TWI876242B - Pharmaceutical formulations comprising a cyclodextrin - Google Patents

Abstract

Disclosed herein is a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist and a cyclodextrin comprising hydroxypropyl substitutions. Said co-formulation may be used for the medical treatment of subjects with overweight or obesity, with or without associated co-morbidities; diabetes, with or without associated comorbidities; cardiovascular diseases, non-alcoholic steatohepatitis (NASH) and cognitive impairment, such as that caused by Alzheimer’s disease.

Description

Pharmaceutical formulations comprising cyclodextrin

The present invention relates to a pharmaceutical formulation, which is a co-formulation of a GLP-1 receptor agonist and an starch receptor agonist. The pharmaceutical formulation can be used for the medical treatment of overweight or obesity with or without one or more associated comorbidities; diabetes with or without one or more associated comorbidities; cardiovascular disease; non-alcoholic steatohepatitis (NASH); and individuals with cognitive impairment, such as diseases caused by Alzheimer's disease.

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.

Semaglutide is also the active pharmaceutical ingredient in Wegovy®. Ozempic® is indicated 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 of 30 kg/ ^m2 or greater than 27 kg/ ^m2 in the presence of at least one weight-related comorbidity.

Ozempic® and Wegovy® are liquid pharmaceutical formulations that include 8 mM phosphate and have a pH of approximately 7.4.

A fixed-dose combination of the starch receptor agonist carglinide and the GLP-1 receptor agonist semaglutide has been studied for the treatment of overweight and obesity (Lancet 2021; 397: 1736–48). The drug products studied are in the form of separate liquid pharmaceutical formulations for subcutaneous administration, which include carglinide or semaglutide, respectively.

To date, the possibility of co-formulating semaglutide and carglinide has not been considered due to the different physicochemical properties of these active pharmaceutical ingredients. Semaglutide, a GLP-1 receptor agonist, has an isoelectric point that is incompatible with the pH optimum of carglinide, a starch receptor agonist. Semaglutide is most stable at pH 7.4 and has previously required formulation in neutral to slightly alkaline solutions at pH 7 to 8 to ensure its solubility in aqueous solutions. Carglinide is most stable at pH 4.0 and has required formulation in acidic solutions, increasing the pH accelerating its rate of chemical degradation. The different physicochemical properties of carglinide and semaglutide preclude a simple mixture of the two peptides. The same applies to other GLP-1 receptor agonist and amylin receptor agonist combinations when the two have incompatible optimal pH ranges.

There remains a need in the art for a simple means to co-administer GLP-1 receptor agonists (such as semaglutide) and amylin receptor agonists (such as capraglinide).

This article discloses a means of co-formulating an amylin receptor agonist and a GLP-1 receptor agonist. This article discloses a liquid pharmaceutical formulation, which includes an amylin receptor agonist, a GLP-1 receptor agonist, and a cyclodextrin having a hydrophilic chemical substitution (such as a hydroxypropyl substitution). The cyclodextrin may be an α-type substituted with a hydroxypropyl group, which includes six cyclically arranged glucose units. The cyclodextrin may be a β-type substituted with a hydroxypropyl group, which includes seven cyclically arranged glucose units. The pharmaceutical formulation may also include a buffer (such as histidine), a tonicity agent (such as sorbitol) and/or a surfactant (such as polysorbate 80); and has a pH value of about 5.5 to 6.5. The pharmaceutical formulations disclosed herein can be administered by parenteral injection, preferably subcutaneous injection.

The pharmaceutical formulations disclosed herein can be used to medically treat individuals with: overweight or obesity with or without associated comorbidities; diabetes with or without associated comorbidities; cardiovascular disease; nonalcoholic steatohepatitis (NASH); and cognitive impairments, such as those caused by Alzheimer's disease. The pharmaceutical formulations disclosed herein can improve convenience, treatment compliance, and ultimately improve clinical outcomes in patients.

The present invention is a liquid pharmaceutical formulation, which comprises an amylin receptor agonist, a GLP-1 receptor agonist and a cyclodextrin substituted with a hydroxypropyl group.

The pharmaceutical formulation disclosed herein includes two active pharmaceutical ingredients, namely a GLP-1 receptor agonist and an amyloid receptor agonist.

Disclosed herein is a means of co-formulating an amylin receptor agonist and a GLP-1 receptor agonist, wherein the GLP-1 receptor agonist has an isoelectric point that excludes co-formulation in a pH range such that the amylin receptor agonist is chemically stable. Disclosed herein is a means of co-formulating a GLP-1 receptor agonist having an isoelectric point (pI) below 6.0 (e.g., 3.5 to 6.0, e.g., 4.0 to 6.0) with an amylin receptor agonist.

The optimal pH of an amylin receptor agonist is the pH at which it is most chemically and physically stable. The optimal pH of a GLP-1 receptor agonist is the pH at which it is most chemically and physically stable. The physical stability of a GLP-1 receptor agonist may reflect its isoelectric point, which may coincide with the pH at which the worst physical stability is expected.

Disclosed herein is a means of formulating an amylin receptor agonist and a GLP-1 receptor agonist whose optimal pH values differ by at least about two pH units, such as 2 to 5 pH units, such as 2 to 4 pH units, such as 3 to 5 pH units.

The GLP-1 receptor agonist may be semaglutide. The amylin receptor agonist may be cagerinide or a biologically active metabolite of cagerinide. The compositions of the formulations disclosed herein improve the chemical and physical stability of those active drug ingredients; retain the pharmacokinetic profile of the bioavailability and exposure of the active drug ingredients; and exhibit acceptable local tolerability upon subcutaneous injection.

The terms "pharmaceutical formulation", "co-formulation" and "drug product" are used interchangeably herein to refer to a liquid pharmaceutical formulation comprising a GLP-1 receptor agonist and an amyloid receptor agonist.

The pharmaceutical formulation disclosed herein is suitable for parenteral injection, preferably subcutaneous injection .

The term "amylin" herein refers to a polypeptide having the same amino acid sequence as endogenous amylin, such as human amylin .

Amylin compounds can target the calcitonin receptor (CTR) and/or the amylin receptor (AMYR). The latter consists of two component isoforms: the calcitonin receptor (CTR) and one of three receptor activity modifying proteins (RAMP1–3) that result in three possible complexes, AMYR1–3. Amylin receptor agonists

The pharmaceutical formulation disclosed in the present invention includes an amyloid receptor agonist. An "amyloid receptor agonist" can be defined as a chemical entity that can bind to and activate an amyloid receptor. In the context of the present invention, an "amyloid receptor agonist" can at least bind to and activate amyloid receptor 3 (AMYR3). Amyloid receptor agonists can also agonize calcitonin receptors and AMYR1-2.

Examples of endogenous amylin receptor agonists are human amylin and human calcitonin. An example of an exogenous amylin receptor agonist is calgrin. An example of an exogenous amylin receptor agonist is pramlintide.

The amylin receptor agonist in the pharmaceutical formulation disclosed herein may be cagerinide or a biologically active metabolite of cagerinide.

The biologically active metabolite of kaglinide may have aspartate (Asp) at position 21 or 22. The biologically active metabolite of kaglinide may have isoaspartate (iso-Asp) at position 21 or 22.

Kaglinide is a starch receptor agonist, also known as AM833. It is the compound of Example 53 in WO2012/168432: N-α-[(S)-4-carboxy-4-(19-carboxynonadecayl)butyryl]-[Glu14, Arg17, Pro37]-pramlintide. Kaglinide can be prepared as described in WO2012/168432, pages 153 to 155.

Kaglinide can be in the form of a salt, preferably a pharmaceutically acceptable salt.

The concentration of calgrin peptide in the pharmaceutical formulations disclosed herein may be from about 0.25 mg/ml to about 22 mg/ml.

The pharmaceutical formulations disclosed herein may include capgranin at a concentration of about 0.33 to 18 mg/ml; such as 0.25 to 0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5 to 1.0 mg/ml, such as about 0.67 mg/ml; such as 1.0 to 1.5 mg/ml, such as about 1.33 mg/ml; such as 1.5 to 2.0 mg/ml, such as about 1.5 mg/ml; such as 2.0 to 2.5 mg/ml; such as 2.5 to 3.0 mg/ml; such as 3.0 to 3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5 to 4.0 mg/ml; such as 4.0 to 5.0 mg/ml; such as 5.0 to 6.0 such as 13 to 22 mg/ml, such as about 18 mg/ml; such as about 20 to 22 mg/ml.

The pharmaceutical formulation disclosed herein may include no more than 22 mg/ml of cagerin. The pharmaceutical formulation disclosed herein may include no more than 12 mg/ml of cagerin. GLP-1

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

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

An example of an exogenous GLP-1 receptor agonist is semaglutide. An example of an exogenous GLP-1 receptor agonist is tesiparatide. semaglutide

Semaglutide is a GLP-1 receptor agonist, also known as N ^6.26 -{18-[N-(17-carboxyheptadecayl)-L-γ-glutamidoyl]-10-oxo-3,6,12,15-tetraoxo-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propionic acid),34-L-arginine] human glucagon peptide 1 (7-37). Semaglutide can be prepared as described in Example 4 of WO2006/097537.

Semaglutide may exist in the composition in a fully or partially ionized form; for example, one or more carboxylic acid groups (-COOH) may be deprotonated to carboxyl groups ( ^-COO- ) and/or one or more amine groups ( _-NH2 ) may be protonated to _-NH3 ⁺ groups.

Semaglutide may be in the form of a salt, preferably a pharmaceutically acceptable salt.

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

The pharmaceutical formulation may include semaglutide at a concentration of about 0.33 to 18 mg/ml; such as 0.25 to 0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5 to 1.0 mg/ml, such as about 0.67 mg/ml; such as 1.0 to 1.5 mg/ml, such as about 1.33 mg/ml; such as 1.5 to 2.0 mg/ml, such as about 1.5 mg/ml; such as 2.0 to 2.5 mg/ml; such as about 2.2 mg/ml; such as 2.5 to 3.0 mg/ml; such as 3.0 to 3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5 to 4.0 mg/ml; such as 4.0 to 5.0 such as about 4.8 mg/ml; such as 5.0 to 6.0 mg/ml; such as 6.0 to 7.0 mg/ml, such as about 6.4 mg/ml; such as 7.0 to 8.0 mg/ml, such as about 8.0 mg/ml; such as 8.0 to 9.0 mg/ml, such as 9.0 to 10.0 mg/ml, such as about 9.6 mg/ml; such as 10 to 11 mg/ml, such as about 10.7 mg/ml; such as 11.0 to 12.0 mg/ml, such as 11 to 13 mg/ml; such as about 12.8 mg/ml; such as 13 to 22 mg/ml, such as about 16 mg/ml; such as about 18 mg/ml; such as about 20 to 22 mg/ml.

The pharmaceutical formulation disclosed herein may include no more than 22 mg/ml of semaglutide. The pharmaceutical formulation disclosed herein may include no more than 12 mg/ml of semaglutide. Isoelectric point

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

The theoretical calculated isoelectric point of a GLP-1 receptor agonist may be in the range of 3.5 to 6.0, such as 4.0 to 6.0, such as 3.8 to 4.9, such as 4.0 to 4.5. Semaglutide has a theoretical calculated isoelectric point of about 4.37.

The theoretical calculated isoelectric point of the amylin receptor agonist may have an isoelectric point (pI) in the range of 8 to 12, such as 8 to 9. Capgranin has a theoretical calculated isoelectric point of about 8.56. Cyclodextrin

The pharmaceutical formulation disclosed herein includes cyclodextrins substituted with hydroxypropyl groups. The pharmaceutical formulation may include about 10% to 25% w/v of cyclodextrins substituted with hydroxypropyl groups. The pharmaceutical formulation may include more than 10% w/v of cyclodextrins substituted with hydroxypropyl groups. The pharmaceutical formulation may include less than 22% w/v of cyclodextrins substituted with hydroxypropyl groups. The pharmaceutical formulation may include about 10% to 20% w/v, about 15% to 25% w/v, about 12% to 18% w/v, about 10% to 17.5% w/v, about 11.25% to 15%, such as about 15% w/v of cyclodextrins substituted with hydroxypropyl groups.

Cyclodextrins are oligosaccharide starch derivatives composed of 6, 7, or 8 α-(1,4)-glucopyranose (glucose) units arranged in a cycle and expressed as α, β, or γ forms, respectively. Cyclodextrins have a wide range of applications as pharmaceutical excipients [P. Breen & S. S. Jambhekar, Cyclodextrins in pharmaceutical formulations II: solubilization, binding constant, and complexation efficiency, Drug Discovery Today, Vol. 21, February 2, 2016]. The European Medicines Agency describes their use as excipients in its guidance [Background review for cyclodextrins used as excipients, 2014, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)], [Cyclodextrins used as excipients, 2017, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)]. Types of cyclodextrins without hydrophilic substitutions have very poor solubility and are rarely used in parenteral medicinal products.

In order to improve the solubility of cyclodextrins, the hydroxyl groups of the glucose units of the cyclodextrins can be hydrophilically chemically substituted by different numbers of, for example, hydroxypropyl groups, resulting in different degrees of substitution, which can be described as the average number of hydroxypropyl groups per cyclodextrin molecule (abbreviated DS) or as the molar substitution, which corresponds to the average number of hydroxypropyl groups per glucose unit present in the relevant cyclodextrin (abbreviated MS). The hydroxypropyl value of each cyclodextrin can be achieved by multiplying the molar substitution by the number of glucose units included in the relevant cyclodextrin. Differences in the degree of substitution can lead to changes in physicochemical properties, such as surface activity and complexing ability. Hydroxyl groups can also be chemically substituted by sulfobutyl ether groups. These primary 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 containing hydroxypropyl substitutions are generally abbreviated as HP-CD, while cyclodextrins containing sulfobutyl ether substitutions are abbreviated as SBE-CD.

Cyclodextrins including hydrophilic substitutions adopt a shape that can be described as a pyramidal shape with a hydrophobic inner cavity and a hydrophilic outer surface formed by many hydrophilic substituents capable of forming hydrogen bonds with adjacent water molecules, thereby improving water solubility [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11].

Their hydrophobic microenvironment within the cavities of these pyramidal structures allows them to form complexes of drugs and cyclodextrins primarily through hydrophobic interactions [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11]. When complexes are formed between cyclodextrins and drug molecules with one or more hydrophobic regions, these regions as well as the hydrophobic regions of the cyclodextrins are shielded by water, thereby increasing the solubility of the complex compared to the solubility of the individual components. Furthermore, once a complex is formed between cyclodextrin and peptide molecules, it impairs the intermolecular interactions that normally lead to aggregation [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11].

Unexpectedly, cyclodextrins with hydroxypropyl substitutions were found to be superior in stabilizing co-formulations of cagriceptide and semaglutide with each other compared to the same cyclodextrin type with sulfobutyl ether substitutions.

Cyclodextrins may be of the hydroxypropyl-substituted α-type, which includes six glucose units arranged in a ring. The α-type hydroxypropyl-substituted cyclodextrin is abbreviated as HP-A-CD. Hydroxypropyl-α-cyclodextrin (CAS: 128446-33-3/99241-24-4) is commercially available with an average molar substitution (MS) of 0.8 and a molar substitution range of 0.5 to 0.9.

The pharmaceutical formulations disclosed herein may include cyclodextrin, which is a hydroxypropyl-substituted beta form comprising seven glucose units arranged in a ring.

The β-hydroxypropyl substituted cyclodextrin is abbreviated as HP-B-CD.

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

According to the European and United States Pharmacopoeias [USP 38 NF 33, Pharm Eur 8, estimated according to the method described in USP〈761〉/Pharm. Eur. 2.2.33], the hydroxypropyl degree of substitution (DS) of commercially available hydroxypropyl-β-cyclodextrins as pharmaceutical excipients ranges between 2.8 and 10.5, corresponding to 0.4 to 1.5 hydroxypropyl groups (MS) per glucose unit. Commercially available cyclodextrins (such as hydroxypropyl-β-cyclodextrin) are usually described by their average molar substitution (MS) over a range of molar substitutions.

Hydroxypropyl-β-cyclodextrin (CAS: 128446-35-5/94035-02-6) is commercially available and can be used as an excipient having an average molar substitution (MS) comprising: MS: 0.62, molar substitution range 0.58 to 0.68; MS: 0.67, molar substitution range from (0.6 to 0.9); MS: 0.68, molar substitution range from (0.58 to 0.72); MS: 0.84, molar substitution range from (0.8 to 1.0); MS: 0.92, molar substitution range from (0.81 to 0.99); MS: 1.08, molar substitution range from (0.86 to 1.14); wherein each number describes the number of hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having a minimum of about 0.4 hydroxypropyl groups per glucose unit. The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having a maximum of about 1.0 hydroxypropyl groups per glucose unit.

The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having a substitution range of 0.58 to 1.0 hydroxypropyl groups per glucose unit. The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) range of about 0.62 to 0.92 hydroxypropyl groups per glucose unit.

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

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.4 to 0.75 hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin, which has about 0.75 hydroxypropyl groups per glucose unit.

The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) of about 0.62 per glucose unit. The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.58 to 0.68 hydroxypropyl groups per glucose unit.

The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) of about 0.68 per glucose unit. The pharmaceutical formulation disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.58 to 0.72 hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) of about 0.67. The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.6 to 0.9 hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) of about 0.84. The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.8 to 1.0 hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having an average molar substitution (MS) of about 0.92. The pharmaceutical formulations disclosed herein may include hydroxypropyl-β-cyclodextrin having about 0.81 to 0.99 hydroxypropyl groups per glucose unit.

The pharmaceutical formulations disclosed herein may include 10% to 25% w/v (e.g., about 10% to 20% w/v, e.g., about 15% to 25% w/v, e.g., about 12% to 18% w/v, e.g., about 10% to 17.5% w/v, e.g., about 11.25% to 15%, e.g., about 15% w/v) hydroxypropyl-β-cyclodextrin having a minimum of about 0.4 hydroxypropyl groups per glucose unit and a maximum of about 1.0 hydroxypropyl groups per glucose unit; such as an average of 0.62 to 0.92 hydroxypropyl groups per glucose unit, such as about 0.75 hydroxypropyl groups per glucose unit; such as 0.62 to 0.84 hydroxypropyl groups per glucose unit; such as about 0.4 to 0.75 hydroxypropyl groups per glucose unit; such as an average of 0.62 hydroxypropyl groups per glucose unit, such as about 0.58 to 0.68 hydroxypropyl groups per glucose unit. Other excipients

The pharmaceutical formulation may include a buffer. The use of buffers in pharmaceutical formulations is well known to those skilled in the art. For convenience, reference is made to Remington: The Science and Practice of Pharmacy , 20th edition, 2000.

pH can be measured at "room temperature", which is usually defined as 15 to 25°C or 15 to 20°C. pH is preferably measured at about 20°C.

The pharmaceutical formulation disclosed herein may include a buffer having a pKa close to the desired pH of the solution. The pharmaceutical formulation may include a buffer having at least one of a pKa of about 5.0 to 7.0. The pharmaceutical formulation may include a buffer having a pKa of about 5.0 to 7.0. The pharmaceutical formulation may include a buffer selected from the group consisting of histidine, citrate, and/or phosphate. The buffer may be citrate at a concentration of 3-30 mM. The buffer may be histidine at a concentration of 3-30 mM. The buffer may be phosphate at a concentration of 3 to 30 mM.

The pharmaceutical formulation may further include one or more reagents for adjusting pH, such as NaOH and/or HCl.

The desired pH of the pharmaceutical formulation may be about 5.5 to 6.5. The pH is preferably 5.6 to 6.0. The pH may be about 5.6, such as about 5.7, such as about pH 5.8, such as about 5.9, such as about 6.0.

The pharmaceutical formulation may include a tonicity agent. The use of tonicity agents in pharmaceutical compositions is well known to those skilled in the art. For convenience, reference is made to Remington: The Science and Practice of Pharmacy , 20th edition, 2000.

The purpose of the tonicity agent is to protect living tissue when the formulation is injected into the body. The tonicity agent can 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.

The concentration of the tonicity agent is such that the formulation is isotonic. Where the tonicity agent is mannitol, it may be present in a concentration of 16.5 to 37.5 mg/ml, such as about 20 mg/ml. Wherein the tonicity agent is sorbitol, it can be present in a concentration of about 10 to 40 mg/ml, such as about 16.5 to 37.5 mg/ml; such as about 10 to 30 mg/ml; such as about 16 to 28 mg/ml, such as about 16.5 to 25 mg/ml, such as about 16 to 26 mg/ml; such as about 16 to 24 mg/ml; such as about 26 mg/ml, such as about 24 mg/ml, such as about 22 mg/ml, such as about 20 mg/ml, such as about 18 mg/ml, such as about 16 mg/ml, such as about 12 mg/ml. Wherein the tonicity agent is trehalose, it can be present in a concentration of 33 to 75 mg/ml, such as about 38 mg/ml.

Pharmaceutical formulations may include surfactants. Surfactants may also be included to increase the physical stability and robustness of the formulation during preparation, storage, and use as a drug; for example, to preserve formulation stability when exposed to air within a container. The use of surfactants in pharmaceutical compositions is well known to those skilled in the art. For convenience, reference is made to Remington: The Science and Practice of Pharmacy , 20th edition, 2000.

The surfactant 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.

The pharmaceutical formulation may include 0.01 mg/ml or more of polysorbate 20 and up to 2.0, such as up to 1.5 mg/ml of polysorbate 20, such as about 0.01 to 1.0 mg/ml of polysorbate 20, such as about 0.05 mg/ml of polysorbate 20.

The pharmaceutical formulation may include 0.01 mg/ml or more of polysorbate 80 and up to 2.0, such as up to 1.5 mg/ml of polysorbate 80, such as about 0.01 to 1.0 mg/ml of polysorbate 80, such as about 0.05 mg/ml of polysorbate 80.

The pharmaceutical formulation includes water for injection (WFI). 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.

The pharmaceutical formulations disclosed herein may not include preservatives. Medical Uses

The pharmaceutical formulations disclosed herein can be used in medicine.

The pharmaceutical formulations disclosed herein can be administered by parenteral injection. The pharmaceutical formulations disclosed herein can be administered by subcutaneous injection.

As used herein, the term "treatment" refers to the administration of medical treatment to any human or other vertebrate individual in need thereof. The individual should have been physically examined by a licensed physician or veterinarian who has made a preliminary or definitive diagnosis 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 person to person, depending on the individual's health status. Thus, the treatment may be preventive (preventable), palliative, symptomatic and/or curative.

In some embodiments, the pharmaceutical formulation is administered to a human subject.

The pharmaceutical formulations disclosed herein can be used to: (i) prevent and/or treat all forms of diabetes and related symptoms, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (maturity-onset diabetes of the young), gestational diabetes, and/or reduce HbA1c; (ii) delay or prevent the progression of diabetic diseases, such as the progression of type 2 diabetes, delay the progression of impaired glucose tolerance (IGT) to type 2 diabetes requiring insulin, and/or delay the progression of type 2 diabetes not requiring insulin to type 2 diabetes requiring insulin; (iii) prevention and/or treatment of eating disorders, such as obesity, for example, by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating and/or preventing binge eating disorder, eating urges, bulimia nervosa and/or obesity induced by administration of antipsychotics or steroids; reducing gastrointestinal motility; and/or delaying gastric emptying; (iv) prevention and/or treatment of cardiovascular disease, such as delaying or reducing the development of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, vascular remodeling, 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.

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

Generally, all individuals suffering from obesity are also considered to be overweight. Disclosed herein is a method for treating or preventing obesity. Disclosed herein is the use of the formulations disclosed herein for treating or preventing obesity. In some embodiments, the individual suffering from obesity is a human, such as an adult or a pediatric (including infants, children, and adolescents).

Body mass index (BMI) is a measurement of body fat based on height and weight. The calculation formula is BMI = weight (kg) / height ( ^m2 ). A human individual with obesity may have a BMI of 30 kg/ ^m2 or higher; the individual may also be referred to as obese. In some embodiments, the human individual with obesity may have a BMI of ≥35 or a BMI range of ≥30 to <40. In some embodiments, obesity is severe obesity or morbid obesity, wherein the human individual may have a BMI of ≥40.

Disclosed herein is a method for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity. Disclosed herein is the use of a formulation disclosed herein for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity.

In some embodiments, the subject suffering from overweight is a human, such as an adult or a pediatric (including infants, children, and adolescents). In some embodiments, the human subject suffering from overweight may have a BMI of 25 kg/m ² or higher, such as a BMI of 27 kg/m ² or higher. In some embodiments, the human subject suffering from overweight has a BMI range of 25 to <30 or range of 27 to <30.

An elevated BMI increases an individual's risk of developing any of a number of diseases or comorbidities. The weight-related comorbidity may be one of the above-mentioned diseases, or a combination thereof. In some embodiments, the weight-related comorbidity is selected from the group consisting of: hypertension, diabetes (such as type 2 diabetes), dyslipidemia, high cholesterol, and obstructive sleep apnea.

Disclosed herein is a method for reducing weight. A human subject to weight reduction may have a BMI of 25 kg/m ² or higher, such as 27 kg/m ² or higher BMI (overweight) or 30 kg/m ² or higher BMI (obesity). In some embodiments, a human subject to weight reduction may have a BMI of 35 kg/m ² or higher or a BMI of 40 kg/m ² or higher. The term "weight reduction" may include treating or preventing obesity and/or overweight.

In some embodiments, administration of the semaglutide and capraglinide pharmaceutical formulations disclosed herein can be used 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) of 30 kg/m ² or higher (obesity) or 27 kg/m ² or higher (overweight) in the presence of at least one weight-related comorbidity (e.g., hypertension, type 2 diabetes, jaundice, or dyslipidemia).

In some embodiments, administration of the semaglutide and capgraminide pharmaceutical formulations disclosed herein may result in >15% weight loss, such as >20% weight loss, such as >25% weight loss, such as >30% weight loss, such as about 15% to 40% weight loss, such as about 20% to 35% weight loss, such as about 25% to 30% weight loss within 26 weeks after the start of treatment.

In some embodiments, administration of the semaglutide and capgraminide pharmaceutical formulations disclosed herein may result in >15% weight loss, such as >20% weight loss, such as >25% weight loss, such as >30% weight loss, such as about 15% to 40% weight loss, such as about 20% to 35% weight loss, such as about 25% to 30% weight loss within 26 weeks after the start of treatment.

In some embodiments, administration of a pharmaceutical formulation of semaglutide and canegrinide disclosed herein results in a higher reduction in _HbA1c in %-points compared to treatment with semaglutide alone as the active ingredient or canegrinide alone as the active ingredient .

The pharmaceutical formulations of the present invention can be administered as a single dose at predetermined time intervals.

A single dose of the pharmaceutical formulation disclosed herein may contain any one of the following doses of an amylin receptor agonist, such as cagricans, and a GLP-1 receptor agonist, such as semaglutide.

An effective amount of an amylin receptor agonist (such as capraglinide) and a GLP-1 receptor agonist (such as semaglutide) can be administered to a subject in need thereof.

In some embodiments, the dose is administered about once a week. In some embodiments, the interval between two fixed doses may be about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In a preferred embodiment, a fixed maintenance dose (once a week) is administered about every 7 days.

In some embodiments, the dose is administered to an individual suffering from any one or more of the diseases or comorbidities listed above. In some preferred embodiments, the dose is administered to an individual suffering from obesity (body mass index [BMI] ≥30 kg/m ² ). In some preferred embodiments, the dose is administered to an individual who is overweight (BMI ≥27 kg/m ² to <30 kg/m ² ) and has at least one weight-related comorbidity (e.g., hypertension, type 2 diabetes, or dyslipidemia).

In some embodiments, once weekly treatment results in statistically significant, dose-dependent weight loss.

In some preferred embodiments, the administered dose is used as an adjunct to diet and physical activity to improve glycemic control in adults with type 2 diabetes.

After initiation of treatment, it may be beneficial to administer increasing doses of an amylin receptor agonist, such as carglinide, and a GLP-1 receptor agonist, such as semaglutide, to an individual in need. Once an individual is acclimated to this treatment, it may be beneficial to administer maintenance doses of an amylin receptor agonist, such as carglinide, and a GLP-1 receptor agonist, such as semaglutide, to an individual in need.

In some embodiments, treatment is once weekly with a dose escalation period of 16 weeks.

In some embodiments, treatment is once weekly, with dose escalations occurring approximately once per week.

In some embodiments, treatment is once a week, with dose escalations occurring approximately every other week.

In some embodiments, treatment is once a week, with dose escalations occurring approximately every three weeks.

In some embodiments, treatment is once a week, with dose escalations occurring approximately every four weeks.

The amount of starch receptor agonist administered may be about 0.25 to 16 mg, such as about 0.25 to 9.0 mg, such as about 0.25 to 4.5 mg, such as about 0.25 to 2.4 mg.

The amount of capgranin administered may be about 0.25-16 mg, such as about 0.25 to 9.0 mg, such as about 0.25 to 4.5 mg, such as about 0.25 to 2.4 mg.

In some embodiments, the dose of capgranin administered is about 0.25 mg.

In some embodiments, the dose of capgranin administered is about 0.5 mg.

In some embodiments, the dose of capgranin administered is about 1.0 mg.

In some embodiments, the dose of capgranin administered is about 1.5 mg.

In some embodiments, the dose of capgranin administered is about 1.7 mg.

In some embodiments, the dose of capgranin administered is about 2.4 mg.

In some embodiments, the dose of capgranin administered is about 3.4 mg.

In some embodiments, the dose of capgranin administered is about 3.6 mg.

In some embodiments, the dose of capgranin administered is about 4.5 mg.

In some embodiments, the dose of capgranin administered is about 7.2 mg.

In some embodiments, the dose of capgranin administered is about 8.0 mg.

In some embodiments, the dose of capgranin administered is about 9.0 mg.

In some embodiments, the dose of capgranin administered is about 16.0 mg.

The amount of GLP-1 receptor agonist administered may be about 0.25 to 16 mg, such as about 0.25 to 9.0 mg, such as about 0.25 to 4.5 mg, such as about 0.25 to 2.4 mg.

The amount of semaglutide administered may be about 0.25 to 16 mg, such as about 0.25 to 9.0 mg, such as about 0.25 to 4.5 mg, such as about 0.25 to 2.4 mg.

In some embodiments, the dose of semaglutide administered is about 0.25 mg.

In some embodiments, the dose of semaglutide administered is about 0.5 mg.

In some embodiments, the dose of semaglutide administered is about 1.0 mg.

In some embodiments, the dose of semaglutide administered is about 1.5 mg.

In some embodiments, the dose of semaglutide administered is about 1.7 mg.

In some embodiments, the dose of semaglutide administered is about 2.4 mg.

In some embodiments, the dose of semaglutide administered is about 3.6 mg.

In some embodiments, the dose of semaglutide administered is about 4.5 mg.

In some embodiments, the dose of semaglutide administered is about 4.8 mg.

In some embodiments, the dose of semaglutide administered is about 6.0 mg.

In some embodiments, the dose of semaglutide administered is about 6.9 mg.

In some embodiments, the dose of semaglutide administered is about 7.2 mg.

In some embodiments, the dose of semaglutide administered is about 8.0 mg.

In some embodiments, the dose of semaglutide administered is about 9.0 mg.

In some embodiments, the dose of semaglutide administered is about 12 mg.

In some embodiments, the dose of semaglutide administered is about 16.0 mg. In some embodiments, the dose of semaglutide administered is about 16.0 mg.

In some embodiments, the ratio of amylin receptor agonist to GLP-1 receptor agonist is about 1: 2. In some embodiments, the ratio of capgranin to semaglutide is about 1: 2.

In some embodiments, the dose of capgraminide is about 0.125 mg and the dose of semaglutide is about 0.25 mg.

In some embodiments, the dose of capraglinide is about 0.25 mg and the dose of semaglutide is about 0.5 mg.

In some embodiments, the dose of capgraminide is about 0.5 mg and the dose of semaglutide is about 1.0 mg.

In some embodiments, the dose of capgraminide is about 0.75 mg and the dose of semaglutide is about 1.5 mg.

In some embodiments, the dose of capgraminide is about 0.85 mg and the dose of semaglutide is about 1.7 mg.

In some embodiments, the dose of capgraminide is about 1.2 mg and the dose of semaglutide is about 2.4 mg.

In some embodiments, the dose of capgraminide is about 2.25 mg and the dose of semaglutide is about 4.5 mg.

In some embodiments, the dose of capgraminide administered is about 3.6 mg and the dose of semaglutide administered is about 7.2 mg.

In some embodiments, the dose of capraglinide is about 4.0 mg and the dose of semaglutide is about 8.0 mg.

In some embodiments, the dose of capraglinide is about 7.2 mg and the dose of semaglutide is about 14.4 mg.

In some embodiments, the dose of capraglinide is about 8.0 mg and the dose of semaglutide is about 16.0 mg.

In some embodiments, the maintenance dose of capraglinide is about 1.2 mg and the maintenance dose of semaglutide is about 2.4 mg.

In some embodiments, the maintenance dose of capraglinide is about 2.25 mg and the dose of semaglutide is about 4.5 mg.

In some embodiments, the maintenance dose of capraglinide is about 4.0 mg and the maintenance dose of semaglutide is about 8.0 mg.

In some embodiments, the maintenance dose of capraglinide is about 8.0 mg and the maintenance dose of semaglutide is about 16.0 mg.

In some embodiments, the ratio of amylin receptor agonist to GLP-1 receptor agonist is about 1: 1. In some embodiments, the ratio of capgranin to semaglutide is about 1: 1.

In some embodiments, the dose of capgraminide is about 0.25 mg and the dose of semaglutide is about 0.25 mg.

In some embodiments, the dose of capraglinide is about 0.5 mg and the dose of semaglutide is about 0.5 mg.

In some embodiments, the dose of capgraminide is about 1.0 mg and the dose of semaglutide is about 1.0 mg.

In some embodiments, the dose of capgraminide is about 1.7 mg and the dose of semaglutide is about 1.7 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 2.4 mg.

In some embodiments, the maintenance dose of capraglinide is about 2.4 mg and the maintenance dose of semaglutide is about 2.4 mg.

In some embodiments, the dose of capgraminide is about 4.5 mg and the dose of semaglutide is about 4.5 mg.

In some embodiments, the dose of capraglinide is about 8.0 mg and the dose of semaglutide is about 8.0 mg.

In some embodiments, the dose of capgraminide is about 16.0 mg and the dose of semaglutide is about 16.0 mg.

In some embodiments, the ratio of amylin receptor agonist to GLP-1 receptor agonist is between 1:1 and 1:7.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 2.4 mg to 16.0 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 3.6 mg to 16.0 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 2.4 mg to 13.5 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 3.6 mg to 13.5 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 3.6 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 4.8 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 6.0 mg.

In some embodiments, the dose of capraglinide is about 2.4 mg and the dose of semaglutide is about 6.9 mg.

In some embodiments, the dose of capgraminide is about 2.4 mg and the dose of semaglutide is about 7.2 mg.

In some embodiments, the dose of capraglinide is about 2.4 mg and the dose of semaglutide is about 8.0 mg.

In some embodiments, the dose of capraglinide is about 2.4 mg and the dose of semaglutide is about 12 mg.

In some embodiments, the dose of capgraminide is about 3.4 mg and the dose of semaglutide is about 13.5 mg.

In some embodiments, capgraminide and semaglutide are administered once weekly at an initial dose of 0.25 mg and then titrated to subsequent dose levels of 0.5 mg, 1.0 mg, and 1.7 mg until a target/maintenance dose of 2.4 mg once weekly is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at 0.25 mg and then titrated every four weeks to subsequent 0.5 mg, 1.0 mg, and 1.7 mg dose levels until a target/maintenance dose of 2.4 mg once weekly is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at 0.25 mg and then titrated every four weeks to subsequent 0.5 mg, 1.0 mg, and 1.7 mg dose levels until a target/maintenance dose of 2.4 mg once weekly is reached.

In some embodiments, 0.25 mg of cargranin and 0.25 mg of semaglutide are administered once a week for four weeks (weeks 0 to 3), and then increased every four weeks to subsequent dose levels of 0.5 mg cargranin and 0.5 mg semaglutide (weeks 4 to 7), 1.0 mg cargranin and 1.0 mg semaglutide (weeks 8 to 11), and 1.7 mg cargranin and 1.7 mg semaglutide (weeks 12 to 15), until a target/maintenance dose of 2.4 mg cargranin and 2.4 mg semaglutide once a week (weeks 16 and thereafter) is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at an initial dose of 0.25 mg and then titrated to subsequent dose levels of 0.5 mg, 1.0 mg, 1.7 mg, and 2.4 mg until a target/maintenance dose of 4.5 mg once weekly is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at an initial dose of 0.25 mg and then titrated to subsequent dose levels of 0.5 mg, 1.0 mg, 1.7 mg, 2.4 mg, 3.6 mg, and 4.5 mg until a target/maintenance dose of 7.2 mg once weekly is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at an initial dose of 0.25 mg, followed by titration to subsequent dose levels of 0.5 mg, 1.0 mg, and 1.7 mg, 2.4 mg, 3.6 mg, 4.5 mg, and 7.2 mg until a target/maintenance dose of 8.0 mg once weekly is reached.

In some embodiments, capgraminide and semaglutide are administered once weekly at an initial dose of 0.25 mg, followed by titration to subsequent dose levels of 0.5 mg, 1.0 mg, 1.7 mg, 2.4 mg, 3.6 mg, 4.5 mg, 7.2 mg, and 8.0 mg until a target/maintenance dose of 16.0 mg once weekly is reached.

As used herein, specific numerical values associated with numbers or intervals may be interpreted as specific numerical values or approximate numerical values (e.g., plus or minus 10%, 15%, or 20% when amounts are provided by weight; plus or minus 0.4 when pH is measured).

The following is a non-limiting list of embodiments of the present invention. Embodiment 1. A liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist, and a cyclodextrin substituted with a hydroxypropyl group. 2. A liquid pharmaceutical formulation according to Embodiment 1, wherein the GLP-1 receptor agonist has an isoelectric point that is incompatible with the optimal pH of the amylin receptor agonist. 3. A liquid pharmaceutical formulation according to any of the foregoing embodiments, wherein the optimal pH of the GLP-1 receptor agonist and the amylin receptor agonist differ by at least about two pH units, such as 2 to 5 pH units, such as 2 to 4 pH units, such as 3 to 5 pH units. 4. A liquid pharmaceutical formulation according to any of the preceding embodiments, wherein the optimal pH of the amylin receptor agonist is 3.5 to 4.5, such as about 4.0. 5. A pharmaceutical formulation according to any of the preceding embodiments, wherein the amylin receptor agonist is cagerinide. 6. A liquid pharmaceutical formulation according to any of the preceding embodiments, wherein the GLP-1 receptor agonist has an isoelectric point of less than 5.0, such as 3.0 to 5.0, such as 3.8 to 4.9. 7. A pharmaceutical formulation according to any of the preceding embodiments, wherein the GLP-1 receptor agonist is semaglutide. 8. A pharmaceutical formulation according to any of the preceding embodiments, wherein the cyclodextrin is an α-type comprising six cyclically arranged glucose units substituted with hydroxypropyl groups, or a β-type comprising seven cyclically arranged glucose units substituted with hydroxypropyl groups. 9. A pharmaceutical formulation according to any of the preceding embodiments, wherein the cyclodextrin is an α-type comprising six cyclically arranged glucose units substituted with hydroxypropyl groups. 10. A pharmaceutical formulation according to any of the preceding embodiments, wherein the cyclodextrin is a β-type comprising seven cyclically arranged glucose units substituted with hydroxypropyl groups. 11. A pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises at most about 1.0 hydroxypropyl group. 12. A pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises a maximum of about 0.92 hydroxypropyl groups. 13. A pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises a maximum of about 0.75 hydroxypropyl groups. 14. A pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises a maximum of about 0.68 hydroxypropyl groups. 15. A pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises a minimum of about 0.4 hydroxypropyl groups. 16. The pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises at least about 0.58 hydroxypropyl groups. 17. The pharmaceutical formulation according to any of the preceding embodiments, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises about 0.58 to 1.0 hydroxypropyl groups. 18. The pharmaceutical formulation according to embodiment 17, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises an average (MS) of 0.62 to 0.92 hydroxypropyl groups. 19. The pharmaceutical formulation according to embodiment 17, wherein each glucose unit of the hydroxypropyl-β-cyclodextrin comprises an average (MS) of about 0.62 to 0.84 hydroxypropyl groups. 20. The pharmaceutical formulation according to embodiment 17, wherein the hydroxypropyl-β-cyclodextrin comprises an average (MS) of about 0.62 hydroxypropyl groups per glucose unit. 21. The pharmaceutical formulation according to embodiment 17, wherein the hydroxypropyl-β-cyclodextrin comprises about 0.4 to 0.75 hydroxypropyl groups per glucose unit, such as about 0.58 to 0.68 hydroxypropyl groups per glucose unit. 22. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 10% to 25% w/v of cyclodextrin. 23. The pharmaceutical formulation according to any of the preceding embodiments, comprising more than 10% cyclodextrin. 24. The pharmaceutical formulation according to any of the preceding embodiments, comprising less than 22% w/v of cyclodextrin. 25. The pharmaceutical formulation according to any of the preceding embodiments, comprising less than 20% w/v of cyclodextrin. 26. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 10% to 20% w/v of the cyclodextrin. 27. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 10% to 17.5% w/v of cyclodextrin. 28. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 12% to 18% w/v of cyclodextrin. 29. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 11.25% to 15% w/v of HP-β-CD. 30. A pharmaceutical formulation according to any of the preceding embodiments, comprising about 15% w/v cyclodextrin. 31. A pharmaceutical formulation according to any of the preceding embodiments, comprising at least about 1 mg/ml of the GLP-1 receptor agonist. 32. A pharmaceutical formulation according to any of the preceding embodiments, comprising up to about 22 mg/ml of the GLP-1 receptor agonist. 33. A pharmaceutical formulation according to any of the preceding embodiments, comprising about 1 to 12 mg/ml of the GLP-1 receptor agonist. 34. A pharmaceutical formulation according to any of the preceding embodiments, comprising at least about 1 mg/ml of the amylin receptor agonist. 35. The pharmaceutical formulation according to any of the preceding embodiments, comprising up to about 22 mg/ml of an amylin receptor agonist. 36. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 1 to 12 mg/ml of an amylin receptor agonist. 37. The pharmaceutical formulation according to any of the preceding embodiments, comprising 0.25 to 22 mg/ml of capgranin. 38. The pharmaceutical formulation according to any of the preceding embodiments, comprising 0.25 to 22 mg/ml of semaglutide. 39. The pharmaceutical formulation according to any of the preceding embodiments, comprising 0.25 to 22 mg/ml of capgranin and 0.25 to 22 mg/ml of semaglutide. 40. A pharmaceutical formulation according to any of the preceding embodiments, comprising an effective amount of capraglinide and semaglutide. 41. A pharmaceutical formulation according to any of the preceding embodiments, further comprising a tonicity agent; provided that the tonicity agent is not sodium chloride. 42. A pharmaceutical formulation according to the preceding embodiments, wherein the tonicity agent is mannitol, sorbitol or trehalose, or a combination thereof. 43. A pharmaceutical formulation according to the preceding embodiments, wherein the tonicity agent is mannitol. 44. A pharmaceutical formulation according to the preceding embodiments, comprising mannitol at a concentration of about 16.5 to 37.5 mg/ml, such as about 20 mg/ml. 45. A pharmaceutical formulation according to embodiment 41, wherein the tonicity agent is sorbitol. 46. The pharmaceutical formulation according to the preceding embodiment, comprising sorbitol at a concentration of about 10 to 40 mg/ml (e.g., about 10 to 30 mg/ml, e.g., about 16 to 28 mg/ml, e.g., about 16.5 to 37.5 mg/ml, e.g., about 16.5 to 25 mg/ml, e.g., about 16 to 24 mg/ml; e.g., about 24 mg/ml, e.g., about 20 mg/ml, e.g., about 16 mg/ml, e.g., about 12 mg/ml). 47. The pharmaceutical formulation according to embodiment 41, wherein the tonicity agent is trehalose. 48. The pharmaceutical formulation according to the preceding embodiments, comprising trehalose at a concentration of about 33 to 75 mg/ml, such as about 33 to 45 mg/ml, such as about 38 mg/ml. 49. The pharmaceutical formulation according to any of the preceding embodiments, further comprising a buffer having a pKa of about 5.0 to 7.0. 50. The pharmaceutical formulation according to any of the preceding embodiments, further comprising a buffer selected from the group consisting of: histidine, citrate and/or phosphate. 51. The pharmaceutical formulation according to any of the preceding embodiments, comprising a buffer of up to 30 mM. 52. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 3 to 30 mM citrate. 53. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 3 to 30 mM histidine, such as 3 to 15 mM histidine, such as 3 to 10 mM histidine, such as about 6 mM histidine. 54. The pharmaceutical formulation according to any of the preceding embodiments, comprising about 3 to 30 mM phosphate. 55. The pharmaceutical formulation according to any of the preceding embodiments, further comprising a surfactant. 56. The pharmaceutical formulation according to the preceding embodiments, wherein the surfactant is polysorbate 20 and/or polysorbate 80. 57. The pharmaceutical formulation according to the preceding embodiments, comprising up to about 2.0 mg/ml of polysorbate 20 and/or polysorbate 80. 58. The pharmaceutical formulation according to the preceding embodiment, comprising up to about 1.5 mg/ml of polysorbate 20 and/or polysorbate 80. 59. The pharmaceutical formulation according to the preceding embodiment, wherein the surfactant is polysorbate 80. 60. The pharmaceutical formulation according to any of the preceding embodiments, wherein the pH is about 5.5 to 6.5, preferably 5.6 to 6.0, such as about 5.7, such as about pH 5.8, such as about 5.9, such as about 6.0. 61. The pharmaceutical formulation according to any of the preceding embodiments, 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. 62. A pharmaceutical formulation according to any of the preceding embodiments, which is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes at least about 0.4 hydroxypropyl groups per glucose unit and at most about 1.0 hydroxypropyl groups per glucose unit, histidine, sorbitol, polysorbate 80, and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 63. A pharmaceutical formulation according to any of the preceding embodiments, which is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl beta-cyclodextrin, which includes a maximum of about 0.58 to 1.0 hydroxypropyl groups per glucose unit, histidine, sorbitol, polysorbate 80, and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 64. A pharmaceutical formulation according to any of the preceding embodiments, which is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl beta-cyclodextrin, which includes an average of 0.62 to 0.92 hydroxypropyl groups per glucose unit, histidine, sorbitol, polysorbate 80, and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 65. The pharmaceutical formulation according to any of the aforementioned embodiments is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes an average of 0.62 to 0.84 hydroxypropyl groups per glucose unit, histidine and/or citrate, sorbitol, polysorbate 80 and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 66. The pharmaceutical formulation according to any of the aforementioned embodiments is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes an average of 0.62 to 0.68 hydroxypropyl groups per glucose unit, histidine and/or citrate, sorbitol, polysorbate 80 and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 67. The pharmaceutical formulation according to any of the aforementioned embodiments is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes an average of 0.62 hydroxypropyl groups per glucose unit, histidine and/or citrate, sorbitol, polysorbate 80 and about 75 to 90% w/w water; and has a pH of 5.6 to 6.0. 68. A pharmaceutical formulation according to any of the foregoing embodiments, which is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes up to about 0.75 hydroxypropyl groups per glucose unit, such as about 0.4 to 0.75 hydroxypropyl groups per glucose unit, histidine, sorbitol, polysorbate 80 and about 75 to 90% w/w water; and has a pH of 5.5 to 6.5. 69. A pharmaceutical formulation according to any of the foregoing embodiments, which is mainly composed of: effective amounts of carglinide and semaglutide, hydroxypropyl β-cyclodextrin, which includes up to about 0.75 hydroxypropyl groups per glucose unit, such as about 0.4 to 0.75 hydroxypropyl groups per glucose unit, histidine and/or citrate, sorbitol, polysorbate 20 and/or 80, and about 75 to 90% w/w water; and has a pH value of 5.6 to 6.0. 70. The pharmaceutical formulation according to any of the preceding embodiments, which is mainly composed of: - effective amounts of carglinide and semaglutide, - more than 10% w/v and less than 22% w/v, such as 10% to 20% w/v of hydroxypropyl-β-cyclodextrin (0.58 to 1.0 hydroxypropyl groups per glucose unit), - about 3 to 30 mM histidine, - about 10 to 40 mg/ml sorbitol, - up to 2.0 mg/ml polysorbate 20 and/or polysorbate 80, - pH 5.6-6.0, preferably pH 5.8, - water for injection. 71. A pharmaceutical formulation according to any of the preceding embodiments, which is mainly composed of: - an effective amount of carglinide and semaglutide, - more than 10% w/v and less than 22% w/v, such as 10% to 20% w/v of hydroxypropyl-β-cyclodextrin, which includes an average of 0.62 to 0.84 hydroxypropyl groups per glucose unit, - about 3 to 30 mM histidine and/or citrate, - about 10 to 40 mg/ml sorbitol, - up to 2.0 mg/ml polysorbate 20 and/or polysorbate 80, - pH 5.6-6.0, preferably pH 5.8, - water for injection. 72. A pharmaceutical formulation according to any of the preceding embodiments, which consists essentially of: - 0.25 to 22 mg/ml of capraglin, - 0.25 to 22 mg/ml of semaglutide, - more than 10% w/v and less than 22% w/v, such as 10% to 20% w/v of hydroxypropyl-β-cyclodextrin (0.58 to 1.0 hydroxypropyl groups per glucose unit), - about 6 mM of histidine, - about 10 to 40 mg/ml of sorbitol, - up to 2.0 mg/ml of polysorbate 20 and/or 80, - pH 5.6-6.0, preferably pH 5.8, - water for injection. 73. A pharmaceutical formulation according to any of the preceding embodiments, which is used as a medicament. 74. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used to treat an individual with an initial body mass index (BMI) of 27 or more (such as 30 or more). 75. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used to treat an individual with an initial body mass index (BMI) of 27 or more and at least one weight-related comorbidity. 76. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, for use as an adjunct to a reduced calorie diet and increased physical activity for chronic weight management in adult individuals with an initial body mass index (BMI) of 30 kg/ ^m2 or more (obesity) or 27 kg/ ^m2 or more (overweight) in the presence of at least one weight-related comorbidity. 77. The use according to any one of the preceding embodiments 73 to 76, wherein the comorbidity is diabetes and/or cardiovascular disease and/or NASH. 78. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, for use in the treatment of individuals suffering from diabetes (such as type II diabetes). 79. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used as an adjunct to diet and exercise to improve glycemic control in adult individuals with type 2 diabetes. 80. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used to treat and/or prevent cardiovascular disease. 81. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used to treat and/or prevent NASH. 82. The pharmaceutical formulation according to any one of the preceding embodiments 1 to 72, which is used to treat and/or prevent cognitive disorders, such as diseases caused by Alzheimer's disease. 83. According to the pharmaceutical formulation of any one of the preceding embodiments 1 to 72 for use according to any one of the preceding embodiments 73 to 82, characterized in that the formulation is administered by parenteral gastrointestinal injection. 84. According to the pharmaceutical formulation of any one of the preceding embodiments 1 to 72 for use according to any one of the preceding embodiments 73 to 82, characterized in that the formulation is administered by subcutaneous injection. 85. According to the pharmaceutical formulation of any one of the preceding embodiments 1 to 72 for use according to any one of the preceding embodiments 73 to 84, characterized in that the formulation is administered approximately once a week. 86. According to the pharmaceutical formulation of any one of embodiments 1 to 72 described above for use according to any one of embodiments 73 to 85, wherein the ratio of the dose of the administered cargranin peptide to the dose of the administered semaglutide is about 1: 1. 87. According to the pharmaceutical formulation of any one of embodiments 1 to 72 described above for use according to any one of embodiments 73 to 85, wherein the ratio of the dose of the administered cargranin peptide to the dose of the administered semaglutide is from 1: 1 to 1: 7. 88. According to the pharmaceutical formulation of any one of embodiments 1 to 72 described above for use according to any one of embodiments 73 to 85, wherein the ratio of the dose of the administered cargranin peptide to the dose of the administered semaglutide is about 1: 2. Example Example 1 : Effect of Hydroxypropyl -β- cyclodextrin (HP-β-CD) on the Chemical Stability of Kaglinide

This example demonstrates the effect of HP-B-CD on the chemical stability of cagerin peptides in terms of stability of degradation products through the parameters of cagerin peptide purity and cagerin peptide-related high molecular weight protein (HMWP). cagerin peptides are most stable at pH 4.0, and their rate of chemical degradation generally accelerates with increasing pH. Unexpectedly, this example shows that stable cagerin peptide formulations can be obtained at pH 6 when formulated with HP-B-CD. Composition

The compositions of the Kaglin peptide formulations 1, 2 and 3 are shown in Table 1. Table 1 Compositions of the Kaglin peptide formulations 1, 2 and 3 Element Kaglin peptide formulation 1 2 3 Carglinide drug substance (mg/ml) 18 18 18 HP-B-CD KLEPTOSE ^® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ 0 0 25 Citrate, 1 H ₂ O ² (mM) 6.1 3.7 3.7 Sodium hydrogen phosphate ( _2H2O2 ⁾ (mM) 7.7 12.6 12.6 HCl qs qs qs NaOH qs qs qs Water for Injection (WFI) Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 4.0 6.0 6.0 ¹ MS: molar substitution, corresponding to ^{hydroxypropyl} per glucose unit. Different buffer concentrations were used in different formulations to ensure that the buffer process was performed at different pH.

The formulation is prepared by dissolving the excipient in water and the drug substance in the excipient solution. The solution pH is adjusted and water is added to achieve the final desired volume before sterilization by filtration through a 0.22 µm sterile filter. After filtration is complete, the formulation is filled into 1 ml pre-filled syringes. Methods

Samples were stored at 37°C for up to 21 days and analyzed after 14 and 21 days to determine the degree of purity of HMWP and caprin.

The extent of covalently bound HMWP was quantified using size exclusion chromatography (SEC). Samples were analyzed using a WATERS HMWP column (7.8 x 300 mm) and isocratically eluted with an eluent 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 divided by the area of the main peak x 100%.

The purity of kagerin peptide was determined by reverse phase high performance liquid chromatography (RP-UHPLC). Samples were analyzed using a Kinetex C18, 1.7 μm, 100 Å column (2.1 x 150 mm) and gradient elution was performed with 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 60°C with UV detection (215 nm) using injection volumes ranging from 2 to 7.5 μl and a flow rate of 0.25 ml/min. Purity was estimated as the area of the main peak divided by the area of all peaks x 100%. Table 2 Chemical purity of cagerin peptide with or without HP-B-CD at pH 4.0 and 6.0 (%) Kaglin peptide formulation pH HP-B-CD (MS: 0.92) ¹ Relationship between HMWP content (%) and time (days) at 37° C Purity of Capgranin (%) vs. time (days) at 37°C 0 14 twenty one 0 14 twenty one 1 4.0 0% w/v 0.0% 0.2% 0.2% 90.4% 88.2% 87.4% 2 6.0 0% w/v 0.1% 7.7% 9.8% 90.1% 74.0% 67.1% 3 6.0 25% w/v 0.1% 0.3% 0.5% 90.2% 79.9% 75.3% ¹ MS: molar substitution, corresponding to the total number of hydroxypropyl groups per glucose unit

Table 2 shows that when carglinide was stored at 37°C and at pH 4.0, very low HMWP was formed and only a slight decrease in the purity of carglinide was seen. In contrast, at pH 6.0, the rate of HMWP formation and the rate of decrease in the purity of carglinide accelerated. Unexpectedly, this rapid chemical degradation was offset by the addition of HP-B-CD to the formulation, making it possible to formulate carglinide at pH 6. Example 2 : Effect of HP-B-CD on the physical stability of semaglutide

This example shows the stabilizing effect of HP-B-CD on the physical stability of semaglutide, i.e. its tendency to form peptide fibers. This effect is evident when semaglutide is formulated at suboptimal pH.

The compositions of semaglutide formulations 1, 2 and 3 are shown in Table 3. Table 3 Compositions of semaglutide formulations 1, 2 and 3 Element Semaglutide formulations 1 2 3 Semaglutide drug substance (mg/ml) 4.8 4.8 4.8 HP-B-CD KLEPTOSE ^® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ 0 25 0 Citrate, 1 H ₂ O ² (mM) 3.7 3.7 0.9 Sodium hydrogen phosphate ( _2H2O2 ⁾ (mM) 12.6 12.6 18.2 HCl qs qs qs NaOH qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 6.0 6.0 7.4 ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit. ² Different buffer concentrations were used in different formulations to ensure that the buffer process was performed at different pH.

The formulations were prepared as described in Example 1.

The aggregation and propensity of semaglutide to form peptide fibers was quantified using the thioflavin T (ThT) fluorescence pressure assay. Analysis of the presence of peptide fibers is based on the fluorescence properties of the ThT probe, which exhibits low fluorescence in the unbound/native peptide-bound state, but high fluorescence when bound to peptide fibers, and a red-shift in the wavelength of the fluorescence maximum upon fiber binding.

Two samples were pooled and 1400 μl of the sample was added to 28 μl of 1 mM ThT stock solution, of which 200 μl was then transferred to 6 different wells of a 96-well microplate containing one glass bead. The assay was run on a BMG CLARIOstar fluorescence plate reader equipped with a monochromator with bi-orbital shaking and a speed of 300 rpm at 40 °C for 169 h, using 450 nm and 480 nm for excitation and emission, respectively. The delay time was measured from the start of the experiment until fibrillation occurred, shown as an increase in ThT fluorescence. Table 4 Physical stability of semaglutide at pH 6.0 and 7.4 Semaglutide formulations pH HP-B-CD (MS: 0.92) ³ Delay time until fiberization 1 6.0 0% w/v 2.35 ^hours1 2 6.0 25% w/v ＞ ¹⁶⁹ hours1,2 3 7.4 0% w/v ＞ ¹⁶⁹ hours1,2 ¹ Results are the average of 6 replicates ² No fibrillation was observed in any of the 6 replicates during the 169-hour experiment ³ MS: molar substitution, corresponding to the total number of hydroxypropyl groups per glucose unit

The semaglutide formulations were subjected to shear stress inducing conditions and measured by the tendency to form peptide fibers. Unexpectedly, the presence of HP-B-CD was found to inhibit the formation of semaglutide peptide fibers. When semaglutide was formulated at pH 6 and lacked HP-B-CD (semaglutide formulation 1), fibrillation occurred after 2.35 hours; that is, semaglutide was physically unstable under such conditions. When semaglutide was formulated at pH 6 and HP-B-CD was present (semaglutide formulation 2), fibrillation was not observed throughout the experiment; that is, semaglutide was physically stable. In addition, when formulated at pH 6 and in the presence of HP-B-CD (Semaglutide Formulation 2), the physical stability of semaglutide was found to be comparable to that of semaglutide in the absence of HP-B-CD but at its optimal formulation conditions at pH 7.4 (Semaglutide Formulation 3). Example 3 : Effect of HP-B-CD on the Chemical Stability of Semaglutide

This example demonstrates the effect of HP-B-CD on the chemical stability of semaglutide in terms of the stability of degradation products through the purity of semaglutide and the high molecular weight protein (HMWP) parameters associated with semaglutide.

The same formulation as in Example 2 was used.

The formulations were prepared as described in Example 1.

The HMWP content and semaglutide purity were measured after storage at 37°C on days 0, 14, and 21.

The purity of semaglutide was determined by reverse phase high performance liquid chromatography (RP-HPLC), wherein the samples were analyzed using a Kinetex C18, 2.6 μm column (4.6 x 150 mm) and gradient elution was performed with 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 to 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 x 100%.

Size exclusion chromatography (SEC) was used to determine the extent of covalently bound HMWP. Samples were analyzed using a Waters SEC 1.7 μm column (4.6 x 150 mm) and isocratically eluted with a plasma eluent consisting of 300 mM sodium chloride, 10 mM sodium dihydrogen phosphate monohydrate, 5 mM orthophosphate, and 50% v/v 2-propanol. Chromatography was performed at 50°C with UV detection (280 nm) using injection volumes of 1 to 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 divided by the area of the main peak x 100%. Table 5 Purity of semaglutide at suboptimal and optimal pH Semaglutide formulations pH HP-B-CD (MS: 0.92) ¹ Relationship between the purity of semaglutide (%) and time (days) at 37°C Relationship between HMWP content (%) and time (days) at 37° C 0 14 twenty one 0 14 twenty one 1 6.0 0% w/v 96.89% 94.58% 93.48% 0.12% 0.25% 0.35% 2 6.0 25% w/v 96.51% 94.53% 93.73% 0.04% 0.11% 0.11% 3 7.4 0% w/v 96.82% 95.56% 94.81% 0.12% 0.32% 0.40% ¹ MS: molar substitution, corresponding to the total number of hydroxypropyl groups per glucose unit

Table 5 shows how the chemical purity of semaglutide decreases over time. When semaglutide (semaglutide formulation 1) was formulated at pH 6.0, the chemical purity decreased more rapidly than when formulated at the optimal pH of 7.4 (semaglutide formulation 3). Unexpectedly, when semaglutide (semaglutide formulation 2) was formulated at pH 6.0, HP-B-CD improved the chemical stability of semaglutide (in terms of purity decrease and HMWP formation). Example 4 : Effect of molar substitution of HP-B-CD on the physical stability of co-formulations of cagerin and semaglutide

This example shows the effect of HP-B-CD molar substitution on the physical stability of capraglinide and semaglutide .

The compositions of co-formulation 1 and co-formulation 2 are shown in Table 6. Table 6 Compositions of co-formulation 1 and co-formulation 2 Element Co-formulation 1 2 Carglinide drug substance (mg/ml) 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ 25 - HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ - 25 Citrate, 1 H ₂ O (mM) 3 3 HCl qs qs NaOH qs qs WFI Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1.

All samples were stored under pressure conditions, defined as: -    Duration: 28 days -    Temperature: 30°C ± 2°C -    Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold room. 20 rotations were performed three days per week and 40 rotations were performed two days per week.

The number of particles present that can only be seen under a microscope quantifies the physical stability of the mixture of capraglin and semaglutide and is obtained by means of microfluidic imaging (MFI, see Sharma, D.K. et al., AAPS J. (2010), 12: 455-464 for the principles of MFI technology). The following process was performed for each syringe sample analyzed: The experiment was performed at ambient temperature. The liquid was first removed from each syringe by removing the plunger and then pipetting the liquid into the sample container. The samples were transferred to a 96-well deep well plate, which was inserted into the sample processing unit (Bot1) on the Protein Simple MFI™ 5200 instrument equipped with a standard Protein Simple MFI™ 100 μm flow cell. The samples were analyzed with the standard MFI system setup, meaning that the liquid was pipetted into a reservoir connected to a flow cell, the liquid was illuminated by a 10 LED light source (470 nm), and the contents of the flow cell were recorded as bright field images with a digital camera (through magnification optics) throughout the experiment. Data acquisition was done with Protein Simple MVSS software. The image stream recorded during the entire run was processed by the validated Novo Nordisk proprietary software MFI Data Validator, from which the number of individual particles was obtained (normalized in counts per ml of analyzed liquid) and presented by size; >5 µm, >10 µm, and >25 µm, which is the standard size range for particles visible under a microscope. It should be noted that the number of particles >5 μm includes all particles with a diameter greater than 5 μm (>5 μm, >10 μm, and >25 μm), and the number of particles >10 μm includes all particles with a diameter greater than 10 μm (>10 μm and >25 μm). Particle size is defined as the equivalent circular diameter (ECD).

The presence of starch-like peptide fibers was analyzed using thioflavin-T (ThT) fluorescence assay. The experiments were performed at 25°C. The liquid was first removed from each syringe by removing the plunger and then pipetting the liquid into a sample container. Subsequently, 500 µl of sample was mixed with approximately 9 µl of ThT stock solution in a separate sample container to give a final ThT concentration of 20 μM. The samples were incubated in the dark at ambient temperature for 25 minutes. 200 µl of sample were transferred to one well of a 96-well microplate. The samples were measured on a BMG CLARIOstar fluorescence disk reader equipped with a monochromator, using 440 nm for excitation and 470 to 550 nm for emission, respectively.

Data collection was performed using CLARIOstar control software. In this assay, the emission maximum was observed to occur at a wavelength of approximately 485 nm; the results are therefore reported as ThT fluorescence at 485 nm, expressed in relative fluorescence units (RFU). Table 8 Physical stability of HP-B-CD co-formulations with high or medium molar substitution Co-formulation HP-B-CD Average Molar Substitution The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 Particle size＞5μm 1 HP-B-CD (MS: 0.92) ¹ 459 334 1754 22433 2 HP-B-CD (MS: 0.62) ¹ 256 379 715 245 Particle size＞10μm 1 HP-B-CD (MS: 0.92) ¹ 52 27 602 10598 2 HP-B-CD (MS: 0.62) ¹ 39 39 132 122 Particle size＞25μm 1 HP-B-CD (MS: 0.92) ¹ 0 0 2 644 2 HP-B-CD (MS: 0.62) ¹ 6 0 8 4 ThT fluorescence at 485 nm 1 HP-B-CD (MS: 0.92) ¹ 3357 3538 3588 13640 2 HP-B-CD (MS: 0.62) ¹ 3784 3720 3967 3463 The results for the number of particles visible under the microscope are the average of three replicates and have been rounded to the nearest integer value. ¹ MS: mole, corresponding to the total number of hydroxypropyl groups per glucose unit

The results in Table 7 show that the least particles were produced in co-formulation 2 containing HP-B-CD (average MS: 0.62). In addition, no increase in the number of microscopic particles or ThT fluorescence was observed in the case of co-formulation 2 during the 28-day experimental period.

In contrast, in the case of co-formulation 1 containing HP-B-CD (average MS: 0.92), an increase in the number of particles visible under a microscope was observed after 21 days, and an increase in ThT fluorescence was observed after 28 days.

In these otherwise identical, citrate-buffered co-formulations of semaglutide and capraglinide, the co-formulation (2) including HP-B-CD (average MS: 0.62) was physically more stable than the co-formulation (1) including HP-B-CD (average MS: 0.92). Example 5 : Effect of Hydroxypropyl -B- 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 .

The compositions of co-formulation 3, co-formulation 4, and co-formulation 5 are shown in Table 8. Table 8 Compositions of co-formulation 3, co-formulation 4, and co-formulation 5 Element Co-formulation 3 4 5 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 11.25 12.5 15 L-Histidine(mM) 6 6 6 Sorbitol ² (mg/ml) 26 twenty four 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 HCl qs qs qs NaOH qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 5.7 ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit ² Due to the different HP-B-CD concentrations tested, different sorbitol concentrations were required to obtain isotonic preparations

The formulations were prepared as described in Example 1.

The samples were stored at 37°C for 28 days and analyzed after 14, 21, and 28 days to determine the chemical purity of semaglutide.

The purity of semaglutide was determined using reverse phase ultra-high performance liquid chromatography (RP-UHPLC) using a Waters Acquity Phenyl-Hexyl 1.7 μm column (2.1 x 150 mm) to analyze the samples and gradient elution with 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 MQ water with 80% acetonitrile. Chromatography was performed at 62°C with UV detection (215 nm) using injection volumes of 2 to 14 μl and a flow rate of 0.25 ml/min. Purity was quantified as the area of the main semaglutide peak divided by the area of all relevant peaks x 100%.

It should be noted that in other experiments, the same method was used to determine the purity of semaglutide. Table 9 Chemical purity of semaglutide with different HP-B-CD concentrations (%) Co-formulation HP-B-CD (MS: 0.62) ¹ Relationship between the purity (%) of semaglutide and time (days) at 37°C 0 14 twenty one 28 3 11.25% w/v 96.90% 90.72% 88.65% 87.52% 4 12.5% w/v 96.87% 92.90% 91.25% 89.81% 5 15% w/v 96.83% 93.61% 92.92% 91.67% ¹ MS: molar substitution, corresponding to the total number of hydroxypropyl groups per glucose unit

The results in Table 9 show that the chemical stability and purity of semaglutide also depend on the HP-B-CD concentration. Semaglutide remained chemically stable in all co-formulations including 11.25% to 15% w/v HP-B-CD. However, the chemical stability and purity of semaglutide were the highest when the co-formulation included 15% w/v HP-B-CD. Example 6 : Effect of different tensile agents on the physical stability of co-formulations

This example shows the stability effects of different tensile agents on the physical stability of otherwise identical co- formulations of capraglin and semaglutide.

The compositions of co-formulations 6 to 12 are shown in Table 10. Table 10 Compositions of co-formulations 6 to 12 Element Co-formulation 6 7 8 9 10 11 12 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 10 10 10 10 10 10 10 L-Histidine(mM) 6 6 6 6 6 6 6 Polysorbate 20 (mg/ml) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Glycerol (mg/ml) - 16 - - - - - Sorbitol (mg/ml) - - 34 - - - - Mannitol (mg/ml) - - - 34 - - - Trehalose (mg/ml) - - - - 63 - - Sucrose (mg/ml) - - - - - 63 - NaCl (mg/ml) - - - - - - 6.0 HCl qs qs qs qs qs qs qs NaOH qs qs qs qs qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 5.7 5.7 5.7 5.7 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1.

All samples were stored under stress conditions defined as: ․ Duration: 18 days ․ Temperature: 37°C ± 2°C ․ Stress conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 100 rotations were performed every week for five days. The number of particles visible only under a microscope was quantified as described in Example 4. Table 11 Effect of different tensile agents on the physical stability of co-formulations Co-formulation Tension agent The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 7 11 14 18 Particle size＞5μm 6 without 256 385 429 952 1296 7 glycerin 292 241 292 466 691 8 Sorbitol 212 341 340 517 345 9 Mannitol 94 438 177 245 360 10 Trehalose 121 155 213 504 472 11 sucrose 59 104 140 539 749 12 NaCl ¹ 36 946 - - - Particle size＞10μm 6 without 4 27 96 216 377 7 glycerin 10 twenty three twenty three 48 106 8 Sorbitol 6 54 42 36 73 9 Mannitol 2 twenty three 12 8 50 10 Trehalose 4 10 40 50 69 11 sucrose 2 8 13 142 253 12 NaCl ¹ 2 403 - - - Particle size＞25μm 6 without 0 4 4 27 75 7 glycerin 0 0 4 6 4 8 Sorbitol 0 0 2 4 0 9 Mannitol 0 0 0 0 0 10 Trehalose 0 0 0 0 4 11 sucrose 0 0 2 15 55 12 NaCl ¹ 0 106 - - - Results are the average of 2 replicates and have been rounded to the nearest integer value (-) No sampling ¹ For co-formulation 12 containing NaCl, sampling was stopped earlier than for the other formulations due to a rapid increase in the number of particles visible under the microscope .

The results in Table 11 show that the number of microscopic particles increased most rapidly in the co-formulation that included NaCl as a tensiometer (Co-formulation 12). After 7 days, the number of particles greatly exceeded the number of particles determined from the other co-formulations. Therefore, sampling of the co-formulation containing NaCl for analysis of the number of microscopic particles was stopped after 7 days.

After 14 days, an increase in the number of microscopic particles was seen in the co-formulations containing glycerol and sucrose, and the two co-formulations were considered comparable in terms of physical stability. The number of particles remained the lowest in the co-formulations containing mannitol, sorbitol, or trehalose. In these co-formulations, almost no increase in the number of microscopic particles was seen during the 18 periods of storage of the co-formulations under stress conditions.

Among the co-formulations tested, those that included mannitol, sorbitol, or trehalose as tonicity agents remained the most stable over time. Example 7 : Effect of different surfactants on the physical stability of co-formulations

This example shows the effect of different surfactants on the physical stability of otherwise identical co- formulations of capraglin and semaglutide.

The compositions of co-formulation 13, co-formulation 14 and co-formulation 15 are shown in Table 12. Table 12 Compositions of co-formulation 13, co-formulation 14 and co-formulation 15 Element Co-formulation 13 14 15 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 10 10 10 L-Histidine(mM) 6 6 6 Polysorbate 20 (mg/ml) 0.05 - - Polysorbate 80 (mg/ml) - 0.05 - Poloxamer 188 (mg/ml) - - 0.5 HCl qs qs qs NaOH qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1.

All samples were stored under pressure conditions, defined as: ․ Duration: 17 days ․ Temperature: 37°C ± 2°C ․ Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 100 rotations were performed every week for five days. The number of particles visible only under a microscope was quantified as described in Example 4. Table 13 Effect of different surfactants on the physical stability of co-formulations Co-formulation Surfactant The relationship between the number of particles that can only be seen under a microscope and the time point ( day ) 0 7 10 14 17 Particle size＞5μm 13 Polysorbate 20 464 304 396 772 2803 14 Polysorbate 80 566 292 520 286 ¹ 348 15 Poloxamer 188 1235 1278 2783 940 ¹ 1704 Particle size＞10μm 13 Polysorbate 20 twenty three twenty three 43 143 297 14 Polysorbate 80 19 31 76 57 ¹ 48 15 Poloxamer 188 100 245 301 290 ¹ 523 Particle size＞25μm 13 Polysorbate 20 0 2 6 11 13 14 Polysorbate 80 0 0 0 4 ¹ 0 15 Poloxamer 188 0 13 17 61 ¹ 52 The result is the average of 2 replicates and has been rounded to the nearest integer value. Only ¹ replicate is summed.

At 17 days of storage under pressure, co-formulation 14 contained the lowest number of microscopic particles. An increase in microscopic particles was observed in co-formulation 13 containing polysorbate 20 after 14 days, while in co-formulation 15 containing poloxamer 188, microscopic particles formed after 7 days under pressure. It is clear that the co-formulation containing polysorbate 80 is the most stable, and the co-formulation containing polysorbate 20 is also acceptably stable. Example 8 : Effect of different buffer substances on the physical stability of co-formulations

This example shows that the buffer species have an effect on the physical stability of otherwise identical co- formulations of capraglin and semaglutide.

The compositions of co-formulation 1 and co-formulation 16 are shown in Table 14. Table 14 Compositions of co-formulation 1 and co-formulation 16 Element Co-formulation 1 16 Carglinide drug substance (mg/ml) 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ 25 25 Citrate, 1 H ₂ O (mM) 3 - L-Histidine(mM) - 6 HCl qs qs NaOH qs qs WFI Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1.

All samples were stored under pressure conditions, defined as: ○ Duration: 21 days ○ Temperature: 37°C ± 2°C ○ Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 100 rotations were performed every week for five days. The number of particles visible only under a microscope was quantified as described in Example 4. Table 15 Effect of buffer substances on the physical stability of co-formulations Co-formulation Buffer material The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 7 11 14 18 twenty one Particle size＞5μm 1 Citrate 459 668 408 409 4717 3115 16 Histidine 386 752 569 571 2495 2392 Particle size＞10μm 1 Citrate 52 117 55 125 2114 1446 16 Histidine 68 80 103 151 969 760 Particle size＞25μm 1 Citrate 0 0 0 16 542 332 16 Histidine 2 0 4 13 162 117 The result is the average of 2 replicates and has been rounded to the nearest integer value .

Until already on day 14 of storage under stress conditions, the physical stability of both coformulations was similar and acceptable. However, after day 18, the number of microscopic particles was greater in the citrate-buffered coformulation (coformulation 1) than in the histidine-buffered coformulation (coformulation 16). Histidine-buffered coformulation 16 was the most stable. Example 9 : Effect of different buffer concentrations on the chemical stability of coformulations

This example shows the effect of buffer concentration on the chemical stability of otherwise identical co-formulations .

The compositions of co-formulations 17 and 18 are shown in Table 16. Table 16 Compositions of co-formulations 17 and 18 Element Co-formulation 17 18 Carglinide drug substance (mg/ml) 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 10 10 L-Histidine(mM) 6 20 HCl qs qs NaOH qs qs WFI Prepare to 1 ml Prepare to 1 ml pH 5.7 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1.

The samples were stored at 30°C for 21 days and analyzed after 7, 14 and 21 days to determine the chemical purity of the cagerin peptide. The purity of the cagerin peptide was determined as described in Example 5 (for semaglutide). Table 17 Effect of buffer concentration on the chemical stability of cagerin peptide in co-formulations Co-formulation Buffer concentration Relationship between the purity of carglinide (%) and time (days) at 30°C 0 7 14 twenty one 17 Histidine: 6 mM 89.2% 88.2% 86.5% 84.2% 18 Histidine: 20 mM 89.2% 87.9% 86.0% 83.5% Summary

The results in Table 17 show that both co-formulations are stable. However, the chemical purity of kagerinide is the highest in co-formulation 17. The purity of kagerinide decreases rapidly over time when the histidine concentration is 20 mM. Example 10 : Effect of different buffer concentrations on the physical stability of co-formulations

This example shows the effect of histidine buffer concentration on the physical stability of the co-formulation .

The compositions of the tested co-formulations are shown in Table 16 .

The formulations were prepared as described in Example 1.

All samples were stored under pressure conditions, defined as: ․ Duration: 18 days ․ Temperature: 37°C ± 2°C ․ Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a refrigerated room. 100 rotations were performed every week for five days. The number of particles visible only under a microscope was quantified as described in Example 4. Table 18 Effect of buffer concentration on physical stability of co-formulations Co-formulation Buffer concentration The relationship between the number of particles that can only be seen under a microscope and the time point ( day ) 0 7 10 14 18 Particle size＞5μm 17 Histidine: 6 mM 239 392 165 189 266 18 Histidine: 20 mM 50 262 197 1029 926 Particle size＞10μm 17 Histidine: 6 mM 4 8 15 17 46 18 Histidine: 20 mM 2 17 17 461 423 Particle size＞25μm 17 Histidine: 6 mM 0 0 2 0 2 18 Histidine: 20 mM 0 0 0 120 104 The result is the average of 2 replicates and has been rounded to the nearest integer.

The difference in physical stability between co-formulations 17 and 18 became most apparent after 14 days. The data in Table 18 show that the number of microscopic particles observed in co-formulation 18 (containing 20 mM histidine) was greater than the number of microscopic particles observed in co-formulation 17 (containing 6 mM histidine). In other words, the co-formulation including 6 mM histidine was the most physically stable. Example 11 : Effect of HP-B-CD concentration on subcutaneous tolerance after subcutaneous injection

This example shows the concentration-dependent effect of HP-B-CD on subcutaneous tissue after subcutaneous injection.

The compositions of the tested co-formulation carriers prepared at different HP-B-CD concentrations are shown in Table 19. Table 19 Compositions of co-formulation carriers containing different concentrations of HP-B-CD Element Co-formulation carrier 1 2 3 4 5 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 10 12.5 15 17.5 20 L-Histidine(mM) 6 6 6 6 6 Sorbitol ² (mg/ml) 28 twenty four 20 16 12 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 0.05 HCl qs qs qs qs qs NaOH qs qs qs qs qs WFI Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml pH 6.0 6.0 6.0 6.0 6.0 ¹ MS: molar substitution, corresponding to the concentration of hydroxypropyl ^2- sorbitol per glucose unit, is varied with the concentration of HP-B-CD to maintain isotonic process conditions

The formulations were prepared as described in Example 1 except that the addition of active pharmaceutical ingredients was omitted.

Local (subcutaneous) tolerance was investigated in 5 Landrace × Yorkshire × Duroc (LYD) pigs by evaluating skin lesions obtained 6 days (necropsy) after subcutaneous administration of 600 µl of formulations containing HP-B-CD using a syringe equipped with a 25 G needle and a 5 mm stopper. Skin samples measuring 2x2 cm were collected at necropsy, fixed in neutral buffered formalin, trimmed using a multi-blade knife, embedded in paraffin, cut into 4 µm slices, mounted on glass slides and subsequently stained with hematoxylin-eosin (HE). The extent of subcutaneous tissue necrosis was assessed using light microscopy and scored on a numerical scale, with code 1 reflecting "no necrosis" and code 4 reflecting "moderate necrosis". A total of 5 skin samples were taken for each co-formulation vehicle. However, due to variations in the sectioning of subcutaneous tissue for successful assessment of necrosis, not all injection sites were assigned a score: 1, no necrosis 2, minimal necrosis 3, mild necrosis 4, moderate necrosis The extent of subcutaneous necrosis induced by isotonic co-formulation vehicle preparations containing 10% w/v to 20% w/v HP-B-CD upon subcutaneous injection was assessed. The results are presented in Table 20. Table 20 Necrotic fraction of subcutaneous tissue necrosis 6 days after injection of co-formulation vehicle with different percentages of HP-B-CD Co-formulation carrier HP-B-CD concentration Necrosis, subcutaneous Number of skin samples successfully scored out of the total 1 10% w/v 2,2,2,2,3 5 out of 5 2 12.5% w/v 3,3 2 out of 5 3 15% w/v 2,2,3 3 out of 5 4 17.5% w/v 2,2,3 3 out of 5 5 20% w/v 2,3,3,4 4 out of 5 Summary

A correlation was observed between increasing HP-B-CD concentrations in the co-formulation carriers and injection site necrosis. In one instance, a co-formulation carrier containing 20% w/v HP-B-CD caused moderate subcutaneous necrosis at the injection site. Formulations containing less than 20% w/v HP-B-CD all produced only mild or minimal subcutaneous necrosis at the injection site. All co-formulation carriers containing 10% to 20% w/v HP-B-CD were tolerated to an acceptable degree, with co-formulation carriers containing 10% to 17.5% w/v HP-B-CD being preferred. Example 12 : Effect of different tensioning agents on subcutaneous tolerability upon subcutaneous injection

This example shows the effect of any of three different tonicity agents (sorbitol, mannitol, and trehalose) in an otherwise identical isotonic co-formulation vehicle on local tolerability .

The composition of the co-formulation carriers tested is shown in Table 21. Table 21 Composition of isotonic co-formulation carriers prepared with different tensile agents Element Co-formulation carrier 6 7 8 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 15 15 L-Histidine(mM) 6 6 6 Polysorbate 20 (mg/ml) 0.1 0.1 0.1 Sorbitol (mg/ml) twenty four - - Mannitol (mg/ml) - 25 - Trehalose (mg/ml) - - 46 HCl qs qs qs NaOH qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 6.0 6.0 6.0 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

The formulations were prepared as described in Example 1 except that the addition of active pharmaceutical ingredients was omitted.

The local tolerance of subcutaneous administration of isotonic vehicle preparations containing HP-B-CD and three different tensors was investigated in two live Landrace × Yorkshire × Duroc (LYD) pigs by evaluating the skin response to subcutaneous administration of 600 µL of vehicle preparations. The preparations were injected using a syringe equipped with a 25 G needle and a 5 mm stopper. Approximately 24 hours after injection, necropsies were performed and skin specimens measuring 2x2 cm were fixed in neutral buffered formalin and trimmed into 4 µm thin sections using a multi-blade, embedded in paraffin and subsequently stained with HE. For both samples, the severity of subcutaneous tissue necrosis, inflammatory cell infiltration, and hemorrhagic distribution was assessed by a trained toxicopathologist using light microscopy and scored on a numerical scale, where code 1 reflects "no abnormality" and code 3 reflects "mild severity": 1, no abnormality 2, minimal severity 3, mild severity Table 22 Severity scores of subcutaneous tissue necrosis, inflammatory cell infiltration, and hemorrhagic distribution 24 hours after subcutaneous injection of co-formulated vehicles containing three types of tensors Co-formulation carrier Tension agent Bad Death Inflammatory cell infiltration Bleeding distribution 6 Sorbitol 2,1 2,1 1,1 7 Mannitol 2,2 2,3 1,2 8 Trehalose 1,1 2,2 1,2 Summary

The data presented in Table 22 show that sorbitol was the tonic agent that resulted in the least severe necrosis, inflammatory cell infiltration, and hemorrhage overall. These observations confirm that sorbitol is the preferred tonic agent to obtain good and acceptable subcutaneous tolerability of co-formulations containing active pharmaceutical ingredients. Example 13 : Confirmation of the Effect of Tonicity Type on Subcutaneous Tolerability in Histidine-Buffered and Citrate-Buffered Compositions Upon Subcutaneous Injection

This experiment examined: (1) the effect of tonicity type on the local tolerance profile following subcutaneous injection of an otherwise identical histidine-buffered coformulation; and (2) the effect of tonicity type on the local tolerance profile following subcutaneous injection of a citrate-buffered coformulation vehicle.

The compositions of the co-formulations evaluated are described in Tables 23a and 23b. Table 23a Composition of isohistidine buffered co-formulation vehicle Element Co-formulation 19 20 Carglinide drug substance (mg/ml) 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 15 L-Histidine(mM) 6 6 Trehalose (mg/ml) 38.6 - Sorbitol (mg/ml) - 20 Polysorbate 80 (mg/ml) 0.05 0.05 HCl qs qs NaOH qs qs WFI Up to 1 ml Up to 1 ml pH 6.0 6.0 ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit Table 23b Composition of isoosmotic citrate buffered co-formulation vehicle Element Co-formulation carrier twenty one HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ 25 Citrate, 1 H ₂ O (mM) 3 HCl qs NaOH qs WFI Up to 1 ml pH 5.7 ¹ MS: molar substitution, corresponding to the hydroxypropyl group per glucose unit

Co-formulations 19 and 21 were prepared as described in Example 1. Formulation carrier 21 was prepared as described in Example 1 except that the addition of active pharmaceutical ingredients was omitted. Methods

The local tolerance of the co-formulations described in Tables 23a and 23b upon subcutaneous administration was studied in 8 live mini pigs by evaluating skin lesions obtained 6 days (necropsy) after subcutaneous administration of a sample size of 750 µl using a syringe equipped with a 25 G size needle and a 5 mm stopper. Skin samples of size 2x2 cm were collected at necropsy, fixed in neutral buffered formalin, trimmed using a multi-blade knife, embedded in paraffin, cut into 4 µm thin slices, mounted on glass slides and subsequently stained with hematoxylin-eosin (HE). For the samples, the severity of subcutaneous tissue necrosis was assessed by a trained toxicopathologist using an optical microscope and scored on a numerical scale, where code 1 reflects "no abnormality" and code 5 reflects "significant severity": 1, no abnormality 2, minimal severity 3, mild severity 4, moderate severity 5, significant severity The results of the necrosis scores are shown in Tables 24a and 24b. Table 24a Severity score of subcutaneous tissue necrosis 6 days after injection of histidine buffered co-formulations Co-formulation Buffer Tension agent Necrotic scores of four skin samples 19 Histidine Trehalose 1,2,3,3 20 Histidine Sorbitol 2,2,2,2 Table 24b Subcutaneous tissue necrosis severity scores 6 days after injection of citrate buffered co-formulation vehicle Co-formulation carrier Buffer Tension agent Necrotic scores of eight skin samples twenty one Citrate without 3,4,4,4,4,5,5,5 Summary

The results presented in Table 24a show that the type of tonicity agent included in the formulation affects its in vivo local tolerance. There was a correlation between the tonicity agent and the subcutaneous necrosis observed at the injection site. Subcutaneous injection of co-formulation 19 including trehalose resulted in two events of mild necrosis (score 3). Subcutaneous injection of co-formulation 20 including sorbitol resulted in only minimal necrosis (score 2), which is a better outcome. These results confirm that, in this otherwise identical co-formulation vehicle scenario, the formulation including 15% w/v HP-B-CD (mean MS: 0.62) and sorbitol is better than the formulation including 15% w/v HP-B-CD (mean MS: 0.62) and trehalose.

The results presented in Table 24b show that three significant necrosis events (score 5) were observed using the co-formulation 21 carrier including 25% w/v HP-B-CD (average MS: 0.92), citrate and no tonicity agent, confirming that this particular co-formulation is not suitable for subcutaneous injection. Example 14 : Effects of different types of hydroxypropyl-substituted cyclodextrins on the physical and chemical stability of co-formulations of cagerinide and semaglutide

This example shows the effects of hydroxypropyl-α-cyclodextrin (HP-A-CD), hydroxypropyl-β-cyclodextrin (HP-B-CD), and hydroxypropyl-γ-cyclodextrin (HP-G-CD) on the formation of particles that can only be seen under a microscope and the chemical degradation of cagerin in otherwise identical cagerin and semaglutide co-formulations .

The compositions of co-formulations 22, 23 and 24 are shown in Table 25. Table 25 Compositions of co-formulations 22 , 23 and 24 Element Co-formulation twenty two twenty three twenty four Carglinide drug substance (mg/ml) 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 HP-A-CD (CycloLab) (% w/v) (average MS: 0.8, MS range: 0.5-0.9) ¹ 15 - - HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ - 15 - HP-G-CD (CycloLab) (% w/v) (average MS: 0.6, MS range: 0.4-0.7) ¹ - - 15 L-Histidine(mM) 6 6 6 Sorbitol (mg/ml) 20 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 HCl qs qs qs NaOH qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1.

The samples for determination of the number of microscopic particles were stored under pressure conditions defined as: -    Duration: 42 days -    Temperature: 30°C ± 2°C -    Pressure conditions: During storage, the samples were inverted 360° to simulate patient use outside a cold room. 20 rotations were performed three days per week and 40 rotations were performed two days per week. The number of microscopic particles was determined as described in Example 4.

Samples for determination of the purity of cagerinide were stored at 37°C for up to 42 days. The purity of semaglutide was determined using the following reverse phase high performance liquid chromatography (RP-HPLC) method, wherein the samples were analyzed using a Kinetex C18, 2.6 μm column (4.6 x 150 mm) and gradient elution was performed with 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 to 100 μl and a flow rate of 0.7 ml/min. The purity of capgranin peptide was quantified as the area of the main peak divided by the area of all related peaks x 100%.

The purity of semaglutide was determined using the same method in other experiments. Table 26 Physical stability of co-formulations of cagerinide and semaglutide formulated with different types of hydroxypropylcyclodextrin Co-formulation Types of Hydroxypropyl-Substituted Cyclocortin The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 35 42 Particle size＞5μm twenty two HP-A-CD 65 196 270 441 523 690 twenty three HP-B-CD 49 122 61 245 324 503 twenty four HP-G-CD ¹ 453537 - - - - - Particle size＞10μm twenty two HP-A-CD 4 7 14 27 47 41 twenty three HP-B-CD 4 8 5 20 20 116 twenty four HP-G-CD ¹ 107013 - - - - - Particle size＞25μm twenty two HP-A-CD 0 0 0 1 3 0 twenty three HP-B-CD 0 1 1 1 4 15 twenty four HP-G-CD ¹ 316 - - - - - The results of the number of particles visible under the microscope are the average of 3 replicates and have been rounded to the nearest integer value (-) No sampling ¹ Regarding the co-formulation 24 containing HP-G-CD, sampling was stopped earlier than other formulations due to the rapid increase in the number of particles. Table 27 Chemical purity of cargelinide in co-formulations of cargelinide and semaglutide formulated with different types of hydroxypropylcyclodextrin (%) Co-formulation Types of Hydroxypropyl Cyclodextrin Relationship between the purity of carglinide (%) and time (days) at 37°C 0 14 28 42 twenty two HP-A-CD 94.0% 87.2% 79.0% 71.1% twenty three HP-B-CD 96.8% 88.9% 81.9% 75.0% Summary

The results presented in Table 26 show that in coformulation 24 (HP-G-CD), a high number of microscopic particles was observed already at time point zero, which ruled out the use of HP-G-CD for coformulation of capgranin and semaglutide. After the initial analysis at time point zero, sampling for microscopic particle number analysis was stopped for coformulation 24 containing HP-G-CD. With respect to coformulation 22 (HP-A-CD) and coformulation 23 (HP-B-CD), almost no increase in the number of microscopic particles was observed.

The chemical purity results of calgrin peptide using HP-A-CD or HP-B-CD presented in Table 27 show that the purity of calgrin peptide in co-formulation 22 containing HP-A-CD decreases slightly more rapidly than that in co-formulation 23 containing HP-B-CD.

Based on the results in Table 26, HP-A-CD or HP-B-CD can be used for the co-formulation of cagerinide and semaglutide. However, based on the results in Table 27, HP-B-CD is preferred over HP-A-CD for the co-formulation of cagerinide and semaglutide due to the higher purity of cagerinide when formulated with HP-B-CD. Example 15 : Effect of the molar substitution degree of HP-B-CD on the physical and chemical stability of the co-formulation of cagerinide and semaglutide

This example shows the effect of molar substitution of HP-B-CD on the formation of microscopic particles, HMWP content, and chemical purity of semaglutide in otherwise identical citrate-buffered co- formulations of cagerinide and semaglutide.

The compositions of co-formulations 25 to 32 are shown in Table 28. Table 28 Compositions of co-formulations of citrate buffered cagrilide and semaglutide containing HP-B-CD excipients with different hydroxypropyl molar substitution Element Co-formulation 25 26 27 28 29 30 31 32 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 - 25 - - - - - HP-B-CD (CycloLab) (% w/v) (average MS: 0.67, MS range: 0.6-0.9) ¹ - - - 25 - - - - HP-B-CD Cavitron ^® (Ashland) (% w/v) (average MS: 0.68, MS range: 0.58-0.72) ¹ - - - - 25 - - - HP-B-CD Trappsol ^® (CTD, Inc.) (% w/v) (Average MS: 0.84, MS range: 0.8-1.0) ¹ - - - - - 25 - - HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ - 15 - - - - 25 - HP-B-CD Cavitron ^® (Ashland) (%w/v) (average MS: 1.08, MS range: 0.86-1.14) ¹ - - - - - - - 25 Citrate, 1 H ₂ O (mM) 3 3 3 3 3 3 3 3 HCl qs qs qs qs qs qs qs qs NaOH qs qs qs qs qs qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1.

Samples for determination of the number of microscopic particles were stored under pressure conditions defined as: -    Duration: 28 days -    Temperature: 30°C ± 2°C -    Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold room. 20 rotations were performed three days per week and 40 rotations were performed two days per week.

The number of particles visible under a microscope was quantified as described in Example 4.

Samples for determination of semaglutide purity and HMWP content were stored at 37°C for up to 28 days. The purity of semaglutide was determined as in Example 14.

The extent of covalently bound HMWP was determined using size exclusion chromatography (SEC). Samples were analyzed using a Waters SEC 1.7 μm column (4.6 x 150 mm) and isocratically eluted with a plasma eluent consisting of 185 mM sodium chloride, 5 mM sodium dihydrogen phosphate monohydrate, 3 mM orthophosphate, and 47% (v/v) isopropanol. Chromatography was performed at 50°C with UV detection (215 nm) using injection volumes of 1 to 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 divided by the area of the main peak x 100%. Table 29 Content of microscopic particles in citrate-buffered calgrin and semaglutide co-formulations containing HP-B-CD excipients with different hydroxypropyl molar substitution degrees Co-formulation HP-B-CD average molar substitution ¹ The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 Particle size＞5μm 25 0.62 82 342 1232 25324 26 0.92 89 10108 13381 49863 27 0.62 126 550 370 448 28 0.67 59 356 191 887 29 0.68 77 27 629 4323 30 0.84 97 374 381 7031 31 0.92 106 244 761 3161 32 1.08 95 32626 54994 114586 Particle size＞10μm 25 0.62 11 73 349 4799 26 0.92 9 4007 5128 20604 27 0.62 13 175 93 131 28 0.67 13 103 53 262 29 0.68 6 10 131 1542 30 0.84 18 129 135 2224 31 0.92 32 59 271 765 32 1.08 6 13421 29840 59104 Particle size＞25μm 25 0.62 0 3 39 330 26 0.92 1 627 830 3461 27 0.62 0 3 8 13 28 0.67 0 1 6 27 29 0.68 0 1 5 245 30 0.84 1 3 34 296 31 0.92 3 4 46 56 32 1.08 1 825 7895 15815 The results of the number of particles visible under the microscope are the average of 3 replicates and have been rounded to the nearest integer value ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit Table 30 HMWP content in citrate-buffered cagrilide and semaglutide co-formulations containing HP-B-CD excipients with different hydroxypropyl molar substitutions Co-formulation HP-B-CD molar substitution ¹ Relationship between HMWP content (%) and time (days) at 37° C 0 14 twenty one 28 25 0.62 0.03% 0.08% 0.13% 0.18% 26 0.92 0.05% 0.12% 0.18% 0.22% 27 0.62 0.04% 0.08% 0.14% 0.15% 28 0.67 0.05% 0.11% 0.17% 0.20% 29 0.68 0.04% 0.09% 0.16% 0.20% 30 0.84 0.05% 0.12% 0.17% 0.21% 31 0.92 0.04% 0.13% 0.22% 0.27% 32 1.08 0.11% 0.47% 0.61% 0.76% ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit Table 31 Chemical purity of semaglutide in citrate-buffered cagrilide and semaglutide co-formulations containing HP-B-CD excipients with different hydroxypropyl molar substitutions (%) Co-formulation HP-B-CD molar substitution ¹ Relationship between the purity (%) of semaglutide and time ( days ) at 37°C 0 14 twenty one 28 25 0.62 97.0% 95.3% 94.4% 93.8% 26 0.92 97.0% 95.1% 93.9% 93.4% 27 0.62 97.0% 95.2% 94.3% 93.4% 28 0.67 96.9% 95.0% 94.1% 93.5% 29 0.68 97.0% 95.3% 94.4% 93.5% 30 0.84 97.1% 95.4% 94.6% 93.8% 31 0.92 96.9% 94.4% 93.4% 92.3% 32 1.08 96.6% 91.4% 89.1% 87.5% ¹ MS: molar substitution, corresponding to the total number of hydroxypropyl groups per glucose unit

The results presented in Tables 29, 30 and 31 show that physical stability, formation of HWMP and chemical purity of semaglutide are dependent on the molar substitution of HP-B-CD when studied in citrate buffered cagerinide and semaglutide co-formulations. After 28 days, the lowest HMWP content and almost no increase in the number of particles were observed in co-formulation 27 containing 25% w/v HP-B-CD (average MS: 0.62). In contrast, in co-formulation 32 containing 25% w/v HP-B-CD (average MS: 1.08), a large increase in the number of particles visible under a microscope was observed only after 14 days, and the highest content of HMWP was also observed after 28 days.

All citrate-buffered co-formulations of capraglin and semaglutide containing 25% w/v HP-B-CD (average MS: 0.92 or less) were physically and chemically stable, with those containing 25% w/v HP-B-CD having an average MS of 0.68 or less being the most stable.

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

Co-formulation 25 containing 15% w/v HP-B-CD (average MS: 0.62) showed acceptable chemical and physical stability.

However, histidine-buffered cagerinide and semaglutide co-formulations 33 to 37 containing 15% w/v HP-B-CD are preferred due to their superior physical stability. Using histidine as the buffer liquid and sorbitol as the tonicity agent, the preferred HP-B-CD molar substitution range is expanded to an average of 0.62 to 0.92 (or 0.58 to 1.0 in total). Example 16 : Effect of the molar substitution degree of HP-B-CD on the physical stability of cagerinide and semaglutide co-formulations

This example shows the effect of the molar substitution of HP-B-CD on the amount of microscopic particles in an otherwise identical histidine-buffered co-formulation of capraglin and semaglutide.

The compositions of co-formulations 33 to 38 are shown in Table 32. Table 32 Compositions of co-formulations 33 to 38 of histidine buffered cagrilide and semaglutide containing HP-B-CD excipients with different hydroxypropyl molar substitution degrees Element Co-formulation 33 34 35 36 37 38 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 - - - - - HP-B-CD (CycloLab) (% w/v) (average MS: 0.67, MS range: 0.6-0.9) ¹ - 15 - - - - HP-B-CD Cavitron ^® (Ashland) (% w/v) (average MS: 0.68, MS range: 0.58-0.72) ¹ - - 15 - - - HP-B-CD Trappsol ^® (CTD, Inc.) (% w/v) (Average MS: 0.84, MS range: 0.8-1.0) ¹ - - - 15 - - HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ - - - - 15 - HP-B-CD Cavitron ^® (Ashland) (%w/v) (average MS: 1.08, MS range: 0.86-1.14) ¹ - - - - - 15 L-Histidine(mM) 6 6 6 6 6 6 Sorbitol (mg/ml) 20 20 20 20 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 0.05 0.05 HCl qs qs qs qs qs qs NaOH qs qs qs qs qs qs WFI Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml Prepare to 1 ml pH 5.8 5.8 5.8 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1.

Samples for determination of the number of microscopic particles were stored under pressure conditions defined as: - Duration: 28 days - Temperature: 30°C ± 2°C - Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 20 rotations were performed for three days per week and 40 rotations were performed for two days per week. The number of microscopic particles was quantified as described in Example 4. Table 33 Number of microscopic particles in histidine-buffered cagrilide and semaglutide co-formulations containing HP-B-CD with different molar substitutions of hydroxypropyl groups Co-formulation HP-B-CD molar substitution ¹ The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 Particle size＞5μm 33 0.62 19 300 252 460 34 0.67 37 176 220 66 35 0.68 twenty two 175 453 290 36 0.84 17 270 221 112 37 0.92 51 873 804 473 38 1.08 71 1311 1765 1621 Particle size＞10μm 33 0.62 1 14 6 61 34 0.67 6 9 6 4 35 0.68 1 5 twenty three 56 36 0.84 1 13 0 6 37 0.92 3 45 twenty four 117 38 1.08 4 143 416 579 Particle size＞25μm 33 0.62 0 0 0 3 34 0.67 0 0 0 0 35 0.68 0 3 0 1 36 0.84 0 0 0 4 37 0.92 0 1 1 20 38 1.08 1 0 14 27 The results for the number of particles visible under the microscope are the average of three replicates and have been rounded to the nearest integer value. ¹ MS: mole, corresponding to the total number of hydroxypropyl groups per glucose unit

The results presented in Table 33 show that co-formulations 33 to 37 including histidine buffer with HP-B-CD having extensive molar substitutions (average MS: 0.62 to 0.92) remained physically stable for 28 days.

In contrast, co-formulation 38 including HP-B-CD (average MS: 1.08) was not physically stable after 14 days.

The results show the synergistic effect between cyclodextrin and other excipients in the co-formulation, expanding the preferred range of molar substitution (average MS: 0.62 to 0.92). Example 17 : Effect of β- cyclodextrin substitution type on the physical stability of co-formulations

This example shows the effect of sulfobutyl ether-β-cyclodextrin (SBE-B-CD) and hydroxypropyl-β-cyclodextrin on the physical stability of otherwise identical co -formulations of capraglinide and semaglutide.

The compositions of co-formulations 39 and 40 are shown in Table 36. Table 36 Compositions of co-formulations containing HP-B-CD or SBE-B-CD Element Co-formulation 39 40 Carglinide drug substance (mg/ml) 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 - SBE-B-CD (CycloLab) (%w/v) (average MS: 0.87, MS range: 0.84-0.99) ¹ - 15 L-Histidine(mM) 6 6 Sorbitol (mg/ml) 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 HCl qs qs NaOH qs qs WFI Up to 1 ml Up to 1 ml pH 5.8 5.8 ¹ MS: molar substitution, corresponding to the sulfobutyl ether/hydroxypropyl process per glucose unit

The formulations were prepared as described in Example 1.

Samples for determination of the number of microscopic particles were stored under pressure conditions defined as: - Duration: 35 days - Temperature: 30°C ± 2°C - Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 20 rotations were performed for three days per week and 40 rotations were performed for two days per week. The number of microscopic particles was quantified as described in Example 4. Table 37 Content of microscopic particles in co-formulations of cagerinide and semaglutide containing HP-B-CD or SBE-B-CD Co-formulation Replacement type The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 35 Particle size＞5μm 39 HP-B-CD 49 122 61 245 324 40 SBE-B-CD 317 1686 6940 20714 280478 Particle size＞10μm 39 HP-B-CD 4 8 5 20 20 40 SBE-B-CD 13 640 3503 10248 138324 Particle size＞25μm 39 HP-B-CD 0 1 1 1 4 40 SBE-B-CD 0 126 738 2008 29834 The results for the number of particles visible under the microscope are the average of three replicates and are rounded to the nearest integer .

The results in Table 37 show that when SBE-B-CD was used to co-formulate carglinide and semaglutide, a large increase in microscopic particles was observed after 14 days; that is, the co-formulation was physically unstable. When HP-B-CD was used instead, almost no increase was observed during the 35-day period of the study; that is, the co-formulation was physically stable.

In contrast to what we have shown for hydroxypropyl-β-cyclodextrin, these results demonstrate that sulfobutyl ether-β-cyclodextrin (SBE-B-CD) is not a suitable cyclodextrin for co-formulation of cagrilide and semaglutide. Example 18 : Effect of pH on the physical and chemical stability of co-formulations

This example shows the effect of pH on the physical and chemical stability of cagerinide in an otherwise identical cagerinide and semaglutide co- formulation .

The compositions of co-formulations 41 to 45 are shown in Table 38. Table 38 Compositions of co-formulations with different pH Element Co-formulation 41 42 43 44 45 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 15 15 15 15 L-Histidine(mM) 6 6 6 6 6 Sorbitol (mg/ml) 20 20 20 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 0.05 HCl qs qs qs qs qs NaOH qs qs qs qs qs WFI (ml) Amount to 1 Amount to 1 Amount to 1 Amount to 1 Amount to 1 pH 5.5 5.6 5.7 5.8 6.0 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulation was prepared as described in Example 1.

The samples for determination of the number of microscopic particles were stored under pressure conditions defined as: -    Duration: 28 days -    Temperature: 30°C ± 2°C -    Pressure conditions: During storage, the samples were inverted 360° to simulate patient use outside a cold room. 20 rotations were performed three days per week and 40 rotations were performed two days per week. The number of microscopic particles was quantified as described in Example 4.

Samples used to determine the purity of cagerinide were stored at 37°C for up to 28 days. The purity of cagerinide was determined as described in Example 14. Table 39 Physical stability of cagerinide and semaglutide co-formulations at different pH in the pH range of 5.5 to 6.0 Co-formulation pH The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 Particle size＞5μm 41 5.5 70 6594 - 523259 42 5.6 69 174 730 1355 43 5.7 120 117 232 297 44 5.8 49 122 61 245 45 6.0 33 178 133 60 Particle size＞10μm 41 5.5 6 851 - 385284 42 5.6 6 9 200 476 43 5.7 3 5 8 18 44 5.8 4 8 5 20 45 6.0 4 6 3 9 Particle size＞25μm 41 5.5 1 4 - 190781 42 5.6 0 0 18 64 43 5.7 0 0 0 0 44 5.8 0 1 1 1 45 6.0 1 0 0 1 The results of the number of particles visible under a microscope are the average of 3 replicates and have been rounded to the nearest integer value (-) No sampling Table 40 Chemical purity of carglinide in carglinide and semaglutide co-formulations with different pH in the pH range of 5.5 to 6.0 (%) Co-formulation pH Relationship between the purity of capraglin (%) and time (days) at 37°C 0 14 28 41 5.5 97.1% 92.1% 85.9% 42 5.6 97.1% 91.6% 85.0% 43 5.7 97.0% 90.6% 83.2% 44 5.8 96.9% 88.9% 81.9% 45 6.0 96.9% 88.2% 78.3% Summary

The results presented in Tables 39 and 40 show that the physical and chemical stability of semaglutide and carglinide depends on the pH of the formulation: the highest pH results in the lowest carglinide purity at 37°C after 28 days; the lowest pH results in an increase in the number of particles that can only be seen under a microscope after 14 days. Based on these physical and chemical stability results, the optimal pH range for this particular carglinide and semaglutide co-formulation is 5.6 to 6.0, while a pH of 5.5 does not result in a co-formulation with acceptable physical stability. Example 19 : Effect of the concentration ratio of carglinide and semaglutide on the physical stability of the co-formulation This example shows the effect of different concentration ratios of carglinide and semaglutide on the content of microscopic particles observed in the formulation.

The compositions of histidine buffer co-formulations 46 to 50 are shown in Table 41, and histidine buffer co-formulations 51 to 61 with slightly modified compositions are shown in Table 42. Table 41 Compositions of histidine buffer co-formulations with different ratios of capraglin and semaglutide concentrations Element Co-formulation 46 47 48 49 50 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 4.8 6.4 8.0 9.6 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 15 15 15 15 L-Histidine(mM) 6 6 6 6 6 Sorbitol (mg/ml) 20 20 20 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 0.05 HCl qs qs qs qs qs NaOH qs qs qs qs qs WFI Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml pH 5.8 5.8 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to hydroxypropyl per glucose unit Table 42 Compositions of histidine buffer co-formulations with modified compositions containing various ratios of capgranin and semaglutide concentrations Element Co-formulation 51 52 53 54 55 56 57 58 59 60 61 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 4.8 6.4 8.0 9.6 9.6 9.6 9.6 10.7 12.8 16 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 20 20 20 20 15 20 20 20 20 20 20 L-Histidine(mM) 6 6 6 6 6 6 6 6 6 6 6 Sorbitol (mg/ml) 35 35 35 35 35 20 35 35 35 35 35 Polysorbate 80 (mg/ml) 1.78 1.78 1.78 1.78 0.05 0.05 0.05 1.78 1.78 1.78 1.78 HCl qs qs qs qs qs qs qs qs qs qs qs NaOH qs qs qs qs qs qs qs qs qs qs qs WFI Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml pH 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1.

Samples for determination of the number of microscopic particles were stored under pressure conditions defined as: - Duration: 28 days - Temperature: 30°C ± 2°C - Pressure conditions: During storage, samples were inverted 360° to simulate patient use outside a cold storage room. 20 rotations were performed for three days per week and 40 rotations were performed for two days per week. The number of microscopic particles was quantified as described in Example 4. Table 43 Content of microscopic particles in co-formulations containing different concentration ratios of carglinide and semaglutide Co-formulation Concentration of Capgranin / Semaglutide ( ratio ) The relationship between the number of particles that can only be seen under a microscope and time ( days ) 0 14 twenty one 28 Particle size＞5μm 46 3.2 / 3.2 mg/ml (1:1) 207 214 542 341 47 3.2 / 4.8 mg/ml (1:1.5) 65 133 214 591 48 3.2 / 6.4 mg/ml (1:2) 55 76 198 134 49 3.2 / 8.0 mg/ml (1:2.5) 44 86 262 139 50 3.2 / 9.6 mg/ml (1:3) 56 184 137 540 51 3.2 / 3.2 mg/ml (1:1) 297 467 650 11684 52 3.2 / 4.8 mg/ml (1:1.5) 377 541 331 14453 53 3.2 / 6.4 mg/ml (1:2) 588 501 980 45916 54 3.2 / 8.0 mg/ml (1:2.5) 452 341 712 13108 55 3.2 / 9.6 mg/ml (1:3) 37 226 121 161 56 3.2 / 9.6 mg/ml (1:3) 55 77 290 315 57 3.2 / 9.6 mg/ml (1:3) 41 181 273 368 58 3.2 / 9.6 mg/ml (1:3) 312 457 445 25494 59 3.2 / 10.7 mg/ml (1:3.33) 1240 594 1043 53301 60 3.2 / 12.8 mg/ml (1:4) 1459 549 320 77697 61 3.2 / 16 mg/ml (1:5) 1025 568 877 5202 Particle size＞10μm 46 3.2 / 3.2 mg/ml (1:1) 18 31 25 51 47 3.2 / 4.8 mg/ml (1:1.5) 5 9 twenty two 106 48 3.2 / 6.4 mg/ml (1:2) 3 9 18 twenty four 49 3.2 / 8.0 mg/ml (1:2.5) 10 9 13 13 50 3.2 / 9.6 mg/ml (1:3) 1 9 8 130 51 3.2 / 3.2 mg/ml (1:1) 38 45 79 1146 52 3.2 / 4.8 mg/ml (1:1.5) 69 94 80 2053 53 3.2 / 6.4 mg/ml (1:2) 74 81 171 7608 54 3.2 / 8.0 mg/ml (1:2.5) 78 57 118 1651 55 3.2 / 9.6 mg/ml (1:3) 5 9 5 14 56 3.2 / 9.6 mg/ml (1:3) 4 8 twenty two 59 57 3.2 / 9.6 mg/ml (1:3) 0 15 27 61 58 3.2 / 9.6 mg/ml (1:3) 50 79 74 3233 59 3.2 / 10.7 mg/ml (1:3.33) 147 72 165 6793 60 3.2 / 12.8 mg/ml (1:4) 155 84 51 10270 61 3.2 / 16 mg/ml (1:5) 80 99 189 382 Particle size＞25μm 46 3.2 / 3.2 mg/ml (1:1) 1 0 4 1 47 3.2 / 4.8 mg/ml (1:1.5) 0 1 0 6 48 3.2 / 6.4 mg/ml (1:2) 0 1 3 3 49 3.2 / 8.0 mg/ml (1:2.5) 1 0 3 3 50 3.2 / 9.6 mg/ml (1:3) 0 1 0 9 51 3.2 / 3.2 mg/ml (1:1) 0 0 1 13 52 3.2 / 4.8 mg/ml (1:1.5) 4 0 3 60 53 3.2 / 6.4 mg/ml (1:2) 8 4 6 157 54 3.2 / 8.0 mg/ml (1:2.5) 8 3 3 25 55 3.2 / 9.6 mg/ml (1:3) 0 0 1 0 56 3.2 / 9.6 mg/ml (1:3) 3 0 0 3 57 3.2 / 9.6 mg/ml (1:3) 0 0 4 0 58 3.2 / 9.6 mg/ml (1:3) 1 5 0 42 59 3.2 / 10.7 mg/ml (1:3.33) 1 0 5 97 60 3.2 / 12.8 mg/ml (1:4) 3 3 1 68 61 3.2 / 16 mg/ml (1:5) 8 4 26 8 The results for the number of particles visible under the microscope are the average of three replicates and are rounded to the nearest integer .

The results presented in Table 43 show that after 21 days, almost no increase in the number of microscopic particles was observed in co-formulations 46 to 60 containing 3.2 mg/ml of capraglin and up to 12 mg/ml of semaglutide.

After 14 days, an increase in the number of microscopic particles was observed in the co-formulation 61 containing 3.2 mg/ml capraglin and 16 mg/ml semaglutide.

All histidine buffered co-formulations 46 to 61 including 3.2 mg/ml capraglin and up to 16 mg/ml semaglutide were physically stable. Example 20 : Effect of HP-B-CD concentration on the physical stability of co-formulations

This example shows the effect of HP-B-CD concentration on the physical stability of co-formulations of capraglinide and semaglutide, where the co -formulations were exposed to physical stress.

The compositions of co-formulations 62 to 65 containing histidine buffer compositions are shown in Table 44. Table 44 Compositions of co-formulations containing different concentrations of HP-B-CD Element Co-formulation 62 63 64 65 Carglinide drug substance (mg/ml) 3.2 3.2 3.2 3.2 Semaglutide drug substance (mg/ml) 3.2 3.2 3.2 3.2 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 7.5 10 12.5 15 L-Histidine(mM) 6 6 6 6 Sorbitol (mg/ml) 20 20 20 20 Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 HCl qs qs qs qs NaOH qs qs qs qs WFI Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml pH 5.8 5.8 5.8 5.8 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1.

The aggregation and tendency of peptide fibers to form of kaglinide and semaglutide in the co-formulations were measured using the thioflavin T (ThT) fluorescence pressure assay as described in Example 2. Table 45 Physical stability of kaglinide and semaglutide co-formulations with different HP-B-CD concentrations Co-formulation HP-B-CD (MS: 0.62) ² Delay time until fiberization 62 7.5% w/v 17.66 ^hours1 63 10% w/v 28.06 ^hours1 64 12.5% w/v 70.97 ^hours1 65 15% w/v 119.0 ^{hours1 1} The results are the average of 6 replicates with ² MS: molar substitutions corresponding to the total number of hydroxypropyl groups per glucose unit.

The results presented in Table 45 show that the physical stability of the co-formulations of cagricans and semaglutide is dependent on the concentration of HP-B-CD, with lower concentrations resulting in shorter lag times for fibrillation to occur. The co-formulations comprising 7.5% w/v HP-B-CD were the least stable. The co-formulations comprising 15% w/v HP-B-CD were the most stable. Example 21 : Local subcutaneous tolerability of carrier formulations with different HP-B- CD content and molar substitution and overall buffer composition upon subcutaneous injection

This experiment examined: (1) the effect of HP-B-CD concentration and mean MS (0.62 vs. 0.92) on the local tolerance profile after subcutaneous injection. (2) the effect of the formulation vehicle on the local tolerance profile after subcutaneous injection in the presence of HP-B-CD.

The composition of the co-formulation vehicles tested is shown in Table 46. Table 46 Composition of co-formulation vehicles with different HP-B-CD content and molar substitution and overall buffer composition Element Co-formulation carrier 9 10 11 12 13 14 15 16 HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.62, MS range: 0.58-0.68) ¹ 15 20 twenty two twenty two 25 20 twenty two - HP-B-CD KLEPTOSE® (Roquette) (% w/v) (Average MS: 0.92, MS range: 0.81-0.99) ¹ - - - - - - - twenty two L-Histidine(mM) 6 6 6 6 6 - - - Citrate, 1 H ₂ O (mM) - - - - - 3 3 3 Sorbitol (mg/ml) 20 20 20 35 20 - - - Polysorbate 80 (mg/ml) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 HCl qs qs qs qs qs qs qs qs NaOH qs qs qs qs qs qs qs qs WFI Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml Up to 1 ml pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 ¹ MS: molar substitution, corresponding to the number of hydroxypropyl groups per glucose unit

The formulations were prepared as described in Example 1 except that no active drug ingredient was added.

Local (subcutaneous) tolerance was investigated in four Landrace × Yorkshire × Duroc (LYD) pigs by evaluating skin lesions obtained 6 days (necropsy) after subcutaneous administration of 200 µl of formulations containing HP-B-CD using a NovoPen 4 (32 G/4 mm) with a NovoFine Plus needle. Skin samples measuring 2x2 cm were collected at necropsy, fixed in neutral buffered formalin, trimmed using a multi-blade knife, embedded in paraffin, cut into 4 µm slices, mounted on glass slides and subsequently stained with hematoxylin-eosin (HE). For the four samples, the severity of subcutaneous tissue necrosis and inflammatory cell infiltration was assessed by a trained toxicopathologist using light microscopy and scored on a numerical scale, where code 1 reflects "no abnormality detected" and code 5 reflects "marked severity": 1, no abnormality detected 2, minimal severity 3, mild severity 4, moderate severity 5, marked severity

The co-formulation vehicle was evaluated for the degree of subcutaneous tissue necrosis and inflammatory cell infiltration caused by subcutaneous injection. The results are presented in Table 47. Table 47 Subcutaneous tissue necrosis severity score and inflammatory cell infiltration 6 days after injection of co-formulation vehicles with different HP-B-CD contents and molar substitutions and buffer compositions Co-formulation carrier Bad Death Inflammatory cell infiltration, granulomatous 9 1,1,1,1 2,2,2,2 10 1,1,2,2 2,2,2,3 11 2,3,4,4 3,3,4,4 12 2,4,4,4 3,3,4,4 13 3,4,4,4 3,3,4,5 14 1,3,3,3 1,3,3,3 15 3,4,5,5 4,4,4,5 16 1,5,4,5 2,4,4,5 Summary

The results in Table 47 show that in vivo topical subcutaneous tolerability depends on the concentration of HP-B-CD and the overall buffer composition. Co-formulation vehicles including histidine and sorbitol showed better tolerability than those co-formulation vehicles including citrate.

Coformulation vehicles including 20% w/v or less HP-B-CD resulted primarily in no or minimal necrotic or inflammatory cell infiltration (scores of 1 or 2) and a single observation of mild inflammatory cell infiltration (score of 3). Coformulation vehicles including 22% w/v or more HP-B-CD resulted in minimal to moderate necrotic and inflammatory cell infiltration (scores up to 4). Based on these results, coformulations containing less than 22% HP-B-CD appear to be suitable for subcutaneous use.

Coformulation vehicles containing 20% w/v and 22% w/v HP-B-CD and citrate (coformulation vehicles 14 and 15) resulted in significant necrosis and inflammatory cell infiltration (scores up to 5). Unexpectedly, coformulation vehicles containing 20% w/v and 22% w/v HP-B-CD, histidine and sorbitol (coformulation vehicles 10 and 11) were better tolerated, resulting in moderate necrosis and inflammatory cell infiltration (scores up to 4).

Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those of ordinary skill in the art. It should be understood, therefore, that the appended patent claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

without

without

Claims (19)

A pharmaceutical formulation comprising: - effective amounts of cagrilintide and semaglutide, - more than 10% w/v and less than 22% w/v of hydroxypropyl-substituted α- or β-cyclodextrin, comprising at least about 0.4 and at most about 1.0 hydroxypropyl groups per glucose unit, - about 3 to 30 mM histidine, - about 10 to 40 mg/ml sorbitol, - up to 2.0 mg/ml polysorbate 20 and/or 80, - about 75% to 90% w/w water and - having a pH of 5.6 to 6.0. A pharmaceutical formulation comprising: - effective amounts of carglinide and semaglutide, - more than 10% w/v and less than 22% w/v of hydroxypropyl-substituted α- or β-cyclodextrin, comprising an average of 0.62 to 0.84 hydroxypropyl groups per glucose unit, - about 3 to 30 mM of histidine and/or citrate, - about 10 to 40 mg/ml of sorbitol, - up to 2.0 mg/ml of polysorbate 20 and/or 80, - about 75% to 90% w/w of water and - having a pH of 5.6 to 6.0. The pharmaceutical formulation as described in claim 1 or 2, comprising no more than 22 mg/ml of cagerin. The pharmaceutical formulation as described in claim 1 or 2, comprising no more than 22 mg/ml of semaglutide. A pharmaceutical formulation as described in claim 1 or 2, wherein the cyclodextrin comprises 0.58 to 1.0 hydroxypropyl groups per glucose unit. A pharmaceutical formulation as described in claim 1 or 2, wherein the cyclodextrin is hydroxypropyl-substituted β-cyclodextrin. A pharmaceutical formulation as described in claim 1 or 2, comprising 10-20% w/v cyclodextrin. A pharmaceutical formulation as described in claim 1 or 2, comprising 12-18% w/v cyclodextrin. A pharmaceutical formulation as described in claim 1 or 2, comprising about 15% w/v cyclodextrin. A pharmaceutical formulation as described in claim 1 or 2, comprising up to 1.5 mg/ml of polysorbate 20 and/or 80. A pharmaceutical formulation as described in any one of claims 1 to 2, which is used as a medicine. A pharmaceutical formulation as described in any one of claims 1 to 2, which is used to treat a human subject suffering from obesity or overweight, with or without one or more related comorbidities, wherein the human subject suffering from overweight has a BMI of 25 kg/ ^m2 or more. A pharmaceutical formulation as described in any one of claims 1 to 2, which is used as an adjunct to a reduced calorie diet and increased physical activity for chronic weight management in adult individuals with an initial body mass index (BMI) of 30 kg/ ^m2 or more (obesity) or 27 kg/ ^m2 or more (overweight) in the presence of at least one weight-related comorbidity. The pharmaceutical formulation as described in claim 11 is characterized in that the formulation is administered by parenteral injection. The pharmaceutical formulation as described in claim 12 is characterized in that the formulation is administered by parenteral injection. The pharmaceutical formulation as described in claim 13 is characterized in that the formulation is administered by parenteral injection. A pharmaceutical formulation as described in claim 11, wherein the ratio of the dose of the administered capraglinide to the dose of the administered semaglutide is from 1:1 to 1:7. A pharmaceutical formulation as described in claim 12, wherein the ratio of the dose of the administered capraglinide to the dose of the administered semaglutide is from 1:1 to 1:7. The pharmaceutical formulation of claim 13, wherein the ratio of the dose of the administered capgranin peptide to the dose of the administered semaglutide is from 1:1 to 1:7.