Anti-inflammatory viscosupplement containing hyaluronic acid

Abstract

The present invention relates to therapeutic compositions comprising hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, as well as methods for making the therapeutic compositions and their use for the treatment of arthritis.

Description

[Technical field]

[0001] The present invention relates to therapeutic compositions comprising hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, as well as methods for making the therapeutic compositions and their use for the treatment of arthritis. [Background technology]

[0002] The most common manifestations of arthritis are osteoarthritis and rheumatoid arthritis. Osteoarthritis (also called arthropathy) is a chronic degenerative joint change with cartilage degeneration accompanied by pain and functional limitations. Synovial fluid present in the joint cavity protects articular cartilage from mechanical stress. Alterations or loss of synovial fluid can lead to cartilage damage and osteoarthritis (OA) in the joint. OA can enhance the degeneration of the joint surface and synovial fluid (Berenbaum, Osteoarthritis and Cartilage, (2013), doi:10.1016 / j.joca.2012.11.012). On the other hand, rheumatoid arthritis (chronic polyarthritis, primary chronic polyarthritis, RA), a widespread joint disease of inflammation, is a chronic autoimmune disease. Currently, the treatment options are injections of hyaluronic acid preparations as viscosupplementation therapy to improve the mechanical properties of synovial fluid and systemic administration of nonsteroidal antirheumatic drugs (NSAIDs) or cortisol. Injections of hyaluronic acid preparations may result in temporary improvement in mobility and pain relief, but do not treat the inflammation causally. Anti-inflammatory drugs treat inflammation but incur side effects. The inflammatory process in the joints is accompanied by the infiltration of immune cells stimulated by chemokines such as IL-8 (CXCL8) and RANTES (CLL5) in the synovial fluid (Vergunst et al., (2005) Scandinavian Journal of Rheumatology, doi:10.1080 / 03009740500439159; Valcamonica et al., Clin. Exp., Rheumatol. (2014); Goldring & Berenbaum, Curr. Opin. Pharmacol. 22, 51-63 (2015); Pierzchala et al., Arch. Immunol. Ther. Exp. (Warsz). (2011) doi:10.1007 / s00005-011-0115-4). Summary of the Invention

[0003] The technical problem underlying the present invention is to overcome the shortcomings of known methods for treating arthritis, in particular OA and RA, and of the therapeutic compositions used in these methods. In particular, the technical problem of the present invention is to provide a therapeutic composition which leads to an improved treatment of arthritis, in particular which makes it possible to treat arthritis symptomatically and causally.

[0004] The invention solves the technical problem underlying the invention in particular through the subject matter of the independent claims as well as the dependent claims and the teachings of this specification. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a therapeutic composition according to the present invention. [Diagram 2] 1 is a photograph of various hydrogel particles used in accordance with the present invention. [Figure 3A] FIG. 1 is a graph showing reduction of LPS-induced knee joint swelling in a mouse model. [Figure 3B] FIG. 1 is a graphical representation of gene expression of the inflammatory marker TNFα. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The present invention relates to a therapeutic composition comprising hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, particularly an aqueous liquid, in which the hydrogel particles comprise at least one polyionic polymer component, which is covalently bound to at least one uncharged polymer component and / or (particularly) at least one non-polymeric crosslinker component to form a network, and in which the at least one polyionic polymer component has sulfate or sulfonate groups. These sulfate or sulfonate groups are present in the hydrogel, particularly in the hydrogel particles, as free, i.e. unbound, ionizable groups, particularly as groups that are negatively charged under physiological conditions. These sulfate or sulfonate groups are also present in the composition according to the invention, i.e. in the presence of the hyaluronic acid component, as free, i.e. unbound, ionizable groups, particularly as groups that are negatively charged under physiological conditions.

[0007] Thus, the present invention relates in particular to a composition, in particular an anti-inflammatory composition, for use as a biolubricant, i.e. as a lubricant for living tissue, in particular in vivo, comprising at least three components. These three components are at least one, in particular one hyaluronic acid component, hydrogel particles and at least one pharma- ceutically acceptable carrier. The hydrogel particles according to the present invention comprise at least two components, namely at least one, in particular one polyionic polymer component, in particular one polyanionic polymer component, in particular one glycosaminoglycan (GAG) component and at least one non-polymeric crosslinker component or (optionally) at least one, in particular one uncharged polymer component, in particular one polyethylene glycol (PEG) component. The at least three components contemplated by the present invention are present in the composition according to the present invention in a mixed form without being chemically bound to each other, in particular the hydrogel particles are not chemically covalently bound to the hyaluronic acid component, rather the hydrogel particles are only present in a physical mixture with the hyaluronic acid component in a pharma- ceutically acceptable carrier, in particular an aqueous liquid. That is, in accordance with the present invention, the hyaluronic acid component is present in a free, unbound, physically associated form with the hydrogel particles, and neither the polyionic polymer component, the uncharged polymer component, nor the non-polymeric crosslinker component of the hydrogel particles are chemically covalently bound to the free hyaluronic acid component.

[0008] In one embodiment according to the invention, at least one non-charged polymer component is covalently bonded to at least one polyionic polymer component, in particular directly or by means of a crosslinker component, so that the non-charged polymer component and the polyionic polymer component covalently bonded thereto are interconnected to form a network of hydrogel particles, optionally in the additional presence of a non-polymeric crosslinker component.

[0009] In a further embodiment according to the invention, the at least one non-polymeric crosslinker component is covalently bonded, in particular directly, to the at least one polyionic polymer component, such that the at least one non-polymeric crosslinker component and the polyionic polymer component covalently bonded thereto are interconnected to form a network of hydrogel particles.

[0010] Thus, the present invention particularly contemplates at least one polyionic polymer component being covalently bonded directly to at least one uncharged polymer component or via at least one crosslinker component to form a network, or in a further embodiment at least one polyionic polymer component being bonded to at least one crosslinker component to form a network.

[0011] According to the present invention, the polyionic polymer component has sulfate or sulfonate groups, particularly sulfate groups. According to the present invention, these sulfate or sulfonate groups are not covalently bonded to the uncharged polymer component, particularly the polyethylene glycol component or the hyaluronic acid component. According to the present invention, these sulfate or sulfonate groups are not covalently bonded to the non-polymeric crosslinker component.

[0012] Without being bound by theory, the sulfate or sulfonate groups in the hydrogel are essentially present in deprotonated form. These deprotonated sulfate or sulfonate groups may be charge compensated or form charge interactions with counterions and / or proteins. Thus, the sulfate or sulfonate groups can form reversible bonds to soluble molecules via non-covalent interactions, particularly charge interactions, particularly preferably upon contact with biological fluids containing soluble molecules.

[0013] The polyionic polymer used to prepare the hydrogel particles preferably has, in addition to sulfate or sulfonate groups, at least two further functional groups, in particular selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof, which functional groups covalently bond the polyionic polymer to the non-charged polymer and / or non-polymeric crosslinker component used to prepare the hydrogel particles, forming a hydrogel, and at least one polyionic polymer component is crosslinked through at least one non-charged polymer component or at least one non-polymeric crosslinker component to form a network. The at least two functional groups of the polyionic polymer may be the same or different, in particular they are the same.

[0014] In a preferred embodiment of the present invention, the uncharged polymers used for the preparation of hydrogel particles have one functional group, particularly a terminal group, selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof at each free end of the polymer. Thus, the uncharged polymers are functionalized, particularly terminal group functionalized. Through these reactive functional groups, particularly terminal groups, the at least one uncharged polymer is preferably bonded to at least one polyionic polymer, thus forming the network structure of the hydrogel particles. The at least two functional groups of the uncharged polymers may be the same or different, particularly they are the same.

[0015] The uncharged polymer may be linear or branched, in particular multi-armed, i.e. in a preferred embodiment of the invention the uncharged polymer consists of several branches, in particular the uncharged polymer is star-shaped, i.e. multi-armed with several, in particular 4 or 8, in particular 4, chains of equal length branching out from one centre, composed of corresponding repeating units.

[0016] In a preferred embodiment of the present invention, the non-polymeric crosslinker molecule used in the preferred embodiment for producing hydrogel particles has at least two functional groups, particularly end groups, selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups, hydroxylated aromatic groups and combinations thereof.Therefore, the non-polymeric crosslinker molecule is functionalized, particularly end group functionalized.Through these reactive functional groups, particularly end groups, at least one non-polymeric crosslinker molecule is preferably bonded with at least one polyionic polymer, thus forming the network structure of the hydrogel particle.The at least two functional groups of the non-polymeric crosslinker molecule may be the same or different, particularly they are the same.

[0017] The therapeutic composition of the invention is characterized by a particularly advantageous use for the treatment of arthritis, in particular rheumatoid arthritis, osteoarthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis. The therapeutic composition according to the invention advantageously binds to proinflammatory cytokines, in particular chemokines such as interleukin-8 (IL-8) from synovial fluid, and thus prevents chemokine-induced immune cell migration. Furthermore, the therapeutic composition according to the invention exhibits a particularly advantageous viscosupplementation effect, i.e. leads to a reduction in frictional resistance in the synovial fluid, thus resulting in a function of improving lubrication in the joints (also referred to herein as lubricating effect), which is preferably at least the same as, but in particular better than, that found in known compositions used in this context, such as hyaluronic acid. The lubricating effect provided by the invention leads to a reduction in pain in the patient and prevents or reduces functional and movement limitations, thus preventing defensive postures and preventing further joint damage. The inventors have surprisingly found that the therapeutic viscosupplementation composition of the present invention is characterized in particular by a functional combination of an advantageous lubricating effect and the ability to bind, i.e. sequester, proinflammatory chemokines, and thus an anti-inflammatory effect, and furthermore, the composition exhibits a particularly good injectability. Advantageously, hyaluronic acid does not interfere with the sequestering effect of the hydrogel particles, but rather provides a surprising advantage. It is particularly advantageous that the lubricating effect is exerted for a longer period of time, since the sequestering effect of the hydrogel by binding proinflammatory cytokines not only results in the reduction and prevention of inflammatory processes in tissues, but also in the protection of the hyaluronic acid component of the composition from degradation, i.e. the slower degradation of the hyaluronic acid component. The combination of anti-inflammatory properties and improved mechanical properties, especially in terms of the prolonged lubricating effect and good injectability, allows a particularly effective intra-articular therapy, and therefore a targeted therapy, of arthritis, in particular at least one of its clinical symptoms, in particular its symptoms, in particular all its manifestations, in a synergistic manner and system.

[0018] In a preferred embodiment according to the invention, the therapeutic composition according to the invention containing hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier is prepared by covalently bonding at least one uncharged polymer with at least one polyionic polymer either directly or via at least one crosslinker component to form a hydrogel, which accordingly has at least one uncharged polymer component covalently bonded to at least one polyionic polymer component to form a network, and this hydrogel, particularly in the form of hydrogel particles, is mixed with at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier.

[0019] In a preferred embodiment according to the invention, a therapeutic composition according to the invention containing hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier is prepared by covalently bonding at least one non-polymeric crosslinker molecule directly with at least one polyionic polymer to form a hydrogel, which correspondingly comprises at least one non-polymeric crosslinker component covalently bonded to at least one polyionic polymer component to form a network, and this hydrogel, particularly in the form of hydrogel particles, is mixed with at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier.

[0020] In a preferred embodiment, the at least one uncharged polymer has an average molecular weight of between 5000 Da and 25000 Da, in particular between 10000 Da and 19000 Da.

[0021] In a preferred embodiment, the repeat unit of at least one uncharged polymer component has an average molecular weight of between 30 Da and 55 Da, in particular between 40 Da and 50 Da.

[0022] In one particularly preferred embodiment, the at least one uncharged polymer component is a linear uncharged polymer component.

[0023] In one particularly preferred embodiment, at least one uncharged polymer moiety is a branched, in particular multi-armed, uncharged polymer moiety.

[0024] In a particularly preferred embodiment, at least one multi-armed uncharged polymer moiety is a 4-arm or 8-armed uncharged polymer moiety.

[0025] In a particularly preferred embodiment, the at least one uncharged polymer component is selected from the group consisting of a polyethylene glycol (PEG) component, a poly(2-oxazoline) (POX) component, a polyvinylpyrrolidone (PVP) component, a polyvinyl alcohol (PVA) component, a polyacrylamide (PAM) component, and combinations thereof.

[0026] In one particularly preferred embodiment, at least one of the multi-arm, uncharged polymer moieties is a PEG moiety.

[0027] In one particularly preferred embodiment, the at least one uncharged polymer moiety is a polyethylene glycol moiety, in particular a linear or multi-armed polyethylene glycol moiety.

[0028] In one particularly preferred embodiment, at least one multi-armed, uncharged polymer moiety is a four-armed polyethylene glycol moiety.

[0029] In a further preferred embodiment, the at least one multi-armed, uncharged polymer moiety is an eight-arm polyethylene glycol moiety.

[0030] In one particularly preferred embodiment, at least one multi-arm, uncharged polymer component is a star-shaped PEG, also known as starPEG.

[0031] In a preferred embodiment, the uncharged polymer used for the preparation of the hydrogel particles according to the invention has at each free end of the polymer chain a functional group, in particular a terminal group, selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof. Thus, the uncharged polymer is in particular terminal-group functionalized. At least two functional groups of the uncharged polymer may be the same or different, in particular they are the same.

[0032] In one particularly preferred embodiment, the uncharged polymer used to prepare the hydrogel particles according to the invention has at least two amino groups, in particular terminal amino groups.

[0033] In one particularly preferred embodiment, the uncharged polymer used to prepare the hydrogel particles according to the invention has at least two carboxyl groups, in particular terminal carboxyl groups.

[0034] In a preferred embodiment, the polyionic polymer component is a polyanionic polymer component, in particular a glycosaminoglycan component.

[0035] In a particularly preferred embodiment, the glycosaminoglycan used for the preparation of the hydrogel particles according to the invention is selected from the group consisting of chondroitin sulfate, dextran sulfate, dermatan sulfate, glucosamine sulfate, heparin, selectively desulfated heparin, heparan sulfate and hyaluronan sulfate. In particular, the glycosaminoglycan is heparin, heparan sulfate or dextran sulfate, in particular heparin or selectively desulfated heparin, in particular selectively N-desulfated heparin.

[0036] In one particularly preferred embodiment, the glycosaminoglycan used in the preparation of the hydrogel particles according to the invention is a desulfated glycosaminoglycan, in particular a selectively N-desulfated glycosaminoglycan, especially selectively N-desulfated heparin.

[0037] In a particularly preferred embodiment, the glycosaminoglycan used for the preparation of the hydrogel particles according to the invention is selectively N-desulfated heparin, which is available according to the synthesis instructions from Atallah et al. (Biomaterials. October 2018; 181:227-239. doi: 10.1016 / j.biomaterials.2018.07.056. Epub July 30, 2018).

[0038] In one preferred embodiment, the polyionic polymer component used in the preparation of hydrogel particles according to the present invention is poly(4-styrenesulfonic acid-co-maleic acid).

[0039] In a preferred embodiment, the average molecular weight of the polyionic polymer is from 3 kDa to 20 kDa, in particular from 4 kDa to 14 kDa, in particular from 10 kDa to 14 kDa, especially from 13 kDa to 14 kDa.

[0040] In one preferred embodiment there are from 40 to 80, especially from 40 to 75, particularly from 40 to 70, especially from 65 to 80, especially from 65 to 75 sulfate or sulfonate groups per polyionic polymer molecule.

[0041] In a preferred embodiment, there are 40 to 55, particularly 40 to 50, and especially 45 to 50 sulfate or sulfonate groups per desulfated glycosaminoglycan, particularly per selectively N-desulfated glycosaminoglycan, in particular per selectively N-desulfated heparin.

[0042] In a preferred embodiment, the repeat units of at least one polyionic polymer component have an average molecular weight of from 400 Da to 550 Da, especially from 420 Da to 470 Da.

[0043] In a preferred embodiment, the repeat units of at least one polyionic polymer component have an average molecular weight of from 400 Da to 550 Da, particularly from 450 Da to 550 Da, especially from 500 Da to 550 Da.

[0044] In one preferred embodiment, there are from 1.0 to 4.5, particularly from 1.5 to 4.5, and especially from 2.0 to 3.0 sulfate or sulfonate groups per repeat unit of at least one polyionic polymer component.

[0045] In a preferred embodiment, there are 1.0 to 4.5, particularly 1.5 to 2.0, sulfate or sulfonate groups per repeat unit of at least one polyionic polymer component, particularly per desulfated glycosaminoglycan, particularly per selectively N-desulfated glycosaminoglycan, particularly per selectively N-desulfated heparin.

[0046] In a preferred embodiment, the non-polymeric crosslinker molecules used for the preparation of the hydrogel particles according to the invention have at least two functional groups suitable for forming one covalent bond each to the polyionic polymer component in a preferred embodiment of the invention, in particular selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups, hydroxylated aromatic groups and combinations thereof.

[0047] Therefore, the non-polymeric crosslinker molecules that represent the non-polymeric crosslinker components in the hydrogel are functionalized, in particular end-group functionalized. Through these reactive functional groups, at least one non-polymeric crosslinker molecule is preferably bonded to at least one polyionic polymer, thus forming the network structure of the hydrogel particles. At least two functional groups of the non-polymeric crosslinker molecule polymers may be the same or different, in particular they are the same.

[0048] In a preferred embodiment, the crosslinker molecule is a non-polymeric bifunctional crosslinker molecule.

[0049] In a preferred embodiment, the crosslinker molecule has at least two amino groups. In a particularly preferred embodiment, the crosslinker molecule having at least two amino groups is selected from the group consisting of ethylenediamine, propylenediamine (1,3-diaminopropane), butane-1,4-diamine, pentane-1,5-diamine (cadaverine), hexamethylene-1,6-diamine, and combinations thereof.

[0050] In a preferred embodiment, the crosslinker molecule has at least two carboxyl groups. In a particularly preferred embodiment, the crosslinker molecule having at least two carboxyl groups is selected from the group consisting of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, and combinations thereof.

[0051] In one particularly preferred embodiment, the crosslinker molecule is a molecule having at least two different functional groups, particularly at least two functional groups capable of crosslinking a polyionic polymer component and an uncharged polymer component, particularly N-(2-aminoethyl)maleimide.

[0052] In a preferred embodiment, the at least one non-polymeric crosslinker component is selected from the group consisting of ethylenediamine, propylenediamine (1,3-diaminopropane), butane-1,4-diamine, pentane-1,5-diamine (cadaverine), hexamethylene-1,6-diamine, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, N-(2-aminoethyl)maleimide, and combinations thereof.

[0053] In a particularly preferred embodiment, the crosslinker molecule is in particular an enzymatically cleavable sequence, in particular a matrix metalloprotease (MMP) response element, such as PQGIWGQ, IPVSLRSG or VPMSMRGG, a cathepsin response element, such as VPMSMRGG, an elastase response element, such as AAPV or APEEIMDRQ, a blood coagulation enzyme response element, such as thrombin responsive GGF-pipecolic acid-RYSWGCG or GG-cyclohexylalanine-ARSWGCG, a FXa response element, such as GGIEGRMGGWCG, a kallikrein response element, such as CGGGPFRIGGWCG or a bacterial protease response element, such as aureolysin responsive ADVFEA or AAEAA, an elastase response element, such as AAPV or a protease IV response sequence MKATKLVLGAVILGSTLLAG, in particular one of the sequences GPQGIAGQ, GPQGIWGQ or GCGGPQGIWGQGGCG. The sequences GPQGIAGQ and GPQGIWGQ are artificially created sequences that are cleavable by a number of matrix metalloproteases (MMPs), in particular MMP1, MMP3, MMP7, MMP9.

[0054] In a preferred embodiment, the enzymatically cleavable sequence is flanked at both the C-terminus and N-terminus, respectively, by a cysteine, in particular the sequence GCG or GCGG. The polyionic polymer and the uncharged polymer or at least two polyionic polymers are crosslinked via the respective cysteines.

[0055] In one preferred embodiment, the polyionic polymer components used in the preparation of hydrogel particles according to the invention are covalently linked with non-polymeric crosslinker components, particularly peptides.

[0056] In one preferred embodiment, the uncharged polymer component and the polyionic polymer component preferably used in the preparation of the hydrogel particles according to the invention are covalently linked to each other by a non-polymeric crosslinker component, in particular a peptide.

[0057] In one preferred embodiment, the non-polymeric crosslinker component and the polyionic polymer component are covalently bonded to one another via at least one amide bond.

[0058] In one preferred embodiment, the uncharged polymer component and the polyionic polymer component are covalently bonded to one another via at least one amide bond by a non-polymeric crosslinker component.

[0059] In a further preferred embodiment, the uncharged polymer component and the polyionic polymer component used for the preparation of the hydrogel particles according to the invention are directly covalently bonded to one another, in particular by an amide bond, a thiol-amine bond, a disulfide bond or a bioorthogonal thioether bond obtained by a thiol-maleimide reaction, a thiol-vinylsulfone reaction or a thiol-acrylate reaction.

[0060] In a preferred embodiment, the uncharged polymeric components, in particular those having amino groups, and the polyionic polymeric components, in particular those having carboxyl groups, used in the preparation of the hydrogel particles according to the invention are directly covalently bonded to each other by amide bonds, which in a preferred embodiment are made possible by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activation of the carboxyl groups of the polyionic polymeric components.

[0061] In a further preferred embodiment, the non-polymeric crosslinker component and the polyionic polymer component used in the preparation of the hydrogel particles according to the invention are directly covalently bonded to one another by means of an amide bond, a thiol-amine bond, a disulfide bond or a bioorthogonal thioether bond, in particular obtained by a thiol-maleimide reaction, a thiol-vinylsulfone reaction or a thiol-acrylate reaction.

[0062] In a preferred embodiment, the non-polymeric crosslinker component, in particular a non-polymeric crosslinker component having amino groups, and the polyionic polymer component, in particular a polyionic polymer component having carboxyl groups, used in the preparation of the hydrogel particles according to the invention are directly covalently bonded to each other by amide bonds, which in a preferred embodiment are made possible by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activation of the carboxyl groups of the polyionic polymer component.

[0063] In a preferred embodiment, the hydrogel particles comprise at least two, in particular two, different non-polymeric crosslinker components. In a particularly preferred embodiment, the hydrogel particles comprise one non-polymeric crosslinker component having at least two carboxyl groups, in particular end-functionalized with one carboxyl group each, and one non-polymeric crosslinker component having at least two amino groups, in particular end-functionalized with one amino group each. Preferably, the non-polymeric crosslinker component having at least two carboxyl groups can be bonded to the non-polymeric crosslinker component having an amino group via at least one amide bond, and the non-polymeric crosslinker component having at least two amino groups can be bonded to the polyionic polymer component having a carboxyl group, in particular heparin, via at least one amide bond, where in a preferred embodiment, the amide bond is enabled by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activation of the carboxyl groups of the non-polymeric crosslinker molecule and the polyionic polymer component.

[0064] In a preferred embodiment, the hydrogel particles comprise at least two, in particular two, different uncharged polymer components. In a particularly preferred embodiment, the hydrogel particles comprise one uncharged polymer component having at least two carboxyl groups, in particular end-functionalized with one carboxyl group each, and one uncharged polymer component having at least two amino groups, in particular end-functionalized with one amino group each. Preferably, the uncharged polymer component having at least two carboxyl groups can be bonded to the uncharged polymer component having an amino group via at least one amide bond, and the uncharged polymer component having at least two amino groups can be bonded to the polyionic polymer component having a carboxyl group, in particular heparin, via at least one amide bond, where in a preferred embodiment, the amide bond is enabled by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activation of the carboxyl groups of the uncharged polymer component and the polyionic polymer component.

[0065] In a preferred embodiment, the hydrogel particles comprise one uncharged polymer component. In a particularly preferred embodiment, the hydrogel particles comprise one uncharged polymer component having at least two amino groups, in particular end-functionalized with one amino group each. Preferably, the uncharged polymer component having at least two amino groups can be bonded to a polyionic polymer component having a carboxyl group, in particular heparin, via at least one amide bond, where in a preferred embodiment, the amide bond is enabled by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activation of the carboxyl group of the polyionic polymer component.

[0066] In one particularly preferred embodiment, the uncharged polymer, especially end-functionalized with one carboxyl group each, is a multi-arm, especially an 8-arm, PEG.

[0067] In one particularly preferred embodiment, the uncharged polymer, especially end-functionalized with one amino group each, is a multi-arm, especially a four-arm, PEG.

[0068] In one particularly preferred embodiment, the hydrogel particles in the therapeutic composition are present in a swollen state, especially in an aqueous solution, especially water or an aqueous buffer solution, especially PBS, in swollen form.

[0069] In a preferred embodiment, the hydrogel particles are capable of swelling 0.75 to 2.5 times the size of the hydrogel particle immediately after hydrogel formation.

[0070] In a preferred embodiment, the content of swollen hydrogel particles in the composition according to the invention is from 5% to 60% by volume, in particular from 9% to 55% by volume, in particular from 10% to 50% by volume (each relative to the total volume of the composition).

[0071] In a preferred embodiment, the hydrogel particles have a storage modulus, especially in the swollen state, of at most 22.0 kPa, especially at most 20.0 kPa, especially at most 15.0 kPa, especially at most 10.0 kPa.

[0072] In a preferred embodiment, the hydrogel particles have a storage modulus, especially in the swollen state, of 0.1 kPa to 22.0 kPa, in particular 0.1 kPa to 20.0 kPa, in particular less than 0.1 kPa to 15.0 kPa, in particular less than 0.1 kPa to 10.0 kPa.

[0073] In a preferred embodiment, the hydrogel particles have an average size, especially in the swollen state, of at most 200 μm, in particular at most 80 μm.

[0074] In a preferred embodiment, the hydrogel particles have an average diameter (size), especially in the swollen state, of 10 μm to 200 μm, especially 5 μm to 150 μm, especially 10 μm to 80 μm. Preferably, the average diameter of the hydrogel particles is determined by fluorescence microscopy, preferably using a dye having a reactive group, especially an amine group.

[0075] In one preferred embodiment, the hydrogel particles have a spherical shape, especially in the swollen state.

[0076] In a preferred embodiment, the hydrogel particles have a size distribution, especially in the swollen state, with a deviation of less than 15%, especially less than 10%, of the average size.

[0077] In a preferred embodiment, the hydrogel particles have a concentration of sulfate or sulfonate groups, in particular in the swollen state, of at least 0.1 mmol / l, in particular at least 10 mmol / l, in particular at least 100 mmol / l, in particular from 0.1 mmol / l to 800 mmol / l, in particular from 10 mmol / l to 800 mmol / l, in particular from 20 mmol / l to 500 mmol / l, in particular from 20 mmol / l to 200 mmol / l, in particular from 50 mmol / l to 200 mmol / l, in particular in the volume of the hydrogel particles.

[0078] In a particularly preferred embodiment, the hydrogel particles have sulfate groups, in particular in a swollen state, in a concentration of at least 0.1 mmol / l, in particular at least 10 mmol / l, in particular at least 100 mmol / l, in particular from 0.1 mmol / l to 800 mmol / l, in particular from 10 mmol / l to 800 mmol / l, in particular from 20 mmol / l to 500 mmol / l, in particular from 20 mmol / l to 200 mmol / l, in particular from 50 mmol / l to 200 mmol / l, in particular from 10 mmol / l to 500 mmol / l, in particular from 10 mmol / l to 2 ...

[0079] In a particularly preferred embodiment, the hydrogel particles have sulfonate groups, in particular in a swollen state, in a concentration of at least 0.1 mmol / l, in particular at least 10 mmol / l, in particular at least 100 mmol / l, in particular from 0.1 mmol / l to 800 mmol / l, in particular from 10 mmol / l to 800 mmol / l, in particular from 20 mmol / l to 500 mmol / l, in particular from 20 mmol / l to 200 mmol / l, in particular from 50 mmol / l to 200 mmol / l, in particular in a volume of the hydrogel particles present in the swollen state.

[0080] In a preferred embodiment, the hydrogel particles have an average mesh width of at least 5 nm, in particular at least 5.7 nm. The minimum average mesh width ensures that all relevant signal molecules with a diameter between 3 nm and 5 nm can penetrate the hydrogel particles rapidly and sterically unhindered, the ability of the signal molecules to diffuse through the particles being essentially dependent on the charge properties of the particles and the resulting interaction with the signal molecules.

[0081] In a preferred embodiment, the hydrogel particles have an average mesh width of 5 nm to 30 nm, in particular 5.7 nm to 30 nm.

[0082] In a preferred embodiment, the composition according to the invention has a hyaluronic acid component content of 0.1% to 3.0% by weight, in particular 0.4% to 2.0% by weight, in particular 0.5% to 2.0% by weight, in particular 0.5% to 1.8% by weight (each relative to the total mass of the composition).

[0083] In a preferred embodiment, the composition according to the invention has a content of hyaluronic acid component of 5% to 20% by volume, in particular 10% to 20% by volume, in particular 10% to 18% by volume (each relative to the total volume of the composition), in particular a content of hyaluronic acid solution of 10% by weight (relative to the total mass of hyaluronic acid solution).

[0084] In one preferred embodiment, the hyaluronic acid component is hyaluronic acid and / or a salt thereof, in particular the hyaluronic acid component is the sodium salt of hyaluronic acid.

[0085] In a preferred embodiment, the hyaluronic acid component is hyaluronic acid having an average molecular weight of 4 kDa to 100,000 kDa, in particular 500 kDa to 100,000 kDa, in particular 1,000 kDa to 7,000 kDa, in particular 1,000 kDa to 5,000 kDa.

[0086] In a preferred embodiment, the composition according to the invention has a content of 30% to 90% by volume, in particular 30% to 85% by volume, in particular 40% to 80% by volume of a pharma- ceutically acceptable carrier (each relative to the total volume of the composition).

[0087] In a particularly preferred embodiment, the at least one pharma- ceutically acceptable carrier is an aqueous liquid, in particular water or an aqueous buffer solution, in particular phosphate buffered saline (PBS).

[0088] In a particularly preferred embodiment, the at least one pharma- ceutically acceptable carrier, in particular the aqueous liquid, has a pH value of 6.5 to 8.0, in particular 6.8 to 7.6, in particular 7.0 to 7.5, in particular 7.4.

[0089] In a particularly preferred embodiment, the therapeutic composition further comprises at least one additive, in particular marine collagen, sorbitol, mannitol, platelet rich plasma (PRP), polyphenols, S-allyl cysteine, sodium pentosan-polyphosphate and / or extracellular vesicles containing curcuminoids, in particular sorbitol.

[0090] In one preferred embodiment, the solids content of the composition according to the invention is between 10% and 25% by weight, in particular between 10% and 17% by weight, in particular between 12% and 15% by weight (each relative to the total mass of the composition).

[0091] In one preferred embodiment, the composition according to the invention has a viscosity of from 10 Pa / s to 100 Pa / s, in particular from 20 Pa / s to 90 Pa / s, in particular from 30 Pa / s to 80 Pa / s.

[0092] In one preferred embodiment, the composition according to the invention is an injectable therapeutic composition.

[0093] In a preferred embodiment, the injection force for injecting a composition according to the invention through a 25G needle at an injection rate of 0.05 ml / s is at most 10 N, in particular 2.5 N to 3.5 N, in particular 2.7 N to 3.5 N. In a particularly preferred embodiment, the hydrogel particles are present in a swollen state when the injection force is measured.

[0094] In a preferred embodiment, the composition according to the invention has a coefficient of friction of at most 0.2, in particular between 0.085 and 0.095, at 10 revolutions / s, respectively.

[0095] In a preferred embodiment, the composition according to the invention is capable of reducing the concentration of at least one free cytokine, particularly a chemokine, especially IL-8, in the surrounding solution, especially synovial fluid. In a preferred embodiment, the composition according to the invention is capable of reducing the concentration of at least one free cytokine by 70% to 80% in a cytokine-containing model synovial fluid.

[0096] In one particularly preferred embodiment of the invention, the free cytokine, in particular a chemokine, is a pro-inflammatory cytokine, in particular a chemokine.

[0097] In a particularly preferred embodiment, the free cytokine is selected from the group consisting of IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES and combinations thereof, in particular IL-8.

[0098] The present invention also relates to a therapeutic composition for use in a therapeutic method for treating arthritis, in particular osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis, in particular for application, in particular for injection, in particular intra-articular injection, in a joint of a human or animal patient.

[0099] The present invention also relates to the use of the hydrogel particles used in the therapeutic composition of the present invention, in particular together with at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier and optionally at least one additive, in a therapeutic method for treating arthritis, in particular osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis.

[0100] The present invention particularly relates to a therapeutic method for treating arthritis, particularly osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis, using the therapeutic composition of the present invention.

[0101] The present invention also relates to a method for treating arthritis, in particular osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis, in which an effective amount of the therapeutic composition of the present invention is applied to a human or animal patient, in particular injected, in particular in a joint, i.e. intra-articularly.

[0102] The present invention particularly relates to a method for treating arthritis, in particular osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis, using a therapeutic composition, in particular a therapeutic composition comprising hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, in which the hydrogel particles comprise at least one polyethylene glycol component covalently bonded to at least one polyionic polymer component, the at least one polyionic polymer component having sulfate or sulfonate groups, and in which an effective amount of the composition according to the invention is administered to a human or animal patient, in particular by intra-articular injection.

[0103] In a particularly preferred embodiment of the present invention, the therapeutic compositions and methods for treating arthritis are characterized in that the treatment of arthritis is achieved by the lubricating effect provided by the present invention, the cytokine reducing effect, particularly the anti-inflammatory effect, provided by the present invention, or both.

[0104] The present invention also relates to therapeutic compositions of the invention for use in therapeutic methods for treating back pain, particularly those applied, especially injected, to a human or animal patient.

[0105] The present invention also relates to the use of the hydrogel particles used in the therapeutic composition of the present invention, in particular together with at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier and, optionally, at least one additive, in a therapeutic method for treating lower back pain.

[0106] In particular, the present invention relates to a therapeutic method for treating lower back pain using the therapeutic composition of the present invention.

[0107] The present invention also relates to a method for treating back pain in which an effective amount of the therapeutic composition of the present invention is administered, particularly by injection, to a human or animal patient.

[0108] The present invention particularly relates to a method for treating lower back pain using a therapeutic composition, particularly a therapeutic composition comprising hydrogel particles, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, wherein the hydrogel particles comprise at least one polyethylene glycol component covalently bonded to at least one polyionic polymer component, the at least one polyionic polymer component having sulfate or sulfonate groups, and wherein an effective amount of the composition according to the invention is administered, particularly by injection, to a human or animal patient.

[0109] The present invention also relates to a method for producing a therapeutic composition, particularly a therapeutic composition of the present invention, comprising the steps of: a) providing at least one uncharged polymer, in particular a functionalized uncharged polymer or / and at least one functionalized non-polymeric crosslinker molecule and at least one polyionic polymer having sulfate or sulfonate groups, at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier and optionally further components, b) a method step of crosslinking non-charged polymer and / or non-polymeric crosslinker molecules with a polyionic polymer to form a network, thereby obtaining a hydrogel, in particular a hydrogel particle, composed of at least one polyionic polymer component covalently bonded to at least one non-charged polymer component and / or at least one non-polymeric crosslinker component, c) a method step of mixing the hydrogel, in particular the hydrogel particles, with at least one hyaluronic acid component and at least one pharma- ceutically acceptable carrier, and d) Method steps for obtaining a therapeutic composition The present invention relates to a method comprising the steps of:

[0110] In a preferred embodiment, the non-charged polymer preferably functionalized has at least two functional groups, particularly terminal groups, selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof, wherein the at least two groups may be the same or different.

[0111] In a preferred embodiment, the non-polymeric crosslinker molecule, which is preferably functionalized, has at least two functional groups, particularly terminal groups, selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups, hydroxylated aromatic groups, and combinations thereof, wherein the at least two groups can be the same or different.

[0112] In a preferred embodiment, the polyionic polymer has at least one sulfate or sulfonate group plus at least two additional functional groups selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups, and combinations thereof, where the at least two groups can be the same or different.

[0113] The at least two functional groups each selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof preferably contemplated in the at least one polyionic polymer and the at least one non-charged polymer and / or non-polymeric crosslinker molecule used according to the present invention are selected for the polyionic polymer and the non-charged polymer and / or non-polymeric crosslinker molecule, respectively, such that the reactive partner functional groups are capable of forming one covalent bond with each other between the polyionic polymer and the at least one non-charged polymer or between the at least one polyionic polymer and the at least one non-polymeric crosslinker molecule to form a network structure.

[0114] The uncharged polymer is preferably selected from the group consisting of polyethylene glycol (PEG), poly(2-oxazoline) (POX), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and / or polyacrylamide (PAM) having at least two or more functional groups, in particular amino groups, which are crosslinkable with the polyionic polymer having carboxyl groups which are preferred in the preferred embodiment as functional groups.

[0115] The non-polymeric crosslinker molecules are preferably bifunctional crosslinker molecules having at least two or more functional groups, particularly amino groups, capable of crosslinking with polyionic polymers having carboxyl groups as preferred functional groups in the preferred embodiment.

[0116] In a particularly preferred embodiment, in method step b) the carboxyl groups of the carboxyl-bearing polymer or non-polymeric crosslinker molecules are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysulfosuccinimide (EDC / sNHS) activated.

[0117] In a preferred embodiment, the uncharged polymer is PEG and the polyionic polymer is glycosaminoglycan. In a preferred embodiment, the molar ratio of polyethylene glycol to glycosaminoglycan during cross-linking in method step b) is 0.75 to 3.0 (PEG / GAG), in particular 0.75, in particular 1.5, in particular 2.25, in particular 3.0.

[0118] In a preferred embodiment, the molar ratio of polyethylene glycol to glycosaminoglycan during crosslinking in method step b) is 0.75 to 3.0 (PEG / GAG), in particular 0.75, in particular for a solids content of 12.0% to 15.0% (w / v), in particular 13.3% (w / v), relative to the total volume of the mixture comprising polyethylene glycol and glycosaminoglycan, respectively, 1.5, in particular for a solids content of 12.0% to 15.0% (w / v), in particular 13.3% (w / v), 2.25, in particular for a solids content of 12.0% to 15.0% (w / v), in particular 13.3% (w / v), and 3.0, in particular for a solids content of 12.0% to 15.0% (w / v), in particular 13.3% (w / v).

[0119] In a preferred embodiment, the concentration of the polyionic polymer in method step b) is between 0.5 mmol / l and 6.0 mmol / l (for the mixture of at least one uncharged polymer and at least one polyionic polymer).

[0120] In a preferred embodiment, the concentration of the uncharged polymer in method step b) is between 5.0 mmol / l and 12.0 mmol / l (for a mixture of at least one uncharged polymer and at least one polyionic polymer).

[0121] In a preferred embodiment, the concentration of the non-polymeric crosslinker molecules in method step b) is between 5.0 mmol / l and 24.0 mmol / l (based on the mixture of at least one non-polymeric crosslinker molecule and at least one polyionic polymer).

[0122] In a preferred embodiment, the hydrogel particles in process step b) are obtained from the hydrogel obtained in process step b) by mechanical comminution, in particular fragmentation, grinding, chopping, high pressure processes, in particular ultrasonication, and subsequent filtration, in particular filtration using a filter device with a pore size of up to 200 μm.

[0123] In a preferred embodiment, the hydrogel particles in process step b) are produced from the hydrogel obtained in process step b) by mechanical comminution, in particular fragmentation, grinding, chopping, a high-pressure process, in particular ultrasonication, followed by filtration, in particular filtration using a filter device with a pore size of at most 200 μm, or are produced by cryogelation of the obtained hydrogel particles and subsequent freeze-drying or by microfluidic co-flow gelation processes.

[0124] In one particular embodiment of the invention, the hydrogel particles produced in process step b) are produced such that they have a granular structure, in particular are present in the form of particles and in particular have a particle size with an average diameter of up to 200 μm in the swollen state, after mechanical comminution, in particular fragmentation, grinding, chopping, high pressure processes, in particular ultrasonication and subsequent filtration from the hydrogel obtained in process step b) or after cryogelation or microfluidic parallel flow gelation processes.

[0125] In a preferred embodiment, the hydrogel, the hyaluronic acid component and the at least one pharma- ceutically acceptable carrier are mixed in process step c) by stirring, in particular at a stirring speed of 500 rev / min.

[0126] In one particularly preferred embodiment, the hydrogel, the hyaluronic acid component and the pharma- ceutically acceptable carrier are mixed in method step c) for a period of from 1 min to 20 min, in particular from 8 min to 12 min, in particular for 10 min.

[0127] In a preferred embodiment, the hydrogel, in particular the hydrogel particles, are swollen, in particular in PBS, prior to mixing in method step c).

[0128] In a preferred embodiment, the hydrogel, in particular the hydrogel particles, are concentrated prior to mixing in method step c), in particular by centrifugation, in particular by centrifugation at 3000 G for 5 minutes, whereby preferably at least a portion of the supernatant is removed after centrifugation.

[0129] In one preferred embodiment, the present invention relates to a method for producing a therapeutic composition, comprising, in method step b), obtaining from the hydrogel obtained in method step b) one or more of the following: b1) a method step of fragmenting the hydrogel by ultrasonic treatment, in particular using an ultrasonic homogenizer, to obtain hydrogel fragments; and b2) A method step of filtering the hydrogel fragments to obtain hydrogel particles. The present invention relates to a method for forming hydrogel particles by

[0130] In one particularly preferred embodiment, the hydrogel is swollen, in particular completely, in PBS prior to fragmentation according to method step b1).

[0131] In a particularly preferred embodiment, in method step b1) the fragmentation of the hydrogel is carried out by grinding, chopping, high pressure treatment or ultrasonic treatment, in particular ultrasonic treatment.

[0132] In a preferred embodiment, the solids content of the hydrogel obtained in process step b1) is between 9% and 13% by weight (based on the total mass of the hydrogel).

[0133] In a particularly preferred embodiment, the fragmentation in method step b1) is carried out for a period of between 1 minute and 15 minutes.

[0134] In a preferred embodiment, the filtration according to method step b2) is carried out by means of a filter having a pore size in the range from 5 μm to 200 μm, from 10 μm to 200 μm, in particular from 10 μm to 80 μm, in particular from 50 μm to 200 μm, in particular from 110 μm to 200 μm, in particular 200 μm.

[0135] In a preferred embodiment, the present invention comprises in process step b) the following: b1') a process step of preparing an emulsion containing at least one polyionic polymer and at least one non-charged polymeric and / or non-polymeric crosslinker molecule; b2') a method step of cooling the emulsion containing at least one polyionic polymer and at least one non-charged polymer and / or non-polymeric crosslinker molecule to obtain cryogelled hydrogel particles; b3') a method step of freeze-drying the hydrogel particles; b4') Method steps for purifying freeze-dried hydrogel particles whereby a hydrogel, in particular a hydrogel particle, is formed; It now relates to a method for producing a therapeutic composition, in which during method steps b1') to b3') at least one polyionic polymer and at least one non-charged polymer and / or non-polymeric crosslinker molecule are crosslinked to obtain a hydrogel.

[0136] In a preferred embodiment, in process step b1') an emulsion is prepared which contains at least one uncharged polymer and at least one polyionic polymer in toluene.

[0137] In a particularly preferred embodiment, the process parameters in method steps b1') to b3') and the cryogelation parameters are adjusted in such a way that the resulting hydrogel particles have a granular morphology, in particular are present as particles with an average particle size (average diameter) of up to 200 μm in the swollen state.

[0138] In a preferred embodiment, the present invention further comprises in process step b) the following additional: b1″) a method step of mixing at least one polyionic polymer and at least one non-charged polymer and / or non-polymeric crosslinker molecule, b2″) a method step of forming hydrogel particles by means of a microfluidic device, in particular a microfluidic chip; b3'') Method steps for purifying hydrogel particles Hydrogel particles are formed by It now relates to a method for producing a therapeutic composition, in which during method step b2″) at least one polyionic polymer and at least one non-charged polymer and / or non-polymeric crosslinker molecule are crosslinked to obtain a hydrogel.

[0139] In a preferred embodiment, the microfluidic device has a crossover configuration, where the mixture of polyionic polymer and non-charged polymer or / and non-polymeric crosslinker molecules flows through a first crossover inlet and an opposite first crossover outlet, and the organic phase flows through a second crossover inlet and an opposite second crossover outlet, where the flow directions of the mixture of polyionic polymer and non-charged polymer or / and non-polymeric crosslinker molecules and the flow direction of the organic phase extend perpendicular to each other. In this embodiment, the microfluidic device preferably has a crossover inlet diameter of 5 μm to 50 μm.

[0140] In a particularly preferred embodiment, the process parameters and the microfluidic device are adjusted so that the resulting hydrogel particles have a granular morphology, in particular are present as particles, in particular particles having an average particle size (average diameter) of up to 200 μm in the swollen state.

[0141] In a particularly preferred embodiment, the mixture of charged polymer and polyionic polymer has a flow rate of 5 μl / min to 25 μl / min, in particular 10 μl / min to 20 μl / min. In a particularly preferred embodiment, the organic phase has a flow rate of 5 μl / min to 25 μl / min, in particular 10 μl / min to 20 μl / min. In a particularly preferred embodiment, the organic phase comprises 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane (also known as Novec™ 7500 from 3M) and / or perfluorinated polyether-polyethylene glycol-perfluorinated polyether-triblock copolymer (PFPE-PEG-PFPE), in particular the triblock copolymer from Krytox™ 157 FSH (DuPont)-Jeffamine™ ED 600 Amine (Huntsman)-Krytox™ 157 FSH (DuPont), obtained according to the synthesis instructions published in Lab Chip, 2008, 8, 1632-1639, in particular at 1.8% (weight / volume).

[0142] In a preferred embodiment, in method step b1'') the at least one polyionic polymer and the at least one non-charged polymer and / or non-polymeric crosslinker molecule are mixed at a temperature between 2°C and 10°C, in particular between 2°C and 8°C, in particular between 2°C and 6°C.

[0143] In a preferred embodiment, the hydrogel particles formed in process step b2'') are stored in a further process step b2a''), in particular for up to 14 hours, before being purified in process step b3'').

[0144] In a preferred embodiment, the hydrogel particles in method step b3″) are first separated by a mixture comprising 1H,1H,2H,2H-perfluoro-1-octanol and 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane, in particular a mixture comprising 1H,1H,2H,2H-perfluoro-1-octanol and 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane in a volume ratio of 1:1, followed by repeated washing with PBS.

[0145] The present invention also relates to therapeutic compositions producible, and in particular produced, by the method according to the invention, optionally containing at least one additive, in particular extracellular vesicles containing marine collagen, sorbitol, mannitol, platelet rich plasma (PRP), polyphenols, S-allyl cysteine, sodium pentosan-polyphosphate and / or curcuminoids.

[0146] In the context of the present invention, the term "hydrogel" is understood to mean a gel composed of water-insoluble polymeric components capable of binding water, the molecules of which are chemically linked by covalent bonds to form a network structure, the hydrophilic polymeric components present in the network allowing the hydrogel to swell in water and increase significantly in volume, without losing the cohesion and integrity of the material.

[0147] In the context of the present invention, the term "hydrogel particles" is to be understood as referring to the physical properties of a "hydrogel" material, in particular a hydrogel in particulate form.

[0148] In the context of the present invention, the term "network" is understood to mean a polymer network, in particular a three-dimensional network of polymer chains which are connected to one another via crosslinking points, preferably configured as a permanent network, in which the polymer chains are bonded to one another in the form of covalent bonds via chemical crosslinking points.

[0149] In the context of the present invention, the "concentration of sulfate or sulfonate groups of the hydrogel particle" is understood to mean the number of free sulfate or sulfonate groups, expressed in moles per volume, present in the volume formed by the entire hydrogel particle. This concentration is preferably expressed in mmol / l. In a preferred embodiment, the concentration of sulfate or sulfonate groups is calculated by multiplying the concentration of the polyionic polymer component in the swollen hydrogel particle by the number of repeating units and the number of sulfate or sulfonate groups per repeating unit.

[0150] In the context of the present invention, "selectively N-desulfated glycosaminglycan" or "selectively N-desulfated heparin" is understood to mean a glycosaminglycan having a lower sulfate group percentage compared to a non-desulfated glycosaminglycan, due to the complete or substantial removal of sulfate groups attached to nitrogen atoms of the glycosaminglycan, the lower sulfate group percentage being based on the elimination or reduction of the sulfate group percentage at the nitrogen atoms of the glycosaminglycan.

[0151] When the term "P1" is used in the context of the present application, this term is preferably interpreted as the concentration of sulfate or sulfonate groups, particularly preferably the charge characteristic resulting from the concentration of sulfate or sulfonate groups, particularly preferably the total charge density resulting from the concentration of sulfate or sulfonate groups, in particular in mmol / l.

[0152] The concentration of the polyionic polymer component in the swollen hydrogel particles is calculated by dividing the concentration of the polyionic polymer used during crosslinking by the degree of swelling.

[0153] In the context of the present invention, a "star PEG" or "star PEG" is understood to mean a polyethylene glycol molecule comprising a central core to which are covalently attached a plurality of, for example 4 or 8, in particular 4, chains of especially equal length.

[0154] In the context of the present invention, a "biological fluid" is understood to be a fluid present in or derived from a living biological system. A biological fluid may be, for example, a human or animal body fluid.

[0155] In the context of the present invention, "lipopolysaccharide" is understood to mean a compound that can be obtained from the outer membrane of gram-negative bacteria and that is composed of a fat-like (lipo) component and a sugar component (polysaccharide) that can act as an allergen, endotoxin and / or a particularly powerful inflammatory factor.

[0156] In the context of the present invention, a "non-polymeric cross-linker moiety" is understood to mean an enzymatically cleavable peptide or short molecule having at least two cross-linkable groups, where the short molecule is a molecule not suitable for polymerization itself or a monomer or an oligomer having 2 to 10 repeat units, in particular 3 to 4 repeat units.

[0157] In the context of the present invention, the term "hydrogel particles in a swollen state" or "hydrogel particles present in a swollen state" is preferably understood as hydrogel particles present in physiological saline (PBS) and absorbing the maximum amount of liquid that they can absorb in this solution, i.e. present in a swollen state. Thus, in the swollen state, the hydrogel particles have reached their maximum volume expansion, absorbing the maximum amount of liquid that they can absorb. This term is therefore understood as the state of the particles in which the volume of the particles is increased compared to the volume present immediately after production in a pharma- ceutical acceptable carrier, in particular PBS. The volume swelling is preferably calculated from the volume change of the volume of the hydrogel present immediately after the formation of the network compared to the volume of the swollen hydrogel obtained after its introduction into PBS and the swelling process therein. The swollen state is characterized by reaching an equilibrium degree of swelling of the hydrogel particles in a solution, i.e. preferably PBS, where PBS has the same ionic strength as the biological fluid in the cartilage, in particular the tissue fluid, in particular the synovial fluid, in particular the model synovial fluid. Thermodynamically, in this state, the swelling forces (due to osmotic / electrostatic forces and excluded volume of polymer chains) and the elastic restoring forces (due to covalently bonded network chains) are in equilibrium (see also Freudenberg et al., DOI: 10.1002 / adfm.201101868). As a result, the state regarding the swelling degree does not change any more for hydrogel particles in the same solution. The swelling state can be preferably established by incubating the hydrogel particles in a solution, in particular PBS, until the swelling degree does not change any more, preferably for 1 hour, in particular for 4 hours, in particular for 12 hours, in particular for 24 hours.

[0158] Swelling is preferably measured according to the invention by polymerizing 67 μl of unpolymerized hydrogel mixture, having the same chemical composition as the hydrogel particles to be prepared, between two 9 mm glass supports (Menzel-Glaeser, Germany) treated with Sigmacote (Sigma-Aldrich, Germany) for 16 hours at room temperature, and then removing the resulting hydrogel disk from the glass supports. The diameter of the hydrogel disk after polymerization was optically determined by a scanner of type FLA-3100 (Fujitsu, Japan) (diameter of the hydrogel immediately after crosslinking / hydrogel formation). The hydrogel disk was subsequently swollen for 24 hours in phosphate-buffered saline (PBS), i.e. 0.9% NaCl buffered to pH 7.4 (Sigma-Aldrich, Germany) (physiological conditions) and again determined by a scanner of type FLA-3100 (Fujitsu, Japan) (diameter in the swollen state). The swelling of the hydrogel (swelling degree) was calculated according to the following formula: swelling degree=(diameter of swollen hydrogel) 3 / (diameter of hydrogel immediately after crosslinking / hydrogel formation) 3 was calculated from the determined diameter.

[0159] Alternatively, the degree of swelling, i.e. the average diameter of the hydrogel particles in the swollen state compared to the size of the hydrogel particles immediately after the hydrogel formation, can be determined by fluorescence microscopy. For this, the hydrogel particles are labeled immediately after the hydrogel formation and then swollen with a fluorescent dye, preferably Atto-488-NH 2 Label with 5% CO (1% relative to the number of amino groups in the hydrogel). Record and measure particle size via image analysis software using a fluorescent microscope.

[0160] The swelling degree determined by the hydrogel discs (x times the size between the swollen state and the state of the hydrogel particles immediately after hydrogel formation) is consistent with the swelling degree of the hydrogel particles determined by fluorescence microscopy when the composition of the hydrogel discs and hydrogel particles is the same.

[0161] In the context of the present invention, the "average mesh width" is understood to be the average distance between two mesh junctions (see Polymer Physics, Michael Rubinstein and Ralph H. Colby, 2006, Oxford University Press, Oxford). This distance corresponds to a certain approximation to the sterically limited resistance of the network to the transport process of molecules through the network, since molecules with dimensions larger than the mesh width are excluded from penetrating the network for steric reasons. The mesh width takes into account an ideal network without any defect structure. The average mesh width of the hydrogel is calculated from the storage modulus of the hydrogel particles in the swollen state, determined experimentally, in particular by oscillatory rheometry, based on the rubber elasticity theory and the assumption of an ideal network without defects, according to the following formula:

[0162]

number

[0163] In the context of the present invention, "coefficient of friction" is understood as a measurement that indicates the ratio of the frictional force to the pressing force. The coefficient of friction can in particular be measured in a friction measurement cell, in particular in an Anton Paar MCR 301 rheometer, in particular in a rheometer oscillating at a speed of 10 revolutions per second, using a soda-lime glass sphere (diameter 12.7 mm) that is pressed against the silicone layer with a normal force of 10 N.

[0164] In this case, the liquid / composition to be tested (in particular 1 ml) is placed in the friction measuring cell and a lubricating film is formed between the glass sphere and the silicone layer, which reduces the sliding friction compared to measurements with pure water. In this method, the friction coefficient is determined by the instrument software at a normal force of 10 N and a rotation speed of 10 revolutions per second. Preferably, an Anton Paar T-PTD 200 type friction measuring cell is used with an Anton Paar MCR 301 rheometer.

[0165] In the context of the present invention, the term "cytokine" is understood to mean proteins which control the growth and / or differentiation of cells. Some cytokines are growth factors, others play an important role in immune reactions and inflammatory processes and are also called mediators. Preferably according to the present invention, cytokines are understood to mean in particular interferons, interleukins, colony stimulating factors, tumor necrosis factors and "chemokines", i.e. small signaling molecules.

[0166] In the context of the present invention, "reduction in the concentration of cytokines, in particular chemokines" is understood as a reduction in the concentration of free cytokines, in particular chemokines, in the synovial fluid, in particular in the model synovial fluid, which essentially occurs, without being bound by theory, by binding, in particular by sequestration, of cytokines, in particular chemokines, to the hydrogel particles of the composition according to the invention.

[0167] The reduction is preferably measured by adding a predetermined amount of a cytokine, particularly a chemokine, to a determined volume of synovial fluid, particularly a model synovial fluid, in which the substance to be tested, particularly the therapeutic composition of the invention, particularly the hydrogel particles, is present.

[0168] Preferably, for the measurement of this decrease, 50 μl of the therapeutic composition to be examined (the composition is at the bottom of the reaction vessel) is incubated for 24 hours at room temperature in a 0.5 ml reaction vessel, in particular a Protein LoBind® reaction vessel (Eppendorf Tubes, Germany). The protein or protein mixture solution is prepared by dissolving one or more proteins (cytokines, chemokines or other signaling molecules) in PBS containing 1% bovine albumin and 0.05% (w / v) Proclin™ 300 (Sigma-Aldrich) to achieve an activation concentration of 10 ng / ml of protein, respectively. As a reference, the same protein solution or protein mixture solution without the 50 μl of therapeutic composition is also incubated for 24 hours at room temperature in a 0.5 ml reaction vessel, in particular a Protein LoBind® reaction vessel. Subsequently, one solution sample each (supernatant without composition) is taken from the reaction vessels, in particular the Protein LoBind® reaction vessels, and the protein concentration is measured using ProcartaPlex™ (Thermo Fisher, Germany) in combination with the corresponding ProcartaPlex™ Simplex kit in a Bioplex 200 (Biorad, Germany) type instrument.

[0169] The reduction in cytokine, particularly chemokine, concentration is determined from the determined concentrations of solutions incubated with and without the therapeutic composition being tested according to the following formula: Reduction (%) = (1 - concentration in solution with composition / concentration in solution without composition) x 100.

[0170] In the context of the present invention, "model synovial fluid" is understood as a fluid containing at least one cytokine, in particular a chemokine, dissolved in a concentration of 10 ng / ml in a solution of 1 mg / ml bovine albumin in PBS.

[0171] In the context of the present invention, "injection force" is understood as the force required to extrude the composition at a rate of 0.05 ml / sec through a 25 gauge needle, i.e. a needle having an outer diameter of 0.5 mm according to EN ISO 9626. In a preferred embodiment, the injection force is determined according to the procedure and measurement instructions according to Example C).

[0172] The injection force is preferably determined by loading 0.5 ml of the composition into a 1 ml syringe and measuring the force required to expel it at 10 ml / sec through a 25 G needle in a ZwickRoell universal testing machine equipped with a 50 N measuring cell.

[0173] In the context of the present invention, the "storage modulus" of a hydrogel particle is understood as the elastic part of the complex shear modulus. The elastic part is proportional to the part of the deformation energy that is stored in the material and can be obtained from the material after the release of the load. This energy is preferably determined by frequency-dependent measurement of the shear modulus using oscillatory rheometry in a plate / plate configuration on hydrogel disks that are chemically / physically identical in these respects. The storage modulus mentioned according to the present invention is the storage modulus of hydrogel particles swollen in physiological saline (PBS). In a preferred embodiment, the storage modulus is determined according to the procedure and measurement instructions according to Example B).

[0174] In the context of the present invention, the term "crosslinking" is understood as the formation of covalent bonds between a polyionic polymer component and at least one non-charged polymer component or at least one non-polymeric crosslinker component, for which these components are preferably mixed with one another.

[0175] In the context of the present invention, the term "arthritis" is understood as cartilage or joint disease, in particular cartilage joint disease or articular cartilage disease. In particular, arthritis is an inflammatory disease of joints and / or cartilage. The inflammation can be due to infection, such as bacterial, viral or fungal infection, immune reaction, in particular autoimmune reaction, metabolic disorder, such as gout, or mechanical causes, in particular wear and tear on articular structures, in particular cartilage tissue, or traumatic effects, such as injuries or accidents on articular structures, in particular cartilage tissue. The inflammation can proceed acutely or chronically.

[0176] Arthritis due to mechanical influences, ie for example due to accidents and / or chronic degenerative processes, is also referred to herein as osteoarthritis (arthropathy or activation arthropathy).

[0177] In the context of the present invention, the term "arthritis" is understood in particular as osteoarthritis (arthropathy or activation arthropathy), rheumatoid arthritis, infectious arthritis (septic arthritis), post-infectious arthritis, psoriatic arthritis and gouty arthritis.

[0178] In the context of the present invention, the term "arthritis" is understood in particular as osteoarthritis (OA) and rheumatoid arthritis (RA). Osteoarthritis (also called arthropathy or active arthropathy) is in particular a degenerative, especially chronic degenerative, joint change with cartilage degeneration caused by destructive mechanical effects on cartilage, leading to inflammation and often accompanied by pain, swelling and functional limitations. Rheumatoid arthritis (chronic polyarthritis, primary chronic polyarthritis, RA), a joint disease with widespread inflammation, is a chronic autoimmune disease with the same or similar clinical signs. It also includes Bechterew's disease (ankylosing spondylitis).

[0179] Arthritis can occur in any area of ​​the human or animal body where joints and cartilage are present, particularly in the toes, fingers, knees, hips, spine, especially the cervical spine or spine, shoulders, elbows and wrists.

[0180] Preferably according to the invention, arthritis is accompanied by symptoms such as pain, swelling, limited function and movement of the affected joints, damage to cartilage structure, increased levels of inflammatory cytokines and inflammatory markers and / or pathological immune responses.

[0181] In one embodiment, arthritis of the spine may occur with symptoms of lower back pain.

[0182] In particularly preferred embodiments, therapy of arthritis according to the invention results in at least some reduction or elimination of the clinical signs of arthritis, in particular reduction or inhibition of swelling in the joint area, relief or inhibition of pain, reduction or inhibition of movement and function limitations, reduction or inhibition of inflammatory processes, in particular reduction of concentrations of cytokines having inflammatory effects and / or reduction or inhibition of pathological immune responses.

[0183] In a preferred embodiment, the beneficial effects of the present invention are seen in the inhibition of knee joint swelling in a mouse model following injection of 100 ng lipopolysaccharide (LPS), as measured using a caliper as knee swelling 72 hours after LPS injection.

[0184] In the context of the present invention, the term "at least 1" is to be understood as a notation of quantity representing a number such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10. In a particularly preferred embodiment, the notation "at least 1" may represent exactly the number 1. In a further preferred embodiment, the term "at least 1" may mean 2 or 3 or 4 or 5 or 6 or 7.

[0185] When quantitative expressions, in particular percentage expressions, of ingredients of a product or composition are given in the context of the present invention, unless expressly indicated otherwise or obvious to the skilled artisan, these together with any other explicitly stated or further ingredients of the composition or product, either added up to 100% of the composition and / or product.

[0186] If in the context of the present invention the "presence", "containment", "possession" or "content" of a component is explicitly mentioned or implied, this means that the respective component is present, in particular in a measurable amount.

[0187] If in the context of the present invention the "presence", "content" or "possession" of a component in an amount of 0 [units], in particular mg / kg, μg / kg or % by weight, is explicitly mentioned or implied, this means that the respective component is not present in a measurable amount, in particular is absent.

[0188] The number of decimal places specified corresponds to the precision of the respective measurement method used.

[0189] In the context of the present invention, the term "and / or" is to be interpreted as disclosing all members of the group connected by the term "and / or" both alternatively and additionally to one another in any combination, respectively. This means that the expression "A, B and / or C" should be interpreted as disclosing: a) (A or B or C) or b) (A and B) or c) (A and C) or d) (B and C) or e) (A and B and C).

[0190] In the context of the present invention, the terms "comprise" and "have" are to be understood as meaning that in addition to the elements expressly covered by these terms, further elements not expressly listed can also be added. In the context of the present invention, these terms are also to be understood as meaning that only the elements expressly listed are covered, and that no further elements are present. In this particular embodiment, the meaning of the terms "comprise" and "have" is synonymous with the term "consist of". Furthermore, the terms "comprise" and "have" also cover compositions that contain, in addition to the elements expressly listed, further elements that are not listed but have functionally and qualitatively subordinate properties. In this embodiment, the terms "comprise" and "have" are synonymous with the term "essentially consisting of". The term "consist of" means that only the elements expressly listed are present, and the presence of further elements is excluded.

[0191] Further embodiments of the invention are the subject matter of the dependent claims and further independent claims.

[0192] The invention will now be explained in more detail on the basis of the following examples and the associated drawings.

[0193] The drawings show: FIG. 1 is a schematic diagram showing the structure of a therapeutic composition according to the present invention. FIG. 2 is a photographic representation of various hydrogel particles that may be used in accordance with the present invention. FIG. 3A is a graph showing reduction in LPS-induced knee joint swelling in a mouse model. FIG. 3B is a graphical representation of gene expression of the inflammatory marker TNFα.

[0194] The sequence listing shows: SEQ ID NO: 1: matrix metalloproteinase (MMP) response sequence PQGIWGQ; SEQ ID NO: 2: matrix metalloproteinase (MMP) response sequence IPVSLRSG; SEQ ID NO: 3: matrix metalloproteinase (MMP) response sequence VPMSMRGG; SEQ ID NO: 4 elastase response sequence AAPV, SEQ ID NO: 5 elastase response sequence APEEIMDRQ, SEQ ID NO: 6 Thrombin response sequence GGF-pipecolic acid-RYSWGCG, SEQ ID NO: 7 Thrombin response sequence GG-cyclohexylalanine-ARSWGCG, SEQ ID NO:8 FXa response sequence GGIEGRMGGWCG, SEQ ID NO: 9 Kallikrein response sequence CGGGPFRIGGWCG, SEQ ID NO: 10 Aureolysin response sequence ADVFEA, SEQ ID NO: 11 Aureolysin response sequence AAEAA, SEQ ID NO: 12 Protease IV response sequence MKATKLVLGAVILGSTLLAG, SEQ ID NO: 13: matrix metalloproteinase (MMP) response sequence GPQGIAGQ; SEQ ID NO: 14: matrix metalloproteinase (MMP) response sequence GPQGIWGQ; SEQ ID NO: 15: Matrix metalloproteinase (MMP) response sequence GCGGPQGIWGQGGCG. EXAMPLES

[0195] The therapeutic composition according to the invention suitable for intra-articular injection comprises, in addition to the hyaluronic acid component, hydrogel particles and at least one pharma- ceutically acceptable carrier, and its preparation and use are described below. The basic structure of this therapeutic composition is shown in Figure 1. Figure 1 shows two therapeutically active elements, the hyaluronic acid component and the star-shaped PEG / glycosaminoglycan (heparin) network that forms the hydrogel particles.

[0196] A) Hyaluronic acid ingredient Hyaluronic acid (sodium salt) with a molecular weight of >1.9 million Da from Contipro was used. Its purity corresponds to pharmaceutical quality and is certified according to ISO 13485 (pharmaceuticals). The concentrations in the composition are listed in section C).

[0197] A particularly advantageous range of the molar mass of hyaluronic acid is the range from 500 kDa to 100 million Da.

[0198] B) Preparation of Hydrogel Particles B1: Description of hydrogel properties The hydrogel according to the invention consists of a charged component, i.e. a polyionic polymer component, such as a glycosaminoglycan, in particular heparin (component A1) or selectively N-desulfated heparin (component A2), and an uncharged component, i.e. a non-polymeric crosslinker component and / or an uncharged polymer component, e.g. an uncharged polymer in the form of a multi-arm polyethylene glycol (PEG) in the amine-terminated (component B) and / or carboxy-terminated (component C) form (see Table 2). In this case, the charged and uncharged components are covalently crosslinked into a polymer network, preferably obtained by activating the carboxyl groups of the charged components with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysulfosuccinimide (S-NHS) and crosslinking them directly with the uncharged component containing amino groups or with at least two amino groups via crosslinker molecules (linkers) to form amide bonds, respectively. The synthesis of selectively N-desulfated heparin is carried out according to the instructions from Atallah et al. (Doi: 10.1016 / j.biomaterials.2018.07.056). To modify the charge properties (particularly parameter P1, total charge density), a second uncharged component bearing a carboxyl group (e.g. component C, 8-ArmPEG, carboxyl-terminated) can additionally be used as the third component of the hydrogel network, which is activated at the carboxyl group via EDC / S-NHS in the same way as the charged components. In this case, the network is formed via crosslinks between component A and component C on the one hand and between component A and component B (amine-terminated) on the other hand.

[0199] The structure of the hydrogel preferred according to the invention is defined by the molar concentrations of components A to C in the hydrogel mixture upon crosslinking (see Table 1). These concentrations represent the concentrations of the network-forming components in the reaction state immediately after mixing. From these concentrations the molar ratios of the hydrogel components upon crosslinking can be calculated (see Table 1). The network formation is carried out over a period of 12 hours, whereby the hydrogel formed after this period corresponds to the hydrogel state after crosslinking, in particular after method step b).

[0200] To produce hydrogels of type 1 to type 9, components A to C are dissolved for this purpose by treatment in ultrasound for 5 minutes (in this case the starting concentrations are selected so that after mixing 1 to 3 parts by volume (for components A to C) in the final reaction mixture the concentrations of components A to C corresponding to those listed in table 1 result). The activating reagent EDC / sNHS is dissolved stoichiometrically in ultrapure water in a ratio of amino groups present in the reaction mixture (4 times molar concentration of component B based on the molecular structure with 4-arm PEG) such that in the final reaction mixture there are 2 mol of EDC per mol of amino groups and 1 mol of sNHS for 1 mol of amino groups. The activating reagent is accordingly combined with components A and C bearing carboxyl groups and activated for 10 minutes at room temperature. Subsequently, component B is added to the mixture of A+C+activating reagent to initiate the covalent network formation of the material.

[0201] In the following calculations, it is assumed that all network components are quantitatively incorporated into the hydrogel and remain within the hydrogel during the subsequent equilibrium swelling step in PBS. Volumetric swelling is calculated from the volume change of the specimen after network formation and subsequent swelling in PBS.

[0202] Swelling was measured by polymerizing 67 μl of unpolymerized hydrogel mixture, with the same chemical composition as the hydrogel particles to be produced, between two 9 mm glass supports (Menzel-Glaeser, Germany) treated with Sigmacote (Sigma-Aldrich, Germany) for 16 h at room temperature, and then removing the resulting hydrogel disk from the glass support. The diameter of the hydrogel disk after polymerization was determined optically using a scanner of type FLA-3100 (Fujitsu, Japan) (diameter of the hydrogel immediately after crosslinking / hydrogel formation). Subsequently, the hydrogel disk was allowed to swell for 24 h (physiological conditions) in phosphate-buffered saline (PBS), i.e., 0.9% NaCl buffered to pH 7.4 (Sigma-Aldrich, Germany) and was again determined (diameter in the swollen state) using a scanner of type FLA-3100 (Fujitsu, Japan). The swelling of the hydrogel was determined according to the following formula: swelling = (diameter of swollen hydrogel) 3 / (diameter of hydrogel immediately after crosslinking / hydrogel formation) 3 The diameter was determined from the

[0203] Only hydrogels swollen in PBS are used for further characterization (rheometry to determine storage modulus, sequestration of IL-8 and further inflammatory proteins) and for preparation of mixtures. Thus, the charge properties of the hydrogel (sulfate concentration / sulfonate concentration P1) can be calculated from the reaction mixture using the concentration of the charged components in the reaction mixture and the volumetric swelling (see Table 1, column H). Calculations were performed from the molar concentrations of the hydrogel components at the time of hydrogel formation (see Table 1, column A) assuming complete incorporation of the polymeric hydrogel components while taking into account the volumetric swelling.

[0204] For this, the concentration of the polyionic polymer component in the swollen hydrogel (Table 1, column G) was calculated by first dividing the concentration of the polyionic polymer used to form the hydrogel (Table 1, column A) by the swelling degree (Table 1, column E). The concentration of sulfate or sulfonate groups in the swollen hydrogel (parameter P1, column H, Table 1) was calculated by multiplying the concentration of the polyionic polymer component (charged components) in the swollen hydrogel (Table 1, column G) by the number of sulfate / sulfonate groups per polymer molecule (Table 2).

[0205] The hydrogel materials are characterized by the mixing ratios corresponding to Table 1 (where the formation of an elastic hydrogel serves as an important criterion, i.e. an elastic hydrogel is obtained that does not dissolve after swelling in PBS and is preferably characterized by a range of 0.1 kPa to 22 kPa for the storage modulus (determined by rheometry) that directly correlates with the elasticity / stiffness of the network). The storage modulus of the hydrogel particles was determined by oscillatory rheometry (in kilopascals) using a shear rheometer of the Ares type from TA Instruments (UK). For this, fully swollen 8 mm diameter punched hydrogel discs (swollen for 24 hours in PBS) were measured at room temperature at low deformations (2%) from 1 rad / s to 100 rad / s at increasing frequencies in a 9 mm plate-plate measuring device and averaged over the entire frequency range (one measurement on one sample). The reported values ​​are the average of four independently produced hydrogel discs and are given ± standard deviation (SD).

[0206] Besides the storage modulus, the second important parameter is the charge density in the hydrogel, which is represented by the concentration of anionically charged sulfate / sulfonate groups in the swollen hydrogel (see parameter P1 in Table 1, column H, calculation see above).

[0207] In this case, a variation in the P1 parameter of 0.1 mmol / l to 800 mmol / l is to be expected, with a particularly advantageous range being 20 mmol / l to 500 mmol / l, in particular 50 mmol / l to 200 mmol / l.

[0208] [Table 1]

[0209] [Table 2]

[0210] B2) Preparation of hydrogel particles The hydrogel network according to the formulation instructions according to B1) can be preferably processed by three different methods to give hydrogel particles in the preferred size range, where the target parameters are the average particle size (average diameter) and the volume fraction of particles with a diameter of up to 200 μm present in the final particle suspension, where concentration of the particle suspension by centrifugation is advantageous.

[0211] The production of particles can be achieved by suitable mechanical comminution of the hydrogel from the bulk gel material produced according to the method according to B1), for example by grinding, chopping, high pressure treatment or ultrasonic treatment according to the method B2.1). For this purpose, ultrasonic disruption is preferably used, in which the hydrogel completely swollen in PBS after the method B1) is treated as a gel body in 5 volumes of PBS in a Bandelin Sonoplus ultrasonic homogenizer (Germany) at full power for 10 minutes. The particle mixture is then filtered through a filter with an average pore size of 200 μm to separate larger fragments still present. Other pore sizes of the filter are also possible. Figure 2A shows such hydrogel particles obtained from the hydrogel by mechanical comminution.

[0212] A particle size (average diameter) of up to 200 μm is important to ensure injectability of the composition (see (C)), with a particularly advantageous range being achieved by the range of 80 μm to 10 μm.

[0213] Alternatively, the production of particles can be carried out by method B2.2 using suitable microfluidic methods, in particular the parallel flow method, as follows: for this, solutions of components A+C+EDC / sNHS and component B (concentrations and mixtures according to the formulation instructions in B1) are premixed at 4° C. and then passed through a microfluidic chip with a cross-over configuration (aqueous phase inlet for the cooled gel mixture and an inlet for the organic phase) that determines the size criteria (diameter) at a flow rate of 10 μl / min to 20 μl / min (aqueous gel phase) in Novec® 7500 Engineered Fluid (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane) from 3M® and 1% PFPE-PEG-PFPE surfactant (Krytox® 157 obtained according to the synthesis instructions published in Lab Chip, 2008, 8, 1632-1639). FSH (DuPont)-Jeffamine™ ED 600 Amine (Huntsman)-Krytox™ 157 FSH (DuPont)) is dispersed into the organic phase to form droplets. The geometry of the microfluidic chip and the ratio of the flow rates of the two phases determine the final particle size. The droplets formed react in the channel and then undergo residence time in a collection vessel over a period of 1-12 hours, followed by phase separation by addition of 1H,1H,2H,2H-perfluoro-1-octanol and Novec™ 7500 in a 1:1 volume fraction, followed by purification and complete swelling by repeated washing in PBS.

[0214] This method results in the formation of spherical particles with a narrow size distribution with a deviation of less than 10% in the mean particle diameter. No filtration or further post-processing is required. Figure 2C shows such hydrogel particles obtained using a microfluidic device.

[0215] A particle size (average diameter) of up to 200 μm is important to ensure injectability of the composition (see (C)), with a particularly advantageous range being between 80 μm and 10 μm.

[0216] FIG. 2B shows the hydrogel particles obtained by cryogelation and subsequent freeze-drying (method B2.3).

[0217] In all variants, but particularly in both methods B2.1 and B2.2, the particle suspension can be centrifuged at 3000 G for 5 minutes to concentrate the volume fraction of hydrogel particles in the suspension and the supernatant removed. The concentrated particle suspension is then collected by a suitable pipette (e.g. a positive displacement pipette) and used to prepare the mixture according to section C).

[0218] C) Preparation of Therapeutic Compositions from Hyaluronic Acid and Sequestering Hydrogels The therapeutic composition for use in treating arthritis, particularly osteoarthritis, is comprised of a mixture of hydrogel particles, a pharma- ceutically acceptable carrier and hyaluronic acid. Figure 1 shows a schematic diagram of an injectable therapeutic composition of the present invention made of a hyaluronic acid carrier matrix and star-shaped PEG-glycosaminglycan (GAG) hydrogel particles.

[0219] Compositions (called F1 to F7, see Table 3) are prepared by mixing three components: 1) hyaluronic acid in various volume fractions, 2) hydrogel particle suspension, and 3) PBS (see Table 3). In this case, for the hyaluronic acid component, a solution containing 5 (g / g)% hyaluronic acid in PBS is used (prepared by dissolving powdered hyaluronic acid in PBS and homogenizing by stirring). Component 2 (hydrogel particle suspension) is determined according to the hydrogel formation instructions according to B1 (types 1 to 8, see Table 1) and the manufacturing process according to B2 (preferably B2.1). Component 3) PBS may optionally contain sorbitol or other antioxidants. Mixing of components 1) to 3) is performed by suitable homogenization (e.g., by stirring for 10 minutes at a stirring speed of 500 rpm) according to the volume fractions determined in Table 3.

[0220] Particularly advantageous mechanical properties of this composition are its easy injectability (characterized by low injection forces) and at the same time its advantageous high lubrication action (characterized by a low coefficient of friction).

[0221] To determine injectability, the injection force was measured as follows: 0.5 ml of the therapeutic composition was loaded into a 1 ml syringe and the force required to extrude it through a 25 G needle at 10 ml / s was measured in a ZwickRoell universal testing machine equipped with a measuring cell of 50 N. The lubrication effect was measured in an Anton Paar MCR 301 rheometer using a T-PTD 200 measuring cell on the silicone layer with a soda lime glass sphere (diameter 12.7 mm) applying a normal force of 10 N at a speed of 10 revolutions per second in a friction measurement test setup.

[0222] As an example, an injection force of 3.1±0.4 N was measured for compositions F2, F4 and F6. This is somewhat lower than the injection force of 3.7±0.8 N for 1% hyaluronic acid alone (carrier matrix) and not too far from the injection force of pure water with 2.4±0.4 N. Similarly, a friction coefficient in the range of 0.091±0.004 was determined for compositions F2, F4 and F6, which is comparable to the friction coefficient for 1% hyaluronic acid alone with 0.154±0.002 and significantly lower than the friction coefficient of water with 0.644±0.157.

[0223] An injection force of less than 10 N was achieved (see above for test performance) and a lubricating effect was achieved which could be explained by a friction coefficient of less than 0.2.

[0224] Furthermore, the composition has as an important property the ability to sequester at least one of the proinflammatory chemokines IL-8, IP-10, MCP-1, MIP-1α, MIP-1β and / or RANTES, and is characterized in that it binds a certain percentage of at least one (or all) of the factors from the group of proinflammatory chemokines from a solution relevant to the application.

[0225] To characterize the binding of these chemokines, artificial synovial fluids are prepared by mixing recombinant chemokines in a 0.1% albumin solution (BSA) in PBS at a range of concentrations of approximately 10 ng / ml of each chemokine. The compositions (0.5 ml each) are then contacted with 0.5 ml of artificial synovial fluid at room temperature for 24 hours, after which the supernatants are removed and the reduction in chemokines in the supernatants compared to untreated control solutions is determined using a multiplex immunoassay (Luminex). The results are shown in Table 3, which shows a graded reduction depending on gel type (Type 2, Type 5, or Type 8) and formulation (F1-F7).

[0226] After accurate determination of parameter P1 for all gel types, further correlation of sequestration with hydrogel composition and mixture composition was performed. Formulation F3 has particularly advantageous strong sequestration of the chemokines IL-8, IP-10, MCP-1, MIP-1α, MIP-1β and RANTES by more than 64%, with a simultaneously advantageous low injection force of 0.03 N and an advantageous low coefficient of friction of 0.053 (Tables 3 and 4).

[0227] A potent sequestration of pro-inflammatory chemokines from artificial synovial fluid by the compositions according to the invention of well over 50% was achieved. Table 5 shows in summary form the basic properties of the therapeutic compositions according to the invention.

[0228] To characterize the anti-inflammatory and alleviating effects of the compositions on intra-articular inflammation, 100 ng of lipopolysaccharide dissolved in 10 μl of physiological saline (PBS) was injected into the knee joint (i.e., intra-articularly) of C57BL / 6J mice to induce joint inflammation. 24 hours later, 4 μl of Composition F8, Composition F9, Composition F10, Composition F11, Composition F12 or Composition F13 (PBS only, control) was injected intra-articularly into the knee joint pre-treated with LPS injection. Knee joints treated with physiological saline (PBS) only (without LPS challenge) served as an additional control group. The diameter of the knee joint was recorded using a caliper as a measure of knee swelling before LPS injection and 72 hours after the first LPS injection, which closely reflects the clinical signs during human joint inflammation and swelling. The change in joint swelling is expressed as a percentage relative to the initial value before LPS-induced inflammation and normalized to an additional control group with pure PBS injection (Figure 3A). At the same time, gene expression of the inflammatory marker TNFα (tumor necrosis factor α) was also investigated after 72 hours using rt-qPCR (quantitative PCR), which indicates the presence and activation of immune cells (Figure 3B).

[0229] Formulation F9 shows a particularly advantageous strong anti-inflammatory and symptom-relieving effect with a knee swelling value of 98.3% and a gene expression of the inflammatory marker TNFα of 55.0% compared to the values ​​without induction of inflammation (see Table 3 and Figure 3A). In this effect, the hyaluronic acid component of the composition plays a positive role, causing a surprising additive effect on the efficacy, since the same composition without hyaluronic acid (F10) has a lower anti-inflammatory and lower knee swelling relieving effect (103.6% knee swelling, 75.6% gene expression of TNF) (see Table 3 and Figure 3B).

[0230] [Table 3]

[0231] [Table 4]

[0232] [Table 5]

Claims

1. A therapeutic composition comprising hydrogel particles, at least one hyaluronic acid component, and at least one pharmaceutically acceptable carrier, particularly an aqueous liquid, wherein the hydrogel particles comprise at least one polyionic polymer component, which is covalently bonded to at least one uncharged polymer component and / or at least one non-polymeric crosslinker component to form a network, and wherein the at least one polyionic polymer component has sulfate or sulfonate groups.

2. 2. The therapeutic composition of claim 1, wherein the at least one uncharged polymer component is selected from the group consisting of a polyethylene glycol (PEG) component, a poly(2-oxazoline) (POX) component, a polyvinylpyrrolidone (PVP) component, a polyvinyl alcohol (PVA) component, a polyacrylamide (PAM) component, and combinations thereof.

3. at least one uncharged polymer moiety is a polyethylene glycol moiety, in particular a linear or multi-arm, in particular a 4-arm or 8-arm polyethylene glycol moiety; and / or at least one polyionic polymer component is a glycosaminoglycan component, in particular heparin or selectively desulfated heparin, in particular selectively N-desulfated heparin, or / and the at least one non-polymeric crosslinker component is selected from the group consisting of ethylenediamine, propylenediamine (1,3-diaminopropane), butane-1,4-diamine, pentane-1,5-diamine (cadaverine), hexamethylene-1,6-diamine, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, N-(2-aminoethyl)maleimide, and combinations thereof; The therapeutic composition of claim 1.

4. the hydrogel particles have a storage modulus of at most 22 kPa, in particular 0.1 kPa to 22 kPa (each measured on hydrogel particles swollen in physiological saline (PBS)); or / and the hydrogel particles have an average diameter of at most 200 μm, in particular at most 80 μm (measured on hydrogel particles swollen in physiological saline (PBS)), or / and the hydrogel particles have a concentration of sulfate or sulfonate groups of at least 0.1 mmol / l, in particular at least 10 mmol / l, in particular at least 100 mmol / l, in particular from 0.1 mmol / l to 800 mmol / l, in particular from 10 mmol / l to 800 mmol / l, in particular from 20 mmol / l to 500 mmol / l, in particular from 20 mmol / l to 200 mmol / l, in particular from 50 mmol / l to 200 mmol / l (each measured in the volume of the hydrogel particles swollen in physiological saline (PBS)), The therapeutic composition of claim 1.

5. 2. The therapeutic composition according to claim 1, wherein the content of the hyaluronic acid component is 0.5% to 2.0% by weight, in particular 1.0% to 2.0% by weight (each relative to the total mass of the composition).

6. 10. The therapeutic composition of claim 1, which is an injectable therapeutic composition, in particular an injectable therapeutic composition having an injection force of up to 10 N, in particular 2.7 N to 3.5 N, at an injection rate of 0.05 ml / sec through a 25 gauge needle.

7. a coefficient of friction of at most 0.2, in particular 0.085 to 0.095, at 10 revolutions per second, respectively, and / or capable of reducing the concentration of at least one free cytokine, in particular chemokine, in particular IL-8, in a biological fluid, in particular in synovial fluid, in particular in a model synovial fluid, in particular by more than 50%, in particular by 70% to 80%; The therapeutic composition of claim 1.

8. 2. The therapeutic composition of claim 1, wherein the uncharged polymer component and the polyionic polymer component are directly covalently linked to each other by at least one crosslinker component, in particular a peptide, or by a bioorthogonal thioether bond, in particular obtained by an amide bond, a thiol-amine bond, a disulfide bond, or a thiol-maleimide reaction, a thiol-vinylsulfone reaction, or a thiol-acrylate reaction.

9. and / or having a solids content of 4% to 25% by weight, in particular 5% to 20% by weight (each relative to the total mass of the composition); Further comprising extracellular vesicles containing at least one additive, in particular marine collagen, sorbitol, mannitol, platelet-rich plasma (PRP), polyphenols, S-allyl cysteine, pentosan-polyphosphate Na and / or curcuminoids, The therapeutic composition of claim 1.

10. 10. The therapeutic composition according to claim 1 for use in a method for treating arthritis, in particular osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, psoriatic arthritis or gouty arthritis, in particular for intra-articular injection into a joint of a human or animal patient.

11. A method for producing a therapeutic composition, in particular a therapeutic composition according to any one of claims 1 to 10, comprising the steps of: a) providing at least one uncharged polymer, in particular a functionalized uncharged polymer or / and at least one functionalized non-polymeric crosslinker molecule and at least one polyionic polymer having sulfate or sulfonate groups, at least one hyaluronic acid component and at least one pharmaceutically acceptable carrier, and optionally further components; b) a method step of crosslinking uncharged polymer or non-polymeric crosslinker molecules with a polyionic polymer to form a network, resulting in a hydrogel, in particular a hydrogel particle, composed of at least one polyionic polymer component covalently bonded to at least one uncharged polymer component or / and at least one non-polymeric crosslinker component, c) a method step of mixing the hydrogel, in particular the hydrogel particles, with at least one hyaluronic acid component and at least one pharmaceutically acceptable carrier, and d) Method steps for obtaining a therapeutic composition A method for producing a therapeutic composition comprising:

12. 12. A method for producing a therapeutic composition according to claim 11, wherein the uncharged polymer, in particular the functionalized uncharged polymer and / or at least one functionalized non-polymeric crosslinker molecule, has at least two functional groups suitable for forming one covalent bond each to the polyionic polymer component, in particular selected from the group consisting of amino groups, thiol groups, maleimide groups, vinylsulfone groups, acrylate groups, carboxyl groups and combinations thereof.

13. 12. The method for producing a therapeutic composition according to claim 11, wherein the polyionic polymer having at least one sulfate or sulfonate group has at least two functional groups selected from the group consisting of an amino group, a thiol group, a maleimide group, a vinyl sulfone group, an acrylate group, a carboxyl group, and combinations thereof.

14. In method step b) the hydrogel particles are further b1) a method step of fragmenting the hydrogel by ultrasonic treatment, in particular using an ultrasonic homogenizer, to obtain hydrogel fragments; and b2) filtering the hydrogel fragments to obtain hydrogel particles. formed by, or b1') a process step of preparing an emulsion containing at least one polyionic polymer and at least one uncharged polymer or / and at least one non-polymeric crosslinker molecule, b2') a method step of cooling an emulsion containing at least one polyionic polymer and at least one uncharged polymer or / and at least one non-polymeric crosslinker molecule to obtain cryogelled hydrogel particles, b3') a method step of freeze-drying the hydrogel particles; and b4') Method steps for purifying freeze-dried hydrogel particles is formed by wherein during process steps b1′) to b3′) at least one polyionic polymer and at least one uncharged polymer and / or at least one non-polymeric crosslinker molecule are crosslinked to obtain a hydrogel, or b1″) a method step of mixing at least one polyionic polymer with at least one uncharged polymer and / or at least one non-polymeric crosslinker molecule, b2″) method steps of forming an emulsion using a microfluidic device and subsequent crosslinking to hydrogel particles; and b3'') Method steps for purifying hydrogel particles is formed by wherein during process step b2″) at least one polyionic polymer and at least one uncharged polymer or / and at least one non-polymeric crosslinker molecule are crosslinked to obtain a hydrogel, A method for producing the therapeutic composition of claim 11.

15. A therapeutic composition producible, in particular produced, by the method of claim 11.

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