Composition comprising an iodide and a derivative of hyaluronic acid with an oxidative effect, method of preparation thereof and use thereof

Abstract

An antimicrobial composition is provided. The composition comprises a chloramid of hyaluronic acid or of modified hyaluronic acid. The chloramid has an amidic group (—NH—CO—). The hydrogens of the amidic group are substituted by chlorine atoms according to the structural formula —NCl—CO—. The composition further comprises an iodide. The substitution degree of the hyaluronic acid or of the modified hyaluronic acid by chlorine is in an amount of from 50% to 100%.

Claims

1. An antimicrobial composition comprising: a chloramide of hyaluronic acid or of modified hyaluronic acid, the chloramide having an amidic group (—NH—CO—), wherein the hydrogens of the amidic group are substituted by chlorine atoms according to the structural formula —NCl—CO—; and an iodide; wherein the substitution degree of the hyaluronic acid or of the modified hyaluronic acid by chlorine is in an amount of from 50% to 100%.

2. The antimicrobial composition according to claim 1, wherein the chloramide has an —OH group, a —CH.sub.2—OH, and a —CO—OH group, and wherein: (A) at least a portion of the —OH groups of chloramide are substituted by a —O—CO—R.sup.2 group, where R.sup.2 is a C.sub.1-C.sub.17 linear or aromatic group; (B) at least a portion of the —CH.sub.2—OH groups are substituted by a —CH═O group; (C) at least a portion of the —CO—OH groups are substituted by a —CO—OR.sup.2 group, where R.sup.2 is a C.sub.1-C.sub.17 linear or aromatic group; or (D) any combination of (A), (B), and (C).

3. The antimicrobial composition according to claim 1, wherein the chloramide is of modified hyaluronic acid, the modified hyaluronic acid is selected from the group of ethyl ester, benzyl ester, lauroyl, formyl, palmitoyl, hexanoyl hyaluronan, and combinations thereof, and the iodide is selected from the group of sodium iodide, potassium iodide, calcium iodide, magnesium iodide, and combinations thereof.

4. The antimicrobial composition according to claim 1, wherein the molecular weight of the chloramide is within the range of 5 to 500 kg.mol.sup.−1.

5. The antimicrobial composition according to claim 1, wherein the molar ratio of the chloramide to the iodide in the composition is within the range of 1:1 to 1:5.

6. The antimicrobial composition according to claim 1, further comprising an additive in an amount of no greater than 90 wt. % based on total weight of the composition.

7. The antimicrobial composition according to claim 6, wherein the additive is selected from the group of water, sodium chloride, calcium chloride, glycerol, hyaluronic acid, chondroitin sulfate, sodium alginate, oxy-cellulose, carboxymethyl cellulose, hydroxy ethyl cellulose, a modified hyaluronic acid, and combinations thereof, with the proviso that when the additive comprises a modified hyaluronic acid; (A) at least a portion of the —OH groups of modified hyaluronic acid are substituted by a —O—CO—R.sup.2 group, where R.sup.2 is a C.sub.1-C.sub.17 linear or aromatic group; (B) at least a portion of the —CH.sub.2—OH groups are substituted by a —CH═O group; (C) at least a portion of the —CO—OH groups are substituted by a —CO—OR.sup.2 group, where R.sup.2 is a C.sub.1-C.sub.17 linear or aromatic group; or (D) any combination of (A), (B), and (C).

8. The antimicrobial composition according to claim 1, wherein the composition is in the form of a solid substrate selected from the group of a self-supporting film, lyophilisate, a layer of staple fibres, an endless fibre, a woven fabric, knitted fabric, braided fabric, a layer of nanofibers, and combinations thereof, and wherein the content of the chloramide in the composition, calculated in terms of the dry matter, is within the range of from 10 to 90%.

9. The antimicrobial composition according to claim 8, wherein the composition is in the form of a solid substrate composed of two or more layers.

10. The antimicrobial composition according to claim 1, wherein the composition is in the form of two separate solutions or gels adapted to be mixed immediately before the application of the composition, wherein the chloramide of hyaluronic acid or chloramide of the modified hyaluronic acid is comprised in the first solution or gel, and the iodide is comprised in the second solution or gel, wherein the content of the chloramide in the composition, calculated in terms of dry matter, is within the range of from 10 to 90% and the content of the iodide in the composition, calculated in terms of dry matter, is within the range of from 10 to 90%.

11. The antimicrobial composition according to claim 1, utilized for the preparation of wound covers or for the preparation of implantable medical devices, preparations against acne, antibacterial fillings, antiadhesive barriers, membranes, pouches, or wrappings.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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:

(2) FIG. 1A is a photograph illustrating antimicrobial activity of the composition having the form of a lyophilisate on the basis of hyaluronan chloramide and potassium iodide as prepared according to Example 20; and

(3) FIG. 1B is a photograph illustrating antimicrobial activity of the composition having the form of a lyophilisate on the basis of hyaluronan chloramide and potassium iodide as prepared according to Example 20.

DETAILED DESCRIPTION

(4) The subject matter of the present invention are compositions containing an iodide and a derivative of hyaluronic acid, in which the majority of the hydrogen atoms contained in the amidic group —NH—CO— is substituted by chlorine atoms —NCl—CO—, the term “majority” referring to the range from 50 to 100%. After having been modified in the above manner, the polymer, namely hyaluronan chloramide, which is present in the form of a solution, rapidly reacts with iodides (the other part of the composition) which results in the formation of iodine compounds with oxidation degrees higher than −1, such as I.sub.3.sup.− or I.sub.2, and the native hyaluronic acid. The reaction can be described by the following schema.

(5) ##STR00002##

(6) The final compositions may also comprise modified hyaluronic acid, the chemical modification involving the carboxyl and/or hydroxyl groups of the latter.

(7) The derivative of hyaluronic acid may also be represented by a chloramide of modified hyaluronic acid, wherein the hydrogens of the amidic group —NH—CO— are substituted by chlorine atoms according to the structural formula —NCl—CO—, and simultaneously some of the —OH groups of chloramide are replaced by a —O—CO—R.sup.2 group and/or some of the —CH.sub.2—OH groups are replaced by a —CH═O group and/or some of the CO—OH groups are replaced by a —CO—OR.sup.2 group, wherein R.sup.2 is a linear or aromatic chain containing C.sub.1-C.sub.17 atoms. The examples of the chloramides of modified hyaluronic acid include ethyl ester, benzyl ester, lauroyl, formyl, palmitoyl or hexanoyl hyaluronan. In certain embodiments, the iodide is selected from the group comprising sodium iodide, potassium iodide, calcium iodide, magnesium iodide and a mixture thereof. In various embodiments, the polysaccharide, i.e. chloramide, has a molecular weight ranging from 5 to 500 kg.mol.sup.−1. In various embodiments, the molar ratio of the chloramide to the iodide in the composition is within the range from 1:1 to 1:5.

(8) The antimicrobial composition may also comprise an additive in the concentration of up to 90% w/w, said additive being selected from the group comprising polyethylene oxide, water, glycerol, hyaluronic acid, chondroitin sulfate, ester forms of modified hyaluronic acid, aldehyde forms of modified hyaluronic acid, sodium alginate, oxy-cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, esters of fatty acids, sodium chloride, potassium chloride, or calcium chloride. Accordingly, the aforesaid modified hyaluronic acid is hyaluronic acid in which some of the —OH groups are replaced by a —OCOR.sup.2 group and/or some of the —CH.sub.2—OH groups are replaced by a —CH═O group and/or some of the CO—OH groups are replaced by a —CO—OR.sup.2 group, wherein R.sup.2 is a linear or aromatic chain containing C.sub.1-C.sub.17 atoms.

(9) The preparation of hyaluronan chloramide takes place utilizing agents containing chlorine bound to nitrogen, in certain embodiments, utilizing trichloroisocyanuric acid or utilizing salts of dichloroisocyanuric acid. The preparation (modification) is carried out in water, the pH value of the solution being within the range from 2.5 to 7.5, in various embodiments, within the range from 4.0 to 6.0. The lower pH value is obtained by adding acetic acid in the amount between 0.2 and 7 eq., in various embodiments, between 2 and 4 eq. The initial substrate can be constituted by hyaluronic acid or by a chemically modified derivative thereof, the molecular weight of such substrate ranging from 40 to 2200 kg.mol.sup.−1. Furthermore, the preparation of the final compositions comprises the formation of: solid forms containing iodides and hyaluronan chloramide, such forms including, for example, self-supporting films, lyophilisates, layers of staple fibres (non-woven fabrics), endless fibres, woven fabrics, knitted fabrics, braided fabrics or nanofibrous layers, all of them optionally with a content of farther additives, the proportion of the chloramide in the final composition, calculated in terms of the dry matter, being within the range of 10 to 90% and the proportion of the iodide, calculated in terms of the dry matter, being within the range of 10 to 90%, the solid form being possibly composed of two or more different layers pressed together; liquid and gel forms, optionally containing further additives, wherein the constituent containing iodides is separated from that containing hyaluronan chloramide, said constituents being mixed together immediately prior to the final application, the proportion of the chloramide in the final composition, calculated in terms of the dry matter, being within the range of 10 to 90% and the proportion of the iodide in the final composition, calculated in terms of the dry matter, being within the range of 10 to 90%.

(10) Furthermore, the invention relates to the use of the final composition, particularly in the fields where the following properties or any combinations of the following properties are required: oxidative or antimicrobial or antifungal or antiviral activity biocompatibility and biodegradability possibility of preparing various forms having a convenient degree of stability a significant contribution to the healing process controlled biodegradation rate

(11) The final composition is usable in biomedical applications, particularly for the preparation of wound dressings, implantable medical devices, preparations against acne, antibacterial fillings, anti-adhesive barriers, membranes, pockets, wrappings, products for the prevention of adhesion following to anastomoses, products for prevention of wound dehiscences, or, in combination with other substances, products for supporting surgical corrections of abdominal wall defects.

(12) A controlled biodegradation rate of the final composition can be achieved either by combining the hyaluronan chloramide with additives exhibiting a slower degradability, such as sodium alginate or carboxymethyl cellulose, and/or by cross-linking the hyaluronan chloramide itself utilizing, for example, polyvalent Ca.sup.2+ cations.

(13) The present invention relates to the preparation and use of stable, biocompatible and biodegradable compositions containing iodides, such as potassium iodide, sodium iodide, calcium iodide or magnesium iodide, or a mixture thereof, along with a hyaluronan chloramide, which compositions exhibit antimicrobial and antiviral effects accompanied by healing effects. Furthermore, a wide range of forms is described, which forms have largely variable surface areas, mechanical or rheological properties and degradation periods.

(14) The practical implementation of the technical solution according to the present invention is not complicated from the technological viewpoint and does not require any less available chemicals, solvents or isolation procedures to be used.

(15) In the presence of water, the aforesaid mixture generates iodine having an oxidation degree higher than −1 and providing a broad-spectrum antimicrobial effects. The final compositions include numerous forms, such as such a thin film, a lyophilisate, a layer of staple fibres, an endless fibre, woven fabric, knitted fabric, braided fabric or a nanofibrous layer, such that iodide and hyaluronan chloramide can be transferred both into medical devices or into medicaments.

(16) Furthermore, the invention relates to the use of the final compositions containing an iodide and hyaluronan chloramide in the fields where the following properties or any combinations of the following properties are required: oxidative or antimicrobial or antifungal or antiviral activity biocompatibility and biodegradability possibility of preparing various forms having a convenient degree of stability a significant contribution to the healing process

(17) The figures show the growth inhibition of the microorganisms Bacillus subtilis, Escherichia coli, Pseudomona aeruginosa, Candida albicans (all of them in FIG. 1A) and Staphylolococus epidermidis (FIG. 1B) in the presence of a composition containing a combination of soluble lyophilisates on the basis of hyaluronan chloramide and soluble lyophilisates on the basis of the mixture of hyaluronan and a potassium iodide in the ratio of 5/1, prepared according to Example 20 (two repeats: the left-hand and middle schemes), in comparison to the control material (the right-hand scheme), namely to the lyophilized native hyaluronic acid supplemented with potassium iodide in the ratio of 5/1 according to Example 19. FIG. 1B shows the comparison between one repeat (the left-hand scheme) and the control material (the right-hand scheme).

(18) The procedure of determining the antimicrobial activity is described in Example 43.

EXAMPLES

(19)
DS=degree of substitution=100%*(the molar amount of modified polymer units)/(the total molar amount of all polymer units)

(20) Unless otherwise specified, the expression “equivalent” (eq) as used herein refers to a repeating unit of the respective polysaccharide, such as a dimer of hyaluronic acid. Unless otherwise specified, percentages are weight percents. As used herein, the molecular weight of polymers refers to a weight average value determined utilizing the SEC-MALLS method.

Example 1

(21) Preparation of an Ethyl Ester of Hyaluronan

(22) NaOH was added to the aqueous hyaluronan solution (1 g, 300 kg.mol.sup.−1, in 40 ml of water) until reaching the pH value of 9. Subsequently, 20 ml of dimethyl sulfoxide and 0.08 ml of ethyl iodide were added and the resulting mixture was stirred at the temperature of 45° C. for 3 days. Afterwards, the resulting mixture was precipitated by 140 ml of 100% isopropanol and the solid matter separated by filtration was washed with isopropanol and vacuum dried. The product (897 mg) was analysed utilizing NMR.

(23) The DS value of the ester was 6% (determined utilizing NMR, lit. Kettou S. et al., PV 2009-399).

Example 2

(24) Preparation of a Benzyl Ester of Hyaluronan

(25) NaOH was added to the aqueous hyaluronan solution (1 g, 300 kg.mol.sup.−1, in 40 ml of water) until reaching the pH value of 9. Subsequently, 20 ml of dimethyl sulfoxide and 0.08 ml of benzyl bromide were added and the resulting mixture was stirred at the temperature of 20° C. for 4 days. Afterwards, the resulting mixture was precipitated by 140 ml of 100% isopropanol and the solid matter separated by filtration was washed with isopropanol and vacuum dried. The final product (obtained in the amount of 920 mg) was analysed utilizing NMR.

(26) The DS value of the ester was 3% (determined utilizing NMR, lit. Kettou et al., PV 2009-399).

Example 3

(27) Preparation of an Lauroyl of Hyaluronan

(28) 70 ml of tetrahydrofuran, 4 equivalents of triethylamine and 0.1 equivalents of 4-dimethylaminopyridine were added to the solution of hyaluronan (5 g, 250 kg.mol.sup.−1) in 100 ml of distilled water. Simultaneously, lauric acid (4 equivalents) was dissolved in the mixture consisting of 30 ml of tetrahydrofuran and 7 ml of triethylamine and the obtained solution was supplemented with 4.8 ml of ethyl chloroformate for 15 minutes at 0 to 5° C. The resulting suspension was filtered into the solution of hyaluronan and the reaction mixture was stirred at 20° C. for 5 hours. The resulting solution was precipitated by adding 400 ml of 100% isopropanol and washed with 80% isopropanol and then with 100% isopropanol. Afterwards, the precipitate was dried at 40° C. for 2 days. The substitution degree was determined as 37% utilizing NMR

Example 4

(29) Preparation of a Formyl of Hyaluronan

(30) A one-percent aqueous solution of HA (1 g, 200 kg.mol.sup.−1) containing NaCl 1%, KBr 1%, N-acetylamino-TEMPO (0.01 eq.) and NaHCO.sub.3 (20 eq.) was gradually supplemented with an aqueous solution of NaClO (0.5 eq.) under nitrogen atmosphere. The mixture was stirred at 10° C. for 12 hours, whereupon 0.1 g of ethanol were added. Subsequently, the final mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to the concentration of 0.2% and dialysed against the mixture (0.1% NaCl, 0.1% NaHCO.sub.3) 3 times litres (1× a day) and then against distilled water 7 times 5 litres (2× a day). The final solution was evaporated and analysed.

(31) DS 9% (determined by NMR).

Example 5

(32) Preparation of a Hyaluronan Chloramide

(33) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 82% (determined by NMR).

Example 6

(34) Preparation of a Hyaluronan Chloramide

(35) 5 g of hyaluronan (Mw 40 kg.mol.sup.−1) were dissolved in 100 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 83% (determined by NMR).

Example 7

(36) Preparation of a Hyaluronan Chloramide

(37) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 1000 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 72% (determined by NMR).

Example 8

(38) Preparation of a Hyaluronan Chloramide

(39) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 0.14 ml of acetic acid (0.2 eq.) were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 53% (determined by NMR).

Example 9

(40) Preparation of a Hyaluronan Chloramide

(41) 5 g of hyaluronan (Mw 180 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 3 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 83% (determined by NMR).

Example 10

(42) Preparation of a Hyaluronan Chloramide

(43) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 5 ml of acetic acid (7 eq.) were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for hours. DS 95% (determined by NMR).

Example 11

(44) Preparation of a Hyaluronan Chloramide

(45) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 1.07 g (0.33 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 51% (determined by NMR).

Example 12

(46) Preparation of a Hyaluronan Chloramide

(47) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 4.8 g (1.5 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 96% (determined by NMR).

Example 13

(48) Preparation of a Hyaluronan Chloramide

(49) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, trichloroisocyanuric acid in the amount of 2.91 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 97% (determined by NMR).

Example 14

(50) Preparation of a Hyaluronan Chloramide

(51) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, trichloroisocyanuric acid in the amount of 0.87 g (0.3 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 71% (determined by NMR).

Example 15

(52) Preparation of a Hyaluronan Chloramide

(53) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 5 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 52% (determined by NMR).

Example 16

(54) Preparation of a Hyaluronan Chloramide

(55) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 48 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 85% (determined by NMR). The NMR solution (7 mg of the product in 0.7 ml of D.sub.2O) was measured after another 5 days of standing at 20° C. The DS value was determined as 84%. The solid portion in the form of a powder was left to stand at 20° C. for 100 days and after that the sample was dissolved in D.sub.2O. The DS value was determined as 84%.

Example 17

(56) Preparation of a Hyaluronan Chloramide

(57) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 5° C. for 72 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol and vacuum dried for 20 hours. DS 64% (determined by NMR).

Example 18

(58) Preparation of a Hyaluronan Chloramide

(59) 5 g of hyaluronan (Mw 2200 kg.mol.sup.−1) were dissolved in 250 ml of distilled water. Subsequently, 2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 3.2 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 40° C. for 5 hours. Subsequently, the mixture was precipitated by 2.5 litres of isopropanol and filtered. The solid portion was washed with 2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 75% (determined by NMR).

Example 19

(60) Preparation of a Lyophilisate Comprising Hyaluronan and Potassium Iodide

(61) A solution of hyaluronan (1 g, Mw 2,200 kg.mol.sup.−1) in 50 ml of distilled water was mixed together with a solution of 0.2 g of potassium iodide in 5 ml of distilled water and the mixture was stirred for 10 minutes. Thereupon, the final solution was deep frozen at the temperature of −50° C. and lyophilized.

Example 20

(62) Preparation of a Lyophilisate (Hyaluronan Chloramide+Hyaluronan+Potassium Iodide)—Iodine Generator

(63) The lyophilisate prepared according to Example 19 in the amount of 15 mg and the lyophilisate prepared according to Example 25 in the amount of 15 mg were placed on each other in a stacked manner (the bottom layer being the lyophilisate according to Example 19) and mechanically pressed together utilizing a smooth glass roller at 20° C., the corresponding pressure being applied for 1 minute. The resulting material was stored under dry conditions.

Example 20a

(64) Preparation of a Lyophilisate (Hyaluronan Chloramide+Hyaluronan+Potassium Iodide)—Iodine Generator

(65) The lyophilisate prepared according to Example 19 in the amount of 30 mg and the lyophilisate prepared according to Example 25 in the amount of 3 mg were placed on each other in a stacked manner (the bottom layer being the lyophilisate according to Example 19) and mechanically pressed together utilizing a smooth glass roller at 20° C., the required pressure being applied for 1 minute. The resulting material was stored under dry conditions.

Example 20b

(66) Preparation of a Lyophilisate (Hyaluronan Chloramide+Hyaluronan+Potassium Iodide)—Iodine Generator

(67) The lyophilisate prepared according to Example 19 in the amount of 3 mg and the lyophilisate prepared according to Example 25 in the amount of 30 mg were placed on each other in a stacked manner (the bottom layer being the lyophilisate according to Example 19) and mechanically pressed together utilizing a smooth glass roller at 20° C., the required pressure being applied for 1 minute. The resulting material was stored under dry conditions.

Example 21

(68) Preparation of a Self-Supporting Film (Hyaluronan Chloramide+Hyaluronan Lauroyl+Potassium Iodide)—Iodine Generator

(69) The self-supporting films prepared according to Examples 31 and 32 were placed on each other in a stacked manner (the bottom layer being hyaluronan lauroyl+potassium iodide) and mechanically pressed together utilizing a smooth glass roller at 20° C., the required pressure being applied for 1 minute. The resulting material was stored under dry conditions.

Example 22

(70) Preparation of a Chloramide of an Ethyl Ester of Hyaluronan

(71) 0.5 g of ethyl ester of hyaluronan prepared according to Example 1 were dissolved in 25 ml of distilled water. Subsequently, 0.2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 0.32 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 250 ml of isopropanol and filtered. The solid portion was washed with 0.2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 80% (determined by NMR).

Example 23

(72) Preparation of a Chloramide of an Benzyl Ester of Hyaluronan

(73) 0.5 g of benzyl ester of hyaluronan prepared according to Example 2 were dissolved in 25 ml of distilled water. Subsequently, 0.2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 0.32 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 0.25 litres of isopropanol and filtered. The solid portion was washed with 0.2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 78% (determined by NMR).

Example 24

(74) Preparation of a Chloramide of Formyl Hyaluronan

(75) 0.5 g of formyl hyaluronan prepared according to Example 4 were dissolved in 25 ml of distilled water. Subsequently, 0.2 ml of acetic acid were added and the solution was stirred at the temperature of 20° C. for 15 minutes. Then, a sodium salt of dichloroisocyanuric acid in the amount of 0.32 g (1 eq.) was added. Then, the mixture was stirred at a temperature of 20° C. for 24 hours. Subsequently, the mixture was precipitated by 0.25 litres of isopropanol and filtered. The solid portion was washed with 0.2 litres of isopropanol, whereupon it was vacuum dried for 20 hours. DS 75% (determined by NMR).

Example 25

(76) Preparation of a Lyophilized Hyaluronan Chloramide

(77) A solution of 1 g of the hyaluronan chloramide, prepared according to Example 16, in 50 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value was determined as 83% utilizing NMR.

Example 26

(78) Preparation of a Lyophilized Chloramide of an Ethyl Ester of Hyaluronan

(79) A solution of 0.2 g of the chloramide of ethyl ester of hyaluronan, prepared according to Example 22, in 10 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value was determined as 68% utilizing NMR.

Example 27

(80) Preparation of a Lyophilized Chloramide of a Benzyl Ester of Hyaluronan

(81) A solution of 0.2 g of the chloramide of a benzyl ester of hyaluronan, prepared according to Example 23, in 10 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value was determined as 67% utilizing NMR.

Example 28

(82) Preparation of a Non-Woven Fabric from Staple Fibres Made of Hyaluronan Chloramide

(83) The preparation of a 2% solution was based on the use of hyaluronan chloramide (Example 14) having the substitution degree of 71% according to NMR. The above constituent was weighed and supplemented with distilled water in order to obtain the required amount. The whole mixture was stirred in a stirring device at room temperature, the set speed of the latter being 500 rpm and the stirring time being 24 hours. The final solution was clear and slightly viscous. The method of preparation of staple fibres is based on the precipitation of a polymer solution in a stream of a mobile coagulation bath containing 100% isopropanol. The solution was dosed at room temperature through extrusion nozzles into a stream of a coagulation bath fed through spinning tubes (1 tube/8 mm diameter), the controlled flow rate of the bath being 1.15 m/s. The formation of staple fibres was obtained utilizing precipitation. The formed fibres were entrained in the bath stream, caught by separation combs and transferred into the maturation bath containing 100% isopropanol. Immediately after entering the maturation bath, the raw fibres were ground up by rotating blades of a mixer, the ratio between the amount of the fibres and that of the bath being 0.5 g/350 ml. The final dispersion of fibres was filtered through a permeable substrate through a filter frame. For the given experiment, a filter frame having the surface area of 64 cm.sup.2 was used. After having been filtered, the fibres were carried over onto a drying device utilizing a PAD knitted fabric, said device adapted for the fixation of non-woven fabrics. Prior to being placed onto the drying device, the fibres were freed from the precipitant residues utilizing a roller. The non-woven fabric was dried at the temperature of 40° C. for 30 minutes. The resulting layer was separated from the substrate as a self-supporting layer and weighed utilizing an analytic balance. The areal weight of the fabric was 50.2 g/m.sup.2. The substitution degree of the formed non-woven fabric was determined as 64% utilizing NMR.

Example 29

(84) Preparation of a Nanofibrous Layer Comprising Hyaluronan Chloramide

(85) For the purpose of preparation of a nanofibrous layer containing hyaluronic acid, an aqueous solution having the following composition was prepared. The concentration of the hyaluronan chloramide, which had been prepared according to Example 5, in the dry matter was 37.5%, the concentration of the native hyaluronan having the molecular weight of 80 kg.mol.sup.−1 was 37.5%, and the amount of polyethylene oxide having the molecular weight of 600 kg.mol.sup.−1 was 25%. The overall concentration of the dry matter was 5%. The solution was transferred into a syringe and spun electrostatically on a plate-type collector using a needleless linear nozzle, the voltage and spacing between the emitter and the collector being 50 kV and 16 cm, respectively. The dimension of the fibres was 110±27 nm. The DS value was determined as 30% utilizing NMR.

Example 29a

(86) Preparation of a Nanofibrous Layer Comprising Hyaluronan and Potassium Iodide

(87) For the purpose of preparation of a nanofibrous layer containing hyaluronic acid, an aqueous solution having the following composition was prepared. The concentration of the native hyaluronan having the molecular weight of 80 kg.mol.sup.−1 was 62.5%, the amount of polyethylene oxide having the molecular weight of 600 kg.mol.sup.−1 was 25%, and the amount of KI was 12.5%. The overall concentration of the dry matter was 5%. The solution was transferred into a syringe and spun electrostatically on a plate-type collector using a needleless linear nozzle, the voltage and spacing between the emitter and the collector being 56 kV and 18 cm, respectively. The dimension of the fibres was 140±35 nm.

Example 30

(88) Preparation of a Self-Supporting Film Comprising Hyaluronan Chloramide

(89) The preparation of the film took place in a special drying device where the film was dried in a closed chamber. The apparatus is equipped with the bottom and top plates, the temperatures thereof being controllable. The detailed description of the apparatus is provided in the publication Foglarova et al., PV2015-166; Foglarova M. Et al., Carbohydrate Polymers 2016, 144, 68-75. The weighed amount of 200 mg of the hyaluronan chloramide described in Example 5 was dissolved in 20 ml of demineralised water whereupon the resulting solution was stirred for 2 hours. Then the final solution was dosed to the drying device onto a pad (hydrophobized glass) and was dried in a closed chamber for 18 hours. The bottom and top plates of the dryer had the temperatures of 50° C. and 20° C., respectively. After having been dried, the film was released from the pad and kept for future use. The DS value was determined as 60% utilizing NMR.

Example 31

(90) Preparation of a Self-Supporting Film from Hyaluronan and Potassium Iodide (10/1)

(91) The preparation of the film took place in the device described in Example 30. The weighed amount of 160 mg of sodium hyaluronate and 16 mg of potassium iodide was dissolved in 16 ml of demineralised water whereupon the resulting solution was stirred for 15 minutes. Then the final mixed solution was dosed to the drying device onto a pad (hydrophobized glass) and was dried in a closed chamber for 7 hours. The bottom and top plates of the dryer had the temperatures of 50° C. and 20° C., respectively. After having been dried, the film was released from the pad and kept for future use.

Example 32

(92) Preparation of a Self-Supporting Film from Lauroyl Hyaluronan and Hyaluronan Chloramide (3/1)

(93) The preparation of the film took place in the device described in Example 30. The weighed amount of 150 mg of the lauroyl derivative of sodium hyaluronate (as described in Example 3) was dissolved in 15 ml of an aqueous solution of 2-propanol (50% w/w) whereupon the resulting solution was stirred for 18 hours. The weighed amount of 50 mg of the hyaluronan chloramide described in Example 5 was dissolved in 5 ml of demineralised water whereupon the resulting solution was stirred for 2 hours. Subsequently, both the solutions were blended for 30 minutes. Then the final mixed solution was dosed to the drying device onto a pad (hydrophobized glass) and was dried in a closed chamber for 7 hours. The bottom and top plates of the dryer had the temperatures of 50° C. and 20° C., respectively. After having been dried, the film was released from the pad and kept for future use. The DS value of the hyaluronan chloramide in the final material was determined as 25% utilizing NMR.

Example 33

(94) Preparation of a Nanofibrous Layer (Hyaluronan Chloramide+Hyaluronan+Potassium Iodide)—Iodine Generator

(95) The nanofibrous layers prepared according to Examples 29 and 29a were placed on each other in a stacked manner (the bottom layer being hyaluronan+potassium iodide). The material was stored under dry conditions.

Example 34

(96) Preparation of a Two-Component Mixture in the Form of a Solution (Hyaluronan Chloramide+Sodium Iodide 1/1)—Iodine Generator

(97) Immediately prior to application, a solution of 100 mg of hyaluronan chloramide, as prepared according to Example 5, in 3 ml of distilled water was mixed with a solution comprising 29 mg of sodium iodide in 1 ml of distilled water utilizing a static mixer. Immediately thereafter, orange-coloured triiodide and iodine were formed in the resulting mixture.

Example 35

(98) Preparation of a Two-Component Mixture in the Form of a Gel (Hyaluronan Chloramide+Calcium Iodide 1/5)—Iodine Generator

(99) Immediately prior to application, a solution of 100 mg of hyaluronan chloramide, as prepared according to Example 5, in 6 ml of distilled water was mixed with a solution comprising 758 mg of the native hyaluronan having the molecular weight of 1,900 kg.mol.sup.−1 and 142 mg of calcium iodide in 35 ml of distilled water utilizing a static mixer. Immediately thereafter, orange-coloured triiodide and iodine were formed in the resulting viscous mixture.

Example 36

(100) Preparation of an Oxycellulose/Hyaluronan Chloramide Lyophilizate

(101) A solution of 0.3 g of oxycellulose (Mw 50 kg.mol.sup.−1) and 0.1 g of the hyaluronan chloramide, prepared according to Example 5, in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 24% utilizing NMR.

Example 37

(102) Preparation of an Alginate/Hyaluronan Chloramide Lyophilizate

(103) A solution of 0.3 g of alginate (Mw 40 kg.mol.sup.−1) and 0.1 g of the hyaluronan chloramide, prepared according to Example 5, in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 26% utilizing NMR.

Example 38

(104) Preparation of a Carboxymethyl Cellulose/Hyaluronan Chloramide Lyophilizate

(105) A solution of 0.3 g of carboxymethyl cellulose (Mw 30 kg.mol.sup.−1) and 0.03 g of the hyaluronan chloramide, prepared according to Example 5, in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 3% utilizing NMR.

Example 39

(106) Preparation of a Chondroitin Sulfate/Hyaluronan Chloramide Lyophilizate

(107) A solution of 0.3 g of chondroitin sulfate (Mw 45 kg.mol.sup.−1) and 0.1 g of the hyaluronan chloramide, prepared according to Example 5, in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 25% utilizing NMR.

Example 40

(108) Preparation of a Hydroxyethyl Cellulose/Hyaluronan Chloramide Lyophilizate

(109) A solution of 0.3 g of hydroxyethyl cellulose (Mw 35 kg.mol.sup.−1) and 0.1 g of the hyaluronan chloramide, prepared according to Example 5, in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 25% utilizing NMR.

Example 41

(110) Preparation of a Formyl Hyaluronan/Hyaluronan Chloramide Lyophilizate

(111) A solution of 0.3 g of hyaluronan formyl (Mw 50 kg.mol.sup.−1) prepared according to Example 4 and 0.1 g of the hyaluronan chloramide prepared according to Example 5 in 100 ml of distilled water were, immediately following the homogenization, deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 25% utilizing NMR.

Example 42

(112) Preparation of a Lyophilizate of Hyaluronan Chloramide Cross-Linked with CaCl.sub.2

(113) A solution of hyaluronan chloramide (0.1 g) prepared according to Example 5 in 100 ml of distilled water was supplemented with 0.01 g of CaCl.sub.2.2H.sub.2O and the mixture was stirred at 20° C. for 1 hour. Following the homogenization, the final viscous solution was deep frozen at the temperature of −50° C. and lyophilized. The DS value of the hyaluronan chloramide was determined as 64% utilizing NMR.

Example 43

(114) In Vitro Antimicrobial Activity Testing (FIGS. 1A and 1B):

(115) For the individual microorganisms subjected to testing, suspensions having the concentration of approximately 10.sup.5 CFU/ml were prepared. The suspension in the amount of 100 μl was inoculated on the surface of tryptone soya agar in Petri dishes (an approximate count of microorganisms applied in each dish was 10.sup.4 CFU). The suspension was uniformly spread over the entire surface of the dish utilizing a sterile loop. After soaking the suspension in the agar, the surface of agar was covered by sterile square-shaped samples selected for testing. The dishes with the tested bacterium strains were cultivated at 37° C. for 24 hours. The tests involved the lyophilizates of hyaluronan chloramide and potassium iodide prepared according to Example 20, in the form of square pieces having a weight ranging between 25 and 35 mg and approximate dimensions of 15 mm×15 mm, the respective control materials being analogous lyophilisates having no content of hyaluronan chloramide, as prepared according to Example 19. For efficacy testing, the diffusion plate method (2D layout) was selected. For the cultivation, a non-selective substrate (tryptone-soya agar) was selected. The square-shaped samples were tested on the following microorganisms: Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Candida Albicans, Pseudomona aeruginosa, Staphylococcus epidermidis. The FIG. 1 clearly shows that the compositions containing hyaluronan chloramide along with an iodide according to the invention provide a significantly higher efficacy in comparison to the control material having no content of hyaluronan chloramide.

(116) 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.

(117) 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.

(118) 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.

(119) 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.