.

40. The use according to Claim 34 wherein the concentration of dermatan sulfate in the composition is approximately 50 milligrams per milliliter.