EP4499127A1 - Formulation method - Google Patents

Formulation method

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Publication number
EP4499127A1
EP4499127A1 EP23716466.0A EP23716466A EP4499127A1 EP 4499127 A1 EP4499127 A1 EP 4499127A1 EP 23716466 A EP23716466 A EP 23716466A EP 4499127 A1 EP4499127 A1 EP 4499127A1
Authority
EP
European Patent Office
Prior art keywords
formulation
semaglutide
receptor agonist
cagrilintide
glp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23716466.0A
Other languages
German (de)
French (fr)
Inventor
Rosa Rebecca Erritzøe HANSEN
Benjamin Troest KJELDSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/EP2022/085558 external-priority patent/WO2023110833A1/en
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP4499127A1 publication Critical patent/EP4499127A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/225—Calcitonin gene related peptide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof

Definitions

  • the current invention relates to a method of formulating a pharmaceutical formulation which is a co-formulation of a GLP-1 receptor agonist and an amylin receptor agonist.
  • the pharmaceutical formulation may be used for the medical treatment of subjects with overweight or obesity, with or without one or more associated co-morbidities; diabetes, with or without one or more associated comorbidities; one or more cardiovascular diseases; non-alcoholic steatohepatitis (NASH); and/or cognitive impairment, such as that caused by Alzheimer’s disease.
  • NASH non-alcoholic steatohepatitis
  • Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist and is the active pharmaceutical ingredient in Ozempic®.
  • Ozempic® is indicated (i) as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus and (ii) to reduce the risk of major adverse cardiovascular events in adults with type 2 diabetes mellitus and established cardiovascular disease.
  • Semaglutide is also the active pharmaceutical ingredient in Wegovy®.
  • Wegovy® 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 (BMI) of greater or equal to 30 kg/m 2 or greater than 27 kg/m 2 , in the presence of at least one weight-related comorbidity.
  • BMI body mass index
  • Ozempic® and Wegovy® are liquid pharmaceutical formulations comprising 8 mM phosphate and having a pH of about 7.4.
  • a fixed-dose combination of an amylin receptor agonist, cagrilintide, and the GLP-1 receptor agonist, semaglutide, has been investigated for the treatment of overweight and obesity (Lancet 2021 ; 397: 1736-48).
  • the drug products investigated were in the form of separate liquid pharmaceutical formulations for subcutaneous use, comprising either cagrilintide or semaglutide.
  • Semaglutide a GLP-1 receptor agonist
  • Semaglutide is optimally stable at pH 7.4 and has previously needed to be formulated in a neutral to slightly basic solution of pH 7-8, to ensure its solubility in aqueous solution.
  • Cagrilintide is optimally stable at pH 4.0 and has needed to be formulated in acidic solution, increasing pH accelerating the rate of its chemical degradation.
  • cagrilintide and semaglutide preclude a simple mixture of these two peptides.
  • GLP-1 receptor agonist and amylin receptor agonist combinations when the two have incompatible optimal pH ranges.
  • the therapeutic protein is subjected to physical stress and agitation from several production steps including mixing, filtration and pumping [Yang, M., 2015. Stress and protein instability during formulation and fill/finish processes. BIOPHARM INTERNATIONAL, 28(6), pp.46]. This stress can ultimately lead to protein denaturation or aggregation, which could compromise product quality by causing loss of therapeutic efficacy or increased immunogenicity.
  • the formulation method by which a protein drug product formulation is manufactured must aim to decrease the total stress the protein is subject to, in order to ensure sufficient physical stability of the product.
  • an amylin receptor agonist such as cagrilintide
  • a GLP-1 receptor agonist such as semaglutide
  • a pharmaceutical formulation comprising an amylin receptor agonist, such as cagrilintide, and a GLP-1 receptor agonist, such as semaglutide, said method comprising:
  • Figures 1-12 depict formulation methods 1-12, wherein formulation methods 5, 9, and 11 are embodiments of the current invention.
  • Figure 1 is a flow diagram depicting formulation method 1.
  • HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 2 is a flow diagram depicting formulation method 2. 1 . HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 3 is a flow diagram depicting formulation method 3.
  • HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 4 is a flow diagram depicting formulation method 4.
  • HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 5 is a flow diagram depicting formulation method 5.
  • HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 6 is a flow diagram depicting formulation method 6.
  • HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 7 is a flow diagram depicting formulation method 7.
  • HP-B-CD, sorbitol and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 8 is a flow diagram depicting formulation method 8.
  • HP-B-CD, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 9 is a flow diagram depicting formulation method 9.
  • HP-B-CD was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution. 3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 10 is a flow diagram depicting formulation method 10.
  • HP-B-CD and sorbitol was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Figure 11 is a flow diagram depicting formulation method 11 .
  • HP-B-CD and histidine was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Sorbitol and polysorbate 80 was added and dissolved.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
  • Figure 12 is a flow diagram depicting formulation method 12.
  • HP-B-CD and polysorbate 80 was dissolved in water for injection (60% of the final volume).
  • Semaglutide drug substance was dissolved in the solution.
  • pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
  • the current invention is a method of formulating a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist and a cyclodextrin comprising hydroxypropyl substitutions.
  • a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist comprising:
  • the GLP-1 receptor agonist may be semaglutide.
  • the amylin receptor agonist may be cagrilintide.
  • the cyclodextrin may be of the hydroxypropyl-substituted alpha type, comprising six ring-arranged glucose units, and/or the hydroxypropyl-substituted beta type, comprising seven ring-arranged glucose units.
  • the aqueous, surfactant-free excipient solution comprising cyclodextrin may comprise one or more further excipients, with the proviso that said one or more further excipients is not a surfactant.
  • One or more further excipients may be added to the mixture of the aqueous, surfactant-free excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
  • One or more further excipients may be added to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
  • non-limiting examples of “further excipients” are a buffer and a tonicity agent.
  • the further excipient may be a buffer having at least one pKa of about 5.0-7.0, such as histidine, citrate or phosphate, or a combination thereof.
  • the further excipient may be a tonicity agent, with the proviso that the tonicity agent is not sodium chloride.
  • concentrations of cyclodextrin, GLP-1 receptor agonist, amylin receptor agonist, surfactant and any further excipient will be such as to arrive at the composition of the pharmaceutical formulation (drug product) also disclosed herein.
  • Also 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 precludes its co-formulation in the pH range enabling chemical stability of the amylin receptor agonist.
  • a means of co-formulating a GLP-1 receptor agonist having an isoelectric point (pl) of less than 6.5, preferably less than 6.0, such as 3.5-6.0, such as 3.0- 5.0, such as 4.0-6.0, and an amylin receptor agonist.
  • the optimal pH of the amylin receptor agonist is the pH at which it is, chemically and physically, most stable.
  • the person skilled in the art can readily determine the amylin receptor agonist’s optimal pH by testing its chemical and physical stability, in an aqueous solution essentially consisting of the amylin receptor agonist, a buffer and water for injection, across the pH range.
  • the optimal pH of the GLP-1 receptor agonist is the pH at which it is, chemically and physically, most stable.
  • the person skilled in the art can readily determine the GLP-1 receptor agonist’s optimal pH by testing its chemical and physical stability, in an aqueous solution essentially consisting of the GLP-1 receptor agonist, a buffer and water for injection, across the pH range.
  • the physical stability of the GLP-1 receptor agonist may be a reflection of its isoelectric point, which may coincide with the pH where poorest physical stability might be expected.
  • any GLP-1 receptor agonist and/or any amylin receptor agonist in any liquid formulation can be quantified by means of, e.g., reversed phase (ultra) high performance liquid chromatography (RP-UHPLC or RP-HPLC) and/or by measuring the percentage of high molecular weight protein (%HMWP) by means of, e.g., size exclusion chromatography (SEC).
  • RP-UHPLC reversed phase high performance liquid chromatography
  • %HMWP percentage of high molecular weight protein
  • SEC size exclusion chromatography
  • the physical stability of a GLP-1 receptor agonist and/or any amylin receptor agonist in any liquid formulation can be quantified by measuring particle formation and/or fibrillation by means of micro-flow imaging (MFI) or a Thioflavin T (ThT) fluorescence stress assay, respectively.
  • MFI micro-flow imaging
  • ThT Thioflavin T fluorescence stress assay
  • an amylin receptor agonist and a GLP-1 receptor agonist whose optimal pHs differ by at least about two pH units, such as 2-5 pH units, such as 2-4 pH units, such as 3-5 pH units.
  • the GLP-1 receptor agonist may be semaglutide.
  • the amylin receptor agonist may be cagrilintide or a biologically active metabolite or degradation product of cagrilintide.
  • the composition of the pharmaceutical formulation disclosed herein preserves/improves the chemical and physical stability of the active pharmaceutical ingredients, even when coformulated at pH 5.5-6.5, such as pH 5.6-6.0; preserves the pharmacokinetic profiles of the active pharmaceutical ingredients in terms of their bioavailability and exposure; and exhibits an acceptable local tolerance upon subcutaneous injection.
  • Production of the drug substances precedes implementation of the formulation method disclosed herein, whose aim is to create the final drug product, a pharmaceutical formulation.
  • Application of the method disclosed herein ensures that the pharmaceutical formulation or “drug product” has an improved shelf-life.
  • drug substance refers to the purified active pharmaceutical ingredient.
  • drug substance is well known in the art as referring to “any [substance or] mixture of substances intended to be used in the manufacture of a drug (medicinal) product and that, when used in the production of a drug, becomes an active ingredient of the drug product” (see, for example, EMA ICH Q7a: “Good manufacturing practice for active pharmaceutical ingredients”). It is well known to the person skilled in the art that a drug substance must be essentially free of impurities, to the extent required by regulatory authorities.
  • the drug substance may be a solid, such as a freeze-dried or spray-dried solid.
  • the amylin receptor agonist drug substance may be a solid.
  • the GLP-1 receptor agonist drug substance may be a solid.
  • a solid or “dried” form of the purified active pharmaceutical ingredient (or drug substance) may be obtained as described in WO2012/168432.
  • the drug substance may be dried by freeze drying (i.e., lyophilization; see, for example, Williams and Rolli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 :12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
  • the terms “pharmaceutical formulation”, “coformulation” and “drug product” may herein be used interchangeably to refer to a liquid pharmaceutical formulation comprising a GLP-1 receptor agonist and an amylin receptor agonist.
  • the pharmaceutical formulation disclosed herein is suitable for parenteral injection, preferably subcutaneous injection.
  • amylin herein refers to a polypeptide having the same amino acid sequence as an endogenous amylin, such as human amylin.
  • An amylin receptor agonist may bind to and activate the calcitonin receptor (CTR) and/or the amylin receptors (AMYRs).
  • CTR calcitonin receptor
  • AYRs amylin receptors
  • the latter consist of heterodimers of two components: the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP1- 3) resulting in three possible complexes, AMYR1-3.
  • amylin receptor agonist may be defined as a chemical entity which is capable of binding to an amylin receptor and is capable of activating or “agonising” it.
  • the amylin receptor agonist is capable of binding to and activating at least the amylin receptor 3 (AMYR3).
  • the amylin receptor agonist may also be capable of agonising the calcitonin receptor, the amylin receptor 1 (AMYR1) and/or the amylin receptor 2 (AMYR2).
  • Examples of endogenous amylin receptor agonists are human amylin and human calcitonin.
  • exogenous amylin receptor agonists are cagrilintide and pramlintide (the active pharmaceutical ingredient in Symlin®).
  • the amylin receptor agonist is capable of activating the amylin receptor; in other words, it is “potent” on the amylin receptor.
  • the in vitro potency of the amylin receptor agonist on amylin receptor 3 may be measured as described in WO/2022129526, Assay 2.
  • the potency of the compound may be described by means of its EC 5 o value, wherein EC 5 o represents the concentration of compound upon which 50% of its maximal effect is observed. The lower the EC 5 o value, the more potent the compound.
  • the amylin receptor agonist as disclosed herein may have an EC 5 o value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 10 pM.
  • amylin receptor agonist in the pharmaceutical formulation disclosed herein may be cagrilintide or a biologically active metabolite or degradation product of cagrilintide.
  • Cagrilintide also known as AM833 is the compound of Example 53 in
  • WO2012/168432 N-alpha-[(S)-4-Carboxy-4-(19-carboxynonadecanoylamino)butyryl]- [Glu14,Arg17,Pro37]-pramlintide.
  • Cagrilintide may be prepared as described in WO2012/168432, pages 153-155.
  • Cagrilintide may be in the form of a salt, preferably a pharmaceutically acceptable salt.
  • a biologically active metabolite or degradation product of cagrilintide may have an aspartate (Asp) in position 21 or 22.
  • a biologically active metabolite or degradation product of cagrilintide may have an iso-aspartate (iso-Asp) in position 21 or 22.
  • cagrilintide had an EC 50 value of about 11 pM (WO/2022/129526, Tables 4b and 4c).
  • the concentration of cagrilintide in the pharmaceutical formulation disclosed herein may be from about 0.25 mg/ml to about 22 mg/ml.
  • the pharmaceutical formulation disclosed herein may comprise cagrilintide in a concentration of about 0.33-18 mg/ml; such as 0.25-0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5-1 .0 mg/ml, such as about 0.67 mg/ml; such as 1 .0-1 .5 mg/ml, such as about 1.33 mg/ml; such as 1.5-2.0 mg/ml, such as about 1.5 mg/ml; such as 2.0-2.5 mg/ml; such as 2.5-3.0 mg/ml; such as 3.0-3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5-4.0 mg/ml; such as 4.0-5.0 mg/ml; such as 5.0-6.0 mg/ml; such as 6.0-7.0 mg/ml, such as 7.0-8.0 mg/ml, such as 8.0-9.0 mg/ml, such as 9.0-10.0 mg/ml, such as about 9.6 mg/ml; such as 10-11 mg/ml,
  • the pharmaceutical formulation disclosed herein may comprise no more than 22 mg/ml cagrilintide.
  • the pharmaceutical formulation disclosed herein may comprise no more than 12 mg/ml cagrilintide.
  • GLP-1 or “native GLP-1” herein refers to human Glucagon-Like Peptide-1 (GLP-1 (7-37)).
  • GLP-1 receptor agonist may be defined as a ligand which is capable of binding to the GLP-1 receptor and producing a biological response similar to that of the endogenous ligand, glucagon-like peptide 1 (GLP-1 (7-37)).
  • GLP-1 (7-37) glucagon-like peptide 1
  • a "full" GLP-1 receptor agonist may be defined as a GLP-1 receptor agonist which is capable of eliciting a biological response of the same magnitude as GLP-1 (7-37).
  • exogenous GLP-1 receptor agonists examples include semaglutide (the active pharmaceutical ingredient in Ozempic®, Rybelsus® and Wegovy®), liraglutide (the active pharmaceutical ingredient in Victoza® and Saxenda®), tirzepatide (the active pharmaceutical ingredient in Mounjaro®) and dulaglutide (the active pharmaceutical ingredient in Trulicity®).
  • the GLP-1 receptor agonist is capable of binding to and activating, or “agonising” the GLP-1 receptor; in other words, it is “potent” on the GLP-1 receptor.
  • the in vitro potency of the GLP-1 receptor agonist on the GLP-1 receptor may be measured as described in WO/2022/129526, Assay 1 .
  • the potency of the compound may be described by means of its EC 5 o values, wherein EC50 represents the concentration of compound upon which 50% of its maximal effect is observed. The lower the EC50 value, the more potent the compound.
  • the GLP-1 receptor agonist disclosed herein may have an EC50 value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, even more preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 10 pM.
  • Semaglutide is a GLP-1 receptor agonist also known as N 626 - ⁇ 18-[N-(17- carboxyheptadecanoyl)-L-y-glutamyl]-10-oxo-3,6, 12, 15-tetraoxa-9, 18-diazaoctadecanoyl ⁇ -[8- (2-amino-2-propanoic acid),34-L-arginine]human glucagon-like peptide 1 (7-37). Semaglutide was described in W02006/097537 and in J. Med. Chem. 2015, 58, 18, 7370-7380 and may be manufactured using methods well known to the person skilled in the art, such as that briefly described in W02006/097537, Example 4.
  • Semaglutide may be present in the current pharmaceutical formulation in its fully or partly ionised form; for example one or more carboxylic acid groups (-COOH) may be deprotonated into the carboxylate group (-COO j and/or one or more amino groups (-NH 2 ) may be protonated into the -NH 3 + group.
  • carboxylic acid groups -COOH
  • -COO j carboxylate group
  • amino groups -NH 2
  • Semaglutide may be in the form of a salt, preferably a pharmaceutically acceptable salt.
  • semaglutide had an EC50 value of about 5.5 pM (see WO/2022/129526, Tables 4b and 4c).
  • concentration of semaglutide in the pharmaceutical formulation disclosed herein may be from about 0.25 mg/ml to about 22 mg/ml.
  • the pharmaceutical formulation may comprise semaglutide in a concentration of about 0.33-18 mg/ml; such as 0.25-0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5-1.0 mg/ml, such as about 0.67 mg/ml; such as 1.0-1.5 mg/ml, such as about 1.33 mg/ml; such as 1 .5-2.0 mg/ml, such as about 1 .5 mg/ml; such as 2.0-2.5 mg/ml; such as about 2.2 mg/ml, such as 2.5-3.0 mg/ml; such as 3.0-3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5-4.0 mg/ml; such as 4.0-5.0 mg/ml; such as about 4.8 mg/ml; such as 5.0-6.0 mg/ml; such as 6.0- 7.0 mg/ml, such as about 6.4 mg/ml; such as 7.0-8.0 mg/ml, such as about 8.0 mg/ml; such as 8.0-9.
  • the pharmaceutical formulation disclosed herein may comprise no more than 22 mg/ml semaglutide.
  • the pharmaceutical formulation disclosed herein may comprise no more than 12 mg/ml semaglutide.
  • the GLP-1 receptor agonist and/or amylin receptor agonist in the pharmaceutical formulation disclosed herein may also, for instance, be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry, or other well established techniques, see e.g. Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Dbrwald, “Organic Synthesis on Solid Phase”, Wiley-VCH Verlag GmbH, 2000; and “Fmoc Solid Phase Peptide Synthesis”, Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.
  • the compounds may be produced by recombinant methods, e.g. by culturing a host cell containing a DNA sequence encoding the peptide sequence and capable of expressing the peptide, in a suitable nutrient medium under conditions permitting the expression of the peptide.
  • host cells suitable for expression of these peptides are Escherichia coli, Saccharomyces cerevisiae and mammalian BHK or CHO cell lines.
  • the isoelectric point (pl) of a molecule is the pH at which the molecule carries no net charge.
  • the pl of a peptide may be theoretically calculated from the pK values of its amino acids and of the terminal amine and carboxyl groups and can be used to predict the solubility of the peptide at a given pH.
  • the theoretically calculated isoelectric point of the GLP-1 receptor agonist may be in the range of 3.5-6.5, such as 3.5-6.0, such as 4.0-6.0, such as 3.8-4.9, such as 4.0-4.5.
  • Semaglutide has a theoretically calculated isoelectric point of about 4.37.
  • the theoretically calculated isoelectric point of the amylin receptor agonist may have an isoelectric point (pl) in the range of 8-12, such as 8-9.
  • Cagrilintide has a theoretically calculated isoelectric point of about 8.56.
  • the method disclosed herein involves preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI).
  • WFI water for injection
  • the excipient solution is an aqueous solution, comprising water for injection (WFI).
  • WFI water for injection
  • the pharmaceutical formulation may comprise 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 excipient solution may further comprise one or more further excipients, with the proviso that such further excipient is not a surfactant.
  • a buffer may be such a further excipient.
  • a tonicity agent may be such a further excipient.
  • the aqueous, surfactant-free excipient solution utilised in the formulation method described herein comprises a cyclodextrin comprising hydroxypropyl substitutions.
  • the pharmaceutical formulation disclosed herein necessarily comprises a cyclodextrin comprising hydroxypropyl substitutions.
  • the pharmaceutical formulation may comprise about 10-25% w/v of a cyclodextrin comprising hydroxypropyl substitutions.
  • the pharmaceutical formulation may comprise more than 10% w/v of a cyclodextrin comprising hydroxypropyl substitutions.
  • the pharmaceutical formulation may comprise less than 22% w/v of a cyclodextrin comprising hydroxypropyl substitutions.
  • the pharmaceutical formulation may comprise about 10-20% w/v, about 15- 25% w/v, about 12-18% w/v, about 10-17.5% w/v, about 11.25-15%, such as about 15% w/v of a cyclodextrin comprising hydroxypropyl substitutions.
  • Cyclodextrins are oligosaccharide starch derivatives consisting of 6, 7 or 8 a-(1 ,4)- linked glucopyranose (glucose) units arranged cyclically and denoted the alpha, beta or gamma type, respectively.
  • the cyclodextrins have a wide range of applications, amongst others as pharmaceutical excipients [P. Breen & S. S.
  • the hydroxyl groups of the glucose units of the cyclodextrins may be substituted by a varying number of hydrophilic chemical substitutions, e.g., by hydroxypropyl groups, leading to differences in degree of substitution which can be described as either the average number of hydroxypropyl per cyclodextrin molecule (abbreviated DS) or the molar substitution degree corresponding to the average number of hydroxypropyl per glucose units present in the cyclodextrin in question (abbreviated MS).
  • DS average number of hydroxypropyl per cyclodextrin molecule
  • MS molar substitution degree corresponding to the average number of hydroxypropyl per glucose units present in the cyclodextrin in question
  • hydroxypropyl per cyclodextrin can be achieved by multiplication of the molar substitution degree by the number of glucose units comprised in the cyclodextrin in question. Difference in degree of substitution can result in alterations in physicochemical properties such as surface activity and complexing abilities.
  • the hydroxyl groups may also be chemically substituted by groups of sulfobutylether. These mostly hydrophilic modifications have yielded cyclodextrin derivates highly suitable for parenteral administration [Cyclodextrins used as excipients, 2017, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)]. Cyclodextrins comprising hydroxypropyl substitutions are commonly abbreviated HP-CDs whereas cyclodextrins comprising sulfobutylether substitutions are abbreviated SBE-CDs.
  • the cyclodextrins comprising hydrophilic substitutions adopt what may be described as cone-liked shapes having a hydrophobic inner cavity and a hydrophilic outer surface formed by the many hydrophilic substitutions capable of forming hydrogen bonds with neighbouring water molecules, thereby improving water solubility [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11], Their hydrophobic microenvironment inside the cavity of these cone-like structures enables them to form drug-to-cyclodextrin complexes mainly through hydrophobic interactions [T.
  • the pharmaceutical formulation disclosed herein preferably comprises a cyclodextrin of the hydroxypropyl-substituted alpha type and/or a cyclodextrin of the hydroxypropyl-substituted beta type.
  • the pharmaceutical formulation disclosed herein may comprise a cyclodextrin of the hydroxypropyl-substituted alpha type, comprising six ring-arranged glucose units.
  • the hydroxypropyl substituted cyclodextrin of the alpha type is abbreviated HP-A-CD.
  • Hydroxypropyl-alpha-cyclodextrins (CAS: 128446-33-3/99241-24-4) are commercially available, with an average molar substitution (MS) of 0.8 and a molar substitution range of 0.5-0.9.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl- alpha-cyclodextrin having a minimum of about 0.4 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-alpha-cyclodextrin having a maximum of about 1.0 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl- alpha-cyclodextrin having a molar substitution range of 0.5-0.9 hydroxy propyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-alpha- cyclodextrin having an average molar substitution (MS) of about 0.8 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise a cyclodextrin of the hydroxypropyl-substituted beta type, comprising seven ring-arranged glucose units.
  • the hydroxypropyl substituted cyclodextrin of the beta type is abbreviated HP-B-CD.
  • Hydroxypropyl-beta-cyclodextrins are well known pharmaceutical excipients, typically used in small molecule pharmaceutical formulations, primarily to increase solubility and bioavailability [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11], Thus far, the use of cyclodextrins and cyclodextrin substituted derivatives in protein and peptide-based pharmaceutical formulations is limited.
  • hydroxypropyl substitution degrees for hydroxypropyl-beta-cyclodextrins as pharmaceutical excipients ranges between 2.8 and 10.5 according to the European and US pharmacopoeia [USP 38 NF 33, Pharm Eur 8, as estimated by methods described in USP ⁇ 761 > /Pharm. Eur. 2.2.33], corresponding to 0.4- 1.5 hydroxypropyl per glucose unit (MS).
  • cyclodextrins such as hydroxypropyl-beta-cyclodextrins are usually described by means of the average molar substitutions (MS) of their molar substitution ranges.
  • Hydroxypropyl-beta-cyclodextrins are commercially available for use as excipients, with average molar substitutions (MS) including: MS 0.62, with a molar substitution range of 0.58-0.68; MS 0.67, with a molar substitution range from (0.6-0.9); MS 0.68, with a molar substitution range from (0.58-0.72); MS 0.84, with a molar substitution range from (0.8-1.0); MS 0.92, with a molar substitution range from (0.81-0.99); MS 1 .08, with a molar substitution range from (0.86-1 .14); each value describing the number of hydroxypropyls per glucose unit.
  • MS average molar substitutions
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having a minimum of about 0.4 hydroxy propyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having a maximum of about 1.0 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having a molar substitution range of 0.58-1.0 hydroxy propyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) range of about 0.62-0.92 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.62-0.84 hydroxypropyls per glucose unit.
  • MS average molar substitution
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having about 0.4-0.75 hydroxy propyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having about 0.75 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.62.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.58-0.68 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.68.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.58-0.72 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.67.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.6-0.9 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.84.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.8-1.0 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.92.
  • the pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.81-0.99 hydroxypropyls per glucose unit.
  • the pharmaceutical formulation disclosed herein may comprise 10-25% w/v, such as more than 10% w/v and less than 22% w/v, such as about 10-20% w/v, such as about 12- 18% w/v, such as about 10-17.5% w/v, such as about 11.25-15%, such as about 15% w/v hydroxypropyl-beta-cyclodextrin having a minimum of about 0.4 hydroxypropyls per glucose unit and a maximum of about 1.0 hydroxypropyls per glucose unit; such as an average of 0.62-0.92 hydroxypropyls per glucose unit, such as about 0.75 hydroxypropyls per glucose unit; such as an average of 0.62-0.84 hydroxy propyls per glucose unit; such as about 0.4- 0.75 hydroxy propyls per glucose unit; such as an average of 0.62 hydroxypropyls per glucose unit, such as about 0.58-0.68 hydroxy propyls per glucose unit.
  • a surfactant may further increase the physical stability and robustness of a formulation during its manufacture, storage and use as a medicament; for example, preserve the stability of a formulation when it is exposed to air inside a container.
  • the use of surfactants in pharmaceutical formulations is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the method disclosed herein involves adding surfactant to a mixture of an aqueous, surfactant-free excipient solution, a GLP-1 receptor agonist and an amylin receptor agonist.
  • the pharmaceutical formulation disclosed herein necessarily comprises a surfactant.
  • the surfactant utilised in the method disclosed herein may be selected from the group consisting of polysorbate 20 and/or polysorbate 80.
  • the surfactant may be polysorbate 20.
  • the surfactant may be polysorbate 80.
  • the pharmaceutical formulation may comprise 0.01 mg/ml or more polysorbate 20 and up to 2.0, such as up to 1 .5 mg/ml polysorbate 20, such as about 0.01-1 .0 mg/ml polysorbate 20, such as about 0.05 mg/ml polysorbate 20.
  • the pharmaceutical formulation may comprise 0.01 mg/ml or more polysorbate 80 and up to 2.0, such as up to 1 .5 mg/ml polysorbate 80, such as about 0.01-1.0 mg/ml polysorbate 80, such as about 0.05 mg/ml polysorbate 80.
  • the aqueous, surfactant-free excipient solution may comprise one or more further excipients, with the proviso that such further excipient is not a surfactant.
  • the method disclosed herein may comprise adding one or more further excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
  • the method disclosed herein may comprise adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
  • Further excipients may include a buffer and a tonicity agent.
  • the pharmaceutical formulation disclosed herein necessarily comprises any further excipient employed during the method disclosed herein.
  • the pharmaceutical formulation may comprise a buffer.
  • the use of a buffer in pharmaceutical formulations is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the pharmaceutical formulation disclosed herein may comprise a buffer having a pKa close to the desired pH of the solution.
  • the pharmaceutical formulation may comprise a buffer having at least one pKa value of about 5.0-7.0.
  • the pharmaceutical formulation may comprise a buffer having a pKa of about 5.0-7.0.
  • the pharmaceutical formulation may comprise a buffer selected from the group consisting of histidine, citrate and/or phosphate.
  • the buffer may be histidine, citrate and/or phosphate in a total concentration of 3-30 mM.
  • the buffer may be citrate, in a concentration of 3-30 mM.
  • the buffer may be histidine, in a concentration of 3-30 mM.
  • the buffer may be phosphate, in a concentration of 3-30 mM
  • the pharmaceutical formulation may further comprise one or more agents for adjusting pH, such as NaOH and/or HCI.
  • the desired pH of the pharmaceutical formulation may be about 5.5-6.5.
  • the pH of the pharmaceutical formulation is preferably 5.6-6.0.
  • the pH of the pharmaceutical formulation 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.
  • pH may be measured at “room temperature”, typically defined as 15-25°C or 15- 20°C. pH is preferably measured at about 20°C.
  • the pharmaceutical formulation disclosed herein may comprise a tonicity agent.
  • a tonicity agent in pharmaceutical formulations is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the purpose of the tonicity agent is to protect living tissue when the formulation is injected into the body.
  • the tonicity agent may be selected from the group consisting of mannitol, sorbitol or trehalose, or a combination thereof.
  • the tonicity agent is mannitol.
  • the tonicity agent is sorbitol.
  • the tonicity agent is trehalose.
  • the concentration of the tonicity agent is such as to render the formulation isotonic.
  • the tonicity agent is mannitol, it may be present in a concentration of 16.5-37.5 mg/ml, such as about 20 mg/ml.
  • the tonicity agent is sorbitol, it may be present in a concentration of about 10-40 mg/ml; such as about 16.5-37.5 mg/ml; such as about 10-30 mg/ml; such as about 16-28 mg/ml, such as about 16.5-25 mg/ml, such as about 16-26 mg/ml; such as about 16-24 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.
  • the tonicity agent is trehalose, it may be present in a concentration of 33-75 mg/ml, such as about 38
  • compositions disclosed herein are for medical use.
  • the pharmaceutical formulation disclosed herein may be administered by parenteral injection.
  • the pharmaceutical formulation disclosed herein may be administered by subcutaneous injection.
  • treatment refers to the medical therapy of any human or other vertebrate subject in need thereof.
  • Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other vertebrate.
  • the timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject’s health.
  • said treatment may be prophylactic (preventative), palliative, symptomatic and/or curative.
  • the pharmaceutical formulation disclosed herein may be administered to a human subject.
  • the pharmaceutical formulation disclosed herein may be used in:
  • diabetes and associated symptoms such as hyperglycaemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, noninsulin dependent diabetes, maturity onset diabetes of the young (MODY), gestational diabetes and/or for the reduction of HbA1c;
  • diabetes the delaying or prevention of diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulinrequiring type 2 diabetes and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin-requiring type 2 diabetes;
  • ITT impaired glucose tolerance
  • eating disorders such as obesity, e.g. by decreasing food intake, suppressing appetite, inducing satiety, reducing body weight; treating or preventing binge eating disorder, food cravings, bulimia nervosa and/or obesity induced by the administration of an antipsychotic or a steroid; reducing gastric motility; and/or delaying gastric emptying;
  • cardiovascular disease such as the delaying or reduction of the development of a major adverse cardiovascular event (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularisation, hospitalisation for unstable angina pectoris, and hospitalisation for heart failure;
  • MACE major adverse cardiovascular event
  • NASH non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • the indication is (i). In some embodiments the indication is (ii). In a still further particular aspect the indication is (iii). In a still further particular aspect, the indication is (iv). In a still further particular aspect, the indication is (v). In a still further particular aspect, the indication is (vi). In some embodiments, the indication is type 2 diabetes and/or obesity.
  • a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist comprising:
  • a drug substance such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient
  • a drug substance such as a solid DS, comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient
  • a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist comprising:
  • a drug substance such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient
  • a drug substance such as a solid DS comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient
  • a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist comprising:
  • a drug substance such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient
  • a drug substance such as a solid DS, comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient; • preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions; a tonicity agent, such as sorbitol; a buffer, such as histidine; and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
  • a drug substance such as a solid DS, comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient
  • preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions
  • a tonicity agent such as sorbitol
  • a buffer such as histidine
  • WFI water for injection
  • amylin receptor agonist is cagrilintide or a biologically active metabolite or degradation product of cagrilintide.
  • a method of preparing a pharmaceutical formulation comprising cagrilintide and a semaglutide comprising:
  • a drug substance such as a solid DS, comprising cagrilintide as the active pharmaceutical ingredient
  • a drug substance such as a solid DS, comprising semaglutide as the active pharmaceutical ingredient
  • preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50- 80%, such as about 65%, of the final, pre-defined volume; optionally, one or more further excipients, with the proviso that one or more further excipient is not a surfactant; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5; • dissolving the semaglutide DS in the excipient solution;
  • a method of preparing a pharmaceutical formulation comprising cagrilintide and a semaglutide comprising:
  • a drug substance such as a solid DS, comprising cagrilintide as the active pharmaceutical ingredient
  • a drug substance such as a solid DS, comprising semaglutide as the active pharmaceutical ingredient
  • preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions, sorbitol, histidine and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
  • cyclodextrin is of the hydroxypropyl-substituted alpha type comprising six ring-arranged glucose units and/or the hydroxypropyl-substituted beta type comprising seven ring-arranged glucose units.
  • aqueous, surfactant-free excipient solution comprising cyclodextrin comprises one or more further excipients, with the proviso that said further excipient is not a surfactant.
  • aqueous, surfactant-free excipient solution comprising cyclodextrin further comprises a buffer. 17. The method according to any one of the preceding embodiments, further comprising adding a buffer to the mixture of the excipient solution and the GLP-1 receptor agonist.
  • aqueous, surfactant-free excipient solution further comprises a tonicity agent, with the proviso that the tonicity agent is not sodium chloride.
  • liquid pharmaceutical formulation according to embodiment 31 wherein the GLP-1 receptor agonist has an isoelectric point that is incompatible with the optimal pH of the amylin receptor agonist.
  • liquid pharmaceutical formulation according to any one embodiments 30-31 wherein the optimal pH of the GLP-1 receptor agonist and the amylin receptor agonist differs by at least about two pH units, such as 2-5 pH units, such as 2-4 pH units, such as 3-5 pH units.
  • liquid pharmaceutical formulation according to any one embodiments 30-32, wherein the optimal pH of the amylin receptor agonist is 3.5-4.5, such as about 4.0.
  • composition according to any one of embodiments 30-59 comprising at least about 1 mg/ml of said GLP-1 receptor agonist.
  • composition according to any one of embodiments 30-69 comprising an effective amount of cagrilintide and semaglutide.
  • the pharmaceutical formulation according to embodiment 73 comprising mannitol in a concentration of about 16.5-37.5 mg/ml, such as about 20 mg/ml.
  • the pharmaceutical formulation according to embodiment 75 comprising sorbitol in a concentration of about 10-40 mg/ml, such as about 10-30 mg/ml, such as about 16-28 mg/ml, such as about 16.5-37.5 mg/ml, such as about 16.5-25 mg/ml, such as about 16-24 mg/ml, such as about 24 mg/ml, such as about 20 mg/ml, such as about 16 mg/ml, such as about 12 mg/ml.
  • said tonicity agent is trehalose.
  • the pharmaceutical formulation according to embodiment 77 comprising trehalose in a concentration of about 33-75 mg/ml, such as about 33-45 mg/ml, such as about 38 mg/ml.
  • the pharmaceutical formulation according to embodiment 79 comprising a buffer selected from the group consisting of histidine, citrate and/or phosphate.
  • the pharmaceutical formulation according to any one of embodiments 80-81 comprising about 3-30 mM histidine, such as 3-15 mM histidine, such as 3-10 mM histidine, such as about 6 mM histidine.
  • the pharmaceutical formulation according to embodiment 86 comprising a maximum of about 2.0 mg/ml polysorbate 20 and/or polysorbate 80.
  • the pharmaceutical formulation according to embodiment 87 comprising a maximum of about 1 .5 mg/ml polysorbate 20 and/or polysorbate 80.
  • the pharmaceutical formulation according to any one of embodiments 30-90 comprising at least 75% w/w water, such as about 80% w/w water, such as about 85% w/w water, such as up to about 90% w/w water.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide, a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type comprising a minimum of about 0.4 hydroxypropyls per glucose unit and a maximum of about 1.0 hydroxypropyls per glucose unit, histidine, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising 0.58-1.0 hydroxypropyls per glucose unit, histidine, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.92 hydroxypropyls per glucose unit; histidine, sorbitol, polysorbate 20 and/or 80 and about 75- 90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.84 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.68 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide, a hydroxypropyl beta cyclodextrin comprising a maximum of about 0.75 hydroxypropyls per glucose unit, such as about 0.4-0.75 hydroxy propyls per glucose unit, histidine, sorbitol, polysorbate 80 and about 75-90% w/w water; and having a pH of 5.5-6.5.
  • the pharmaceutical formulation according to any one of embodiments 30-91 essentially consisting of: an effective amount of cagrilintide and semaglutide, a hydroxypropyl beta cyclodextrin comprising a maximum of about 0.75 hydroxypropyls per glucose unit, such as about 0.4-0.75 hydroxy propyls per glucose unit, histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
  • the pharmaceutical formulation according to any one of embodiments 30-91 which essentially consists of: an effective amount of cagrilintide and semaglutide, more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type (0.58-1.0 hydroxypropyls per glucose unit), about 3-30 mM histidine, about 10-40 mg/ml sorbitol, up to 2.0 mg/ml polysorbate 20 and/or 80, pH 5.6-6.0, preferably pH 5.8, water for injection.
  • an effective amount of cagrilintide and semaglutide more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type (0.58-1.0 hydroxypropyls per glucose unit)
  • about 3-30 mM histidine about
  • the pharmaceutical formulation according to any one of embodiments 30-91 which essentially consists of: an effective amount of cagrilintide and semaglutide, more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62- 0.84 hydroxypropyls per glucose unit, about 3-30 mM histidine and/or citrate, about 10-40 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.
  • an effective amount of cagrilintide and semaglutide more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62- 0.84 hydroxy
  • EXAMPLE 1 EFFECT OF HYDROXYPROPYL-BETA-CYCLODEXTRIN (HP-B-CD) ON THE CHEMICAL STABILITY OF CAGRILINTIDE
  • HP-B-CD chemically stabilise cagrilintide
  • chemical stability being measured in terms of cagrilintide purity and cagrilintide-related high molecular weight protein (HMWP).
  • HMWP high molecular weight protein
  • Cagrilintide is optimally stable at pH 4.0, the rate of its chemical degradation typically accelerating with an increase in pH. Surprisingly, a stable cagrilintide formulation was obtained at pH 6 when it was formulated with HP-B-CD.
  • compositions of cagrilintide formulations 1 , 2 and 3 are shown in table 1 .
  • Each cagrilintide formulation was prepared by first dissolving the excipients in water and then dissolving cagrilintide drug substance in the excipient solution.
  • the solution was pH adjusted and water was added to reach the final desired volume before being sterilised by filtration through a 0.22 pm sterile filter. After filtration, the formulation was filled in a 1 ml prefilled syringe.
  • Samples were stored at 37°C for up to 21 days. After 14 days and 21 days, samples were analysed to determine the HMWP and cagrilintide purity levels.
  • HMWP size exclusion chromatography
  • T able 2 shows that when cagrilintide was stored at 37°C and at a pH of 4.0, very little HMWP was formed and only a minor decrease in cagrilintide purity was seen. In contrast, when the pH was 6.0 the rate of HMWP formation and decrease in cagrilintide purity accelerated. Surprisingly, this rapid chemical degradation was counteracted by the addition of HP-B-CD to the formulation, making it possible to formulate cagrilintide at pH 6.
  • compositions of semaglutide formulations 1 , 2 and 3 are shown in table 3.
  • ThT Thioflavin T
  • ThT fluorescence assay Two samples were pooled and 1400 pl sample was added to 28 pl 1 mM ThT stock solution, of which 200 pl was then transferred to 6 different wells on a 96 well microtiter plate with a glass bead in.
  • the assay was run with double orbital shaking and a speed of 300 rpm at 40°C for 169 hours on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 450 nm and 480 nm, respectively.
  • the lag time was measured from the start of the experiment until fibrillation occurs, shown as an increase in ThT fluorescence.
  • HP-B-CD chemically stabilise semaglutide, chemical stability being measured in terms of semaglutide purity and semaglutide-related high molecular weight protein (HMWP).
  • HMWP high molecular weight protein
  • HMWP and semaglutide purity were determined after 0 days, 14 days, and 21 days’ storage at 37°C.
  • Semaglutide purity was determined using reversed phase high performance liquid chromatography (RP-HPLC) where the samples were analysed using a Kinetex C18, 2.6 pm column (4.6 x 150 mm) with a gradient elution of eluent A consisting of 90% v/v 0.09 M phosphate solution, pH 3.6 and 10% v/v acetonitrile, and eluent B consisting of 60% v/v acetonitrile and 20% v/v isopropanol. Chromatography was conducted with UV detection (210 nm) at 30°C using a 10-100 pl injection volume and a flow rate of 0.7 ml/min. Purity was quantified as being the area of the main peak divided by the area of all peaks x 100%.
  • RP-HPLC reversed phase high performance liquid chromatography
  • HMWP size exclusion chromatography
  • Samples were analysed using a Waters SEC 1 .7 pm column (4.6 x 150 mm) with an isocratic elution consisting of 300 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 5 mM ortho-phosphate and 50% v/v 2-propanol.
  • Chromatography was conducted with UV detection (280 nm) at 50°C using a 1-10 pl injection volume and a flow rate of 0.3 ml/min.
  • HMWP was quantified as being the area of all components eluting before the main peak divided by the area of the main peak x 100%.
  • EXAMPLE 4 EFFECT OF THE MOLAR SUBSTUTION OF HP-B-CD ON CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATION PHYSICAL STABILITY
  • This example shows the effect of HP-B-CD molar substitution on the physical stability of cagrilintide and semaglutide.
  • compositions of co-formulation 1 and co-formulation 2 are shown in table 6.
  • the number of sub-visible particles present quantifies the physical stability of cagrilintide and semaglutide combined and were obtained by means of micro-flow imaging (MFI, see e.g. Sharma, D.K. et al. AAPS J. (2010), 12: 455-464 for principles of the MFI technique).
  • MFI micro-flow imaging
  • the sample was analysed by standard MFI system settings implying 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 a digital camera (via magnification optics) recorded the contents of the flow cell as bright field images throughout the experiment. Data acquisition was accomplished using Protein Simple MVSS software. The recorded image stream from the entire run was processed by validated Novo Nordisk proprietary software MFI Data Validator whereby the number (normalised to counts per ml analysed liquid) of individual particles was obtained and presented by size; >5 pm, >10 pm, and >25 pm which are standard size ranges for sub-visible particles.
  • the number of particles >5 pm includes all particles greater than 5 pm in diameter (>5 pm, >10 pm and >25 pm) and the number of particles > 10 pm includes all particles greater than 10 pm in diameter (>10 pm and >25 pm).
  • the particle size is defined as the equivalent circular diameter (ECD).
  • the presence of amyloid peptide fibrils was analysed with a Thioflavin-T (ThT) fluorescence assay.
  • the experiment was performed at 25°C.
  • the liquid from each syringe was taken out by first removing the plunger and then pipetting the liquid into the sample container.
  • 500 pl of the sample was mixed with approximately 9pl of ThT stock solution in a separate sample container, to give a final ThT concentration of 20 pM.
  • the sample was left to incubate in the dark for 25 min at ambient temperature. 200 pl sample was transferred to a well in a 96-well microtiter plate. Samples were measured on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 440 nm and 470-550 nm, respectively.
  • Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
  • EXAMPLE 5 EFFECT OF HYDROXYPROPYL-B-CYCLODEXTRIN CONCENTRATION
  • This example shows the concentration-dependent effect of HP-B-CD on the chemical stability of semaglutide.
  • the purity of semaglutide was determined using reversed phase ultra-high performance liquid chromatography (RP-UHPLC), where the samples were analysed using a Waters Acquity phenyl-hexyl 1.7pm column (2.1 x 150mm) with a gradient elution of eluent A consisting of 0.09% TFA in MQ water, and eluent B consisting of 0.09% TFA in MQ water 0.09% TFA in 80% acetonitrile in MQ water. Chromatography was conducted with UV detection (215nm) at 62°C using 2-14pl injection volume and a flow rate of 0.25ml/min. Purity was evaluated as the area of the main peak of semaglutide divided by the area of all related peaks x 100%. Note that, in other experiments, the same method was used to determine cagrilintide purity.
  • EXAMPLE 6 EFFECT OF DIFFERENT TONICITY AGENTS ON CO-FORMULATION PHYSICAL STABILITY
  • This example shows the stabilising effect of different tonicity agents on the physical stability of otherwise identical cagrilintide and semaglutide co-formulations.
  • compositions of co-formulation 6 to co-formulation 12 are shown in in table 10.
  • Results are the mean of 2 replicates and has been rounded to nearest integer value (-) Sampling not performed
  • EXAMPLE 7 EFFECT OF DIFFERENT SURFACTANTS ON CO-FORMULATION PHYSICAL STABILITY
  • This example shows the effect of different surfactants on the physical stability of otherwise identical cagrilintide and semaglutide co-formulations.
  • compositions of co-formulation 13, co-formulation 14, and co-formulation 15 are shown in table 12.
  • Results are the mean of 2 replicates and has been rounded to nearest integer value
  • Co-formulation 14 contained the lowest number of sub-visible particles when stored for 17 days under stressed conditions. In co-formulation 13, containing polysorbate 20, an increase in sub-visible particles was observed after 14 days, while in co-formulation 15 containing poloxamer 188 sub-visible particles are formed after 7 days at stressed conditions. It is evident that the co-formulation containing polysorbate 80 was the most stable and that the co-formulation containing polysorbate 20 was also acceptably stable.
  • EXAMPLE 8 EFFECT OF DIFFERENT BUFFER SUBSTANCES ON CO-FORMULATION PHYSICAL STABILITY
  • This example shows that the buffer substance has an effect on the physical stability of an otherwise identical cagrilintide and semaglutide co-formulation.
  • the number of sub-visible particles was quantified as described in example 4.
  • Results are the mean of 2 replicates and has been rounded to nearest integer value
  • compositions of co-formulation 17 and co-formulation 18 are shown in table 16.
  • EXAMPLE 10 EFFECT OF DIFFERENT BUFFERS CONCENTRATION ON COFORMULATION PHYSICAL STABILITY
  • This example shows the effect of histidine buffer concentration on co-formulation physical stability.
  • compositions of the tested co-formulations are as shown in table 16
  • the number of sub-visible particles was quantified as described in example 4.
  • Results are the mean of 2 replicates and has been rounded to nearest integer value.
  • This example shows the effect of hydroxypropyl-alpha-cyclodextrin (HP-A-CD), hydroxypropyl-beta-cyclodextrin (HP-B-CD) and hydroxypropyl-gamma-cyclodextrin (HP-G- CD) on the formation of sub-visible particles and chemical degradation of cagrilintide in an otherwise identical cagrilintide and semaglutide co-formulation.
  • HP-A-CD hydroxypropyl-alpha-cyclodextrin
  • HP-B-CD hydroxypropyl-beta-cyclodextrin
  • HP-G- CD hydroxypropyl-gamma-cyclodextrin
  • compositions of co-formulation 22, 23 and 24 are shown in table 19.
  • the number of sub-visible particles was determined as described in example 4. Samples used to determine the purity of cagrilintide were stored at 37°C for up to 42 days. Purity of cagrilintide was determined using the following reversed phase high performance liquid chromatography (RP-HPLC) where the samples were analysed using a Kinetex C18, 2.6 pm column (4.6 x 150 mm) with a gradient elution of eluent A consisting of 90% v/v 0.09 M phosphate solution, pH 3.6 and 10% v/v acetonitrile, and eluent B consisting of 60% v/v acetonitrile and 20% v/v isopropanol.
  • RP-HPLC reversed phase high performance liquid chromatography
  • Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
  • HP-A-CD or HP-B-CD is acceptable for coformulations of cagrilintide and semaglutide.
  • HP-B- CD is preferred compared to HP-A-CD for a cagrilintide and semaglutide co-formulation, due to the superior purity of cagrilintide when formulated with HP-B-CD.
  • EXAMPLE 12 EFFECT OF THE MOLAR SUBSTITUTION DEGREE OF HP-B-CD ON THE PHYSICAL AND CHEMICAL STABILITY OF CAGRILINTIDE AND SEMAGLUTIDE COFORMULATIONS
  • This example shows the effect of the molar substitution of HP-B-CD on the formation of sub-visible particles, HMWP level and chemical purity of semaglutide in otherwise identical, citrate-buffered cagrilintide and semaglutide co-formulations.
  • compositions of co-formulations 25 to 32 are shown in table 22.
  • Table 22 Compositions of citrate-buffered cagrilintide and semaglutide co- formulations containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree
  • the number of sub-visibles was quantified as described in example 4.
  • HMWP size exclusion chromatography
  • Samples were analysed using a Waters SEC 1 .7 pm column (4.6 x 150 mm) with an isocratic elution consisting of 185 mM sodium chloride, 5 mM sodium dihydrogen phosphate monohydrate, 3mM ortho-phosphate and 47% (v/v) isopropanol.
  • Chromatography was conducted with UV detection (215 nm) at 50°C using a 1-8 pl injection volume and a flow rate of 0.3 ml/min.
  • HMWP was quantified as being the area of all components eluting before the main peak divided by the area of the main peak x 100%.
  • the histidine-buffered cagrilintide and semaglutide co-formulations 33 to 37, containing 15% w/v HP-B-CD, are preferred due to their superior physical stability.
  • Histidine as buffer and sorbitol as tonicity agent the preferred HP-B-CD molar substitution range was widened to an average of 0.62-0.92 (or a total of 0.58-1 .0).
  • EXAMPLE 13 EFFECT OF MOLAR SUBSTITUTION DEGREE OF HP-B-CD ON PHYSICAL STABILITY OF CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATIONS
  • This example shows the effect of the molar substitution degree of HP-B-CD on the levels of sub-visible particles in otherwise identical, histidine-buffered cagrilintide and semaglutide co-formulations.
  • compositions of co-formulation 33 to 38 are shown in table 26.
  • Table 26 Compositions of histidine-buffered cagrilintide and semaglutide coformulation 33 to 38 containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree
  • the number of sub-visible particles was quantified as described in example 4.
  • Results for number of sub-visible particles are the mean of 3 replicates and have been rounded to nearest integer value
  • compositions of co-formulations 39 and 40 are shown in table 28.
  • Table 28 Composition of co-formulation containing either HP-B-CD or SBE-B-
  • Samples used to determine the number of sub-visible particles were stored at stressed conditions, defined as: - Duration: 35 days
  • the number of sub-visible particles was quantified as described in example 4.
  • Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
  • This example shows the effect of pH on the physical and chemical stability of cagrilintide in otherwise identical cagrilintide and semaglutide co-formulations.
  • compositions of co-formulation 41 to 45 are shown in table 30.
  • the number of sub-visible particles was quantified as described in example 4. Samples used to determine the purity of cagrilintide was stored at 37°C for up to 28 days. The purity of cagrilintide was determined as described in example 14.
  • Table 31 Physical stability of the cagrilintide and semaglutide co-formulations with varying pH within the pH-range 5.5 to 6.0 Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value (-) sampling not performed.
  • Table 32 Chemical purity (%) of cagrilintide in the cagrilintide and semaglutide co-formulations with varying pH within the pH-range 5.5 to 6.0
  • EXAMPLE 16 EFFECT OF CAGRILINTIDE AND SEMAGLUTIDE CONCENTRATION RATIOS ON THE PHYSICAL STABILITY OF THE CO-FORMULATION
  • This example shows the effect of different concentration ratios of cagrilintide and semaglutide on the levels of sub-visible particles observed in the co-formulation.
  • composition of histidine-buffered co-formulations 46 to 50 are shown in table 33, and the composition of histidine-buffered co-formulations 51 to 61 are shown in table 34.
  • Table 33 Composition of histidine-buffered co-formulations with varying cagrilintide and semaglutide concentration ratios
  • Table 34 Composition of histidine-buffered co-formulations with modified composition with varying cagrilintide and semaglutide concentration ratios
  • the number of sub-visible particles was quantified as described in example 4.
  • Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
  • This example shows the effect of HP-B-CD concentration on the physical stability of the cagrilintide and semaglutide co-formulation, when the co-formulation is exposed to physical stress.
  • composition of co-formulation 62 to 65 with the histidine-buffered composition is shown in table 36.
  • results presented in table 37 show that the physical stability of the cagrilintide and semaglutide co-formulation is dependent upon the concentration of HP-B-CD, with lower concentrations resulting in shorter lag time until fibrillation occurs.
  • the co-formulation comprising 7.5% w/v HP-B-CD was the least stable.
  • the co-formulation comprising 15% w/v HP-B-CD was the most stable.
  • EXAMPLE 18 EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE CO- FORMULATION
  • This example shows how the sequence of adding semaglutide and cagrilintide drug substances during co-formulation manufacture affects the co-formulation physical stability in form of subvisible particle counts.
  • the co-formulation was manufactured using formulation either method 1 or formulation method 3 shown in Table 39 and depicted in figures 1 and 3.
  • the number of sub-visible particles was quantified as described in example 4.
  • Results for content of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
  • This example shows the effect, on drug substance dissolution time, of the presence or absence of polysorbate 80.
  • composition of the cagrilintide and semaglutide co-formulation is as shown in Example 18.
  • the co-formulation was manufactured using either formulation method 1 or formulation method 5, shown in Table 41 and depicted in figures 1 and 5.
  • the dissolution time of semaglutide drug substance is affected by the presence of polysorbate 80 in process step 1.
  • the dissolution time of semaglutide drug substance can be significantly reduced by omitting polysorbate 80 from process step 1 and adding it in process step 5 instead (i.e. formulation method 5).
  • EXAMPLE 20 EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTIONS COMPRISING HP-B-CD (AVERAGE MS: 0.62)
  • This example shows how the sequence of adding semaglutide or cagrilintide drug substance first during the co-formulation manufacture affects the drug substance dissolution time.
  • composition of the cagrilintide and semaglutide co-formulation 67 was as shown in table 43.
  • Table 44 Dissolution time of semaglutide drug substance and cagrilintide drug substance in co-formulation 67 comprising HP-B-CD (Average MS: 0.62) manufactured using different formulation methods
  • the dissolution time of semaglutide and cagrilintide drug substance was affected by the sequence of adding the drug substances.
  • the total dissolution time of cagrilintide and semaglutide drug substance can be significantly reduced by adding semaglutide before cagrilintide during manufacture of the cagrilintide and semaglutide co-formulation.
  • EXAMPLE 21 EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.92)
  • composition shows how the sequence of adding semaglutide or cagrilintide drug substance first during the co-formulation manufacture affects the drug substance dissolution time.
  • composition of the cagrilintide and semaglutide co-formulation 68 was as shown in table 45.
  • Table 46 Dissolution time of semaglutide drug substance and cagrilintide drug substance in co-formulation 68 comprising HP-B-CD (Average MS: 0.92) manufactured using different formulation methods
  • the dissolution time of semaglutide and cagrilintide drug substance was affected by the sequence of adding the drug substances when polysorbate 80 is present in the excipient solution.
  • the total dissolution time of cagrilintide and semaglutide drug substance can be significantly reduced by adding semaglutide before adding cagrilintide in formulation method 2. No effect of the sequence of adding drug substances were observed when comparing formulation method 5 and 6 where polysorbate 80 is not present in excipient solution.
  • EXAMPLE 22 EFFECT OF THE SEQUENCE OF ADDING THE EXCIPIENTS ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.62)
  • This example shows how the sequence of adding the excipients during the coformulation manufacture affects the drug substance dissolution time. The effect of each excipient being present in the initial excipient solution contra being added after the addition of drug substances are shown.
  • composition of the cagrilintide and semaglutide co-formulation 67 was as presented in example 20.
  • the dissolution time of both cagrilintide and semaglutide and thereby also the total dissolution time was greatly increase by the presence of polysorbate 80 in the initial excipient solution compared to when polysorbate 80 was added after the dissolution of both drug substances.
  • formulation method 4 where polysorbate 80 was added after semaglutide and before cagrilintide, the dissolution time for semaglutide was reduced, while the dissolution time for cagrilintide was unchanged, compared to that of formulation method 1 .
  • the sequence of adding the other excipients than polysorbate 80 did not affect the dissolution time of cagrilintide and semaglutide.
  • EXAMPLE 23 EFFECT OF THE SEQUENCE OF ADDING THE EXCIPIENTS ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.92)
  • This example shows how the sequence of adding the excipients during the coformulation manufacture affects the drug substance dissolution time. The effect of each excipient being present in the initial excipient solution contra being added after the addition of drug substances are shown.
  • composition of the cagrilintide and semaglutide co-formulation 68 was as presented in example 21.
  • Table 48 Dissolution time for semaglutide drug substance and cagrilintide drug substance in co-formulation 68 comprising HP-B-CD (Average MS: 0.92) manufactured using different formulation methods
  • the dissolution time of cagrilintide and semaglutide drug substance and thereby also the total dissolution time was greatly increased by the presence of polysorbate80 in the initial excipient solution compared to when polysorbate80 is added after the dissolution of drug substance.
  • the sequence of adding the other excipients than polysorbate 80 does not affect the dissolution time of cagrilintide and semaglutide.
  • EXAMPLE 24 EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE COFORMULATION.
  • This example shows how the sequence of adding semaglutide and cagrilintide drug substances during co-formulation manufacture affects the co-formulation physical stability measured as lag time until fibrillation occurs.
  • composition of the cagrilintide and semaglutide co-formulation was shown in example 20.
  • the propensity of cagrilintide and semaglutide in the co-formulation to aggregate and form peptide fibrils was measured using a Thioflavin T (ThT) fluorescence stress assay as described in example 2.
  • Table 49 Physical stability of co-formulation 67 comprising HP-B-CD (Average MS: 0.62) manufactured adding semaglutide before cagrilintide or adding cagrilintide before semaglutide
  • the physical stability of the final cagrilintide and semaglutide co-formulation is affected by the sequence of added the two drug substances. Increased physical stability measured as longer lag time from initiation of the ThT fibrillation assay until fibrillation occurred was achieved by adding semaglutide drug substance before adding the cagrilintide drug substance. This observation was seen regardless of polysorbate 80 being added in the initial excipient solution or after the dissolution of both drug substances.
  • EXAMPLE 25 EFFECT OF WHEN DURING THE FORMULATION METHOD THE IN- PROCESS PH ADJUSTMENT IS PERFORMED ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATION.
  • This example shows the effect of in-process pH adjustment before or after the addition of cagrilintide on co-formulation physical stability, measured as lag time until fibrillation occurs
  • composition of the cagrilintide and semaglutide co-formulation was shown in example 20.
  • the propensity of cagrilintide and semaglutide in the co-formulation to aggregate and form peptide fibrils was measured using a Thioflavin T (ThT) fluorescence stress assay as described in example 2.
  • Results show that the physical stability measured as lag time until fibrillation was increased when the in-process pH adjustment was performed prior to adding cagrilintide in formulation method 3, compared to formulation 2 where pH was adjusted after the addition of cagrilintide.

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Abstract

Disclosed herein is a method of co-formulating an amylin receptor agonist and a GLP-1 receptor agonist. Disclosed herein is a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist and a cyclodextrin comprising hydroxypropyl 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

FORMULATION METHOD
TECHNICAL FIELD
The current invention relates to a method of formulating a pharmaceutical formulation which is a co-formulation of a GLP-1 receptor agonist and an amylin receptor agonist. The pharmaceutical formulation may be used for the medical treatment of subjects with overweight or obesity, with or without one or more associated co-morbidities; diabetes, with or without one or more associated comorbidities; one or more cardiovascular diseases; non-alcoholic steatohepatitis (NASH); and/or cognitive impairment, such as that caused by Alzheimer’s disease.
BACKGROUND
Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist and is the active pharmaceutical ingredient in Ozempic®. Ozempic® is indicated (i) as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus and (ii) to reduce the risk of major adverse cardiovascular events in adults with type 2 diabetes mellitus and established cardiovascular disease.
Semaglutide is also the active pharmaceutical ingredient in Wegovy®. Wegovy® 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 (BMI) of greater or equal to 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 comprising 8 mM phosphate and having a pH of about 7.4.
A fixed-dose combination of an amylin receptor agonist, cagrilintide, and the GLP-1 receptor agonist, semaglutide, has been investigated for the treatment of overweight and obesity (Lancet 2021 ; 397: 1736-48). The drug products investigated were in the form of separate liquid pharmaceutical formulations for subcutaneous use, comprising either cagrilintide or semaglutide.
Thus far, it has not been considered possible to co-formulate semaglutide and cagrilintide, 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 optimal pH of cagrilintide, an amylin receptor agonist. Semaglutide is optimally stable at pH 7.4 and has previously needed to be formulated in a neutral to slightly basic solution of pH 7-8, to ensure its solubility in aqueous solution. Cagrilintide is optimally stable at pH 4.0 and has needed to be formulated in acidic solution, increasing pH accelerating the rate of its chemical degradation. The different physicochemical properties of cagrilintide and semaglutide preclude a simple mixture of these two peptides. The same applies to other GLP-1 receptor agonist and amylin receptor agonist combinations when the two have incompatible optimal pH ranges.
During the manufacture of biopharmaceuticals the therapeutic protein is subjected to physical stress and agitation from several production steps including mixing, filtration and pumping [Yang, M., 2015. Stress and protein instability during formulation and fill/finish processes. BIOPHARM INTERNATIONAL, 28(6), pp.46]. This stress can ultimately lead to protein denaturation or aggregation, which could compromise product quality by causing loss of therapeutic efficacy or increased immunogenicity.
The formulation method by which a protein drug product formulation is manufactured must aim to decrease the total stress the protein is subject to, in order to ensure sufficient physical stability of the product.
There is a need in the art for a simple method of producing a stable pharmaceutical co-formulation comprising a GLP-1 receptor agonist and an amylin receptor agonist.
SUMMARY OF THE INVENTION
Disclosed herein is a method of co-formulating an amylin receptor agonist, such as cagrilintide, and a GLP-1 receptor agonist, such as semaglutide.
Disclosed herein is a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist, such as cagrilintide, and a GLP-1 receptor agonist, such as semaglutide, said method comprising:
• obtaining or preparing a drug substance (DS) comprising an amylin receptor agonist as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS) comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the GLP-1 receptor agonist DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably to about 6.0;
• dissolving the amylin receptor agonist DS in the mixture of the excipient solution and the GLP-1 receptor agonist;
• adding surfactant to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, further adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
DESCRIPTION OF THE DRAWINGS
Figures 1-12 depict formulation methods 1-12, wherein formulation methods 5, 9, and 11 are embodiments of the current invention.
Formulation method 1 :
Figure 1 is a flow diagram depicting formulation method 1.
1 . HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Water for injection was added to the solution to reach 95% of the final volume.
6. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
7. Water for injection was added to reach 100% of the final volume before being sterile filtered and filled into pre-fillable syringes.
Formulation method 2:
Figure 2 is a flow diagram depicting formulation method 2. 1 . HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. Cagrilintide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Semaglutide drug substance was dissolved in the solution.
5. Water for injection was added to the solution to reach 95% of the final volume.
6. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
7. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 3
Figure 3 is a flow diagram depicting formulation method 3.
1 . HP-B-CD, sorbitol, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
3. Cagrilintide drug substance was dissolved in the solution.
4. Semaglutide drug substance was dissolved in the solution.
5. Water for injection was added to the solution to reach 95% of the final volume.
6. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
7. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 4
Figure 4 is a flow diagram depicting formulation method 4.
1 . HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Polysorbate 80 was added and dissolved.
5. Cagrilintide drug substance was dissolved in the solution. 6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 5
Figure 5 is a flow diagram depicting formulation method 5.
1 . HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Polysorbate 80 was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 6
Figure 6 is a flow diagram depicting formulation method 6.
1 . HP-B-CD, sorbitol and histidine was dissolved in water for injection (60% of the final volume).
2. Cagrilintide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Semaglutide drug substance was dissolved in the solution.
5. Polysorbate80 was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes. Formulation method 7
Figure 7 is a flow diagram depicting formulation method 7.
1 . HP-B-CD, sorbitol and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Histidine was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 8
Figure 8 is a flow diagram depicting formulation method 8.
1 . HP-B-CD, histidine and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Sorbitol was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 9
Figure 9 is a flow diagram depicting formulation method 9.
1 . HP-B-CD was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution. 3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Sorbitol, histidine and polysorbate 80 was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 10
Figure 10 is a flow diagram depicting formulation method 10.
1 . HP-B-CD and sorbitol was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Histidine and polysorbate80 was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 11
Figure 11 is a flow diagram depicting formulation method 11 .
1. HP-B-CD and histidine was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Sorbitol and polysorbate 80 was added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide. 8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
Formulation method 12
Figure 12 is a flow diagram depicting formulation method 12.
1 . HP-B-CD and polysorbate 80 was dissolved in water for injection (60% of the final volume).
2. Semaglutide drug substance was dissolved in the solution.
3. pH was adjusted to pH 6.0 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
4. Cagrilintide drug substance was dissolved in the solution.
5. Sorbitol and histidine were added and dissolved.
6. Water for injection was added to the solution to reach 95% of the final volume.
7. pH was adjusted to pH 5.8 using 0.05N hydrochloride acid and/or 0.05N sodium hydroxide.
8. Water for injection was added to reach 100% of the final volume the formulation before being sterile filtered and filled into pre-fillable syringes.
DESCRIPTION
The current invention is a method of formulating a liquid pharmaceutical formulation comprising an amylin receptor agonist, a GLP-1 receptor agonist and a cyclodextrin comprising hydroxypropyl substitutions.
Application of this method is simple and improves the stability of the active pharmaceutical ingredients, namely the GLP-1 receptor agonist and the amylin receptor agonist.
Disclosed herein is a method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, said method comprising:
• obtaining or preparing a drug substance (DS) comprising an amylin receptor agonist as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS) comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the GLP-1 receptor agonist DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably to about 6.0;
• dissolving the amylin receptor agonist DS in the mixture of the excipient solution and the GLP-1 receptor agonist;
• adding surfactant to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist;
• adding water to about 80-99% of the final, pre-defined volume;
• adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
The GLP-1 receptor agonist may be semaglutide.
The amylin receptor agonist may be cagrilintide.
The cyclodextrin may be of the hydroxypropyl-substituted alpha type, comprising six ring-arranged glucose units, and/or the hydroxypropyl-substituted beta type, comprising seven ring-arranged glucose units.
The aqueous, surfactant-free excipient solution comprising cyclodextrin may comprise one or more further excipients, with the proviso that said one or more further excipients is not a surfactant.
One or more further excipients may be added to the mixture of the aqueous, surfactant-free excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
One or more further excipients may be added to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
In this context, non-limiting examples of “further excipients” are a buffer and a tonicity agent. The further excipient may be a buffer having at least one pKa of about 5.0-7.0, such as histidine, citrate or phosphate, or a combination thereof. The further excipient may be a tonicity agent, with the proviso that the tonicity agent is not sodium chloride.
The concentrations of cyclodextrin, GLP-1 receptor agonist, amylin receptor agonist, surfactant and any further excipient will be such as to arrive at the composition of the pharmaceutical formulation (drug product) also disclosed herein.
Also 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 precludes its co-formulation in the pH range enabling chemical stability of the amylin receptor agonist. Disclosed herein is a means of co-formulating a GLP-1 receptor agonist having an isoelectric point (pl) of less than 6.5, preferably less than 6.0, such as 3.5-6.0, such as 3.0- 5.0, such as 4.0-6.0, and an amylin receptor agonist.
The optimal pH of the amylin receptor agonist is the pH at which it is, chemically and physically, most stable. The person skilled in the art can readily determine the amylin receptor agonist’s optimal pH by testing its chemical and physical stability, in an aqueous solution essentially consisting of the amylin receptor agonist, a buffer and water for injection, across the pH range.
The optimal pH of the GLP-1 receptor agonist is the pH at which it is, chemically and physically, most stable. The person skilled in the art can readily determine the GLP-1 receptor agonist’s optimal pH by testing its chemical and physical stability, in an aqueous solution essentially consisting of the GLP-1 receptor agonist, a buffer and water for injection, across the pH range. The physical stability of the GLP-1 receptor agonist may be a reflection of its isoelectric point, which may coincide with the pH where poorest physical stability might be expected.
As will be apparent to the person skilled in the art, the chemical stability and purity of any GLP-1 receptor agonist and/or any amylin receptor agonist in any liquid formulation can be quantified by means of, e.g., reversed phase (ultra) high performance liquid chromatography (RP-UHPLC or RP-HPLC) and/or by measuring the percentage of high molecular weight protein (%HMWP) by means of, e.g., size exclusion chromatography (SEC).
As will be apparent to the person skilled in the art, the physical stability of a GLP-1 receptor agonist and/or any amylin receptor agonist in any liquid formulation can be quantified by measuring particle formation and/or fibrillation by means of micro-flow imaging (MFI) or a Thioflavin T (ThT) fluorescence stress assay, respectively.
Disclosed here is a means of formulating an amylin receptor agonist and a GLP-1 receptor agonist whose optimal pHs differ by at least about two pH units, such as 2-5 pH units, such as 2-4 pH units, such as 3-5 pH units.
The GLP-1 receptor agonist may be semaglutide. The amylin receptor agonist may be cagrilintide or a biologically active metabolite or degradation product of cagrilintide. The composition of the pharmaceutical formulation disclosed herein preserves/improves the chemical and physical stability of the active pharmaceutical ingredients, even when coformulated at pH 5.5-6.5, such as pH 5.6-6.0; preserves the pharmacokinetic profiles of the active pharmaceutical ingredients in terms of their bioavailability and exposure; and exhibits an acceptable local tolerance upon subcutaneous injection.
Production of the drug substances precedes implementation of the formulation method disclosed herein, whose aim is to create the final drug product, a pharmaceutical formulation. Application of the method disclosed herein ensures that the pharmaceutical formulation or “drug product” has an improved shelf-life.
The term “drug substance” refers to the purified active pharmaceutical ingredient. “Drug substance” is well known in the art as referring to “any [substance or] mixture of substances intended to be used in the manufacture of a drug (medicinal) product and that, when used in the production of a drug, becomes an active ingredient of the drug product” (see, for example, EMA ICH Q7a: “Good manufacturing practice for active pharmaceutical ingredients”). It is well known to the person skilled in the art that a drug substance must be essentially free of impurities, to the extent required by regulatory authorities.
The drug substance may be a solid, such as a freeze-dried or spray-dried solid. The amylin receptor agonist drug substance may be a solid. The GLP-1 receptor agonist drug substance may be a solid.
A solid or “dried” form of the purified active pharmaceutical ingredient (or drug substance) may be obtained as described in WO2012/168432. The drug substance may be dried by freeze drying (i.e., lyophilization; see, for example, Williams and Rolli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 :12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
In the context of the present invention, the terms “pharmaceutical formulation”, “coformulation” and “drug product” may herein be used interchangeably to refer to a liquid pharmaceutical formulation comprising a GLP-1 receptor agonist and an amylin 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 an endogenous amylin, such as human amylin.
An amylin receptor agonist may bind to and activate the calcitonin receptor (CTR) and/or the amylin receptors (AMYRs). The latter consist of heterodimers of two components: the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP1- 3) resulting in three possible complexes, AMYR1-3.
Amylin receptor agonists
The pharmaceutical formulations disclosed herein comprise an amylin receptor agonist. An “amylin receptor agonist” may be defined as a chemical entity which is capable of binding to an amylin receptor and is capable of activating or “agonising” it. In the context of the current invention, the amylin receptor agonist is capable of binding to and activating at least the amylin receptor 3 (AMYR3). The amylin receptor agonist may also be capable of agonising the calcitonin receptor, the amylin receptor 1 (AMYR1) and/or the amylin receptor 2 (AMYR2).
Examples of endogenous amylin receptor agonists are human amylin and human calcitonin. Examples of exogenous amylin receptor agonists are cagrilintide and pramlintide (the active pharmaceutical ingredient in Symlin®).
The amylin receptor agonist is capable of activating the amylin receptor; in other words, it is “potent” on the amylin receptor. The in vitro potency of the amylin receptor agonist on amylin receptor 3 may be measured as described in WO/2022129526, Assay 2. The potency of the compound may be described by means of its EC5o value, wherein EC5o represents the concentration of compound upon which 50% of its maximal effect is observed. The lower the EC5o value, the more potent the compound.
When tested as described as described in WO/2022129526, Assay 2, the amylin receptor agonist as disclosed herein may have an EC5o value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 10 pM.
Cagrilintide
The amylin receptor agonist in the pharmaceutical formulation disclosed herein may be cagrilintide or a biologically active metabolite or degradation product of cagrilintide.
Cagrilintide, also known as AM833, is the compound of Example 53 in
WO2012/168432: N-alpha-[(S)-4-Carboxy-4-(19-carboxynonadecanoylamino)butyryl]- [Glu14,Arg17,Pro37]-pramlintide. Cagrilintide may be prepared as described in WO2012/168432, pages 153-155.
Cagrilintide may be in the form of a salt, preferably a pharmaceutically acceptable salt.
A biologically active metabolite or degradation product of cagrilintide may have an aspartate (Asp) in position 21 or 22. A biologically active metabolite or degradation product of cagrilintide may have an iso-aspartate (iso-Asp) in position 21 or 22.
When the potency of cagrilintide was tested using the procedure described in WO/2022129526, Assay 2, cagrilintide had an EC50 value of about 11 pM (WO/2022/129526, Tables 4b and 4c).
The concentration of cagrilintide in the pharmaceutical formulation disclosed herein may be from about 0.25 mg/ml to about 22 mg/ml.
The pharmaceutical formulation disclosed herein may comprise cagrilintide in a concentration of about 0.33-18 mg/ml; such as 0.25-0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5-1 .0 mg/ml, such as about 0.67 mg/ml; such as 1 .0-1 .5 mg/ml, such as about 1.33 mg/ml; such as 1.5-2.0 mg/ml, such as about 1.5 mg/ml; such as 2.0-2.5 mg/ml; such as 2.5-3.0 mg/ml; such as 3.0-3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5-4.0 mg/ml; such as 4.0-5.0 mg/ml; such as 5.0-6.0 mg/ml; such as 6.0-7.0 mg/ml, such as 7.0-8.0 mg/ml, such as 8.0-9.0 mg/ml, such as 9.0-10.0 mg/ml, such as about 9.6 mg/ml; such as 10-11 mg/ml, such as 11.0-12.0 mg/ml, such as 11-13 mg/ml; such as 13-22 mg/ml, such as about 18 mg/ml; such as about 20-22 mg/ml.
The pharmaceutical formulation disclosed herein may comprise no more than 22 mg/ml cagrilintide. The pharmaceutical formulation disclosed herein may comprise no more than 12 mg/ml cagrilintide.
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 agonist
The pharmaceutical formulations disclosed herein comprise a GLP-1 receptor agonist. A “GLP-1 receptor agonist” may be defined as a ligand which is capable of binding to the GLP-1 receptor and producing a biological response similar to that of the endogenous ligand, glucagon-like peptide 1 (GLP-1 (7-37)). A "full" GLP-1 receptor agonist may be defined as a GLP-1 receptor agonist which is capable of eliciting a biological response of the same magnitude as GLP-1 (7-37).
Examples of exogenous GLP-1 receptor agonists include semaglutide (the active pharmaceutical ingredient in Ozempic®, Rybelsus® and Wegovy®), liraglutide (the active pharmaceutical ingredient in Victoza® and Saxenda®), tirzepatide (the active pharmaceutical ingredient in Mounjaro®) and dulaglutide (the active pharmaceutical ingredient in Trulicity®).
The GLP-1 receptor agonist is capable of binding to and activating, or “agonising” the GLP-1 receptor; in other words, it is “potent” on the GLP-1 receptor. The in vitro potency of the GLP-1 receptor agonist on the GLP-1 receptor may be measured as described in WO/2022/129526, Assay 1 . The potency of the compound may be described by means of its EC5o values, wherein EC50 represents the concentration of compound upon which 50% of its maximal effect is observed. The lower the EC50 value, the more potent the compound.
When tested as described in WO/2022/129526, Assay 1 , the GLP-1 receptor agonist disclosed herein may have an EC50 value of less than 300 pM, such as less than 200 pM, such as less than 150 pM, preferably less than 100 pM, such as less than 75 pM, even more preferably less than 50 pM, such as less than 40 pM, such as less than 30 pM, such as less than 20 pM, such as less than 10 pM.
Semaglutide
Semaglutide is a GLP-1 receptor agonist also known as N626-{18-[N-(17- carboxyheptadecanoyl)-L-y-glutamyl]-10-oxo-3,6, 12, 15-tetraoxa-9, 18-diazaoctadecanoyl}-[8- (2-amino-2-propanoic acid),34-L-arginine]human glucagon-like peptide 1 (7-37). Semaglutide was described in W02006/097537 and in J. Med. Chem. 2015, 58, 18, 7370-7380 and may be manufactured using methods well known to the person skilled in the art, such as that briefly described in W02006/097537, Example 4.
Semaglutide may be present in the current pharmaceutical formulation in its fully or partly ionised form; for example one or more carboxylic acid groups (-COOH) may be deprotonated into the carboxylate group (-COO j and/or one or more amino groups (-NH2) may be protonated into the -NH3 + group.
Semaglutide may be in the form of a salt, preferably a pharmaceutically acceptable salt.
When the potency of semaglutide was tested according to the procedure described in WO/2022/129526, Assay 1 , semaglutide had an EC50 value of about 5.5 pM (see WO/2022/129526, Tables 4b and 4c). The concentration of semaglutide in the pharmaceutical formulation disclosed herein may be from about 0.25 mg/ml to about 22 mg/ml.
The pharmaceutical formulation may comprise semaglutide in a concentration of about 0.33-18 mg/ml; such as 0.25-0.5 mg/ml, such as about 0.33 mg/ml; such as 0.5-1.0 mg/ml, such as about 0.67 mg/ml; such as 1.0-1.5 mg/ml, such as about 1.33 mg/ml; such as 1 .5-2.0 mg/ml, such as about 1 .5 mg/ml; such as 2.0-2.5 mg/ml; such as about 2.2 mg/ml, such as 2.5-3.0 mg/ml; such as 3.0-3.5 mg/ml; such as about 3.2 mg/ml; such as 3.5-4.0 mg/ml; such as 4.0-5.0 mg/ml; such as about 4.8 mg/ml; such as 5.0-6.0 mg/ml; such as 6.0- 7.0 mg/ml, such as about 6.4 mg/ml; such as 7.0-8.0 mg/ml, such as about 8.0 mg/ml; such as 8.0-9.0 mg/ml, such as 9.0-10.0 mg/ml, such as about 9.6 mg/ml; such as 10-11 mg/ml, such as about 10.7 mg/ml; such as 11.0-12.0 mg/ml, such as 11-13 mg/ml; such as about 12.8 mg/ml; such as 13-22 mg/ml, such as about 16 mg/ml; such as about 18 mg/ml; such as about 20-22 mg/ml.
The pharmaceutical formulation disclosed herein may comprise no more than 22 mg/ml semaglutide. The pharmaceutical formulation disclosed herein may comprise no more than 12 mg/ml semaglutide.
Methods of manufacture of the drug substance(s)
The GLP-1 receptor agonist and/or amylin receptor agonist in the pharmaceutical formulation disclosed herein may also, for instance, be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry, or other well established techniques, see e.g. Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Dbrwald, “Organic Synthesis on Solid Phase”, Wiley-VCH Verlag GmbH, 2000; and “Fmoc Solid Phase Peptide Synthesis”, Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.
Alternatively, the compounds may be produced by recombinant methods, e.g. by culturing a host cell containing a DNA sequence encoding the peptide sequence and capable of expressing the peptide, in a suitable nutrient medium under conditions permitting the expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides are Escherichia coli, Saccharomyces cerevisiae and mammalian BHK or CHO cell lines.
Isoelectric point
The isoelectric point (pl) of a molecule is the pH at which the molecule carries no net charge. The pl of a peptide may be theoretically calculated from the pK values of its amino acids and of the terminal amine and carboxyl groups and can be used to predict the solubility of the peptide at a given pH.
The theoretically calculated isoelectric point of the GLP-1 receptor agonist may be in the range of 3.5-6.5, such as 3.5-6.0, such as 4.0-6.0, such as 3.8-4.9, such as 4.0-4.5. Semaglutide has a theoretically calculated isoelectric point of about 4.37.
The theoretically calculated isoelectric point of the amylin receptor agonist may have an isoelectric point (pl) in the range of 8-12, such as 8-9. Cagrilintide has a theoretically calculated isoelectric point of about 8.56.
The method disclosed herein involves preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI).
The excipient solution is an aqueous solution, comprising water for injection (WFI). Application of the method disclosed herein ultimately results in the pharmaceutical formulation disclosed herein. The pharmaceutical formulation may comprise 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 excipient solution may further comprise one or more further excipients, with the proviso that such further excipient is not a surfactant. A buffer may be such a further excipient. A tonicity agent may be such a further excipient.
The aqueous, surfactant-free excipient solution utilised in the formulation method described herein comprises a cyclodextrin comprising hydroxypropyl substitutions.
Application of the method disclosed herein ultimately results in the pharmaceutical formulation disclosed herein. Therefore, the pharmaceutical formulation disclosed herein necessarily comprises a cyclodextrin comprising hydroxypropyl substitutions.
The amounts recited below relate to the pharmaceutical formulation.
The pharmaceutical formulation may comprise about 10-25% w/v of a cyclodextrin comprising hydroxypropyl substitutions. The pharmaceutical formulation may comprise more than 10% w/v of a cyclodextrin comprising hydroxypropyl substitutions. The pharmaceutical formulation may comprise less than 22% w/v of a cyclodextrin comprising hydroxypropyl substitutions. The pharmaceutical formulation may comprise about 10-20% w/v, about 15- 25% w/v, about 12-18% w/v, about 10-17.5% w/v, about 11.25-15%, such as about 15% w/v of a cyclodextrin comprising hydroxypropyl substitutions.
Cyclodextrins are oligosaccharide starch derivatives consisting of 6, 7 or 8 a-(1 ,4)- linked glucopyranose (glucose) units arranged cyclically and denoted the alpha, beta or gamma type, respectively. The cyclodextrins have a wide range of applications, amongst others as pharmaceutical excipients [P. Breen & S. S. Jambhekar, Cyclodextrins in pharmaceutical formulations II: solubilization, binding constant, and complexation efficiency, Drug Discovery Today, Volume 21 , Number 2 February 2016], Guidelines on their use as pharmaceutical excipients have been described by the European Medicines Agency [Background review for cyclodextrins used as excipients, 2014, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)], [Cyclodextrins used as excipients, 2017, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)]. Cyclodextrin types that do not carry hydrophilic substitutions have poor solubility and are rarely used for parenteral drug products.
In order to improve the solubility of cyclodextrins, the hydroxyl groups of the glucose units of the cyclodextrins may be substituted by a varying number of hydrophilic chemical substitutions, e.g., by hydroxypropyl groups, leading to differences in degree of substitution which can be described as either the average number of hydroxypropyl per cyclodextrin molecule (abbreviated DS) or the molar substitution degree corresponding to the average number of hydroxypropyl per glucose units present in the cyclodextrin in question (abbreviated MS). The value of hydroxypropyl per cyclodextrin can be achieved by multiplication of the molar substitution degree by the number of glucose units comprised in the cyclodextrin in question. Difference in degree of substitution can result in alterations in physicochemical properties such as surface activity and complexing abilities. The hydroxyl groups may also be chemically substituted by groups of sulfobutylether. These mostly hydrophilic modifications have yielded cyclodextrin derivates highly suitable for parenteral administration [Cyclodextrins used as excipients, 2017, EMA/CHMP/333892/2013, Committee for Human Medicinal Products (CHMP)]. Cyclodextrins comprising hydroxypropyl substitutions are commonly abbreviated HP-CDs whereas cyclodextrins comprising sulfobutylether substitutions are abbreviated SBE-CDs.
The cyclodextrins comprising hydrophilic substitutions adopt what may be described as cone-liked shapes having a hydrophobic inner cavity and a hydrophilic outer surface formed by the many hydrophilic substitutions capable of forming hydrogen bonds with neighbouring water molecules, thereby improving water solubility [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11], Their hydrophobic microenvironment inside the cavity of these cone-like structures enables them to form drug-to-cyclodextrin complexes mainly through hydrophobic interactions [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11], As a complex is formed between cyclodextrin and a drug molecule carrying one or more hydrophobic regions, these as well as the hydrophobic cavity of cyclodextrin become shielded from water, thereby increasing the solubility of the complex compared to the solubility of the individual constituents. Also, once the complex between cyclodextrin and peptide molecules is formed, it impairs the intermolecular interactions that typically leads to aggregation [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11],
The pharmaceutical formulation disclosed herein preferably comprises a cyclodextrin of the hydroxypropyl-substituted alpha type and/or a cyclodextrin of the hydroxypropyl-substituted beta type.
Unexpectedly, such cyclodextrin carrying hydroxypropyl substitutions was found superior, in its ability to stabilise a co-formulation of cagrilintide and semaglutide, than the same cyclodextrin type carrying sulfobutylether substitutions.
The pharmaceutical formulation disclosed herein may comprise a cyclodextrin of the hydroxypropyl-substituted alpha type, comprising six ring-arranged glucose units. The hydroxypropyl substituted cyclodextrin of the alpha type is abbreviated HP-A-CD. Hydroxypropyl-alpha-cyclodextrins (CAS: 128446-33-3/99241-24-4) are commercially available, with an average molar substitution (MS) of 0.8 and a molar substitution range of 0.5-0.9.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl- alpha-cyclodextrin having a minimum of about 0.4 hydroxypropyls per glucose unit. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-alpha-cyclodextrin having a maximum of about 1.0 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl- alpha-cyclodextrin having a molar substitution range of 0.5-0.9 hydroxy propyls per glucose unit. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-alpha- cyclodextrin having an average molar substitution (MS) of about 0.8 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise a cyclodextrin of the hydroxypropyl-substituted beta type, comprising seven ring-arranged glucose units. The hydroxypropyl substituted cyclodextrin of the beta type is abbreviated HP-B-CD. Hydroxypropyl-beta-cyclodextrins are well known pharmaceutical excipients, typically used in small molecule pharmaceutical formulations, primarily to increase solubility and bioavailability [T. Loftsson, Cyclodextrins in Parenteral Formulations, Journal of Pharmaceutical Sciences, 2020, 1-11], Thus far, the use of cyclodextrins and cyclodextrin substituted derivatives in protein and peptide-based pharmaceutical formulations is limited.
The commercially available hydroxypropyl substitution degrees (DS) for hydroxypropyl-beta-cyclodextrins as pharmaceutical excipients ranges between 2.8 and 10.5 according to the European and US pharmacopoeia [USP 38 NF 33, Pharm Eur 8, as estimated by methods described in USP <761 > /Pharm. Eur. 2.2.33], corresponding to 0.4- 1.5 hydroxypropyl per glucose unit (MS). Commercially available cyclodextrins such as hydroxypropyl-beta-cyclodextrins are usually described by means of the average molar substitutions (MS) of their molar substitution ranges.
Hydroxypropyl-beta-cyclodextrins (CAS: 128446-35-5/94035-02-6) are commercially available for use as excipients, with average molar substitutions (MS) including: MS 0.62, with a molar substitution range of 0.58-0.68; MS 0.67, with a molar substitution range from (0.6-0.9); MS 0.68, with a molar substitution range from (0.58-0.72); MS 0.84, with a molar substitution range from (0.8-1.0); MS 0.92, with a molar substitution range from (0.81-0.99); MS 1 .08, with a molar substitution range from (0.86-1 .14); each value describing the number of hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having a minimum of about 0.4 hydroxy propyls per glucose unit. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having a maximum of about 1.0 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having a molar substitution range of 0.58-1.0 hydroxy propyls per glucose unit. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) range of about 0.62-0.92 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.62-0.84 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having about 0.4-0.75 hydroxy propyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having about 0.75 hydroxypropyls per glucose unit. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.62. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.58-0.68 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.68. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.58-0.72 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.67. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.6-0.9 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.84. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.8-1.0 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta- cyclodextrin having an average molar substitution (MS) of about 0.92. The pharmaceutical formulation disclosed herein may comprise hydroxypropyl-beta-cyclodextrin having about 0.81-0.99 hydroxypropyls per glucose unit.
The pharmaceutical formulation disclosed herein may comprise 10-25% w/v, such as more than 10% w/v and less than 22% w/v, such as about 10-20% w/v, such as about 12- 18% w/v, such as about 10-17.5% w/v, such as about 11.25-15%, such as about 15% w/v hydroxypropyl-beta-cyclodextrin having a minimum of about 0.4 hydroxypropyls per glucose unit and a maximum of about 1.0 hydroxypropyls per glucose unit; such as an average of 0.62-0.92 hydroxypropyls per glucose unit, such as about 0.75 hydroxypropyls per glucose unit; such as an average of 0.62-0.84 hydroxy propyls per glucose unit; such as about 0.4- 0.75 hydroxy propyls per glucose unit; such as an average of 0.62 hydroxypropyls per glucose unit, such as about 0.58-0.68 hydroxy propyls per glucose unit.
Surfactant
A surfactant may further increase the physical stability and robustness of a formulation during its manufacture, storage and use as a medicament; for example, preserve the stability of a formulation when it is exposed to air inside a container. The use of surfactants in pharmaceutical formulations is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.
The method disclosed herein involves adding surfactant to a mixture of an aqueous, surfactant-free excipient solution, a GLP-1 receptor agonist and an amylin receptor agonist.
Application of the method disclosed herein ultimately results in the pharmaceutical formulation disclosed herein. Therefore, the pharmaceutical formulation disclosed herein necessarily comprises a surfactant.
The surfactant utilised in the method disclosed herein may be selected from the group consisting of polysorbate 20 and/or polysorbate 80. The surfactant may be polysorbate 20. The surfactant may be polysorbate 80.
The amounts recited below relate to the pharmaceutical formulation.
The pharmaceutical formulation may comprise 0.01 mg/ml or more polysorbate 20 and up to 2.0, such as up to 1 .5 mg/ml polysorbate 20, such as about 0.01-1 .0 mg/ml polysorbate 20, such as about 0.05 mg/ml polysorbate 20.
The pharmaceutical formulation may comprise 0.01 mg/ml or more polysorbate 80 and up to 2.0, such as up to 1 .5 mg/ml polysorbate 80, such as about 0.01-1.0 mg/ml polysorbate 80, such as about 0.05 mg/ml polysorbate 80.
Further excipients
The aqueous, surfactant-free excipient solution may comprise one or more further excipients, with the proviso that such further excipient is not a surfactant.
The method disclosed herein may comprise adding one or more further excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
The method disclosed herein may comprise adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
Further excipients may include a buffer and a tonicity agent.
Application of the method disclosed herein ultimately results in the pharmaceutical formulation disclosed herein. Therefore, the pharmaceutical formulation disclosed herein necessarily comprises any further excipient employed during the method disclosed herein.
The pharmaceutical formulation may comprise a buffer. The use of a buffer in pharmaceutical formulations is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000. The pharmaceutical formulation disclosed herein may comprise a buffer having a pKa close to the desired pH of the solution. The pharmaceutical formulation may comprise a buffer having at least one pKa value of about 5.0-7.0. The pharmaceutical formulation may comprise a buffer having a pKa of about 5.0-7.0. The pharmaceutical formulation may comprise a buffer selected from the group consisting of histidine, citrate and/or phosphate. The buffer may be histidine, citrate and/or phosphate in a total concentration of 3-30 mM. The buffer may be citrate, in a concentration of 3-30 mM. The buffer may be histidine, in a concentration of 3-30 mM. The buffer may be phosphate, in a concentration of 3-30 mM.
The pharmaceutical formulation may further comprise one or more agents for adjusting pH, such as NaOH and/or HCI.
The desired pH of the pharmaceutical formulation may be about 5.5-6.5. The pH of the pharmaceutical formulation is preferably 5.6-6.0. The pH of the pharmaceutical formulation 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. pH may be measured at “room temperature”, typically defined as 15-25°C or 15- 20°C. pH is preferably measured at about 20°C.
The pharmaceutical formulation disclosed herein may comprise a tonicity agent. The use of a tonicity agent in pharmaceutical formulations is well-known to the skilled person. 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 may be selected from the group consisting of mannitol, sorbitol or trehalose, or a combination thereof. In some embodiments, the tonicity agent is mannitol. In some embodiments, the tonicity agent is sorbitol. In some embodiments, the tonicity agent is trehalose.
The concentration of the tonicity agent is such as to render the formulation isotonic. Where the tonicity agent is mannitol, it may be present in a concentration of 16.5-37.5 mg/ml, such as about 20 mg/ml. Where the tonicity agent is sorbitol, it may be present in a concentration of about 10-40 mg/ml; such as about 16.5-37.5 mg/ml; such as about 10-30 mg/ml; such as about 16-28 mg/ml, such as about 16.5-25 mg/ml, such as about 16-26 mg/ml; such as about 16-24 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. Where the tonicity agent is trehalose, it may be present in a concentration of 33-75 mg/ml, such as about 38 mg/ml. Medical utility of the pharmaceutical formulation
The pharmaceutical formulations disclosed herein are for medical use.
The pharmaceutical formulation disclosed herein may be administered by parenteral injection. The pharmaceutical formulation disclosed herein may be administered by subcutaneous injection.
The term “treatment”, as used herein, refers to the medical therapy of any human or other vertebrate subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other vertebrate. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject’s health. Thus, said treatment may be prophylactic (preventative), palliative, symptomatic and/or curative.
The pharmaceutical formulation disclosed herein may be administered to a human subject.
The pharmaceutical formulation disclosed herein may be used in:
(i) the prevention and/or treatment of any form of diabetes and associated symptoms, such as hyperglycaemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, noninsulin dependent diabetes, maturity onset diabetes of the young (MODY), gestational diabetes and/or for the reduction of HbA1c;
(ii) the delaying or prevention of diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulinrequiring type 2 diabetes and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin-requiring type 2 diabetes;
(iii) the prevention and/or treatment of eating disorders, such as obesity, e.g. by decreasing food intake, suppressing appetite, inducing satiety, reducing body weight; treating or preventing binge eating disorder, food cravings, bulimia nervosa and/or obesity induced by the administration of an antipsychotic or a steroid; reducing gastric motility; and/or delaying gastric emptying;
(iv) the prevention and/or treatment of cardiovascular disease, such as the delaying or reduction of the development of a major adverse cardiovascular event (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularisation, hospitalisation for unstable angina pectoris, and hospitalisation for heart failure; (v) the prevention and/or treatment of non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH);
(vi) the 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 a still further particular aspect the indication is (iii). In a still further particular aspect, the indication is (iv). In a still further particular aspect, the indication is (v). In a still further particular aspect, the indication is (vi). In some embodiments, the indication is type 2 diabetes and/or obesity.
Herein, specific values given in relation to numbers or intervals may be construed as being the specific value or as being the approximate value (such as plus or minus 10, 15 or 20 percent of the specific value, when amounts can be provided by weight; such as plus or minus 0.4, when pH is measured).
Following is a non-limiting list of embodiments of the present invention.
EMBODIMENTS
1. A method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50- 80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5- 8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the GLP-1 receptor agonist DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably to about 6.0;
• dissolving the amylin receptor agonist in the mixture of the excipient solution and the GLP-1 receptor agonist;
• adding surfactant to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist; • optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, further adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
2. A method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions; optionally, one or more further excipients, with the proviso that the one or more further excipient is not a surfactant; and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the GLP-1 receptor agonist DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as about 6.0;
• dissolving the amylin receptor agonist DS in the mixture of the excipient solution and the GLP-1 receptor agonist;
• adding surfactant to the mixture of the GLP-1 receptor agonist DS, the excipient solution and the amylin receptor agonist DS;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
3. A method of preparing a pharmaceutical formulation comprising an amylin receptor agonist and a GLP-1 receptor agonist, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising an amylin receptor agonist as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising a GLP-1 receptor agonist as the active pharmaceutical ingredient; • preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions; a tonicity agent, such as sorbitol; a buffer, such as histidine; and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the GLP-1 receptor agonist DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably to about 6.0;
• dissolving the amylin receptor agonist DS in the mixture of the excipient solution and the GLP-1 receptor agonist DS;
• adding surfactant to the mixture of the excipient solution, the GLP-1 receptor agonist DS and the amylin receptor agonist DS;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, further adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
4. The method according to any one of the preceding embodiments, wherein said GLP-1 receptor agonist is semaglutide.
5. The method according to any one of the preceding embodiments, wherein said amylin receptor agonist is cagrilintide or a biologically active metabolite or degradation product of cagrilintide.
6. A method of preparing a pharmaceutical formulation comprising cagrilintide and a semaglutide, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising cagrilintide as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising semaglutide as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50- 80%, such as about 65%, of the final, pre-defined volume; optionally, one or more further excipients, with the proviso that one or more further excipient is not a surfactant; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5; • dissolving the semaglutide DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably about 6.0;
• dissolving the cagrilintide DS in the mixture of the excipient solution and the semaglutide;
• adding polysorbate 20 and/or polysorbate 80 to the mixture of the excipient solution, the semaglutide and the cagrilintide;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, adjusting the pH to 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to about 5.8;
• adding water to reach the final, pre-defined volume (100%).
7. A method of preparing a pharmaceutical formulation comprising cagrilintide and a semaglutide, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising cagrilintide as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising semaglutide as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions, sorbitol, histidine and water for injection (WFI) to about 50-80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5-8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the semaglutide DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably about 6.0;
• dissolving the cagrilintide DS in the mixture of the excipient solution and the semaglutide DS;
• adding polysorbate 20 and/or polysorbate 80 to the mixture of the semaglutide DS, the excipient solution and the cagrilintide DS;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, adjusting the pH to about 5.5-6.1 , such as 5.7-6.1 , such as 5.7-5.9, such as 5.8;
• adding water to reach the final, pre-defined volume (100%).
8. The method according to any one of the preceding embodiments, wherein said cyclodextrin is of the hydroxypropyl-substituted alpha type comprising six ring-arranged glucose units and/or the hydroxypropyl-substituted beta type comprising seven ring-arranged glucose units.
9. The method according to any one of the preceding embodiments, wherein said cyclodextrin is of the hydroxypropyl-substituted beta type.
10. The method according to any one of the preceding embodiments, wherein said cyclodextrin is of the hydroxypropyl-substituted beta type having a minimum of about 0.4 and a maximum of about 1 .0 hydroxypropyls per glucose unit.
11 . The method according to any one of the preceding embodiments, wherein said cyclodextrin is of the hydroxypropyl-substituted beta type having an average molar substitution (MS) range of about 0.62-0.92 hydroxypropyls per glucose unit, such as an average MS of 0.62, such as an average MS of 0.92.
12. The method according to any one of the preceding embodiments, wherein said aqueous, surfactant-free excipient solution comprising cyclodextrin comprises one or more further excipients, with the proviso that said further excipient is not a surfactant.
13. The method according to any one of the preceding embodiments, comprising adding one or more further excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
14. The method according to any one of the preceding embodiments, comprising adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
15. The method according to any one of the preceding embodiments, comprising adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the surfactant.
16. The method according to any one of the preceding embodiments, wherein said aqueous, surfactant-free excipient solution comprising cyclodextrin further comprises a buffer. 17. The method according to any one of the preceding embodiments, further comprising adding a buffer to the mixture of the excipient solution and the GLP-1 receptor agonist.
18. The method according to any one of the preceding embodiments, further comprising adding a buffer to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
19. The method according to any one of the preceding embodiments, further comprising adding a buffer to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the surfactant.
20. The method according to any one of the preceding embodiments, wherein said buffer has at least one pKa of about 5.0-7.0.
21. The method according to any one of the preceding embodiments, wherein said buffer is histidine, citrate or phosphate, or a combination thereof.
22. The method according to any one of the preceding embodiments, wherein said buffer is histidine.
23. The method according to any one of the preceding embodiments, wherein said surfactant is polysorbate 20 and/or polysorbate 80.
24. The method according to any one of the preceding embodiments, wherein said aqueous, surfactant-free excipient solution further comprises a tonicity agent, with the proviso that the tonicity agent is not sodium chloride.
25. The method according to any one of the preceding embodiments, further comprising adding a tonicity agent to the mixture of the excipient solution and the GLP-1 receptor agonist; with the proviso that said tonicity agent is not sodium chloride.
26. The method according to any one of the preceding embodiments, further comprising adding a tonicity agent to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist. 27. The method according to any one of the preceding embodiments, further comprising adding a tonicity agent to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the surfactant; with the proviso that said tonicity agent is not sodium chloride.
28. The method according to embodiment 27, wherein said tonicity agent is mannitol, sorbitol or trehalose, or a combination thereof.
29. The method according to embodiment 28, wherein said tonicity agent is sorbitol.
30. The liquid pharmaceutical formulation obtained by the method according to any one of the preceding embodiments.
31 . The liquid pharmaceutical formulation according to embodiment 31 , wherein the GLP-1 receptor agonist has an isoelectric point that is incompatible with the optimal pH of the amylin receptor agonist.
32. The liquid pharmaceutical formulation according to any one embodiments 30-31 , wherein the optimal pH of the GLP-1 receptor agonist and the amylin receptor agonist differs by at least about two pH units, such as 2-5 pH units, such as 2-4 pH units, such as 3-5 pH units.
33. The liquid pharmaceutical formulation according to any one embodiments 30-32, wherein the optimal pH of the amylin receptor agonist is 3.5-4.5, such as about 4.0.
34. The pharmaceutical formulation according to any one embodiments 30-33, wherein said amylin receptor agonist is cagrilintide.
35. The pharmaceutical formulation according to any one embodiments 30-34, wherein said GLP-1 receptor agonist has an isoelectric point of less than 6.5, such as less than 6.0, such as 3.5-6.0, such as 3.0-5.0, such as 3.8-4.9.
36. The pharmaceutical formulation according to any one of the preceding embodiments, wherein said GLP-1 receptor agonist is semaglutide. 37. The pharmaceutical formulation according to any one embodiments 30-36, wherein said cyclodextrin is of the hydroxypropyl-substituted alpha type comprising six ring-arranged glucose units and/or the hydroxypropyl-substituted beta type comprising seven ring-arranged glucose units.
38. The pharmaceutical formulation according to any one of embodiments 30-37, wherein said cyclodextrin is of the hydroxypropyl-substituted alpha type comprising six ring- arranged glucose units.
39. The pharmaceutical formulation according to any one of embodiments 30-38, wherein said cyclodextrin is of the hydroxypropyl-substituted beta type comprising seven ring-arranged glucose units.
40. The pharmaceutical formulation according to any one of embodiments 30-39, wherein said cyclodextrin comprises a maximum of about 1 .0 hydroxypropyls per glucose unit.
41 . The pharmaceutical formulation according to any one of embodiments 30-40, wherein said cyclodextrin comprises a maximum of about 0.92 hydroxypropyls per glucose unit.
42. The pharmaceutical formulation according to any one of embodiments 30-41 , wherein said cyclodextrin comprises a maximum of about 0.75 hydroxypropyls per glucose unit.
43. The pharmaceutical formulation according to any one of embodiments 30-42, wherein said cyclodextrin comprises a maximum of about 0.68 hydroxypropyls per glucose unit.
44. The pharmaceutical formulation according to any one of embodiments 30-43, wherein said cyclodextrin comprises a minimum of about 0.4 hydroxypropyls per glucose unit. 45. The pharmaceutical formulation according to any one of embodiments 30-44, wherein said cyclodextrin comprises a minimum of about 0.58 hydroxypropyls per glucose unit.
46. The pharmaceutical formulation according to any one of embodiments 30-45, wherein said cyclodextrin comprises about 0.58-1.0 hydroxy propyls per glucose unit.
47. The pharmaceutical formulation according to any one of embodiments 30-46, wherein said cyclodextrin comprises an average (MS) of 0.62-0.92 hydroxypropyls per glucose unit.
48. The pharmaceutical formulation according to any one of embodiments 30-47, wherein said cyclodextrin comprises an average (MS) of about 0.62-0.84 hydroxypropyls per glucose unit.
49. The pharmaceutical formulation according to any one of embodiments 30-48, wherein said cyclodextrin comprises an average (MS) of about 0.62 hydroxy propyls per glucose unit.
50. The pharmaceutical formulation according to any one of embodiments 30-49, wherein said cyclodextrin comprises about 0.4-0.75 hydroxypropyls per glucose unit, such as about 0.58-0.68 hydroxy propyls per glucose unit.
51 . The pharmaceutical formulation according to any one of embodiments 30-50, comprising about 10-25% w/v cyclodextrin.
52. The pharmaceutical formulation according to any one of embodiments 30-51 , comprising more than 10% w/v cyclodextrin.
53. The pharmaceutical formulation according to any one of embodiments 30-52, comprising less than 22% w/v cyclodextrin.
54. The pharmaceutical formulation according to any one of embodiments 30-53, comprising less than 20% w/v cyclodextrin. 55. The pharmaceutical formulation according to any one of embodiments 30-54, comprising about 10-20% w/v of said cyclodextrin.
56. The pharmaceutical formulation according to any one of embodiments 30-55, comprising about 10-17.5% w/v cyclodextrin.
57. The pharmaceutical formulation according to any one of embodiments 30-56, comprising about 12-18% w/v cyclodextrin.
58. The pharmaceutical formulation according to any one of embodiments 30-57, comprising about 11 .25-15% w/v cyclodextrin.
59. The pharmaceutical formulation according to any one of embodiments 30-58, comprising about 15% w/v cyclodextrin.
60. The pharmaceutical formulation according to any one of embodiments 30-59, comprising at least about 1 mg/ml of said GLP-1 receptor agonist.
61 . The pharmaceutical formulation according to any one of embodiments 30-60, comprising a maximum of about 22 mg/ml of said GLP-1 receptor agonist.
62. The pharmaceutical formulation according to any one of embodiments 30-61 , comprising about 1-12 mg/ml GLP-1 receptor agonist.
63. The pharmaceutical formulation according to any one of embodiments 30-62, wherein said amylin receptor agonist is cagrilintide.
64. The pharmaceutical formulation according to any one of embodiments 30-63, comprising at least about 1 mg/ml of said amylin receptor agonist.
65. The pharmaceutical formulation according to any one of embodiments 30-64, comprising a maximum of about 22 mg/ml amylin receptor agonist.
66. The pharmaceutical formulation according to any one of embodiments 30-65, comprising about 1-12 mg/ml amylin receptor agonist. 67. The pharmaceutical formulation according to any one of embodiments 30-66, comprising 0.25-22 mg/ml cagrilintide.
68. The pharmaceutical formulation according to any one of embodiments 30-67, comprising 0.25-22 mg/ml semaglutide.
69. The pharmaceutical formulation according to any one of embodiments 30-68, comprising 0.25-22 mg/ml cagrilintide and 0.25-22 mg/ml semaglutide.
70. The pharmaceutical formulation according to any one of embodiments 30-69, comprising an effective amount of cagrilintide and semaglutide.
71 . The pharmaceutical formulation according to any one of embodiments 30-70, further comprising a tonicity agent; with the proviso that the tonicity agent is not sodium chloride.
72. The pharmaceutical formulation according to embodiment 71 , wherein said tonicity agent is mannitol, sorbitol or trehalose, or a combination thereof.
73. The pharmaceutical formulation according to embodiment 72, wherein said tonicity agent is mannitol.
74. The pharmaceutical formulation according to embodiment 73, comprising mannitol in a concentration of about 16.5-37.5 mg/ml, such as about 20 mg/ml.
75. The pharmaceutical formulation according to embodiment 72, wherein said tonicity agent is sorbitol.
76. The pharmaceutical formulation according to embodiment 75, comprising sorbitol in a concentration of about 10-40 mg/ml, such as about 10-30 mg/ml, such as about 16-28 mg/ml, such as about 16.5-37.5 mg/ml, such as about 16.5-25 mg/ml, such as about 16-24 mg/ml, such as about 24 mg/ml, such as about 20 mg/ml, such as about 16 mg/ml, such as about 12 mg/ml. 77. The pharmaceutical formulation according to embodiment 72, wherein said tonicity agent is trehalose.
78. The pharmaceutical formulation according to embodiment 77, comprising trehalose in a concentration of about 33-75 mg/ml, such as about 33-45 mg/ml, such as about 38 mg/ml.
79. The pharmaceutical formulation according to any one of embodiments 30-78, further comprising a buffer having at least one pKa of about 5.0-7.0.
80. The pharmaceutical formulation according to embodiment 79, comprising a buffer selected from the group consisting of histidine, citrate and/or phosphate.
81 . The pharmaceutical formulation according to any one of embodiments 79-80, comprising a maximum of 30 mM buffer.
82. The pharmaceutical formulation according to any one of embodiments 80-81 , comprising about 3-30 mM citrate.
83. The pharmaceutical formulation according to any one of embodiments 80-81 , comprising about 3-30 mM histidine, such as 3-15 mM histidine, such as 3-10 mM histidine, such as about 6 mM histidine.
84. The pharmaceutical formulation according to any one of embodiments 80-81 , comprising about 3-30 mM phosphate.
85. The pharmaceutical formulation according to any one of embodiments 30-84, further comprising a surfactant.
86. The pharmaceutical formulation according to embodiment 85, wherein the surfactant is polysorbate 20 and/or polysorbate 80.
87. The pharmaceutical formulation according to embodiment 86, comprising a maximum of about 2.0 mg/ml polysorbate 20 and/or polysorbate 80. 88. The pharmaceutical formulation according to embodiment 87, comprising a maximum of about 1 .5 mg/ml polysorbate 20 and/or polysorbate 80.
89. The pharmaceutical formulation according to embodiment 86, wherein said surfactant is polysorbate 80.
90. The pharmaceutical formulation according to any one of embodiments 30-89, wherein the pH is about 5.5-6.5, preferably 5.6-6.0, such as about 5.7, such as about pH 5.8, such as about 5.9, such as about 6.0.
91 . The pharmaceutical formulation according to any one of embodiments 30-90, comprising at least 75% w/w water, such as about 80% w/w water, such as about 85% w/w water, such as up to about 90% w/w water.
92. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide, a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type comprising a minimum of about 0.4 hydroxypropyls per glucose unit and a maximum of about 1.0 hydroxypropyls per glucose unit, histidine, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
93. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising 0.58-1.0 hydroxypropyls per glucose unit, histidine, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
94. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.92 hydroxypropyls per glucose unit; histidine, sorbitol, polysorbate 20 and/or 80 and about 75- 90% w/w water; and having a pH of 5.6-6.0.
95. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.84 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
96. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62-0.68 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
97. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide; a cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62 hydroxypropyls per glucose unit; histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
98. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide, a hydroxypropyl beta cyclodextrin comprising a maximum of about 0.75 hydroxypropyls per glucose unit, such as about 0.4-0.75 hydroxy propyls per glucose unit, histidine, sorbitol, polysorbate 80 and about 75-90% w/w water; and having a pH of 5.5-6.5.
99. The pharmaceutical formulation according to any one of embodiments 30-91 , essentially consisting of: an effective amount of cagrilintide and semaglutide, a hydroxypropyl beta cyclodextrin comprising a maximum of about 0.75 hydroxypropyls per glucose unit, such as about 0.4-0.75 hydroxy propyls per glucose unit, histidine and/or citrate, sorbitol, polysorbate 20 and/or 80 and about 75-90% w/w water; and having a pH of 5.6-6.0.
100. The pharmaceutical formulation according to any one of embodiments 30-91 , which essentially consists of: an effective amount of cagrilintide and semaglutide, more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type (0.58-1.0 hydroxypropyls per glucose unit), about 3-30 mM histidine, about 10-40 mg/ml sorbitol, up to 2.0 mg/ml polysorbate 20 and/or 80, pH 5.6-6.0, preferably pH 5.8, water for injection.
101 . The pharmaceutical formulation according to any one of embodiments 30-91 , which essentially consists of: an effective amount of cagrilintide and semaglutide, more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type, comprising an average of 0.62- 0.84 hydroxypropyls per glucose unit, about 3-30 mM histidine and/or citrate, about 10-40 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.
102. The pharmaceutical formulation according to any one of embodiments 30-91 , which essentially consists of:
0.25-22 mg/ml cagrilintide,
0.25-22 mg/ml semaglutide, more than 10% w/v and less than 22% w/v, such as 10-20% w/v cyclodextrin of the hydroxypropyl-substituted alpha and/or beta type (0.58-1.0 hydroxypropyls per glucose unit), about 6 mM histidine, about 10-40 mg/ml sorbitol, up to 2.0 mg/ml Polysorbate 20 and/or 80, pH 5.6-6.0, preferably pH 5.8, water for injection. EXAMPLES
EXAMPLE 1 : EFFECT OF HYDROXYPROPYL-BETA-CYCLODEXTRIN (HP-B-CD) ON THE CHEMICAL STABILITY OF CAGRILINTIDE
This example demonstrated the ability of HP-B-CD to chemically stabilise cagrilintide, chemical stability being measured in terms of cagrilintide purity and cagrilintide- related high molecular weight protein (HMWP).
Cagrilintide is optimally stable at pH 4.0, the rate of its chemical degradation typically accelerating with an increase in pH. Surprisingly, a stable cagrilintide formulation was obtained at pH 6 when it was formulated with HP-B-CD.
Composition
The compositions of cagrilintide formulations 1 , 2 and 3 are shown in table 1 .
Table 1 Composition of cagrilintide formulations 1, 2 and 3
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit 2 Different buffer concentrations across formulations are used to ensure buffering at different pH
Preparation process Each cagrilintide formulation was prepared by first dissolving the excipients in water and then dissolving cagrilintide drug substance in the excipient solution. The solution was pH adjusted and water was added to reach the final desired volume before being sterilised by filtration through a 0.22 pm sterile filter. After filtration, the formulation was filled in a 1 ml prefilled syringe.
Methods
Samples were stored at 37°C for up to 21 days. After 14 days and 21 days, samples were analysed to determine the HMWP and cagrilintide purity levels.
Levels of covalently bound HMWP were quantified using size exclusion chromatography (SEC). Samples were analysed using a WATERS HMWP column (7.8 x 300mm) with an isocratic elution consisting of 500 mM sodium chloride, 10 mM sodium dihydrogen phosphate monohydrate, 5 mM ortho-phosphate and 50% (v/v) isopropanol. Chromatography was conducted with UV detection (215 nm) at 50°C using a 10 pl injection volume and a flow rate of 0.5 ml/min. HMWP was quantified as being the area of all components eluting before the main peak divided by the area of the main peak x 100%.
Cagrilintide purity was determined using reversed phase ultra-high performance liquid chromatography (RP-UHPLC). Samples were analysed using a Kinetex C18, 1.7 pm, 100 A, column (2.1 x 150 mm) with a gradient elution of eluent A consisting of 90% v/v 0.09 M phosphate solution, pH 3.6 and 10% v/v acetonitrile, and eluent B consisting of 60% v/v acetonitrile and 20% v/v isopropanol. Chromatography was conducted with UV detection (215 nm) at 60°C using 2-7.5 pl injection volume and a flow rate of 0.25 ml/min. Purity was evaluated as the area of the main peak divided by the area of all peaks x 100%.
Table 2 Chemical purity (%) of cagrilintide at pH 4.0 and 6.0 with and without HP-B-CD
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit Concluding Remarks
T able 2 shows that when cagrilintide was stored at 37°C and at a pH of 4.0, very little HMWP was formed and only a minor decrease in cagrilintide purity was seen. In contrast, when the pH was 6.0 the rate of HMWP formation and decrease in cagrilintide purity accelerated. Surprisingly, this rapid chemical degradation was counteracted by the addition of HP-B-CD to the formulation, making it possible to formulate cagrilintide at pH 6.
EXAMPLE 2: EFFECT OF HP-B-CD ON SEMAGLUTIDE PHYSICAL STABILITY
This example demonstrates the ability of HP-B-CD to physically stabilise semaglutide, which has a propensity to form peptide fibrils. The effect was evident when semaglutide was formulated at suboptimal pH.
Composition
The compositions of semaglutide formulations 1 , 2 and 3 are shown in table 3.
Table 3 Composition of semaglutide formulations 1, 2 and 3
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
2 Different buffer concentrations across formulations are used to ensure buffering at different pH
Preparation process Formulations were prepared as described in example 1.
Methods
The propensity of semaglutide to aggregate and form peptide fibrils, parameters used to quantify physical stability, was measured using a Thioflavin T (ThT) fluorescence stress assay. The analysis for presence of peptide fibrils is based on the fluorescence characteristics of the ThT probe, which displays low fluorescence in the unbound state/native peptide-bound state but high fluorescence when bound to peptide fibrils as well as a red shift in the wavelength of maximum fluorescence upon fibril binding.
Two samples were pooled and 1400 pl sample was added to 28 pl 1 mM ThT stock solution, of which 200 pl was then transferred to 6 different wells on a 96 well microtiter plate with a glass bead in. The assay was run with double orbital shaking and a speed of 300 rpm at 40°C for 169 hours on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 450 nm and 480 nm, respectively. The lag time was measured from the start of the experiment until fibrillation occurs, shown as an increase in ThT fluorescence.
Table 4 Physical stability for semaglutide at pH 6.0 and 7.4
1 Result is the mean of 6 replicates
2 Fibrillation was not observed in any of the 6 replicates within the 169 hours’ duration of the experiment
3 MS: molar substitution, corresponds to hydroxypropyls per glucose unit
Concluding Remarks
The semaglutide formulations were subjected to shear stress-inducing conditions and the propensity of semaglutide to form peptide fibrils was measured. Surprisingly, the presence of HP-B-CD was found to inhibit semaglutide peptide fibril formation. When semaglutide was formulated at pH 6 and in the absence of HP-B-CD (semaglutide formulation 1), fibrillation occurred after 2.35 hours; that is, semaglutide was not physically stable. However, when semaglutide was formulated at pH 6 and in the presence of HP-B-CD (semaglutide formulation 2), no fibrillation was observed throughout the duration of the experiment; that is, semaglutide was physically stable. Furthermore, the physical stability of semaglutide, when formulated at pH 6 and in the presence of HP-B-CD (semaglutide formulation 2), was found comparable to the physical stability of semaglutide when formulated in the absence of HP-B-CD but at its optimal formulation conditions in terms of pH 7.4 (semaglutide formulation 3).
EXAMPLE 3: EFFECT OF HP-B-CD ON SEMAGLUTIDE CHEMICAL STABILITY
This example demonstrated the ability of HP-B-CD to chemically stabilise semaglutide, chemical stability being measured in terms of semaglutide purity and semaglutide-related high molecular weight protein (HMWP).
Composition
The same formulations were used as in example 2.
Preparation process
Formulations were prepared as described in example 1.
Methods
Levels of HMWP and semaglutide purity were determined after 0 days, 14 days, and 21 days’ storage at 37°C.
Semaglutide purity was determined using reversed phase high performance liquid chromatography (RP-HPLC) where the samples were analysed using a Kinetex C18, 2.6 pm column (4.6 x 150 mm) with a gradient elution of eluent A consisting of 90% v/v 0.09 M phosphate solution, pH 3.6 and 10% v/v acetonitrile, and eluent B consisting of 60% v/v acetonitrile and 20% v/v isopropanol. Chromatography was conducted with UV detection (210 nm) at 30°C using a 10-100 pl injection volume and a flow rate of 0.7 ml/min. Purity was quantified as being the area of the main peak divided by the area of all peaks x 100%.
The level of covalently bound HMWP was determined using size exclusion chromatography (SEC). Samples were analysed using a Waters SEC 1 .7 pm column (4.6 x 150 mm) with an isocratic elution consisting of 300 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 5 mM ortho-phosphate and 50% v/v 2-propanol. Chromatography was conducted with UV detection (280 nm) at 50°C using a 1-10 pl injection volume and a flow rate of 0.3 ml/min. HMWP was quantified as being the area of all components eluting before the main peak divided by the area of the main peak x 100%.
Table 5 Semaglutide purity at suboptimal and optimal pH
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit
Concluding Remarks
The results in table 5 show that the chemical purity of semaglutide decreased over time. The chemical purity of semaglutide decreased more rapidly when it was formulated at pH 6.0 (semaglutide formulation 1) than when it was formulated at its optimal pH 7.4 (semaglutide formulation 3). Surprisingly, HP-B-CD improved the chemical stability of semaglutide (in terms of purity decline and HMWP formation) when it was formulated at pH 6.0 (semaglutide formulation 2).
EXAMPLE 4: EFFECT OF THE MOLAR SUBSTUTION OF HP-B-CD ON CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATION PHYSICAL STABILITY
This example shows the effect of HP-B-CD molar substitution on the physical stability of cagrilintide and semaglutide.
Composition
The compositions of co-formulation 1 and co-formulation 2 are shown in table 6.
Table 6 Composition of co-formulation 1 and co-formulation 2
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
All samples were stored at stressed conditions, defined as:
- Duration: 28 days
- Temperature: 30°C ± 2°C
- Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles present quantifies the physical stability of cagrilintide and semaglutide combined and were obtained by means of micro-flow imaging (MFI, see e.g. Sharma, D.K. et al. AAPS J. (2010), 12: 455-464 for principles of the MFI technique). The following procedure was employed for each analysed syringe sample: The experiment was performed at ambient temperature. The liquid from each syringe was taken out by first removing the plunger and then pipetting the liquid into the sample container. The sample was transferred to a 96 deep-well plate which was inserted into the sample handling unit (Bot1) of a Protein Simple MFI™ 5200 apparatus equipped with a standard Protein Simple MFI™ 100 pm flow cell. The sample was analysed by standard MFI system settings implying 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 a digital camera (via magnification optics) recorded the contents of the flow cell as bright field images throughout the experiment. Data acquisition was accomplished using Protein Simple MVSS software. The recorded image stream from the entire run was processed by validated Novo Nordisk proprietary software MFI Data Validator whereby the number (normalised to counts per ml analysed liquid) of individual particles was obtained and presented by size; >5 pm, >10 pm, and >25 pm which are standard size ranges for sub-visible particles. Note that the number of particles >5 pm includes all particles greater than 5 pm in diameter (>5 pm, >10 pm and >25 pm) and the number of particles > 10 pm includes all particles greater than 10 pm in diameter (>10 pm and >25 pm). The particle size is defined as the equivalent circular diameter (ECD).
The presence of amyloid peptide fibrils was analysed with a Thioflavin-T (ThT) fluorescence assay. The experiment was performed at 25°C. The liquid from each syringe was taken out by first removing the plunger and then pipetting the liquid into the sample container. Subsequently 500 pl of the sample was mixed with approximately 9pl of ThT stock solution in a separate sample container, to give a final ThT concentration of 20 pM. The sample was left to incubate in the dark for 25 min at ambient temperature. 200 pl sample was transferred to a well in a 96-well microtiter plate. Samples were measured on a BMG CLARIOstar fluorescence plate reader equipped with monochromators for both excitation and emission using 440 nm and 470-550 nm, respectively.
Data acquisition was accomplished using CLARIOstar Control software. Emission maximum in the present assay was observed to occur at a wavelength of approximately 485 nm; the result for each analysis was therefore reported as the ThT fluorescence at 485 nm, expressed in Relative Fluorescence Units (RFU).
Table 7 Co-formulation physical stability with HP-B-CD with a high or medium molar substitution
Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit
Concluding Remarks
The results in table 7 show that fewest particles were generated in co-formulation 2 containing HP-B-CD (Average MS: 0.62). Furthermore, during the 28 days’ duration of the experiment, no increase in the number of sub-visible particles or ThT fluorescence was seen in the case of co-formulation 2.
In contrast, an increase in sub-visible particle count was seen after 21 days, and an increase in ThT fluorescence was seen after 28 days, in the case of co-formulation 1 containing HP-B-CD (Average MS: 0.92).
In this otherwise identical, citrate-buffered co-formulation of semaglutide and cagrilintide, the co-formulation (2) comprising HP-B-CD (Average MS: 0.62) was more physically stable than the co-formulation (1) comprising HP-B-CD (Average MS: 0.92).
EXAMPLE 5: EFFECT OF HYDROXYPROPYL-B-CYCLODEXTRIN CONCENTRATION
ON SEMAGLUTIDE CHEMICAL STABILITY
This example shows the concentration-dependent effect of HP-B-CD on the chemical stability of semaglutide.
Composition
The composition of co-formulation 3, co-formulation 4, and co-formulation 5 are shown in table 8. Table 8 Composition of co-formulation 3, co-formulation 4, and co-formulation 5
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
2 Different sorbitol concentrations are needed to obtain isotonicity because of the varying HP-
B-CD concentrations tested
Preparation
Formulations were prepared as described in example 1.
Method Samples were stored at 37°C for 28 days at which samples were analysed to determine the chemical purity of semaglutide after 14, 21 , and 28 days.
The purity of semaglutide was determined using reversed phase ultra-high performance liquid chromatography (RP-UHPLC), where the samples were analysed using a Waters Acquity phenyl-hexyl 1.7pm column (2.1 x 150mm) with a gradient elution of eluent A consisting of 0.09% TFA in MQ water, and eluent B consisting of 0.09% TFA in MQ water 0.09% TFA in 80% acetonitrile in MQ water. Chromatography was conducted with UV detection (215nm) at 62°C using 2-14pl injection volume and a flow rate of 0.25ml/min. Purity was evaluated as the area of the main peak of semaglutide divided by the area of all related peaks x 100%. Note that, in other experiments, the same method was used to determine cagrilintide purity.
Table 9 Chemical purity (%) for semaglutide with different HP-B-CD concentrations
Concluding Remarks
The results in table 9 show that the chemical stability and thus purity of semaglutide also depended on HP-B-CD concentration. Semaglutide remained chemically stable in all of the co-formulations (comprising 11.25-15% w/v HP-B-CD). However, semaglutide chemical stability and thus purity was highest when the co-formulation comprised 15% w/v HP-B-CD.
EXAMPLE 6: EFFECT OF DIFFERENT TONICITY AGENTS ON CO-FORMULATION PHYSICAL STABILITY
This example shows the stabilising effect of different tonicity agents on the physical stability of otherwise identical cagrilintide and semaglutide co-formulations.
Composition
The compositions of co-formulation 6 to co-formulation 12 are shown in in table 10.
Table 10 Composition of co-formulation 6 to co-formulation 12
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
All samples were stored for at stressed condition defined as:
• Duration: 18 days
• Temperature: 37°C ± 2°C • Stress condition: During storage, samples were invented 360° to simulate patient use out of refrigerated storage. The rotations were performed 100 times, five days a week. The number of sub-visible particles was quantified as described in example 4.
Table 11 The effect of different tonicity agents on co-formulation physical stability
Results are the mean of 2 replicates and has been rounded to nearest integer value (-) Sampling not performed
1 For co-formulation 12 with NaCI, sampling was discontinued earlier than for the other formulations because of the rapid increase in sub-visible particle counts. Concluding Remarks
The results in table 11 show that the sub-visible particle count increased most rapidly in the co-formulation comprising NaCI as tonicity agent (co-formulation 12). After 7 days, the particle count vastly exceeded the particle count determined for the other coformulations. Therefore, sampling for the analysis of number of sub-visible particles was discontinued for the NaCI-containing co-formulation after 7 days.
After 14 days an increase in sub-visible particle count was seen in the coformulations containing glycerol and sucrose and the two co-formulations were deemed comparable regarding physical stability. The particle count remained lowest in the coformulations that contained mannitol, sorbitol or trehalose. In these co-formulations, virtually no increase in the number of sub-visible particles was seen during the 18 days that the co- formulations were stored at stressed conditions.
Of the co-formulations tested, those comprising mannitol, sorbitol or trehalose as tonicity agent remained the most stable over time.
EXAMPLE 7: EFFECT OF DIFFERENT SURFACTANTS ON CO-FORMULATION PHYSICAL STABILITY
This example shows the effect of different surfactants on the physical stability of otherwise identical cagrilintide and semaglutide co-formulations.
Composition
The compositions of co-formulation 13, co-formulation 14, and co-formulation 15 are shown in table 12.
Table 12 Composition of co-formulation 13, co-formulation 14, and co-formulation 15
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
All samples were stored for at stressed condition defined as:
• Duration: 17 days
• Temperature: 37°C ± 2°C • Stress condition: During storage, samples were invented 360° to simulate patient use out of refrigerated storage. The rotations were performed 100 times, five days every week.
The number of sub-visible particles was quantified as described in example 4. Table 13 The effect of different surfactants on co-formulation physical stability
Results are the mean of 2 replicates and has been rounded to nearest integer value
1 Only one replicate was performed
Concluding Remarks Co-formulation 14 contained the lowest number of sub-visible particles when stored for 17 days under stressed conditions. In co-formulation 13, containing polysorbate 20, an increase in sub-visible particles was observed after 14 days, while in co-formulation 15 containing poloxamer 188 sub-visible particles are formed after 7 days at stressed conditions. It is evident that the co-formulation containing polysorbate 80 was the most stable and that the co-formulation containing polysorbate 20 was also acceptably stable.
EXAMPLE 8: EFFECT OF DIFFERENT BUFFER SUBSTANCES ON CO-FORMULATION PHYSICAL STABILITY
This example shows that the buffer substance has an effect on the physical stability of an otherwise identical cagrilintide and semaglutide co-formulation.
Composition
The composition of co-formulation 1 and co-formulation 16 are shown in table 14.
Preparation process Formulations were prepared as described in example 1 .
Methods
All samples were stored for at stressed condition defined as: o Duration: 21 days o Temperature: 37°C ± 2°C o Stress condition: During storage, samples were invented 360° to simulate patient use out of refrigerated storage. The rotations were performed 100 times, five days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 15 The effect of buffer substance on co-formulation physical stability
Results are the mean of 2 replicates and has been rounded to nearest integer value
Concluding Remarks
Until day 14 of having been stored at stressed conditions, the physical stability of the two co-formulations was similar and acceptable. However, after 18 days, the number of sub- visible particles in the citrate-buffered co-formulation (co-formulation 1) was much greater than that in the histidine-buffered co-formulation (co-formulation 16). The histidine-buffered co-formulation 16 was the most stable. EXAMPLE 9: EFFECT OF DIFFERENT BUFFERS CONCENTRATION ON CO¬
FORMULATION CHEMICAL STABILITY
This example shows the effect of buffer concentration on the chemical stability of otherwise identical co-formulations. Composition
The compositions of co-formulation 17 and co-formulation 18 are shown in table 16.
Table 16 Composition of co-formulation 17 and co-formulation 18
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
Samples were stored at 30°C for 21 days and analysed to determine the chemical purity of cagrilintide after 7, 14, and 21 days. Purity of cagrilintide was determined as described in example 5 (for semaglutide).
Table 17 The effect of buffer concentration on cagrilintide chemical stability in coformulation
Concluding Remarks
The results in table 17 show that both co-formulations were stable. However, the chemical purity of cagrilintide was highest in co-formulation 17. The purity of cagrilintide decreased more rapidly over time when the histidine concentration was 20mM.
EXAMPLE 10: EFFECT OF DIFFERENT BUFFERS CONCENTRATION ON COFORMULATION PHYSICAL STABILITY
This example shows the effect of histidine buffer concentration on co-formulation physical stability.
Composition The compositions of the tested co-formulations are as shown in table 16
Preparation process
Formulations were prepared as described in example 1.
Methods
All samples were stored under stressed conditions, defined as:
• Duration: 18 days
• Temperature: 37°C ± 2°C
• Stress condition: During storage, samples were invented 360° to simulate patient use out of refrigerated storage. The rotations were performed 100 times, five days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 18 The effect of buffer concentration on co-formulation physical stability
Results are the mean of 2 replicates and has been rounded to nearest integer value.
Concluding Remarks
The difference in the physical stability of co-formulations 17 and 18 became most apparent after 14 days. The data in table 18 show that the number of sub-visible particles seen in co-formulation 18 (containing 20 mM histidine) was greater than the number of sub- visible particles seen in co-formulation 17 (containing 6 mM histidine). That is, the coformulation comprising 6 mM histidine was the most physically stable. EXAMPLE 11 : EFFECT OF HYDROXYPROPYL-SUBSTITUTED CYCLODEXTRINS OF VARYING TYPE ON CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATION PHYSICAL AND CHEMICAL STABILITY
This example shows the effect of hydroxypropyl-alpha-cyclodextrin (HP-A-CD), hydroxypropyl-beta-cyclodextrin (HP-B-CD) and hydroxypropyl-gamma-cyclodextrin (HP-G- CD) on the formation of sub-visible particles and chemical degradation of cagrilintide in an otherwise identical cagrilintide and semaglutide co-formulation.
Composition
The compositions of co-formulation 22, 23 and 24 are shown in table 19.
Table 19 Composition of co-formulation 22, 23 and 24
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
Samples used to determine sub-visible particle count were stored at stressed conditions, defined as:
Duration: 42 days
Temperature: 30°C ± 2°C
Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles was determined as described in example 4. Samples used to determine the purity of cagrilintide were stored at 37°C for up to 42 days. Purity of cagrilintide was determined using the following reversed phase high performance liquid chromatography (RP-HPLC) where the samples were analysed using a Kinetex C18, 2.6 pm column (4.6 x 150 mm) with a gradient elution of eluent A consisting of 90% v/v 0.09 M phosphate solution, pH 3.6 and 10% v/v acetonitrile, and eluent B consisting of 60% v/v acetonitrile and 20% v/v isopropanol. Chromatography was conducted with UV detection (210 nm) at 30°C using a 10-100 pl injection volume and a flow rate of 0.7 ml/min. Purity of cagrilintide was quantified as being the area of the main peak divided by the area of all related peaks x 100%.
The same method was used to determine the purity of semaglutide in other experiments.
Table 20 Physical stability of cagrilintide and semaglutide co-formulations formulated with hydroxypropyl cyclodextrins of varying type
Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
(-) Sampling not performed
1 For co-formulation 24 with HP-G-CD, sampling was discontinued earlier than for the other formulations because of rapid increases in particle counts.
Table 21 Chemical purity (%) of cagrilintide in cagrilintide and semaglutide coformulations formulated with hydroxypropyl cyclodextrins of varying type
Concluding Remarks
The results presented in table 20 show that in co-formulation 24 (HP-G-CD), high numbers of sub-visible particles were observed already at time zero, which preclude the use of HP-G-CD to co-formulate cagrilintide and semaglutide. The sampling for the analysis of sub-visible particle counts was discontinued for co-formulation 24 containing HP-G-CD after the initial analysis at time zero. For co-formulation 22 (HP-A-CD) and co-formulation 23 (HP- B-CD) virtually no increase in the number of sub-visible particles was observed.
The results presented in table 21 for the chemical purity of cagrilintide with either HP-A-CD or HP-B-CD, show a slightly more rapid decrease in cagrilintide purity in co- formulation 22, containing HP-A-CD, than in co-formulation 23 containing HP-B-CD.
Based on results in table 20, either HP-A-CD or HP-B-CD is acceptable for coformulations of cagrilintide and semaglutide. However, based on results in table 21 , HP-B- CD is preferred compared to HP-A-CD for a cagrilintide and semaglutide co-formulation, due to the superior purity of cagrilintide when formulated with HP-B-CD.
EXAMPLE 12: EFFECT OF THE MOLAR SUBSTITUTION DEGREE OF HP-B-CD ON THE PHYSICAL AND CHEMICAL STABILITY OF CAGRILINTIDE AND SEMAGLUTIDE COFORMULATIONS
This example shows the effect of the molar substitution of HP-B-CD on the formation of sub-visible particles, HMWP level and chemical purity of semaglutide in otherwise identical, citrate-buffered cagrilintide and semaglutide co-formulations.
Composition
The compositions of co-formulations 25 to 32 are shown in table 22.
Table 22 Compositions of citrate-buffered cagrilintide and semaglutide co- formulations containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
Samples used to determine sub-visible particle count were stored at stressed conditions, defined as:
- Duration: 28 days
- Temperature: 30°C ± 2°C
- Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visibles was quantified as described in example 4.
Samples used to determine the purity of semaglutide and HMWP levels were stored at 37°C for up to 28 days. Purity of semaglutide was determined as in Example 14.
The level of covalently bound HMWP was determined using size exclusion chromatography (SEC). Samples were analysed using a Waters SEC 1 .7 pm column (4.6 x 150 mm) with an isocratic elution consisting of 185 mM sodium chloride, 5 mM sodium dihydrogen phosphate monohydrate, 3mM ortho-phosphate and 47% (v/v) isopropanol. Chromatography was conducted with UV detection (215 nm) at 50°C using a 1-8 pl injection volume and a flow rate of 0.3 ml/min. HMWP was quantified as being the area of all components eluting before the main peak divided by the area of the main peak x 100%.
Table 23 Levels of sub-visible particles in citrate-buffered cagrilintide and semaglutide co-formulations containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit Table 24 Levels of HMWP in citrate-buffered cagrilintide and semaglutide coformulations containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit Table 25 Chemical purity (%) of semaglutide in citrate-buffered cagrilintide and semaglutide co-formulations containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree 1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit
Concluding Remarks
The results presented in tables 23, 24 and 25 show that the physical stability, the formation of HMWP, and the chemical purity of semaglutide is dependent on the molar substitution of the HP-B-CD when studied in a citrate-buffered cagrilintide and semaglutide co-formulation. After 28 days, HMWP levels were lowest and there was virtually no increase in the particle count 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 sub-visible particle count was observed after only 14 days and it was also in this formulation that the highest levels of HMWP were observed after 28 days.
All citrate-buffered cagrilintide and semaglutide co-formulations containing 25% w/v HP-B-CD (Average MS: 0.92 or less), were physically and chemically stable, 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 sub-visible particle count 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, the histidine-buffered cagrilintide and semaglutide co-formulations 33 to 37, containing 15% w/v HP-B-CD, are preferred due to their superior physical stability. With histidine as buffer and sorbitol as tonicity agent, the preferred HP-B-CD molar substitution range was widened to an average of 0.62-0.92 (or a total of 0.58-1 .0).
EXAMPLE 13: EFFECT OF MOLAR SUBSTITUTION DEGREE OF HP-B-CD ON PHYSICAL STABILITY OF CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATIONS
This example shows the effect of the molar substitution degree of HP-B-CD on the levels of sub-visible particles in otherwise identical, histidine-buffered cagrilintide and semaglutide co-formulations.
Composition
The compositions of co-formulation 33 to 38 are shown in table 26. Table 26 Compositions of histidine-buffered cagrilintide and semaglutide coformulation 33 to 38 containing HP-B-CD excipients of varying hydroxypropyl molar substitution degree
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
Samples used to determine the number of sub-visible particles were stored at stressed conditions, defined as:
- Duration: 28 days - Temperature: 30°C ± 2°C
- Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 27 Number of sub-visible particles in histidine-buffered cagrilintide and semaglutide co-formulations containing HP-B-CD of varying hydroxypropyl molar substitution degree
Results for number of sub-visible particles are the mean of 3 replicates and have been rounded to nearest integer value
1 MS: molar substitution, corresponds to hydroxypropyls per glucose unit Concluding Remarks
The results presented in table 27 show that the histidine-buffered co-formulations 33-37, comprising HP-B-CD with a wide range of molar substitutions (Average MS: 0.62- 0.92), remained physically stable for 28 days.
In contrast, in co-formulation 38, comprising HP-B-CD (Average MS 1.08), was not physically stable after 14 days.
The results indicate a synergistic effect between the cyclodextrin and the other excipients in the co-formulation, widening the preferred range of molar substitution (Average MS 0.62-0.92). EXAMPLE 14: EFFECT OF TYPE OF BETA-CYCLODEXTRIN SUBSTITUTION ON THE PHYSICAL STABILITY OF THE CO-FORMULATION
This example shows the effect of sulfobutylether-B-cyclodextrin (SBE-B-CD) and hydroxypropyl-beta-cyclodextrin on the physical stability of otherwise identical cagrilintide and semaglutide co-formulations. Composition
The compositions of co-formulations 39 and 40 are shown in table 28. Table 28 Composition of co-formulation containing either HP-B-CD or SBE-B-
CD
Preparation process Formulations were prepared as described in example 1 .
Methods
Samples used to determine the number of sub-visible particles were stored at stressed conditions, defined as: - Duration: 35 days
- Temperature: 30°C ± 2°C - Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 29 Levels of sub-visible particles in cagrilintide and semaglutide coformulations containing either HP-B-CD or SBE-B-CD
Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
Concluding Remarks
The results in table 29 show that when using SBE-B-CD to co-formulate cagrilintide and semaglutide, a large increase in the number of sub-visible particles is observed after 14 days; that is, the co-formulation is physically unstable. When using a HP-B-CD instead, virtually no increase is observed during the 35 days duration of the study; that is, the coformulation is physically stable.
In contrast to what we have shown for hydroxypropyl-beta-cyclodextrin, these results demonstrate that sulfobutylether-B-cyclodextrin (SBE-B-CD) is not a suitable cyclodextrin to use for co-formulating cagrilintide and semaglutide. EXAMPLE 15: EFFECT OF PH ON THE PHYSICAL AND CHEMICAL STABILITY OF THE CO-FORMULATION
This example shows the effect of pH on the physical and chemical stability of cagrilintide in otherwise identical cagrilintide and semaglutide co-formulations.
Composition
The compositions of co-formulation 41 to 45 are shown in table 30.
Table 30 Composition of co-formulation with varying pH 1 MS: molar substitution, corresponds to hydroxy propyls per g ucose unit
Preparation process
Formulations were prepared as described in example 1. Methods
Samples used to determine the number of sub-visible particles were stored at stressed conditions, defined as:
Duration: 28 days
Temperature: 30°C ± 2°C - Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles was quantified as described in example 4. Samples used to determine the purity of cagrilintide was stored at 37°C for up to 28 days. The purity of cagrilintide was determined as described in example 14.
Table 31 Physical stability of the cagrilintide and semaglutide co-formulations with varying pH within the pH-range 5.5 to 6.0 Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value (-) sampling not performed Table 32 Chemical purity (%) of cagrilintide in the cagrilintide and semaglutide co-formulations with varying pH within the pH-range 5.5 to 6.0
Concluding Remarks
The results presented in tables 31 and 32 show that the physical and chemical stability of semaglutide and cagrilintide depend on the pH of the formulation: the highest pH results in the lowest cagrilintide purity after 28 days at 37°C; the lowest pH results in an increase in the number of sub-visible particles after 14 days. Based on these physical and chemical stability results, the preferred pH range for this particular cagrilintide and semaglutide co-formulation is 5.6-6.0, whilst the pH of 5.5 did not result in a co-formulation of acceptable physical stability.
EXAMPLE 16: EFFECT OF CAGRILINTIDE AND SEMAGLUTIDE CONCENTRATION RATIOS ON THE PHYSICAL STABILITY OF THE CO-FORMULATION
This example shows the effect of different concentration ratios of cagrilintide and semaglutide on the levels of sub-visible particles observed in the co-formulation.
Composition
The composition of histidine-buffered co-formulations 46 to 50 are shown in table 33, and the composition of histidine-buffered co-formulations 51 to 61 are shown in table 34.
Table 33 Composition of histidine-buffered co-formulations with varying cagrilintide and semaglutide concentration ratios
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Table 34 Composition of histidine-buffered co-formulations with modified composition with varying cagrilintide and semaglutide concentration ratios
1 MS: molar substitution, corresponds to hydroxy propyls per g ucose unit
Preparation process
Formulations were prepared as described in example 1.
Methods
Samples used to determine the number of sub-visible particles were stored at stressed conditions, defined as:
Duration: 28 days - Temperature: 30°C ± 2°C
Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times three days every week, and 40 times two days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 35 Levels of sub-visible particles in the co-formulation containing different concentration ratios of cagrilintide and semaglutide
Results for number of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
Concluding Remarks The results presented in table 35 show that after 21 days, virtually no increase in sub-visible particle count was seen in co-formulation 46-60 containing 3.2 mg/ml cagrilintide and up to 12 mg/ml semaglutide.
After 14, an increase in sub-visible particle count was seen for co-formulation 61 containing 3.2 mg/ml cagrilintide and 16 mg/ml semaglutide. All histidine-buffered co-formulations 46 to 61 comprising 3.2 mg/ml cagrilintide and up to 16 mg/ml semaglutide was physically stable. EXAMPLE 17: EFFECT OF CONCENTRATION OF HP-B-CD ON THE PHYSICAL STABILITY OF THE CO-FORMULATION
This example shows the effect of HP-B-CD concentration on the physical stability of the cagrilintide and semaglutide co-formulation, when the co-formulation is exposed to physical stress.
Composition
The composition of co-formulation 62 to 65 with the histidine-buffered composition is shown in table 36.
Table 36 Composition of co-formulation with varying HP-B-CD concentrations
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Preparation process
Formulations were prepared as described in example 1.
Method
The propensity of cagrilintide and semaglutide in the co-formulation to aggregate and form peptide fibrils was measured using a Thioflavin T (ThT) fluorescence stress assay as described in example 2. Table 37 Physical stability for cagrilintide and semaglutide co-formulation with varying HP-B-CD concentrations
1 Result is the mean of 6 replicates
2 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Concluding Remarks
The results presented in table 37 show that the physical stability of the cagrilintide and semaglutide co-formulation is dependent upon the concentration of HP-B-CD, with lower concentrations resulting in shorter lag time until fibrillation occurs. The co-formulation comprising 7.5% w/v HP-B-CD was the least stable. The co-formulation comprising 15% w/v HP-B-CD was the most stable.
EXAMPLE 18: EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE CO- FORMULATION
This example shows how the sequence of adding semaglutide and cagrilintide drug substances during co-formulation manufacture affects the co-formulation physical stability in form of subvisible particle counts.
Composition
The composition of the cagrilintide and semaglutide co-formulation is shown in
Table 38.
Table 38 Composition of co-formulation 66
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Formulation method
The co-formulation was manufactured using formulation either method 1 or formulation method 3 shown in Table 39 and depicted in figures 1 and 3.
Table 39 Co-formulation formulation method
Methods
After manufacture of co-formulation, all samples were stored at stressed condition defined as:
Duration: 6 weeks
Temperature: 30°C ± 2°C
Stress condition: During storage, samples were inverted 360° to simulate patient use out of refrigerated storage. The rotations were performed 20 times for three days, and 40 times for two days every week.
The number of sub-visible particles was quantified as described in example 4.
Table 40 Physical stability of co-formulation manufactured using formulation method 1 or formulation method 3
Results for content of sub-visible particles are the mean of 3 replicates and has been rounded to nearest integer value
Conclusion
The physical stability of the final drug product is affected by the sequence of drug substance addition. The formation of subvisible particles as a function of storage time is minimized in formulation method 1 where semaglutide drug substance is added prior to cagrilintide drug substance. EXAMPLE 19: EFFECT OF POLYSORBATE 80 ON DRUG SUBSTANCE DISSOLUTION TIME
This example shows the effect, on drug substance dissolution time, of the presence or absence of polysorbate 80.
Composition
The composition of the cagrilintide and semaglutide co-formulation is as shown in Example 18. Formulation method
The co-formulation was manufactured using either formulation method 1 or formulation method 5, shown in Table 41 and depicted in figures 1 and 5.
Table 41 Co-formulation formulation method (-) No action is performed at this process step
Methods Excipients were dissolved in water according to the formulation methods shown in Table 41. Once the excipient solution is clear, semaglutide drug substance is added. The time until semaglutide drug substance is fully dissolved and the solution is clear is measured.
The results in Table 42 show a long dissolution time for semaglutide drug substance when formulation method 1 is used, where polysorbate 80 is added to the excipient solution in step 1. In contrast, the semaglutide drug substance dissolution time is significantly reduced when using formulation method 5, where polysorbate 80 is added in step 5 after both drug substances have been added.
Table 42 Semaglutide drug substance dissolution time is dependent on the formulation method
Conclusion
The dissolution time of semaglutide drug substance is affected by the presence of polysorbate 80 in process step 1. The dissolution time of semaglutide drug substance can be significantly reduced by omitting polysorbate 80 from process step 1 and adding it in process step 5 instead (i.e. formulation method 5).
EXAMPLE 20: EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTIONS COMPRISING HP-B-CD (AVERAGE MS: 0.62)
This example shows how the sequence of adding semaglutide or cagrilintide drug substance first during the co-formulation manufacture affects the drug substance dissolution time.
Composition
The composition of the cagrilintide and semaglutide co-formulation 67 was as shown in table 43.
Table 43 Composition of co-formulation 67
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Formulation method
Four different co-formulations with the composition shown in table 43 were manufactured using the formulation methods 1 , 2, 5, and 6 described in the description of the drawings and depicted in Figs 1 , 2, 5 and 6.
Methods
During the manufacture of the co-formulations using the above formulation methods, the time from addition of semaglutide and cagrilintide drug substance until the drug substances were fully dissolved and the solution clear, was measured.
Table 44 Dissolution time of semaglutide drug substance and cagrilintide drug substance in co-formulation 67 comprising HP-B-CD (Average MS: 0.62) manufactured using different formulation methods
Concluding remarks
The dissolution time of semaglutide and cagrilintide drug substance was affected by the sequence of adding the drug substances. The total dissolution time of cagrilintide and semaglutide drug substance can be significantly reduced by adding semaglutide before cagrilintide during manufacture of the cagrilintide and semaglutide co-formulation.
EXAMPLE 21 : EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.92)
This example shows how the sequence of adding semaglutide or cagrilintide drug substance first during the co-formulation manufacture affects the drug substance dissolution time. Composition
The composition of the cagrilintide and semaglutide co-formulation 68 was as shown in table 45.
Table 45 Composition of co-formulation 68
1 MS: molar substitution, corresponds to hydroxy propyls per glucose unit
Formulation method
Four different co-formulations with the composition shown in table 45 were manufactured using the formulation methods 1 , 2, 5, and 6 described in the description of the drawings and depicted in Figs 1 , 2, 5 and 6.
Methods
During the manufacture of the co-formulations using the above formulation methods, the time from addition of semaglutide and cagrilintide drug substance until the drug substances were fully dissolved and the solution clear was measured
Table 46 Dissolution time of semaglutide drug substance and cagrilintide drug substance in co-formulation 68 comprising HP-B-CD (Average MS: 0.92) manufactured using different formulation methods
Concluding remarks
The dissolution time of semaglutide and cagrilintide drug substance was affected by the sequence of adding the drug substances when polysorbate 80 is present in the excipient solution. When comparing formulation method 1 and 2, the total dissolution time of cagrilintide and semaglutide drug substance can be significantly reduced by adding semaglutide before adding cagrilintide in formulation method 2. No effect of the sequence of adding drug substances were observed when comparing formulation method 5 and 6 where polysorbate 80 is not present in excipient solution.
EXAMPLE 22: EFFECT OF THE SEQUENCE OF ADDING THE EXCIPIENTS ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.62)
This example shows how the sequence of adding the excipients during the coformulation manufacture affects the drug substance dissolution time. The effect of each excipient being present in the initial excipient solution contra being added after the addition of drug substances are shown.
Composition
The composition of the cagrilintide and semaglutide co-formulation 67 was as presented in example 20.
Formulation method
Nine different co-formulations with the composition presented in example 20 were manufactured using formulation methods 1 , 4, 5, 7, 8, 9, 10, 11 , and 12, described in the description of the drawings and depicted in Figs 1 , 4, 5, 7, 8, 9, 10, 11 , and 12.
Methods
During the manufacture of the co-formulations using the above formulation methods, the time from addition of semaglutide and cagrilintide drug substance until the drug substances were fully dissolved and the solution clear was measured Table 47 Dissolution time of semaglutide drug substance and cagrilintide drug substance in co-formulation 67 comprising HP-B-CD (Average MS: 0.62) manufactured using different formulation methods
Concluding remarks
The dissolution time of both cagrilintide and semaglutide and thereby also the total dissolution time was greatly increase by the presence of polysorbate 80 in the initial excipient solution compared to when polysorbate 80 was added after the dissolution of both drug substances. In formulation method 4 where polysorbate 80 was added after semaglutide and before cagrilintide, the dissolution time for semaglutide was reduced, while the dissolution time for cagrilintide was unchanged, compared to that of formulation method 1 . The sequence of adding the other excipients than polysorbate 80 did not affect the dissolution time of cagrilintide and semaglutide.
EXAMPLE 23: EFFECT OF THE SEQUENCE OF ADDING THE EXCIPIENTS ON THE DRUG SUBSTANCE DISSOLUTION TIME IN CAGRILINTIDE AND SEMAGLUTIDE COFORMUALTION COMPRISING HP-B-CD (AVERAGE MS: 0.92)
This example shows how the sequence of adding the excipients during the coformulation manufacture affects the drug substance dissolution time. The effect of each excipient being present in the initial excipient solution contra being added after the addition of drug substances are shown.
Composition
The composition of the cagrilintide and semaglutide co-formulation 68 was as presented in example 21.
Formulation method
Eight different co-formulations with the composition presented in example 21 were manufactured using the formulation methods 1 , 5, 7, 8, 9, 10, 11 , and 12 described in the description of the drawings and depicted in Figs 1 , 5, 7, 8, 9, 10, 11 , and 12. Methods
During the manufacture of the co-formulations using the above formulation methods, the time from addition of semaglutide and cagrilintide drug substance until the drug substances were full dissolved and the solution clear was measured
Table 48 Dissolution time for semaglutide drug substance and cagrilintide drug substance in co-formulation 68 comprising HP-B-CD (Average MS: 0.92) manufactured using different formulation methods
Concluding remarks
The dissolution time of cagrilintide and semaglutide drug substance and thereby also the total dissolution time was greatly increased by the presence of polysorbate80 in the initial excipient solution compared to when polysorbate80 is added after the dissolution of drug substance. The sequence of adding the other excipients than polysorbate 80 does not affect the dissolution time of cagrilintide and semaglutide.
EXAMPLE 24: EFFECT OF THE SEQUENCE OF DRUG SUBSTANCE ADDITION ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE COFORMULATION.
This example shows how the sequence of adding semaglutide and cagrilintide drug substances during co-formulation manufacture affects the co-formulation physical stability measured as lag time until fibrillation occurs.
Composition
The composition of the cagrilintide and semaglutide co-formulation was shown in example 20.
Formulation method
Four different co-formulations with the composition shown in example 20 were manufactured using the manufacturing methods 1 , 2, 5, and 6 described in the description of the drawings and depicted in Figs 1 , 2, 5, and 6.
Methods
The propensity of cagrilintide and semaglutide in the co-formulation to aggregate and form peptide fibrils was measured using a Thioflavin T (ThT) fluorescence stress assay as described in example 2.
Table 49 Physical stability of co-formulation 67 comprising HP-B-CD (Average MS: 0.62) manufactured adding semaglutide before cagrilintide or adding cagrilintide before semaglutide
1 Result is the mean of 6 replicates
Concluding remarks
The physical stability of the final cagrilintide and semaglutide co-formulation is affected by the sequence of added the two drug substances. Increased physical stability measured as longer lag time from initiation of the ThT fibrillation assay until fibrillation occurred was achieved by adding semaglutide drug substance before adding the cagrilintide drug substance. This observation was seen regardless of polysorbate 80 being added in the initial excipient solution or after the dissolution of both drug substances.
EXAMPLE 25: EFFECT OF WHEN DURING THE FORMULATION METHOD THE IN- PROCESS PH ADJUSTMENT IS PERFORMED ON THE PHYSICAL STABILITY OF THE CAGRILINTIDE AND SEMAGLUTIDE CO-FORMULATION.
This example shows the effect of in-process pH adjustment before or after the addition of cagrilintide on co-formulation physical stability, measured as lag time until fibrillation occurs
Composition
The composition of the cagrilintide and semaglutide co-formulation was shown in example 20.
Formulation method Two different co-formulations with the composition shown in example 20 were manufactured using the manufacturing methods 2 and 3 described in the description of the drawings and depicted in Figs 2 and 3.
Methods
The propensity of cagrilintide and semaglutide in the co-formulation to aggregate and form peptide fibrils was measured using a Thioflavin T (ThT) fluorescence stress assay as described in example 2.
Table 50 Physical stability of co-formulation 67 comprising HP-B-CD (Average
MS: 0.62) manufactured with pH adjustment before or after the addition of cagrilintide
1 Result is the mean of 6 replicates
Concluding remarks
Results show that the physical stability measured as lag time until fibrillation was increased when the in-process pH adjustment was performed prior to adding cagrilintide in formulation method 3, compared to formulation 2 where pH was adjusted after the addition of cagrilintide.
While 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 is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A method of preparing a pharmaceutical formulation comprising cagrilintide and a semaglutide, comprising:
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising cagrilintide as the active pharmaceutical ingredient;
• obtaining or preparing a drug substance (DS), such as a solid DS, comprising semaglutide as the active pharmaceutical ingredient;
• preparing an aqueous, surfactant-free excipient solution comprising a cyclodextrin comprising hydroxypropyl substitutions and water for injection (WFI) to about 50- 80%, such as about 65%, of the final, pre-defined volume; and having a pH of 6.5- 8.5, such as a pH of about 7.0-8.0, preferably a pH of about 7.5;
• dissolving the semaglutide DS in the excipient solution;
• adjusting the pH to 5.5-6.5, such as to about 5.9-6.1 , preferably about 6.0;
• dissolving the cagrilintide DS in the mixture of the excipient solution and the semaglutide DS;
• adding polysorbate 20 and/or polysorbate 80 to the mixture of the semaglutide DS, the excipient solution and the cagrilintide DS;
• optionally, adding water to about 80-99% of the final, pre-defined volume;
• optionally, adjusting the pH to about 5.5-6.1 , such as to 5.7-6.1 , such as to 5.9-6.1 , such as to 5.7-5.9, such as 5.8;
• adding water to reach the final, pre-defined volume (100%).
2. The method according to any one of the preceding claims, wherein said cyclodextrin is of the hydroxypropyl-substituted alpha type, comprising six ring-arranged glucose units, and/or the hydroxypropyl-substituted beta type, comprising seven ring-arranged glucose units.
3. The method according to any one of the preceding claims, wherein said aqueous, surfactant-free excipient solution comprising cyclodextrin comprises one or more further excipients, with the proviso that said further excipient is not a surfactant.
4. The method according to any one of the preceding claims, comprising adding one or more further excipients to the mixture of the excipient solution and the GLP-1 receptor agonist, with the proviso that said further excipient is not a surfactant.
5. The method according to any one of the preceding claims, comprising adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
6. The method according to any one of the preceding claims, comprising adding one or more further excipients to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the polysorbate 20 and/or polysorbate 80.
7. The method according to any one of the preceding claims, wherein said aqueous, surfactant-free excipient solution comprising cyclodextrin further comprises a buffer.
8. The method according to any one of the preceding claims, further comprising adding a buffer to the mixture of the excipient solution and the GLP-1 receptor agonist.
9. The method according to any one of the preceding claims, further comprising adding a buffer to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist.
10. The method according to any one of the preceding claims, further comprising adding a buffer to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the polysorbate 20 and/or polysorbate 80.
11 . The method according to any one of the preceding claims, wherein said buffer has at least one pKa of about 5.0-7.0; such as histidine, citrate or phosphate, or a combination thereof; preferably, histidine.
12. The method according to any one of the preceding claims, wherein said aqueous, surfactant-free excipient solution further comprises a tonicity agent, with the proviso that the tonicity agent is not sodium chloride.
13. The method according to any one of the preceding claims, further comprising adding a tonicity agent to the mixture of the excipient solution and the GLP-1 receptor agonist; with the proviso that said tonicity agent is not sodium chloride.
14. The method according to any one of the preceding claims, further comprising adding a tonicity agent to the mixture of the excipient solution, the GLP-1 receptor agonist and the amylin receptor agonist; with the proviso that said tonicity agent is not sodium chloride.
15. The method according to any one of the preceding claims, further comprising adding a tonicity agent to the mixture of the excipient solution, the GLP-1 receptor agonist, the amylin receptor agonist and the surfactant; with the proviso that said tonicity agent is not sodium chloride.
EP23716466.0A 2022-03-30 2023-03-30 Formulation method Pending EP4499127A1 (en)

Applications Claiming Priority (7)

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EP22165607 2022-03-30
EP22191564 2022-08-22
PCT/EP2022/085558 WO2023110833A1 (en) 2021-12-13 2022-12-13 Pharmaceutical formulations comprising a cyclodextrin
ARP220103410A AR127945A1 (en) 2021-12-13 2022-12-13 PHARMACEUTICAL FORMULATIONS COMPRISING AN AMYLIN RECEPTOR AGONIST, A GLP-1 RECEPTOR AGONIST AND A CYCLODEXTRIN
TW111147761A TWI876242B (en) 2021-12-13 2022-12-13 Pharmaceutical formulations comprising a cyclodextrin
PK8362022 2022-12-13
PCT/EP2023/058317 WO2023187067A1 (en) 2022-03-30 2023-03-30 Formulation method

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TW202500183A (en) 2023-06-15 2025-01-01 丹麥商諾佛 儂迪克股份有限公司 Pharmaceutical formulations comprising a cyclodextrin
WO2025141472A1 (en) * 2023-12-27 2025-07-03 Alembic Pharmaceuticals Limited Stable pharmaceutical compositions comprising tirzepatide
WO2025172605A1 (en) 2024-02-16 2025-08-21 Adocia Composition comprising a peptide having an isoelectric point of less than 6.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4721760A1 (en) 2024-10-07 2026-04-08 Adocia Composition comprising a peptide having an isoelectric point of less than 6.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
WO2025172606A1 (en) 2024-02-16 2025-08-21 Adocia Composition comprising semaglutide, cagrilintide and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4684802A1 (en) 2024-07-23 2026-01-28 Adocia Composition comprising a peptide having an isoelectric point of less than 6.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4721761A1 (en) 2024-10-07 2026-04-08 Adocia Composition comprising semaglutide, cagrilintide and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4684801A1 (en) 2024-07-23 2026-01-28 Adocia Composition comprising semaglutide, cagrilintide and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4603079A1 (en) 2024-02-16 2025-08-20 Adocia Composition comprising a peptide having an isoelectric point of less than 6.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
EP4603080A1 (en) 2024-02-16 2025-08-20 Adocia Composition comprising semaglutide, cagrilintide and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals
DK182293B1 (en) 2025-01-16 2026-02-25 Novo Nordisk As Semaglutide for use in medical therapy including weight management
CN120305390B (en) * 2025-06-18 2025-10-03 杭州思诺达医药科技有限责任公司 Medicinal preparation for reducing blood sugar

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US8389472B2 (en) * 2005-08-19 2013-03-05 Amylin Pharmaceuticals, Llc Exendin-4 to treat nonalcoholic steatohepatitis and nonalcoholic fatty liver disease
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KR102066987B1 (en) 2011-06-10 2020-01-16 노보 노르디스크 에이/에스 Polypeptides
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TWI850611B (en) 2020-12-18 2024-08-01 丹麥商諾佛 儂迪克股份有限公司 Co-agonists of the glp-1 and amylin receptors

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