ANTIMICROBIAL AND/OR EPITHELIAL CELL GROWTH STIMULATING SUBSTANCE AND COMPOSITION AND TISSUE DRESSING MATERIAL

Abstract

A chitosan for use in an antimicrobial treatment of a patient's tissue and a pharmaceutical composition comprising a chitosan for use in an antimicrobial treatment of a patient's tissue. A method of treating a microbial infection, the method comprising the step of administering to a patient an effective amount of a chitosan and an aqueous solution comprising chitosan. A chitosan or a pharmaceutical composition comprising a chitosan for use in an epithelial cell growth stimulating treatment of a patient's tissue. A method of stimulating the growth of epithelial cells the method comprising the step of administering to a patient or to an epithelial cells containing cell culture an effective amount of a chitosan or a pharmaceutical composition comprising chitosan. A tissue dressing material characterized in that it consists of chitosan or a chitosan comprising composition.

Claims

1. A method for stimulating epithelial growth of a tissue, wherein the tissue comprises cuts, abrasions, burns, razor burn, irritated skin, malar rash, wounds caused by viruses, insect bites and tissues affected by acne, comprising: administering to the tissue an effective amount of a spray comprising chitosan with a degree of acetylation of greater than 0% but equal to or less than 2.5%.

2. The method according to claim 1, wherein the chitosan is provided as liquid composition.

3. The method according to claim 1, wherein the chitosan is in aqueous solution.

4. The method according to claim 3, wherein the chitosan is in aqueous solution in a concentration of less than 15% by weight.

5. The method according to claim 2, wherein the chitosan-containing composition comprises an organic acid selected from the group of monobasic or multibasic organic acids having 2 to 12 carbon atoms and a first pKa value between 1 and 5.

6. The method according to claim 2, wherein the chitosan-containing composition is free of organic solvents.

7. The method according to claim 2, wherein the chitosan-containing composition allows for formation of a film after administration to the tissue and wherein the film is less than 1 mm thick.

8. The method according to claim 1 wherein the chitosan is native chitosan.

9. The method according to claim 1, wherein the degree of acetylation of the chitosan is less than 2%.

10. The method according to claim 1, wherein the chitosan is preparable by a method that involves at least two deacetylation steps.

11. The method according to claim 1, wherein a fraction of more than 10% of the chitosan is present in a form that is insoluble at a pH of 6.5 or greater.

12. The method according to claim 1, wherein the chitosan-containing composition is provided as a kit in combination with a detachment solution.

13. The method according to claim 3, wherein the chitosan is in aqueous solution in a concentration of less than 10% by weight.

14. The method according to claim 3, wherein the chitosan is in aqueous solution in a concentration of less than 7.5% by weight.

15. The method according to claim 3, wherein the chitosan is in aqueous solution in a concentration of less than 5% by weight.

16. The method according to claim 1, wherein the chitosan is removed with an acidic detachment solvent.

17. The method according to claim 1, wherein the chitosan is removed with an aqueous detachment solvent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The invention is illustrated in greater detail with the aid of the following figures:

[0070] FIG. 1 shows an .sup.1H NMR spectrum of native chitosan as purchased;

[0071] FIG. 2 shows an .sup.1H NMR spectrum of native chitosan essentially deacetylated after further hydrolysis steps;

[0072] FIG. 3 Illustrates the effect of a chitosan solution according to the invention on Escherichia coli cultivated on an agar plate after 12 hours of incubation;

[0073] FIG. 4 Illustrates the effect of a chitosan solution according to the invention on Staphylococcus carnosus cultivated on an agar plate after 12 hours of incubation;

[0074] FIG. 5 Shows centrifuge tubes with samples of a Tryptic Soy Broth medium containing a chitosan preparation according to the invention (A) and free of chitosan (B);

[0075] FIG. 6 Shows the samples of FIG. 3 after 16 hours of incubation following the inoculation with Escherichia coli;

[0076] FIG. 7 Illustrates the effect of a chitosan fiber according to the invention on Staphylococcus carnosus cultivated on an agar plate after overnight incubation; and

[0077] FIG. 8 illustrates the controlled dissolution of a chitosan according to the invention by applying gauze soaked with acetate buffered solution;

[0078] FIG. 9 shows a tissue dressing comprising a chitosan according to the invention before (9a), during (9b), and after (9c) the application of the detachment solvent;

[0079] FIG. 10 schematically illustrates a wound to which a liquid chitosan containing composition according to the inventions has been applied;

[0080] FIG. 11 schematically illustrates a wound to which a solid chitosan material according to the inventions has been applied;

[0081] FIG. 12 schematically illustrates a wound to which a non-perforated wound dressing according to the invention has been applied;

[0082] FIG. 13 schematically illustrates a wound to which a perforated wound dressing according to the invention has been applied;

[0083] FIG. 14 illustrates the cell viability of keratinocytes on chitosan materials of various degrees of acetylation, relative to tissue culture polystyrene controls (PS=100%); and

[0084] FIG. 15 shows microscopic images of human keratinocytes grown on chitosan materials of various degrees of acetylation (A: 1.5%; B: 14.5%).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

1. 1H NMR Spectroscopy

[0085] The chitosan used in the examples below was obtained in the form of fine flakes from Cognis (Germany). The degree of acetylation (DA) was determined by 1H NMR spectroscopy. FIG. 1 shows an 1H NMR spectrum obtained from this commercially available chitosan. FIG. 2 shows a corresponding 1H NMR spectrum obtained from chitosan deacetylated after further hydrolysis steps applied to the commercial product as described further below. In both cases, chitosan was analyzed in a mixture of 0.25% DCI in 020 at a chitosan concentration of approximately 0.5% (w/v). The spectra were recorded using a Bruker AC200 spectrometer. NMR chemical shifts (6, in ppm) were referenced to the signal of HDO (6=4.8 ppm). The DA, calculated by comparing the integrated area under the peaks associated with H2-H6 of the D-glucosamine subunit with that of the methyl group, was determined as 14.5% for the native chitosan as purchased, and 1.5% for the deacetylated native chitosan.

2. Synthesis of Low-DA Chitosan

[0086] For further hydrolysis, 50 g (grams) of the chitosan flakes as obtained from the supplier Cognis were placed in a glass container, and 500 g of a 45% aqueous sodium hydroxide solution were added. The glass container was well shaken to mix the components, and placed in an oven for 2 hours at 100° C. It was then removed from the oven, and 500 mL (milliliters) of distilled water were added. The mixture was filtered through a glass frit. Then, the chitosan was washed with distilled water until the pH of the filtrate reached 6.5, and dried at 100° C. for 4 h (hours). This hydrolysis treatment was then repeated, resulting in 42 g of deacetylated native chitosan having a degree of acetylation of 1.5% as determined by 1H NMR spectroscopy.

3. Preparation of Chitosan Solutions (Solutions A1, A2 and A3)

[0087] 15 g of the thus obtained native chitosan having a DA of 1.5% were dissolved in 500 mL of a 2% aqueous acetic acid by gently shaking for 24 h. Below, the material is referred to as chitosan solution A1. A second chitosan solution with a chitosan concentration of 1.5% was obtained by addition of 500 ml of distilled water to 500 ml of solution A1. Below, the material is referred to as chitosan solution A2.

[0088] A third chitosan solution with a chitosan concentration of 0.75% was obtained by addition of 1500 ml of distilled water to 500 ml of solution A1. Below, the material is referred to as chitosan solution A3.

4. Preparation of a First Example of a Solid Film-Type Chitosan (Chitosan B)

[0089] Two portions of 144 mL each of solution A2 were poured into two square-shaped moulds, 24×24 cm.sup.2 (square centimetres) in size, and left in a dust-free environment for drying at room temperature. The resulting film was removed from the first mould, and sterilized using a 10 kGy (kilogray) electron beam. An approximately 80 pm thick transparent film essentially consisting entirely of deacetylated native chitosan acetate salt was obtained. Below, the material is referred to as chitosan B.

5. Preparation of a Second Example of a Solid Film-Type Chitosan (Chitosan C)

[0090] The dried film from the second mould was placed for 2 hours in a bath containing a solution of 1.5% ammonia in methanol/water 90/10 (v/v). The film was then removed from the bath and dried by storage at room temperature. The film was sterilized using a 10 kGy electron beam. An approximately 80 pm thick transparent film essentially consisting entirely of deacetylated native chitosan base was obtained. Below, the material is referred to as chitosan C.

6. Preparation of a Third Example of a Solid Film-Type Chitosan (Chitosan D1)

[0091] 144 mL of solution A2 was filtered first through a glass fiber filter (pore size approximately 1 pm), and then through a 0.22 μm filter for sterilization, poured into a square-shaped mould, 24×24 cm.sup.2 in size, and left in a dust-free environment for drying at room temperature. After 3 days of storage, the resulting film was removed from the mould, transferred in a plastic bag that was then tightly sealed, and sterilized using a 25 kGy (kilogray) electron beam. An approximately 80 pm thick transparent film essentially consisting entirely of deacetylated chitosan acetate salt was obtained. Below, the material is referred to as chitosan

7. Preparation of a Fourth Example of a Solid Film-Type Chitosan (Chitosan D2)

[0092] In a slightly modified procedure, 4% (w/w) glycerol was added to the filtered solution of the previous example before pouring it into the square-shaped mould. Subsequent treatment as described above for chitosan D1 resulted in a transparent film essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol. Below, the material is referred to chitosan D2.

8. Preparation of a Fifth Example of a Solid Film-Type Chitosan (Chitosan D3)

[0093] In a further modified procedure, the glycerol containing solution of deacetylated chitosan was poured into a square-shaped mould which was covered with a two-layered film consisting of polyurethane/polyethylene (Platilon U073 PE, Epurex, Bomlitz/Germany), with the polyurethane side up and the polyethylene side fixed to the bottom of the mould. Subsequent treatment as described above for chitosan D1 resulted in a transparent film essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol which was attached to the polyurethane/polyethylene support film. Below, the material is referred to as chitosan D3. Upon use, the polyethylene layer is removed. The remaining polyurethane layer is gas-permeable.

9. Preparation of a Sixth Example of a Solid Film-Type Chitosan (Chitosan D4)

[0094] In a slightly modified procedure to the preparation of chitosan film D2, 1% (w/w) glycerol was added to the filtered solution before pouring it into the square-shaped mould. Subsequent treatment as described above for chitosan D1 resulted in a transparent film essentially consisting entirely of a mixture of deacetylated chitosan acetate salt and glycerol. Below, the material is referred to chitosan D4.

0. Preparation of Two Examples of Chitosan with Higher DA (Chitosans E1 and F1)

[0095] Two further examples of chitosan were produced by the procedure leading to chitosan D1 with the only modification that in one case the hydrolysis step was shortened, leading to a DA of 4% (chitosan E1), and in the other case the hydrolysis step was entirely omitted, leading to a DA of 16% (chitosan F1).

11. Inhibition of Escherichia coli Growth on Agar

[0096] An agar plate was coated with 200 μL of a suspension (appr. 10.sup.8 cells per ml) of the gram-positive bacterium Escherichia coli. A circular cavity was made in the plate and 100 μL of solution A1 was pipetted into the cavity. The plate was then incubated at 37° C. for 12 hours. The result of the experiment is shown in FIG. 3. A clear circular inhibition zone can be seen around the hole that contains the chitosan solution, indicating antibiotic activity of the chitosan solution against Escherichia coli.

12. Inhibition of Staphylococcus carnosus Growth on Agar

[0097] The above experiment was repeated with the gram-negative bacterium Staphylococcus carnosus. An agar plate was coated with 200 μL of a suspension (appr. 10.sup.8 cells per ml) of the bacterium and the solution A was pipetted into a cavity in the plate. The plate was then incubated at 37° C. for 12 hours. The result of the experiment is shown in FIG. 4. A clear circular inhibition zone can be seen around the hole that contains the chitosan solution, indicating antibiotic activity of the chitosan solution against Staphylococcus carnosus.

13. Inhibition of Escherichia coli Growth in Liquid Medium

[0098] In order to test the antibiotic activity of the chitosan preparation according to the invention, 5 mL of solution A1 was inoculated with 100 pL of overnight activated E. coli (optical cell density 00500=5) at room temperature in a Tryptic Soy Broth liquid medium. The sample was filled in a centrifuge tube and is shown in FIG. 5 on the left (sample A). For comparison, 5 mL of TSB medium free of chitosan was inoculated with 100 pL of overnight activated E. coli (optical cell density 00500=5). A centrifuge tube filled with the chitosan-fee sample is shown on the right in FIG. 5 (sample B). The inoculation was followed by 16 hours of incubation at room temperature under shaking.

[0099] The result of the experiment is shown in FIG. 6. In the medium without chitosan (sample B), a considerable increase in the turbidity of the solution was observed, indicating bacteria growth. In contrast, the chitosan containing solution (sample A) shows no discernable increase in the turbidity. The experiment thus indicates antibiotic activity of the chitosan solution against Escherichia coli in the Tryptic Soy Broth liquid medium.

14. Inhibition of Escherichia coli Growth in Liquid Medium

[0100] In another experiment to test the antibiotic activity of the chitosan preparation according to the invention, solution A1 was inoculated with E. coli K 12 (OD 0.2) in a lysogene broth medium for 15 hours at 37 deg C. under shaking. Solution A1 was added in different volumes to yield final chitosan concentrations as specified in Table 1.

[0101] The results of this experiment are summarized in Table 1

TABLE-US-00001 TABLE 1 Chitosan concentration Control 0.75% 0.3% 0.1% 0.03% 0.01% (no chitosan) Observation − − − + ++ +++ − no growth; +, ++ , +++ growth in increasing order

[0102] The addition of 0.1% Chitosan to E. coli in exponential growth phase starting with an OD of 0.2 for 15 h at 37° C. and shaking (in E. coli optimal growth media) led to a clearing of the solution, the solution remained sterile for an additional observation period of 5 days (RT without shaking).

15. Efficacy of Antimicrobial Preservation

[0103] The efficacy of antimicrobial preservation was tested according to the norm as described in the Ph. Eur. 7.sup.th Edition, Chapter 5.1.3, with solution A3. Two containers were each filled with 20 mL of solution A3 and inoculated with a suspension of either Pseudomonas aeruginosa ATCC 9027 or Staphylococcus aureus ATCC 6538 to give an inoculum of 10.sup.5 to 10.sup.6 microorganisms per mL. The suspension was mixed thoroughly to ensure homogeneous distribution. The inoculated product was maintained at 20° C. to 25° C. under protection from light. A 1 mL sample was removed from each container at zero hour and at the intervals specified in Table 2, and the number of viable microorganisms determined by plate count.

TABLE-US-00002 TABLE 2 CFU per ml of sample Bacteria 0 h 2 d 7 d 14 d 28 d Pseudomonas 8.6 × 10.sup.5 <10 <10 <10 <10 aeruginosa ATCC 9027 Staphylococus aureus 9.2 × 10.sup.5 <10 <10 <10 <10 ATCC 6538

16. Antibiotic Activity of Chitosan Fibers

[0104] To demonstrate the antibiotic activity of chitosan fibers, a chitosan fiber was produced by extrusion of 50 mL of a solution of 4% chitosan in 2% acetic acid, mixed with an equal amount of N-methylpyrrolidone (NMP) through a needle of 50 mm in length and an inner diameter of 1.0 mm. The needle was dipped into a coagulation bath containing a mixture of 2 L (liters) of NMP and 3 mL of 25% aqueous ammonia solution. After completion of the extrusion, the fiber was left in the coagulation bath over night. It was then washed twice in distilled water containing 0.1% by weight of a 25% aqueous ammonia solution for 2 hours, and then dried at room temperature.

[0105] Staphylococcus carnosus was cultured over night at room temperature in a Tryptic Soy Broth growth medium. Subsequently, 500 μL of the thus obtained bacteria suspension was used to coat a Mueller-Hinton Agar plate at pH 5.5. A chitosan fiber section of 4 centimeters in length and 0.2 millimeters in diameter was placed on the plate and incubated over night at 37° C.

[0106] The result of the experiment is shown in FIG. 7. A clear inhibition zone around the chitosan fiber was observed, indication antibiotic activity of the fiber against Staphylococcus carnosus.

17. Treatment of a MRSA Infection

[0107] A 39 year old male patient who suffers from cerebro-orbito-facial arterio-venous malformation associated with recurrent severe bleeding from facial wounds was diagnosed with MRSA wound infection. He was treated by spraying chitosan solution A3 daily for 3 weeks onto the facial wounds. After the treatment MRSA viable microorganisms could not be detected anymore and no other microbial infection could be found at the site of the chitosan treatment.

18. Water Uptake of Chitosan C

[0108] Chitosan C, produced as described in the above example, was weighted, and then placed in distilled water for 15 min. The weight of the wet film was compared to the weight of the dry film, and the water uptake was determined to be 72% by weight.

19. Water Uptake of Chitosan D4

[0109] Chitosan D4, produced as described in the above example, was weighted, and then placed in distilled water for 60 min. The weight of the wet film was compared to the weight of the dry film, and the water uptake was determined to be 1217% by weight 7 days after film preparation, and 475% by weight 14 days after film preparation.

20. Dissolution of Chitosan

[0110] Controlled dissolution of chitosans B and C was tested in dissolution experiments using distilled water, 0.9% aqueous sodium chloride solution, and 0.5% acetic acid/acetate buffered solution, respectively. The pH of the solutions was adjusted to the values indicated in Table 2 using appropriate amounts of 1 N hydrochloric acid or sodium hydroxide solutions. Chitosans B and C were cut into rectangular samples having dry weights between 5 and 10 mg each. A gauze soaked with a 100-fold per volume excess of the respective solution to the dry weight of the film was applied to each sample film and the time for complete film dissolution was recorded.

TABLE-US-00003 TABLE 3 Material B Material C Material C pH of the Material B (0.9% aqueous (0.9% aqueous (0.5% acetic dissolution (distilled sodium sodium acid/acetate mixture water) chloride) chloride) buffer) 4.0 n.a. n.a. n.d. 0.5 h 4.5 n.a. n.a. n.d. 0.5 h 5.0 n.a. n.a. n.d. 2 h 5.5 0.1 h 0.5 h n.d. 4 h n.a. = not analyzed n.d. = no dissolution observed after 24 h

[0111] The controlled dissolution experiment with chitosan material C and a mixture of 0.5% acetic acid/sodium acetate (right column in Table 3), is illustrated in FIG. 8. The material has been stained by storage in 0.01% aqueous indigocarmine solution for 1 hour for better visualization. Complete dissolution was observed after 30 minutes at pH 4.0 and 4.5, after 2 hours at pH 5.0, and after 4 hours at pH 5.5, respectively.

21. In Situ Conversion of Water-Soluble Chitosan into Water-Insoluble Chitosan

[0112] Samples of solution A2 and chitosans D1, D2, D3 and D4 were left unsealed on air at room temperature and a humidity of 20-40%. Under these conditions, solution A2 was drying to a solid film within several hours. Complete dissolution in distilled water was analyzed at days 3, 7, and 14. Results are summarized in Table 4.

TABLE-US-00004 TABLE 4 Chitosan Day 3 Day 7 Day 14 A2 soluble insoluble insoluble D1 soluble soluble insoluble D2 soluble insoluble insoluble D3 soluble insoluble insoluble D4 soluble insoluble insoluble

[0113] Similarly, conversion of the water-soluble into the water-insoluble form of dried solution A2 and chitosans D1, D2, D3 and D4 was observed after application of the wound dressing on human skin. In the case of D3, the chitosan was applied to the skin with its chitosan side. Conversion of the water-soluble into the water-insoluble form dried solution A2 and chitosans D1, D2, D3 and D4 was also observed after alkaline treatment or storage in an alkaline atmosphere.

22. Dissolution of Chitosan with Detachment Solvent

[0114] Chitosans D1, E1 and F1 were dissolved by storage in a 2% acetic acid/acetate buffered solution. The pH of the storing solutions was adjusted to the values indicated in Table 5 using appropriate amounts of 10% sodium hydroxide solutions. Films D1, E1 and F1 made from chitosans with different degrees of acetylation (DA) were left on air for 14 days for conversion into the water-insoluble form, cut into rectangular samples of 1×1 cm.sup.2 size and stored in approximately 10 mL of the respective solution, and the time for complete film dissolution was recorded.

TABLE-US-00005 TABLE 5 DA = 16% DA = 4% DA = 1.5.sup.1)/o pH Time for complete dissolution (min) 4.0 5 10 1 4.5 15 15 2 5.0 30 15 15 5.5 60 60 30 6.0 60 overnight overnight

[0115] In another dissolution experiment, a film C (3×1 cm.sup.2) was fixed on the inside of a commercial perforated band-aid (5×2 cm) which was then fixed on a Petri dish. An acetic acid/acetate buffered solution (pH 5.5) was added dropwise through the perforations of the band-aid causing the wound dressing material film to dissolve. The side of the tissue dressing comprising the film is shown in FIG. 9a before and in FIG. 9c after application of the solution. The application of the solution to the band-aid side of the wound dressing is shown in FIG. 9b.

23. Analysis of Non-Soluble and Soluble Fractions of Low-DA Chitosan

[0116] 0.1 g samples of chitosan with a DA of 1.5%, prepared as described in Example 2, were placed each in 10 ml of phosphate-buffered solution and kept at room temperature under gentle shaking at pH 7.4. At the time points given in Table X, the mixture was filtered, and the non-soluble chitosan remaining in the filter was thoroughly washed and finally dried. The amount of the non-soluble chitosan was determined gravimetrically on a laboratory scale. The results are summarized in Table 6:

TABLE-US-00006 TABLE 6 Time storage PBS 1 hour of 8 hours in 1 day 3 days 10 days non-soluble chitosan 95.5 93.5 84 85 84.5 fraction (%) soluble chitosan 4.5 6.5 16 15 15.5 fraction (%)

24. Cell Viability on Low-DA Chitosan

[0117] Chitosan films having DAs of 1.5, 4.0, and 14.5%, respectively, were placed in 24-well cell culture plates, and human HaCaT keratinocytes were seeded at a density of 5×10.sup.4 cells per cm.sup.2 and cultured for 2 days. Cell viability was determined using the MTS assay (Promega). After 4 h of MTS incubation with the cells, the light absorbance at 490 nm was measured by an ELISA plate reader and subtracted from that of the controls (without cells) to yield the corrected absorbance. Five samples of each DA were studied. FIG. 14 shows the relative light absorbencies a at 490 nm (PS=100%) for the three samples and a control using polystyrene (PS).

25. Cell Growth on Chitosans of Different DA

[0118] Human keratinocytes (5×10.sup.4 cells per well) were grown in a 24 well plate for 2 days at 37° C. on chitosan films having DAs of 1.5 and 14.5%, respectively. For the tests cells were isolated from human skin and grown until subconfluency was reached. After 24 h the medium (Keratinocyte medium with Supplement Mix, Promocell) was changed in order to remove non-adherent cells. FIG. 15 gives representative examples of cells grown on chitosan films after 1 day of incubation.

[0119] In FIG. 10, schematically a tissue 1 comprising a wound 2 is shown. For better illustration, FIGS. 5 to 8 are not drawn to scale. The liquid chitosan containing composition according to the invention has been applied to the tissue 2 and the constituent water has been allowed to evaporate, leaving behind a film 3 that dresses the tissue 2 including the wound 3. In general, the film 3 is about 10 to 20 pm thick. Advantageously, the film 3 tightly snuggles to the tissue surface 4, including the wound surface 5.

[0120] FIG. 11 schematically shows a chitosan in the form of a solid film 6 that is applied to a tissue 1, comprising a wound 2. The solid film is about 80 pm thick. Cavities 7, 8 between the tissue 1 and the chitosan 6 may be filled with water or exudative fluid.

[0121] In FIG. 12, a tissue dressing 9 comprising a chitosan film 6 of FIG. 11 as a first layer and a silicon film 10 as a second layer is applied to a tissue 1, comprising a wound 2. The silicon film 10 is about 50 pm tick. Again, cavities 7, 8 between the tissue 1 and the tissue dressing material 6 may be filled with water or exudative fluid. Finally, FIG. 13 shows a tissue dressing 11 applied to a tissue 1 comprising a wound 2, the tissue dressing 11 differing from that 9 of FIG. 12 in that the silicon film 10 is perforated to allow an exchange of air between the tissue 1 and the surrounding though the wound dressing material 6. The perforations have a diameter if between 50 and 100 pm.

[0122] The features described in the above description, claims and figures can be relevant to the invention in any combination. The reference numerals in the claims have merely been introduced to facilitate reading of the claims and are by no means meant to be limiting.