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Title:
KIT OF GEL-FORMING SOLUTIONS INTENDED FOR PREPARATION OF HYDROGEL BASED ON COVALENTLY CROSSLINKED HYDROXYPHENYL DERIVATIVE OF HYALURONAN FOR PREVENTION OF POSTOPERATIVE COMPLICATIONS RELATED TO FORMATION OF COLORECTAL ANASTOMOSIS, USE OF KIT, METHOD OF PREPARATION OF HYDROGEL AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2021/228292
Kind Code:
A1
Abstract:
The kit of at least two aqueous gel-forming solutions for the preparation of a biodegradable hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan, comprising at least two aqueous solutions A and B, of which the solution A contains horseradish peroxidase and the solution B contains hydrogen peroxide, wherein the solution A and/or the solution B comprises a hydroxyphenyl derivative of a hyaluronan of the general formula I, wherein n is in the range 2 to 5000, M is H+ or a cation of a pharmaceutically acceptable salt selected from the group containing an alkali metal cation, alkaline earth metal cation, and wherein R is OH or a substituent NHR2CONHR1ArOH of the general formula II, wherein Ar is phenylene and R1 is ethylene, or Ar is indolydene and R1 is ethylene, or Ar is hydroxyphenylene and R1 is carboxyethylene, and R2 is alkylene of 3 to 7 carbons, and at the same time the solution A and/or B contains triclosan and hydroxypropyl β-cyclodextrin. It further relates to the use of the kit, the method of preparation of the hydrogel and use thereof.

Inventors:
PARAL JIRI (CZ)
PRAVDA MARTIN (CZ)
KOVAROVA LENKA (CZ)
TOROPITSYN EVGENIY (CZ)
SCIGALKOVA IVANA (CZ)
VELEBNY VLADIMIR (CZ)
Application Number:
PCT/CZ2021/050051
Publication Date:
November 18, 2021
Filing Date:
May 11, 2021
Export Citation:
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Assignee:
CONTIPRO AS (CZ)
International Classes:
A61L27/20; A61K47/36; A61L26/00; A61L27/52; A61L27/54
Domestic Patent References:
WO2011002249A22011-01-06
WO2015054125A12015-04-16
WO2009148405A12009-12-10
Attorney, Agent or Firm:
KANIA,SEDLAK,SMOLA, S.R.O. (CZ)
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Claims:
CLAIMS

1. A kit of at least two aqueous gel-forming solutions for a preparation of a biodegradable hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan, characterized in that it comprises at least two aqueous solutions A and B, of which the solution A contains horseradish peroxidase and the solution B contains hydrogen peroxide, the solution A and/or the solution B containing a hydroxyphenyl derivative of a hyaluronan of a general formula I wherein n is in the range of 2 to 5000, M is H+ or a cation of a pharmaceutically acceptable salt selected from the group comprising of an alkali metal cation, an alkaline earth metal cation, and wherein R is OH or substituent NHR2CONHR 1 ArOH of a general formula II,

(II), wherein Ar is phenylene and Ri is ethylene, or Ar is indolydene and Ri is ethylene, or Ar is hydroxyphenylene and Ri is carboxyethylene, and R2 is alkylene of 3 to 7 carbons, and at the same time the solution A and/or the solution B contains triclosan and hydroxypropyl -b-cyclodextrin.

2. The kit according to claim 1, characterized in that the horseradish peroxidase activity is in the range of 0.5 to 1.5 U/mL, preferably 0.9 to 1.35 U/mL, more preferably 0.8 to 1.2 U/mL, the concentration of hydrogen peroxide is in the range of 1 to 6 mmol/L, preferably 3 to 5 mmol/L, the hydroxyphenyl derivative of hyaluronan of the general formula I has a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; a degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 % and a concentration of 10 to 50 mg/mL, preferably 15 to 25 mg/mL, more preferably 20 mg/mL; and the concentration of triclosan is in the range of 0.2 to 2.2 mg/mL, preferably 1 to 2.2 mg/mL, more preferably 2 mg/mL and the concentration of hydroxypropyl-β-cyclodextrin is in the range of 4 to 100 mg/mL, preferably 25 to 80 mg/mL, more preferably 60 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8.

3. The kit according to claim 1 or claim 2, characterized in that the solution A contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration 10 to 50 mg/mL,

• Horseradish peroxidase with an activity of 0.5 to 1.5 U/mL,

• Triclosan, which is at a concentration of 0.2 to 2.2 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 4 to 100 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL,

• Hydrogen peroxide, in the concentration range of 1 to 6 mmol/L.

4. The kit according to any one of claims 1 to 3, characterized in that the solution A contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.9 to 1.35 U/mL, Triclosan at a concentration of 1 to 2.2 mg/mL, • Hydroxypropyl-β-cyclodextrin at a concentration of 25 to 80 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 5 to 1 : 8, and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL,

• Hydrogen peroxide, which is in the concentration range of 3 to 5 mmol/L.

5. The kit according to any one of claims 1 to 4, characterized in that the solution A contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.8 to 1.2 U/mL,

• Triclosan at a concentration of 2 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is at a concentration of 60 mg/mL, and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL,

• Hydrogen peroxide in the concentration range of 4 to 5 mmol/L.

6. The kit according to claim 1, characterized in that it comprises aqueous solutions A, B and at least one solution C, wherein the solution A comprises a horseradish peroxidase, a hydroxyphenyl derivative of hyaluronan of the general formula I as defined in claim 1, triclosan and hydroxypropyl-β-cyclodextrin , the solution B contains hydrogen peroxide and the solution C comprises a hydroxyphenyl derivative of a hyaluronan of the general formula I as defined in claim 1.

7. The kit according to claim 6, characterized in that the horseradish peroxidase activity in the solution A is in the range of 1 to 3 U/mL, preferably 1.6 to 2.7 U/mL, more preferably 1.8 to 2.4 U/mL, the concentration of hydrogen peroxide in the solution B is in the range of 2 to 12 mmol/L, preferably 6 to 10 mmol/L, the hydroxyphenyl derivative of hyaluronan according to the general formula I as defined in claim 1, that has a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; the degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 %, is present in the solution A in a concentration of 1 to 20 mg/mL, preferably 5 to 15 mg/mL, more preferably 10 mg/mL, in the solution C at a concentration of 10 to 50 mg/mL, preferably 30 to 40 mg/mL, more preferably 35 mg/mL, the concentration of triclosan in the solution A is in the range of 0.2 to 4.4 mg/mL, preferably 2 to 4.4 mg/mL, more preferably 4 mg/mL, the concentration of hydroxypropyl-β-cyclodextrin in the solution A is in the range of 8 to 200 mg/mL, preferably 100 to 160 mg/mL, more preferably 120 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

8. The kit according to claim 6 or claim 7, characterized in that it comprises the solution A which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration 1 to 20 mg/mL,

• Horseradish peroxidase with an activity of 1 to 3 U/mL,

• Triclosan at a concentration of 0.4 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 8 to 200 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B that contains: • Hydrogen peroxide, with a concentration in the range of 2 to 12 mmol/L. and the solution C, that contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL.

9. The kit according to any one of claims 6 to 8, characterized in that it comprises the solution A, which contains:

• Hydroxyphenyl derivative of hyaluronan of the general formula I with weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, in a concentration of 5 to 15 mg/mL,

• Horseradish peroxidase with an activity of 1.6 to 2.7 U/mL,

• Triclosan, which is at a concentration of 3.6 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 108 to 132 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-b- cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B, which contains:

• Hydrogen peroxide in the concentration range of 7 to 12 mmol/L. and the solution C, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 30 to 40 mg/mL.

10. The kit according to any one of claims 6 to 9, characterized in that it comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 10 mg/mL,

• Horseradish peroxidase with an activity of 1.8 to 2.4 U/mL, • Triclosan, which is at a concentration of 3.6 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 108 to 132 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-b- cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B, which contains:

• Hydrogen peroxide in the concentration range of 7 to 12 mmol/L. and the solution C, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molecular weight in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 35 mg/mL.

11. The kit according to any one of claims 1 to 10 for use in the preparation of a biodegradable hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan for the prevention of postoperative complications associated with the formation of a colorectal anastomosis.

12. A method of the preparation of a hydrogel containing a covalently crosslinked hydroxyphenyl derivative of hyaluronan, characterized in that at least two solutions A and B as defined in claim 1 are prepared separately, wherein the solution A and/or the solution B comprises a hydroxyphenyl hyaluronan derivative of the general formula I as defined in claim 1, and at the same time, the solution A and/or the solution B contains triclosan and h ydro x y pro py 1 - b -c yc 1 odcx t ri n , whereupon the solution A is mixed with the solution B to form a hydrogel containing a covalently crosslinked hydroxyphenyl derivative of hyaluronan, which is intended for prevention of postoperative complications associated with colorectal anastomosis.

13. for the method of the preparation of a hydrogel according to claim 12, characterized in that at least two solutions A and B as defined in any one of claims 1 to 6 are prepared separately, of which the solution A contains horseradish peroxidase with an activity in the range of 0.5 to 1.5 U/mL and the solution B contains hydrogen peroxide in a concentration ranging from 1 to 6 mmol/L, wherein the solution A and/or the solution B contains a hydroxyphenyl derivative of hyaluronan of the general formula I as defined in claim 1, wherein its weight average molar weight is in the range of 60 000 g/mol to 2 000 000 g/mol, the degree of substitution is in the range of 1 % to 10 %, and its concentration is in the range of 10 to 50 mg/mL, and at the same time, the solution A and/or the solution B contains triclosan and hydroxypropyl-β-cyclodextrin , whereupon the solution A is mixed with the solution B to form the hydrogel containing a covalently crosslinked hydroxyphenyl derivative hyaluronan, which is intended for prevention of postoperative complications associated with colorectal anastomosis.

14. The method according to claim 12 or claim 13, characterized in that the horseradish peroxidase activity is in the range of 0.5 to 1.5 U/mL, preferably 0.9 to 1.35 U/mL, more preferably 0.8 to 1, 2 U/mL, the concentration of hydrogen peroxide is in the range of 1 to 6 mmol/L, preferably 3 to 5 mmol/L, the hydroxyphenyl derivative of hyaluronan according to the general formula I has a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; a degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 % and a concentration of 10 to 50 mg/mL, preferably 15 to 25 mg/mL, more preferably 20 mg/mL; and the concentration of triclosan is in the range of 0.2 to 2.2 mg/mL, preferably 1 to 2.2 mg/mL, more preferably 2 mg/mL and the concentration of hydroxypropyl-β-cyclodextrin is in the range of 4 to 100 mg/mL, preferably 25 to 80 mg/mL, more preferably 60 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

15. The method according to any one of claims 12 to 14, characterized in that the solution A contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration of 10 to 50 mg/mL,

• Horseradish peroxidase with an activity of 0.5 to 1.5 U/mL,

• Triclosan, which is at a concentration of 0.2 to 2.2 mg/mL, • Hydroxypropyl-β-cyclodextrin at a concentration of 4 to 100 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10. and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL,

• Hydrogen peroxide, in the concentration range of 1 to 6 mmol/L.

16. The method according to any one of claims 12 to 15, characterized in that the solution A contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.9 to 1.35 U/mL,

• Triclosan at a concentration of 1 to 2.2 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 25 to 80 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 5 to 1 : 8, and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL,

• Hydrogen peroxide, which is in the concentration range of 3 to 5 mmol/L.

17. The method according to any one of claims 12 to 16, characterized in that the solution A contains: • Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.8 to 1.2 U/mL,

• Triclosan at a concentration of 2 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is at a concentration of 60 mg/mL. and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL

• Hydrogen peroxide in the concentration range of 4 to 5 mmol/L.

18. The method according to any one of claims 13 to 17, characterized in that the solution A is mixed with the solution B in a volume ratio of 1 : 1.

19. The method according to claim 12, characterized in that solutions A, B and at least one solution C as defined in any one of claims 6 to 10 are prepared separately, wherein the solution A comprises horseradish peroxidase, hydroxyphenyl derivative of hyaluronan of the general formula I as defined in claim 1, triclosan and hydroxypropyl-β-cyclodextrin , the solution B contains hydrogen peroxide and the solution C contains the hydroxyphenyl derivative of hyaluronan of the general formula I as defined in claim 1, after which solutions A, B and C are mixed to form a hydrogel containing a covalently crosslinked hydroxyphenyl derivative of hyaluronan, which is intended for prevention of postoperative complications associated with the formation of colorectal anastomosis.

20. The method according to claim 19, characterized in that the horseradish peroxidase activity in the solution A is in the range of 1 to 3 U/mL, preferably 1.6 to 2.7 U/mL, more preferably 1.8 to 2.4 U/mL, the concentration of hydrogen peroxide in the solution B is in the range of 2 to 12 mmol/L, preferably 6 to 10 mmol/L, hydroxyphenyl derivative of hyaluronan of the general formula I having a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; the degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 %, is present in the solution A in a concentration of 1 to 20 mg/mL, preferably 5 to 15 mg/mL, more preferably 10 mg/mL, and in the solution C at a concentration of 10 to 50 mg/mL, preferably 30 to 40 mg/mL, more preferably 35 mg/mL, the concentration of triclosan in the solution A is in the range of 0.2 to 4.4 mg/mL, preferably 2 to 4.4 mg/mL, more preferably 4 mg/mL, the concentration of hydroxypropyl-β-cyclodextrin in the solution A is in the range of 8 to 200 mg/mL, preferably 100 to 160 mg/mL, more preferably 120 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

21. The method according to claim 19 or claim 20, characterized in that solutions A, B and C are mixed in a volume ratio of 1 : 1 : 2.

22. A hydrogel preparable by the method according to any one of claims 12 to 21, which comprises: covalently crosslinked hydroxyphenyl derivative in a concentration of 10 to 50 mg/mL, which is formed by crosslinking a hydroxyphenyl derivative of hyaluronan of the general formula I

(I), wherein n is in the range of 2 to 5000, M is H+ or a cation of a pharmaceutically acceptable salt selected from the group comprising of an alkali metal cation, alkaline earth metal cation, and wherein R is OH or substituent NHR2CONHR1 ArOH of the general formula II,

(II), wherein Ar is phenylene and Ri is ethylene, or Ar is indolydene and Ri is ethylene, or Ar is hydroxyphenylene and Ri is carboxyethylene, and R2 is alkylene of 3 to 7 carbons, wherein after mixing at least two solutions A and B it reaches a gelation point within 5 to 70 s, preferably 15 to 60 s, more preferably 25 to 50 s, while the value of its elastic module reaches 100 to 1000 Pa, no later than 3 min after mixing the solutions, preferably 100 to 600 Pa, more preferably 100 to 500 Pa, and upon completion of the solidification process, its elastic module is in the range of 500 to 2000 Pa, preferably 600 to 1300 Pa, more preferably 700 to 1200 Pa.

23. The hydrogel according to claim 22, characterized in that it comprises:

• Covalently crosslinked hydroxyphenyl derivative 10 to 50 mg/mL

• Horseradish peroxidase 0.25 to 0.75 U/mL

• Triclosan 0.1 to 1.1 mg/mL

• Hydroxypropyl-β-cyclodextrin 2 to 50 mg/mL.

24. The hydrogel according to claim 22 or claim 23, characterized in that it comprises:

• Covalently crosslinked hydroxyphenyl derivative at a concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.45 to 0.675 U/mL,

• Triclosan at a concentration of 0.5 to 1.1 mg/mL,

• Hydroxypropyl-β-cyclodextrin at concentration of 12 to 38 mg/mL,

25. The hydrogel according to any one of claims 22 to 24, characterized in that it comprises:

• Covalently cross-linked hydroxyphenyl derivative at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.4 to 0.6 U/mL,

• Triclosan at a concentration of 1 mg/mL, Hydroxypropyl-β-cyclodextrin of concentration 30 mg/mL.

26. The hydrogel according to any one of claims 22 to 24 for use in the prevention of postoperative complications associated with the formation of a colorectal anastomosis.

Description:
Kit of gel-forming solutions intended for preparation of hydrogel based on covalently crosslinked hydroxyphenyl derivative of hyaluronan for prevention of postoperative complications related to formation of colorectal anastomosis, use of kit, method of preparation of hydrogel and use thereof

Field of invention

The present invention relates to a kit of gel-forming solutions for a preparation of a hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan for the prevention of postoperative complications associated with colorectal anastomosis resulting from anastomic leakage and including, in particular, dehiscence of colorectal anastomosis and development of inflammation. It further relates to an use of the kit, a method of the preparation of the hydrogel and use thereof.

Background of the invention

Colorectal carcinoma (CRC) is a disease of affluence. Malignant neoplasm of colorectum is one of the most common oncological diagnoses [1], The relative five-year survival of patients with colorectal cancer in cases diagnosed in 2001-2005 was about 50% in both sexes (calculated from all reported cases, i.e. treated and for various reasons untreated). In the European Union, the incidence of rectal cancer is 15-25 newly diagnosed tumors per population of 100,000 per year. Mortality is reported among 4-10 patients per population of 100,000 per year with a slight predominance of the male population [2].

Procedures used in the surgical treatment of CRC include resection of the affected part of the intestine and subsequent formation of an anastomosis (connection). One of the most serious early complications of this procedure is an anastomic leak, which leads to leakage of the contents of the digestive tract outside the intestinal lumen. The presence of intestinal bacteria in the small pelvic area can cause infection with localized (pelvic abscess) or generalized (peritonitis, sepsis) manifestations. Leakage of the anastomosis can also lead to dehiscence of the anastomosis. It may be a localized problem that does not affect most of the circumference of the anastomosis, but it may also be a complete disintegration of the anastomosis. Such a condition endangers the patient's life, it is necessary to solve it operatively and there is a real risk that the patient will be dependent on an artificial intestinal outlet for the rest of his life. Among the risk factors for the development of anastomic leakage or dehiscence of the anastomosis includes intestinal ischemia in the suture line, excessive tension in the anastomosis, the presence of local sepsis, etc. Complications arise either due to a poorly technically performed connection (usually within 48 hours of surgery) or more often from poor healing of the anastomosis. This usually occurs between the 4 th and 6 th postoperative day, with very low rectal resections even later [1],

There is currently a number of surgical procedures that try to minimize the onset of this complication, yet anastomosis dehiscence occurs in 5 to 20 % of operated patients [1], So-called staplers are used to reconnect the digestive tract, that are designed for fast and even suturing of the tissue [3]. Various types of tissue adhesives are often used as a supplement or replacement for suture material to increase the resistance of gastrointestinal anastomoses [4- 6]. Their task is to strengthen the joint of the digestive tract and their presence is to reduce the leakage of intestinal contents into the peritoneum. Fibrin adhesives, cyanoacrylates, polyethylene glycol-based hydrogels and gelatin-based hydrogels are most commonly used in this indication [7, 8].

Clinically, fibrin glue is currently the most commonly used to support the healing of anastomosis, the use of which probably has a really positive effect on the healing process of colorectal anastomosis and increase of the resistance of the joint to anastomosis leakage [9]. However, if the intestine is perforated, despite the increased resistance of the anastomosis, e.g. due to its ischemization due to inappropriate surgical techniques, the mere presence of a tissue adhesive does not provide any additional protection against the possible development of infection.

Despite the large number of preclinical studies performed to verify the effectiveness of the use of tissue adhesives in the formation of colorectal anastomosis, their role is not entirely clear. Studies generally agree that the presence of tissue adhesive will reduce unwanted leakage of intestinal contents out of the intestinal lumen and increase the strength of the tissue connection in the short term [8]. However, greater joint rigidity may prevent peristaltic bowel movements, which may increase the risk of bowel obstruction. In addition, the use of cyanoacrylates can negatively affect the healing of the surgical wound [6, 10]. From a longer- term perspective, the use of tissue adhesives, especially cyanoacrylate -based materials, may not be advantageous in a given indication. Ustek et al. [11] published the results of a preclinical study describing the use of liposomal iodinated povidone (PVP-I) combined with a polyacrylate gel [12]. The publication describes the positive effect of the presence of a hydrogel on the healing of an anastomosis, which is attributed to the combination of wet covering of the inner wound and the broad- spectrum antimicrobial effect of the used PVP-I complex. However, said hydrogel does not provide mechanical support for the anastomosis, nor is mentioned its possible barrier function, preventing the contents of the digestive tract from leaking out of its lumen.

The use of a combination of a hydrogel with fibroblast growth factor [13] or with a platelet-rich plasma (PRP) fraction [14] has also been preclinically tested as a possible way to accelerate the healing of colorectal anastomosis. In both studies, the beneficial effect of increased concentrations of the observed factors on the healing of the anastomosis was described. However, even these hydrogels do not provide protection against the spread of infection in the event of dehiscence of the anastomosis.

A suture material containing the antiseptic triclosan (TCS; e.g. VICRYL® Plus Antibacterial Suture) can currently be used as a means of preventing postoperative infection at the site of the procedure. Indeed, meta- analyzes of the results of clinical studies comparing the incidence of postoperative infections using sutures containing and lacking TCS show a reduction in the likelihood of infection when sutures containing this antimicrobial are used [15, 16]. In the case of colorectal surgery, the included studies described the use of antimicrobial suture material to close the abdominal cavity, resp. suturing of the abdominal fascia [17-20].

Triclosan (2,4,4 '-trichloro-2'-hydroxydiphenyl ether) is an antimicrobial synthetic substance, poorly soluble in water and well soluble in polar organic solvents (ethanol, chloroform, isopropanol). It is a chemically stable substance that can be stored under normal conditions for many years [21].

Triclosan (trade name Irgasan®, TCS) has been used as an ingredient in a number of cosmetic and pharmaceutical formulations for almost 50 years. It was originally used as an additive in soaps, shower gels, oral hygiene products, but also as an antiseptic for the production of functional fabrics (surgical gowns) and plastics (kitchen utensils, children's toys, antimicrobial surface treatment of medical devices). Due to its extensive use, TCS has been extensively described in terms of antimicrobial efficacy, acute and chronic toxicity, mutagenicity, reproductive toxicity, and teratogenicity. [22-25] Triclosan has a broad spectrum of biocidal activity, which includes Gram positive and Gram negative non-sporulating bacteria, some species of fungi and yeasts. It also has antiviral effects [22, 23]. TCS exhibits both bacteriostatic and bactericidal effects in a concentration- dependent manner [26]. At lower concentrations, the inhibitory effect of TCS on the activity of enoyl-acyl carrier protein (ACP) reductase (Fabl), which is a key enzyme for the synthesis of fatty acids in bacteria, is particularly evident. [27, 28] At higher concentrations of TCS, non-specific mechanisms of action of bisphenols, such as damage to membrane integrity, participate on the biocidal effect [29, 30].

The possibility of incorporating TCS into the hydrogel structure is limited by its low solubility in water. A possible solution is to prepare the inclusion of TCS with cyclodextrins (CD) [31, 32]. For example, triclosan containing supramolecular hydrogels based on pluronic acid F-127 and a-cyclodextrin have been prepared by this procedure. [33] Based on structural studies and due to its very favorable pharmacological profile, 2 -hydroxypropyl-β-cyclodextrin (HR-β-CD) appears to be the most suitable candidate for the preparation of absorbable hydrogels containing TCS / CD inclusion [34, 35]. US20170281781A1 describes that cyclodextrins are able to interact with mucosal surface proteins and the presence of water- soluble cyclodextrin derivatives increases the mucoadhesive properties of hydrogels [36].

Hyaluronan is a polysaccharide that consists of disaccharide units composed of D- glucuronic acid and D-N -acetylglucosamine linked by alternating b-1,4 and b-1,3 glycosidic bonds. The weight average molecular weight (if molecular weight is mentioned below, it will always be the weight average molecular weight) in vivo is in the range of 3,000 g/mol to 20,000,000 g/mol. It is a polysaccharide that is easily soluble in aqueous media, where, depending on molecular weight and concentration, it forms very viscous solutions. Hyaluronan is a component of almost all tissues and body fluids of vertebrates, and is abundant, especially in connective tissues. It is a highly hygroscopic molecule, hyaluronan solutions are strongly osmotically active and the presence of hyaluronan is, among other things, important for tissue hydration [37].

In addition, hyaluronan is able to modulate inflammatory responses of tissues, both by influencing the production of cytokines and by its effect on the adhesion of cytokine-activated lymphocytes. Its antioxidant properties and ability to scavenge free radicals reduce the activity of proteinases acting during inflammation, whereby hyaluronan contributes to the stabilization of the affected tissue and promotes its granulation [37]. In addition to their use in the treatment of chronic wounds, hyaluronan-based products are widely used in the prevention of postoperative adhesions. Hyaluronan solutions are used to fill the abdominal cavity after surgery. The presence of the hyaluronan solution is intended to mechanically separate the traumatized surfaces of the internal organs and thus prevent their adhesions. The disadvantage of using these solutions is the short biological half-life of unmodified hyaluronan, which does not provide long-lasting protection against adhesions. In order to solve this problem, hyaluronan containing gels and foils have been proposed in the past, which at the site of action act as a barrier against the formation of adhesions for a longer period of time [37].

Various types of hyaluronan derivatives have also been developed in the past that are able to undergo a sol-gel transition under physiological conditions in situ [38, 39]. Phenolic hyaluronan derivatives, for example, can be used for this purpose. Calabro et al. [40-42] describe in EP1587945B1 and EP1773943B1 a method for the preparation of hydroxyphenyl derivatives of hyaluronan by the reaction of carboxyls present in the structure of D-glucuronic acid of hyaluronan, with aminoalkyl derivatives of phenol, e.g. tyramine. Products of this reaction are hyaluronan amides [43]. The same document also discloses that crosslinking of hydroxyphenyl derivatives of hyaluronan can be initiated by the addition of peroxidase (e.g. horseradish peroxidase) and diluted hydrogen peroxide solution. Horseradish peroxidase (HRP, E.C.1.11.1.7) is currently widely used as a catalyst for organic and biotransformation reactions [44-48]. Hydrogels based on hydroxyphenyl derivatives of hyaluronan can be used as injectable matrices for controlled release of substances or as materials suitable for culturing and implanting cells [49]. WO/2017/197262 describes the use of a tyraminated hyaluronan derivative for the preparation of a hydrogel matrix containing several types of reservoirs of biologically active substances. It also uses a horseradish peroxidase-mediated reaction to crosslink the hydrogel. Wolfova et al. disclose in CZ303879 a conjugate of hyaluronan and tyramine comprising an aliphatic linker inserted between a polymer chain and tyramine. The presence of an aliphatic linker allows a higher efficiency of the crosslinking reaction and gives the network higher elasticity. This document also describes the possible use of hydrogels based on the respective derivative as a biodegradable barrier preventing the formation of postoperative adhesions.

Although hyaluronan is widely used as a material for the prevention of postoperative adhesions, in the case of prevention of complications associated with colorectal anastomosis, where there is an increased risk of infection and development of peritonitis, the use of materials based on it is not always advantageous. Hyaluronan hydrogel crosslinked with polyvalent iron ions (Intergel) has been evaluated in the past as a material that increases the virulence of some bacterial strains and increases the risk of postoperative complications [37, 50]. Antiadhesive membranes based on a mixture of hyaluronan and carboxymethylcellulose are contraindicated when used in direct contact with intestinal anastomoses, for direct contact with the suture line of the anastomosis and in the case of clinically manifested infection [51].

Summary of the invention

The object of the invention is to overcome the shortcomings of the prior art and to develop a means for the preparation of a biodegradable hydrogel based on hyaluronan containing an antiseptic agent in order to prevent postoperative complications associated with the formation of colorectal anastomosis. Such means is a kit of gel-forming solutions intended for the preparation of a hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan, which essentially comprises at least two aqueous solutions A and B, of which the solution A contains horseradish peroxidase and the solution B contains hydrogen peroxide, the solution A and/or the solution B containing a hydroxyphenyl derivative of a hyaluronan of a general formula I wherein n is in the range of 2 to 5000, M is H + or a cation of a pharmaceutically acceptable salt selected from a group containing alkali metal cation, alkaline earth metal cation, and wherein R is OH or substituent NHR2CONHR 1 ArOH of a general formula II, wherein Ar is phenylene and Ri is ethylene, or Ar is indolydene and Ri is ethylene, or Ar is hydroxyphenylene and Ri is carboxyethylene, and R2 is alkylene of 3 to 7 carbons, and at the same time the solution A and/or the solution B contains triclosan and hydroxypropyl-b- cyclodextrin. The cation of the pharmaceutically acceptable salt is preferably selected from the group containing of Na + , K + , Mg 2+ or Li + .

Preferably, in the kit of the invention, the horseradish peroxidase activity is in the range of 0.5 to 1.5 U/mL, preferably 0.9 to 1.35 U/mL, more preferably 0.8 to 1.2 U/mL, the concentration of hydrogen peroxide is in the range of 1 to 6 mmol/L, preferably 3 to 5 mmol/L, the hydroxyphenyl derivative of hyaluronan according to the general formula I has a weight average molecular weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; a degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 % and a concentration of 10 to 50 mg/mL, preferably 15 to 25 mg/mL, more preferably 20 mg/mL; and the concentration of triclosan is in the range of 0.1 to 2.2 mg/mL, preferably 1 to 2.2 mg/mL, more preferably 2 mg/mL and the concentration ofhydroxypropyl-β-cyclodextrin is in the range of 4 to 100 mg/mL, preferably 25 to 80 mg/mL, more preferably 60 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

Polysaccharides, including hyaluronan and derivatives prepared thereof, belong to polymers formed by a mixture of macromolecules of different lengths and thus form non- uniform (polydisperse) systems. The molar mass of such polymers can be expressed as numerical average molar mass (Mn) or weight average molar mass (Mw). The ratio of these two types of average molecular weights of the polymer chains (Mw/Mn) expresses the degree of non-amorphous (polydispersity) of the polymer sample and is referred to as the polydispersity index (PI). The PI of hydroxyphenyl derivative of hyaluronan of the general formula (I) as mentioned above is in the range of 1 to 3.

More preferably, the kit of the invention comprises the solution A, which contains:

• A hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight- average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration 10 to 50 mg/mL,

• Horseradish peroxidase with an activity of 0.5 to 1.5 U/mL,

• Triclosan, which is at a concentration of 0.2 to 2.2 mg/mL, • Hydroxypropyl-β-cyclodextrin , which is in a concentration of 4 to 100 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10. and the solution B, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL,

• Hydrogen peroxide, in the concentration range of 1 to 6 mmol/L.

Further, more preferably, the kit of the invention comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, whereas it is at concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.9 to 1.35 U/mL,

• Triclosan at a concentration of 1 to 2.2 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 25 to 80 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 5 to 1 : 8. and the solution B, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, whereas it is at concentration 15 to 25 mg/mL,

• Hydrogen peroxide, which is in the concentration range of 3 to 5 mmol/L. And even more preferably, the kit of the invention comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.8 to 1.2 U/mL,

• Triclosan at a concentration of 2 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is at a concentration of 60 mg/mL. and the solution B, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL

• Hydrogen peroxide in the concentration range of 4 to 5 mmol/L.

According to yet another preferred embodiment of the invention, the kit comprises aqueous solutions A, B and at least one solution C, wherein the solution A comprises horseradish peroxidase, a hydroxyphenyl derivative of hyaluronan of the general formula I as defined above, triclosan and hydroxypropyl-β-cyclodextrin , the solution B comprises hydrogen peroxide and the solution C comprises a hydroxyphenyl derivative of a hyaluronan of the general formula I as defined above.

Further preferably, the horseradish peroxidase activity in the solution A is in the range of 1 to 3 U/mL, preferably 1.6 to 2.7 U/mL, more preferably 1.8 to 2.4 U/mL, the concentration of hydrogen peroxide in the solution B is in the range of 2 to 12 mmol/L, preferably 6 to 10 mmol/L, a hydroxyphenyl derivative of hyaluronan according to the general formula I that has a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; the degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 %, is present in the solution A in a concentration of 1 to 20 mg/mL, preferably 5 to 15 mg/mL, more preferably 10 mg/mL, and in the solution C at a concentration of 10 to 50 mg/mL, preferably 30 to 40 mg/mL, more preferably 35 mg/mL, the concentration of triclosan in the solution A is in the range of 0.2 to 4.4 mg/mL, preferably 2 to 4.4 mg/mL, more preferably 4 mg/mL, the concentration of hydroxypropyl-β-cyclodextrin in the solution A is in the range of 8 to 200 mg/mL, preferably 100 to 160 mg/mL, more preferably 120 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

Further, more preferably, the kit of the invention comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight- average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration 1 to 20 mg/mL,

• Horseradish peroxidase with an activity of 1 to 3 U/mL,

• Triclosan at a concentration of 0.4 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin, which is in a concentration of 8 to 200 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl^-cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B, which contains:

• Hydrogen peroxide, whereas the concentration is in the range 2 to 12 mmol/L. and the solution C, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL,

Further, more preferably, the kit of the invention comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, in a concentration of 5 to 15 mg/mL,

• Horseradish peroxidase with an activity of 1.6 to 2.7 U/mL,

• Triclosan, which is at a concentration of 3.6 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 108 to 132 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-b- cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B, which contains:

• Hydrogen peroxide in the concentration range of 7 to 12 mmol/L. and the solution C, which contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 30 to 40 mg/mL.

And even more preferably, the kit of the invention comprises the solution A, which comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 10 mg/mL,

• Horseradish peroxidase with an activity of 1.8 to 2.4 U/mL,

• Triclosan, which is at a concentration of 3.6 to 4.4 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is in a concentration of 108 to 132 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-b- cyclodextrin is in the range of 1 : 4 to 1 : 10, the solution B, which contains:

• Hydrogen peroxide in the concentration range of 7 to 12 mmol/L. and the solution C, which contains: • Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molecular weight in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 35 mg/mL.

Furthermore, the invention also relates to the use of a kit according to the invention for the preparation of a biodegradable hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan at its site of action in the small pelvic region, where the formed hydrogel serves as prevention of postoperative complications related to formation of colorectal anastomosis. Postoperative complications associated with the formation of a colorectal anastomosis are selected from the group containing of anastomic leakage, opening (dehiscence) of the colorectal anastomosis, development of infection in the small pelvis and peritoneum.

A method of preparation of a hydrogel comprising a covalently crosslinked hydroxyphenyl hyaluronan derivative, wherein at least two solutions A and B as defined in claim 1 are prepared separately, wherein the solution A and/or the solution B comprises a hydroxyphenyl hyaluronan derivative of the general formula I as defined above and, at the same time, the solution A and/or the solution B contains triclosan and hydroxypropyl-β-cyclodextrin , after which the solution A is mixed with the solution B to form a hydrogel containing a covalently crosslinked hydroxyphenyl derivative of hyaluronan, which is intended to prevent postoperative complications associated with colorectal anastomosis.

Another embodiment of the invention is a method of preparation of a hydrogel comprising a covalently crosslinked hydroxyphenyl derivative of hyaluronan, whose essence lies in that at least two solutions A and B are prepared separately as described above, wherein the solution A and/or the solution B comprises a hydroxyphenyl derivative of hyaluronan of the general formula I as defined above, and at the same time the solution A and/or the solution B contains triclosan and hydroxypropyl-β-cyclodextrin , after which the solution A is mixed with the solution B to form a hydrogel containing a covalently crosslinked hydroxyphenyl derivative of hyaluronan, which is intended to prevent postoperative complications associated with colorectal anastomosis. Preferably, the solution A contains horseradish peroxidase with an activity in the range of 0.5 to 1.5 U/mL and the solution B contains hydrogen peroxide in a concentration in the range of 1 to 6 mmol/L, wherein the solution A and/or the solution B contains a hydroxyphenyl derivative of hyaluronan of the general formula I as described above, wherein its weight average molar weight is in the range of 60,000 g/mol to 2,000,000 g/mol, the degree of substitution is in the range of 1 % to 10 %, and its concentration is in the range of 10 to 50 mg/mL, while the solution A and/or the solution B contains triclosan and hydroxypropyl-b- cyclodextrin, after which the solution A is mixed with the solution B to form a hydrogel containing a covalently cross-linked hydroxyphenyl derivative of hyaluronan, which is intended to prevent postoperative complications associated with the formation of colorectal anastomosis.

Postoperative complications associated with the formation of colorectal anastomosis are selected from the group comprising of colorectal anastomosis disintegration, anastomic leakage, spread of infection.

In a preferred embodiment of the method according to the invention, the horseradish peroxidase activity is in the range of 0.5 to 1.5 U/mL, preferably 0.9 to 1.35 U/mL, more preferably 0.8 to 1.2 U/mL, the hydrogen peroxide concentration is in the range of 1 to 6 mmol/L, preferably 3 to 5 mmol/L, the hydroxyphenyl derivative of hyaluronan according to the general formula I has a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100 000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; a degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 % and a concentration of 10 to 50 mg/mL, preferably 15 to 25 mg/mL, more preferably 20 mg/mL; and the concentration of triclosan is in the range of 0.2 to 2.2 mg/mL, preferably 1 to 2.2 mg/mL, more preferably 2 mg/mL and the concentration of hydroxypropyl-β-cyclodextrin is in the range of 4 to 100 mg/mL, preferably 25 to 80 mg/mL, more preferably 60 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

According to yet another preferred embodiment of the method according to the invention, the solution A comprises: • Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol and a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, at a concentration of 10 to 50 mg/mL,

• Horseradish peroxidase with an activity of 0.5 to 1.5 U/mL,

• Triclosan, that is at a concentration of 0.2 to 2.2 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 4 to 100 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10. and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 60,000 g/mol to 2,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 10 %, in a concentration of 10 to 50 mg/mL,

• Hydrogen peroxide, in the concentration range of 1 to 6 mmol/L.

According to yet another preferred embodiment of the method according to the invention, the solution A comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar weight in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.9 to 1.35 U/mL,

• Triclosan at a concentration of 1 to 2.2 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 25 to 80 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 5 to 1 : 8. and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 100,000 g/mol to 1,000,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 1 % to 5 %, at a concentration of 15 to 25 mg/mL, • Hydrogen peroxide, which is in the concentration range of 3 to 5 mmol/L.

According to yet another preferred embodiment of the method according to the invention, the solution A comprises:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.8 to 1.2 U/mL,

• Triclosan at a concentration of 2 mg/mL,

• Hydroxypropyl-β-cyclodextrin , which is at a concentration of 60 mg/mL. and the solution B contains:

• Hydroxyphenyl derivative of hyaluronan according to the general formula I with a weight average molar mass in the range of 200,000 g/mol to 400,000 g/mol and with a polydispersity index of 1 to 3, with a degree of substitution in the range of 2 % to 4 %, at a concentration of 20 mg/mL

• Hydrogen peroxide in the concentration range of 4 to 5 mmol/L.

According to yet another preferred embodiment of the method according to the invention, the solution A is mixed with the solution B in a volume ratio of 1 : 1.

Yet another preferred embodiment of the method according to the invention is to prepare solutions A, B and at least one solution C, defined above for a kit comprising solutions A, B and at least one solution C, wherein the solution A comprises horseradish peroxidase, a hydroxyphenyl derivative of hyaluronan of the general formula I, as defined above, triclosan and hydroxypropyl-β-cyclodextrin , the solution B contains hydrogen peroxide and the solution C contains a hydroxyphenyl derivative of hyaluronan of the general formula I as defined above, whereupon solutions A, B and C are mixed to form a hydrogel containing covalently crosslinked hydroxyphenyl derivative of hyaluronan, which is intended to prevent postoperative complications associated with the formation of colorectal anastomoses. Further preferably, the horseradish peroxidase activity in the solution A is in the range of 1 to 3 U/mL, preferably 1.6 to 2.7 U/mL, more preferably 1.8 to 2.4 U/mL, the concentration of hydrogen peroxide in the solution B is in the range of 2 to 12 mmol/L, preferably 6 to 10 mmol/L, a hydroxyphenyl derivative of hyaluronan according to the general formula I having a weight average molar weight in the range of 60,000 g/mol to 2,000,000 g/mol, preferably 100,000 g/mol to 1,000,000 g/mol, more preferably 200,000 g/mol to 400,000 g/mol; the degree of substitution in the range of 1 % to 10 %, preferably 1 % to 5 %, more preferably 2 % to 4 %, is present in the solution A in a concentration of 1 to 20 mg/mL, preferably 5 to 15 mg/mL, more preferably 10 mg/mL, and in the solution C at a concentration of 10 to 50 mg/mL, preferably 30 to 40 mg/mL, more preferably 35 mg/mL, the concentration of triclosan in the solution A is in the range of 0.2 to 4.4 mg/mL , preferably 2 to 4.4 mg/mL, more preferably 4 mg/mL, the concentration of h ydro x y p ro py I-b-cycl odcx t ri n in the solution A is in the range of 8 to 200 mg/mL, preferably 100 to 160 mg/mL, more preferably 120 mg/mL, wherein the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin is in the range of 1 : 4 to 1 : 10, preferably in the range of 1 : 5 to 1 : 8, more preferably 1 : 6.

Preferably, solutions A, B, C are mixed in a volume ratio of 1: 1: 2.

Yet another embodiment of the invention is a hydrogel preparable by the method of the invention as set forth above, comprising: covalently crosslinked hydroxyphenyl derivative in a concentration of 10 to 50 mg/mL, which is formed by crosslinking a hydroxyphenyl derivative of hyaluronan of the general formula I wherein n is in the range of 2 to 5000, M is H + or a cation of a pharmaceutically acceptable salt selected from the group containing of an alkali metal cation, alkaline earth metal cation, and wherein R is OH or substituent NHRiCONHR 1 ArOH of the general formula

II, wherein Ar is phenylene and Ri is ethylene, or Ar is indolydene and Ri is ethylene, or Ar is hydroxyphenylene and Ri is carboxyethylene, and R2 is alkylene of 3 to 7 carbons, wherein after mixing at least two solutions A and B it reaches a gelation point within 5 to 70 s, preferably 15 to 60 s, more preferably 25 to 50 s, while the value of its elastic module reaches 100 to 1000 Pa, no later than 3 min after mixing the solutions preferably 100 to 600 Pa, more preferably 100 to 500 Pa, and upon completion of the solidification process, its elastic module is in the range of 500 to 2000 Pa, preferably 600 to 1300 Pa, more preferably 700 to 1200 Pa.

The cation of the pharmaceutically acceptable salt is preferably selected from the group containing of Na + , K + , Mg 2+ or Li + .

Preferably, the hydrogel of the invention comprises:

• Covalently crosslinked hydroxyphenyl derivative 10 to 50 mg/mL

• Horseradish peroxidase 0.25 to 0.75 U/mL

• Triclosan 0.1 to 1.1 mg/mL

• Hydroxypropyl-β-cyclodextrin 2 to 50 mg/mL.

More preferably, the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin (TCS : HR-β-CD ratio) is in the range of 1 : 4 to 1 : 10, whereas it reaches the gelation point within s to 70 s after mixing solutions A and B, furthermore, no later than 3 minutes after mixing the solutions, the value of its elastic module reaches 100 to 1000 Pa, and after the completion of the solidification process, its elastic module is in the range of 500 to 2000 Pa.

More preferably, the hydrogel of the invention comprises:

• Covalently crosslinked hydroxyphenyl derivative at a concentration of 15 to 25 mg/mL,

• Horseradish peroxidase with an activity of 0.45 to 0.675 U/mL,

• Triclosan at a concentration of 0.5 to 1.1 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 12 to 38 mg/mL. Even more preferably, the molar ratio of triclosan to hydroxypropyl-β-cyclodextrin (TCS : HR-β-CD ratio) is in the range of 1 : 5 to 1 : 8, whereas it reaches the gelation point within 15 s to 60 s after mixing solutions A and B, furthermore, no later than 3 minutes after mixing the solutions the value of its elastic module reaches 100 to 600 Pa and after the completion of the solidification process its elastic module is in the range of 600 to 1300 Pa

Even more preferably, the hydrogel of the invention comprises:

• Covalently cross-linked hydroxyphenyl derivative at a concentration of 20 mg/mL,

• Horseradish peroxidase with an activity of 0.4 to 0.6 U/mL,

• Triclosan at a concentration of 1 mg/mL,

• Hydroxypropyl-β-cyclodextrin at a concentration of 30 mg/mL.

Even more preferably, after mixing solutions A and B it reaches the gelation point within 25 s to 50 s, furthermore, within 3 min after mixing the solutions the value of its elastic module reaches 100 to 500 Pa and after completion of the solidification process, its elastic module is in the range of 600 to 1200 Pa.

According to yet another preferred embodiment of the invention, the hydrogel according to the invention is used as a filling material around the colorectal anastomosis to prevent colorectal anastomosis from opening, anastomic leakage and the spread of infection due to anastomic leakage.

The hydrogel formed using the kit of the invention contains an antiseptic agent and serves as a barrier to the growth of bacteria in the small pelvic region, thereby helping to prevent postoperative complications resulting from anastomic leakage and opening of the colorectal anastomosis.

Hydrogels belong to viscoelastic materials, whose complex rheological behavior, i.e. the behavior of substances, which include in part both viscous and elastic components, can be expressed by the so-called complex module G*. In rheology, the elastic component of deformation is expressed by the so-called elastic (memory) module (G') and the viscous component by the so-called viscous (lossy) module (G")· Mathematically, it is possible to express a complex module as a complex number containing of a real and an imaginary component: and mutual relation between G", G" and G* is given by the equation:

The process of gel formation is called gelation. In the case described, a hydrogel is formed from a polymer precursor solution which contains linear polysaccharide chains. During the chemical reaction, cross-links are formed between the individual polymer chains and thus a polymer network is formed. We call the gelation point the moment when an infinite three-dimensional network just appears in the system. The word "infinite" is to be understood as meaning that the dimensions of the resulting network are identical to the dimensions of the macroscopic gel phase. The weight fraction of the network is still insignificant at the gelation point, but in the further course it increases rapidly (the weight of the gel fraction increases at the expense of the weight of the soluble fraction), which is reflected in a gradual increase in the elastic module of the resulting hydrogel.

Rheologically, this change is expressed as follows: at the gelation point, the viscosity of the liquid is limited to infinity, while the module of elasticity assumes non-zero values, as can be seen from Fig. 9. At the gelation point, the elastic and viscous modules have the same value. Once the gelation point is reached, the gel formation process is not complete. As the chemical reaction continues, the polymer network increases and its rigidity increases, which is also reflected in the increase in G'. After some time, however, the crosslinking reaction stops (e.g., due to depletion of the reactants) and the polymer network stabilizes, the gel solidification process is completed. We refer to this point as solidification and the time to reach it is referred to in this text as T solid . The size of the elastic module after the completion of the gel formation process is referred to herein as G's.

The object of the present invention is a kit for the preparation of a biodegradable hyaluronan-based hydrogel containing the antibacterial agent triclosan in the form of an inclusion with hydroxypropyl-β-cyclodextrin .

The kit according to the invention comprises at least two aqueous solutions A and B, one of which contains horseradish peroxidase (solution A) and the other hydrogen peroxide (solution B), whereas at least one of the solutions contains a hydroxyphenyl derivative of hyaluronan, and at least one of the precursor solutions contains triclosan in the form of an inclusion with hydroxypropyl-β-cyclodextrin. Mixing solutions A and B of the described composition in a ratio of 1 : 1 forms a hydrogel based on a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), which is characterized in that its elastic module value reaches 100 to 1000 Pa no later than 3 minutes after mixing the solutions and after completion of the solidification process, its elastic module is in the range of 500 to 2000 Pa. The hydrogel is formed at the site of application {in situ ) and undergoes a sol-gel transition under physiological conditions. The hydrogel is intended for use during surgery, when it is able to completely fill the space of the small pelvis in the immediate vicinity of the colorectal anastomosis. The rate of solidification of the described composition allows the application of a sufficient amount of gel-forming mixture to the small pelvic area, perfect filling of the application site and the formation of a homogeneously crosslinked hydrogel in a period of time that does not excessively prolong the surgery. The resulting hydrogel provides sufficient mechanical support for the created colorectal anastomosis, but at the same time does not prevent peristaltic movements of the intestinal wall.

The use of the kit according to the invention allows the hydrogel used to be able to ideally fill the entire space of the small pelvis and thus surround the formed anastomosis. For this purpose, it is appropriate to use hydrogels that are able to undergo a sol-gel transition directly at the site of application under physiological conditions. The rate of the hydrogel formation process at the site of application, the homogeneity of crosslinking of the resulting hydrogel and its final viscoelastic properties are important parameters that contribute to the effectiveness of the composition. Too rapid hydrogel formation may not lead to perfect filling of the anatomically segmented area of the small pelvis and homogeneous crosslinking of the material. Inhomogeneous crosslinking can cause some of the material to leak from the application site and limit the barrier function of the device. Conversely, the slow formation of a gel can unnecessarily delay the course of surgery. Insufficient crosslinking of the hydrogel, which manifests itself as low stiffness of the hydrogel, can lead to failure of its barrier function, as it can migrate from the site of application. On the contrary, too strongly crosslinked hydrogels, showing high rigidity, can hinder the natural peristaltic movements of the intestine, and thus disrupt its function. The kit according to the invention thus allows the formation of a hydrogel which homogeneously fills the space of the small pelvis, surrounds the formed anastomosis and reaches a value of its elastic modulus of 100 to 1000 Pa within 3 minutes after application and its elastic module is 500 to 2000 Pa after completion of the solidification process. These conditions can be met by hydrogels prepared from hydroxyphenyl hyaluronan derivatives of the general formula I as specified above, which can be crosslinked by a horseradish peroxidase catalyzed reaction, even though the presence of hydroxypropyl-β-cyclodextrin has been found to slow down the gelation rate and efficiency of the crosslinking reaction (see Example 16, Fig. 8).

To achieve the desired properties, it was therefore necessary to choose a suitable combination of the concentration of the hyaluronan derivative, its molecular weight, the degree of substitution, the concentration of hydrogen peroxide and the activity of horseradish peroxidase. The reaction is initiated by the addition of hydrogen peroxide so that the preparation of the hydrogel can be carried out by mixing two solutions of the hydroxyphenyl derivative of hyaluronan of the general formula I as specified above, where one of them contains hydrogen peroxide and the other horseradish peroxidase. Large volumes of homogeneously crosslinked hydrogel can be prepared by ensuring sufficient mixing of both precursor solutions (e.g. by using a static mixer).

Hydrogels in general may not themselves be an effective barrier against the spread of infection, as it is generally known that some types of hydrogels are used as suitable substrates for culturing bacteria [52, 53]. Colonization of the hydrogel by bacteria would in fact lead to a failure of the composition, as it would not prevent the development of infection in the small pelvis and its possible spread into the peritoneum. Hydrogel colonization can be prevented by combination with suitable antibacterial agents [54] . In this case, triclosan was selected as the antimicrobial agent in the form of an inclusion with 2 -hydroxypropyl-β-cyclodextrin . In vitro experiments have shown that hydrogels containing triclosan in the form of inclusions have shown in vitro antimicrobial effects and are not colonized by microorganisms.

The absorbability (biodegradability) of the material is considered to be a technically advantageous solution in the field of the development of medical devices intended for implantation into the patient's body [55]. Even in this case, it is advantageous for the hydrogel to be absorbed after fulfilling its purpose and not to require further surgery necessary to remove it. Hydrogels based on enzymatically crosslinked hydroxyphenyl derivatives of hyaluronan according to the invention also show this property.

On the other hand, the hydrogel according to the present invention is intended to act for several days (2 to 6 days, or even more days after surgery [1]) as a barrier to the spread of infection in the small pelvic region, outside of lumen of the digestive tract or during dehiscence of the anastomosis, exposed to the intestinal microflora. It produces a number of different types of enzymes (proteases [56], glycosidases - heparinases, chondroitinases, hyaluronidases), which can cause the degradation of biopolymers, including glycosaminoglycans, including hyaluronan [57]. Premature degradation of the hydrogel at the site of application would lead to loss of its barrier function and loss of its effect. The action of enzymes of the intestinal microflora does not prevent the use of the combination with antibacterial substances, because they themselves do not prevent the person from the action of bacterial hydrolases, which can penetrate into the affected area from the damaged digestive tract. During the development of the hydrogel according to the invention, it has been shown that the resistance of the developed material to the action of hydrolytic enzymes (e.g. hyaluronidase) and thus its rate of degradation can be controlled by the crosslink density of the hydrogel polymer network. It was found that with increasing elastic module G', the value of which reflects the crosslinking density of the gel, the resistance of the prepared hydrogels to the action of hydrolytic enzymes (e.g. hyaluronidase) increases and their degradation time increases. Furthermore, the presence of TCS/HP-β-CD inclusion in the hydrogel was found to further increase the resistance of hydrogels to hydrolytic enzymes (e.g. hyaluronidase) compared to hydrogels of comparable degree of crosslinking (comparable G' and swelling coefficient Q) without TCS/HP-β-CD content. This is an advantageous property because it prolongs the time for which the hydrogel can fulfill its barrier function even in hydrogels with relatively lower G", the consistency of which does not prevent peristaltic bowel movements.

During experiments performed on a model of dehiscence (opening or spacing) of porcine colorectal anastomosis, the presence of the above-described hydrogel of the present invention surrounding the collateral anastomosis and filling the small pelvis was found to prevent postoperative complications associated with development of sepsis), even if an intestinal perforation simulating partial dehiscence (disintegration) of the anastomosis was created in the vicinity of the formed anastomosis for experimental reasons. In contrast to the prior art compositions (tissue adhesives) which are applied to increase the mechanical resistance of the formed gastrointestinal tract connection, the hydrogel according to the invention also acts in the event of anastomosis leakage or partial anastomosis dehiscence, because in addition to its action as a support for the anastomosis and partial prevention of its mechanical damage, it also, above all, acts as a barrier preventing the escape of intestinal contents from the lumen of the colon and its spread in the area of the small pelvis. The antiseptic triclosan present prevents the colonization of the hydrogel and the small pelvic region by bacteria of the intestinal microflora, the development of peritonitis and other postoperative complications. The degree of crosslinking of the described hydrogel prevents premature degradation of the hydrogel by the action of hydrolytic enzymes produced by bacteria of the intestinal microflora but does not prevent the gradual complete absorption of the hydrogel after fulfilling its function.

The term "hyaluronan" means hyaluronic acid, or a pharmaceutically acceptable salt thereof. Detailed description of the drawings

Fig. 1: The graph shows the differences in the rate of weight loss of hydrogels upon degradation by hyaluronidase depending on the efficiency of their crosslinking.

Fig. 2: Antimicrobial effect of test samples on the bacterial strain Staphylococcus aureus. Fig. 3: Antimicrobial effect of test samples on the bacterial strain Escherichia coli.

Fig. 4: Antimicrobial effect of test samples on the yeast Candida albicans.

Fig. 5: Antimicrobial effect of test samples on the yeast Clostridium sporogenes.

Fig. 6: 6A - a prototype of a device consisting of the solution A and the solution B; 6B - colorectal anastomosis in a porcine model; 6C - porcine small pelvis filled with hydrogel Fig. 7: Application of hydrogel into a porcine small pelvis

Fig. 8: Influence of HR-β-CD on the preparation of gels based on HA-TA

Fig. 9: Change in elastic and viscous module of the gel during gelation.

Examples of embodiments of the invention DS = degree of substitution = 100 % * molar amount of modified disaccharide units of hyaluronan / molar amount of all disaccharide units of the hyaluronan derivative. The degree of substitution was determined by 1 H NMR spectroscopy. The weight average molar mass (Mw) and polydispersity index (PI) were determined by the SEC-MALLS method. Triclosan concentrations and HRP activity were determined spectrophotometrically.

Gelation kinetics

The gelation kinetics was determined using an AR-G2 rotary rheometer (TA instruments) using a plate-plate arrangement with a top geometry with a diameter of 40 mm and a gap setting of 400 pm. Precursor solutions A (250 μL) and B (250 μL) are applied onto the bottom stationary plate and pre-shear 2000 1/s for 1 s is used for their homogenization. The gelation kinetics is determined by the oscillation time sweep method at a frequency of 1 Hz and a shift of 0.001 rad at 37 ° C. The gelation time is determined as the intersection of the elastic and viscous module, and the elastic module for comparing the individual samples with each other is subtracted at 3 minutes from the start of the experiment (n = 3-5).

Viscoelastic properties

Hydrogels with a total volume of 1.7 ± 0.3 mL (n = 3-5) were prepared for testing and aged for 1 hour. The viscoelastic properties of the hydrogels were determined using an AR- G2 rotary rheometer using a cross-hatch geometry with a roughened surface to prevent the prepared hydrogel from slipping. The measurement was performed in strain sweep mode at a frequency of 1 Hz and a shift in the range of 0.001 to 2 rad. For the purposes of this application, the measurement was used to determine the elastic module of the gels after solidification (G's).

Example 1 Synthesis of tyraminated HA derivative (HA-TA)

Example 1A: Synthesis of 6-amino-/V- G2- (4-hydroxyphenyl) ethyl] hexanamide

6[(/er/-butoxycarbonyl) amino] hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 mL of tetrahydrofuran (THF). 1 , 1 '-carbodiimidazolc (0.70 g, 4.3 mmol) was added to the acid solution. The mixture was heated to 50 °C for sixty minutes. The reaction vessel was then purged with inert gas. To the reaction mixture was added tyramine (0.59 g, 4.3 mmol). The mixture was further heated for another 2 hours. The THF was then removed by distillation under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate. The solution was washed with 150 mL of purified water (divided into three parts). The organic layer was dried over a molecular sieve. The ethyl acetate was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of MeOH and 2 mL of trifluoroacetic acid (TFA) was added to the solution. The solution was heated to reflux for 6 hours. The solvent was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate. The solution was washed with 150 mL of purified water (divided into three parts). The organic layer was dried over a molecular sieve. The ethyl acetate was removed by distillation under reduced pressure. m = 0.75 g (70% of theory)

Hylauronan (10.00 g, M w = 2,000,000 g/mol) was dissolved in 750 mL of a 2.5% (w/w) Na 2 HP0 4 .12 H2O solution. The solution was cooled to 5 °C. To the resulting solution were added 2.60 g of NaBr and 0.05 g of 4-acetamido-2,2,6,6-tetramethylpiperidine-l-oxyl. After thorough homogenization of the solution, 3 mL of NaCIO solution (10-15% of available CI2) were added to the reaction mixture. The reaction was continued with stirring for 15 min. The reaction was quenched by the addition of 100 mL of 40% propan-2-ol solution. The product was purified by ultrafiltration and isolated by precipitation with propan-2-ol.

IR (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm -1 . 1H NMR (D2O) d: 2,01 (s, 3 H, CH3-), 3,37 - 3,93 (m, skeleton of hyaluronan), 4,46 (s, 1H, anomer), 4,54 (s, 1H anomer., -O-CH(OH)-), 5,27 (geminal glycol -CH-(OH)2).

Example 1 C: Pi a tyraminated HA derivative with a ~ 80,000 g/mol, DS ~ 3%)

The aldehyde derivative of HA (~ 60,000 g/mol, DS = 9%) (5.00 g) was dissolved in 500 mL of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-amino- N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (1.25 g, 5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm '1 .

Example ID: Prt a tyraminated HA derivative with a

The aldehyde derivative of was dissolved in 500 mL of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-amino- N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm -1 . hyaluronan), 4,46 (s, 1H, anomer), 4,54 (s, 1H anomer., -O-CH(OH)-), 6,59 (d, 2H, arom.), 7,01 (d, 2H. arom).

Example IE: Preparation of tyraminated derivative o

The aldehyde derivative of was dissolved in 500 mL demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-ami no- A- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm '1 .

Example IF: Prt a tyraminated HA derivative with a

The aldehyde derivative of was dissolved in 500 mL of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-ami no- N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm '1 . 1H NMR (D 2 0) d: 1,25 (t, 2 H, g -CH 2 - aminohexane acids), 1,48 (m, 2 H, d -CH 2 - aminohexane acids)l,51 (m, 2 H, b -CH 2 - aminohexane acids), 2,01 (s, 3 H, CH3-), 2,65 (m, 2H, Ph-CH 2 -), 2,73 (m, 2H, e-CH 2 - aminohexane acids), 3,37 - 3,93 (m, skeleton of hyaluronan), 4,46 (s, 1H, anomer), 4,54 (s, 1H anomer., -O-CH(OH)-), 6,59 (d, 2H, arom.), 7,01 (d, 2H. arom).

SEC MALLS: Mw = 1 800000 kDa; PI = 1,55

DS ( 1 H NMR): 1,0 %

Example 1G: P reparation a tyraminated HA derivative with a

The aldehyde derivative of (5.00 g) was dissolved in 500 mL demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-ami no- A- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm -1 . DS ( 1 H NMR): 3,2 %

Example II: Preparat of a tyraminated HA derivative with a C6

The aldehyde derivative of HA (-2,500,000 g/mol, DS = 4%) (5.00 g) was dissolved in 500 mL demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To a solution of HA-CHO was added 6-amino- N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Then picoline-borane complex (0.270 g, 2.5 mmol) was added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by propan-2-ol precipitation. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

IR (KBr):: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945 ,893 cm -1 . 1H NMR (D 2 0) d: 1,25 (t, 2 H, γ -CH 2 - aminohexane acids), 1,48 (m, 2 H, δ -CH 2 - aminohexane acids)1,51 (m, 2 H, β -CH 2 - aminohexane acids), 2,01 (s, 3 H, CH3-), 2,65 (m, 2H, Ph-CH 2 -), 2,73 (m, 2H, ε-CH 2 - aminohexane acids), 3,37 - 3,93 (m, skeleton of hyaluronan), 4,46 (s, 1H, anomer), 4,54 (s, 1H anomer., -O-CH(OH)-), 6,59 (d, 2H, arom.), 7,01 (d, 2H. arom).

SEC MALLS: Mw = 91 000 g/mol; PI = 1,65 DS (Ή NMR): 7,2 %

Example 2: Preparation of a hydrogel by mixing solutions A and B of the means

A HA-TA derivative prepared according to the procedure of Example ID was used to prepare solutions of the means for hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in table 1.

Table 1: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 2.

Table 2: Composition and parameters of the prepared hydrogel

Example 3: Preparation of a hydrogel by mixing solutions A and B of the menas

The HA-derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 3

Table 3: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 4.

Example 4: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative (prepared according to Example ID) was used to prepare solutions of the means for hydrogel preparation. Concentration of individual components of the solution A and the solution B are given in Table 5.

Table 5: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 6. Example 5: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 7.

Table 7: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 8. Example 6: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 9.

Table 9: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 10.

Table 10: Composition and parameters of the prepared hydrogel Example 7: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 11.

Table 11: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 12.

Table 12: Composition and parameters of the prepared hydrogel

Example 8: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 13.

Table 13: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 14.

Table 14: Composition and parameters of the prepared hydrogel

Example 9: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 15.

Table 15: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 16.

Table 16: Composition and parameters of the prepared hydrogel

Example 10: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of solution A and B are given in Table 17.

Table 17: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 18.

Table 18: Composition and parameters of the prepared hydrogel

Example 11: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example IF was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 19.

Table 19: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 20.

Table 20: Composition and parameters of the prepared hydrogel

Example 12: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example IE was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 21

Table 21: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 22.

Table 22: Composition and parameters of the prepared hydrogel Example 13: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-derivative prepared according to Example 1G was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 23.

Table 23: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 24.

Table 24: Composition and parameters of the prepared hydrogel

Example 14: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative (prepared according to Example II) was used to prepare solutions of the means for hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 25.

Table 25: Composition of precursor solutions A and B for hydrogel preparation The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G' 3 min ) and after the completion of solidification (G' s ) are given in Table 26.

Table 26: Composition and parameters of the prepared hydrogel Example 15: Preparation of a hydrogel by mixing solutions A and B of the means

The HA-TA derivative prepared according to Example 1C was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 27.

Table 27: Composition of precursor solutions A and B for hydrogel preparation

The hydrogel was prepared by mixing solutions A and B in a ratio of 1 : 1. The hydrogel thus prepared contains the enzyme horseradish peroxidase, a covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic modulus G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 28.

Table 28: Composition and parameters of the prepared hydrogel Example 16: Influence of hydrogel crosslink density and presence of TCS/HP-p-CD inclusion on the rate of its degradation by hyaluronidase in vitro

Three types of hydrogels without inclusion of TCS/HP-β-CD (Type A, B, C) were prepared, which differed in their viscoelastic properties and swelling coefficient. A hydroxyphenyl derivative of hyaluronan with a Mw of 281,000 g/mol and a DS of 2.2% was used for preparation of the hydrogels. The concentrations of the reagents used for the preparation of hydrogels are given in Table 29. The crosslinking density and thus the viscoelastic properties of the hydrogels were regulated by the concentration of crosslinking agents - HRP and H2O2. Next, a hydrogel containing TCS/HP-β-CD inclusion (Type D) was prepared according to Example 8. Viscoelastic properties

The viscoelastic properties of the hydrogels were determined using an AR-G2 rotary rheometer (TA Instruments) using the strain sweep method at a constant frequency value of 1 Hz and a displacement between 10 -3 and 2 radians at 25 °C. In order to prevent the prepared hydrogels from slipping during the measurement, cross-hatched geometry was used. Hydrogels with a diameter of 17.5 mm were prepared for the determination. Hydrogels were evaluated 60 minutes after preparation, i.e. after solidification. The following table shows the viscoelastic properties of the (elastic G) hydrogels.

Swelling The hydrogels were immersed in saline (0.9% NaCl) and allowed to swell for 24 h in an incubator at 37 °C. The hydrogels were weighed. The degree of swelling was determined on the basis of a calculation according to a formula where Q is the swelling coefficient, m 0 is the weight of the gel after preparation and m s is the weight of the gel after disintegration into equilibrium.

Table 29: Values of elastic modulus and degree of swelling of prepared hydrogels.

Degradation rate

The swollen gels were transferred to other vials to which degradation medium (1 mL of a solution containing bovine testicular hyaluronidase with an activity of 480 U/mL) was added. Degradation of the gels was performed at 37 ° C with stirring. During the experiment, the weight of the hydrogels was determined every 30 minutes until they were completely degraded.

Results Hydrogel A shows the lowest value of G' and the highest swelling coefficient, from which it can be deduced that it also achieves the lowest degree of crosslinking. On the other hand, hydrogel C shows the highest degree of crosslinking. Although hydrogels B and D differ in the content of TCS/HP-β-CD inclusion, on the basis of comparable values of G" and Q, it can be deduced that they also show a comparable degree of crosslinking.

The design of the experiment allows a relative comparison of the resistance of the prepared hydrogels to the action of the hydrolytic enzyme in vitro. It is clear from the graph (see Fig. 1) that hydrogel A was the fastest to undergo enzymatic degradation, while hydrogel C was the most resistant. The results confirm that the rate of hydrogel degradation depends on the crosslink density of hydrogels, the measure thereof are viscoelastic properties of hydrogels and their degree of swelling. The rate of degradation of hydrogel D was significantly lower compared to hydrogel B, even though both types of hydrogels showed a similar degree of crosslinking. A possible cause is the presence of TCS/HP-β-CD inclusion in the structure of hydrogel D, which can slow down the enzymatic degradation of the hydrogel.

Example 17: Effect of the presence of HR-β-CD on the gelation rate and the elastic modulus of a hydrogel prepared from HA-TA

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Tables 30 - 33:

Table 30: Composition of solutions A and B for the preparation of hydrogel I without HR-b- CD

Table 31: Composition of solutions A and B for the preparation of hydrogel CD content of 0.3 mg/ml

Table 32: Composition of solutions A and B for the preparation of hydrogel III containing HR-β-CD 3 mg/ml

Table 33: Composition of solutions A and B for the preparation of hydrogel IV containing HR-β-CD 30 mg/ml

Results:

Table 34: Composition and parameters of the prepared hydrogels I to IV formed by mixing solutions A with B according to Tables 30 to 33, as above. With increasing concentration of HR-β-CD in the gel-forming mixture, the rate and efficiency of the crosslinking reaction decreases, which is reflected in the prolongation of Tg and G' 3 min . Example 18: Antimicrobial action of triclosan-containing hydrogels in vitro

Preparation of hydrogels for antimicrobial tests:

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Tables 30 - 38:

Table 34: Composition of precursor solutions A and B for the preparation of a hydrogel without TCS

TCS content of 0.1 mg/mL

Table 37: Composition of precursor solutions A and B for the preparation of a hydrogel with a TCS content of 0.8 mg/mL

Table 38: Composition of precursor solutions A and B or the preparation of a hydrogel with a TCS content of 1 mg/mL

Hydrogels were prepared by mixing solutions A and B in a volume ratio of 1 : 1.

Antimicrobial activity test The diffusion plate method (2D arrangement) was chosen to test the effectiveness of the hydrogels. Non-selective medium - tryptone-soy agar - was used for cultivation. Blood agar was used to cultivate Clostridium sporogenes under anaerobic conditions.

Gel samples were tested in 4 microorganisms:

• Staphylococcus aureus (G + cocci ),

• Escherichia coli (G-rods),

• Clostridium sporogenes (G + anaerobic rod ),

• Candida albicans (yeast).

Inoculum preparation:

An approximately 48-hour-old culture was used to prepare the inoculum, from which a bacterial suspension of 0.5 McFarland was prepared, corresponding to a concentration of the order of 10 7 -10 8 CFU/mL.

In the case of yeast, a suspension of 4.0 McFarland was prepared, which corresponds to a concentration of the order of 10 7 CFU/mL.

Microbiological analysis:

For testing itself, the suspension was further diluted to approximately 10 4 CFU/mL and then inoculated with 100 μL onto the surface of tryptone soy agar in Petri dishes, and the suspension was spread evenly over the surface of the entire dish with a sterile stick. The approximate number of microorganisms applied to the dish was of the order of 10 3 CFU. After soaking the suspension in the agar, the test samples were sterile transferred to its surface.

Plates with test strains and samples were stored for culture at 37 ° C for 24 hours. Clostridium sporogenes was cultured under anaerobic conditions.

After culturing for 24 hours, all plates inoculated with the microorganisms and test samples were evaluated. The comparison was made against a negative control, which consisted of a hydrogel without TCS/CD inclusion. A sample showing an antimicrobial effect on the test microorganism was manifested by the formation of an inhibition zone in close proximity to the test sample.

Table 39: Overview of the antimicrobial action of the prepared gels. *Note:

• (+) The inhibition zone in close proximity to the sample / sample shows an antimicrobial effect · (-) The growth of microorganisms in the immediate vicinity of the sample does not show an antimicrobial effect

The antimicrobial effect of hydrogels on the Stapyhlococcus aureus strain was demonstrated for all TCS concentrations tested. The antimicrobial effect of hydrogels on Escherichia coli strain was demonstrated from a TCS concentration of 0.5 mg/mL. The antimicrobial effect of hydrogels on the yeast Candida albicans and Clostridium sporogenes was observed only from a TCS concentration of 0.8 mg/mL. None of the hydrogels containing TCS in the concentration range of 0.1 to 1 mg/mL was colonized by bacteria, which confirms the possibility of their use as a barrier against the spread of infection.

Example 19: Preparation of a hydrogel by mixing solutions A, B and C from a kit The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of the solution A and the solution B are given in Table 40

Table 40: Composition of precursor solutions A, B and C for hydrogel preparation

The hydrogel was prepared by mixing solutions A, B and C in a ratio of 1 : 1 : 2. The hydrogel thus prepared contains the enzyme horseradish peroxidase, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 41.

Table 41: Composition and parameters of the prepared hydrogel

Example 20: Preparation of a hydrogel by mixing solutions A, B and C of the kit

The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of solution A and B are given in Table 42

Table 42: Composition of precursor solutions A, B and C for hydrogel preparation

The hydrogel was prepared by mixing solutions A, B and C in a ratio of 1 : 1 : 2. The hydrogel thus prepared contains the enzyme horseradish peroxidase, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G'3 min) and after the completion of solidification (G' s ) are given in Table 43.

Table 43: Composition and parameters of the prepared hydrogel

Example 21: Preparation of a hydrogel by mixing solutions A, B C of the kit The HA-TA derivative prepared according to Example ID was used to prepare solutions of the means for the hydrogel preparation. The concentrations of the individual components of solution A and B are given in Table 44

Table 44: Composition of precursor solutions A, B and C for hydrogel preparation

The hydrogel was prepared by mixing solutions A, B and C in a ratio of 1 : 1 : 2. The hydrogel thus prepared contains the enzyme horseradish peroxidase, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA), hydroxypropyl-β-cyclodextrin and triclosan. The final composition of the hydrogel, including the values of the gelation time (Tg) and the value of the elastic module G' after 3 min (G T m in) and after the completion of solidification (G' s ) are given in Table 45.

Table 45: Composition and parameters of the prepared hydrogel

Example 22: Preclinical testing on a porcine colorectal anastomosis dehiscence model The in vivo study was divided into two phases. In the first phase of in vivo tests, a hydrogel of a defined composition was implanted in the small pelvic area. The aim was to determine the relationship between the gelation time (Tg) and determined by rheological measurement and the time required to solidify (T solid ) the appropriate amount (V gel ) of material in vivo (see Table 46). T solid does not agree with the in vitro determined Tg, because it does not describe the moment of hydrogel formation, but the moment when a macroscopic ally homogeneous hydrogel filling the small pelvic region is obtained in vivo. The hydrogel prepared according to Example 2 was used for this experiment.

Table 46: Parameters of the hydrogel implanted in the small pelvic area

The second phase examined the effect of the presence of the hydrogel on the healing process of the colorectal anastomosis dehiscence model, which was created by perforation of the intestinal wall near the anastomosis. The severity of the condition was simulated by the size of the perforation in the range of 5 - 15 mm. The hydrogels of Examples 3 to 8, the properties of which are summarized in Table 47, were used to fill the small pelvic area.

Table 47: Parameters of hydrogels implanted in the small pelvic region after creating a model of colorectal anastomosis dehiscence.

At 14 days postoperatively, the animals were sacrificed and the healing status of the anastomosis was assessed. The results were evaluated by macroscopic and histological examination. An analogy with the classification of manifestations of complications associated with the healing of colorectal anastomosis in human medicine was used to evaluate the clinical condition [58]:

Table 48: Definition of the severity degrees of clinical manifestations of postoperative complications associated with colorectal anastomosis healing [58].

The preclinical study performed included a total of 21 pigs with a model of colorectal anastomosis with varying degrees of damage. The onset of dehiscence was simulated by perforation of the colon near the anastomosis. The hydrogel (composition according to Table 41; 20 to 40 mL/animal) was applied to 18 animals at the end of the procedure. The condition of the animals was evaluated for 14 days, after which they were sacrificed. In some cases, it was possible to identify gel residues at the application site even after two weeks. It is completely absorbed in less than 30 days. In none of the 18 cases when the hydrogel was applied, there were clinical signs of the development of sepsis, or signs of intestinal obstruction, or other side effects of the use of the developed hydrogel. The clinical condition of the animals was assessed by classification A. In contrast, in two of the three animals in the control group, in which the gel was not used during the operation, there were complications when it was necessary to use additional antibiotic treatment. The condition of these animals was classified as category B.

Table 49: Phase 2 evaluation of a preclinical study N - no finding

Y - macroscopic or histological finding References

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[1] Zavoral M, Grega T, Suchanek S. Komplikace lecby kolorektalniho karcinomu. Onkologie. 2016;10:41-7.

[2] Miroslav Trubac ML. Chimrgicka lecba karcinomu tlusteho stfeva a konccnfku. dostupny na https://zdravieurocz/clanek/postgradualni-medicina/chimrgick a-lecba-karcinomu-tlusteho- streva-a-konecniku-478291.

[3] Gresham RD, Manzo SE, Aranyi E, Geiste RJ, Jankowski BK, Milliman K. Surgical stapling device for performing circular anastomoses US6945444B2. Google Patents; 2005.

[4] Argyra E, Polymeneas G, Karvouni E, Kontorravdis N, Theodosopoulos T, Arkadopoulos N. Sutureless Pancreatojejunal Anastomosis Using an Absorbable Sealant: Evaluation in a Pig Model. Journal of Surgical Research. 2009;153:282-6.

[5] de la Portilla F, Garcia-Cabrera AM, Pereira S, de Marco F, Molero M, Muntane J, et al. An Experimental Study on the Use of Calcium Alginate to Heal Colonic Anastomoses. Journal of Investigative Surgery. 2016;29:32-9.

[6] Giingor G, Demiral G, §enol M, Bayraktar B, Cclik Y, Boliik S. Cyanoacrylate application on colonic anastomosis: is it safe or not? Przeglad Gastroenterologiczny. 2016;11:206-10.

[7] Lauto A, Mawad D, Foster LJR. Adhesive biomaterials for tissue reconstruction. Journal of Chemical Technology & Biotechnology. 2008;83:464-72.

[8] Vakalopoulos KA, Daams F, Wu Z, Timmermans L, Jeekel JJ, Kleinrensink G-J, et al. Tissue adhesives in gastrointestinal anastomosis: a systematic review. Journal of Surgical Research. 2013;180:290-300.

[9] Huh JW, Kim HR, Kim YJ. Anastomotic leakage after laparoscopic resection of rectal cancer: the impact of fibrin glue. The American Journal of Surgery. 2010;199:435-41.

[10] Costales AB, Patil D, Mulya A, Kirwan JP, Michener CM. 2-Octylcyanoacrylate for the prevention of anastomotic leak. The Journal of surgical research. 2018;226:166-72.

[11] Ustek S, Kismet K, Akkus MA, Ozcan AH, Aydogan A, Renda N. Effect of Povidone- Iodine Liposome Hydrogel on Colonic Anastomosis. European Surgical Research. 2005;37:242-5.

[12] Reimer K, Vogt PM, Broegmann B, Hauser J, Rossbach O, Kramer A, et al. An Innovative Topical Drug Formulation for Wound Healing and Infection Treatment: In vitro and in vivo Investigations of a Povidone-Iodine Liposome Hydrogel. Dermatology. 2000;201:235-41.

[13] Hirai K, Tabata Y, Hasegawa S, Sakai Y. Enhanced intestinal anastomotic healing with gelatin hydrogel incorporating basic fibroblast growth factor. Journal of tissue engineering and regenerative medicine. 2016; 10.

[14] Yol S, Tekin A, Yilmaz H, Kucukkartallar T, Esen H, Caglayan O, et al. Effects of platelet rich plasma on colonic anastomosis. Journal of Surgical Research. 2008;146:190-4.

[15] de Jonge SW, Atema JJ, Solomkin JS, Boermeester MA. Meta-analysis and trial sequential analysis of triclosan-coated sutures for the prevention of surgical-site infection. British Journal of Surgery. 2017;104:E118-E33.

[16] Wu X, Kubilay NZ, Ren J, Allegranzi B, Bischoff P, Zayed B, et al. Antimicrobial- coated sutures to decrease surgical site infections: a systematic review and meta- analysis. European Journal of Clinical Microbiology & Infectious Diseases. 2017;36:19-32.

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