Formulation Method

Abstract

Disclosed herein is a method for compounding an amylin receptor agonist and a GLP-1 receptor agonist.Disclosed herein is a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist, and a cyclodextrin comprising hydroxypropyl substitution.The formulation can be used for medical treatment of subjects with overweight or obesity with or without associated comorbidities, diabetes with or without associated comorbidities, cardiovascular disease, non-alcoholic steatohepatitis (NASH), and cognitive impairment such as that caused by Alzheimer's disease.

Description

[Technical field]

[0001] The present invention relates to a method of formulating a pharmaceutical formulation that is a combination of a GLP-1 receptor agonist and an amylin receptor agonist. The pharmaceutical formulation may be used for the medical treatment of subjects with cognitive impairment, such as those caused by overweight or obesity with or without one or more associated comorbidities, diabetes with or without one or more associated comorbidities, one or more cardiovascular diseases, non-alcoholic steatohepatitis (NASH), and / or Alzheimer's disease. [Background technology]

[0002] Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist and is the active pharmaceutical ingredient in Ozempic®, which is indicated (i) as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes, and (ii) to reduce the risk of major adverse cardiovascular events in adults with type 2 diabetes and established cardiovascular disease.

[0003] Semaglutide is also the active pharmaceutical ingredient in Wegovy®. Wegovy® is used to treat weight-related heart disease and strokes over 30 kg / m2 in the presence of at least one weight-related comorbidity. 2 or more than 27kg / m 2 It is presented as an adjunct to a reduced calorie diet and increased physical activity for chronic weight management in adult patients with an initial body mass index (BMI) above 18.

[0004] Ozempic® and Wegovy® are liquid pharmaceutical formulations that contain 8 mM phosphate and have a pH of about 7.4.

[0005] A fixed-dose combination of the amylin receptor agonist caglilintide and the GLP-1 receptor agonist semaglutide has been investigated for the treatment of overweight and obesity (Non-Patent Document 1). The medicinal products studied were in the form of separate liquid pharmaceutical formulations for subcutaneous use, containing either caglilintide or semaglutide.

[0006] So far, it has not been thought possible to combine semaglutide and caglilintide due to the different physicochemical properties of these pharmaceutical active ingredients. Semaglutide, a GLP-1 receptor agonist, has an isoelectric point that is incompatible with the optimal pH of caglilintide, an amylin receptor agonist. Semaglutide is optimally stable at pH 7.4 and previously required to be formulated in neutral to slightly basic solutions with a pH of 7-8 to ensure solubility in aqueous solutions. Caglilintide is optimally stable at pH 4.0 and required to be formulated in acidic solutions, accelerating its rate of chemical degradation as the pH increased. The different physicochemical properties of caglilintide and semaglutide preclude a simple mixture of these two peptides. The same is true for other GLP-1 receptor agonist and amylin receptor agonist combinations where the two have optimal pH ranges where they are incompatible.

[0007] During biopharmaceutical manufacturing, therapeutic proteins are subjected to physical stress and agitation from several production steps, including mixing, filtration, and pumping [2]. This stress can ultimately lead to protein denaturation or aggregation, which can impair product quality by causing a loss of therapeutic efficacy or increased immunogenicity.

[0008] The formulation methods by which protein pharmaceutical preparations are produced must aim to reduce the total stresses to which the protein is subjected in order to ensure sufficient physical stability of the product.

[0009] There is a need in the art for simple methods of producing stable pharmaceutical formulations containing a GLP-1 receptor agonist and an amylin receptor agonist. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Lancet 2021;397:1736-48 [Non-Patent Document 2] Yang, M., 2015. Stress and protein instability during formulation and fill / finish processes. BIOPHARM INTERNATIONAL,28(6),pp.46 Summary of the Invention

[0011] Disclosed herein are methods of combining amylin receptor agonists, such as caglilintide, and GLP-1 receptor agonists, such as semaglutide.

[0012] Disclosed herein is a method for preparing a pharmaceutical formulation comprising an amylin receptor agonist, such as caglilintide, and a GLP-1 receptor agonist, such as semaglutide, the method comprising: Obtaining or preparing a drug substance (DS) containing an amylin receptor agonist as an active pharmaceutical ingredient; Obtaining or preparing a drug substance (DS) containing a GLP-1 receptor agonist as an active pharmaceutical ingredient; preparing a surfactant-free aqueous excipient solution comprising a cyclodextrin containing hydroxypropyl substitution to about 50-80%, e.g., about 65%, of a final predetermined volume, and water for injection (WFI), and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; Dissolving the GLP-1 receptor agonist DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example to about 5.9 to 6.1, preferably about 6.0; dissolving the amylin receptor agonist DS in a mixture of the excipient solution and the GLP-1 receptor agonist; adding a surfactant to a mixture of an excipient solution, a GLP-1 receptor agonist, and an amylin receptor agonist; Optionally, adding water to about 80-99% of the final predetermined volume; Optionally, further adjusting the pH to about 5.5-6.1, e.g., 5.7-6.1, e.g., 5.9-6.1, e.g., 5.8; and Adding water to reach the final desired volume (100%). [Brief description of the drawings]

[0013] [Figure 1] 1-12 depict formulation methods 1-12, with formulation methods 5, 9, 10, and 11 being embodiments of the present invention. [Diagram 2] See legend to Figure 1. [Diagram 3] See legend to Figure 1. [Figure 4] See legend to Figure 1. [Diagram 5] See legend to Figure 1. [Figure 6] See legend to Figure 1. [Figure 7] See legend to Figure 1. [Figure 8] See legend to Figure 1. [Figure 9] See legend to Figure 1. [Figure 10] See legend to Figure 1. [Figure 11] See legend to Figure 1. [Figure 12] See legend to Figure 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Formulation Method 1: FIG. 1 is a flow diagram depicting formulation method 1. 1. HP-B-CD, sorbitol, histidine, and polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Water for injection was added to the solution to reach 95% of the final volume. 6. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 7. Water for injection was added to reach 100% of the final volume before sterile filtering and filling into pre-fillable syringes.

[0015] Formulation method 2: FIG. 2 is a flow diagram depicting formulation method 2. 1. HP-B-CD, sorbitol, histidine, and polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. Caglilintide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. The semaglutide drug substance was dissolved in the solution. 5. Water for injection was added to the solution to reach 95% of the final volume. 6. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 7. Water for injection was added to reach 100% of the final formulation volume before sterile filtering and filling into prefillable syringes.

[0016] Formulation method 3 FIG. 3 is a flow diagram depicting formulation method 3. 1. HP-B-CD, sorbitol, histidine, and polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 3. Caglilintide drug substance was dissolved in the solution. 4. The semaglutide drug substance was dissolved in the solution. 5. Water for injection was added to the solution to reach 95% of the final volume. 6. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 7. Water for injection was added to reach 100% of the final formulation volume before sterile filtering and filling into prefillable syringes.

[0017] Formulation method 4 FIG. 4 is a flow diagram depicting formulation method 4. 1. HP-B-CD, sorbitol, and histidine were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Polysorbate 80 was added and dissolved. 5. Caglilintide drug substance was dissolved in the solution. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0018] Formulation Method 5 FIG. 5 is a flow diagram depicting formulation method 5. 1. HP-B-CD, sorbitol, and histidine were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Polysorbate 80 was added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0019] Formulation Method 6 FIG. 6 is a flow diagram depicting formulation method 6. 1. HP-B-CD, sorbitol, and histidine were dissolved in water for injection (60% of the final volume). 2. Caglilintide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. The semaglutide drug substance was dissolved in the solution. 5. Polysorbate 80 was added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0020] Formulation Method 7 FIG. 7 is a flow diagram depicting formulation method 7. 1. HP-B-CD, sorbitol, and polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Histidine was added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0021] Formulation Method 8 FIG. 8 is a flow diagram depicting formulation method 8. 1. HP-B-CD, histidine, and polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Sorbitol was added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0022] Formulation Method 9 FIG. 9 is a flow diagram depicting formulation method 9. 1. HP-B-CD was dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Sorbitol, histidine, and polysorbate 80 were added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0023] Formulation Method 10 FIG. 10 is a flow diagram depicting formulation method 10. 1. HP-B-CD and sorbitol were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Histidine and polysorbate 80 were added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0024] Formulation Method 11 FIG. 11 is a flow diagram depicting formulation method 11. 1. HP-B-CD and histidine were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Sorbitol and polysorbate 80 were added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0025] Formulation Method 12 FIG. 12 is a flow diagram depicting formulation method 12. 1. HP-B-CD and Polysorbate 80 were dissolved in water for injection (60% of the final volume). 2. The semaglutide drug substance was dissolved in the solution. 3. The pH was adjusted to pH 6.0 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 4. Caglilintide drug substance was dissolved in the solution. 5. Sorbitol and histidine were added and dissolved. 6. Water for injection was added to the solution to reach 95% of the final volume. 7. The pH was adjusted to pH 5.8 using 0.05N hydrochloric acid and / or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final formulation volume prior to sterile filtering and filling into prefillable syringes.

[0026] The present invention is a method of formulating a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist, and a cyclodextrin that includes hydroxypropyl substitutions.

[0027] The application of this method is simple and improves the stability of the pharmaceutical active ingredients, namely GLP-1 receptor agonists and amylin receptor agonists.

[0028] Disclosed herein is a method for preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, the method comprising: Obtaining or preparing a drug substance (DS) containing an amylin receptor agonist as an active pharmaceutical ingredient; Obtaining or preparing a drug substance (DS) containing a GLP-1 receptor agonist as an active pharmaceutical ingredient; preparing a surfactant-free aqueous excipient solution comprising a cyclodextrin containing hydroxypropyl substitution to about 50-80%, e.g., about 65%, of a final predetermined volume, and water for injection (WFI), and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; Dissolving the GLP-1 receptor agonist DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example to about 5.9 to 6.1, preferably about 6.0; dissolving the amylin receptor agonist DS in a mixture of the excipient solution and the GLP-1 receptor agonist; adding a surfactant to a mixture of an excipient solution, a GLP-1 receptor agonist, and an amylin receptor agonist; adding water to about 80-99% of the final desired volume; adjusting the pH to about 5.5-6.1, such as 5.7-6.1, such as 5.9-6.1, such as 5.8; Adding water to reach the final desired volume (100%).

[0029] The GLP-1 receptor agonist may be semaglutide.

[0030] The amylin receptor agonist can be caglilintide.

[0031] The cyclodextrins are of the hydroxypropyl-substituted alpha type, which contains glucose units in a six-membered ring structure, and / or of the hydroxypropyl-substituted beta type, which contains glucose units in a seven-membered ring structure.

[0032] The surfactant-free aqueous excipient solution comprising a cyclodextrin can include one or more additional excipients, provided that the one or more additional excipients are not surfactants.

[0033] One or more additional excipients may be added to the mixture of the surfactant-free aqueous excipient solution and the GLP-1 receptor agonist, provided that the additional excipient is not a surfactant.

[0034] One or more additional excipients may be added to the mixture of the excipient solution, the GLP-1 receptor agonist, and the amylin receptor agonist.

[0035] In this context, non-limiting examples of "additional excipients" are buffers and tonicity agents. The additional excipients have at least one pKa of about 5.0 to 7.0 and may be, for example, buffers such as histidine, citrate or phosphate, or combinations thereof. The additional excipients may be tonicity agents, with the proviso that the tonicity agent is not sodium chloride.

[0036] The concentrations of the cyclodextrin, GLP-1 receptor agonist, amylin receptor agonist, surfactant, and any further excipients will likewise be such to arrive at the composition of the pharmaceutical formulation (medicament) disclosed herein.

[0037] Also disclosed herein is a means for combining an amylin receptor agonist and a GLP-1 receptor agonist, the GLP-1 receptor agonist having an isoelectric point that precludes its combination within a pH range that allows the amylin receptor agonist to be chemically stable. Disclosed herein is a means for combining a GLP-1 receptor agonist and an amylin receptor agonist, the GLP-1 receptor agonist having an isoelectric point (pI) of less than 6.5, preferably less than 6.0, e.g., 3.5-6.0, e.g., 3.0-5.0, e.g., 4.0-6.0.

[0038] The optimal pH of an amylin receptor agonist is the pH at which it is most chemically and physically stable. Those skilled in the art can easily determine the optimal pH of an amylin receptor agonist by testing its chemical and physical stability in an aqueous solution consisting essentially of the amylin receptor agonist, a buffer, and water for injection over a range of pH values.

[0039] The optimal pH of a GLP-1 receptor agonist is the pH at which it is most chemically and physically stable.Those skilled in the art can easily determine the optimal pH of a GLP-1 receptor agonist by testing its chemical and physical stability in an aqueous solution that essentially consists of a GLP-1 receptor agonist, a buffer, and water for injection over a pH range.The physical stability of a GLP-1 receptor agonist can be a reflection of its isoelectric point, which can coincide with the pH at which the poorest physical stability can be expected.

[0040] As will be apparent to one of skill in the art, the chemical stability and purity of any GLP-1 receptor agonist and / or any amylin receptor agonist in any liquid formulation can be quantified, for example, by reverse phase (ultra) high performance liquid chromatography (RP-UHPLC or RP-HPLC) and / or by measuring the percentage of high molecular weight protein (% HMWP), for example, by size exclusion chromatography (SEC).

[0041] As will be apparent to one of skill in the art, the physical stability of a GLP-1 receptor agonist and / or any amylin receptor agonist in any liquid formulation can be quantified by measuring particle formation and / or fibrillation by microflow imaging (MFI) or thioflavin T (ThT) fluorescence stress assay, respectively.

[0042] Disclosed herein are means of formulating amylin receptor agonists and GLP-1 receptor agonists whose pH optima differ by at least about 2 pH units, e.g., 2-5 pH units, e.g., 2-4 pH units, e.g., 3-5 pH units.

[0043] The GLP-1 receptor agonist can be semaglutide. The amylin receptor agonist can be caglilintide, or a biologically active metabolite or degradation product of caglilintide. The pharmaceutical formulation composition disclosed herein maintains / improves the chemical and physical stability of the pharmaceutical active ingredients, maintains the pharmacokinetic profile of the pharmaceutical active ingredients in terms of their bioavailability and exposure, and exhibits acceptable local tolerance upon subcutaneous injection, even when formulated at pH 5.5-6.5, e.g., pH 5.6-6.0.

[0044] The production of the drug substance precedes the practice of the formulation methods disclosed herein, the purpose of which is to create a pharmaceutical formulation that is the final drug product. Application of the methods disclosed herein ensures that the pharmaceutical formulation or "drug product" has an improved shelf life.

[0045] The term "drug substance" refers to a purified active pharmaceutical ingredient. "Drug substance" is well known in the art to refer to "any [substance or] mixture of substances that is intended to be used in the manufacture of a drug (medicinal) article and that, when used in the production of a drug product, becomes an active pharmaceutical ingredient" (see, e.g., EMA ICH Q7a: "Good manufacturing practice for active pharmaceutical ingredients"). It is well known to those skilled in the art that, to the extent required by regulatory authorities, a drug substance must be essentially free of impurities.

[0046] The drug substance may be a solid, such as a freeze-dried or spray-dried solid. The amylin receptor agonist drug substance may be a solid. The GLP-1 receptor agonist drug substance may be a solid.

[0047] A solid or "dry" form of the purified active pharmaceutical ingredient (or drug substance) may be obtained as described in WO2012 / 168432. The drug substance may be dried by lyophilization (i.e., freeze-drying, see, e.g., Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray-drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, UK), pp. 491-676, Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206, and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), or air-drying (Carpenter and Crowe (1988) Cryobiology 25:459-470, and Roser (1991) Biopharm. 4:47-53).

[0048] In the context of the present invention, the terms "pharmaceutical formulation," "combination," and "medicament" may be used interchangeably herein to refer to a liquid pharmaceutical formulation comprising a GLP-1 receptor agonist and an amylin receptor agonist.

[0049] The pharmaceutical formulations disclosed herein are suitable for parenteral injection, preferably subcutaneous injection.

[0050] Amylin As used herein, the term "amylin" refers to a polypeptide having the same amino acid sequence as endogenous amylin, such as human amylin.

[0051] Amylin Receptors Amylin receptor agonists can bind to and activate the calcitonin receptor (CTR) and / or the amylin receptor (AMYR), the latter of which consists of a heterodimer of two components, the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP1-3), resulting in three possible complexes, AMYR1-3.

[0052] Amylin receptor agonists The pharmaceutical preparation disclosed herein comprises an amylin receptor agonist. An "amylin receptor agonist" can be defined as a chemical substance that can bind to and activate or "agonize" an amylin receptor. In the context of the present invention, an amylin receptor agonist can bind to and activate at least amylin receptor 3 (AMYR3). An amylin receptor agonist can also agonize calcitonin receptor, amylin receptor 1 (AMYR1), and / or amylin receptor 2 (AMYR2).

[0053] Examples of endogenous amylin receptor agonists are human amylin and human calcitonin. Examples of exogenous amylin receptor agonists are caglilintide and pramlintide (the pharmaceutical active ingredient of Symlin®).

[0054] Amylin receptor agonists are capable of activating the amylin receptor, or in other words, have a "potency" on the amylin receptor. The in vitro potency of amylin receptor agonists on the amylin receptor 3 can be measured as described in Assay 2 of WO / 2022129526. The potency of a compound is determined by its EC 50 It can be described by the value, EC 50 represents the concentration of a compound at which 50% of its maximal effect is observed. EC 50 The lower the value, the more potent the compound.

[0055] When tested as described in Assay 2 of WO / 2022129526, the amylin receptor agonists disclosed herein have an EC of less than 300 pM, such as less than 200 pM, for example less than 150 pM, preferably less than 100 pM, for example less than 75 pM, preferably less than 50 pM, for example less than 40 pM, for example less than 30 pM, for example less than 20 pM, for example less than 10 pM. 50 It may have a value.

[0056] Caglirintide The amylin receptor agonist in the pharmaceutical formulations disclosed herein may be caglilintide, or a biologically active metabolite or breakdown product of caglilintide.

[0057] Caglilintide, also known as AM833, is the compound of Example 53 of WO2012 / 168432: N-alpha-[(S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-[Glu14,Arg17,Pro37]-pramlintide. Caglilintide can be prepared as described on pages 153-155 of WO2012 / 168432.

[0058] Caglilintide may be in the form of a salt, preferably a pharma- ceutically acceptable salt.

[0059] A biologically active metabolite or degradation product of caglilintide may have an aspartate (Asp) at position 21 or 22. A biologically active metabolite or degradation product of caglilintide may have an iso-aspartate (iso-Asp) at position 21 or 22.

[0060] When the potency of caglilintide was tested using the procedure described in Assay 2 of WO / 2022129526, caglilintide had an EC 50 values ​​(WO / 2022 / 129526, Tables 4b and 4c).

[0061] The concentration of caglilintide in the pharmaceutical formulations disclosed herein may be from about 0.25 mg / ml to about 22 mg / ml.

[0062] The pharmaceutical formulation disclosed herein may have a concentration of about 0.33 to 18 mg / ml, for example, 0.25 to 0.5 mg / ml, for example, about 0.33 mg / ml, for example, 0.5 to 1.0 mg / ml, for example, about 0.67 mg / ml, for example, 1.0 to 1.5 mg / ml, for example, about 1.33 mg / ml, for example, 1.5 to 2.0 mg / ml, for example, about 1.5 mg / ml, for example, 2.0 to 2.5 mg / ml, for example, 2.5 to 3.0 mg / ml, for example, 3.0 to 3.5 mg / ml, for example, about 3.2 mg / ml, for example, 3.5 to 4. The composition may contain caglirintide at a concentration of about 100 mg / ml, for example, 4.0 to 5.0 mg / ml, for example, 5.0 to 6.0 mg / ml, for example, 6.0 to 7.0 mg / ml, for example, 7.0 to 8.0 mg / ml, for example, 8.0 to 9.0 mg / ml, for example, 9.0 to 10.0 mg / ml, for example, about 9.6 mg / ml, for example, 10 to 11 mg / ml, for example, 11.0 to 12.0 mg / ml, for example, 11 to 13 mg / ml, for example, 13 to 22 mg / ml, for example, about 18 mg / ml, for example, about 20 to 22 mg / ml.

[0063] The pharmaceutical formulations disclosed herein may contain no more than 22 mg / ml of caglilintide.The pharmaceutical formulations disclosed herein may contain no more than 12 mg / ml of caglilintide.

[0064] GLP-1 The term "GLP-1" or "native GLP-1" herein refers to human glucagon-like peptide-1 (GLP-1(7-37)).

[0065] GLP-1 receptor agonists The pharmaceutical formulations disclosed herein include a GLP-1 receptor agonist. A "GLP-1 receptor agonist" can be defined as a ligand that can bind to the GLP-1 receptor and produce a biological response similar to that of the endogenous ligand, glucagon-like peptide 1 (GLP-1(7-37)). A "full" GLP-1 receptor agonist can be defined as a GLP-1 receptor agonist that can induce a biological response of the same magnitude as GLP-1(7-37).

[0066] Examples of exogenous GLP-1 receptor agonists include semaglutide (the active pharmaceutical ingredient in Ozempic®, Rybelsus®, and Wegovy®), liraglutide (the active pharmaceutical ingredient in Victoza® and Saxenda®), tirzepatide (the active pharmaceutical ingredient in Mounjaro®), and dulaglutide (the active pharmaceutical ingredient in Trulicity®).

[0067] A GLP-1 receptor agonist is capable of binding to and activating or "agonizing" the GLP-1 receptor, in other words, having "potency" at the GLP-1 receptor. The in vitro potency of a GLP-1 receptor agonist for the GLP-1 receptor may be measured as described in Assay 1 of WO / 2022 / 129526. The potency of a compound is determined by its EC 50 It can be described by the value, EC 50 represents the concentration of a compound at which 50% of its maximal effect is observed. EC 50 The lower the value, the more potent the compound.

[0068] When tested as described in Assay 1 of WO / 2022 / 129526, the GLP-1 receptor agonists disclosed herein have an EC of less than 300 pM, such as less than 200 pM, for example less than 150 pM, preferably less than 100 pM, for example less than 75 pM, even more preferably less than 50 pM, for example less than 40 pM, for example less than 30 pM, for example less than 20 pM, for example less than 10 pM. 50 It may have a value.

[0069] Semaglutide Semaglutide is a 6.26 -{18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propanoic acid),34-L-arginine] human glucagon-like peptide 1 (7-37) is also known as a GLP-1 receptor agonist.Semaglutide is described in WO2006 / 097537 and J.Med.Chem.2015,58,18,7370-7380 and can be prepared using methods well known to those skilled in the art, such as those briefly described in Example 4 of WO2006 / 097537.

[0070] Semaglutide may be present in the pharmaceutical formulation in its fully or partially ionized form, e.g. one or more carboxylic acid groups (-COOH) may be replaced by carboxylate groups (-COO - ), and / or one or more amino groups (-NH2) may be deprotonated to -NH3 + The group may be protonated.

[0071] Semaglutide may be in the form of a salt, preferably a pharma- ceutically acceptable salt.

[0072] When the potency of semaglutide was tested using the procedure described in Assay 1 of WO / 2022 / 129526, semaglutide had an EC 50 values ​​(see WO / 2022 / 129526, Tables 4b and 4c).

[0073] The concentration of semaglutide in the pharmaceutical formulations disclosed herein may be from about 0.25 mg / ml to about 22 mg / ml.

[0074] The pharmaceutical preparation may have a concentration of about 0.33 to 18 mg / ml, for example, 0.25 to 0.5 mg / ml, for example, about 0.33 mg / ml, for example, 0.5 to 1.0 mg / ml, for example, about 0.67 mg / ml, for example, 1.0 to 1.5 mg / ml, for example, about 1.33 mg / ml, for example, 1.5 to 2.0 mg / ml, for example, about 1.5 mg / ml, for example, 2.0 to 2.5 mg / ml, for example, about 2.2 mg / ml, for example, 2.5 to 3.0 mg / ml, for example, 3.0 to 3.5 mg / ml, for example, about 3.2 mg / ml, for example, 3.5 to 4.0 mg / ml, for example, 4.0 to 5.0 mg / ml, for example, about 4.8 mg / ml, for example For example, it may contain semaglutide at a concentration of 5.0 to 6.0 mg / ml, for example, 6.0 to 7.0 mg / ml, for example, about 6.4 mg / ml, for example, 7.0 to 8.0 mg / ml, for example, about 8.0 mg / ml, for example, 8.0 to 9.0 mg / ml, for example, 9.0 to 10.0 mg / ml, for example, about 9.6 mg / ml, for example, 10 to 11 mg / ml, for example, about 10.7 mg / ml, for example, 11.0 to 12.0 mg / ml, for example, 11 to 13 mg / ml, for example, about 12.8 mg / ml, for example, 13 to 22 mg / ml, for example, about 16 mg / ml, for example, about 18 mg / ml, for example, about 20 to 22 mg / ml.

[0075] The pharmaceutical formulations disclosed herein may contain no more than 22 mg / ml of semaglutide.The pharmaceutical formulations disclosed herein may contain no more than 12 mg / ml of semaglutide.

[0076] Drug substance manufacturing method The GLP-1 receptor agonists and / or amylin receptor agonists in the pharmaceutical formulations disclosed herein can also be produced, for example, by classical peptide synthesis, e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry, or other well-established techniques, see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999; Florencio Zaragoza Dorwald, "Organic Synthesis on Solid Phase", Wiley-VCH Verlag GmbH, 2000; and "Fmoc Solid Phase Peptide Synthesis", Edited by WC Chan and PD White, Oxford University Press, 2000.

[0077] Alternatively, the compounds can be produced by recombinant methods, for example by culturing a host cell that contains a DNA sequence encoding the peptide sequence and is capable of expressing the peptide in a suitable nutrient medium under conditions that permit expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides are Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines.

[0078] isoelectric point The isoelectric point (pI) of a molecule is the pH at which the molecule carries no net charge. The pI of a peptide may be theoretically calculated from the pK values ​​of its amino acids and terminal amine and carboxyl groups and can be used to predict the solubility of a peptide at a given pH.

[0079] The theoretically calculated isoelectric point of the GLP-1 receptor agonist may be in the range of 3.5 to 6.5, such as 3.5 to 6.0, for example 4.0 to 6.0, for example 3.8 to 4.9, for example 4.0 to 4.5. Semaglutide has a theoretically calculated isoelectric point of about 4.37.

[0080] The theoretically calculated isoelectric point of the amylin receptor agonist may have an isoelectric point (pI) in the range of 8 to 12, for example, 8 to 9. Caglilintide has a theoretically calculated isoelectric point of about 8.56.

[0081] Excipient Solutions The methods disclosed herein involve preparing a surfactant-free aqueous excipient solution comprising a cyclodextrin that includes hydroxypropyl substitution and water for injection (WFI).

[0082] The excipient solution is an aqueous solution that includes water for injection (WFI). The application of the method disclosed herein ultimately results in the pharmaceutical formulation disclosed herein. The pharmaceutical formulation may include more than 75% w / w water, such as 80% w / w water, such as about 85% w / w water, such as up to 90% w / w water.

[0083] The excipient solution may further comprise one or more additional excipients, provided that such additional excipient is not a surfactant. A buffer may be such an additional excipient. A tonicity agent may be such an additional excipient.

[0084] Cyclodextrin The surfactant-free aqueous excipient solutions utilized in the formulation methods described herein include cyclodextrins that contain hydroxypropyl substitutions.

[0085] Application of the methods disclosed herein ultimately results in the pharmaceutical formulations disclosed herein. Thus, the pharmaceutical formulations disclosed herein necessarily include cyclodextrins that include hydroxypropyl substitution.

[0086] The amounts listed below relate to pharmaceutical formulations.

[0087] The pharmaceutical formulation may comprise about 10-25% w / v of a cyclodextrin containing hydroxypropyl substitutions. The pharmaceutical formulation may comprise more than 10% w / v of a cyclodextrin containing hydroxypropyl substitutions. The pharmaceutical formulation may comprise less than 22% w / v of a cyclodextrin containing hydroxypropyl substitutions. The pharmaceutical formulation may comprise about 10-20% w / v, about 15-25% w / v, about 12-18% w / v, about 10-17.5% w / v, about 11.25-15%, e.g., about 15% w / v of a cyclodextrin containing hydroxypropyl substitutions.

[0088] Cyclodextrins are oligosaccharide starch derivatives consisting of 6, 7, or 8 α-(1,4) linked glucopyranose (glucose) units arranged periodically and exhibiting alpha, beta, or gamma type, respectively. Cyclodextrins have a wide range of applications, especially as pharmaceutical excipients [P.Breen&S.S.Jambhekar,Cyclodextrins in pharmaceutical formulations II:solubilization,binding constant,and complexation efficiency,Drug Discovery Today,Volume 21,Number 2 February 2016]. Guidelines for their use as pharmaceutical excipients have been described by the European Medicines Agency [Background review for cyclodextrins used as excipients, 2014, EMA / CHMP / 333892 / 2013, Committee for Human Medicinal Products (CHMP)] and [Cyclodextrins used as excipients, 2017, EMA / CHMP / 333892 / 2013, Committee for Human Medicinal Products (CHMP)]. Cyclodextrin types that do not carry hydrophilic substitution have poor solubility and are rarely used in parenteral medicines.

[0089] To improve the solubility of cyclodextrins, the hydroxyl groups of the glucose units of cyclodextrins may be replaced by different numbers of hydrophilic chemical substitutions, for example by hydroxypropyl groups, leading to differences in the degree of substitution that can be described either as the average number of hydroxypropyls per cyclodextrin molecule (abbreviated DS) or as the molar degree of substitution (abbreviated MS) corresponding to the average number of hydroxypropyls per glucose unit present in the cyclodextrin in question. The value of hydroxypropyl per cyclodextrin can be achieved by multiplying the molar degree of substitution by the number of glucose units contained in the cyclodextrin in question. The differences in the degree of substitution can lead to changes in physicochemical properties such as surface activity and complexation ability. The hydroxyl groups can also be chemically replaced by sulfobutyl ether groups. These mainly hydrophilic modifications have resulted in cyclodextrin derivatives that are highly suitable for parenteral administration [Cyclodextrins used as excipients, 2017, EMA / CHMP / 333892 / 2013, Committee for Human Medicinal Products (CHMP)]. Cyclodextrins that contain hydroxypropyl substitutions are commonly abbreviated as HP-CD, while cyclodextrins that contain sulfobutyl ether substitutions are abbreviated as SBE-CD.

[0090] Cyclodextrins containing hydrophilic substitutions adopt what may be described as a cone-like shape with a hydrophobic interior cavity and a hydrophilic exterior surface formed by many hydrophilic substitutions that can form hydrogen bonds with adjacent water molecules, thereby improving aqueous solubility [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11].

[0091] The hydrophobic microenvironment inside the cavities of these cone-like structures allows the formation of complexes between drugs and cyclodextrins primarily through hydrophobic interactions [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11]. As complexes form between cyclodextrins and drug molecules carrying one or more hydrophobic regions, these as well as the hydrophobic cavities of the cyclodextrins are shielded from water, thereby increasing the solubility of the complex compared to the solubility of the individual components. Also, when complexes are formed between cyclodextrin molecules and peptide molecules, the intermolecular interactions that typically lead to aggregation are impaired [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11].

[0092] The pharmaceutical formulations disclosed herein preferably contain hydroxypropyl-substituted alpha-cyclodextrins and / or hydroxypropyl-substituted beta-cyclodextrins.

[0093] Unexpectedly, it has been found that such cyclodextrins bearing hydroxypropyl substitutions are superior in their ability to stabilise formulations of caglilintide and semaglutide than the same cyclodextrin types bearing sulfobutyl ether substitutions.

[0094] The pharmaceutical formulations disclosed herein may include hydroxypropyl-substituted alpha-cyclodextrins, which contain glucose units with a six-membered ring structure. Alpha-hydroxypropyl-substituted cyclodextrins are abbreviated as HP-A-CD. Hydroxypropyl-alpha-cyclodextrins (CAS: 128446-33-3 / 99241-24-4) are commercially available with an average molar degree of substitution (MS) of 0.8 and a molar substitution range of 0.5 to 0.9.

[0095] The pharmaceutical formulations disclosed herein may include hydroxypropyl-alpha-cyclodextrins having a minimum of about 0.4 hydroxypropyl per glucose unit.The pharmaceutical formulations disclosed herein may include hydroxypropyl-alpha-cyclodextrins having a maximum of about 1.0 hydroxypropyl per glucose unit.

[0096] The pharmaceutical formulations disclosed herein may include hydroxypropyl-alpha-cyclodextrins having a molar substitution range of hydroxypropyl of 0.5 to 0.9 per glucose unit. The pharmaceutical formulations disclosed herein may include hydroxypropyl-alpha-cyclodextrins having an average molar substitution (MS) of hydroxypropyl of about 0.8 per glucose unit.

[0097] The pharmaceutical formulations disclosed herein may include hydroxypropyl-substituted beta-type cyclodextrins, which contain a glucose unit with a seven-membered ring structure.

[0098] Hydroxypropyl-substituted cyclodextrin of the beta form is abbreviated as HP-B-CD.

[0099] Hydroxypropyl-beta-cyclodextrin is a well-known pharmaceutical excipient, typically used in small molecule pharmaceutical formulations, primarily to increase solubility and bioavailability [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11]. So far, the use of cyclodextrins and cyclodextrin substituted derivatives in protein and peptide-based pharmaceutical formulations has been limited.

[0100] The commercially available hydroxypropyl degree of substitution (DS) of hydroxypropyl-beta-cyclodextrin as a pharmaceutical excipient ranges from 2.8 to 10.5 according to the methods described in the European and US Pharmacopoeias [USP 38 NF 33, Pharm Eur 8, USP〈761〉 / Pharm. Eur.2.2.33], corresponding to 0.4 to 1.5 hydroxypropyls per glucose unit (MS). Commercially available cyclodextrins such as hydroxypropyl-beta-cyclodextrin are usually described by the average molar substitution (MS) of their molar substitution range.

[0101] Hydroxypropyl-beta-cyclodextrin (CAS: 128446-35-5 / 94035-02-6) is commercially available for use as an excipient, with average molar substitution (MS) ranging from MS0.62 with a molar substitution range of 0.58-0.68, MS0.67 with a molar substitution range of (0.6-0.9), MS0.68 with a molar substitution range of (0.58-0.72), MS0.84 with a molar substitution range of (0.8-1.0), MS0.92 with a molar substitution range of (0.81-0.99), and MS1.08 with a molar substitution range of (0.86-1.14), where each value describes the number of hydroxypropyl groups per glucose unit.

[0102] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having a minimum of about 0.4 hydroxypropyl per glucose unit.The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having a maximum of about 1.0 hydroxypropyl per glucose unit.

[0103] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having a molar substitution range of hydroxypropyl per glucose unit of 0.58 to 1.0.The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) range of hydroxypropyl per glucose unit of about 0.62 to 0.92.

[0104] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrin having an average molar substitution (MS) of hydroxypropyl of about 0.62 to 0.84 per glucose unit.

[0105] The pharmaceutical formulations disclosed herein can include hydroxypropyl-beta-cyclodextrin having about 0.4 to 0.75 hydroxypropyl groups per glucose unit.

[0106] The pharmaceutical formulations disclosed herein may include a hydroxypropyl-beta-cyclodextrin having about 0.75 hydroxypropyl per glucose unit.

[0107] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) of about 0.62. The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having about 0.58 to 0.68 hydroxypropyls per glucose unit.

[0108] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) of about 0.68. The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having about 0.58 to 0.72 hydroxypropyls per glucose unit.

[0109] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) of about 0.67.The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having about 0.6 to 0.9 hydroxypropyls per glucose unit.

[0110] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) of about 0.84.The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having about 0.8 to 1.0 hydroxypropyls per glucose unit.

[0111] The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having an average molar substitution (MS) of about 0.92. The pharmaceutical formulations disclosed herein may include hydroxypropyl-beta-cyclodextrins having about 0.81 to 0.99 hydroxypropyls per glucose unit.

[0112] The pharmaceutical formulations disclosed herein may comprise 10-25% w / v, such as greater than 10% w / v and less than 22% w / v, such as about 10-20% w / v, such as about 12-18% w / v, such as about 10-17.5% w / v, such as about 11.25-15%, such as about 15% w / v, of hydroxypropyl-beta-cyclodextrin having a minimum of about 0.4 hydroxypropyl per glucose unit and a maximum of about 1.0 hydroxypropyl per glucose unit, such as an average of 0.62-0.92 hydroxypropyl per glucose unit, such as about 0.75 hydroxypropyl per glucose unit, such as an average of 0.62-0.84 hydroxypropyl per glucose unit, such as about 0.4-0.75 hydroxypropyl per glucose unit, such as an average of 0.62 hydroxypropyl per glucose unit, such as about 0.58-0.68 hydroxypropyl per glucose unit.

[0113] Surfactants Surfactants can further increase the physical stability and robustness of a formulation during its manufacture, storage, and pharmaceutical use, for example, maintaining the stability of the formulation when exposed to air inside a container. The use of surfactants in pharmaceutical formulations is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 2006. th edition,2000 is referred to.

[0114] The methods disclosed herein involve adding a surfactant to a mixture of a surfactant-free aqueous excipient solution, a GLP-1 receptor agonist, and an amylin receptor agonist.

[0115] Application of the methods disclosed herein ultimately results in the pharmaceutical formulations disclosed herein. Thus, the pharmaceutical formulations disclosed herein necessarily include a surfactant.

[0116] The surfactant utilized in the methods disclosed herein may be selected from the group consisting of polysorbate 20 and / or polysorbate 80. The surfactant may be polysorbate 20. The surfactant may be polysorbate 80.

[0117] The amounts listed below relate to pharmaceutical formulations.

[0118] The pharmaceutical formulation may comprise greater than or equal to 0.01 mg / ml polysorbate 20 and up to 2.0, such as up to 1.5 mg / ml polysorbate 20, for example, about 0.01 to 1.0 mg / ml polysorbate 20, for example, about 0.05 mg / ml polysorbate 20.

[0119] The pharmaceutical formulation may comprise greater than or equal to 0.01 mg / ml polysorbate 80 and up to 2.0, such as up to 1.5 mg / ml polysorbate 80, for example, about 0.01 to 1.0 mg / ml polysorbate 80, for example, about 0.05 mg / ml polysorbate 80.

[0120] Further excipients The surfactant-free aqueous excipient solution may contain one or more additional excipients, provided that such additional excipients are not surfactants.

[0121] The methods disclosed herein may include adding one or more additional excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, provided that the additional excipient is not a surfactant.

[0122] The methods disclosed herein may include adding one or more additional excipients to the mixture of the excipient solution, the GLP-1 receptor agonist, and the amylin receptor agonist.

[0123] Additional excipients may include buffers and tonicity agents.

[0124] The application of the methods disclosed herein ultimately results in the pharmaceutical formulations disclosed herein, which therefore necessarily include any additional excipients employed during the methods disclosed herein.

[0125] Pharmaceutical formulations may include buffers. The use of buffers in pharmaceutical formulations is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 20 th edition,2000 is referred to.

[0126] The pharmaceutical formulations disclosed herein may include a buffering agent having a pKa close to the desired pH of the solution. The pharmaceutical formulation may include a buffering agent having at least one pKa value between about 5.0 and 7.0. The pharmaceutical formulation may include a buffering agent having a pKa between about 5.0 and 7.0. The pharmaceutical formulation may include a buffering agent selected from the group consisting of histidine, citrate, and / or phosphate. The buffering agent may be histidine, citrate, and / or phosphate at a total concentration between 3 and 30 mM. The buffering agent may be citrate at a concentration between 3 and 30 mM. The buffering agent may be histidine at a concentration between 3 and 30 mM. The buffering agent may be phosphate at a concentration between 3 and 30 mM.

[0127] The pharmaceutical formulation may further include one or more agents to adjust the pH, such as NaOH and / or HCl.

[0128] The desired pH of the pharmaceutical formulation may be about 5.5 to 6.5. The pH of the pharmaceutical formulation is preferably 5.6 to 6.0. The pH of the pharmaceutical formulation may be about 5.6, for example, about 5.7, for example, about pH 5.8, for example, about 5.9, for example, about 6.0.

[0129] The pH may be measured at "room temperature", which is typically defined as 15-25° C. or 15-20° C. The pH is preferably measured at about 20° C.

[0130] The pharmaceutical formulations disclosed herein may include an isotonicity agent. The use of isotonicity agents in pharmaceutical formulations is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 2006. th edition,2000 is referred to.

[0131] The purpose of the tonicity agent is to protect living tissue when the formulation is injected into the body. The tonicity agent may be selected from the group consisting of mannitol, sorbitol, or trehalose, or a combination thereof. In some embodiments, the tonicity agent is mannitol. In some embodiments, the tonicity agent is sorbitol. In some embodiments, the tonicity agent is trehalose.

[0132] The concentration of the isotonicity agent is such as to make the formulation isotonic. When the isotonicity agent is mannitol, it may be present at a concentration of 16.5-37.5 mg / ml, for example, about 20 mg / ml. When the isotonicity agent is sorbitol, it may be present at a concentration of about 10-40 mg / ml, for example, about 16.5-37.5 mg / ml, for example, about 10-30 mg / ml, for example, about 16-28 mg / ml, for example, about 16.5-25 mg / ml, for example, about 16-26 mg / ml, for example, about 16-24 mg / ml, for example, about 26 mg / ml, for example, about 24 mg / ml, for example, about 22 mg / ml, for example, about 20 mg / ml, for example, about 18 mg / ml, for example, about 16 mg / ml, for example, about 12 mg / ml. When the tonicity agent is trehalose, it may be present in a concentration of 33 to 75 mg / ml, for example about 38 mg / ml.

[0133] Medical usefulness of pharmaceutical preparations The pharmaceutical formulations disclosed herein are for medical use.

[0134] The pharmaceutical formulations disclosed herein may be administered by parenteral injection.The pharmaceutical formulations disclosed herein may be administered by subcutaneous injection.

[0135] As used herein, the term "treatment" refers to the medical therapy of any human or other vertebrate subject in need thereof. The subject is expected to have undergone a physical examination by a physician or veterinarian who has given a provisional or definitive diagnosis indicating that the use of the particular treatment will be beneficial to the health of the human or other vertebrate. The timing and purpose of the treatment may vary from individual to individual according to the current state of the subject's health. Thus, the treatment may be prophylactic (preventative), palliative, symptomatic, and / or curative.

[0136] The pharmaceutical formulations disclosed herein can be administered to human subjects.

[0137] The pharmaceutical formulations disclosed herein may be used in: (i) prevention and / or treatment of any form of diabetes and related conditions, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, maturity-onset diabetes of the young (MODY), gestational diabetes, and / or reduction of HbA1c; (ii) delaying or preventing the progression of diabetic disease, such as the progression of type 2 diabetes, delaying the progression from impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, and / or delaying the progression from insulin-nonrequiring type 2 diabetes to insulin-requiring type 2 diabetes; (iii) prevention and / or treatment of eating disorders such as obesity, for example by reducing food intake, suppressing appetite, inducing satiety, reducing body weight; treating or preventing antipsychotic or steroid-induced binge eating disorders, food cravings, bulimia nervosa, and / or obesity; and / or slowing gastric emptying, (iv) prevention and / or treatment of cardiovascular disease, such as delaying or reducing the occurrence of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization for unstable angina, and hospitalization for heart failure; (v) prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH); (vi) prevention and / or treatment of cognitive disorders, such as Alzheimer's disease.

[0138] In some embodiments, the indication is (i). In some embodiments, the indication is (ii). In still further particular aspects, the indication is (iii). In still further particular aspects, the indication is (iv). In still further particular aspects, the indication is (v). In still further particular aspects, the indication is (vi). In some embodiments, the indication is type 2 diabetes and / or obesity.

[0139] As used herein, a particular value given in terms of a numerical value or interval may be interpreted as being the particular value or as an approximation (e.g., plus or minus 10, 15, or 20 percent of the particular value when amounts may be provided by weight; e.g., plus or minus 0.4 when pH is measured).

[0140] The following is a non-limiting list of embodiments of the present invention.

[0141] Embodiment 1. A method for preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising: Obtaining or preparing a solid drug substance (DS), such as a DS, which contains an amylin receptor agonist as an active pharmaceutical ingredient; Obtaining or preparing a DS, such as a solid drug substance (DS), which contains a GLP-1 receptor agonist as a pharmaceutical active ingredient; preparing a surfactant-free aqueous excipient solution comprising a cyclodextrin containing hydroxypropyl substitution to about 50-80%, e.g., about 65%, of a final predetermined volume and water for injection (WFI), and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; dissolving a GLP-1 receptor agonist DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example, about 5.9 to 6.1, preferably about 6.0; dissolving an amylin receptor agonist in a mixture of an excipient solution and a GLP-1 receptor agonist; adding a surfactant to a mixture of an excipient solution, a GLP-1 receptor agonist, and an amylin receptor agonist; Optionally, adding water to about 80-99% of the final predetermined volume; Optionally, further adjusting the pH to about 5.5-6.1, e.g., 5.7-6.1, e.g., 5.9-6.1, e.g., 5.8; and adding water to reach a final predetermined volume (100%). 2. A method for preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising: Obtaining or preparing a solid drug substance (DS), such as a DS, which contains an amylin receptor agonist as an active pharmaceutical ingredient; Obtaining or preparing a DS, such as a solid drug substance (DS), which contains a GLP-1 receptor agonist as a pharmaceutical active ingredient; preparing a surfactant-free aqueous excipient solution comprising about 50-80%, e.g., about 65%, of a final predetermined volume of cyclodextrin containing hydroxypropyl substitution, optionally one or more additional excipients, provided that the one or more additional excipients are not surfactants, and water for injection (WFI), and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; dissolving a GLP-1 receptor agonist DS in an excipient solution; Adjusting the pH to 5.5-6.5, for example, about 6.0; dissolving an amylin receptor agonist DS in a mixture of an excipient solution and a GLP-1 receptor agonist; adding a surfactant to a mixture of the GLP-1 receptor agonist DS, the excipient solution, and the amylin receptor agonist DS; Optionally, adding water to about 80-99% of the final predetermined volume; adjusting the pH to about 5.5-6.1, for example 5.7-6.1, for example 5.9-6.1, for example 5.8; adding water to reach a final predetermined volume (100%). 3. A method for preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising: Obtaining or preparing a solid drug substance (DS), such as a DS, which contains an amylin receptor agonist as an active pharmaceutical ingredient; Obtaining or preparing a DS, such as a solid drug substance (DS), which contains a GLP-1 receptor agonist as a pharmaceutical active ingredient; preparing a surfactant-free aqueous excipient solution comprising cyclodextrin with hydroxypropyl substitution, an isotonicity agent such as sorbitol, a buffer such as histidine, and water for injection (WFI) to about 50-80%, e.g., about 65%, of a final predetermined volume, and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably about 7.5; dissolving a GLP-1 receptor agonist DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example about 5.9 to 6.1, preferably about 6.0; dissolving the amylin receptor agonist DS in a mixture of the excipient solution and the GLP-1 receptor agonist DS; adding a surfactant to a mixture of an excipient solution, a GLP-1 receptor agonist DS, and an amylin receptor agonist DS; Optionally, adding water to about 80-99% of the final predetermined volume; Optionally, further adjusting the pH to about 5.5-6.1, e.g., 5.7-6.1, e.g., 5.9-6.1, e.g., 5.8; and adding water to reach a final predetermined volume (100%). 4. The method of any one of embodiments 1-3, wherein the GLP-1 receptor agonist is semaglutide. 5. The method according to any one of embodiments 1 to 4, wherein the amylin receptor agonist is caglilintide or a biologically active metabolite or degradation product of caglilintide. 6. A method for preparing a pharmaceutical formulation containing caglilintide and semaglutide, comprising: Obtaining or preparing a solid drug substance (DS), such as a DS, containing caglirintide as an active pharmaceutical ingredient; Obtaining or preparing a solid drug substance (DS), such as a DS, containing semaglutide as an active pharmaceutical ingredient; preparing a surfactant-free aqueous excipient solution comprising cyclodextrin containing hydroxypropyl substitution and water for injection (WFI) to about 50-80%, e.g., about 65%, of a final predetermined volume, optionally with one or more additional excipients, provided that the one or more additional excipients are not surfactants, and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; dissolving semaglutide DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example, about 5.9 to 6.1, preferably about 6.0; dissolving caglilintide DS in a mixture of excipient solution and semaglutide; adding polysorbate 20 and / or polysorbate 80 to a mixture of excipient solution, semaglutide and caglilintide; Optionally, adding water to about 80-99% of the final predetermined volume; optionally adjusting the pH to 5.5-6.1, such as 5.7-6.1, such as 5.9-6.1, such as 5.8; adding water to reach a final predetermined volume (100%). 7. A method for preparing a pharmaceutical formulation containing caglilintide and semaglutide, comprising: Obtaining or preparing a solid drug substance (DS), such as a DS, containing caglirintide as an active pharmaceutical ingredient; Obtaining or preparing a solid drug substance (DS), such as a DS, containing semaglutide as an active pharmaceutical ingredient; preparing a surfactant-free aqueous excipient solution comprising cyclodextrin containing hydroxypropyl substitution to about 50-80%, e.g., about 65%, of a final predetermined volume, sorbitol, histidine, and water for injection (WFI), and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably a pH of about 7.5; dissolving semaglutide DS in an excipient solution; adjusting the pH to 5.5 to 6.5, for example about 5.9 to 6.1, preferably about 6.0; dissolving caglilintide DS in a mixture of excipient solution and semaglutide DS; adding polysorbate 20 and / or polysorbate 80 to a mixture of semaglutide DS, an excipient solution and caglirintide DS; Optionally, adding water to about 80-99% of the final predetermined volume; optionally adjusting the pH to about 5.5-6.1, e.g., 5.7-6.1, e.g., 5.7-5.9, e.g., 5.8; adding water to reach a final predetermined volume (100%). 8. The method according to any one of the preceding embodiments, wherein the cyclodextrin is a hydroxypropyl-substituted alpha-type cyclodextrin containing a glucose unit with a 6-membered ring structure, and / or a hydroxypropyl-substituted beta-type cyclodextrin containing a glucose unit with a 7-membered ring structure. 9. The method of any one of the preceding embodiments, wherein the cyclodextrin is a hydroxypropyl-substituted beta-type. 10. The method of any one of the preceding embodiments, wherein the cyclodextrin is a hydroxypropyl-substituted beta type having a minimum of about 0.4 and a maximum of about 1.0 hydroxypropyls per glucose unit. 11. The method of any one of the preceding embodiments, wherein the cyclodextrin is a hydroxypropyl-substituted beta-type having an average molar substitution (MS) range of about 0.62 to 0.92 hydroxypropyl per glucose unit, e.g., an average MS of 0.62, e.g., an average MS of 0.92. 12. The method of any one of the preceding embodiments, wherein the surfactant-free aqueous excipient solution comprising a cyclodextrin comprises one or more additional excipients, with the proviso that the additional excipients are not surfactants. 13. The method of any one of embodiments 1-12, comprising adding one or more additional excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, with the proviso that the additional excipient is not a surfactant. 14. The method of any one of embodiments 1-13, comprising adding one or more additional excipients to the mixture of the excipient solution, the GLP-1 receptor agonist, and the amylin receptor agonist. 15. The method of any one of the preceding embodiments, comprising adding one or more additional excipients to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and surfactant. 16. The method of any one of the preceding embodiments, wherein the surfactant-free aqueous excipient solution comprising a cyclodextrin further comprises a buffer. 17. The method of any one of embodiments 1-16, further comprising adding a buffering agent to the mixture of excipient solution and GLP-1 receptor agonist. 18. The method of any one of embodiments 1-17, further comprising adding a buffering agent to the mixture of the excipient solution, the GLP-1 receptor agonist, and the amylin receptor agonist. 19. The method of any one of the preceding embodiments, further comprising adding a buffering agent to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and surfactant. 20. The method of any one of the preceding embodiments, wherein the buffer has at least one pKa of about 5.0 to 7.0. 21. The method of any one of the preceding embodiments, wherein the buffer is histidine, citrate, or phosphate, or a combination thereof. 22. The method of any one of the preceding embodiments, wherein the buffer is histidine. 23. The method of any one of embodiments 1-22, wherein the surfactant is polysorbate 20 and / or polysorbate 80. 24. The method of any one of embodiments 1-23, wherein the surfactant-free aqueous excipient solution further comprises an isotonicity agent, with the proviso that the isotonicity agent is not sodium chloride. 25. The method of any one of embodiments 1-24, further comprising adding an isotonicity agent to the mixture of excipient solution and GLP-1 receptor agonist, with the proviso that the isotonicity agent is not sodium chloride. 26. The method of any one of embodiments 1-25, further comprising adding an isotonicity agent to the mixture of the excipient solution, the GLP-1 receptor agonist, and the amylin receptor agonist. 27. The method of any one of embodiments 1-26, further comprising adding an isotonicity agent to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and surfactant, with the proviso that the isotonicity agent is not sodium chloride. 28. The method of embodiment 27, wherein the tonicity agent is mannitol, sorbitol, or trehalose, or a combination thereof. 29. The method of embodiment 28, wherein the tonicity agent is sorbitol. 30. A liquid pharmaceutical formulation obtained by the method according to any one of embodiments 1 to 29. 31. The liquid pharmaceutical formulation according to embodiment 31, wherein the GLP-1 receptor agonist has an isoelectric point that is incompatible with the pH optimum of the amylin receptor agonist. 32. The liquid pharmaceutical formulation according to embodiment 30 or 31, wherein the pH optima of the GLP-1 receptor agonist and the amylin receptor agonist differ by at least about 2 pH units, such as 2 to 5 pH units, such as 2 to 4 pH units, such as 3 to 5 pH units. 33. The liquid pharmaceutical formulation according to any one of embodiments 30-32, wherein the amylin receptor agonist has an optimal pH of 3.5 to 4.5, for example about 4.0. 34. A pharmaceutical formulation according to any one of embodiments 30 to 33, wherein the amylin receptor agonist is caglilintide. 35. A pharmaceutical formulation according to any one of embodiments 30 to 34, wherein the GLP-1 receptor agonist has an isoelectric point of less than 6.5, such as less than 6.0, for example, between 3.5 and 6.0, for example, between 3.0 and 5.0, for example, between 3.8 and 4.9. 36. A pharmaceutical formulation according to any one of embodiments 30 to 35, wherein the GLP-1 receptor agonist is semaglutide. 37. A pharmaceutical formulation according to any one of embodiments 30 to 36, wherein the cyclodextrin is a hydroxypropyl-substituted alpha type containing a glucose unit with a 6-membered ring structure, and / or a hydroxypropyl-substituted beta type containing a glucose unit with a 7-membered ring structure. 38. A pharmaceutical formulation according to any one of embodiments 30 to 37, wherein the cyclodextrin is a hydroxypropyl-substituted alpha type containing a glucose unit with a six-membered ring structure. 39. A pharmaceutical formulation according to any one of embodiments 30 to 38, wherein the cyclodextrin is a hydroxypropyl-substituted beta-type containing a glucose unit with a seven-membered ring structure. 40. A pharmaceutical formulation according to any one of embodiments 30-39, wherein the cyclodextrin contains up to about 1.0 hydroxypropyl per glucose unit. 41. A pharmaceutical formulation according to any one of embodiments 30 to 40, wherein the cyclodextrin contains up to about 0.92 hydroxypropyl groups per glucose unit. 42. A pharmaceutical formulation according to any one of embodiments 30 to 41, wherein the cyclodextrin contains up to about 0.75 hydroxypropyl groups per glucose unit. 43. A pharmaceutical formulation according to any one of embodiments 30 to 42, wherein the cyclodextrin contains up to about 0.68 hydroxypropyl groups per glucose unit. 44. A pharmaceutical formulation according to any one of embodiments 30 to 43, wherein the cyclodextrin contains a minimum of about 0.4 hydroxypropyl groups per glucose unit. 45. A pharmaceutical formulation according to any one of embodiments 30 to 44, wherein the cyclodextrin contains a minimum of about 0.58 hydroxypropyl groups per glucose unit. 46. ​​A pharmaceutical formulation according to any one of embodiments 30 to 45, wherein the cyclodextrin contains about 0.58 to 1.0 hydroxypropyl per glucose unit. 47. A pharmaceutical formulation according to any one of embodiments 30 to 46, wherein the cyclodextrin contains an average (MS) of 0.62 to 0.92 hydroxypropyl groups per glucose unit. 48. A pharmaceutical formulation according to any one of embodiments 30 to 47, wherein the cyclodextrin contains an average (MS) of about 0.62 to 0.84 hydroxypropyl groups per glucose unit. 49. A pharmaceutical formulation according to any one of embodiments 30 to 48, wherein the cyclodextrin contains an average (MS) of about 0.62 hydroxypropyl groups per glucose unit. 50. The pharmaceutical formulation of any one of embodiments 30-49, wherein the cyclodextrin comprises about 0.4 to 0.75 hydroxypropyl groups per glucose unit, for example, about 0.58 to 0.68 hydroxypropyl groups per glucose unit. 51. A pharmaceutical formulation according to any one of embodiments 30 to 50, comprising about 10 to 25% w / v cyclodextrin. 52. The pharmaceutical formulation of any one of embodiments 30-51, comprising more than 10% w / v cyclodextrin. 53. The pharmaceutical formulation of any one of embodiments 30-52, comprising less than 22% w / v cyclodextrin. 54. The pharmaceutical formulation of any one of embodiments 30-53, comprising less than 20% w / v cyclodextrin. 55. A pharmaceutical formulation according to any one of embodiments 30 to 54, comprising about 10 to 20% w / v of said cyclodextrin. 56. A pharmaceutical formulation according to any one of embodiments 30 to 55, comprising about 10 to 17.5% w / v cyclodextrin. 57. A pharmaceutical formulation according to any one of embodiments 30 to 56, comprising about 12 to 18% w / v cyclodextrin. 58. A pharmaceutical formulation according to any one of embodiments 30 to 57, comprising about 11.25 to 15% w / v cyclodextrin. 59. A pharmaceutical formulation according to any one of embodiments 30 to 58, comprising about 15% w / v cyclodextrin. 60. A pharmaceutical formulation according to any one of embodiments 30 to 59, comprising at least about 1 mg / ml of said GLP-1 receptor agonist. 61. A pharmaceutical formulation according to any one of embodiments 30 to 60, comprising up to about 22 mg / ml of said GLP-1 receptor agonist. 62. A pharmaceutical formulation according to any one of embodiments 30 to 61, comprising about 1 to 12 mg / ml of a GLP-1 receptor agonist. 63. A pharmaceutical formulation according to any one of embodiments 30 to 62, wherein the amylin receptor agonist is caglilintide. 64. A pharmaceutical formulation according to any one of embodiments 30 to 63, comprising at least about 1 mg / ml of said amylin receptor agonist. 65. A pharmaceutical formulation according to any one of embodiments 30 to 64, comprising up to about 22 mg / ml of amylin receptor agonist. 66. A pharmaceutical formulation according to any one of embodiments 30 to 65, comprising about 1 to 12 mg / ml of an amylin receptor agonist. 67. A pharmaceutical formulation according to any one of embodiments 30 to 66, comprising 0.25 to 22 mg / ml of caglilintide. 68. A pharmaceutical formulation according to any one of embodiments 30 to 67, comprising 0.25 to 22 mg / ml of semaglutide. 69. A pharmaceutical formulation according to any one of embodiments 30 to 68, comprising 0.25 to 22 mg / ml of caglilintide and 0.25 to 22 mg / ml of semaglutide. 70. A pharmaceutical formulation according to any one of embodiments 30 to 69, comprising an effective amount of caglilintide and semaglutide. 71. The pharmaceutical formulation of any one of embodiments 30-70, further comprising an isotonicity agent, with the proviso that the isotonicity agent is not sodium chloride. 72. A pharmaceutical formulation according to embodiment 71, wherein the tonicity agent is mannitol, sorbitol, or trehalose, or a combination thereof. 73. The pharmaceutical formulation according to embodiment 72, wherein the tonicity agent is mannitol. 74. The pharmaceutical formulation of embodiment 73, comprising mannitol in a concentration of about 16.5 to 37.5 mg / ml, for example, about 20 mg / ml. 75. The pharmaceutical formulation of embodiment 72, wherein the tonicity agent is sorbitol. 76. The pharmaceutical formulation according to embodiment 75, comprising sorbitol at a concentration of about 10 to 40 mg / ml, for example, about 10 to 30 mg / ml, for example, about 16 to 28 mg / ml, for example, about 16.5 to 37.5 mg / ml, for example, about 16.5 to 25 mg / ml, for example, about 16 to 24 mg / ml, for example, about 24 mg / ml, for example, about 20 mg / ml, for example, about 16 mg / ml, for example, about 12 mg / ml. 77. The pharmaceutical formulation of embodiment 72, wherein the tonicity agent is trehalose. 78. The pharmaceutical formulation of embodiment 77, comprising trehalose at a concentration of about 33 to 75 mg / ml, for example, about 33 to 45 mg / ml, for example, about 38 mg / ml. 79. The pharmaceutical formulation of any one of embodiments 30-78, further comprising a buffer having at least one pKa of about 5.0 to 7.0. 80. The pharmaceutical formulation according to embodiment 79, comprising a buffer selected from the group consisting of histidine, citrate, and / or phosphate. 81. A pharmaceutical formulation according to any one of embodiments 79-80, comprising up to 30 mM of a buffering agent. 82. A pharmaceutical formulation according to any one of embodiments 80-81, comprising about 3-30 mM citrate. 83. A pharmaceutical formulation according to any one of embodiments 80-81, comprising about 3-30 mM histidine, such as 3-15 mM histidine, such as 3-10 mM histidine, for example about 6 mM histidine. 84. A pharmaceutical formulation according to any one of embodiments 80-81, comprising about 3-30 mM phosphate. 85. A pharmaceutical formulation according to any one of embodiments 30 to 84, further comprising a surfactant. 86. A pharmaceutical formulation according to embodiment 85, wherein the surfactant is polysorbate 20 and / or polysorbate 80. 87. The pharmaceutical formulation of embodiment 86, comprising up to about 2.0 mg / ml of polysorbate 20 and / or polysorbate 80. 88. The pharmaceutical formulation of embodiment 87, comprising up to about 1.5 mg / ml of polysorbate 20 and / or polysorbate 80. 89. The pharmaceutical formulation of embodiment 86, wherein the surfactant is Polysorbate 80. 90. A pharmaceutical formulation according to any one of embodiments 30 to 89, wherein the pH is about 5.5 to 6.5, preferably 5.6 to 6.0, for example about 5.7, for example about pH 5.8, for example about 5.9, for example about 6.0. 91. A pharmaceutical formulation according to any one of embodiments 30 to 90, comprising at least 75% w / w water, such as about 80% w / w water, such as about 85% w / w water, such as up to about 90% w / w water. 92. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of an effective amount of caglilintide and semaglutide, a hydroxypropyl-substituted alpha and / or beta cyclodextrin containing a minimum of about 0.4 hydroxypropyl per glucose unit and a maximum of about 1.0 hydroxypropyl per glucose unit, histidine, sorbitol, polysorbate 20 and / or 80, and about 75-90% w / w water, and having a pH of 5.6 to 6.0. 93. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl-substituted alpha and / or beta cyclodextrin containing 0.58 to 1.0 hydroxypropyl per glucose unit, histidine, sorbitol, polysorbate 20 and / or 80, and about 75 to 90% w / w water, and having a pH of 5.6 to 6.0. 94. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl-substituted alpha and / or beta cyclodextrins containing an average of 0.62 to 0.92 hydroxypropyl per glucose unit, histidine, sorbitol, polysorbate 20 and / or 80, and about 75 to 90% w / w water, and having a pH of 5.6 to 6.0. 95. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl-substituted alpha and / or beta cyclodextrins containing an average of 0.62 to 0.84 hydroxypropyl per glucose unit, histidine and / or citrate, sorbitol, polysorbate 20 and / or 80, and about 75 to 90% w / w water, and having a pH of 5.6 to 6.0. 96. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl-substituted alpha and / or beta cyclodextrins containing an average of 0.62 to 0.68 hydroxypropyl per glucose unit, histidine and / or citrate, sorbitol, polysorbate 20 and / or 80, and about 75 to 90% w / w water, and having a pH of 5.6 to 6.0. 97. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl-substituted alpha and / or beta cyclodextrins containing an average of 0.62 hydroxypropyl per glucose unit, histidine and / or citrate, sorbitol, polysorbate 20 and / or 80, and about 75-90% w / w water, and having a pH of 5.6 to 6.0. 98. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl beta cyclodextrin containing up to about 0.75 hydroxypropyl per glucose unit, for example about 0.4 to 0.75 hydroxypropyl per glucose unit, histidine, sorbitol, polysorbate 80, and about 75 to 90% w / w water, and having a pH of 5.5 to 6.5. 99. A pharmaceutical formulation according to any one of embodiments 30 to 91, consisting essentially of effective amounts of caglilintide and semaglutide, hydroxypropyl beta cyclodextrin containing up to about 0.75 hydroxypropyl per glucose unit, for example about 0.4 to 0.75 hydroxypropyl per glucose unit, histidine and / or citrate, sorbitol, polysorbate 20 and / or 80, and about 75 to 90% w / w water, and having a pH of 5.6 to 6.0. 100. In essence, - an effective amount of caglilintide and semaglutide, - more than 10% w / v and less than 22% w / v, e.g. 10-20% w / v of hydroxypropyl-substituted alpha and / or beta cyclodextrins (0.58-1.0 hydroxypropyl per glucose unit); - approximately 3 to 30 mM histidine, - sorbitol at about 10-40 mg / ml, - polysorbate 20 and / or 80 up to 2.0 mg / ml; - pH 5.6 to 6.0, preferably pH 5.8; - the pharmaceutical formulation according to any one of embodiments 30 to 91, consisting of water for injection. 101. In essence, - an effective amount of caglilintide and semaglutide, - hydroxypropyl-substituted alpha and / or beta cyclodextrins containing an average of 0.62 to 0.84 hydroxypropyl units per glucose unit, more than 10% w / v and less than 22% w / v, e.g. 10 to 20% w / v; - about 3-30 mM histidine and / or citrate; - sorbitol at about 10-40 mg / ml, - polysorbate 20 and / or polysorbate 80 up to 2.0 mg / ml; - pH 5.6 to 6.0, preferably pH 5.8; - the pharmaceutical formulation according to any one of embodiments 30 to 91, consisting of water for injection. 102. In essence, - 0.25-22 mg / ml of caglirintide, - 0.25 to 22 mg / ml of semaglutide, - more than 10% w / v and less than 22% w / v, e.g. 10-20% w / v of hydroxypropyl-substituted alpha and / or beta cyclodextrins (0.58-1.0 hydroxypropyl per glucose unit); - approximately 6 mM histidine, - sorbitol at about 10-40 mg / ml, - polysorbate 20 and / or 80 up to 2.0 mg / ml; - pH 5.6 to 6.0, preferably pH 5.8; - the pharmaceutical formulation according to any one of embodiments 30 to 91, consisting of water for injection. EXAMPLES

[0142] Example 1: Effect of Hydroxypropyl-beta-cyclodextrin (HP-B-CD) on the Chemical Stability of Caglirintide This example demonstrates the ability of HP-B-CD to chemically stabilize caglilintide, where the chemical stability is measured in terms of caglilintide purity and caglilintide-related high molecular weight protein (HMWP).

[0143] Caglilintide is optimally stable at pH 4.0, and its chemical decomposition rate typically accelerates with increasing pH. Surprisingly, a stable caglilintide formulation was obtained at pH 6 when formulated with HP-B-CD.

[0144] composition The compositions of caglirintide formulations 1, 2, and 3 are shown in Table 1. [Table 1]

[0145] Preparation process Each caglilintide formulation was prepared by first dissolving the excipients in water, then dissolving the caglilintide drug substance in the excipient solution. The solution was pH adjusted and water was added to reach the desired final volume, and then sterilized by filtration through a 0.22 μm sterile filter. After filtration, the formulation was filled into 1 ml pre-filled syringes.

[0146] method Samples were stored for up to 21 days at 37° C. After 14 and 21 days, samples were analyzed to determine HMWP and caglirintide purity levels.

[0147] The levels of covalently bound HMWP were quantified using size exclusion chromatography (SEC). Samples were analyzed using a WATERS HMWP column (7.8 x 300 mm) with an isocratic elution consisting of 500 mM sodium chloride, 10 mM sodium dihydrogen phosphate monohydrate, 5 mM orthophosphate, and 50% (v / v) isopropanol. Chromatography was performed at 50°C with UV detection (215 nm) using an injection volume of 10 μl and a flow rate of 0.5 ml / min. HMWP was quantified as the area of ​​all components eluting before the main peak x 100% divided by the area of ​​the main peak.

[0148] Reverse phase ultra-high performance liquid chromatography (RP-UHPLC) was used to determine the purity of kaglilintide. Samples were analyzed using a Kinetex C18, 1.7 μm, 100 Å, column (2.1 × 150 mm) with gradient elution of eluent A consisting of 90% v / v of 0.09 M phosphate solution, pH 3.6 and 10% v / v of acetonitrile, and eluent B consisting of 60% v / v of acetonitrile and 20% v / v of isopropanol. Chromatography was performed at 60 °C with UV detection (215 nm) using an injection volume of 2-7.5 μl and a flow rate of 0.25 ml / min. Purity was assessed as the area of ​​the main peak divided by the area of ​​all peaks × 100%. [Table 2]

[0149] conclusion Table 2 shows that when kagurilintide was stored at 37°C and pH 4.0, almost no HMWPs were formed and only a slight decrease in kagurilintide purity was observed. In contrast, when the pH was 6.0, the rate of HMWP formation and the decrease in kagurilintide purity accelerated. Surprisingly, this rapid chemical degradation was countered by the addition of HP-B-CD to the formulation, making it possible to formulate kagurilintide at pH 6.

[0150] Example 2: Effect of HP-B-CD on the physical stability of semaglutide This example demonstrates the ability of HP-B-CD to physically stabilize semaglutide, which has a tendency to form peptide fibrils. The effect was evident when semaglutide was formulated at a suboptimal pH.

[0151] composition The compositions of semaglutide formulations 1, 2, and 3 are shown in Table 3. [Table 3]

[0152] Preparation process The formulations were prepared as described in Example 1.

[0153] method The tendency of semaglutide to aggregate and form peptide fibrils, a parameter used to quantify physical stability, was measured using a Thioflavin T (ThT) fluorescence stress assay. Analysis for the presence of peptide fibrils is based on the fluorescent properties of the ThT probe, which shows low fluorescence in the unbound / native peptide-bound state but high fluorescence when bound to peptide fibrils, as well as a red shift in the wavelength of maximum fluorescence upon fibril binding.

[0154] The two samples were pooled and 1400 μl of sample was added to 28 μl of 1 mM ThT stock solution, of which 200 μl was then transferred to six different wells on a 96-well microtiter plate containing glass beads. The assay was carried out on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 450 nm and 480 nm, respectively, for 169 h at 40°C with dual orbital shaking and a speed of 300 rpm. The lag time was measured from the start of the experiment until fibrillation occurred, indicated as an increase in ThT fluorescence. [Table 4]

[0155] conclusion Semaglutide formulations were subjected to shear stress-inducing conditions to measure the tendency of semaglutide to form peptide fibrils. Surprisingly, the presence of HP-B-CD was found to inhibit semaglutide peptide fibril formation. When semaglutide was formulated at pH 6 in the absence of HP-B-CD (semaglutide formulation 1), fibrillation occurred after 2.35 hours, i.e. semaglutide was not physically stable. However, when semaglutide was formulated at pH 6 in the presence of HP-B-CD (semaglutide formulation 2), no fibrillation was observed throughout the duration of the experiment, i.e. semaglutide was physically stable. Furthermore, the physical stability of semaglutide when formulated in the presence of HP-B-CD at pH 6 (semaglutide formulation 2) was found to be equivalent to the physical stability of semaglutide when formulated in the absence of HP-B-CD but at its optimal formulation conditions for pH 7.4 (semaglutide formulation 3).

[0156] Example 3: Effect of HP-B-CD on the chemical stability of semaglutide This example shows the ability of HP-B-CD to chemically stabilise semaglutide, where chemical stability is measured in terms of semaglutide purity and semaglutide-associated high molecular weight proteins (HMWPs).

[0157] composition The same formulation as in Example 2 was used.

[0158] Preparation process The formulations were prepared as described in Example 1.

[0159] method HMWP and semaglutide purity levels were determined after 0, 14 and 21 days of storage at 37°C.

[0160] Semaglutide purity was determined using reversed-phase high performance liquid chromatography (RP-HPLC) where samples were analysed using a Kinetex C18, 2.6 μm, column (4.6×150 mm) with gradient elution of eluent A consisting of 90% v / v 0.09 M phosphate solution, pH 3.6 and 10% v / v acetonitrile, and eluent B consisting of 60% v / v acetonitrile and 20% v / v isopropanol. Chromatography was performed at 30° C. with UV detection (210 nm) using an injection volume of 10-100 μl and a flow rate of 0.7 ml / min. Purity was quantified as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0161] The levels of covalently bound HMWP were determined using size exclusion chromatography (SEC). Samples were analyzed using a Waters SEC 1.7 μm column (4.6 × 150 mm) with an isocratic elution consisting of 300 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 5 mM orthophosphate, and 50% v / v 2-propanol. Chromatography was performed with UV detection (280 nm) at 50 °C using an injection volume of 1-10 μl and a flow rate of 0.3 ml / min. HMWP was quantified as the area of ​​all components eluting before the main peak × 100% divided by the area of ​​the main peak. [Table 5]

[0162] conclusion The results in Table 5 show that the chemical purity of semaglutide decreased over time. The chemical purity of semaglutide decreased more rapidly when formulated at pH 6.0 (semaglutide formulation 1) than when formulated at its optimal pH of 7.4 (semaglutide formulation 3). Surprisingly, HP-B-CD improved the chemical stability (in terms of purity loss and HMWP formation) of semaglutide when formulated at pH 6.0 (semaglutide formulation 2).

[0163] Example 4: Effect of molar substitution of HP-B-CD on the physical stability of caglilintide and semaglutide formulations This example shows the effect of molar substitution of HP-B-CD on the physical stability of caglilintide and semaglutide.

[0164] composition The compositions of Formulation 1 and Formulation 2 are shown in Table 6. [Table 6]

[0165] Preparation process The formulations were prepared as described in Example 1.

[0166] method All samples were stored under stress conditions defined as follows: - Duration: 28 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0167] The number of subvisible particles present quantifies the physical stability of the combined caglilintide and semaglutide and was obtained by microflow imaging (MFI, see for example Sharma, DK et al. AAPS J. (2010), 12: 455-464 for the principle of MFI technique). The following procedure was adopted for each analyzed syringe sample. The experiment was carried out at ambient temperature. The liquid from each syringe was removed by first removing the plunger and then pipetting the liquid into the sample container. The samples were transferred to a 96-deep well plate, which was inserted into the sample handling unit (Bot1) of the Protein Simple MFI™ 5200 instrument equipped with a standard Protein Simple MFI™ 100 μm flow cell. Samples were analyzed by standard MFI system settings, meaning that liquid was pipetted into a reservoir connected to a flow cell, the liquid was illuminated by a 10 LED light source (470 nm), and a digital camera (through magnification optics) recorded the contents of the flow cell as bright-field images throughout the experiment. Data collection was achieved using Protein Simple MVSS software. Recorded image streams from the entire experiment were processed by the validated Novo Nordisk proprietary software MFI Data Validator to obtain individual particle counts (normalized to counts per ml of analyzed liquid) and presented by size for the standard size ranges of subvisible particles: >5 μm, >10 μm, and >25 μm. Note that the number of particles that are >5 μm includes all particles with a diameter of >5 μm (>5 μm, >10 μm, and >25 μm), and the number of particles that are >10 μm includes all particles with a diameter of >10 μm (>10 μm and >25 μm). Particle size is defined as the equivalent circular diameter (ECD).

[0168] The presence of amyloid peptide fibrils was analyzed with a Thioflavin-T (ThT) fluorescence assay. Experiments were performed at 25 °C. Liquid from each syringe was removed by first removing the plunger and then pipetting the liquid into a sample container. Approximately 500 μl of sample was subsequently mixed with approximately 9 μl of ThT stock solution in a separate sample container to obtain a final ThT concentration of 20 μM. Samples were allowed to incubate for 25 min in the dark at ambient temperature. 200 μl of sample was transferred to wells in a 96-well microtiter plate. Samples were measured on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 440 nm and 470-550 nm, respectively.

[0169] Data collection was accomplished using CLARIOstar Control software. The emission maximum in this assay was: It was observed to occur at a wavelength of approximately 485 nm, and therefore the results of each assay were reported as ThT fluorescence at 485 nm, expressed in relative fluorescence units (RFU). [Table 7]

[0170] conclusion The results in Table 7 show that the fewest particles were produced in Formulation 2 containing HP-B-CD (average MS: 0.62). Furthermore, no increase in the number of subvisible particles or ThT fluorescence was observed for Formulation 2 over the 28 day period of the experiment.

[0171] In contrast, formulation 1 containing HP-B-CD (average MS: 0.92) showed an increase in the number of subvisible particles after 21 days and an increase in ThT fluorescence after 28 days.

[0172] In these otherwise identical citrate-buffered formulations of semaglutide and caglilintide, formulation (2) containing HP-B-CD (mean MS: 0.62) was more physically stable than formulation (1) containing HP-B-CD (mean MS: 0.92).

[0173] Example 5: Effect of hydroxypropyl-β-cyclodextrin concentration on the chemical stability of semaglutide This example shows the concentration-dependent effect of HP-B-CD on the chemical stability of semaglutide.

[0174] composition The compositions of Formulation 3, Formulation 4, and Formulation 5 are shown in Table 8. [Table 8]

[0175] preparation The formulations were prepared as described in Example 1.

[0176] method Samples were stored at 37°C for 28 days, whereupon they were analysed to determine the chemical purity of semaglutide after 14, 21 and 28 days.

[0177] The purity of semaglutide was determined using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) where samples were analysed using a Waters Acquity phenyl-hexyl 1.7 μm column (2.1 × 150 mm) with gradient elution of eluent A consisting of 0.09% TFA in MQ water and eluent B consisting of 0.09% TFA in MQ water, 0.09% TFA in 80% acetonitrile in MQ water. Chromatography was performed with UV detection (215 nm) at 62 °C using an injection volume of 2-14 μl and a flow rate of 0.25 ml / min. Purity was assessed as the area of ​​the main semaglutide peak divided by the area of ​​all relevant peaks × 100%.

[0178] It should be noted that in other experiments, the same method was used to determine caglirintide purity. [Table 9]

[0179] conclusion The results in Table 9 show that the chemical stability, and therefore the purity, of semaglutide also depended on the HP-B-CD concentration. Semaglutide was chemically stable in all formulations (containing 11.25 to 15% w / v HP-B-CD). However, the chemical stability, and therefore the purity, of semaglutide was highest when the formulation contained 15% w / v HP-B-CD.

[0180] Example 6: Effect of different tonicity agents on the physical stability of the formulations This example shows the stabilizing effect of different tonicity agents on the physical stability of otherwise identical caglilintide and semaglutide formulations.

[0181] composition The compositions of Compounds 6 to 12 are shown in Table 10. [Table 10]

[0182] Preparation process The formulations were prepared as described in Example 1.

[0183] method All samples were stored under stress conditions defined as follows: Duration: 18 days · Temperature: 37℃±2℃ Stress conditions: During storage, the specimens were rotated 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 100 times, 5 days per week.

[0184] The number of sub-visible particles was quantified as described in Example 4. [Table 11]

[0185] conclusion The results in Table 11 show that the subvisible particle count increased most rapidly in the formulation containing NaCl as a tonicity agent (Formulation 12). After 7 days, the particle count significantly exceeded that determined for the other formulations. Therefore, sampling for analysis of the number of subvisible particles was discontinued for the NaCl-containing formulations after 7 days.

[0186] After 14 days, an increase in the number of subvisible particles was observed in the formulations containing glycerol and sucrose, and the two formulations were considered equivalent in terms of physical stability. The particle count remained lowest in the formulations that contained mannitol, sorbitol, or trehalose. These formulations showed virtually no increase in the number of subvisible particles over the 18 days that the formulations were stored under stress conditions.

[0187] Of the formulations tested, those containing mannitol, sorbitol, or trehalose as tonicity agents remained the most stable over time.

[0188] Example 7: Effect of different surfactants on the physical stability of the formulations This example shows the effect of different surfactants on the physical stability of otherwise identical caglilintide and semaglutide formulations.

[0189] composition The compositions of Formulations 13, 14, and 15 are shown in Table 12. [Table 12]

[0190] Preparation process The formulations were prepared as described in Example 1.

[0191] method All samples were stored under stress conditions defined as follows: Duration: 17 days · Temperature: 37℃±2℃ Stress conditions: During storage, the specimens were rotated 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 100 times, 5 days per week.

[0192] The number of sub-visible particles was quantified as described in Example 4. [Table 13]

[0193] conclusion Formulation 14 contained the lowest number of subvisible particles when stored for 17 days under stress conditions. In formulation 13 containing polysorbate 20, an increase in subvisible particles was observed after 14 days, while in formulation 15 containing poloxamer 188, subvisible particles were formed after 7 days under stress conditions. It is clear that the formulation containing polysorbate 80 was the most stable, and that the formulation containing polysorbate 20 was also acceptably stable.

[0194] Example 8: Effect of different buffer substances on the physical stability of the formulations This example shows that buffering substances have an effect on the physical stability of otherwise identical caglilintide and semaglutide formulations.

[0195] composition The compositions of Formulation 1 and Formulation 16 are shown in Table 14. [Table 14]

[0196] Preparation process The formulations were prepared as described in Example 1.

[0197] method All samples were stored under stress conditions defined as follows: Duration: 21 days o Temperature: 37℃±2℃ o Stress conditions: During storage, the specimens were rotated 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 100 times, 5 days per week.

[0198] The number of sub-visible particles was quantified as described in Example 4. [Table 15]

[0199] conclusion Up to 14 days after storage under stress conditions, the physical stability of the two formulations was similar and acceptable. However, after 18 days, the number of subvisible particles in the citrate buffered formulation (Formulation 1) was much higher than in the histidine buffered formulation (Formulation 16). The histidine buffered formulation 16 was the most stable.

[0200] Example 9: Effect of Different Buffer Concentrations on the Chemical Stability of Formulations This example shows the effect of buffer concentration on the chemical stability of an otherwise identical formulation.

[0201] composition The compositions of Formulations 17 and 18 are shown in Table 16. [Table 16]

[0202] Preparation process The formulations were prepared as described in Example 1.

[0203] method Samples were stored at 30° C. for 21 days and analyzed to determine the chemical purity of caglilintide after 7, 14, and 21 days. The purity of caglilintide was determined as described in Example 5 (for semaglutide). [Table 17]

[0204] conclusion The results in Table 17 show that both formulations were stable. However, the chemical purity of caglilintide was highest in formulation 17. The purity of caglilintide decreased more rapidly over time when the histidine concentration was 20 mM.

[0205] Example 10: Effect of Different Buffer Concentrations on the Physical Stability of Formulations This example demonstrates the effect of histidine buffer concentration on the physical stability of the formulation.

[0206] composition The compositions of the tested formulations are shown in Table 16.

[0207] Preparation process The formulations were prepared as described in Example 1.

[0208] method All samples were stored under stress conditions defined as follows: Duration: 18 days · Temperature: 37℃±2℃ Stress conditions: During storage, the specimens were rotated 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 100 times, 5 days per week.

[0209] The number of sub-visible particles was quantified as described in Example 4. [Table 18]

[0210] conclusion The difference in physical stability between Formulations 17 and 18 was most evident after 14 days. The data in Table 18 show that the number of subvisible particles seen in Formulation 18 (containing 20 mM histidine) was greater than the number of subvisible particles seen in Formulation 17 (containing 6 mM histidine). That is, the formulation containing 6 mM histidine was the most physically stable.

[0211] Example 11: Effect of different types of hydroxypropyl-substituted cyclodextrins on the physical and chemical stability of formulations of caglilintide and semaglutide This example shows the effect of hydroxypropyl-alpha-cyclodextrin (HP-A-CD), hydroxypropyl-beta-cyclodextrin (HP-B-CD), and hydroxypropyl-gamma-cyclodextrin (HP-G-CD) on the formation of subvisible particles and the chemical degradation of caglilintide in otherwise identical caglilintide and semaglutide formulations.

[0212] composition The compositions of Formulations 22, 23, and 24 are shown in Table 19. [Table 19]

[0213] Preparation process The formulations were prepared as described in Example 1.

[0214] method The samples used to determine the subvisible particle count were stored under stress conditions defined as follows. - Duration: 42 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0215] The number of subvisible particles was determined as described in Example 4. Samples used to determine the purity of kaglilintide were stored at 37° C. for up to 42 days. The purity of kaglilintide was determined using the following reversed-phase high-performance liquid chromatography (RP-HPLC): samples were analyzed using a Kinetex C18, 2.6 μm, column (4.6×150 mm) with gradient elution of eluent A consisting of 90% v / v 0.09 M phosphate solution, pH 3.6 and 10% v / v acetonitrile, and eluent B consisting of 60% v / v acetonitrile and 20% v / v isopropanol. Chromatography was performed at 30° C. with UV detection (210 nm) using an injection volume of 10-100 μl and a flow rate of 0.7 ml / min. The purity of kaglilintide was quantified as the area of ​​the main peak divided by the area of ​​all relevant peaks × 100%.

[0216] The same method was used to determine the purity of semaglutide in other experiments. [Table 20] [Table 21]

[0217] conclusion The results presented in Table 20 show that in formulation 24 (HP-G-CD), a large number of subvisible particles were observed already at time zero, which precludes the use of HP-G-CD to formulate caglilintide and semaglutide. Sampling for analysis of the number of subvisible particles was stopped for formulation 24 containing HP-G-CD after the first analysis at time zero. For formulation 22 (HP-A-CD) and formulation 23 (HP-B-CD), virtually no increase in the number of subvisible particles was observed.

[0218] The results presented in Table 21 for the chemical purity of caglilintide using either HP-A-CD or HP-B-CD show a slightly more rapid decrease in caglilintide purity in formulation 22 containing HP-A-CD than in formulation 23 containing HP-B-CD.

[0219] Based on the results in Table 20, either HP-A-CD or HP-B-CD is acceptable for the formulation of caglilintide and semaglutide. However, based on the results in Table 21, HP-B-CD is preferred compared to HP-A-CD for the formulation of caglilintide and semaglutide due to the superior purity of caglilintide when formulated with HP-B-CD.

[0220] Example 12: Effect of molar substitution of HP-B-CD on the physical and chemical stability of caglilintide and semaglutide formulations This example shows the effect of molar substitution of HP-B-CD on subvisible particle formation, HMWP levels, and chemical purity of semaglutide in otherwise identical citrate-buffered caglilintide and semaglutide formulations.

[0221] composition The compositions of Formulations 25 to 32 are shown in Table 22. [Table 22]

[0222] Preparation process The formulations were prepared as described in Example 1.

[0223] method The samples used to determine the subvisible particle count were stored under stress conditions defined as follows. - Duration: 28 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0224] The number of subvisibles was quantified as described in Example 4.

[0225] Samples used to determine semaglutide purity and HMWP levels were stored for a maximum of 28 days at 37° C. Semaglutide purity was determined as in Example 14.

[0226] The levels of covalently bound HMWP were determined using size exclusion chromatography (SEC). Samples were analyzed using a Waters SEC 1.7 μm column (4.6 × 150 mm) with an isocratic elution consisting of 185 mM sodium chloride, 5 mM sodium dihydrogen phosphate monohydrate, 3 mM orthophosphate, and 47% (v / v) isopropanol. Chromatography was performed with UV detection (215 nm) at 50 °C using an injection volume of 1-8 μl and a flow rate of 0.3 ml / min. HMWP was quantified as the area of ​​all components eluting before the main peak × 100% divided by the area of ​​the main peak. [Table 23] [Table 24] [Table 25]

[0227] conclusion The results presented in Tables 23, 24, and 25 show that the physical stability, formation of HMWPs, and chemical purity of semaglutide, when studied in citrate-buffered caglilintide and semaglutide formulations, depend on the molar substitution of HP-B-CD. After 28 days, the HMWP levels were lowest, with virtually no increase in particle count in formulation 27 containing 25% w / v HP-B-CD (average MS: 0.62). In contrast, a large increase in subvisible particle count was observed in formulation 32 containing 25% w / v HP-B-CD (average MS: 1.08) after only 14 days, and it was also in this formulation that the highest levels of HMWPs were observed after 28 days.

[0228] All citrate-buffered caglilintide and semaglutide formulations containing 25% w / v HP-B-CD (mean MS: ≦0.92) were physically and chemically stable, with the formulation containing 25% w / v HP-B-CD being the most stable, with a mean MS ≦0.68.

[0229] After only 14 days, an increase in the number of subvisible particles was observed in formulation 26 containing 15% w / v HP-B-CD (average MS: 0.92), indicating physical instability of this particular formulation.

[0230] Formulation 25, containing 15% w / v HP-B-CD (average MS: 0.62), demonstrated acceptable chemical and physical stability.

[0231] However, histidine-buffered caglilintide and semaglutide formulations33-37 containing 15% w / v HP-B-CD are preferred due to their superior physical stability. With histidine as the buffer and sorbitol as the tonicity agent, the preferred HP-B-CD molar substitution range was extended to an average of 0.62-0.92 (or a total of 0.58-1.0).

[0232] Example 13: Effect of molar substitution of HP-B-CD on the physical stability of caglilintide and semaglutide formulations This example shows the effect of molar substitution of HP-B-CD on the level of subvisible particles in otherwise identical histidine-buffered caglilintide and semaglutide formulations.

[0233] composition The compositions of Formulations 33-38 are shown in Table 26. [Table 26]

[0234] Preparation process The formulations were prepared as described in Example 1.

[0235] method The samples used to determine the number of subvisible particles were stored under stress conditions defined as follows. - Duration: 28 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0236] The number of sub-visible particles was quantified as described in Example 4. [Table 27]

[0237] conclusion The results presented in Table 27 show that histidine-buffered formulations 33-37 containing HP-B-CD with a wide range of molar substitution (average MS: 0.62-0.92) remained physically stable for 28 days.

[0238] In contrast, formulation 38, which contained HP-B-CD (average MS 1.08), was not physically stable after 14 days.

[0239] The results indicate synergy between cyclodextrins and other excipients in the formulation, extending the preferred range of molar substitution (average MS 0.62-0.92).

[0240] Example 14: Effect of beta-cyclodextrin substitution on the physical stability of formulations This example shows the effect of sulfobutylether-B-cyclodextrin (SBE-B-CD) and hydroxypropyl-beta-cyclodextrin on the physical stability of otherwise identical caglilintide and semaglutide formulations.

[0241] composition The compositions of Formulations 39 and 40 are shown in Table 28. [Table 28]

[0242] Preparation process The formulations were prepared as described in Example 1.

[0243] method The samples used to determine the number of subvisible particles were stored under stress conditions defined as follows. - Duration: 35 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0244] The number of sub-visible particles was quantified as described in Example 4. [Table 29]

[0245] conclusion The results in Table 29 show that when SBE-B-CD is used to formulate caglilintide and semaglutide, a large increase in the number of subvisible particles is observed after 14 days, i.e., the formulation is physically unstable. When HP-B-CD is used instead, virtually no increase is observed over the 35 day period of the study, i.e., the formulation is physically stable.

[0246] In contrast to those shown for hydroxypropyl-beta-cyclodextrin, these results indicate that sulfobutylether-B-cyclodextrin (SBE-B-CD) is not a suitable cyclodextrin for use in formulating caglilintide and semaglutide.

[0247] Example 15: Effect of pH on the physical and chemical stability of formulations This example shows the effect of pH on the physical and chemical stability of otherwise identical caglilintide and semaglutide formulations.

[0248] composition The compositions of Formulations 41 to 45 are shown in Table 30. [Table 30]

[0249] Preparation process The formulations were prepared as described in Example 1.

[0250] method The samples used to determine the number of subvisible particles were stored under stress conditions defined as follows. - Duration: 28 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0251] The number of sub-visible particles was quantified as described in Example 4.

[0252] The samples used to determine the purity of caglirintide were stored for a maximum of 28 days at 37° C. The purity of caglirintide was determined as described in Example 14. [Table 31] [Table 32]

[0253] conclusion The results presented in Tables 31 and 32 show that the physical and chemical stability of semaglutide and caglilintide is dependent on the pH of the formulation, with the highest pH resulting in the lowest caglilintide purity after 28 days at 37° C. and the lowest pH resulting in an increase in the number of subvisible particles after 14 days. Based on these physical and chemical stability results, the preferred pH range for this particular caglilintide and semaglutide formulation is 5.6-6.0, although a pH of 5.5 did not result in a formulation with acceptable physical stability.

[0254] Example 16: Effect of concentration ratio of caglilintide and semaglutide on the physical stability of the formulation This example shows the effect of different concentration ratios of caglilintide and semaglutide on the level of sub-visible particles observed in the formulation.

[0255] composition The compositions of histidine buffered formulations 46-50 are shown in Table 33, and the compositions of histidine buffered formulations 51-61 are shown in Table 34. [Table 33] [Table 34]

[0256] Preparation process The formulations were prepared as described in Example 1.

[0257] method The samples used to determine the number of subvisible particles were stored under stress conditions defined as follows. - Duration: 28 days - Temperature: 30℃±2℃ - Stress conditions: During storage, the specimens were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times on 3 days per week and 40 times on 2 days per week.

[0258] The number of sub-visible particles was quantified as described in Example 4. [Table 35-1] [Table 35-2]

[0259] conclusion The results presented in Table 35 show that after 21 days, formulations 46-60 containing 3.2 mg / ml caglilintide and up to 12 mg / ml semaglutide showed virtually no increase in the number of subvisible particles.

[0260] After 14, an increase in the number of subvisible particles was seen for formulation 61 containing 3.2 mg / ml caglilintide and 16 mg / ml semaglutide.

[0261] All histidine-buffered formulations 46 - 61 containing 3.2 mg / ml caglilintide and up to 16 mg / ml semaglutide were physically stable.

[0262] Example 17: Effect of HP-B-CD concentration on the physical stability of the formulations This example shows the effect of HP-B-CD concentration on the physical stability of caglilintide and semaglutide formulations when the formulations are exposed to physical stress.

[0263] composition The compositions of formulations 62-65 with histidine buffer composition are shown in Table 36. [Table 36]

[0264] Preparation process The formulations were prepared as described in Example 1.

[0265] method The tendency of caglilintide and semaglutide in the formulations to aggregate and form peptide fibrils was measured using the Thioflavin T (ThT) fluorescent stress assay described in Example 2. [Table 37]

[0266] conclusion The results presented in Table 37 show that the physical stability of caglilintide and semaglutide formulations depends on the concentration of HP-B-CD, with lower concentrations resulting in a shorter lag time before fibrillation occurs. The formulation containing 7.5% w / v HP-B-CD was the least stable. The formulation containing 15% w / v HP-B-CD was the most stable.

[0267] Example 18: Effect of order of drug substance addition on the physical stability of caglilintide and semaglutide formulations This example shows how the order of addition of semaglutide and caglilintide drug substances during formulation manufacture affects the physical stability of the formulation in the form of subvisible particle count.

[0268] composition The composition of the semaglutide and caglilintide combination is shown in Table 38. [Table 38]

[0269] Formulation Method The formulations were prepared using either formulation method 1 or formulation method 3, shown in Table 39 and depicted in Figures 1 and 3. [Table 39]

[0270] method After preparation of the formulations, all samples were stored under stress conditions defined as follows: - Duration: 6 weeks - Temperature: 30℃±2℃ - Stress conditions: During storage, the samples were turned 360° to simulate patient use after removal from refrigerated storage. Rotations were performed 20 times over 3 days and 40 times over 2 days, weekly.

[0271] The number of sub-visible particles was quantified as described in Example 4. [Table 40]

[0272] conclusion The physical stability of the final drug product is affected by the order of drug substance addition. The formation of sub-visible particles as a function of storage time is minimized in formulation method 1, where the semaglutide drug substance is added before the caglilintide drug substance.

[0273] Example 19: Effect of Polysorbate 80 on Drug Substance Dissolution Time This example shows the effect of the presence or absence of polysorbate 80 on drug substance dissolution time.

[0274] composition The composition of the caglilintide and semaglutide combination is as shown in Example 18.

[0275] Formulation Method The formulations were prepared using either formulation method 1 or formulation method 5 shown in Table 41 and depicted in FIGS. [Table 41]

[0276] method The excipients were dissolved in water according to the formulation method shown in Table 41. Once the excipient solution is clear, the semaglutide drug substance is added. The time until the semaglutide drug substance is completely dissolved and the solution is clear is measured.

[0277] The results in Table 42 show a long dissolution time for the semaglutide drug substance when formulation method 1 is used, in which polysorbate 80 is added to the excipient solution in step 1. In contrast, the semaglutide drug substance dissolution time is significantly shortened when using formulation method 5, in which polysorbate 80 is added in step 5 after both drug substances have been added. [Table 42]

[0278] conclusion The dissolution time of semaglutide drug substance is affected by the presence of polysorbate 80 in process step 1. By omitting polysorbate 80 from process step 1 and instead adding it in process step 5 (i.e. formulation method 5), the dissolution time of the semaglutide drug substance can be significantly reduced.

[0279] Example 20: Effect of order of drug substance addition on drug substance dissolution time in caglilintide and semaglutide formulations containing HP-B-CD (average MS: 0.62) This example shows how the order of adding the semaglutide or caglilintide drug substance first during formulation manufacture affects drug substance dissolution time.

[0280] composition The composition of caglilintide and semaglutide formulation 67 was as shown in Table 43. [Table 43]

[0281] Formulation Method Four different formulations having the compositions shown in Table 43 were produced using formulation methods 1, 2, 5, and 6, as described in the figure legends and depicted in Figures 1, 2, 5, and 6.

[0282] method During preparation of the formulations using the formulation methods described above, the time from addition of semaglutide and caglilintide drug substances to when the drug substances were completely dissolved and the solution was clear was measured. [Table 44]

[0283] conclusion The dissolution times of semaglutide and caglilintide drug substances were affected by the order of addition of the drug substances. By adding semaglutide before caglilintide during the manufacture of caglilintide and semaglutide formulations, the total dissolution time of caglilintide and semaglutide drug substances can be significantly reduced.

[0284] Example 21: Effect of order of drug substance addition on drug substance dissolution time in caglilintide and semaglutide formulations containing HP-B-CD (average MS: 0.92) This example shows how the order of adding the semaglutide or caglilintide drug substance first during formulation manufacture affects drug substance dissolution time.

[0285] composition The composition of caglilintide and semaglutide formulation 68 was as shown in Table 45. [Table 45]

[0286] Formulation Method Four different formulations having the compositions shown in Table 45 were produced using formulation methods 1, 2, 5, and 6, as described in the figure legends and depicted in Figures 1, 2, 5, and 6.

[0287] method During the manufacture of the formulations using the formulation methods described above, the time from addition of semaglutide and caglilintide drug substances to when the drug substances were completely dissolved and the solution was clear was measured. [Table 46]

[0288] conclusion The dissolution times of semaglutide and caglilintide drug substances were affected by the order of adding the drug substances when polysorbate 80 was present in the excipient solution. Comparing formulation methods 1 and 2, the total dissolution times of caglilintide and semaglutide drug substances can be significantly shortened by adding semaglutide before adding caglilintide in formulation method 2. Comparing formulation methods 5 and 6, where polysorbate 80 was not present in the excipient solution, no effect of the order of adding the drug substances was observed.

[0289] Example 22: Effect of order of excipient addition on drug substance dissolution time in caglilintide and semaglutide formulations with HP-B-CD (average MS: 0.62) This example shows how the order of excipient addition during formulation manufacture affects drug substance dissolution time. The effect of each excipient present in the initial excipient solution versus being added after the drug substance addition is shown.

[0290] composition The composition of caglilintide and semaglutide formulation 67 was as presented in Example 20.

[0291] Formulation Method Nine different formulations having the composition presented in Example 20 were prepared using formulation methods 1, 4, 5, 7, 8, 9, 10, 11, and 12 described in the figure legends and depicted in Figures 1, 4, 5, 7, 8, 9, 10, 11, and 12.

[0292] method During the manufacture of the formulations using the formulation methods described above, the time from addition of semaglutide and caglilintide drug substances to when the drug substances were completely dissolved and the solution was clear was measured. [Table 47]

[0293] conclusion The dissolution times of both caglilintide and semaglutide, and thereby the total dissolution time, were significantly increased by the presence of polysorbate 80 in the initial excipient solution compared to when polysorbate 80 was added after dissolution of both drug substances. In formulation method 4, where polysorbate 80 was added after semaglutide and before caglilintide, the dissolution time of semaglutide was reduced compared to the dissolution time of formulation method 1, while the dissolution time of caglilintide remained unchanged. The order of addition of excipients other than polysorbate 80 did not affect the dissolution times of caglilintide and semaglutide.

[0294] Example 23: Effect of order of excipient addition on drug substance dissolution time in caglilintide and semaglutide formulations with HP-B-CD (average MS: 0.92) This example shows how the order of excipient addition during formulation manufacture affects drug substance dissolution time. The effect of each excipient present in the initial excipient solution versus being added after the drug substance addition is shown.

[0295] composition The composition of caglilintide and semaglutide formulation 68 was as presented in Example 21.

[0296] Formulation Method Eight different formulations having the composition presented in Example 21 were prepared using formulation methods 1, 5, 7, 8, 9, 10, 11, and 12 described in the figure legends and depicted in Figures 1, 5, 7, 8, 9, 10, 11, and 12.

[0297] method During the manufacture of the formulations using the formulation methods described above, the time from addition of semaglutide and caglilintide drug substances to when the drug substances were completely dissolved and the solution was clear was measured. [Table 48]

[0298] conclusion The dissolution times of caglilintide and semaglutide drug substances, and thereby also the total dissolution time, were significantly increased by the presence of polysorbate 80 in the initial excipient solutions compared to when polysorbate 80 was added after dissolution of the drug substances. The order of addition of excipients other than polysorbate 80 does not affect the dissolution times of caglilintide and semaglutide.

[0299] Example 24: Effect of order of drug substance addition on the physical stability of caglilintide and semaglutide formulations. This example shows how the order of addition of semaglutide and caglilintide drug substances during formulation manufacture affects the physical stability of the formulation, measured as the lag time until fibrillation occurs.

[0300] composition The composition of the caglilintide and semaglutide combination is shown in Example 20.

[0301] Formulation Method Four different formulations having the compositions shown in Example 20 were prepared using preparation methods 1, 2, 5, and 6 described in the figure legends and depicted in FIGS.

[0302] method The tendency of caglilintide and semaglutide in the formulations to aggregate and form peptide fibrils was measured using the Thioflavin T (ThT) fluorescent stress assay described in Example 2. [Table 49]

[0303] conclusion The physical stability of the final caglilintide and semaglutide formulations is affected by the order in which the two drug substances are added. By adding the semaglutide drug substance before adding the caglilintide drug substance, increased physical stability was achieved, measured as a longer lag time from the start of the ThT fibrillation assay until fibrillation occurred. This observation was seen regardless of whether polysorbate 80 was added to the initial excipient solution or after dissolution of both drug substances.

[0304] Example 25: Effect of in-process pH adjustment during formulation process on the physical stability of caglilintide and semaglutide formulations. This example shows the effect of in-process pH adjustment before or after the addition of caglirintide on the physical stability of the formulation, measured as the lag time until fibrillation occurs.

[0305] composition The composition of the caglilintide and semaglutide combination is shown in Example 20.

[0306] Formulation Method Two different formulations having the compositions shown in Example 20 were prepared using Preparation Methods 2 and 3, described in the Figure Legend and depicted in FIGS.

[0307] method The tendency of caglilintide and semaglutide in the formulations to aggregate and form peptide fibrils was measured using the Thioflavin T (ThT) fluorescent stress assay described in Example 2. [Table 50]

[0308] conclusion The results show that physical stability, measured as lag time to fibrillation, was increased when in-process pH adjustment was performed before adding caglilintide in formulation method 3 compared to formulation 2 where the pH was adjusted after the addition of caglilintide.

[0309] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art, and it is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A method for preparing a pharmaceutical formulation comprising caglilintide and semaglutide, comprising the steps of: Obtaining or preparing a drug substance (DS), e.g., a solid DS, comprising caglirintide as a pharmaceutical active ingredient; - obtaining or preparing a drug substance (DS), e.g. a solid DS, comprising semaglutide as an active pharmaceutical ingredient; preparing a surfactant-free aqueous excipient solution comprising cyclodextrin containing hydroxypropyl substitutions and water for injection (WFI) to about 50-80%, e.g., about 65%, of a final predetermined volume, and having a pH of 6.5-8.5, e.g., a pH of about 7.0-8.0, preferably about 7.5; - dissolving the semaglutide DS in the excipient solution; adjusting the pH to 5.5-6.5, for example about 5.9-6.1, preferably about 6.0; - dissolving the caglilintide DS in the mixture of the excipient solution and the semaglutide DS; adding polysorbate 20 and / or polysorbate 80 to the mixture of semaglutide DS, the excipient solution and the caglirintide DS; Optionally, adding water to about 80-99% of the final predetermined volume; Optionally adjusting the pH to about 5.5-6.1, such as 5.7-6.1, such as 5.9-6.1, such as 5.7-5.9, such as 5.8; and - adding water to reach the final predetermined volume (100%).

2. 2. The method of claim 1, wherein the cyclodextrin is a hydroxypropyl-substituted alpha type containing glucose units with a six-membered ring structure and / or a hydroxypropyl-substituted beta type containing glucose units with a seven-membered ring structure.

3. 3. The method of claim 1 or 2, wherein the surfactant-free aqueous excipient solution comprising cyclodextrin comprises one or more additional excipients, with the proviso that the additional excipients are not surfactants.

4. 4. The method of any one of claims 1 to 3, comprising adding one or more further excipients to the mixture of excipient solution and GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.

5. The method of any one of claims 1 to 4, comprising adding one or more further excipients to the mixture of excipient solution, GLP-1 receptor agonist, and amylin receptor agonist.

6. 6. The method of any one of claims 1 to 5, comprising adding one or more further excipients to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and polysorbate 20 and / or polysorbate 80.

7. The method of any one of claims 1 to 6, wherein the surfactant-free aqueous excipient solution comprising a cyclodextrin further comprises a buffering agent.

8. The method of any one of claims 1 to 7, further comprising adding a buffering agent to the mixture of excipient solution and GLP-1 receptor agonist.

9. The method of any one of claims 1 to 8, further comprising adding a buffering agent to the mixture of excipient solution, GLP-1 receptor agonist, and amylin receptor agonist.

10. 10. The method of any one of claims 1 to 9, further comprising adding a buffering agent to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and polysorbate 20 and / or polysorbate 80.

11. 11. The method of any one of claims 1 to 10, wherein the buffer, such as histidine, citrate or phosphate, or a combination thereof, preferably histidine, has at least one pKa of about 5.0 to 7.

0.

12. 12. The method of any one of claims 1 to 11, wherein the surfactant-free aqueous excipient solution further comprises an isotonicity agent, with the proviso that the isotonicity agent is not sodium chloride.

13. 13. The method of any one of claims 1 to 12, further comprising adding an isotonicity agent to the mixture of excipient solution and GLP-1 receptor agonist, with the proviso that said isotonicity agent is not sodium chloride.

14. 14. The method of any one of claims 1 to 13, further comprising adding an isotonicity agent to the mixture of excipient solution, GLP-1 receptor agonist, and amylin receptor agonist, with the proviso that said isotonicity agent is not sodium chloride.

15. 15. The method of any one of claims 1 to 14, further comprising adding an isotonicity agent to the mixture of excipient solution, GLP-1 receptor agonist, amylin receptor agonist, and surfactant, with the proviso that said isotonicity agent is not sodium chloride.

Images