INJECTABLE GEL PRODUCT

Abstract

A method of producing an injectable gel product is provided, comprising (a) cross-linking a first glycosaminoglycan (GAG) with a first crosslinking agent to produce a gel, wherein the charging ratio of crosslinking agent to disaccharide unit is below 0.15; (b) preparing particles of the gel; (c) mixing the glycosaminoglycan (GAG) gel particles with a second GAG to provide a mixture; (d) cross-linking the mixture with a second crosslinking agent to obtain cross-linking between the GAGs of the second, outer phase, thereby providing a gel having a first, inner phase of the cross-linked GAG gel particles, embedded in a gel of the second GAG outer phase; and (e) preparing injectable particles, each such particle containing a plurality of the cross-linked GAG gel particles of the first, inner phase. An injectable gel product, an aqueous composition, and a pre-filled syringe as also provided.

Claims

1.-31. (canceled)

32. A method of producing an injectable gel product, comprising: (a) cross-linking a first glycosaminoglycan (GAG) with a first crosslinking agent at a first charging ratio of crosslinking agent to disaccharide unit below 0.15 to produce a gel; (b) preparing particles of the gel; (c) mixing the GAG gel particles with a second GAG to provide a mixture; (d) cross-linking the second GAG with a second crosslinking agent to obtain a gel having a first, inner phase of the cross-linked GAG gel particles embedded in a second, outer phase comprising the second GAG, and (e) preparing injectable particles of the gel from (d), each such particle containing a plurality of the cross-linked GAG gel particles of the first, inner phase; wherein the inner phase, the outer phase, or both the inner phase and the outer phase are crosslinked via one or more polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

33. The method according to claim 32, wherein the one or more polyfunctional cross-linking agent is BDDE.

34. The method according to claim 32, wherein both the first GAG and the second GAG are covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

35. The method according to claim 32, wherein the first GAG is covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

36. The method according to claim 35, wherein the second GAG is covalently crosslinked via crosslinks comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

37. The method according to claim 36, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

38. The method according to claim 32, wherein the second GAG is covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

39. The method according to claim 38, wherein the first GAG and the second GAG are covalently crosslinked via crosslinks comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

40. The method according to claim 39, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

41. The method according to claim 32, wherein the second GAG is cross-linked with the second crosslinking agent at a second charging ratio of crosslinking agent to disaccharide unit that is less than the first charging ratio.

42. The method according to claim 41, wherein the second charging ratio is less than 0.05.

43. The method according to claim 32, wherein the cross-linking (d) further comprises cross-linking between the gels of the first, inner phase and the second, outer phase.

44. The method according to claim 32, wherein the GAG gel particles and the second GAG are both in a dry state when mixed.

45. The method according to claim 32, wherein the preparation of particles comprise precipitating and drying.

46. The method according to claim 32, wherein the mixture (c) comprises at least 50% by dry weight of the GAG gel particles.

47. The method according to claim 32, wherein the crosslinking (a) and/or (b) results in ether bonds.

48. The method according to claim 32, wherein the GAG is hyaluronic acid.

49. An injectable gel product comprising a first, inner phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second, outer phase of a cross-linked GAG gel, wherein the second, outer phase is in the form of particles, wherein the gel of the first, inner phase has a Degree of Modification (MoD) of 0.15 or lower, wherein the gel of the second, outer phase has a MoD that is lower than the MoD of the gel of the first, inner phase, and wherein the MoD is the molar amount of bound cross-linking agent(s) relative to the total molar amount of repeating GAG disaccharide units; wherein the inner phase, the outer phase, or both the inner phase and the outer phase are crosslinked via one or more polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

50. The injectable gel product according to claim 49, wherein the one or more polyfunctional cross-linking agent is BDDE.

51. The injectable gel product according to claim 49, wherein each of the plurality of crosslinked GAG gel particles in the first, inner phase and the plurality of crosslinked GAG gel particles in the second, outer phase are independently covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

52. The injectable gel product according to claim 49, wherein the plurality of crosslinked GAG gel particles in the first, inner phase are covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

53. The injectable gel product according to claim 52, wherein the plurality of crosslinked GAG gel particles in the second, outer phase are independently covalently crosslinked via crosslinks comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

54. The injectable gel product according to claim 53, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

55. The injectable gel product according to claim 49, wherein the plurality of crosslinked GAG gel particles in the second, outer phase are covalently crosslinked via a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

56. The injectable gel product according to claim 55, wherein the plurality of crosslinked GAG gel particles in the second, outer phase are independently covalently crosslinked via crosslinks comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

57. The injectable gel product according to claim 56, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

59. The injectable gel product according to claim 49, wherein the MoD of the gel of the second, outer phase is 0.10 or lower.

60. The injectable gel product according to claim 49, wherein the gel of the second, outer phase has a Swelling factor (SwF) above 3.0.

61. The injectable gel product according to claim 49, wherein the gels of the first, inner phase and the second, outer phase are cross-linked to each other.

62. The injectable gel product according to claim 49, wherein the particles of the second, outer phase have a particle size at least three times the size of an inner particle size.

63. The injectable gel product according to claim 49, wherein the cross-linked GAG of the first, inner phase have a dry weight content of that is at least 50% of the total dry weight content of GAGs in the inner and outer phase.

64. The injectable gel product according to claim 49, wherein the GAG is hyaluronic acid.

65. The injectable gel product according to claim 49, wherein the cross-linking is with ether bonds.

66. The injectable gel product according to claim 49, further comprising a buffering agent.

67. A pre-filled syringe, comprising an injectable gel product according to claim 49.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0167] FIG. 1 shows a graphical outline of an embodiments of the method of the present disclosure. PSR=particle size reduction

[0168] FIG. 2 shows results from degradation tests.

[0169] FIG. 3 shows results from degradation tests.

[0170] FIG. 4 shows gel particles with embedded gel particles at a magnification of 10×.

[0171] FIG. 5 shows a microscope image of gel particles obtained in Example 6 at a magnification of 10×.

[0172] FIG. 6 shows a microscope image of gel particles obtained in Example 9.

EXPERIMENTAL EXAMPLES

[0173] The following non-limiting examples will further illustrate the present invention. In these examples, the GAG used is hyaluronic acid, denoted HA and referring to sodium hyaluronate.

Analytical Test Methods

[0174] Gel Content (GelC)

[0175] The GelC describes in % the proportion of the total HA that is bound in gel form. Gel content is defined as the amount of HA in a sample that does not pass through a 0.22 μm filter. GelC is calculated from the amount of HA that is collected in the filtrate and is given in percent of the total amount of HA in the gel sample.

[0176] MoD (Degree of Modification)

[0177] MoD describes the molar amount of bound cross-linking agent(s) relative to the total number of repeating HA disaccharide units. This measure does not distinguish between mono-linked and actually cross-linked cross-linking agent(s), i.e. all cross-linking agent(s) that is bound to HA via at least one covalent bond is included. For instance, a MoD of 1% for a HA gel cross-linked with BDDE means that there is 1 bound (monolinked or cross-linked) molecule of BDDE per 100 disaccharide units in the HA gel.

[0178] MoD may be determined using NMR spectroscopy on enzymatically degraded gel product. Soluble HA, residual (non-bound) cross-linking agent(s) and derivatives thereof are washed away prior to the degradation of the gel by filtration on a 0.22 μm filter. The gel product is degraded at 37° C. by enzymatic treatment using Chondroitinase AC from Arthrobacter aurescens. The degraded gel product is subjected to NMR spectroscopy by recording one-dimensional .sup.1H NMR spectra on a 400 MHz spectrometer, equipped with a standard 5 mm probe.

[0179] The NMR spectra may be evaluated by integration of the signal at δ.sub.H 1.6 ppm, which origins from four protons in the linked BDDE molecule, and the signals at δH 2.0 ppm, which is from the three protons in the CH.sub.3 groups on the N-acetylglucosamine residues of the HA disaccharides. The ratio between the integrals for these two signals is proportional to the ratio between the molar amount of bound BDDE and disaccharides after correction for the number of protons responsible for each signal, hence giving MoD.

[00001]$\mathrm{MoD}=\frac{n_{\mathrm{bound}\mathrm{crosslinking}\mathrm{agent}}}{n_{\mathrm{disaccharide}\mathrm{units}}}$

[0180] Charging Ratio

[0181] Another quantity for measuring the degree of cross-linking is the charging ratio, i.e. the ratio between the number of crosslinking agents added to the reaction vessel and the total number of disaccharide units added to the reaction vessel when performing the cross-linking reaction. The charging ratio may not be the same as the MoD, since the crosslinking reaction may be incomplete, i.e. all crosslinkers may not have reacted to form crosslinks.

[0182] Swelling Factor (SwF)

[0183] The strength or density of the gel network after crosslinking can be estimated/determined e.g. by allowing the gel to absorb water or saline to equilibrium. As a result of chain breaks during degradation of the gel, the network becomes weaker and less dense, which can be detected by the increasing swelling factor (SwF). The SwF is measured according to the following protocol: About 1 g of gel product is weighed into a measuring glass. Saline is added, the gel is thoroughly dispersed and allowed to absorb water until equilibrium. After sedimentation, the volume of the saturated gel is read. The ratio final volume to initial volume is denoted Swelling Factor (SwF).

Example 1—General Process Outline for Producing a Combination Gel

[0184] An embodiment of a method for producing an injectable gel product crosslinked with BDDE is graphically outlined in FIG. 1. In the process, the bulk is precipitated and dried after the first particle size reduction step (PSR). The dry, crosslinked, gel powder is subsequently mixed with HA, BDDE and NaOH for a second crosslinking, where the previously crosslinked gel particles become embedded into a second gel material. The second material is then subjected to the same process steps as in the normal process. The particles from the first PSR are smaller than those from the second PSR, to allow room for the inner gel into the outer gel.

[0185] The first and second cross-linking are typically carried out at a temperature of 10-75 degrees centigrade, e.g. 10-40 degrees centigrade, such as 10-35 degrees centigrade or 10-30 degrees centigrade but it is preferred that the step is carried out at 15-35 degrees centigrade, such as 15-30 degrees centigrade, and especially at room temperature, e.g. 20-25 degrees centigrade. Preferred temperature ranges are 10-50 degrees centigrade, such as 18-40 degrees centigrade.

[0186] The reaction time is suitably in the range of 2-40 h, such as 4-36 h. A longer reaction time than 2 h is useful for reproducibility, especially at larger scale. Longer reaction time than 40 h yields a gel with lower gel strength or may even disrupt the gel entirely. The reaction time is preferably in the range of 8-30 h, such as 12-24 h, e.g. 16-24 h.

[0187] As an example, the cross-linking step may be performed at 15-35 degrees centigrade for 2-40 h, such as at room temperature for 16-24 h.

[0188] FIG. 4 shows a microscope image at a magnification of 10× of gel particles obtained by the method of the present disclosure, with smaller particles embedded in the large particles.

Example 2—Preparation of One-Component Gel

[0189] A one-component gel was prepared as follows: 100 g HA (Mw 1MDa) was mixed and allowed to react with 200 g NaOH 3% w/w and 1.8 g BDDE—resulting in a charging ratio of 0.036. After crosslinking, the gel was neutralized and heat-treated to inactivate any residual crosslinker. Particle size reduction (PSR) was done using an 80 μm mesh. The gel was precipitated in EtOH to obtain a gel powder, which was washed and dried.

Example 3—Preparation of One-Component Gel

[0190] Performed as example 2, but using 30 g HA, 73.5 g NaOH 2.5% w/w and 0.22 g BDDE—resulting in a charging ratio of 0.015.

Example 4—Preparation of Two-Component Gel

[0191] A two-component gel was prepared as follows: 5 g of the gel powder from example 2 was mixed and allowed to react with 5 g HA (mw 1MDa), 45.5 g NaOH 1.33% w/w and 90 mg BDDE—resulting in a charging ratio of 0.036. The PSR from example 2 was repeated, but using a 315 μm mesh. The two-component gel powder was hydrated to a final concentration of 20 mg/ml in phosphate buffer and autoclaved. The gel was subjected to 90° C., and the gel content was measured at 16 h intervals (se FIG. 2). Thus, in Example 4, the charging ratio in both the first and second crosslinking reaction was 0.036.

Example 5—Preparation of Two-Component Gel

[0192] Performed as example 4, but with 7 g of the gel powder from example 2 and 3 g HA, resulting in a charging ratio of 0.043. The gel was subjected to 90° C., and the gel content was measured at 16 h intervals (FIG. 2). Restylane Lyft (from Galderma) was used as a reference. Thus, in Example 5, the charging ratio was 0.036 in the first crosslinking reaction and 0.043 in the second crosslinking reaction.

[0193] The gels of Examples 4 and 5 had the following initial characteristics:

TABLE-US-00001 Dry weight content of GelC [HA] Sample inner gel % (mg/ml) Example 4 50% 83 20 Example 5 70% 91 19 Restylane Lyft — 86 20

[0194] As seen in FIG. 2, the 70% combination gel degrades until the outer gel is gone, and then levels out at about 70% remaining gel which is degraded at a lower rate. The same type of behaviour was observed with the 50% combination gel, but this gel levels out at 50%. The reference gel, containing only a single gel type, is degraded at a more constant rate. These results clearly demonstrate the combination gel concept, i.e. a combination gel having an outer gel that degrades first before the inner gel starts to degrade. The same type of behaviour is expected in vivo.

Example 6—Preparation of Two-Component Gel

[0195] Performed as example 4, but with 2.5 g of the gel powder from example 3 and 2.5 g HA (Mw 1MDa), 15 g NAOH 2% w/w and 35 mg BDDE, resulting in a charging ratio of 0.022. The gel was subjected to 90° C., and the swelling factor was measured at 16 h intervals (FIG. 3). As a reference, gel powder from example 3 was crosslinked a second time to correspond to the inner gel of examples 6 and 7. FIG. 5 shows a microscope image of gel particles obtained in Example 6 at a magnification of 10×.

[0196] Thus, in Example 6, the charging ratio was 0.015 in the first crosslinking reaction and 0.022 in the second crosslinking reaction.

Example 7—Preparation of Two-Component Gel

[0197] Performed as example 6, but with 3.5 g of the gel powder from example 3 and 1.5 g HA, resulting in a charging ratio of 0.025. The gel was subjected to 90° C., and the swelling factor was measured at 16 h intervals (FIG. 3). Thus, in Example 7, the charging ratio was 0.015 in the first crosslinking reaction and 0.025 in the second crosslinking reaction.

The gels of Examples 6 and 7 had the following initial characteristics:

TABLE-US-00002 Dry weight content of [HA] GelC Sample inner gel SwF (mg/ml) % Example 6 50% 4.0 20 93 Example 7 70% 3.7 20 94 Ref — 3.1 20 96

[0198] The 50% combination gel increases its swelling more than the other gels since this gel comprises a large portion of the softer outer gel, that mostly contributes to the swelling. After a certain period of time (about 40 hours for the 50% combination gel), the outer gel is gone and only the firmer inner gel may contribute to the swelling. This gives a drop in measured SwF, that then slowly rises again due to swelling of the inner gel. The 70% combination gel shows a similar, but less distinct behaviour, since it contains a lower amount of the outer gel. The gel having only an inner gel (single gel) shows a more constant increase in swelling behaviour. Thus, the inner gel will, after degradation of the outer gel, display a swelling factor similar to that of the initial combination gel.

[0199] The below Table summarizes the charging ratios used for preparing the two-component gels in experimental Examples 4-7 above:

TABLE-US-00003 Charging ratio Charging ratio 1st crosslinking 2nd crosslinking Sample reaction reaction Example 4 0.036 0.036 Example 5 0.036 0.043 Example 6 0.015 0.022 Example 7 0.015 0.025

[0200] Further, performing the first and second crosslinking reactions having the charging ratios according to the Table above resulted in the below degree of modification (MoD) for the inner and outer gels in the prepared two-component gels in experimental Examples 4-7:

TABLE-US-00004 MoD inner MoD outer gel in the two- gel in the two- Sample component gel (%) component gel (%) Example 4 3.9 1.1 Example 5 3.9 1.1 Example 6 2.2 1.0 Example 7 2.2 1.0

[0201] Thus, the MoD of the inner gels in all Examples are higher than the MoD of the outer gels, which is due to crosslinkers diffusing into the inner gel during the second crosslinking reaction to crosslink this gel further, thereby increasing the MoD of the inner gel during the second crosslinking step.

Example 8—Preparation of One-Component Gels Crosslinked with Amide Bonds

[0202] Gels were also prepared using crosslinking with amide bonds, in which hyaluronic acid (HA), DATH (diaminotrehalose) and DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) are mixed and allowed to react. DMTMM functions as a coupling agent whereas DATH functions as a crosslinker.

[0203] A one-component gel was prepared as follows: HA was mixed and allowed to react with proper amounts of DATH and DMTMM solved in water. After crosslinking, the gel was diluted to 20 mg/g in Saline and heated to 70° C. for about 20 hours. Particle size reduction (PSR) was done using 125 μm mesh. The gel was precipitated in EtOH to obtain a gel powder, which was washed and dried.

Example 9—Preparation of Two-Component Gels Crosslinked with Amide Bonds

[0204] Dry, crosslinked, gel powder of HA crosslinked with amide bonds prepared as in Example 8 was subsequently mixed and allowed to react with proper amounts of HA, DATH and DMTMM solved in water for a second crosslinking, where the previously crosslinked gel particles became embedded into a second gel material.

[0205] After the second crosslinking, the gel was diluted to 20 mg/g in Saline and heated to 70° C. for about 20 hours. Particle size reduction (PSR) was done using 315 μm mesh. The gel was precipitated in EtOH to obtain a gel powder, which was washed and dried. The two-component gel powder was hydrated to a final concentration of 20 mg/ml in phosphate buffer and autoclaved.

[0206] A micrograph of the prepared two-component gel from Example 9 is shown in FIG. 6.

[0207] Gel characteristics of the gels crosslinked with amide bonds are shown in the below Table:

TABLE-US-00005 HA-conc GelC SwF G′ G″ Example (mg/g) (%) (ml/g) (Pa) (Pa) Tanδ 8 20 96 2.9 1261 115 0.09 9 20 82 4.4  288  36 0.12

[0208] The person skilled in the art realizes that the present invention by no means is limited to the preferred embodiments described above. On the contrary, many modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage.