Combination with Albumin, in Particular for Treating a Cartilage Defect

Abstract

A combination comprising, spatially separate from one another, a first component and a second component, where the first component comprises crosslinkable albumin and the second component comprises a polymer, wherein non-terminal monomer units of the polymer comprise at least partially, more particularly only partially, an albumin-crosslinking group. Additionally disclosed is a reaction product obtainable by means of the combination, to a medical device, to a medicinal product for innovative therapies, to a kit, to a discharge apparatus, and to a functionalized hyaluronic acid.

Claims

1.-21. (canceled)

22. A combination product comprising a first component and a second component, wherein the first component comprises crosslinkable albumin and the second component comprises a polymer, wherein non-terminal monomer units of the polymer comprise at least partially an albumin-crosslinking group and further wherein the first component and the second component are spatially separate.

23. The combination product of claim 22, wherein less than 20% of the non-terminal monomer units comprise an albumin-crosslinking group.

24. The combination product of claim 22, wherein every third to thousandth non-terminal monomer unit comprises an albumin-crosslinking group.

25. The combination product of claim 22, wherein the polymer is a polymer functionalized by the albumin-crosslinking groups.

26. The combination product of claim 22, wherein the polymer is selected from the group consisting of polysaccharide functionalized by albumin-crosslinking groups, muco-polysaccharide functionalized by albumin-crosslinking groups, protein functionalized by albumin-crosslinking groups, synthetic polymer functionalized by albumin-crosslinking groups, and combinations of at least two thereof.

27. The combination product of claim 22, wherein the polymer is selected from the group consisting of hyaluronic acid functionalized by albumin-crosslinking groups, carboxymethylcellulose functionalized by albumin-crosslinking groups, dextran functionalized by albumin-crosslinking groups, polyvinyl alcohol functionalized by albumin-crosslinking groups, polyvinylpyrrolidone functionalized by albumin-crosslinking groups, and combinations of at least two thereof.

28. The combination product of claim 22, wherein the polymer is a hyaluronic acid functionalized by albumin-crosslinking groups.

29. The combination product of claim 27, wherein glucuronic acid units of the hyaluronic acid are functionalized by albumin-crosslinking groups.

30. The combination product of claim 29, wherein the albumin-crosslinking groups are each linked covalently via a linker unit to a carboxy carbon atom of the glucuronic acid units.

31. The combination product of claim 30, wherein the linker unit possesses formula I or formula II below:
NHNHCO(CH.sub.2).sub.n(formula I)
NH(CH.sub.2).sub.n(formula II) where n is an integer from 1 to 12.

32. The combination product of claim 22, wherein the albumin-crosslinking group and/or the albumin-crosslinking groups is or are a nucleophilic group and/or nucleophilic groups.

33. The combination product of claim 22, wherein the crosslinkable albumin is a functionalized albumin.

34. The combination product of claim 22, wherein the crosslinkable albumin is functionalized by at least one group selected from the group consisting of maleimide groups, vinyl sulfone groups, acrylate groups, alkyl halide groups, azirine groups, pyridyl groups, thionitrobenzoic acid groups, arylating groups, and combinations of at least two thereof.

35. The combination product of claim 22, wherein the crosslinkable albumin is maleimide-functionalized albumin.

36. The combination product of claim 22, wherein the first component further comprises cells, more particularly mammalian cells, preferably selected from the group consisting of musculoskeletal cells, metabolism-regulating glandular cells, islet cells, melatonin-producing cells, progenitor cells, stem cells, more particularly mesenchymal stem cells, and combinations of at least two thereof.

37. The combination product of claim 22, wherein the first component is free from hyaluronic acid.

38. The combination product of claim 22, wherein the combination product is a hydrogel.

39. The combination product of claim 22, wherein the combination product is a medical device.

40. A kit comprising a combination product, wherein the combination product comprises a first component and a second component, wherein the first component comprises crosslinkable albumin and the second component comprises a polymer, wherein non-terminal monomer units of the polymer comprise at least partially an albumin-crosslinking group and further wherein the first component and the second component are spatially separate.

41. A use of a polymer as a crosslinking agent for crosslinking albumin, wherein non-terminal monomer units of the crosslinking polymer are partially functionalized with albumin-reactive groups.

Description

EXAMPLES SECTION

1. Preparation of a Thiol-Functionalized Hyaluronic Acid

[0118] A thiol-functionalized hyaluronic acid with low loading was synthesized.

[0119] For this purpose, hyaluronic acid (average molecular weight 57 kDa) was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) at a pH of 4.75 and then modified with an excess of 3,3-dithiobis(propanoic dihydrazide). Following removal of excess 3,3-dithiobis(propanoic dihydrazide) by means of dialysis, the disulphide of the hydrazide coupled to the hyaluronic acid was reduced with tris(2-carboxyethyl)phosphine. Then low molecular mass components were removed by dialysis and the resulting functionalized hyaluronic acid was concentrated by ultrafiltration. In the hyaluronic acid ultimately obtained, approximately every 35th repeating disaccharide unit had a thiol group.

2. Preparation of Maleimide-Functionalized Serum Albumin of Bovine Origin

[0120] 250 mg of human, leporine, bovine or ovine serum albumin (Sigma-Aldrich) were dissolved in 5 ml of 1 M sodium borate (pH 8.2).

[0121] Additionally, 106 mg of 3-maleimidopropionic acid N-hydroxysuccinimide ester (SMP, Obiter Research, Urbana, Ill., USA) were dissolved in 950 l of dimethylformamide (DMF). Insoluble material was separated off by centrifugation. Then 500 l of the supernatant were added to the albumin solution. This was followed by incubation at room temperature for a further 60 minutes. After that, 500 l of 3 M sodium acetate (pH 4.7) were added and dialysis was carried out three times against 1 l of PBS on ice. The dialysate was subsequently concentrated by ultrafiltration (YM-3 membrane, Millipore) to a volume of 3.5 ml and subjected to sterilizing filtration.

3. Preparation of a Hydrogel

[0122] The thiol-functionalized hyaluronic acid prepared according to example 1 was added to the maleimide-functionalized albumin prepared according to example 2. Within a few minutes a hydrogel was obtained, which exhibited no separation tendency whatsoever.

4. Preparation of Thiol-Functionalized Carboxymethylcellulose

[0123] Thiol-functionalized carboxymethylcelluloses with low loading were synthesized.

[0124] For this purpose, carboxymethylcellulose (average molecular weight 90 kDa, degree of functionalization 0.75, i.e. 0.75 carboxyl groups per anhydroglucose unit) was dissolved in water and in various batches was activated respectively with 0.30 or 0.15 or 0.075 molar equivalent (based on carboxyl groups) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, CAS 3945-69-5) and then reacted with an excess of cystamine dihydrochloride at a pH of 6.5-7. Following removal of excess cystamine dihydrochloride by means of dialysis, the disulphide of the cystamine coupled to carboxymethylcellulose was reduced to the thiol using TCEP (tris(2-carboxyethyl)phosphine). Then low molecular mass components were removed by dialysis and the purified thiol-functionalized carboxymethylcellulose was concentrated by ultrafiltration. The thiol-functionalized carboxymethylcelluloses ultimately obtained had degrees of substitution of 3.6 and 2.8 and 2.1 thiol groups, respectively, per 100 anhydroglucose units.

5. Preparation of Hydrogels from Thiol-Functionalized Carboxymethylcelluloses

[0125] The thiol-functionalized carboxymethylcellulose prepared according to example 4 with a degree of substitution of 3.6 was added to a maleimide-functionalized albumin, prepared according to example 2, and PBS. The reactive groups (thiol and maleimide groups, respectively) were each calculated to a concentration of 2 mmol/L in the mixture. Within a few minutes, a hydrogel was obtained which exhibited no separation tendency whatsoever.

[0126] It was similarly possible in the same way, using the thiol-functionalized carboxymethylcellulose prepared according to example 4 with a degree of substitution of 2.1, to produce a hydrogel which exhibited no separation tendency whatsoever.