MEDICAL PREPARATION WITH A CARRIER BASED ON HYALURONAN AND/OR DERIVATIVES THEREOF, METHOD OF PREPARATION AND USE THEREOF

Abstract

Disclosed is a method of preparing a medical preparation with a carrier based on hyaluronan and/or its derivatives, which can be used in the field of medicine and cosmetics. The medical preparation comprises a conjugate of hyaluronic acid and/or its derivative with a medical substance, according to the general formula A-SN, where: A is the medical substance; S is the way of linking the medical substance with the carrier; and N is the carrier based on hyaluronic acid and/or its derivatives.

Claims

1.-26. (canceled)

27. A method of preparing a medical preparation based on hyaluronic acid and/or palmitoyl hyaluronan or formyl hyaluronan, said method comprising binding a medical substance A comprising a terminal N-protecting group and comprising amino acids and/or peptides to a carrier N in an aprotic polar solvent, the carrier N comprising hyaluronic acid, palmitoyl hyaluronan, and/or formyl hyaluronan and having a molecular weight of from 1.510.sup.4 to 2.510.sup.6 g.Math.mol.sup.1, and then cleaving off the terminal N-protecting group of the medical substance Abound on the carrier in a basic environment to form a conjugate; wherein the carrier N is in solid phase during the whole preparation, is insoluble in the reaction environment, and is biodegradable; and wherein the conjugate has the general formula X:
A-SN(X), which is also represented by the formula: ##STR00003## where A is a peptide or an amino acid; S is OC(O), NH, or a linear linker based on a peptide comprising hydrophobic amino acids Xaa; m is from 10 to 1250; and n is from 100 to 12500.

28. The method according to claim 27, wherein the carrier N is in the form of an endless fibre, thread, textile, thin film, staple fibre, and/or nonwoven fabric.

29. The method according to claim 27, wherein the formation of the conjugate is performed at the temperature of from 20 C. to 40 C. for a time of from 2 to 48 hours.

30. The method according to claim 27, wherein the aprotic polar solvent is selected from the group consisting of N,N-dimethyl formamide, dimethyl sulphoxide, N-methyl-2-pyrrolidone, acetonitrile, dichloro methane, tetrahydrofuran, 1,4-dioxane, and combinations thereof and/or the basic environment in which the N-terminal protecting group is cleaved off is selected from the group consisting of 20% piperidine in DMF, 2% DBU in DMF, and 30% tert-butylamine in DMF.

31. The method of preparation according to claim 27, wherein binding the medical substance A to the carrier N comprises esterification; wherein the medical substance A is an amino acid or peptide comprising the terminal N-protecting group and a carboxyl group; wherein the medical substance A is first subjected to activation of the carboxyl group via a condensation agent in an aprotic polar solvent to form a reactive intermediate that is subsequently reacted with the carrier N in the presence of an organic base and a catalyst; and wherein the terminal N-protecting group of the medical substance A bound on the carrier is then cleaved off in a basic environment to form the conjugate according to the general formula X, where S is OC(O) or a linear linker based on a peptide comprising hydrophobic amino acids Xaa.

32. The method according to claim 31, wherein the condensation agent comprises N,N-diisopropyl carbodiimide, N,N-dicyclohexyl carbodiimide, 1-[bis(dimethylamino) methylen]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluoro phosphate, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide, propylphosphonic acid anhydride, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro phosphate, ethyl chloroformiate, benzotriazole-1-yl-oxy-tris(dimethylamino) phosphonium hexafluoro phosphate, O-(benzotriazole-1-yl)-N,N,N,N-tetramethyl uronium tetrafluoroborate, 4-triethylamino-dicyclohexyl carbodiimide p-toluen sulfonate, 4-nitrophenol, N-hydroxy succinimide, 2,4,5-trichloro phenol, 2,3,4,5,6-pentafluoro phenol, and/or 2,3,4,5,6-pentachloro phenol.

33. The method according to claim 31, wherein the organic base is selected from the group consisting of triethylamine, pyridine, morpholine, N-methyl morpholine, N,N-diisopropyl ethylamine, imidazole, and/or the catalyst is selected from the group consisting of ethyl(hydroxyimino) cyanoacetate, hydroxybenzo triazole, 1-hydroxy-7-azabenzotriazole, or N,N-dimethylamino pyridine.

34. The method according to claim 31, wherein the conjugate is further repeatedly reacted with the reactive intermediate which can be the same or different from the reactive intermediate of the previous step.

35. The method according to claim 31, wherein the condensation agent is N,N-diisopropyl carbodiimide, the aprotic polar solvent is N,N-dimethyl formamide, the catalyst is a combination of ethyl (hydroxyimino) cyanoacetate and N,N-dimethylamino pyridine, and the basic environment is 20% piperidine in N,N-dimethyl formamide.

36. The method according to claim 27, wherein the amount of the medical substance A corresponds to 1 to 5 equivalents, with respect to a dimer of hyaluronic acid or a derivative thereof.

37. The method according to claim 31, wherein the amount of the condensation agent corresponds to 0.1 to 5 equivalents, with respect to a dimer of hyaluronic acid or a derivative thereof, and/or the amount of the organic base corresponds up to 10 equivalents, with respect to a dimer of hyaluronic acid or a derivative thereof, and/or the amount of the catalyst corresponds 0.1 to 5 equivalents, with respect to a dimer of hyaluronic acid or a derivative thereof.

38. The method according to claim 31, wherein 3 equivalents of ethyl(hydroxyimino) cyanoacetate as the catalyst and 0.3 equivalents of N,N-dimethylamino pyridine as the organic base are used, each with respect to a dimer of hyaluronic acid or a derivative thereof.

39. The method according to claim 31, wherein the amount of the medical substance A corresponds to 3 equivalents, the amount of the condensation agent corresponds to 3 equivalents, and the amount of the catalyst corresponds to 3 equivalents, each with respect to a dimer hyaluronic acid or a derivative thereof.

40. The method according to claim 31, wherein the amount of the medical substance A corresponds to 1 equivalent, the amount of the condensation agent corresponds to 1 equivalent, and the amount of the catalyst corresponds to 1 equivalent, with respect to a dimer of hyaluronic acid or a derivative thereof.

41. The method of preparation according to claim 27, wherein binding the medical substance A to the carrier N comprises reductive amination in the presence of a polar aprotic solvent and a reduction agent; and wherein the terminal N-protecting group of the medical substance A bound on the carrier is then cleaved off in a basic environment to form the conjugate according to the general formula X, where S is NH, or a linear linker based on a peptide comprising hydrophobic amino acids Xaa.

42. The method according to claim 31, wherein the conjugate is further repeatedly reacted with the medical substance A that can be the same or different from the medical substance A of the previous step.

43. The method of preparation according to claim 31, wherein the polar aprotic solvent is DMF, the reduction agent is picBH.sub.3, and the basic environment is 20% solution of piperidine in DMF.

44. The method according to claim 27, wherein the medical substance A is bound to the carrier via a linear linker having the general formula Xaa-Ahx-Ahx-Xaa.

45. A medical preparation with a carrier based on hyaluronic acid and/or palmitoyl hyaluronan or formyl hyaluronan, the medical preparation comprising a conjugate according to the general formula:
A-SN(X), which is also represented by the formula: ##STR00004## where A is a peptide or an amino acid; S is OC(O), NH, or a linear linker based on a peptide comprising hydrophobic amino acids Xaa; m is from 10 to 1250; and n is from 100 to 12500.

46. The medical preparation according to claim 45 for use in medical applications as a dosage form of an active peptide or amino acid, wherein the medical preparation is formulated for administration dermally, sublingually, buccallly, or locally into an open wound.

47. The medical preparation according to claim 45, wherein the medical preparation is formulated for dermal application and comprises the peptide Dalargin as the medical substance A.

48. The medical preparation according to the claim 45, wherein the medical preparation is formulated for oral or sublingual application and comprises, as the medical substance A, a peptide selected from the group consisting of Desmopressin, Lysipressin, and Glypressin.

49. The medical preparation according to claim 45, wherein the medical preparation is formulated for buccal application and comprises, as the medical substance, a peptide selected from the group consisting of antivirotics and adjuvants, releasing factor for luteinizing and follicle-stimulating hormones, and combinations thereof.

50. The medical preparation according to claim 45, wherein the medical preparation is formulated for direct application into an open wound and comprises, as the medical substance, a peptide selected from the group consisting of Glypressin, Dalargin, and AdDP.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The enclosed FIG. 1 shows hyaluronan where R=H+ or Na+ and n is equal to 100 to 12500.

[0048] FIG. 2 shows the structural formula of alginate.

[0049] FIG. 3 shows the structural formula of cellulose.

[0050] FIG. 4 shows the structural formula of the medical preparation according to the invention, where A is the medical substance, S is the way of linking the medical substance with a carrier, N is the carrier based on hyaluronic acid and its derivatives, subscript m is from 10 to 1250, and subscript n is from 100 to 12500.

[0051] FIG. 5 shows the structural formula of hyaluronic acid or hyaluronic acid derivative with the bound peptide, where HYA is hyaluronic acid or its derivative, and the peptide comprises 7 lysine units arranged in a dendrimer structure.

[0052] FIG. 6 shows DiI stained NHDF cells adhered to a fibre of native HYA comprising RGD motives. The images were obtained by means of fluorescent microscope after 48 hours of cultivation.

[0053] FIG. 7 shows changes in the presence of non-adhered DiI stained NHDF cells population. The images were obtained by means of fluorescent microscope after 1, 2, 4, and 7 days of cultivation.

DESCRIPTION OF THE EMBODIMENTS

[0054] The conjugate of peptide with the material based on hyaluronan according to the invention was successfully prepared and tested by the authors in the applicant's facilitiesContipro a.s., Doln Dobru, CZ.

LIST OF ABBREVIATIONS

[0055] AdDP 1-adamantylamide-(L)-alanyl-(D)-isoglutamine [0056] Ahx aminohexanoic acid [0057] Boc tert-butyloxycarbonyl [0058] Castro reagent benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluoro phosphate [0059] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene [0060] DCC N,N-dicyclohexylcarbodiimide [0061] DCM dichloromethane [0062] DEE diethylether [0063] DIC N,N-diisopropylcarbodiimide [0064] DiI 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine perchlorate [0065] DIPEA N,N-diisopropyl ethylamine [0066] DMAP N,N-dimethyl aminopyridine [0067] DMF N,N-dimethyl formamide [0068] EDC 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide [0069] Fmoc fluorenyl methyloxy carbonyl [0070] HATU 1-[bis(dimethylamino) methylen]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluoro phosphate [0071] HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro phosphate [0072] HOAt 1-hydroxy-7-azabenzo triazole [0073] HOBt N-hydroxy benztriazole [0074] HOSu N-hydroxy succinimide [0075] IPA isopropyl alcohol [0076] OxymaPure ethyl (hydroxyimino) cyanoacetate [0077] picBH.sub.3 picolin borane complex [0078] T3P propyl phosphonic acid anhydride [0079] TBTU O-(benzotriazole-1-yl)-N,N,N,N-tetramethyl uronium tetrafluoro borate [0080] WSCD 4-triethylamino dicyclohexyl carbodiimide p-toluen sulphonate

[0081] The fibre from the native HYA and nonwoven fabric from the native HYA were prepared according to the process described in the patent WO/2013/167098. The fibre from palmitoyl HYA was prepared according to the process described in the patent WO/2014/082611.

[0082] In the following examples, the term equivalent (eq.) relates to a repetitive unit of the respective form of hyaluronan. The percentage is per volume, if not stated otherwise.

[0083] The molecular weight of hyaluronan starting forms is the weight average molecular weight determined by the SEC-MALLS method.

[0084] In the examples, for the purposes of subsequent analysis only, norleucin, as the non-essential amino acid, was bound first to easily determine proportional occurrence of amino acids. However, norleucin bonding is not the necessary condition and is not intended to limit the scope of the invention.

Example 1

Anchoring of Fmoc-Nle-OH to the Fibre of Native HYA

[0085] 1 m of the fibre of native HYA (12 mg, 0.03 mmol, Mw=3.10 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. The solution of 3 eq. of Fmoc-Nle-OH, 3 eq. of OxymaPure and 0.3 eq. of DMAP in 0.5 ml anhydrous DMF was prepared outside the reactor; after dissolving all of the components 3 eq. of DIC were added to activate the carboxyl group of an amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml DMF, 31.5 ml DCM, 31.5 ml IPA, 31.5 ml DCM, 31.5 ml DEE.

[0086] The yield of the condensation reaction was determined by Fmoc-release test as the substitution S=0.01862 mmol/g.

Example 2

Preparation of H-Nle-O-HYA

[0087] 1 m of the fibre of Fmoc-Nle-O-HYA prepared according to the Example 1 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. Fmoc protecting group was cleaved off by means of adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the time of cleavage to 20 min and then it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0088] The reaction is quantitative. Releasing of the amino group was confirmed by performing the ninhydrin confirmation reaction (Kaiser test).

Example 3

Anchoring of Fmoc-Nle-OH to the Fibre of Native HYA

[0089] 1 m of fibre of native HYA (12 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit, the fibre was repeatedly washed with THF. A solution of 3 eq. of Fmoc-Nle-OH, 3 eq. of OxymaPure and 0.3 eq. of DMAP in 0.5 ml THF; after dissolving all the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor with the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of THF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE. The yield of the condensation reaction was determined by Fmoc-release test as the substitution S0,01759 mmol/g.

Example 4

Anchoring of Fmoc-Nle-OH to the Fibre of Native HYA

[0090] 1 m of the fibre of native HYA (12 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 1 eq. of Fmoc-Nle-OH, 1 eq. of OxymaPure, and 0.3 eq. of DMAP in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components 1 eq. of DIC was added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0091] The yield of the condensation reaction was determined by Fmoc-release test as the substitution S=0.01804 mmol/g.

Example 5

Preparation of Fmoc-[Lys(Boc)].SUB.4.-Nle-O-HYA

[0092] 1 m of H-Nle-O-HYA prepared according to the Example 2 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. The solution of 3 eq. of Fmoc-Lys(Boc)-OH, and 3 eq. of OxymaPure in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. The Fmoc protecting group was cleaved off by means of adding 1.5 ml of 20% solution of piperidine in DMF into the reactor with the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min and then it was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. This procedure was repeated 4 times.

[0093] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The product composition (nfound) was confirmed by amino acids analysis as presented in Table 2. It is a serial synthesis performed by a step-by-step method; however, this procedure was repeated for all said derivatives. The individual rows show the composition of the prepared conjugate after finishing said cycle; i.e., after finishing the procedure described in Example 3 the product having the composition described in the first row is obtained. Then the procedure is repeated, and the product described in the second row is obtained, etc.

TABLE-US-00002 TABLE 2 S n.sub.calculated n.sub.found Compound [mmol/g] [nmol] [nmol] H-Lys(Boc)-Nle-O-HYA 0.01839 0.290 0.295 H-Lys(Boc)-Lys(Boc)-Nle-O-HYA 0.01863 0.576 0.584 H-Lys(Boc)-Lys(Boc)-Lys(Boc)- 0.01888 0.870 0.876 Nle-O-HYA H-Lys(Boc)-Lys(Boc)-Lys(Boc)- 0.01856 1.189 1.194 Lys(Boc)-Nle-O-HYA

Example 6

Preparation of a Peptide Comprising 7 Lysine Units Arranged in a Dendrimer StructureUse of a Native HYA Fibre

[0094] 1 m of H-Nle-O-HYA prepared according to the Example 2 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. The solution of 3 eq. of Fmoc-Lys(Fmoc)-OH, and 3 eq. of OxymaPure in 0.5 ml anhydrous DMF was prepared outside the reactor; after dissolving all the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor with the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. The reaction was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. The Fmoc protecting group was cleaved off by means of adding 1.5 ml of 20% solution of piperidine in DMF into the reactor with the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min. The reaction was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. This procedure was repeated 2 times.

[0095] The reaction was monitored by the Fmoc-release test. The yield was determined by the Fmoc-release test. The composition of the product, shown in FIG. 5, was confirmed by an amino acid analysis, as presented in Table 3. It is a serial synthesis performed by step-by-step method; however, this procedure was repeated for all said derivatives. The individual rows show the composition of the prepared conjugate after finishing said cycle; i.e., after finishing the procedure described in Example 3 the product having the composition described in the first row is obtained. Then the procedure is repeated, and the product described in the second row is obtained, etc.

TABLE-US-00003 TABLE 3 S n.sub.calculated n.sub.found Compound [mmol/g] [nmol] [nmol] H-Lys-Nle-O-HYA 0.03381 0.25 0.262 H-Lys.sub.3-Nle-O-HYA 0.07169 0.77 0.799 H-Lys.sub.7-Nle-O-HYA 0.14211 1.84 1.876

Example 7

Preparation of a Peptide Comprising 7 Alanine Units on the Fibre of Native HYA

[0096] 1 m of H-Nle-O-HYA prepared according to the Example 2 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of Fmoc-Lys(Boc)-OH, and 3 eq. of OxymaPure in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. The reaction was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. Cleavage of Fmoc protecting group was performed by adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. This procedure was repeated 7 times for Fmoc-Ala-OH.

[0097] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The product composition was confirmed by an amino acids analysis as the proportional occurrence of individual amino acids. The purity of the material was determined by means of MS-HPLC after degradation of the material. The results of the individual analysis are shown in the following Table 4:

TABLE-US-00004 TABLE 4 S Amino acid Purity Compound [mmol/g] Ala Lys Nle [%] H-(Ala).sub.7-Lys(Boc)- 0.01798 7.28 1.03 1 86 Nle-O-HYA

Example 8

Anchoring of Fmoc-Nle-OH to the Fibre of Palmitoyl HYA

[0098] 1 m of the fibre from palmitoyl HYA (15 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol-) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of Fmoc-Nle-OH, and 3 eq. of OxymaPure and 0.3 eq. of DMAP in 0.5 ml anhydrous DMF was prepared outside the reactor; after dissolving all of the components, 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF.

[0099] The yield of the condensation reaction was determined by Fmoc-release test as the substitution S=0.01029 mmol/g.

Example 9

Preparation of H-Nle-O-Palmitoyl HYA

[0100] 1 m of Fmoc-Nle-O-palmitoylHYA prepared according to Example 8 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. Fmoc protecting group was cleaved off by means of adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for 5 min. The reaction was repeated while extending the time of cleavage to 20 min and then it was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0101] The reaction is quantitative. Releasing of the amino group was confirmed by performing the ninhydrin confirmation reaction (Kaiser test).

Example 10

Preparation of Fmoc-[Lys(Boc)]4-Nle-O-palmitoylHYA

[0102] 1 m of H-Nle-O-palmitoylHYA prepared according to the Example 9 was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of Fmoc-Lys(Boc)-OH, and 3 eq. of OxymaPure in 0.5 ml anhydrous DMF was prepared outside the reactor; after dissolving all of the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours and it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. Cleavage of Fmoc protecting group was performed by adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min and then it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. This procedure was repeated 4 times.

[0103] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The composition of the product (nfound) was confirmed by amino acid analysis, as presented in Table 5 below. It is a serial synthesis performed by the step-by-step method; however, this procedure was repeated for all the derivatives. The individual rows show the composition of the prepared conjugate after finishing the said cycle; i.e., after finishing the procedure described in the Example 3 the product having the composition described in the first row is obtained. Then the procedure is repeated, and the product described in the second row is obtained, etc.

TABLE-US-00005 TABLE 5 S N.sub.calculated N.sub.foundo Compound [mmol/g] [nmol] [nmol] H-Lys(Boc)-Nle-O-palmitoylHYA 0.01044 0.157 0.152 H-Lys(Boc)-Lys(Boc)-Nle-O- 0.01057 0.314 0.318 palmitoylHYA H-Lys(Boc)-Lys(Boc)-Lys(Boc)- 0.01061 0.478 0.469 Nle-O-palmitoylHYA H-Lys(Boc)-Lys(Boc)-Lys(Boc)- 0.01053 0.631 0.619 Lys(Boc)-Nle-O-palmitoylHYA

Example 11

Preparation of a Peptide Comprising 7 Lysine Units Arranged in a Dendrimer StructureUse of the Palmitoyl HYA Fibre

[0104] 1 m of H-Nle-O-palmitoylHYA prepared according to the Example 9 was placed into the 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of Fmoc-Lys(Boc)-OH, and 3 eq. of OxymaPure in 0.5 ml anhydrous DMF was prepared outside the reactor; after dissolving all components, 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. The reaction was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. Cleavage of Fmoc protecting group was performed by adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the fibre. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min. The reaction was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DMF. This procedure was repeated 2 times.

[0105] The reaction was monitored by Fmoc-release test. The yield was determined by Fmoc-release test. The composition of the product, shown in FIG. 5, where HYA denotes palmitoyl hyaluronan, was confirmed by amino acid analysis, as presented in Table 6. It is a serial synthesis performed by the step-by-step method; however, this procedure was repeated for all the said derivatives. The individual rows show the composition of the prepared conjugate after finishing said cycle; i.e., after finishing the procedure described in the Example 3 the product having the composition described in the first row is obtained. Then the procedure is repeated, and the product described in the second row is obtained, etc.

TABLE-US-00006 TABLE 6 S N.sub.calculated N.sub.found Compound [mmol/g] [nmol] [nmol] H-Lys-Nle-O-palmitoylHYA 0.00984 0.16 0.154 H-Lys.sub.3-Nle-O-palmitoylHYA 0.02130 0.49 0.501 H-Lys.sub.7-Nle-O-palmitoylHYA 0.04265 1.12 1.113

Example 12

Anchoring Fmoc-Lys-NH.SUB.2 .to formylHYA Fibre

[0106] 1 m of formylHYA fibre (11 mg, 0.028 mmol, Mw=3.10.sup.5 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. The solution of 3 eq. of Fmoc-Lys-NH.sub.2 in 0.5 ml of anhydrous DMF was prepared outside the reactor and transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. Then 3 eq. of picBH3 were added and the reaction proceeded at the temperature of 18 to 23 C. for the next 24 hours, then it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE. Cleavage of Fmoc protecting group was performed by adding 1.5 ml of 20% solution of piperidine in DMF into the reactor to the solution. The reaction proceeded for 5 min at the temperature of 18 to 23 C. The reaction was repeated while extending the cleavage time to 20 min and then it was terminated by filtering the reaction solution off. The fibre was then washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0107] Releasing of the amino group was confirmed by performing a ninhydrin confirmation reaction (Kaiser test).

Example 13

Anchoring the Peptide with RGD Motif to the Fibre of Native HYA

[0108] 1 m of native HYA fibre (12 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol-) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of a peptide having the appropriate sequence, 3 eq. of OxymaPure in 0.3 eq. of DMAP in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all the components 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. The reaction was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0109] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The composition of the product was confirmed by amino acid analysis as the proportion of individual amino acids. The purity of the material was determined by means of MS-HPLC after the degradation of the material. The results of the individual analyses are shown in the following Table 7:

TABLE-US-00007 TABLE 7 S Amino acid Purity Compound [mmol/g] Ahx Asp Gly Nle Arg [%] Ac-Arg-Gly-Asp-Gly-Gly-Gly-Nle-O-HYA 1.04 3.89 1 1.03 90 H-Arg-Gly-Asp-Gly-Gly-Gly-Nle-O-HYA 0.01802 1.01 3.91 1 0.97 82 H-Gly-Gly-Gly-Arg-Gly-Asp-Nle-O-HYA 0.01832 1.04 4.07 1 1.08 85 Ac-Arg-Gly-Asp-Ahx-Ahx-Nle-O-HYA 2.20 1.04 1.06 1 1.01 95 H-Arg-Gly-Asp-Ahx-Ahx-Nle-O-HYA 0.01843 2.27 0.93 1.05 1 1.00 90 Ac-Ahx-Ahx-Arg-Gly-Asp-Nle-O-HYA 2.21 1.03 1.26 1 1.01 86 H-Ahx-Ahx-Arg-Gly-Asp-Nle-O-HYA 0.01831 2.31 0.98 1.05 1 1.02 89

Example 14

Anchoring the Peptide Having RGD Motif to the Native HYA Nonwoven Fabric

[0110] 5 squares of the side of 5 mm made of nonwoven fabric of native HYA (10.5 mg, 0.03 mmol, Mw=1.0410.sup.6 g.Math.mol.sup.1) were placed into a 2 ml syringe reactor equipped with a frit; the material was repeatedly washed with DMF. A solution of 3 eq. of a peptide having the appropriate sequence and 3 eq. of OxymaPure in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components, 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the material. The reaction proceeded at the temperature of 18 to 23 C. for the next 20 hours. The reaction was terminated by filtering the reaction solution off. Then the fabric was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0111] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The composition of the product was confirmed by an amino acid analysis as the proportional occurence of the individual amino acids. The purity of the material was determined by means of MS-HPLC after the degradation of the material. The results of the individual analysis are presented in the following Table 8:

TABLE-US-00008 TABLE 8 S Amino acid Purity Compound [mmol/g] Ahx Asp Gly Nle Arg [%] H-Arg-Gly-Asp-Gly-Gly-Gly-Nle-O-HYA 0.01802 1.04 3.94 1 1.01 85 H-Gly-Gly-Gly-Arg-Gly-Asp-Nle-O-HYA 0.01832 1.06 4.14 1 1.07 76 Ac-Arg-Gly-Asp-Ahx-Ahx-Nle-O-HYA 2.17 0.96 1.05 1 1.04 95 H-Arg-Gly-Asp-Ahx-Ahx-Nle-O-HYA 0.01843 2.15 1.01 1.12 1 1.06 88

Example 15

Anchoring the Peptide Fmoc-(VPGLG).SUB.2.-OH to a Fibre of Native HYA

[0112] 1 m of native HYA fibre (12 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of the peptide Fmoc-(VPGLG).sub.2-OH, 3 eq. of OxymaPure, and 0.3 eq. of DMAP in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components, 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for further 20 hours, and it was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0113] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The composition of the product was confirmed by an amino acid analysis as the proportional occurrence of the individual amino acids. The purity of the material was determined by means of MS-HPLC after the degradation of the material. The results of the individual analysis are shown in the following Table 9:

TABLE-US-00009 TABLE 9 S Amino acid Purity Compound [mmol/g] Gly Leu Pro Val [%] H-(Val-Pro-Gly-Leu-Gly).sub.2-O-HYA 0.01809 2.37 1.26 1.13 1 85

Example 16

Anchoring Dalagrin to the Fibre of Native HYA

[0114] 1 m of native HYA fibre (12 mg, 0.03 mmol, Mw=3.10.sup.5 g.Math.mol.sup.1) was placed into a 2 ml syringe reactor equipped with a frit; the fibre was repeatedly washed with DMF. A solution of 3 eq. of the peptide Fmoc-Dalagrin, 3 eq. of OxymaPure, and 0.3 eq. of DMAP in 0.5 ml of anhydrous DMF was prepared outside the reactor; after dissolving all of the components, 3 eq. of DIC were added to activate the carboxyl group of the amino acid. This solution was transferred into the reactor to the fibre. The reaction proceeded at the temperature of 18 to 23 C. for further 20 hours, and it was terminated by filtering the reaction solution off. Then the fibre was washed with 31.5 ml of DMF, 31.5 ml of DCM, 31.5 ml of IPA, 31.5 ml of DCM, 31.5 ml of DEE.

[0115] The yield of the condensation reaction was determined by Fmoc-release test as the substitution in mmol/g. The composition of the product was confirmed by an amino acid analysis as the proportional occurrence of the individual amino acids. The purity of the material was determined by means of MS-HPLC after the degradation of the material. The results of the individual analysis are shown in the following Table 10:

TABLE-US-00010 TABLE 10 S Amino acid Purity Compound [mmol/g] ala Arg Gly Nle Leu Phe Tyr [%] H-Tyr-ala-Gly-Phe-Leu-Arg- 0.01894 0.99 0.99 0.96 1 0.98 0.96 0.99 93 Nle-O-HYA

Example 17

In Vitro Cell Adhesion on the Fibre Comprising RGD Motif

[0116] The fibre of native HYA comprising RGD motif, prepared according to the procedure described in Example 14, and control fibres were cut into 1 cm pieces and transferred onto a non-adhesive panel with 24 wells. Primary human fibroblasts (NHDF) were pre-marked with the fluorescent dye DiI (Ex.sub.max/Em.sub.max 549/565) and inoculated, in the amount of 10.sup.5 cells, into the wells filled with the tested samples. Then the panel was shaken by a shaker (5 hrs/160 rpm) to keep the cells in suspension, under cultivation conditions (37 C., 5% CO.sub.2). After 24 h of cultivation the fibres were transferred into a well with fresh cultivation medium. Then the cells were observed and detected by fluorescent microscope Nikon Ti-Eclipse with the use of TRITC filter (Ex.sub.max/Em.sub.max, 549/565). The fibroblasts proliferation was monitored for 7 days of cultivation.

[0117] Human dermal fibroblasts adhered and proliferated during the time only on the fibre with the peptide H-Arg-Gly-Asp-Ahx-Ahx-Nle-OH bonded to HYA via 2 units of 6-aminohexanoic acid that form a firm and elastic linker and thus make the RGD peptide accessible for cell adhesion receptors. On the contrary, the peptide H-Ahx-Ahx-Arg-Gly-Asp-Nle-OH, as well as the linking of peptides H-Arg-Gly-Asp-Gly-Gly-Gly-Nle-OH and H-Gly-Gly-Gly-Arg-Gly-Asp-Nle-OH via triglycine linker, did not support the cell adhesion, as can be seen in FIG. 6. Based on these data we can assume that it is the RGD motif which supports the cell adhesion, being the minimum domain that can be recognized by cell receptors, eventually the Ahx-linker. The positive effect of Ahx-linker was then displaced by testing the fibres with the bonded peptide comprising RGD or RGD motif, where NHDF adhered exclusively on the peptide with RGD.

Example 18

In Vitro Cell Adhesion on a Nonwoven Fabric of Native HYA Comprising RGD Motif

[0118] Nonwoven fabric of HYA with an anchored peptide H-Arg-Gly-Asp-Ahx-Ahx-Nle-OH prepared according to the procedure described in Example 14, and a control fabric were cut into squares with a side length of 0.5 cm, and were transferred onto 24 wells plate of non-adhesive panel. Primary human fibroblasts (NHDF) were pre-marked with the fluorescent dye DiI (Ex.sub.max/Em.sub.max 549/565) and inoculated, in the amount of 10.sup.5 cells, into the wells filled with the tested samples. Then the panel was shaken by a shaker (5 hrs/160 rpm) to keep the cells in suspension, under cultivation conditions (37 C., 5% CO.sub.2). After 24 h of cultivation the fabric was transferred into a well with a fresh culture media. Then the cells were observed and detected by the fluorescent microscope Nikon Ti-Eclipse with the use of TRITC filter (Ex.sub.max/Em.sub.max, 549/565). The fibroblasts proliferation was monitored for 7 days of cultivation, as can be seen in FIG. 7.

[0119] NHDF adhered uniformly onto the fabric surface and after 48 hours they elongated in a characteristic manner.

INDUSTRIAL APPLICABILITY

[0120] The new medical preparation with a carrier based on hyaluronan and its derivatives can be used as a suitable drug form for treatment with the attached medical substance. This new dosage form brings an improved prolonged activity of the medical substance bound on the solid carrier of the complex system. Suitable forms of hyaluronan with the bound medical substances can be used in human and veterinary medicine.