Compositions for wound treatment

Abstract

A wound dressing composition comprising a chitosan and an oxidized cellulose. For example, the composition may be in the form of a sponge formed by freeze drying an aqueous dispersion of chitosan and oxidized regenerated cellulose (ORC). The composition is especially suitable for the treatment of chronic wounds.

Claims

1. A wound dressing composition comprising an intimate mixture of: a chitosan; an oxidized regenerated cellulose, wherein the weight ratio of the chitosan to the oxidized regenerated cellulose is in the range of about 2:1 to 1:2; and from about 0.01% to about 5% by weight on a dry weight basis of platelet derived growth factor (PDGF); wherein the pH of the intimate mixture is in the range of about 2 to 8.

2. A wound dressing composition according to claim 1, wherein said oxidized regenerated cellulose is in the form of dispersed fibers or powder.

3. A wound dressing composition according to claim 1, wherein said oxidized regenerated cellulose and chitosan are dispersed in a semi-solid or solid vehicle for topical application.

4. A wound dressing composition according to claim 1, wherein the oxidized regenerated cellulose and chitosan together make up at least 25% by weight of the intimate mixture on a dry weight basis.

5. A wound dressing composition according to claim 4, wherein the oxidized regenerated cellulose and the chitosan together make up at least 50% by weight of the intimate mixture on a dry weight basis.

6. A wound dressing composition according to claim 1, wherein the composition is a flexible film.

7. A wound dressing comprising a wound dressing composition according to claim 1.

8. A wound dressing according to claim 7, which is sterile and packaged in a microorganism-impermeable container.

9. A method of preparing an active wound dressing material comprising the steps of: (i) preparing a material comprising a composition according to claim 1; and (ii) washing and drying the material to form said active wound dressing material.

10. A wound dressing composition according to claim 1, wherein the pH of the intimate mixture is in the range of about 3 to 7.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) Specific embodiments of the present invention will now be described further, by way of example, with reference to the accompanying drawings, in which:

(2) FIG. 1 shows a graph of elastase activity against time for a wound dressing material according to the present invention as compared with positive and negative controls; and

(3) FIG. 2 shows a graph of collagenase activity against time for a wound dressing material according to the present invention as compared with the same positive and negative controls.

Reference Example 1: Preparation of a Collagen/Fibrous ORC Sponge

(4) A freeze-dried collagen/ORC sponge is prepared as follows.

(5) First, the collagen component is prepared from bovine corium as follows. Bovine corium is split from cow hide, scraped and soaked in sodium hypochlorite solution (0.03% w/v) to inhibit microbial activity pending further processing. The corium is then washed with water and treated with a solution containing sodium hydroxide (0.2% w/v) and hydrogen peroxide (0.02% w/v) to swell and sterilize the corium at ambient temperature. The corium splits then undergo an alkali treatment step in a solution containing sodium hydroxide, calcium hydroxide and sodium bicarbonate (0.4% w/v, 0.6% w/v and 0.05% w/v, respectively) at pH greater than 12.2, ambient temperature, and for a time of 10-14 days, with tumbling, until an amide nitrogen level less than 0.24 mmol/g is reached. The corium splits then undergo an acid treatment step with 1% hydrochloric acid at ambient temperature and pH 0.8-1.2. The treatment is continued with tumbling until the corium splits have absorbed sufficient acid to reach a pH less than 2.5. The splits are then washed with water until the pH value of corium splits reaches 3.0-3.4. The corium splits are then comminuted with ice in a bowl chopper first with a coarse comminution and then with a fine comminution setting. The resulting paste, which is made up in a ratio of 650 g of the corium splits to 100 g of water, as ice, is frozen and stored before use in the next stage of the process. However, the collagen is not freeze-dried before admixture with the ORC in the next stage.

(6) The ORC component of the freeze-dried pad is prepared as follows. A SURGICEL cloth (Johnson & Johnson Medical, Arlington) is milled using a rotary knife cutter through a screen-plate, maintaining the temperature below 60 C.

(7) The milled ORC powder and the required weight (according to solids content) of frozen collagen paste are then added to a sufficient amount of water acidified with acetic acid to obtain a pH value of 3.0 and a total solids content of 1.0%. The mixture is homogenized through a Fryma MZ130D homogenizer, progressively diminishing the settings to form a homogeneous slurry. The pH of the slurry is maintained at 2.9-3.1. The slurry temperature is maintained below 20 C., and the solids content is maintained at 1%0.07.

(8) The resulting slurry is pumped to a degassing vessel. Vacuum is initiated for a minimum of 30 minutes, with intermittent stirring, to degas the slurry. The slurry is then pumped into freeze-drier trays to a depth of 25 mm. The trays are placed onto freezer shelves where the temperature has been preset to 40 C. The freeze-drier programme is then initiated to dry and dehydrothermally cross-link the collagen and ORC to form thick sponge pads. On completion of the cycle, the vacuum is released, the freeze-dried blocks are removed, and are then split to remove the top and bottom surface layers, and to divide the remainder of the blocks into 3 mm-thick pads. The step of splitting the freeze-dried blocks into pads is carried out with a Fecken Kirfel K1 slitter. Finally, the pads are die-cut to the desired size and shape on a die-cutter, packaged, and sterilized with 18-29 KGy of cobalt 60 gamma-irradiation. Surprisingly, this irradiation does not cause significant denaturation of the collagen, which appears to be stabilized by the presence of ORC. The resulting freeze-dried collagen ORC pads have a uniform, white, velvety appearance. The thickness of the pads is 3.20.17 mm (N=8 batches). These pads are used as the positive control in the Procedures described below.

Reference Example 2: Preparation of an Alginate/Fibrous ORC Sponge

(9) An alginate/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of alginate

(10) Sodium alginate was obtained from Pronova Biomedical in a powdered form. The powder was dissolved in ice cold water at a concentration of 2% w/v by mixing with a paddle stirrer. The solution was then diluted to 1% solids by the addition of an equal volume of 0.1M acetic acid. A known weight of the sodium alginate solution was then added to the ORC to give a final ratio of 45% ORC/55% sodium alginate in the final material. The sponges were then prepared as in example 1.

Reference Example 3: Preparation of a Hyaluronate/Fibrous ORC Sponge

(11) A hyaluronate/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of alginate

(12) Sodium Hyaluronate with an average molecular weight distribution of 500,000 daltons was obtained from Lifecore Biomedical Inc. in a powdered form. The powder was dissolved in ice cold water at a 2% w/v concentration with mixing overnight. A known weight of the sodium hyaluronate solution was then added to the ORC to give a final ratio of 45% ORC/55% sodium hyaluronate in the final material. The sponges were then prepared as in example 1.

Reference Example 4: Preparation of a Pectin/Fibrous ORC Sponge

(13) A pectin/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of pectin

(14) Apple derived pectin was obtained from the Sigma Chemical Co. The powder was dissolved in ice cold water at 2% w/v with stirring overnight. A known weight of the pectin solution was then added to the ORC to give a final ratio of 45% ORC/55% pectin in the final material. The sponges were then prepared as in example 1.

Reference Example 5: Preparation of a Beta-Glucan/Fibrous ORC Sponge

(15) A beta-glucan/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of a beta-glucan

(16) B-Glucan was obtained in a powdered form from Sigma Chemical Company, and was dissolved in ice cold water at 2% w/v by stirring overnight. The final solution was diluted with an equal volume of 0.1M Acetic acid to give a solution with a final concentration of 1% w/v in 0.05M acetic acid. A known weight of the b-glucan solution was then added to the ORC to give a final ratio of 45% ORC/55% b-glucan in the final material. The sponges were then prepared as in example 1.

Reference Example 6: Preparation of a Locust Bean Gum/Fibrous ORC Sponge

(17) A locust bean gum/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of locust bean gum

(18) Locust Bean Gum was obtained in a granular form from the Sigma Chemical Company. The granules were suspended in ice cold water at 2% w/v. The suspension was then slowly heated to 95 C. to solubilise the gum. The resulting solution was centrifuged at 5000 g to clarify the solution and remove the insoluble fragments of seed coat. The supernatant was then removed and solids content calculated, the solution was then diluted to a final concentration of 1% w/v by the addition of 0.05M acetic acid. A known weight of the locust bean gum solution was then added to the ORC to give a final ratio of 45% ORC/55% locust bean gum in the final material. The sponges were then prepared as in example 1.

EXAMPLE 1: PREPARATION OF A CHITOSAN/FIBROUS ORC SPONGE

(19) A chitosan/fibrous ORC sponge was prepared as described in Reference Example 1, but with replacement of the collagen by an equal weight fraction of a chitosan

(20) Chitosan practical grade powder was obtained from the Sigma Chemical Company. The powder was dissolved in ice cold water at a 2% w/v concentration. The solution was then diluted to 1% w/v chitosan by the addition of an equal volume of 0.1M acetic acid. A known weight of the chitosan solution was then added to the ORC to give a final ratio of 45% ORC/55% chitosan in the final material. The sponges were then prepared as in example 1.

EXAMPLE 2: PREPARATION OF A CHITOSAN/ORC FILM

(21) A chitosan/ORC film for application to a wound is made as follows.

(22) 15 grams of chitosan powder was mixed in 1.5 liters of water until blended. 2 grams of glycerol were blended into the mixture. 15 grams of ORC fibers prepared as described in Example 1 were then added with high shear mixing. The resulting mixture was then poured into the bottom of a PTFE tray to a thickness of about 5 mm and air-dried to form films of the ORC/chitosan complex.

EXAMPLE 3

(23) A wound treatment gel for topical application to a wound was prepared as follows.

(24) ORC fibers were prepared as described in Example 1 and the resulting fibers are dispersed at 2% w/w concentration in a 3% w/w carboxymethyl cellulose (CMC) aqueous gel containing 2 wt. % of dissolved chitosan chloride.

(25) Procedure I: Binding of Platelet Derived Growth Factor

(26) PDGF binding studies were carried out as follows:

(27) Small sections of test material (approximately 1 cm.sup.2 squares of INTERCEED ORC fabric, and approximately 1 cm0.5 cm0.4 cm sections of the freeze-dried sponges) were weighed and soaked in 100 mM sodium phosphate dibasic buffer containing 150 mM sodium chloride (total volume 1 ml) for at least one hour at room temperature. Samples were then incubated with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours at room temperature. 22 ng of PDGF was then added to each sample in 250 l of PBS containing 2% BSA, and samples were then incubated for a further hour at 37 C. Each sample was then washed three times with 250 l PBS, followed by increasing concentrations of sodium chloride. Finally, each sample was washed with 4.0M urea. PDGF ELISA analyses of the original PDGF preparation and the various washings were carried out. The results were as follows:

(28) TABLE-US-00001 Polysaccharide + Protection Release ORC of PDGF of PDGF Comments Chitosan yes yes 50% released & retains activity over 96 hr Alginate ? no Since no GF released could not tell if it was protected Hyaluronate no yes 80% released over 96 hr but not active - not protected Pectin no yes 40% released over 96 hr but not active - not protected Beta-glucan ? no Since no GF released could not determine if protected Locust ? no Since no GF bean gum released could not determine if protected

(29) It can be seen that the chitosan/ORC materials bind PDGF well, and release the bound PDGF with relatively little loss of activity. This is useful for wound healing, since it enables the materials to act as PDGF reservoirs at the wound site, by binding PDGF and then releasing it back into the wound as the material biodegrades in vivo None of the other ORC/polysaccharide complexes studied has this characteristic.

(30) Procedure 2: Elastase Inhibition

(31) The levels of neutrophil-derived elastase present in the wound fluid samples were measured spectrofluorimetrically using substrate activity assays. The substrates comprise short peptides synthesised to mimic the appropriate enzyme cleavage site and contain a fluorescent reporter group which is released upon hydrolysis. Enzyme activity was determined by measuring the rate of production of the fluorimetric compound, 7-amino 4-methyl coumarin. Activity was expressed either as relative fluorescence units per minute (RFU/min) or change in fluorescence when corrected for total protein (RFU/min/mg protein). Each sample was tested times 6 and the average value calculated. The substrate was prepared at a 10 mM-stock concentration, and diluted to a working concentration of 0.5 mM in the appropriate assay buffer. The reaction mixture, combined in a microtiter well (black, flat bottomed) comprised 5 l wound fluid, 175 l assay buffer and 20 l substrate (final concentration 50 M). The microtiter plate was read immediately at 455 nm (excitation 383 nm) and at timed intervals over the next hour; between readings the plate was covered and incubated at 37 C. Neutrophil-derived elastase-like activity was estimated using the fluorimetric substrate Methoxy-Alanine-Alanine-Proline-Valine 7-amino 4-methyl coumarin (Bachem UK, Ltd.) solubilised in methanol. The assay buffer required for optimal activity of this enzyme was 0.1M Hepes, pH 7.5 containing 0.5M NaCl and 10% dimethyl sulphoxide.

(32) A sample of the collagen/ORC sponge prepared as described in Reference Example 1 was used as a positive control. A sample of SOF-WICK (Registered Trade Mark) gauze was used as a negative control.

(33) The results are shown in FIG. 1. It can be seen that the complex according to the present invention provides a significant inhibition of elastase activity after 2 and 24 hours.

(34) Procedure 3: Collagenase Inhibition

(35) The levels of matrix metalloproteinases present in the wound fluid samples were measured spectrofluorimetrically using substrate activity assays. The substrates comprise short peptides synthesised to mimic the appropriate enzyme cleavage site and contain a fluorescent reporter group which is released upon hydrolysis. Enzyme activity was determined by measuring the rate of production of the fluorimetric compound, 7-amino 4-methyl coumarin. Activity was expressed either as relative fluorescence units per minute (RFU/min) or change in fluorescence when corrected for total protein (RFU/min/mg protein). Each sample was tested times 6 and the average value calculated. The substrate was prepared at a 10 mM-stock concentration, and diluted to a working concentration of 0.5 mM in the appropriate assay buffer. The reaction mixture, combined in a microtiter well (black, flat bottomed) comprised 5 l wound fluid, 1750 assay buffer and 20 l substrate (final concentration 50 M). The microtiter plate was read immediately at 455 nm (excitation 383 nm) and at timed intervals over the next hour; between readings the plate was covered and incubated at 37 C. Matrix metalloproteinase-like activity was estimated utilising the substrate Succinyl-Glycine-Proline-Leucine-Glycine-Proline 7-amino 4-methyl coumarin (Bachem, UK, Ltd.) solubilised in methanol. The assay buffer necessary for maximal MMP activity was 40 mM Tris/HCl, pH 7.4 containing 200 mM NaCl and 10 mM CaCl.sub.2.

(36) A sample of the collagen/ORC sponge prepared as described in Reference Example 1 was used as a positive control. A sample of SOF-WICK (Registered Trade Mark) gauze was used as a negative control.

(37) The results are shown in FIG. 2. It can be seen that the complex according to the present invention provides rapid inhibition of collagenase activity, and almost complete inhibition after 2 and 24 hours.

(38) The above examples are intended for the purpose of illustration only. Many other embodiments falling within the scope of the accompanying claims will be apparent to the skilled reader.