ANTIMICROBIAL COMPOSITION COMPRISING A POLYSACCHARIDE, A STABILIZING AGENT AND TRIIODIDE, METHOD OF PREPARATION THEREOF AND USE THEREOF

Abstract

A composition having antimicrobial activity is provided. The composition comprises a polysaccharide, a stabilizer, and a triiodide, where the triiodide decomposition to iodide and volatile iodine can be significantly suppressed by the presence of the stabilizer. Methods of preparing and using the composition and devices prepared therewith are also disclosed. As compared to liquid forms comprising triiodide, stabilized solid forms can be used for a much wider range of applications due to their shape stability and a significantly smaller volume and weight of total material.

Claims

1.-14. (canceled)

15. An antimicrobial composition, comprising: a polysaccharide component comprising a polysaccharide selected from the group of hyaluronic acid, sodium alginate, oxycellulose, carboxymethyl cellulose, and hydroxyethyl cellulose or a chemically modified hyaluronic acid derivative comprising at least one of a substituted OH group of formula OC(O)R.sup.2 and a substituted C(O)OH group of formula C(O)OR.sup.2 with each R.sup.2 independently being an a linear or aromatic group having 1-15 carbon atoms, the polysaccharide or chemically modified hyaluronic acid derivative having a molecular weight in a range from 510.sup.3 to 110.sup.6 g.mol.sup.1; a thiazole-based stabilizer selected from the group of thiamine, oxythiamine hydrochloride, 5-(2-hydroxyethyl)-3,4-dimethyl thiazolium iodide, 3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium bromide, and combinations thereof; and sodium or potassium triiodide.

16. The composition of claim 15, wherein the composition is in a solid form selected from the group of lyophilizate, self-supporting film, non-woven fabric, endless fiber, woven fabric, knitted fabric, plaited fabric, a nanofiber layer, and combinations thereof.

17. A method of preparing the composition of claim 15, the method comprising: combining the thiazole-based stabilizer, the sodium or potassium triiodide, and the polysaccharide component to form the composition.

18. The method of claim 17, comprising: combining the thiazole-based stabilizer and the sodium or potassium triiodide in a molar ratio of the thiazole-based stabilizer to the sodium or potassium triiodide in a range from 1:1 to 5:1 to form a stabilizer/triiodide mixture; and combining the stabilizer/triiodide mixture and the polysaccharide component to form the composition; wherein the sodium or potassium triiodide is utilized in an amount of from 0.2 to 10% by weight, based on a total weight of the polysaccharide component.

19. The method claim 18, further comprising: combining the polysaccharide or chemically modified hyaluronic acid derivative and a water/acetic acid solvent mixture comprising water and acetic acid in a volume ratio of from 20:1 to 200:1, thereby give the polysaccharide component as a polysaccharide/solvent mixture, where the polysaccharide or chemically modified hyaluronic acid derivative is present in an amount of from 0.2 to 6 wt. % based on a total weight of the polysaccharide/solvent mixture; wherein combining the stabilizer/triiodide mixture and the polysaccharide component is further defined as combining the stabilizer/triiodide mixture and the polysaccharide/solvent mixture to form the composition.

20. The method of claim 19, wherein the water/acetic acid solvent mixture comprises a volume ratio of water to acetic acid solvent of 100:1.

21. The method of claim 18, further comprising: combining the stabilizer/triiodide mixture and an ethanol/water solvent mixture to form a stabilizer/triiodide/solvent solution; wherein combining the stabilizer/triiodide mixture and the polysaccharide component is further defined as applying the stabilizer/triiodide/solvent solution to the polysaccharide or chemically modified derivative thereof to form the composition.

22. The method of claim 21, wherein the stabilizer/triiodide/solvent solution is applied to the polysaccharide component over an application time of from 10 minutes to 72 hours and at a temperature of from 5 to 40 C.

23. The method of claim 21, wherein applying the stabilizer/triiodide/solvent solution to the polysaccharide component comprises spraying the stabilizer/triiodide/solvent solution onto the polysaccharide or chemically modified hyaluronic acid derivative or immersing the polysaccharide or chemically modified hyaluronic acid derivative in the stabilizer/triiodide/solvent solution for a time period of from 5 to 15 hours.

24. The method of claim 21, wherein the sodium or potassium triiodide is present in the stabilizer/triiodide/solvent solution in an amount of from 0.2 to 10% by weight based on a total weight of the stabilizer/triiodide/solvent solution, wherein the thiazole-based stabilizer and the sodium or potassium triiodide are combined in a molar ratio of from 1:1 to 5:1, and wherein the volume ratio of ethanol to water of the ethanol/water solvent mixture is from 3:1 to 9:1.

25. The method of claim 24, wherein the thiazole-based stabilizer and the sodium or potassium triiodide are combined in a molar ratio of from 1.1:1.

26. A medical device comprising the composition prepared according to claim

17.

27. The medical device of claim 26, wherein the composition is in a final form selected from the group of lyophilizate, self-supporting film, nanofibre layer, non-woven fabric, fiber, knitted fabric, woven fabric, plaited fabric, and combinations thereof.

28. The medical device of claim 26, wherein the medical device is in a form of a wound dressing or an implantable medical device.

29. A method of utilizing the composition of claim 15 for preparing a wound dressings, the method comprising: providing the composition; and preparing the wound dressing utilizing the composition.

30. A method of utilizing the composition of claim 15 for preparing an implantable medical device, the method comprising: providing the composition; and preparing the implantable medical device utilizing the composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other advantages of the disclosed subject matter will be readily appreciated, as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

[0040] FIG. 1 is an image illustrating use of a non-limiting embodiment of an antimicrobial composition;

[0041] FIG. 2 is another image illustrating use of a non-limiting embodiment of the antimicrobial composition; and

[0042] FIG. 3 is another image illustrating use of a non-limiting embodiment of the antimicrobial composition.

DETAILED DESCRIPTION

[0043] An antimicrobial composition comprising a polysaccharide or a derivative thereof or a mixture of polysaccharides and/or derivatives thereof, a stabilizing agent and sodium or potassium triiodide is provided. In various embodiments, the composition results in the stabilization of various types of solid forms containing the polysaccharide and/or its chemically modified derivative and/or their mixture and the iodine in the form of a triiodide anion (I.sub.3.sup.). As stabilizing agents, or stabilizers, significantly suppressing the decomposition of the triiodide anion to iodine (I.sub.2) and iodide (I.sup.), the cationic heterocyclic compounds of general formula X may be successfully used,

##STR00004## [0044] wherein [0045] R is -alkyl, aromatic, heteroaromatic, linear or branched chain C.sub.1-C.sub.30, optionally containing N or O atoms, [0046] R.sup.1 is -alkyl, aromatic, heteroaromatic, linear or branched chain C.sub.1-C.sub.30, optionally containing N or O atoms, or H, where R.sup.1 in the compound of the formula X are independently the same or different, and [0047] Y is a chloride, bromide or iodide anion.

[0048] A method of preparation is also provide. The method may be to prepare solid forms. Two procedures can be used. However, the method of preparation is not limited to these two procedures. Procedure 1: The triiodide and the stabilizer are sorbed on the surface of the finished final form. Procedure 2: The triiodide and the stabilizer are added to the system before producing the final form.

[0049] In certain embodiments, the difference between Procedures 1 and 2 is that in the Procedure 2 the triiodide anion with the stabilizer are distributed in the bulk of material more homogeneously, whereas in the Procedure 1 the triiodide anion with the stabilizer are preferentially distributed on the surface of the respective form.

[0050] As used herein, the term form refers to types of materials, such as, for example, thin film, lyophilizate, staple fiber layer, endless fiber, woven fabric, plaited fabric or nanofibrous layer.

[0051] Furthermore, the invention relates to the applications of the prepared solid forms in the fields where a biocompatible and biodegradable material with an antiseptic effect is required. These areas include, but are not limited to, wound dressings or implantable medical devices. FIGS. 1 and 2 provide non-limiting and exemplary comparisons of antimicrobial activity of hyaluronic acid (HA) based lyophilizates prepared by Procedure 2 (the triiodide and the stabilizer are distributed more homogeneously). Triiodide-free materials (HA-TH, HA-BTH, HA-B1, a HA) were tested as controls, exhibiting no inhibitory activity. Materials with the antimicrobial triiodide (HA-TH-I.sub.3, HA-BTH-I.sub.3, a HA-B1-I.sub.3) inhibited the growth of microorganisms. All materials were tested for Escherichia coli (FIG. 1) and Staphylococcus aureus (FIG. 2) strains.

[0052] FIG. 3 provides another non-limiting and exemplary comparison of wound healing effect of the HA-vitamin B1-triiodide lyophilizate prepared in Example 13 (in the figure the portion of the wound healed by this preparation is indicated as HyB.sub.1) and of an antimicrobial octenidine-containing lyophilizate based on hyaluronan (in the figure is indicated as SL) at 0, 2 and 5 days in a patient with an open wound on a leg (process described in Example 42). The figure shows a comparable efficacy of both materials.

EXAMPLES

[0053] DS=degree of polysaccharide substitution=100%*(molar amount of a modified polysaccharide unit)/(molar amount of polysaccharide repeating units), as determined by NMR (see, e.g. Kettou et al. CZ Application No. PV 2009-399).

[0054] The term equivalent (equiv) used herein refers to the repeating unit of the respective polysaccharide, unless otherwise indicated.

[0055] Percentages are reported as percentage by weight, unless otherwise indicated.

[0056] The amount of active iodine in %means an equivalent of oxidation activity rate of the material, which is equivalent to the oxidation activity of the material with the corresponding weight percentage of I.sub.2. Determined by standard redox titration with sodium thiosulphate.

[0057] The molecular weight of polysaccharides is weight average molecular weight determined by SEC-MALLS method.

Example 1

[0058] Preparation of Hyaluronan Ethyl Ester

[0059] To a solution of hyaluronan (1 g, 300 kg.mol.sup.1) in 40 mL of water, NaOH was added until pH =9. Then 20 mL of dimethyl sulfoxide and 0.08 mL of ethyl iodide were added and the mixture was stirred for 3 days at 45 C. Subsequently, the resulting mixture was precipitated by 140 mL of 100% isopropanol, the solids were filtered off, washed by isopropanol and dried under vacuum. The product (897 mg) was analyzed by NMR.

[0060] DS of ester was 6% (determined by NMR).

Example 2

[0061] Preparation of Hyaluronan Benzyl Ester

[0062] To a solution of hyaluronan (1 g, 300 kg.mol.sup.1) in 40 mL of water, NaOH was added until pH =9. Then 20 mL of dimethyl sulfoxide and 0.08 mL of benzyl bromide were added and the mixture was stirred for 4 days at 20 C. Subsequently, the resulting mixture was precipitated by 140 mL of 100% isopropanol, the solids were filtered off, washed by isopropanol and dried under vacuum. The product (920 mg) was analyzed by NMR.

[0063] DS of ester was 3% (determined by NMR).

Example 3

[0064] Preparation of Lauroyl Hyaluronan

[0065] To a solution of hyaluronan (5 g, 250 kg.mol.sup.1) in 100 mL of distilled water, 70 mL of tetrahydrofurane, 4 equivalents of triethylamine and 0.1 equivalents of 4-dimetylaminopyridine were added. Concurrently, lauric acid (4 equivalents) was dissolved in 30 mL of tetrahydrofurane and 7 mL of triethylamine and to this solution 4.8 mL of ethyl-chloroformiate was added at 0-5 C. in 15 minutes. The suspension formed was filtered into the hyaluronan solution and the reaction was stirred for 5 hours at 20 C. Subsequently, the resulting solution was precipitated by an addition of 400 mL of 100% isopropanol, washed with 80% isopropanol, then with 100% isopropanol. The precipitate was dried at 40 C. for 2 days. The degree of substitution was determined by NMR to be 37%.

Example 4

[0066] Preparation of Palmitoyl Hyaluronan

[0067] To a solution of hyaluronan (10 g, 250 kg.mol.sup.1) in 300 mL of distilled water, 300 mL of tetrahydrofurane was added. Subsequently 2.5 equivalents of triethylamine, 0.04 equivalents of 4-dimethylaminopyridine and 2 equivalents of palmitic acid anhydride were added to this solution. The resulting solution was stirred at laboratory temperature for 3 hours, then was precipitated by 1 L of 100% isopropanol, washed with 80% isopropanol and dried at 40 C. for 2 days. The degree of substitution was 30% (determined by NMR).

Example 5

[0068] Preparation of Thiamine-KI.sub.3 Solution in Ethanol/Water 3/1

[0069] 150 mg of I.sub.2 and 225 mg of KI were dissolved in 21 mL of ethanol. 210 mg of thiamine hydrochloride were dissolved in 7 mL of distilled water. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 6

[0070] Preparation of Thiamine-KI.sub.3 Solution in Ethanol/Water 6/1

[0071] 150 mg of I.sub.2 and 225 mg of KI were dissolved in 25.7 mL of ethanol. In 4.3 mL of distilled water, 210 mg of thiamine hydrochloride were dissolved. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 7

[0072] Preparation of Thiamine-KI.sub.3 Solution in Ethanol/Water 9/1

[0073] 150 mg of I.sub.2 and 225 mg of KI were dissolved in 27 mL of ethanol. 210 mg of thiamine hydrochloride were dissolved in 3 mL of distilled water. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 8

[0074] Preparation of Thiamine-NaI.sub.3 Solution in Ethanol/Water 3/1

[0075] 150 mg of I.sub.2 and 203 mg of NaI were dissolved in 21 mL of ethanol. 210 mg of thiamine hydrochloride were dissolved in 7 mL of distilled water. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 9

[0076] Preparation of Thiamine-NaI.sub.3 Solution in Ethanol/Water 6/1

[0077] 150 mg of I.sub.2 and 203 mg of NaI were dissolved in 25.7 mL of ethanol. 210 mg of thiamine hydrochloride were dissolved in 4.3 mL of distilled water. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 10

[0078] Preparation of Thiamine-NaI.sub.3 Solution in Ethanol/Water 9/1

[0079] 150 mg of I.sub.2 and 203 mg of NaI were dissolved in 27 mL of ethanol. 210 mg of thiamine hydrochloride were dissolved in 3 mL of distilled water. Both solutions were mixed at 20 C. and stored at 0-5 C.

Example 11

[0080] Preparation of Hyaluronan Ethyl Ester-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0081] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of the hyaluronan derivative prepared according to the Example 1 (0.4 g) in 100 mL of distilled water and 0.5 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4.2%.

Example 12

[0082] Preparation of Hyaluronan Benzyl Ester-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0083] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of the hyaluronan derivate prepared according to the Example 2 (0.4 g) in 100 mL of distilled water and 5 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4%.

Example 13

[0084] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0085] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4%.

Example 14

[0086] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0087] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 200 mL of distilled water and 2 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 179 mg of thiamine hydrochloride in 3 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 3.5%.

Example 15

[0088] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0089] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 36 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 3.5%.

Example 16

[0090] Preparation of Haluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) Lyophilizate

[0091] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 80 kg.mol.sup.1) in 20 mL of distilled water and 0.1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 36 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 2%.

Example 17

[0092] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) LyophilizateCoating

[0093] Hyaluronan in the form of lyophilizate was completely immersed in a solution of NaI.sub.3 in ethanol/water 3/1 (Example 8) for 24 hours at 20 C. Then the lyophilizate was immersed in isopropanol for 2 seconds, pulled out and dried by applying the filter paper from both sides of the material. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1.5%.

Example 18

[0094] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) LyophilizateCoating

[0095] Hyaluronan in the form of lyophilizate was completely immersed in a solution of NaI.sub.3 in ethanol/water 9/1 (Example 10) for 24 hours at 40 C. Then the lyophilizate was immersed in isopropanol for 2 seconds, pulled out and dried by applying the filter paper from both sides of the material. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 2%.

Example 19

[0096] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) LyophilizateCoating

[0097] Hyaluronan in the form of lyophilizate was completely immersed in a solution of Kb in ethanol/water 6/1 (Example 6) for 10 minutes at 40 C. Then the lyophilizate was immersed in isopropanol for 2 seconds, pulled out and dried by applying the filter paper from both sides of the material. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1.5%.

Example 20

[0098] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) LyophilizateCoating

[0099] Hyaluronan in the form of lyophilizate was completely immersed in a solution of Kb in ethanol/water 9/1 (Example 7) for 48 hours at 5 C. Then the lyophilizate was immersed in isopropanol for 2 seconds, pulled out and dried by applying the filter paper from both sides of the material. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 2%.

Example 21

[0100] Preparation of Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) LyophilizateCoating

[0101] Hyaluronan in the form of lyophilizate was completely immersed in a solution of KI.sub.3 in ethanol/water 3/1 (Example 5) for 10 hours at 20 C. Then the lyophilizate was immersed in isopropanol for 2 seconds, pulled out and dried by applying the filter paper from both sides of the material. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1%.

Example 22

[0102] Preparation of Hyaluronan-Thiazolium Iodide-I.sub.3 (HA-TH-I.sub.3) Lyophilizate

[0103] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 35 mg of 5-(2-hydroxyethyl)-3,4-dimethyl thiazolium iodide in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 3%.

Example 23

[0104] Preparation of Hyaluronan-Benzyl Thiazolium Bromide-I.sub.3 (HA-BTH-I.sub.3) Lyophilizate

[0105] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 37 mg of 3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium bromide in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 3.5%.

Example 24

[0106] Preparation of Hyaluronan-Oxythiamine-I.sub.3 (HA-OB1-I.sub.3) Lyophilizate

[0107] 4.0 mg of KI and 2.7 mg of I.sub.2 were added to a solution of hyaluronan (0.4 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 45 mg of oxythiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 0.5%.

Example 25

[0108] Preparation of a Non-Woven Fabric from Staple Fibers Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3)Coating

[0109] 1% aqueous HA solution was extruded through a nozzle with an inner diameter of 0.6 mm into a non-stationary coagulation bath consisting of 100% isopropanol at room temperature, which circumfluents the nozzle at 3 m.s.sup.1. The solution is precipitated into 3-4 cm long fibers. The crude fibers are shortened in a blender for 30 seconds at a ratio of 1 g of fibers per 1 liter of coagulation bath. The resulting fibrous dispersion having a fiber length of 3-4 mm is filtered through a substrate consisting of PAD knitted fabric and dried on a drying plate allowing fixation of the shape of the resulting fabric during drying. The resulting layer was separated from the substrate as a self-supporting layer. The fabric so formed was formatted to the desired size and immersed in a solution of NaI.sub.3+B1 in ethanol/water 9/1 (Example 10). The fabric was placed on a shaker and subjected to NaI.sub.3+B1 solution for 60 minutes at 20 C. and shaking speed of 80 oscillations per minute. The treated fabric is dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1.8%.

Example 26

[0110] Preparation of a Non-Woven Fabric from Staple Fibers from Palmitoyl Hyaluronan-Thiamin-I.sub.3 (HA-B1-I.sub.3)Coating

[0111] 1% palmitoyl HA solution (prepared as described in Example 4), dissolved in a mixture of water and isopropanol in volume ratio 1:1, was extruded through a nozzle with an inner diameter of 0.6 mm into a non-stationary coagulation bath consisting of 90% isopropanol at room temperature, which circumfluents the nozzle at 3 m.s.sup.1. The solution is precipitated into 3-4 cm long fibers. The crude fibers are dehydrated in 100% acetone and shortened in a blender for 10 seconds at a ratio of 0.9 g of fibers per 1 liter of 100% isopropanol. The resulting fibrous dispersion having a fiber length of 3-4 mm is filtered through a substrate consisting of PAD knitted fabric and dried at 40 C. on a drying plate allowing fixation of the shape of the resulting fabric during drying. The resulting layer was separated from the substrate as a self-supporting layer. The fabric so formed was formatted to the desired size and immersed in a solution of NaI.sub.3+B1 in ethanol/water 9/1 (Example 10). The fabric was placed on a shaker and exposed to NaI.sub.3+B1 solution for 70 minutes at 20 C. and shaking speed of 80 oscillations per minute.

[0112] The treated fabric is dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1.5%.

Example 27

[0113] Preparation of the Nanofiber Layer of Hyaluronan-Thiamin-I.sub.3 (HA-B1-I.sub.3)Coating

[0114] An aqueous solution of the following composition was prepared to prepare a nanofibre layer containing hyaluronic acid. The concentration of HA having the molecular weight of 82 kg.mol.sup.1 in the dry matter was 80%, the concentration of polyethylene oxide with the molecular weight of 400 kg. mol.sup.1 was 5%, the concentration of polyvinyl alcohol with a molecular weight of 200 kg.mol.sup.1 was 15%, the concentration of the total dry matter was 6%. The solution was filled into a syringe and electrostatically spun onto a plate collector using a needle-free linear nozzle, voltage of 45 kV and distance of 18 cm between the emitter and the collector. The fibers have the dimension of 11027 nm. This material was completely immersed in a solution of NaI.sub.3+B1 in ethanol/water 6/1 (Example 9) for 48 hours at 20 C. Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 8%.

Example 28

[0115] Preparation of a Self-Supporting Film from Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3)Coating

[0116] Preparation of the film was carried out in a specialized drying apparatus where the film was dried in closed space. The device is equipped with a bottom and top plate with adjustable temperature. The device is further described in (Foglarova et al., CZ Application No. PV2015-166, Foglarova M. et al., Carbohydrate Polymers 2016, 144, 68-75). 240 mg of sodium hyaluronate having the molecular weight of 330 kg.mol.sup.1 was dissolved in 24 mL, of demineralized water and the mixture was stirred for at least 18 hours. The solution was then charged on a pad of the drying apparatus (hydrophobized glass) and dried in closed space at the bottom plate temperature of 50 C. and the top plate temperature of 20 C. The drying time was 20 hours. After drying, the film was removed from the pad and stored for further use. This material was then completely immersed in a solution of NaI.sub.3+B1 in ethanol/water 6/1 (Example 9) for 72 hours at 20 C. Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 0.1%.

Example 29

[0117] Preparation of a Self-Supporting Film from Palmitoyl Hyaluronan-Thiamine-I.sub.3 (palmHA-B1-I.sub.3)Coating

[0118] The film preparation device is described in the Example 28. 240 mg of palmitoyl derivative of sodium hyaluronan, described in Example 4, was dissolved in 24 mL of an aqueous solution of 2-propanol (50% w/w) and the mixture was stirred for at least 18 hours. The solution was then dispensed on a pad of the drying apparatus (hydrophobized glass) and dried in closed space at the bottom plate temperature of 50 C. and the top plate temperature of 40 C. The drying time was 20 hours. After drying, the film was removed from the pad and stored for further use. This material was then completely immersed in a solution of NaI.sub.3+B1 in ethanol/water 6/1 (Example 9) for 72 hours at 20 C. Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be be 0.2%.

Example 30

[0119] Preparation of a Self-Supporting Film from Lauroyl Hyaluronan-Thiamine-I.sub.3 (laurHA-B1-I.sub.3)Coating

[0120] The film preparation device is described in the Example 28.240 mg of lauroyl derivative of sodium hyaluronan, described in Example 3, was dissolved in 24 mL of an aqueous solution of 2-propanol (50% w/w) and the mixture was stirred for at least 18 hours. The solution was then charged on a pad, of the drying apparatus (hydrophobized glass) and dried in closed space at the bottom plate temperature of 50 C. and the top plate temperature of 40 C. The drying time was 20 hours. After drying, the film was removed from the pad and stored for further use. This material was then completely immersed in a solution of NaI.sub.3+B1 in ethanol/water 6/1 (Example 9) for 24 hours at 20 C. Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 0.3%.

Example 31

[0121] Preparation of the Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) Staple Fiber LayerCoating

[0122] The non-woven fabric was produced by combining staple microfibers that are prepared by the wet spinning method in a non-stationary coagulation bath. Hyaluronic acid of the molecular weight 1,000 kg.mol.sup.1 was used. The coagulation bath consists of isopropanol. The precipitated fibers were then shortened by grinding, filtered to a substrate, dried and compressed. The resulting layer was separated from the substrate as a self-supporting layer, This material was then completely immersed in a solution of NaI.sub.3B 1 in ethanol/water 9/1 (Example 10) for 1 hour at 20 C. Then it was dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 1.8%.

Example 32

[0123] Preparation of Knitted Fabric from Hyaluronan-Thiamine-I.sub.3 (HA-B1-I.sub.3) FibersCoating

[0124] An endless fiber of hyaluronan having the molecular weight of 600 kDa was used to produce the knitted fabric; the fiber fineness was 10 tex, the strength 1.1 N and the ductility 9.8%. Three fibers were pooled and twisted on a ring machine at feeding 10 m/min and spindle speeds of 3,000 min.sup.1; the resulting twist had the value of 300 m.sup.1. A two-sided tricot knitted fabric with closed stitches was knitted from threads on a double bed warp knitting machine. The knitted fabric was then washed in ethanol at 40 C. for 20 minutes. The resulting knitted fabric strip was 11 mm wide, had a mas per unit area of 99 g.m.sup.2 and stitches density 36 cm.sup.2. This material was then completely immersed in a solution of KI.sub.3+B1 in ethanol/water 6/1 (Example 6) for 24 hours at 20 C. Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 0.1%.

Example 33

[0125] Preparation of Knitted Fabric from Palmitoyl Hyaluronan-Thiamine-I.sub.3 (palmHA-B1-I.sub.3) FibersCoating

[0126] An endless fiber of palmitoyl hyaluronan having the molecular weight of 320 kDa and the degree of substitution 30% (determined by NMR) was used to produce a knitted fabric; the fiber fineness was 9 tex, the strength of 0.6 N and the ductility of 21%. Three fibers were pooled and twisted on a ring machine at feeding 10 m/min and spindle speeds 3,000 min.sup.1; the resulting twist had the value 300 m.sup.1. A two-sided tricot knitted fabric with closed stitches was knitted from threads on a double bed warp knitting machine. The knitted fabric was then washed in ethanol at 40 C. for 20 minutes. The resulting knitted fabric strip was 11 mm wide, had a mas per unit area of 91 g.m.sup.2 and stitches density 36 cm.sup.2. This material was then completely immersed in a solution of KI.sub.3+B1 in ethanol/water 9/1 (Example 7) for 15 hours at 20 C.

[0127] Then the material was collected and immersed in isopropanol for 2 seconds, collected and dried at laboratory temperature. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 0.3%.

Example 34

[0128] Preparation of Alginate-Thiamine-I.sub.3 Lyophilizate

[0129] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of sodium alginate (0.4 g, Mw 400 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4.4%.

Example 35

[0130] Preparation of Oxycellulose-Thiamine-I.sub.3 Lyophilizate

[0131] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of oxycellulose (0.4 g, Mw 50 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 3.3%.

Example 36

[0132] Preparation of Hydroxyethyl Cellulose-Thiamine-I.sub.3 Lyophilizate

[0133] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of hydroxyethyl cellulose (0.4 g, Mw 720 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 6.1%.

Example 37

[0134] Preparation of Carboxymethyl Cellulose-Thiamine-I.sub.3 Lyophilizate

[0135] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of carboxymethyl cellulose (0.4 g, Mw 250 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4.6%.

Example 38

[0136] Preparation of Oxycellulose/Hyaluronan-Thiamine-I.sub.3 Lyophilizate

[0137] 40 mg of KI and 27 mg of 12 were added to a solution of oxycellulose (0.3 g, Mw 50 kg.mol.sup.1) and hyaluronic acid (0.1 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4%.

Example 39

[0138] Preparation of Alginate/Hyaluronan-Thiamine-I.sub.3 Lyophilizate

[0139] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of sodium alginate (0.3 g, Mw 400 kg.mol.sup.1) and hyaluronic acid (0.1 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid, and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4.3%.

Example 40

[0140] Preparation of Carboxymethyl Cellulose/Hyaluronan-Thiamine-I.sub.3 Lyophilizate

[0141] 40 mg of KI and 27 mg of I.sub.2 were added to a solution of carboxymethyl cellulose (0.3 g, Mw 250 kg.mol.sup.1) and hyaluronic acid (0.1 g, Mw 500 kg.mol.sup.1) in 100 mL of distilled water and 1 mL of acetic acid and the resulting mixture was stirred for 24 hours at laboratory temperature. A solution of 38 mg of thiamine hydrochloride in 1 mL of distilled water was then added, the resulting solution was homogenized, immediately frozen at 50 C. and lyophilized. The amount of the active iodine was determined by reductive titration with sodium thiosulphate to be 4.2%.

Example 41

[0142] In Vitro Antimicrobial Activity Assay (FIGS. 1 and 2):

[0143] Suspensions of individual tested microorganisms were prepared at an approximate concentration of 10.sup.5 CFU/mL. Onto the surface of tryptone soya agar in Petri dishes, 100 L of suspension (approximately 10.sup.4 CFU of microorganisms on the dish) was applied. The suspension was evenly spread over the entire surface of the dish with a sterile loop. After the suspension was absorbed by agar, the tested samples were transferred in a sterile way onto the surface of the agar in the form of squares. The dishes with bacterial test strains were cultured at 37 C. for 24 hours. Lyophilizates with the antimicrobial substance HA-B1-I.sub.3, HA-TH-I.sub.3 and HA-BTH-I.sub.3 (prepared according to the Examples 13, 22, 23) were tested, where analogous lyophilizates without the active substance HA-TH, HA-BTH and lyophilizates with HA alone were used as controls. Squares of the weight of 15-20 mg and approximate dimensions of 15152 mm were prepared, with 0.7-1.3 mg of potassium triiodide or without potassium triiodide. For efficiency testing, a diffusion plate method (2D layout) was chosen. A non-selective soil (tryptone soya agar) was used for cultivation. The square samples were tested on 2 microorganismsEscherichia coli (G-rod) and Staphylococcus aureus (G+coccus). Figures 1 and 2 clearly show a considerably higher efficiency of lyophilizates of the invention in comparison with lyophilizates without triiodide or with HA itself.

Example 42

[0144] Testing of the Tolerance and the Effect on Wound Healing (FIG. 3).

[0145] A one-week analysis was conducted to compare the effect of HA-B1-I.sub.3 lyophilizate (prepared according to the Example 13) on the course of the wound healing. The study was focused primarily on the tolerance of the preparation and the comparison of its efficacy with the standard wound healing agent with a proven effect, which is a dressing containing an active-layer, which is a combination of hyaluronan and the antimicrobial substance octenidine (HA-octenidine). For testing, a bandage of the same composition as HA-octenidine dressing was used, but the active layer was replaced with the HA-B1-I.sub.3 lyophilizate. The study was conducted in a patient where half of the wound was always treated with a HA-B1-I.sub.3 lyophilizate bandage (indicated as HyB.sub.1 in FIG. 3), the second half with a standard HA-octenidine dressing.

[0146] In the patient, the bandage was tolerated without any negative subjective or objective problems. The wound healing course during the observed one-week period was comparable to the healing when HA-octenidine preparation was used. On the wounds covered by HA-octenidine and HA-B1-I.sub.3 lyophilizate no signs of infectious or inflammatory complications were recorded. Thus, it can be concluded that the effect of the new HA-B.sub.1-I.sub.3 complex is comparable to that of the HA-octenidine standard dressing. The preparation according to the invention is advantageous in comparison with the octenidine preparation especially because iodine is considerably more biocompatible compared to octenidine, and therefore much more suitable, for example, for implantable materials.

[0147] The terms comprising or comprise are used herein in their broadest sense to mean and encompass the notions of including, include, consist(ing) essentially of, and consist(ing) of. The use of for example, e.g., such as, and including to list illustrative examples does not limit to only the listed examples. Thus, for example or such as means for example, but not limited to or such as, but not limited to and encompasses other similar or equivalent examples. The term about as used herein serves to reasonably encompass or describe minor variations in numerical values measured by instrumental analysis or as a result of sample handling. Such minor variations may be in the order of 0-25, 0-10, 0-5, or 0-2.5, % of the numerical values. Further, The term about applies to both numerical values when associated with a range of values. Moreover, the term about may apply to numerical values even when not explicitly stated.

[0148] Generally, as used herein a hyphen - or dash - in a range of values is to or through; a > is above or greater-than; a is at least or greater-than or equal to; a < is below or less-than; and a is at most or less-than or equal to. On an individual basis, each of the aforementioned applications for patent, patents, and/or patent application publications, is expressly incorporated herein by reference in its entirety in one or more non-limiting embodiments.

[0149] It is to be understood that the appended claims are not limited to express and particular compounds, compositions, or methods described in the detailed description, which may vary between particular embodiments which fall within the scope of the appended claims. With respect to any Markush groups relied upon herein for describing particular features or aspects of various embodiments, it is to be appreciated that different, special, and/or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of a Markush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims.

[0150] The present invention has been described herein in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. The present invention may be practiced otherwise than as specifically described within the scope of the appended claims. The subject matter of all combinations of independent and dependent claims, both single and multiple dependent, is herein expressly contemplated.