Pharmaceutical compositions containing insulin derivatives and their uses.

Abstract

The present invention is in the field of pharmaceutical compositions for the treatment of medical conditions related to diabetes. More specifically, the invention provides pharmaceutical compositions comprising a long-acting acylated derivative of a human insulin analogue, and to the medical use of these compositions for basal insulin administration therapy.

Description

1 / 44 Pharmaceutical compositions containing insulin derivatives and their uses. TECHNICAL FIELD

[001] The present invention pertains to the field of pharmaceutical compositions for the treatment of medical conditions related to diabetes. More specifically, the invention provides pharmaceutical compositions comprising a long-acting acylated derivative of a human insulin analogue, and to the medical use of such compositions for basal insulin administration therapy. PREVIOUS TECHNIQUE

[002] The main goal of insulin therapy in the treatment of metabolic disorders is to produce sustained near-normal blood glucose levels by replacing or supplementing endogenous insulin secretion in the most physiological way possible, postprandially, as well as between meals and during the night. Separating basal and meal-related (bolus) insulin requirements represents a systematic approach to subcutaneous insulin therapy.

[003] The most relevant pharmacological characteristics of any given insulin - onset of action, peak effect profile, duration of action, etc. - are largely determined by its absorption kinetics from the subcutaneous injection site to the systemic circulation.

[004] Non-covalent zinc-mediated oligomerization is a well-described property of insulin products. Under physiological pH, human insulin is soluble but has a tendency to self-associate into well-defined hexamers (i.e., one unit of six insulin molecules) by the coordination of two zinc ions (Zn++) to high-affinity binding sites (B10His). It is also well known that phenolic ligands, especially phenol, bind specifically to the insulin hexamer and promote Petition 870220115194, dated 09 / 12 / 2022, page 16 / 64 2 / 44 the formation of the R-state hexamer. After injection, the phenolic ligands diffuse rapidly from the injection site. In the absence of phenol at the injection site, the conformation and size of the insulin oligomer can change, as well as the viscosity of the insulin-containing solution, contributing to a prolonged action profile.

[005] Jonassen et al., Pharm. Res. 2012 29 2104-2114 describes the prolongation mechanism of insulin degludec, an insulin derivative with an acylated fatty acid chain for once-daily administration, and describes the correlation between high zinc concentration and prolongation. WO 2009 / 115469 describes several long-acting insulin derivatives with an acylated fatty acid chain. WO 2013 / 153000 describes the formulation of these long-acting insulin derivatives for subcutaneous administration, which contain a high zinc content (not less than 3.5 Zn++ / six moles of insulin derivatives). This was designed to achieve the prolonged duration of action compatible with a once-weekly administration profile. The high Zn++ content in the formulations described in WO 2009 / 115469 leads to a prolonged PK profile.

[006] WO 2009 / 063072 discloses pharmaceutical compositions for parenteral administration comprising a basal insulin derivative (e.g., degludec) and a GLP-1 derivative (e.g., liraglutide). Because the liraglutide monomer binds to zinc to form the diheptamer, a higher zinc content than the degludec monoformulation is required in the combined formulation to achieve a comparable PK profile to degludec and to achieve acceptable physical stability. SUMMARY OF THE INVENTION

[007] According to the present invention, a new formulation of long-acting insulin derivatives has been developed, which is capable of promoting a conformational state and an oligomerization pattern that more closely resembles that of human insulin, i.e., hexamers, Petition 870220115194, dated 09 / 12 / 2022, page 17 / 64 3 / 44 especially R6 hexamers.

[008] In another aspect, the invention provides a pharmaceutical composition comprising a long-acting insulin derivative selected in a unique combination of carefully formulated excipients in order to reduce oligomer formation at the injection site, while still demonstrating PK / PD properties suitable for once-weekly administration.

[009] In another aspect, the invention provides a pharmaceutical composition with lower viscosity after injection and, consequently, less propensity to create any discomfort after injection.

[0010] In another aspect, the invention provides a pharmaceutical composition with improved stability.

[0011] In another aspect, the invention provides a pharmaceutical composition for use as a medicament for the treatment of a metabolic disorder.

[0012] Other objects of the invention will be apparent to those skilled in the art from the detailed description and examples that follow. DETAILED DISCLOSURE OF THE INVENTION

[0013] In its first aspect, the invention provides a pharmaceutical composition comprising an insulin derivative selected from the group consisting of

[0014] A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30; (Compound 1);

[0015] A14E, B16H, B25H, B29K(N£-Hexadecandioyl-YGlu), human insulin desB30 (Compound 2);

[0016] A14E, B16H, B25H, B29K(N£-Eicosanedioyl-YGlu), human insulin desB30 (Compound 3); and Petition 870220115194, dated 09 / 12 / 2022, page 18 / 64 4 / 44

[0017] A14E, B25H, desB27, B29K(N£-Octadecandioyl-YGlu), human insulin desB30 (Compound 4); and further comprising about 1 to about 2% (w / w) of glycerol; about 45 to about 75 mM of phenol; about 0 to about 19 mM of m-cresol; about 1.5 to about 2.5 moles of zinc ions per six moles of said insulin derivative; not more than about 75 mM of sodium chloride; and having a pH value in the range of 7.2 to 8.0.

[0018] In another aspect, the invention provides pharmaceutical compositions that further comprise an insulinotropic GLP-1 compound and, in particular, the insulinotropic GLP-1 compound known as semaglutide.

[0019] Semaglutide can be described by the structure Aib8,Lys26(OEG-OEG-gamma-Glu-C18-diacid),Arg34)GLP-1 H(7-37)-OH, which can also be designated as (N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4Carboxi-4-(17-carboxi-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Aib8,Arg34]GLP-1-(7-37), disclosure cf in WO 2006 / 097537.

[0020] The present invention may be further characterized by reference to one or more of the following features or embodiments: 1. A pharmaceutical composition of the invention comprising an insulin derivative, which is A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 2. A pharmaceutical composition of the invention comprising an insulin derivative, which is A14E, B16H, B25H, B29K (Ni:-HexadecandioylYGlu), human insulin desB30 (Compound 2). 3. A pharmaceutical composition of the invention comprising an insulin derivative, which is A14E, B16H, B25H, B29K (Ni:-Eicosanedioyl-YGlu), human insulin desB30 (Compound 3). Petition 870220115194, dated 09 / 12 / 2022, page 19 / 64 5 / 44 4. A pharmaceutical composition of the invention comprising an insulin derivative, which is A14E, B25H, desB27, B29K (N£-Octadecandioyl-YGlu), human insulin desB30 (Compound 4). 5. The pharmaceutical composition of the aforementioned embodiments, in which the insulin derivative is in the range of approximately 3.5 to approximately 5.0 mM. 6. The pharmaceutical composition of the aforementioned embodiments, in which the insulin derivative is in the range of approximately 4.0 to approximately 4.5 mM. 7. The pharmaceutical composition of the previous embodiments, in which the insulin derivative is approximately 4.2 mM. 8. The pharmaceutical composition of the foregoing embodiments comprises from about 1 to about 2% (weight / weight) of glycerol. 9. The pharmaceutical composition of the foregoing embodiments comprising from about 1.4 to about 1.8% (weight / weight) of glycerol. 10. The pharmaceutical composition of the foregoing embodiments comprising from about 1.5% to about 1.6% (weight / weight) of glycerol. 11. The pharmaceutical composition of the foregoing embodiments comprising from about 45 to about 75 mM of phenol. 12. The pharmaceutical composition of the foregoing embodiments comprising from about 50 to about 70 mM of phenol. 13. The pharmaceutical composition of the foregoing embodiments comprising from about 55 to about 65 mM of phenol. 14. The pharmaceutical composition of the foregoing embodiments comprising approximately 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mM of phenol. 15. The pharmaceutical composition of the foregoing embodiments comprising from about 0 to about 19 mM of m-cresol. 16. The pharmaceutical composition of the foregoing embodiments comprising 0 mM, 1 mM, 2 mM, 3 mM, 4 mM of m-cresol. Petition 870220115194, dated 09 / 12 / 2022, page 20 / 64 6 / 44 17. The pharmaceutical composition of the foregoing embodiments comprises from about 0 to about 15 mM of m-cresol. 18. The pharmaceutical composition of the foregoing embodiments comprising approximately 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM or 19 mM of m-cresol. 19. The pharmaceutical composition of the foregoing embodiments comprises from about 1.5 to about 2.5 moles of zinc ions per six moles of insulin derivative. 20. The pharmaceutical composition of the foregoing embodiments comprises from about 2.0 to about 2.4 moles of zinc ions per six moles of insulin derivative. 21. The pharmaceutical composition of the foregoing embodiments comprises approximately 2.0 or 2.1 moles of zinc ions per six moles of insulin derivative. 22. The pharmaceutical composition of the foregoing embodiments comprises approximately 2.2 or 2.3 moles of zinc ions per six moles of insulin derivative. 23. The pharmaceutical composition of the foregoing embodiments comprises approximately 2.4 or 2.5 moles of zinc ions per six moles of insulin derivative. 24. The pharmaceutical composition of the foregoing embodiments comprising less than about 75 mM of sodium chloride. 25. The pharmaceutical composition of the foregoing embodiments comprises from about 5 to about 50 mM of sodium chloride. 26. The pharmaceutical composition of the foregoing embodiments comprises from about 10 to about 25 mM of sodium chloride. 27. The pharmaceutical composition of the foregoing embodiments comprising from about 15 to about 25 mM of sodium chloride. Petition 870220115194, dated 09 / 12 / 2022, p. 21 / 64 7 / 44 28. The pharmaceutical composition of the foregoing embodiments comprising approximately 20 mM, 50 mM or 75 mM of sodium chloride. 29. The pharmaceutical composition of the aforementioned modalities has a pH value in the range of 7.2 to 8.0. 30. The pharmaceutical composition of the aforementioned modalities has a pH value in the range of 7.2 to 7.6. 31. The pharmaceutical composition of the foregoing embodiments having a pH value of approximately 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9. 32. The pharmaceutical composition of the foregoing embodiments comprising from about 4.0 to about 4.5 mM of insulin derivative; from about 1 to about 2% (w / w) glycerol; from about 50 to about 70 mM phenol; from about 0 to about 15 mM m-cresol; from about 2.0 to about 2.5 moles of zinc ions per six moles of insulin derivative; less than about 50 mM of sodium chloride; and with a pH value in the range of 7.2 to 7.6. 33. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; approximately 0 mM of m-cresol; approximately 2.0 moles of zinc ions per six moles of insulin derivative; of approximately 20 mM sodium chloride; and with a pH value of approximately 7.4. Petition 870220115194, dated 09 / 12 / 2022, page 22 / 64 8 / 44 34. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; of about 10 mM m-cresol; approximately 2.0 moles of zinc ions per six moles of insulin derivative; of approximately 20 mM sodium chloride; and with a pH value of approximately 7.4. 35. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; approximately 0 mM of m-cresol; approximately 2.2 moles of zinc ions per six moles of insulin derivative; of approximately 20 mM sodium chloride; and with a pH value of approximately 7.4. 36. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; of about 10 mM m-cresol; approximately 2.2 moles of zinc ions per six moles of insulin derivative; Petition 870220115194, dated 09 / 12 / 2022, page 23 / 64 9 / 44 of approximately 20 mM sodium chloride; and with a pH value of approximately 7.4. 37. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; approximately 0 mM of m-cresol; approximately 2.4 moles of zinc ions per six moles of insulin derivative; of approximately 20 mM sodium chloride; and with a pH value of approximately 7.4. 38. The pharmaceutical composition of the foregoing embodiments comprises approximately 4.2 mM of insulin derivative; of about 1.5% (w / w) glycerol; approximately 60 mM of phenol; of about 10 mM m-cresol; approximately 2.4 moles of zinc ions per six moles of insulin derivative; of approximately 20 mM sodium chloride; and with a pH value around 7.4. 39. The pharmaceutical composition of the foregoing embodiments comprising about 4.2 mM of insulin derivative; about 1 to about 2% (w / w) of glycerol; about 45 to about 75 mM of phenol; about 0 to about 15 mM of m-cresol; about 1.5 to about 2.5 moles of zinc ions per six moles of said insulin derivative; not more than about 50 mM of sodium chloride; and with a pH value in the range of 7.2 to 8.0. Petition 870220115194, dated 09 / 12 / 2022, p. 24 / 64 10 / 44 40. The pharmaceutical composition of the foregoing embodiments comprising approximately 0 mM of m-cresol. 41. The pharmaceutical composition of the foregoing embodiments comprising from about 5 to about 10 mM of m-cresol. 42. The pharmaceutical composition of the foregoing embodiments comprising approximately 10 mM of m-cresol. 43. The pharmaceutical composition, according to the previous embodiments, which also includes semaglutide. 44. A pharmaceutical composition comprising A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1); and semaglutide; and further comprising about 1 to about 2% (w / w) of glycerol; about 45 to about 75 mM of phenol; 0-15 mM of m-cresol; about 1.5 to about 2.5 moles of zinc ions per six moles of said insulin derivative; not more than about 25 mM of sodium chloride; and with a pH value in the range of 7.2 to 8.0 45. The pharmaceutical composition, according to the previous embodiments, comprising from about 0.20 to about 0.70 mM of semaglutide. 46. ​​The pharmaceutical composition, according to the previous embodiments, comprising from about 0.30 to about 0.70 mM of semaglutide. 47. The pharmaceutical composition, according to the previous embodiments, comprising from about 3.5 mM to about 5.0 mM of A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 48. The pharmaceutical composition, according to the previous embodiments, comprising from about 0.30 to about 0.70 mM of Petition 870220115194, dated 09 / 12 / 2022, p. 25 / 64 11 / 44 semaglutide, and from about 3.5 mM to about 5.0 mM of A14E, B16H, B25H, B29K ((N£-Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 49. The pharmaceutical composition, according to the foregoing embodiments, comprising about 0.30 mM of semaglutide, and from about 3.5 mM to about 5.0 mM of A14E, B16H, B25H, B29K((N£-EicosanedioylYGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 50. The pharmaceutical composition, according to the previous embodiments, comprising approximately 0.40 mM of semaglutide, and 4.2 mM of A14E, B16H, B25H, B29K((N^Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 51. The pharmaceutical composition, according to the previous embodiments, comprising approximately 0.49 mM or 0.50 mM of semaglutide, and 4.2 mM of A14E, B16H, B25H, B29K((N^Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 52. The pharmaceutical composition, according to the previous embodiments, comprising approximately 0.60 mM of semaglutide, and 4.2 mM of A14E, B16H, B25H, B29K((N^Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1). 53. A pharmaceutical composition (co-formulation) comprising A14E, B16H, B25H, B29K((N^Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1); and semaglutide; and further comprising about 1.5% (w / w) of glycerol; about 60 mM of phenol; about 0 mM of m-cresol; about 2.2 moles of zinc ions per six moles of said insulin derivative; about 20 mM of sodium chloride; and having a pH value around 7.4. Petition 870220115194, dated 09 / 12 / 2022, p. 26 / 64 12 / 44 54. A pharmaceutical composition (co-formulation) comprising A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1); and semaglutide; and further comprising about 1.5% (w / w) of glycerol; about 60 mM of phenol; about 10 mM of m-cresol; about 2.2 moles of zinc ions per six moles of said insulin derivative; about 20 mM of sodium chloride; and having a pH value around 7.4. 55. The pharmaceutical composition of the foregoing modalities for administration to a subject in need at intervals less frequent than once a day (i.e., at intervals greater than 24 hours), for a period of at least 3 months, at least 6 months, or at least 1 year. 56. The pharmaceutical composition of the previous modalities for administration to a subject in need of administration to the subject with a frequency in the range of every 2 days to every 11 days, on average. 57. The pharmaceutical composition of the previous modalities for administration to a subject in need of administration to the subject with a frequency in the range of every 3 to 10 days, on average. 58. The pharmaceutical composition of the previous modalities for administration to a subject in need of administration to the subject with a frequency in the range of every 4 to every 9 days, on average. 59. The pharmaceutical composition of the previous modalities for administration to a subject in need of administration to the subject with a frequency in the range of every 5 to every 8 days, on average. 60. The pharmaceutical composition of the previous modalities for administration to a subject in need of administration to the subject with a frequency in the range of every 6 to every 7 days, on average. 61. The pharmaceutical composition of the previous versions for Petition 870220115194, dated 09 / 12 / 2022, page 27 / 64 13 / 44 administration to a subject in need of administration to the subject once a week, that is, every 7 days, on average, for a period of time of at least 3 months, at least 6 months, or at least 1 year. 62. A method for producing an injectable pharmaceutical composition comprises: (i) Prepare a solution by dissolving A14E, B16H, B25H, B29K((N£Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 in water (ii) Prepare a solution by dissolving preservatives and isotonicity agents in water (iii) Prepare a solution by dissolving zinc ions in water (iv) Mix solution a) and solution b) (v) Add solution c) to solution a+b (vi) Dissolve semaglutide in the combined solution a+b+c (vii) Adjust the pH of mixture f) to the desired pH, followed by sterile filtration.

[0021] Any combination of two or more of the embodiments described in this document is considered to be within the scope of the present invention. Biological Activity

[0022] In another aspect, the invention provides pharmaceutical compositions useful as medicaments for the treatment of metabolic diseases, disorders or conditions and, in particular, diseases, disorders or conditions related to diabetes.

[0023] In one embodiment, the pharmaceutical composition of the invention is for use in the treatment or relief of a disease, disorder or condition related to diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome. Petition 870220115194, dated 09 / 12 / 2022, p. 28 / 64 14 / 44 (metabolic syndrome X, insulin resistance syndrome).

[0024] In another embodiment, the pharmaceutical composition of the invention is for use in the treatment or relief of a disease, disorder or condition related to diabetes and, in particular, type 1 diabetes, or type 2 diabetes.

[0025] The actual dosage depends on the nature and severity of the disease being treated and is at the discretion of the physician, and may be varied by titrating the dosage to the specific circumstances of this invention to produce the desired therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further illustrated by reference to the accompanying drawings, in which:

[0027] Figure 1 shows the oligomerization of monoformulations A, B, and C of Compound 1 under simulated injection site conditions:

[0028] Figure 1A: Apparent mean hydrodynamic radius (rH) [nm] measured by DLS;

[0029] Figure 1B: Apparent mean molecular weight measured by GC-MALS;

[0030] Figure 1C: Apparent mean sedimentation coefficient recnte (S) measured by AUC;

[0031] White bars: Compound 1, Formulation C with 2,2 Zn++ per six insulins (mol:mol);

[0032] Gray bars: Compound 1, Formulation B with 2.4 Zn++ per six insulins (mol:mol);

[0033] Black bars: Compound 1, Formulation A containing 4.5 Zn++ per six insulins (mol:mol);

[0034] Figure 2 shows the oligomerization of the 01-06 monoformulations of Compound 1 under simulated injection site conditions: Petition 870220115194, dated 09 / 12 / 2022, page 29 / 64 15 / 44

[0035] Figure 2A: Apparent average hydrodynamic radius (Rh, average) [nm] measured by DLS;

[0036] Figure 2B: Apparent average sedimentation coefficient (S*) measured by AUC

[0037] Figure 3 shows the apparent dynamic viscosity [cP] (Figure 3A and Figure 3C) and specific viscosity [nspec] (Figure 3B, Figure 3D) of different buffer-exchanged formulations of Compound 1 and interstitial fluid buffer (ISF) as a function of temperature [°C];

[0038] Figure 3A and Figure 3B: Formulation A; Formulation B; Formulation C; ISF buffer;

[0039] Figure 3C and Figure 3D: Formulation 02; Formulation 03; Formulation 04; ISF buffer;

[0040] Figure 4 shows the conformational state of Compound 1 in the following formulations:

[0041] Figure 4A: CD near UV ^251nm(M-1 cm-1)] showing the conformational changes (T state; mixed TR state; R state) as a function of Zn++ by six insulins (mol:mol) for Formulations 01-06;

[0042] Figure 4B: CD near UV ^e251nm(M-1 cm-1)] showing the conformational changes (T state; mixed TR state; R state) as a function of Zn++ for six insulins (mol:mol) for Formulations B1-B6, D1-D7, and human insulin formulations with various zinc contents:

[0043] Open circles: Human insulin (600 nmol / mL human insulin, 30 mM phenol, 150 mM NaCl, pH 7.4);

[0044] Black squares: Compound 1 formulated with 25 mM phenol, 25 mM m-cresol and 20 mM NaCl;

[0045] Gray circles: Compound 1 formulated with 60 mM phenol, 10 mM m-cresol and 20 mM NaCl;

[0046] Figure 5 shows the oligomer distribution of Petition 870220115194, dated 09 / 12 / 2022, page 30 / 64 16 / 44 Compound 1 in the SEC formulations:

[0047] Figure 5A shows the native SEC chromatogram of Formulations 01, 02, 03, 04, 05 and 06;

[0048] Figure 5B shows the native SEC chromatogram of Formulations A and B1-B6;

[0049] Figure 6 shows the SAXS scattering data [Intensity (au) vs. s(A-1)] of Compound 1 and human insulin in the formulated state:

[0050] Figure 6A: Scattering curves of Formulation A of Compound 1 shown in black and human insulin shown in gray (Compound 1, 4.2 mM, 4.5 Zn / hexamer; Human insulin 0.6 mM, 2.2 Zn / hexamer);

[0051] Figure 6B: Scattering curves of Formulation C of Compound 1 shown in black and human insulin shown in gray (Compound 1, 4.2 mM, 2.2 Zn / hexamer; Human insulin 0.6 mM, 2.2 Zn / hexamer (R6-hexamer));

[0052] Figure 7 shows the purity of Compound 1 in the formulations:

[0053] Figure 7A shows the purity (% of total peptide) under storage at 30°C [Time point (Month)] for Formulation A (black line), Formulation B (gray line) and Formulation C (dashed line) of Compound 1;

[0054] Figure 7B shows the purity (% of total peptide) under storage at 37°C [Time point (weeks)] for Compound 1 for Formulations 01, 04, 05 and 06;

[0055] White circles: Formulation 01, Black circles: Formulation 04, White squares: Formulation 05, Black squares: Formulation 06; Petition 870220115194, dated 09 / 12 / 2022, page 31 / 64 17 / 44

[0056] Figure 8 shows the purity of Compound 1 (% of total Compound 1) under storage at 37°C [Time point (weeks)] in formulations combined with semaglutide:

[0057] White circles: combination-Formulation I, black circles: combination-Formulation II, white triangles: combination-Formulation III, black triangles: combination-Formulation IV, white squares: combination-Formulation V, black squares: combination-Formulation VI;

[0058] Figure 9 shows the oligomerization of the combined formulations under simulated injection site conditions:

[0059] Figure 9A: Apparent mean hydrodynamic radius (rH) [nm] measured by DLS;

[0060] Figure 9B: shows the size of oligomers of the combined formulations after the buffer is changed, as observed by AUC (S*). EXAMPLES

[0061] The invention is further illustrated with reference to the following examples, which are not intended to be, in any way, limiting to the scope of the invention as claimed. Example 1 Improved biophysical properties under simulated injection site conditions. Protocol

[0062] The API of the formulations is A14E, B16H, B25H, B29K((N£-Eicosanedioyl-YGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1), obtained as described in, for example, WO 2009 / 115469, see Example 33.

[0063] The oligomerization of Compound 1 was determined under simulated injection site conditions. The simulated injection site conditions Petition 870220115194, dated 09 / 12 / 2022, page 32 / 64 18 / 44 injections were obtained by depleting phenol and / or metacresol, respectively, and switching the buffer to simulated interstitial fluid buffer. This procedure removed virtually all phenolic binders but maintained the zinc / insulin ratio of the original formulation.

[0064] The formulations had their buffer changed to interstitial fluid buffer (ISF-) using the PD-MidiTrap G-25 column according to the manufacturer's protocol.

[0065] The columns were equilibrated with the target buffer by washing the column with a sufficient volume of target buffer, and the formulation was applied in an appropriate volume and eluted with the target buffer. The ISF buffer consists of 140 mM NaCl, 4 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 10 mM Hepes, pH 7.4.

[0066] Unless otherwise indicated, the procedure was as follows: Replace the cap at 22°C; Subsequent incubation at 37°C for 14-18 hours; and Subsequent measurement at 22°C (unless otherwise indicated).

[0067] Typically, measurements were taken approximately 1 hour after the end of incubation at 37°C. If this was not possible, samples were stored at 22°C until measurement.

[0068] Unless otherwise indicated, samples were measured undiluted. It should be noted that the size of the oligomer resulting from the described phenol depletion procedure depends on the process and, for a given formulation, may vary with factors such as time, temperature, and column batch. Therefore, it is desirable that measurements for a given set of formulations be compared within the same experiment and not between experiments. Therefore, for the same reference formulation, it was tested together with several formulations of the invention in Petition 870220115194, dated 09 / 12 / 2022, page 33 / 64 19 / 44 different experiments. For example, Formulation A vs. Formulation B and C; Formulation 01 vs. Formulations 02-06.

[0069] The apparent mean hydrodynamic radius (γη) was measured by Dynamic Light Scattering (DLS) using a DynaPro PR™ (Wyatt technology, Santa Barbara, CA, USA). Prior to analysis, the samples were centrifuged for 5 minutes at 1200 rpm to remove any dust particles in the solution. Measurements were performed at 25°C using 40 acquisitions and a 2-second acquisition time.

[0070] Apparent average molecular weight (kDa) measured by Composition Gradient Multi-Angle Static Light Scattering (CGMALS) using a system consisting of a Wyatt Technology Corporation (WTC) Calypso II titrator unit coupled to a WTC DAWN8+ light scattering detector (operating at 664 nm) and a WTC Optilab T-rEX refractometer (operating at 660 nm) at 25°C. Samples were filtered through a 0.45 pm filter followed by a 0.22 pm filter before measurement.

[0071] Sedimentation velocity (SV) experiments were performed using an XL-I Analytical Ultracentrifuge (BeckmanCoulter, Brea, CA) on 12-mm or 3-mm double-sector centerpieces lined with sapphire windows. Samples were spun at 40,000 rpm and 20°C until sedimentation was complete and monitored with the instrument's interference optics. Sedimentation Coefficient (SCD) Distributions were calculated using SedFit, version 11.8 (www.analyticalultracentrifugation.com) using the c(s) model with a grid of 100 s values ​​over a range sufficient to describe all sedimentation material, as assessed by rmsd and residual run pattern. The frictional ratio f / fo was treated as a variable to be optimized during the adjustment (P Schuck, MA Perugini, NR Gonzales, GJ Howlett and D Schubert: Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems; (Biophys. J. Petition 870220115194, dated 09 / 12 / 2022, page 34 / 64 20 / 44 2002 82:1096). The average values ​​of the sedimentation coefficient were obtained by integrating the resulting c(s) distributions.

[0072] Temperature-dependent dynamic viscosities were measured using a Lovis2000 rolling ball viscometer (Anton Paar, Graz, Austria). The temperature was decreased from 40°C to 4°C in 2°C steps, allowing 5 minutes for temperature equilibration between steps. The buffer density was measured simultaneously using a DMA5000 densitometer, also from Anton Paar.

[0073] Three monoformulations were prepared, namely, Formulation A representative of the state of the art (see, for example, WO 2013 / 153000), and Formulations B, C representative of the invention (60 mM phenol / 10 mM m-cresol). Table 1A: Comparative monoformulations A, B and C Ingredient Formulation A Representative of the state of the art Formulation B Representative of the invention Formulation C Representative of the invention Compound 1 4200 nmol / mL (4.2 mM) 4200 nmol / mL (4.2 mM) 4200 nmol / mL (4.2 mM) Zinc (as zinc acetate) 206 pg / mL (~4.5 Zn++ / hexamer) 110 pg / mL (~2.4 Zn++ / hexamer) 101 pg / mL (~2.2 Zn++ / hexamer) Glycerol 16 mg / mL (1.6%) 15 mg / mL (1.5%) 15 mg / mL (1.5%) Phenol 2.35 mg / mL (25 mM) 5.65 mg / mL (60 mM) 5.65 mg / mL (60 mM) Meta-cresol 2.70 mg / mL (25 mM) 1.08 mg / mL (10 mM) 1.08 mg / mL (10 mM) Sodium chloride 1.17 mg / mL (20 mM) 1.17 mg / mL (20 mM) 1.17 mg / mL (20 mM) pH 7.4 7.4 7.4 Petition 870220115194, dated 09 / 12 / 2022, page 35 / 64 21 / 44

[0074] Six other monoformulations, namely Formulation 01 (identical to Formulation A) and Formulations 02, 03, 04, 05 and 06 representative of the invention were produced (Table 1B). The zinc content varied from 4.5 Zn++ / six insulins to 2.4, 2.2, and 2.0 Zn++ / six insulins. In addition, preservative systems of 25 / 25 mM phenol / m-cresol or 60 / 0 mM phenol / m-cresol were tested. Table 1B: Comparative Formulations Ingredient Formulation 01* 02 03 04 05 06 Compound 1 (mM) 4.2 4.2 4.2 4.2 4.2 4.2 Zn(acetate)2 (pg / mL) 206 206 101 101 110 92 n Zn / 6 insulin (mol / mol) ~4.5 ~4.5 ~2.2 ~2.2 ~2.4 ~2.0 Phenol 25 mM 60 mM 25 mM 60 mM 60 mM 60 mM m-cresol 25 mM 0 25 mM 0 0 0 glycerol 1.6% 1.5% 1.6% 1.5% 1.5% 1.5% Vehicle (all formulations) 20 mM NaCl pH 7.4 * Same as Formulation A Improved oligomer size of the exchanged buffer monoformulations containing 60 mM phenol and 10 mM cresol (Table 2A; Figures 1A, 1B, 1C) Table 2A: Oligomer size at simulated injection site AB Ç Rh(average) [nm] 37.31 9.21 8.37 S*(average) [SI] 17.32 10.53 9.41 Mw (average) [kPa] 1059.8 190.5 184.7 Petition 870220115194, dated 09 / 12 / 2022, page 36 / 64 22 / 44 Improved oligomer size of the buffer-exchanged monoformulations containing 60 mM phenol and 0 m-cresol (Table 2B; Figures 2A and 2B) Table 2B Oligomer size at simulated injection site 01 02 03 04 05 06 Rh(average) [nm] 19.42 14.20 5.83 4.51 5.20 3.98 S*(average) [S] 8.46 11.00 5.39 4.78 5.33 4.32 Conclusion

[0075] The zinc content was reduced from 4.5 Zn++ / six insulins in Formulation A to 2.4 and 2.2 Zn++ / six insulins in Formulations B and C (with increased phenol and reduced metacresol) respectively. This decrease in zinc was accompanied by a reduction in oligomer size (see Figures 1A, 1B and 1C), as determined under simulated injection site conditions by the method described above.

[0076] The results of formulation 01-06 further confirm that the oligomer size is reduced when Zn is reduced from 4.5 Zn++ / six insulins to 2.2 ± 0.2 Zn++ / six insulins. For 2.2 ± 0.2 Zn++ / six insulins, the oligomer size is further reduced when phenol / cresol is reduced from 25mM / 25mM to 60mM / 0mM. See Figures 2A and 2B. Improved viscosity of monoformulations with exchanged buffer having 60 mM phenol and 10 mM m-cresol (see Table 3A; Figures 3A and 3B) Table 3A: Viscosity of Formulations A, B, C with buffer change Dynamic viscosity (cP) Specific viscosity Formulation Formulation temp (C) ABC Buffer ISF ABC 40 1.194 0.633 0.628 0.605 0.9735 5 0.0462 8 0.0380 2 38 1.359 0.659 0.653 0.627 1.1674 6 0.0510 4 0.0414 7 36 1.548 0.687 0.68 0.651 1.3778 0.0553 0.0445 Petition 870220115194, dated 09 / 12 / 2022, page 37 / 64 23 / 44 Dynamic viscosity (cP) Specific viscosity Formulation Formulation temp (C) ABC Buffer ISF ABC 8 5 34 1.758 0.718 0.709 0.677 1.5967 5 0.0605 6 0.0472 7 32 1.989 0.751 0.74 0.704 1.8252 8 0.0667 6 0.0511 4 30 2.237 0.787 0.773 0.733 2.0518 4 0.0736 7 0.0545 7 28 2.506 0.825 0.809 0.765 2.2758 2 0.0784 3 0.0575 2 26 2.792 0.866 0.847 0.798 2.4987 5 0.0852 1 0.0614 24 3.098 0.91 0.889 0.835 2.7101 8 0.0898 2 0.0646 7 22 3.409 0.958 0.934 0.874 2.9004 6 0.0961 1 0.0686 5 20 3.729 1.01 0.982 0.916 3.0709 6 0.1026 2 0.0720 5 18 4.051 1.066 1.035 0.962 3.2110 2 0.1081 1 0.0758 8 16 4.321 1.126 1.092 1.012 3.2697 6 0.1126 5 0.0790 5 14 4.597 1.191 1.153 1.066 3.3123 8 0.1172 6 0.0816 1 12 4.865 1.262 1.22 1.125 3.3244 4 0.1217 8 0.0844 4 10 5.131 1.338 1.293 1.19 3.3117 6 0.1243 7 0.0865 5 8 5.369 1.421 1.372 1.26 3.2611 0.1277 0.0888 Petition 870220115194, dated 09 / 12 / 2022, page 38 / 64 24 / 44 Dynamic viscosity (cP) Specific viscosity Formulation Formulation temp (C) ABC Buffer ISF ABC 1 8 9 6 5.593 1.514 1.458 1.338 3.1801 2 0.1315 4 0.0896 9 4 5.795 1.612 1.556 1.424 3.0695 2 0.1320 2 0.0927 Improved viscosity of monoformulations with exchanged buffer containing 60 mM phenol and 0 m-cresol (see Table 3B; Figures 3C and 3D) Table 3B: Viscosity of Formulations with buffer changes 02, 03 and 04. Dynamic viscosity (cP) Specific viscosity Formulation Formulation ISF _02 _03 _04 _02 _03 _04 3.99 1.5447 2.7441 1.9996 1.6278 0.77646 0.29449 0.0538 6 1.4513 2.5087 1.8529 1.5296 0.72859 0.27672 0.05395 8 1.3668 2.3492 1.7355 1.4414 0.71876 0.26975 0.05458 10 1.2917 2.2389 1.6335 1.3615 0.7333 0.26461 0.05404 12 1.2209 2.1624 1.5448 1.2894 0.77115 0.2653 0.05611 14 1.1555 2.1078 1.4678 1.2215 0.82415 0.27027 0.05712 16 1.095 2.06 1.4063 1.1575 0.88128 0.28429 0.05708 18 1.04 2.0041 1.3516 1.0984 0.92702 0.29962 0.05615 20 0.9893 1.927 1.3014 1.043 0.94784 0.31548 0.05428 22 0.9423 1.8351 1.247 0.9916 0.94747 0.32336 0.05232 24 0.8991 1.7221 1.1941 0.9447 0.91536 0.32811 0.05072 26 0.8588 1.597 1.1404 0.9003 0.85957 0.3279 0.04832 28 0.8216 1.4748 1.085 0.8594 0.79503 0.32059 0.04601 30 0.7869 1.3608 1.0249 0.8219 0.72932 0.30245 0.04448 32 0.7544 1.2594 0.9713 0.7867 0.66941 0.28751 0.04282 34 0.7239 1.1654 0.9215 0.754 0.60989 0.27297 0.04158 Petition 870220115194, dated 09 / 12 / 2022, page 39 / 64 25 / 44 Dynamic viscosity (cP) Specific viscosity Formulation Formulation ISF _02 _03 _04 _02 _03 _04 36 0.7004 1.0795 0.8749 0.7236 0.54126 0.24914 0.03312 38 0.6951 1.0027 0.8321 0.6948 0.44253 0.19709 -4.32E- 04 40 0.7107 0.9345 0.7927 0.6682 0.3149 0.11538 -0.0598 Conclusion

[0077] These experiments show that the viscosity of the formulation under simulated injection site conditions according to this method is highly dependent on the zinc content, such that decreasing the zinc ratio leads to lower viscosity. Example 2 tmax and ti / 2 elimination

[0078] tmax represents the time to maximum concentration (maximum plasma exposure), et% represents the elimination half-life, in which half of the compound disappears from the plasma after administration.

[0079] The altered biophysical properties described in Example 1 are consistent with a PK profile showing the earlier tmax in pigs (see Table 3). This indicates that the residence time of Compound 1 in the subcutis is reduced when formulated according to the invention, and in contrast to the same compound being provided in a formulation according to the prior art.

[0080] Surprisingly, the reduced zinc content had almost no impact on the duration of action (i.e., the elimination t1 / 2 was not affected), which makes the formulation according to the invention and the prior art formulation equally suitable for once-weekly administration. Petition 870220115194, dated 09 / 12 / 2022, pp. 40 / 64 26 / 44 Table 4 Formulation Species tmax (hours) ti / 2 (hours) Compound 1, Formulation A 4.5 zinc / hexamer, 25 mM phenol, 25 mM m-cresol Pig 20 47 Human 42 185 Compound 1, Formulation C 2.2 zinc / hexamer, 60 mM phenol, 10 mM m-cresol, 20 mM NaCl Pig 8 45 Conclusion

[0081] These experiments show that the residence time in the subcutis (tmax) of Compound 1 was significantly reduced as a result of the reduction in Zn++ / hexamer. This observation is consistent with the data presented in Example 1, which show a reduction in the size of the oligomers formed under the simulated injection site conditions.

[0082] Thus, when formulated according to the invention, Compound 1 forms smaller oligomers at the injection site, resulting in a PK / PD profile with a shorter residence time in the subcutis (reduced tmax).

[0083] But surprisingly, although the subcutaneous residence time (tmax) of Compound 1 was significantly reduced, the formulations of the invention maintained the same elimination half-life (the elimination ti / 2 was not affected). This means that the formulations of the invention could facilitate long-acting insulin derivatives reaching maximum circulating concentration more quickly, while still maintaining maximum concentration for a longer duration.

[0084] This unexpected finding also makes it possible to limit the formation of large oligomers at the injection site, while still maintaining a long duration of action compatible with a once-weekly administration profile. The formation of large oligomers with high Petition 870220115194, dated 09 / 12 / 2022, page 41 / 64 27 / 44 viscosity at the injection site may cause discomfort after injection. Example 3 Improved conformal state in the formulation.

[0085] With the presence of zinc, human insulin exists as hexamers in the formulation. Human insulin hexamers can adopt two different conformational states, depending on the conformation of the monomers. The eight N-terminal amino acid residues of the B chain of the insulin monomer can be in an extended conformation (T state) or in an α-helical conformation (R state). In the presence of phenol and NaCl, human insulin adopts the R conformation, which is the favorable conformation with respect to physical and chemical stability (Dunn MF. Zinc-ligand interactions modulate assembly and stability of the insulin hexamer: A review. Biometals 2005; 18; 295-303). Protocol

[0086] Conformational changes of the insulin hexamer were tracked by the CD signature at 251 nm (Krüger P, Gilge G, Cabuk Y and Wollmer A; Biol. Chem. Hoppe-Seyler 1990 371 669-673). Samples were measured using a Jasco 815 instrument and a path length of 0.02 cm. Blank titrations were subtracted and the resulting Δε 251nm was plotted graphically vs Zn / 6Ins (mol / mol). Conformational state of monoformulations containing 60 mM phenol / 0 m-cresol

[0087] The conformational state of Formulations 01-06 is shown in Table 5A and Figure 4A. Table 5A: Propensity of the conformational state in Formulation 01-06 with 60 mM phenol / 0 m-cresol Formulation 01 02 03 04 05 06 Propensity of the state R Δε (M-1cm-1) at 251 nm -6.15 -6.65 -7.63 -8.12 -7.98 -8.19 Petition 870220115194, dated 09 / 12 / 2022, page 42 / 64 28 / 44

[0088] Figure 4A shows the conformational changes (T state; mixed TR state; R state) as a function of Zn++ for six insulins (mol:mol) for Formulations 01-06. Conformational state of the monoformulations containing 60 mM phenol and 10 mM mcresol.

[0089] Two series of formulations were prepared. All formulations contained 4.2 mM of Compound 1, 16 mg / mL (1.6%) glycerol, 20 mM NaCl, with a pH of 7.4. Formulations in series B contained 60 mM phenol and 10 mM m-cresol, with varying zinc content as 1.5 (B1), 2.0 (B2), 2.3 (B3), 2.5 (B4), 3.0 (B5), and 4.0 (B6) for 6 insulins. Series D contained 25 mM phenol and 25 mM m-cresol, with varying zinc content as 1.5, 2.0, 2.3, 2.5, 3.0, 4.0, and 4.5 for 6 insulins. A series of human insulin formulations with varying zinc contents was also prepared. The conformational state is shown in Table 5B and Figure 4B. Table 5B. Propensity of the conformational state in Formulations B1-B6 with 60 mM phenol and 10 m-cresol, and Formulations D1-D7 with 25 mM phenol and m-cresol. Propensity of the R-state Δε (M-1cm-1) at 251 nm Series B Series D Zn / 6Ins 60 / 10 fe / cre 25 / 25 fe / cre *1.5 -6.83 (B1) -6.58 (D1) 2.0 -7.67 (B2) -7.06 (D2) 2.3 -7.42 (B3) -6.88 (D3) 2.5 -7.23 (B4) -6.67 (D4) 3.0 -7.54 (B5) -6.45 (D5) 4.0 -6.72 (B6) -5.58 (D6) 4.5 na -5.03 (D7) * Not included in Figure 4B

[0090] Figure 4B shows the conformational changes Petition 870220115194, dated 09 / 12 / 2022, pp. 43 / 64 29 / 44 (T state; mixed TR state; R state) as a function of Zn++ for six insulins (mol:mol) for Series B and Series D formulations, and for human insulin formulations. Conclusion

[0091] Near-UV CD data show that the T / R conformation of Compound 1 was dependent on zinc concentration. A reduction in zinc was accompanied by a change in the conformational state of Compound 1 in the formulation from a mixed T / R state to the R state, which was also accompanied by a higher hexamer content formed in the formulations (see Example 4 below). The R state and hexamer content were further increased by a change in the phenol / meta-cresol ratio from 25 / 25 mM to 60 / 10 mM (Figure 4B).

[0092] The data from Formulations 01-06 show that the R state of Compound 1 in the formulations is further enriched by the reduction of Zn from 4.5 to 2.2Zn ± 0.2Zn / 6 of Compound 1 and by the omission of m-cresol (Figure 4A). Example 4 Improved distribution of oligomers in the formulation Protocol

[0093] Size exclusion chromatography is a sensitive method for quantifying the distribution of non-covalent oligomers in insulin formulations. SEC was performed using a BEH200 column, 1.7 µm, 4.6 x 150 mm, with a running buffer consisting of 8.0 mM phenol, 140 mM NaCl, 10 mM Tris-HCl, pH 7.4. Chromatography was performed at 22°C using an injection volume of 2 µL and a flow rate of 0.3 mL / min. Albumin, a covalent insulin hexamer, and a monomeric insulin were used as molecular weight standards. Chromatograms were analyzed by integration to represent species larger than hexamer (3.0-3.8 min), hexamer (3.8 Petition 870220115194, dated 09 / 12 / 2022, pp. 44 / 64 30 / 44 4.3 min), and species smaller than hexamer (4.3-5.5 min). Note that the exact integration limits for each dataset will vary slightly due to variations in column performance.

[0094] Small-angle X-ray scattering (SAXS) data were collected using a BioSAXS-2000 instrument equipped with a flow cell and a Dectris 100K detector covering a q-range of 0.0080.661 A-1. Buffer measurements were subtracted from sample measurements to obtain protein scattering profiles. Data from a reference sample of human insulin in a hexameric state with the R conformation were collected according to the same procedure, from a sample of 0.6 mM human insulin, 3 Zn++ / six insulins, 16 mM phenol, 20 mM NaCl, and 7 mM phosphate buffer at pH 7.4. Distribution of oligomers for formulations comprising 60 mM phenol and 0 m-cresol.

[0095] The native SEC chromatogram comparing Formulations 01, 02, 03, 04, 05 and 06 was generated (see Table 6 and Figure 5A). Table 6: Species distribution as obtained by native SEC by chromatogram integration Peak Assignment Formulation 01 02 03 04 05 06 % of species in the formulation (3.0-3.8 min) Greater than hexamer 8.8 9.4 1.1 0.6 0.7 0.2 (3.8-4.3 min) Hexamer 34.5 48.9 95.1 98.1 95.9 98.9 (4.3-5.5 min) Less than hexamer 56.8 41.7 3.8 1.3 3.5 0.9 Distribution of oligomers for formulations comprising 60 mM phenol and 10 mM m-cresol. Petition 870220115194, dated 09 / 12 / 2022, pp. 45 / 64 31 / 44

[0096] The native SEC chromatogram comparing Formulations B1-B6 and Formulation A was generated (see Table 6 and Figure 5B). The area under the curve is similar for all chromatograms. The chromatograms were analyzed by integration to represent species larger than hexamer (3.0-3.8 min), hexamer (3.8-4.3 min), and species smaller than hexamer (4.3-5.5 min). Note that the exact integration limits for each dataset will vary slightly due to variations in column performance. Table 7: Distribution of oligomers by SEC Peak Assignment Formulation B1 B2 B3 B4 B5 B6 A % of species in the formulation Greater than hexamer 0.2 0.5 0.8 0.7 2.2 4.9 18.2 Hexamer 80.3 94.8 86.7 82.1 65.5 41.8 22.0 Less than hexamer 19.5 4.8 12.5 17.2 32.3 53.3 59.8 Conclusion

[0097] SEC data for Formulations 01-06 (Table 6, Figure 5A) show that the oligomer distribution of Formulation 01 of Compound 1 is characterized by broad bands without clear dominant oligomeric species. The oligomer distribution of Compound 1 in Formulations 03-06 is narrower compared to Formulations 01 and 02. The retention time of the main peak in Formulations 03-06 is consistent with a hexamer, and the small peak with a retention time of 5 minutes is consistent with a monomer or dimer.

[0098] SEC data from Formulations B1-B6 and A (Table 7, Figure 5B) also show that, with 2.0-2.5 zinc / 6 insulins, the hexamer peak is enriched, compared to 4.0 or 4.5 Zn. Petition 870220115194, dated 09 / 12 / 2022, pp. 46 / 64 32 / 44

[0096] Therefore, the hexamer peak is enriched at low zinc content (e.g., 2.0Zn to 2.5Zn) compared to high zinc content (e.g., 4.0 or 4.5Zn). For 4.5Zn and 2.2Zn, the hexamer is enriched at 60mM / 0 or 10mM phenol / m-cresol relative to 25 / 25 mM phenol / m-cresol. When formulated according to the invention, the hexamer content is increased and the oligomerization becomes more well-defined.

[0099] SAXS data also confirm that the oligomerization pattern of Formulation A of Compound 1 with 4,5 Zn++ / hexamer does not resemble the classic human insulin hexamer, while a hexamer-based structure of Compound 1 is dominant in Formulation C (Figure 6). When formulated according to the invention (Formulation C), the oligomerization pattern becomes more well-defined and consistent with a hexamer-based structure similar to human insulin. Example 5 Improved chemical and physical stability

[00100] The experimental results in this example showed that both the chemical and physical stability of Compound 1 in the new formulations are improved compared to the reference Formulation A. In particular, with the high phenol content and low m-cresol content, both the chemical and physical stability of Compound 1 increase as the zinc concentration decreases. Protocol

[00101] Purity was determined by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC), in which samples were analyzed using an Acquity CSH Fluoro Phenyl, 130A, 1.7 μm, 2.1x150 mm column, acetonitrile gradient elution in an acetonitrile mobile phase and phosphoric buffer in water with subsequent UV detection (215 nm) at a flow rate of 0.30 mL / min with a sample injection volume of Petition 870220115194, dated 09 / 12 / 2022, page 47 / 64 33 / 44 2-3 pL. Purity was assessed as the area of ​​the main peak divided by the area of ​​all peaks x 100%. Chemical stability of the Compound 1 formulation with 60 mM phenol and 10 mM m-cresol.

[00102] Stability measured as % purity of total peptides with RP-UHPLC shows a significantly increased stability of Compound 1 in Formulation B and Formulation C, which contained 60 mM phenol and 10 mM m-cresol, compared to Formulation A of Compound 1, which contained 25 mM phenol and 25 mM m-cresol. See Table 8 and Figure 7A. Table 8: % Purity of Compound 1 Formulation Storage time at 30°C (months) 0 1 2 3 A 95.4 93.8 93.3 91.3 B 95.3 94.3 95.0 94.3 C 95.7 94.7 95.0 94.6 Chemical stability of Compound 1 formulations with 60 mM phenol and 0 m-cresol.

[00103] The stability of the formulations presented in the table, measured as % purity of total peptides with RP-UHPLC, further confirms that the chemical stability of Compound 1 increases as a function of zinc concentration in formulations with relatively high phenol content and low m-cresol content. The results show increased stability of Compound 1 when zinc was reduced from 4.5 Zn++ / six insulins to 2.4, 2.2, and 2.0 Zn++ / six insulins in formulations with a change in the preservative system from 25 / 25 mM phenol / m-cresol to 60 mM phenol and 0 m-cresol. See Table 9 and Figure 7B. Petition 870220115194, dated 09 / 12 / 2022, pp. 48 / 64 34 / 44 Table 9: % Purity of Compound 1 Formulation Storage time at 37°C (weeks) 0 2 4 6 01 97.5 96.1 94.3 92.8 04 97.4 96.7 96.0 95.3 05 97.6 96.7 96.0 95.3 06 97.3 96.7 96.0 95.2 Conclusion

[00104] When formulated according to the invention (see Formulations B, C, 04, 05 and 06; Tables 8 and 9, and Figure 7A and Figure 7B), the chemical stability increases compared with the prior art (Formulations A and Formulation 01). Physical stability Protocol:

[00105] The formulations of Compound 1 were tested in a 96-well microtiter plate with 4 replicates of 200 pL. To 1.0 mL of each formulation, ThT (thioflavin T) was added at 1 μM. The thioflavin T (ThT) assay for amyloid fibril formation propensity was performed in a Thermo Fluoroskan, with agitation at 960 rpm, 37°C, for 45 hours. ThT emission was examined before and after the assay. The lag time until the onset of ThT fluorescence emission is a measure of physical stability. Lag times were determined from fluorescence curves averaged over 4 replicates. A longer lag time is indicative of greater physical stability.

[00106] It should be noted that the ThT results obtained from the described protocol may vary between experiments. Therefore, it is desirable that measurements for a given set of formulations be compared within the same experiment and not between experiments. In this example, the same reference formulation was tested together. Petition 870220115194, dated 09 / 12 / 2022, page 49 / 64 35 / 44 with various formulations of the invention in different experiments. For example, Formulation A vs. Formulation C; Formulation 01 vs. Formulations 04-06.

[00107] Latency times are shown in Table 10 below. Formulation A or Formulation 01 as the reference formulation for comparison.

[00108] The results are shown in Table 10A and Table 10B. Physical stability of Compound 1 in formulations with 60 mM phenol and 10 mcresol. Conclusion

[00109] The latency times obtained in the ThT assay indicate that the physical stability of Compound 1 is improved in formulations with low zinc content, high phenol content, and low m-cresol content. The data showed that reducing zinc from 4.5Zn / 6lns to 2.2 ±0.2Zn / 6lns and concomitantly increasing phenol from 25 mM to 60 mM and reducing m-cresol from 25 mM to 10 mM lead to a longer latency time and thus improved physical stability. With m-cresol removed, the physical stabilization of Compound 1 was further improved in formulations with low zinc content (see Table 10B). Example 6 Combined formulations and their chemical and physical stabilities

[00110] Compound 1 can be co-formulated together with Petition 870220115194, dated 09 / 12 / 2022, pages 50 / 64 36 / 44 a semaglutide GLP-1 analogue once weekly for a fixed-ratio combination.

[00111] The following combined formulations 1 to 6 of Compound 1 and semaglutide were prepared. The monoformulation of Compound 1 was also prepared as a reference monoformulation 1. The intended target values ​​are shown in Table 11 below. Table 11: Combined formulations of Compound 1 and semaglutide Monoformulation Reference 1 Combination 1 Combination 2 Combination 3 Combination 4 Combination 5 Combination 6 Compound 1 (mM) 4.2 4.2 4.2 4.2 4.2 4.2 4.2 Semaglutide (mM) / (mg / mL) na 0.49 / 2.0 0.4 / 1.6 0.6 / 2.4 0.3 / 1.2 5 0.49 / 2.0 0.49 / 2.0 Zn(acetate)2 (mM) 1.54 1.54 1.54 1.54 1.54 1.4 1.75 n Zn / 6 insulin ~2.2 ~2.2 ~2.2 ~2.2 ~2.2 ~2.0 ~2.5 Vehicle (all formulations) 60 mM phenol 10 mM m-cresol 1.5% glycerol 20 mM NaCl pH 7.4

[00112] The concentrations of Compound 1 and semaglutide in the produced formulations were measured using RP-HPLC and reference materials. These concentrations are indicated in Tables 12A and 12B below. Petition 870220115194, dated 09 / 12 / 2022, pp. 51 / 64 37 / 44 Table 12A: Measured concentrations of Compound 1 and semaq utida Monoformulation Reference-1 Combination 1 Combination 2 Combination 3 Combination 4 Compound 1 (mM) Measured 4.1 4.1 4.1 4.1 4.1 Semaglutide (mM) Measured 0.5 0.4 0.6 0.3 Table 12B: Measured concentrations of Compound 1 and semaqualide (a separate batch from Table 12A) Reference Monoformulation-1 Combination 5 Combination 6 Compound 1 (mM) Measured 4.2 4.3 4.3 Semaglutide (mM) Measured 0.49 0.49

[00113] The measured concentrations, therefore, deviated by less than 3% from the intended target values.

[00114] The following combined l-VI formulations of Compound 1 and semaglutide were also subsequently prepared. The Compound 1 monoformulation was prepared as a reference for monoformulation 2. The intended target values ​​are shown in Table 13 below. Petition 870220115194, dated 09 / 12 / 2022, pages 52 / 64 38 / 44 Table 13 Monoformulation Reference 2 Combination I Combination II Combination III Combination IV Combination V Combination VI Compound 1 (mM) 4.2 4.2 4.2 4.2 4.2 4.2 4.2 Semaglutide (mM) / (mg / mL) na 0.49 / 2.0 0.49 / 2.0 0.3 / 1.25 0.49 / 2.0 0.49 / 2.0 0.3 / 1.25 Zn(acetate)2 (pg / mL) 101 206 101 101 101 110 110 n Zn / 6 insulin ~2.2 ~4.5 ~2.2 ~2.2 ~2.2 ~2.4 ~2.4 Phenol 60 mM 25 mM 60 mM 60 mM 25 mM 60 mM 60 mM m-cresol 0 25 mM 0 0 25 mM 0 0 Vehicle (all formulations) 1.5% glycerol 20 mM NaCl pH 7.4 Physical stabilization Protocol:

[00115] The formulations were tested in a 96-well microtiter plate with 8 replicates of 200 pL. To 1.0 mL of each formulation, ThT (thioflavin T) was added at 1 pM. The thioflavin T (ThT) assay for propensity to form amyloid fibrils was performed on a Thermo Fluoroskan, with Petition 870220115194, dated 09 / 12 / 2022, pp. 53 / 64 39 / 44 agitation at 960 rpm, 37°C, for 45 hours. ThT emission was examined before and after the assay. The latency time until the onset of ThT fluorescence emission (formation of amyloid fibrils) is a measure of physical stability. Latency times were determined from fluorescence curves averaged over 8 replicates. Latency times were tested twice for combined formulations 1-4 and combined formulations 5 and 6, respectively; and each with a tested monoformulation as a reference, to make the results comparable. A longer latency time is indicative of greater physical stability.

[0097] The reference monoformulation of Compound 1 and the combined formulations were tested in a 96-well microtiter plate Thioflavin T (ThT) assay for amyloid fibril formation propensity.

[00116] Latency times are shown in Tables 14A, 14B, and 14C below. Table 14A: Combined formulation latency times Monoformulation Reference 1 Combination 1 Combination 2 Combination 3 Combination 4 Latency time (hours) 11.0 19.3 18.0 21.6 23 Table 14B: Combined formulation latency times Monoformulation Reference 1 Combination 5 Combination 6 Latency time (hours) 9.9 20.6 45 Petition 870220115194, dated 09 / 12 / 2022, pages 54 / 64 40 / 44 Table 14C: Combined formulation latency times Reference monoformulation 2 Combination 1 Combination II Combination III Combination IV Combination V Combination VI Latency time (hours) 29.3 44.3 45 45 28.6 45 45 Conclusion:

[00117] The ThT assay indicated that, without increasing the zinc content, the combined formulations of Compound 1 and semaglutide did not compromise the physical stability of Compound 1 compared to that of the Compound 1 monoformulation. In fact, the latency times of the combined formulations were much longer than that of the Compound 1 monoformulation, showing that the co-formulation of Compound 1 with semaglutide actually stabilizes the formulation with respect to the undesirable formation of amyloid fibrils. Compared to the combined formulation of another long-acting insulin derivative and a GLP-1 derivative (e.g., degludec and liraglutide), this finding is unexpected and surprising.

[00118] The results in Table 14C show that decreasing the m-cresol content can further improve the physical stability of the combined formulation of Compound 1 and semaglutide; and increasing the phenol content also improves the physical stability of the combined formulation of Compound 1 and semaglutide.

[00119] The results in Table 14C also show that, Petition 870220115194, dated 09 / 12 / 2022, pages 55 / 64 41 / 44 when co-formulating Compound 1 with semaglutide in a formulation according to the invention, the physical stability of Compound 1 increases compared to using the prior art formulation (Formulation I) for the combined formulation of Compound 1 and semaglutide. Chemical stabilization Protocol

[00120] Purity was determined by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC), in which samples were analyzed using an Acquity CSH Fluoro Phenyl, 130A, 1.7 µm, 2.1x150 mm column, acetonitrile gradient elution in an acetonitrile mobile phase and phosphoric buffer in water with subsequent UV detection (215 nm) at a flow rate of 0.30 mL / min with a sample injection volume of 2-3 µL. Purity was assessed as the area of ​​the main peak divided by the area of ​​all peaks x 100%.

[00121] The stability of the formulations presented in Table 15 is measured as % purity of total Compound 1 using RP-UHPLC.

[00122] The results confirm greater chemical stability of Compound 1 in the combined formulations when zinc is reduced from 4.5 Zn++ / six insulins to 2.4, 2.2, and 2.0 Zn++ / six insulins (Table 15, Figure 8). A change in the preservative system from 25 / 25 mM phenol / m-cresol to 60 mM phenol and 0 m-cresol results in a further improvement in the chemical stability of Compound 1 in the combined formulation. Table 15: Purity of Compound 1 in the combined formulation Combined Formulation Purity with storage time at 37°C (weeks) 0 2 4 6 I 97.5% 96.1% 94.4% 92.9% II 97.6% 96.6% 95.8% 95.0% III 97.6% 96.7% 95.9% 95.2% IV 97.2% 96.4% 95.4% 94.4% Petition 870220115194, dated 09 / 12 / 2022, pages 56 / 64 42 / 44 Combined formulation Purity with storage time at 37°C (weeks) 0 2 4 6 V 97.3% 96.6% 95.9% 95.1% VI 97.3% 96.6% 95.9% 95.2% Conclusion

[00123] By co-formulating Compound 1 with semaglutide in a formulation according to the invention, the chemical stability of Compound 1 increases compared with the use of the prior art formulation (Formulation I) for the combined formulation of Compound 1 and semaglutide. Example 7 PK properties of co-formulations with semaglutide in the LYD porcine PK model

[00124] Of the formulations produced in Example 6, the monoformulation and reference of Compound 1 and Combination 1 and Combination 2 were characterized in the LYD pig PK animal model. Importantly, the PK parameters, tmax and t% of Compound 1, as well as the mean residence time (MRT) of Compound 1, were not significantly altered after coformulation with semaglutide.

[00125] A crossover study with 16 animals (n=8 for each formulation) was performed. Table 16: PK Parameters of Compound 1 Reference Monoformulation 1 Combination 1 Combination 2 tmax (hours) 13.0 ±7.0 9.0 ±2.0 8.0 ±2.0 t% (hours) 48.0 ±4.1 48.6 ±3.2 47.9 ±5.3 MRT (hours) 71 ±6 69.0 ±4.0 68.0 ±4.0 Mean values ​​plus standard deviation are shown.

[00126] The PK parameters for Compound 1, when Petition 870220115194, dated 09 / 12 / 2022, pages 57 / 64 43 / 44 co-formulated with semaglutide in Combination 1 and Combination 2, were not significantly altered compared to Compound 1 administered as a monoformulation. The tmax values ​​were slightly lower for the co-formulations, but with the standard deviation to the reference of Compound 1, the values ​​overlap. The t% and MRT were very similar for Compound 1 in the co-formulations compared to the reference monoformulation. In conclusion, the PK properties of Compound 1 were not significantly affected by co-formulation with semaglutide. Example 8 Improved size of oligomers in co-formulations with 60 / 0 buffer exchanged

[00127] The oligomerization of the combined I-VI formulations formed under simulated conditions at the Compound 1 injection site was determined according to the protocol described in Example 1. The results are shown in Table 17 and Figure 9A and Figure 9B. Table 17: Oligomer size of the combined formulations Combination I Combination II Combination III Combination IV Combination V Combination VI Rh(average) [nm] 13.7 4.1 4.0 4.6 4.5 4.7 S*(average) [S] 8.32 4.30 4.40 4.72 4.67 4.96 Conclusion

[00128] These experiments show that the size of the oligomers formed under the simulated injection site conditions is highly dependent on the zinc content.

[00129] The average size of oligomers formed from combined formulations under simulated injection site conditions is significantly reduced in formulations with low zinc content (by Petition 870220115194, dated 09 / 12 / 2022, pages 58 / 64 44 / 44 example, 2.4 and 2.2 Zn++ / six insulins) compared to formulations with high zinc content (e.g., 4.5 Zn++ / six insulins). Increasing the phenol content and decreasing the m-cresol content further reduces the average size of oligomers formed from the combined formulations under simulated injection site conditions. Petition 870220115194, dated 09 / 12 / 2022, pages 59 / 64

Claims

1 / 4 CLAIMS 1. Pharmaceutical composition CHARACTERIZED in that it comprises an insulin derivative selected from the group consisting of: A14E, B16H, B25H, B29K((Nε-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1); A14E, B16H, B25H, B29K(Nε-Hexadecandioyl-γGlu), human insulin desB30 (Compound 2); A14E, B16H, B25H, B29K(Nε-Eicosanedioyl-γGlu), human insulin desB30 (Compound 3); and A14E, B25H, desB27, B29K (Nε-Octadecandioyl-γGlu), human insulin desB30 (Compound 4); and further comprising 1 to 2% (w / w) of glycerol; 45 to 75 mM of phenol; 0 to 19 mM of m-cresol; 1.5 to 2.5 moles of zinc ions per six moles of said insulin derivative; 5 to 50 mM of sodium chloride; and having a pH value in the range of 7.2 to 8.

0.

2. Pharmaceutical composition, according to claim 1, CHARACTERIZED in that the insulin derivative is A14E, B16H, B25H, B29K((Nε-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1).

3. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that the amount of the insulin derivative is in the range of 3.5 to 5.0 mM.

4. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises 45 to 75 mM of phenol, such as 55 mM to 65 mM of phenol; or comprising 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, or 70 mM of phenol.

5. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises from 0 to 19 mM of m-cresol, such as from 0 mM to 15 mM of m-cresol; or comprising 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM of m-cresol.

6. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises less than 25 mM of sodium chloride.

7. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises: 4.0 to 4.5 mM of insulin derivative; 1 to 2% (w / w) of glycerol; 50 to 70 mM of phenol; 0 to 15 mM of m-cresol; 2.0 to 2.5 moles of zinc ions per six moles of insulin derivative; not more than 25 mM of sodium chloride; and having a pH value in the range of 7.2 to 7.

6.

8. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises: 4.2 mM of insulin derivative; 1.5% (w / w) of glycerol; 60 mM of phenol; 0 mM of m-cresol; 2.2 moles of zinc ions per six moles of insulin derivative; 20 mM of sodium chloride; and having a pH value of 7.

4.

9. Pharmaceutical composition, according to claim 1 or 2, Petition 870260062449, dated 06 / 25 / 2026, page 9 / 15 3 / 4 CHARACTERIZED in that it comprises: 4.2 mM of insulin derivative; 1.5% (weight / weight) of glycerol; 60 mM of phenol; 10 mM of m-cresol; 2.2 moles of zinc ions per six moles of insulin derivative; 20 mM of sodium chloride; and having a pH value of 7.

4.

10. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that it comprises: 4.2 mM of A14E, B16H, B25H, B29K((Nε-Eicosanedioyl-γGlu-[2-(2{2-[2-(2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl)), human insulin desB30 (Compound 1); 1.5% (w / w) of glycerol; 60 mM of phenol; 10 mM of m-cresol; 2.2 moles of zinc ions per six moles of insulin derivative; 20 mM of sodium chloride; and having a pH value of 7.

4.

11. Pharmaceutical composition, according to any one of claims 1 to 10, CHARACTERIZED in that it is for use as a medicament for the treatment of a metabolic disorder.

12. Pharmaceutical composition, according to any one of claims 1 to 10, CHARACTERIZED in that it is for use as a medicament for the treatment or relief of a disease, disorder or condition related to diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome (metabolic syndrome X, insulin resistance syndrome). Petition 870260062449, dated 06 / 25 / 2026, page 10 / 15 4 / 4 13. Use of a pharmaceutical composition, as defined in any one of claims 1 to 10, CHARACTERIZED in that it is for the preparation of a medicament for the treatment of a metabolic disorder.

14. Use of a pharmaceutical composition, as defined in any one of claims 1 to 10, CHARACTERIZED by the fact that it is for the preparation of a medicament for the treatment of a disease, disorder or condition related to diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome (metabolic syndrome X, insulin resistance syndrome). Petition 870260062449, dated 06 / 25 / 2026, page 11 / 15

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