Preserved preparation

Abstract

Described herein are preserved formulations of insulin-Fc fusions that have sufficiently long-term pharmacokinetic and pharmacodynamic profiles for once-weekly administration in the treatment of diabetes, and are sufficiently stable to allow storage and use without unacceptable loss of chemical or physical stability.

Description

[Technical field]

[0001] The present invention relates to preserved formulations of insulin-Fc fusions, which contain insulin-Fc fusions with sufficiently long-term pharmacokinetic and pharmacodynamic profiles for once-weekly administration in the treatment of diabetes, and are sufficiently stable to allow storage and use without unacceptable loss of chemical or physical stability. [Background technology]

[0002] Diabetes is a chronic disease characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. Type 1 diabetes (T1D) is characterized by little or no insulin secretory capacity, and T1D patients require insulin therapy for survival. Type 2 diabetes (T2D) is characterized by elevated blood glucose levels due to defective insulin secretion, insulin resistance, excessive hepatic glucose production, and / or all of the above contributing factors. Many T2D patients require insulin therapy as the disease progresses.

[0003] Because T1D patients produce little or no insulin, effective insulin therapy generally involves the use of two types of exogenous insulin: a fast-acting prandial insulin provided by bolus injection, and a long-acting basal insulin administered once or twice daily to control blood glucose levels between meals. Treatment for T2D patients typically begins with prescribed weight loss, exercise, and a diabetic diet, but if these measures fail to control elevated blood glucose, oral medications and incretin-based therapies may be required. If these medications remain insufficient, treatment with insulin is considered. T2D patients whose disease has progressed to the point where they require insulin therapy generally begin daily injections of long-acting basal insulin.

[0004] Currently available basal insulins include insulin glargine, sold under the trade name LANTUS®, insulin detemir, sold under the trade name LEVEMIR®, and insulin degludec, sold under the trade name TRESIBA®. Each of these insulins is indicated for once-daily administration and is available in a preserved formulation with sufficient antimicrobial efficacy to allow multiple dosing from a single container or device.

[0005] Existing insulin therapy treatment regimes involving daily injections can be complex and painful to administer, and can result in undesirable side effects such as hypoglycemia and weight gain. Research is ongoing to develop insulin products that have a longer duration of action and thus require less frequent injections, such as once a week, than currently available insulin products.

[0006] One category of such insulin products contains an insulin receptor activating moiety linked to the Fc region of an antibody, referred to herein as insulin-Fc fusions. Examples of such products are described in U.S. Patent No. 9,855,318, which describes compounds and their formulations, including formulations containing the phenolic preservative m-cresol commonly used in insulin products, including the once-daily basal insulin described above.

[0007] However, it has been found that formulations of insulin-Fc fusions containing preservatives as described in US Patent No. 9,855,318 and / or at the concentrations of currently available insulin products can result in unacceptable stability penalties. Thus, new formulations containing preservatives that provide sufficient antimicrobial efficacy but do not result in unacceptable stability penalties are needed.

[0008] The present invention aims to meet these needs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Thus, in one aspect, the present invention provides an aqueous, sterile pharmaceutical composition comprising: a) an insulin-Fc fusion; b) phenol, c) one or more additional preservatives selected from the group consisting of phenoxyethanol and benzyl alcohol; d) a tonicity agent; and e) a surfactant; and f) a buffer solution, It has a pH of 6 to 7.5. An aqueous, sterile pharmaceutical composition is provided in which phenol and one or more additional preservatives are present in concentrations that allow an in-use shelf life of at least 12 weeks without unacceptable loss of stability.

[0010] In another aspect, the present invention provides an aqueous, sterile pharmaceutical composition comprising: a) Basal BIF with a concentration of 5 to 30 mg / mL; b) Phenol at a concentration of 1.5 to 4 mg / mL; c) benzyl alcohol at a concentration of 4 to 14 mg / mL; d) glycerin having a concentration of 15 to 35 mg / mL; e) Poloxamer 188 at a concentration of 0.01 to 0.5 mg / mL; f) a phosphate concentration of 5 to 10 mM; The composition has a pH of 6 to 7.5.

[0011] In another aspect, the present invention provides a method of improving glycemic control comprising administering to a human in need of improved glycemic control an effective amount of an aqueous, sterile pharmaceutical composition of the present invention.

[0012] Additionally, the present invention provides an aqueous, sterile pharmaceutical composition of the present invention for use in therapy. More particularly, the present invention provides a pharmaceutical composition for use in improving glycemic control. The present invention also provides the use of the pharmaceutical composition in the manufacture of a medicament for improving glycemic control.

[0013] Additionally, the present invention provides an article of manufacture comprising the aqueous sterile pharmaceutical composition of the present invention. More specifically, in certain embodiments, the article of manufacture is a multi-use vial, a cartridge, a reusable pen syringe, a disposable pen device, a pump device for continuous subcutaneous insulin infusion therapy, or a container closure system for use in a pump device for continuous subcutaneous insulin infusion therapy.

[0014] The present invention relates to a preserved formulation of insulin-Fc fusions with long duration of action. Insulin-Fc fusions are described, for example, in US Patent No. 9,855,318, CN103509118, WO2011 / 122921, US2015 / 0196643, WO2018 / 185131, WO2020 / 006529, WO2020 / 074544, WO2021126584, US20210300983, US2021 / 0324033, and US2021340212.

[0015] In certain preferred embodiments, the insulin-Fc fusion is a compound described in US Pat. No. 9,855,318 known as basal insulin Fc (BIF) or insulin efsitora alfa (CAS Registry No. 2131038-11-2). BIF comprises a dimer of an insulin receptor agonist fused to a human IgG Fc region, the insulin receptor agonist comprising an insulin B chain analog fused to an insulin A chain analog by use of a first peptide linker, the C-terminal residue of the insulin A chain analog being fused directly to the N-terminal residue of a second peptide linker, and the C-terminal residue of the second peptide linker being fused directly to the N-terminal residue of the human IgG Fc region. Each monomer of BIF has the amino acid sequence set forth in SEQ ID NO:1. FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSL DQLENYCGGGGGQGGGGQGGGGQGGGGECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:1). Each monomer contains intrachain disulfide bonds between cysteine ​​residues at positions 7 and 44, 19 and 57, 43 and 48, 114 and 174, and 220 and 278. Two monomers are linked to form a dimer by a disulfide bond between cysteine ​​residues at positions 80 and 83. The structure, function and production of BIF are described in more detail in U.S. Pat. No. 9,855,318.

[0016] As used herein, the term "BIF" refers to any insulin receptor agonist composed of two monomers having the amino acid sequence of SEQ ID NO:1, and includes any protein that is the subject of a regulatory submission seeking approval of an insulin receptor agonist product that relies in whole or in part on data submitted to a regulatory agency by Eli Lilly and Company regarding a BIF, regardless of whether the party seeking approval of such product actually identifies the insulin receptor agonist as a BIF or uses some other terminology.

[0017] The concentration of insulin-Fc fusion in the composition of the invention should be sufficient to allow administration of the range of insulin doses required by patients with T2DM and T1DM who have a wide range of insulin requirements. Currently available basal insulin products suitable for once-daily administration, such as LANTUS (insulin glargine), TOUJEO (insulin glargine), TRESIBA (insulin degludec) and LEVEMIR (insulin detemir), are available in concentrations ranging from 100 insulin units (IU) / mL to 300 IU / mL. In certain embodiments of the invention, the insulin-Fc fusion is present in a concentration ranging from about 100 to about 2000 insulin units (IU) / mL. In certain embodiments, the insulin-Fc fusion is present in a concentration of about 250 IU / mL, 500 IU / mL or 1000 IU / mL. The concentration of the insulin-Fc fusion can also be expressed as mass per volume. For example, in certain embodiments where the insulin-Fc fusion is BIF, the concentration of BIF is about 5-30 mg / mL, hi certain embodiments, the concentration of BIF is selected from the group consisting of 7.15, 14.3, and 28.6 mg / mL.

[0018] The formulations of the invention are sterile when initially manufactured, however, if the compositions are provided in multi-use vials or cartridges, an antimicrobial preservative that is compatible with the insulin-Fc fusion and any other components of the formulation is added in a strength sufficient to meet regulatory and pharmacopoeial antimicrobial preservative requirements for multi-use products. These requirements include tests designed to challenge the ability of the preservative to inhibit or kill microorganisms that may be inadvertently introduced into the product. Guidelines for performing these tests can be found in the United States Pharmacopeia (USP). <51> "Antimicrobial Effectiveness Testing" and the European Pharmacopoeia (Ph.Eur. or EP) 5.1.3 "Efficacy of Antimicrobial Preservation". See, e.g., Meyer, BD et al., Antimicrobial preservative use in parenteral products: Past and present, JOURNAL OF PHARMACEUTICAL SCIENCES 2007, 96, (12), 3155-3167; Moser, CL, Meyer, BK, Comparison of compendial antimicrobial effectiveness tests: A review. AAPS PHARM.SCI.TECH, 2011, 12, (1), pp. 222-226.

[0019] The acceptance criteria referred to above are log of microbial counts at various defined time points. 10The reductions are evaluated and the numbers are compared to the initial time point zero inoculum level. For example, the USP standard requires a 1.0 log reduction or more from the initial bacterial count after 7 days, a 3.0 log reduction or more from the initial bacterial count after 14 days, and no increase from the 14 day bacterial count after 28 days. The EP B standard is considered mandatory by EU regulatory agencies and requires at least a 1 log reduction after 24 hours and a 3 log reduction after 7 days compared to the initial bacterial count. Since the EP B standard is more stringent than the USP standard, any formulation that meets the EP B standard is also required to meet the USP <51> The EP A standard is the most stringent, requiring a 2 log reduction after 6 hours and a 3 log reduction after 24 hours. With many preservative systems, the EP A standard is difficult to achieve, and the preservatives added to achieve EP A are often at levels that have deleterious effects on the product and / or are toxic to the patient, but are considered more achievable.

[0020] Therapeutic insulin products currently available for subcutaneous administration are multi-dose products and therefore must meet regulatory requirements for antimicrobial effectiveness, including USP and EP B standards. Commonly used preservatives to meet these requirements include phenol (CAS number 108-95-2, molecular formula CH), as in the products listed in Table 1 below. 50 H, molecular weight 94.11), and m-cresol (CAS number 108-39-4, molecular formula CH 80 , molecular weight 108.14). [Table 1]

[0021] However, in formulations of insulin-Fc fusions such as BIF, these concentrations of m-cresol and phenol result in protein precipitation and therefore cannot be used to provide sufficient antimicrobial efficacy to meet USP and EP requirements. Therefore, the formulations of the present invention use a different preservative: phenoxyethanol (CAS number 122-99-6, molecular formula CH 10The antimicrobial efficacy criteria are dependent on the use of phenol (CAS number 100-51-6, molecular formula CHO, molecular weight 138.16 g / mol) and / or benzyl alcohol (CAS number 100-51-6, molecular formula CHO, molecular weight 108.14 g / mol). Specifically, it has been found that the use of certain concentrations of phenol in combination with benzyl alcohol and / or phenoxyethanol allows the antimicrobial efficacy criteria to be met in formulations within the desired pH range of BIF without causing unacceptable loss of physical stability.

[0022] The phenol and benzyl alcohol and / or phenoxyethanol in the formulations of the invention must be in sufficient concentrations to ensure that the formulation meets the minimum sterility requirements for parenteral products as set forth in the USP and EP B guidelines. As used herein, the term "sterile" refers to formulations that meet these minimum sterility requirements.

[0023] However, the concentration of these preservatives should not be so high as to cause unacceptable physical or chemical stability problems for the insulin-Fc fusion protein. The compositions of the present invention are sufficiently stable to allow storage and multiple weeks of use (referred to herein as "in-use" shelf life) without unacceptable loss of stability. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of at least 12 weeks. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of 12 weeks under refrigeration for 2 weeks at 30°C. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of 8 weeks at 25°C. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of 12 weeks at 25°C. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of 8 weeks at 30°C. In certain embodiments, the compositions are sufficiently stable to allow an in-use shelf life of 12 ...

[0024] With regard to phenol, some multi-dose parenteral drug products use 5 mg / mL phenol as a preservative, but this concentration has been found to result in protein precipitation in BIF formulations, so the concentration should be less than 5 mg / mL. In certain embodiments of the invention, the concentration of phenol ranges from 1.5 to 4 mg / mL. In certain embodiments of the invention, the concentration of phenol is about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mg / mL. In certain preferred embodiments, the concentration of phenol ranges from 1.8 to 3.5 mg / mL. In certain preferred embodiments, the concentration of phenol is about 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5 mg / mL. In certain preferred embodiments, the concentration of phenol is about 1.8, 2, 2.5, 3 or 3.5 mg / mL. In a particularly preferred embodiment, the concentration of phenol is about 1.8 mg / mL. It should be noted that due to its physical properties, phenol is typically added to aqueous compositions such as those described herein in the form of a 90% aqueous solution. For example, in many of the tests described below, phenol was added as "liquefied distilled phenol", which is 90% phenol + 10% water. In these tests, the phenol concentrations listed refer to the concentration of the 90% solution added to the composition. Thus, the absolute phenol content in a composition prepared using 2 mg / mL of a 90% phenol solution is 1.8 mg / mL. Unless otherwise indicated, such as in the tests described below using a 90% phenol solution, the concentration of phenol contained in the compositions of the present invention refers to the absolute phenol content.

[0025] The concentration of either phenoxyethanol or benzyl alcohol in the formulation of the present invention depends on the concentration of phenol, but must be present in a concentration sufficient to make the formulation sterile at the desired pH.For example, in a particular embodiment at pH 6.5, 9 mg / mL of phenoxyethanol is not sufficient to pass the EP B standard in the absence of phenol, but a concentration as low as 4 mg / mL can be used in combination with a phenol concentration as low as 1.8 mg / mL to meet the EP B standard.Similarly, in a particular embodiment, 9 mg / mL of benzyl alcohol is not sufficient to even pass the USP standard, but a concentration as low as 5 mg / mL can be used in combination with phenol at 1.8 mg / mL to meet the USP standard.

[0026] In certain embodiments, the concentration of phenoxyethanol ranges from 4 mg / mL to 14 mg / mL. In certain embodiments, the concentration of phenoxyethanol is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mg / mL. In certain preferred embodiments, the concentration of phenoxyethanol is about 4 or about 8 mg / mL.

[0027] In certain embodiments, the concentration of benzyl alcohol ranges from 5 to 10 mg / mL. In certain embodiments, the concentration of benzyl alcohol is about 5, 6, 7, 8, 9, or 10 mg / mL. In certain preferred embodiments, the concentration of benzyl alcohol is about 9 mg / mL.

[0028] The pH of the formulation of the invention is in the range of 5.5 to 7.5. In certain embodiments, the pH is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In certain embodiments, the pH is in the range of 6 to 7. In certain embodiments, the pH is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. Preferably, the pH of the formulation of the invention is at least the PI of the insulin-Fc fusion. For formulations containing BIF, the pH is preferably at least about 6.1. In certain embodiments containing BIF, the pH is in the range of 6.2 to 7.4. In certain embodiments containing BIF, the pH is in the range of 6.2 to 6.9. In certain embodiments containing BIF, the pH is in the range of 6.3 to 6.8. In particularly preferred embodiments containing BIF, the pH is about 6.5.

[0029] If desired, a buffering agent may be included. Examples of such buffering agents are phosphates, such as dibasic sodium phosphate, citrate, sodium acetate, and tris(hydroxymethyl)aminomethane, or TRIS. If a buffering compound is required, a citrate buffer or a phosphate buffer is preferred. In certain embodiments, the compositions of the present invention include a citrate buffer at a concentration in the range of 5-10 mM. In certain embodiments, the compositions of the present invention include a phosphate at a concentration in the range of 5-10 mM. In certain preferred embodiments, the compositions of the present invention include a phosphate at a concentration of about 5, 6, 7, 8, 9, or 10 mM. In certain preferred embodiments, the compositions of the present invention include a phosphate at a concentration of either about 5 mM or about 10 mM.

[0030] When administering the composition, it is desirable to match the tonicity (i.e., osmolarity) of the body fluid at the injection site as closely as possible, since solutions that are not nearly isotonic with body fluids will produce a painful stinging sensation upon administration. It is therefore desirable for the composition to be nearly isotonic with the body fluid at the site of injection. If the osmolarity of the composition in the absence of a tonicity agent is sufficiently lower than the osmolarity of the tissue (for blood, approximately 300 mOsmol / kg; the European Pharmacopoeia requirement for osmolarity is >240 mOsmol / kg), then an isotonicity agent should generally be added to increase the tonicity of the composition to approximately 300 mOsmol / kg. The osmolarity of the composition is determined by the identity and concentration of the other excipients in the composition, including the stabilizer. Thus, to determine whether an isotonicity agent must be added, the concentrations of all of the various excipients in the composition must be evaluated, and such evaluation and determination is easily made using standard techniques. See Remington: The Science and Practice of Pharmacy, edited by David B. Troy and Paul Beringer, Lippincott Williams & Wilkins, 2006, pp. 257-259; Remington: Essentials of Pharmaceutics, Linda Ed Felton, Pharmaceutical Press, 2013, pp. 277-300. Exemplary tonicity agents include glycerol (glycerin), mannitol, and sodium chloride. When the addition of an isotonicity agent is required, glycerin is preferred. In certain embodiments, the concentration of glycerol is about 10 to about 50 mg / mL. In certain embodiments, the concentration of glycerol is about 15 to about 35 mg / mL. In certain embodiments, the concentration of glycerol is selected from the group consisting of about 15, 17, 20, 21, and 35 mg / mL. In certain preferred embodiments, the concentration of glycerin is about 17 mg / mL.

[0031] The compositions of the present invention may also include other excipients, including stabilizers such as surfactants. Examples of surfactants disclosed for use in parenteral pharmaceutical compositions include polysorbates such as polysorbate 20 (TWEEN® 20) and polysorbate 80 (TWEEN 80), polyethylene glycols such as PEG 400, PEG 3000, TRITON® X-100, polyethylene glycols such as polyoxyethylene (23) lauryl ether (CAS number: 9002-92-0, sold under the trade name BRIJ®), alkoxylated fatty acids such as MYRJ®, polysorbate 80 (PBA), polysorbate 80 (PBA), polysorbate 90 (PBA), polysorbate 10 ... Included are propylene glycol, block copolymers such as Poloxamer 188 (CAS number 9003-11-6, sold under the trade name PLURONIC® F-68) and Poloxamer 407 (PLURONIC® F127), sorbitan alkyl esters (e.g., SPAN®), polyethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®), and its derivatives such as trehalose and trehalose laurate ester.

[0032] In certain preferred embodiments, the composition comprises a surfactant selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188. Poloxamer 188 is most preferred. In certain embodiments, the concentration of the surfactant ranges from 0.01 to 10 mg / mL or 0.1 to 0.5 mg / mL. In preferred embodiments where the surfactant is poloxamer 188, the concentration of poloxamer 188 is about 0.4 mg / mL.

[0033] In certain embodiments, the compositions of the present invention are provided in products such as multi-use vials, cartridges, reusable pen injectors, disposable pen devices, pump devices for continuous subcutaneous insulin infusion therapy, or other container closure systems for use in pump devices for continuous subcutaneous insulin infusion therapy.In certain embodiments, the compositions are provided in reusable pen injectors that can be used to provide variable doses of insulin that can be adjusted in specific increments.For example, in certain embodiments, such pen injectors contain 1500 units of insulin and can be adjusted in 5 unit increments to deliver a dose of up to 400 units per injection.In other embodiments, such pen injectors contain 3000 units of insulin and can be adjusted in 10 unit increments to deliver a dose of up to 800 units per injection.

[0034] As used herein, the term "about" is intended to refer to the degree of acceptable error for a given amount or quantity, given the nature or precision of the measurement. For example, the degree of error can be indicated by the number of significant figures provided for a measurement, as understood in the art, including, but not limited to, a variation of ±1 of the most precise significant figure reported for the amount or quantity. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Numerical values ​​provided herein are approximate unless otherwise stated, and the term "about" means that they can be inferred when not expressly stated. EXAMPLES

[0035] Conformational stability in the presence of preservatives The conformational stability of BIF when formulated with various phenolic preservatives was studied, the compositions of which are shown in Table 2 below. [Table 2]

[0036] Extrinsic fluorescence measurements were performed to assess the conformational stability of BIF. Extrinsic fluorescent dyes such as 1-anilinonaphthalene-8-sulfonate (ANS) are minimally fluorescent in aqueous environments but become highly fluorescent in polar organic solvents. These fluorescent dyes have been used to detect the exposure of hydrophobic patches on protein surfaces and provide information about protein folding and unfolding processes. See, for example, Hawe, A. et al., Extrinsic fluorescent dyes as tools for protein characterization. PHARMACEUTICAL RESEARCH, 2008, 25(7), 1487-1499. The extrinsic fluorescence method is a plate-based method that uses a Bis-ANS fluorescent probe to measure the surface hydrophobicity of proteins in solution. Fluorescence spectra were measured using a SpectraMax i3x multimode microplate reader (Molecular Devices, San Jose, USA). Samples were placed in black polypropylene 96-well corning half-area flat plates. Approximately 100 μL of the sample composition containing 5 μM dye was transferred to each well and measured at 25° C. The excitation wavelength (λ Ex ) was 390 nm, and the emission spectrum was scanned from 420 to 600 nm in 2 nm increments.

[0037] The peak fluorescent signals of the BIF-containing compositions are shown in Table 3 below. [Table 3]

[0038] As shown in Table 3, in the absence of preservative, some fluorescence is detected, indicating that there are hydrophobic patches on the surface of BIF, even in its native folded state. When preservative is added, the fluorescence intensity increases. In the absence of BIF, Bis-ANS and preservative do not generate any fluorescent signal. Thus, the observed intensity is due to the interaction between BIF and preservative molecules and the resulting partial unfolding of the protein, leading to the exposure of more hydrophobic patches.

[0039] Furthermore, the intensity of the fluorescent signal correlated with the hydrophobicity of the preservative, with m-cresol being the most hydrophobic and producing the strongest signal, followed by phenol and benzyl alcohol. Benzyl alcohol, the least hydrophobic of the three preservatives, caused the least perturbation to the BIF conformation.

[0040] Physical stability in the presence of preservatives used in currently available insulin products A formulation containing 28.6 mg / mL BIF and m-cresol and / or phenol at concentrations used in insulin products sold as multi-dose was prepared at pH 6.5. The formulation was filled into glass vials, stored at room temperature, and tested by visual inspection. The results are shown in Table 4 below: [Table 4]

[0041] Formulations 1 and 2 each resulted in precipitation of the BIF drug substance, indicating physical instability. Formulations 3 and 4 remained clear, indicating that the BIF drug substance remained physically stable.

[0042] Stability as a function of pH Biophysical developability / high throughput profiling studies were performed on 2 mg / mL formulations of BIF in different buffer matrices and pH conditions. Differential scanning calorimetry (DSC) was used to determine the onset melting temperature, T m (T m,Onset) was measured. m,Onset is the temperature at which a folded protein begins to lose its native conformation, i.e., T m,Onset The higher the , the less susceptible the protein is to denaturation. The results are shown in Table 5 below. [Table 5]

[0043] As the solution pH increases from 5.5 to 7.5, regardless of buffer type or ionic strength, T m,Onset increases accordingly.

[0044] The colloidal stability of BIF with and without preservatives was also tested at pH conditions below and above the pI of BIF, approximately 6.1. BIF drug substance prepared in citrate buffer was used for pH titration using 1.5 N citric acid or 1 N NaOH.

[0045] The compositions were visually inspected for opalescence, which may be a precursor to potential liquid phase separation. Raut, AS; Kalonia, DS, Pharmaceutical perspective on opalescence and liquid-liquid phase separation in protein solutions. Molecular Pharmaceutics, 2016, 13(5), pp. 1431-1444. The compositions, both with and without preservatives, appeared opalescent as the pH approached the pI of the drug substance and became clear at a pH above the pI.

[0046] These studies indicate that a pH higher than the pI of BIF favors the conformational and colloidal stability of BIF.

[0047] Preservative concentration and antibacterial efficacy The study was designed to test the antimicrobial efficacy of formulations of the BIF drug product containing various concentrations of phenoxyethanol and benzyl alcohol with or without phenol. The materials used to prepare the compositions are shown in Table 7 below. [Table 6]

[0048] Compositions containing BIF and mixtures of phenol with either phenoxyethanol or benzyl alcohol were prepared as described in Tables 8 and 9 below. [Table 7] [Table 8]

[0049] Approximately 150 mL of the solution was filtered through a 0.22 μm PVDF filter and immediately transferred into a sterile glass container. Samples were stored at 5° C. until antimicrobial efficacy testing.

[0050] The AET was performed by inoculating the test solutions with pure cultures of various microorganisms to represent common potential microbial contaminants. <51> The vials were inoculated with the following microorganisms listed in EP 5.1.3: Aspergillus brasiliensis spores, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The inoculated solutions were stored at controlled room temperature (20°C-25°C) in a refrigerated incubator. The viable cell concentrations in the inoculated vials were measured by plate counting at initial, 6 hours, 24 hours, 7 days, 14 days, and 28 days after inoculation. The results were compared in accordance with EP 5.1.3 and USP <51> The formulations were judged as either passing or failing each test criterion, compared to the acceptance criteria set forth in . EP "A" criteria are considered to be the most stringent, followed by EP "B" criteria, and then USP criteria. The objective of this study is to identify BIF drug product formulations that meet the EP B and USP criteria.

[0051] The results for the phenoxyethanol containing compositions are shown in Table 10 below: [Table 9]

[0052] As shown in Table 10, phenoxyethanol alone can be an effective preservative. At concentrations of 10 mg / mL and above, the EP B and USP criteria are met. In addition, a combination of 4 mg / mL phenoxyethanol and 2 mg / mL or more liquefied phenol can also meet the EP B and USP criteria. Finally, the EP B and USP criteria are met over a range of pH.

[0053] The results for the benzyl alcohol containing compositions are shown in Table 11 below. [Table 10]

[0054] As shown in Table 11, solutions containing 9 mg / mL benzyl alcohol and no phenol fail to meet the EP B and USP criteria, likely due to the fact that they are beyond the pH range considered optimal for benzyl alcohol. See Meyer, BD et al., Antimicrobial preservative use in parenteral products: Past and present. JOURNAL OF PHARMACEUTICAL SCIENCES, 2007, 96, (12), pp. 3155-3167. However, when combined with phenol, solutions across the pH range of BIF drug products meet the USP criteria and, with the exception of the formulation containing the lowest tested concentrations of benzyl alcohol and phenol, meet the EP B criteria. In addition, surprisingly, formulations containing 9 mg / mL benzyl alcohol and various concentrations of liquefied phenol meet the more stringent EP A criteria.

[0055] Comparison of preservatives – chemical and physical stability The study was designed to examine the physical and chemical stability of two BIF formulations containing a combination of phenol with either phenoxyethanol or benzyl alcohol. The drug substances and excipients used are listed in Table 12 below. [Table 11]

[0056] A solution containing 50 mg / mL BIF and a buffer containing a combination of sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate was prepared to give a buffer strength of 5 mM phosphate and other components as shown in Table 13 below. [Table 12]

[0057] The solution was filtered through a 0.22 μm PVDF filter and immediately transferred into sterile glass containers and then filled into 5 mL glass vials. The vials were capped and stored at 5° C., 25° C., and 30° C. for up to 6 months. Samples were tested by various stability assays at various time points.

[0058] The results are shown in Tables 14 to 19 below. [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18]

[0059] Solution pH remained constant throughout the study period, as shown in Table 14. Results from SEC and subvisible particles also confirmed that the samples were physically stable at each pH, ​​as shown in total aggregates (Table 15) and particulate matter by HIAC or MFI (Tables 16 and 17). Chemical stability of the formulations was evaluated using RP-HPLC and AEX. The formulations exhibited comparable chemical stability, as reflected by total impurities (Table 18) and total acidic variants (Table 19), and were most stable at pH 6.4 in this study.

[0060] stability The study was designed to evaluate the stability and functionality of the multi-use BIF drug product in a 3 mL cartridge. All compositions were made with 5 mM phosphate, 21 mg / mL glycerin, and 0.4 mg / mL poloxamer. Other characteristics of the compositions are shown in Table 20 below. [Table 19]

[0061] The compositions were filled into 3 mL glass cartridges and closed with siliconized chlorobutyl plungers and DNR-free disk seals. The cartridges were stored at 5° C., 25° C., 30° C. or 35° C. for up to 6 months. Samples were submitted for testing as shown in Table 21 below. [Table 20]

[0062] No clear trends were observed in the HIAC or MFI data with respect to pH, storage conditions, or formulation composition. All HIAC particle counts were within specification for measurements ≥10 μm and ≥25 μm.

[0063] The results of SEC, RP-HPLC and AEX are shown in Tables 22 to 24 below. [Table 21] [Table 22] [Table 23] [Table 24]

[0064] As shown in Table 22, all formulations performed well at 5° C., 25° C., and 30° C., with no notable differences between control and test samples, suggesting that the tested concentrations of benzyl alcohol and phenol did not induce significant protein denaturation. With no clear trends between samples, the aggregate growth observed at 35° C. is primarily driven by heat stress.

[0065] As shown in Table 23, at 5° C. there is no difference between the control and test samples. Differences are seen at 25° C., 30° C., and 35° C. At these temperatures, the growth of total impurities is promoted as the pH increases.

[0066] Similarly, at 5° C., there is negligible growth of TAV, as shown in Table 24. TAV ​​growth is significant at elevated temperatures (25° C., 30° C., and 35° C.) and correlates with increasing pH.

[0067] In summary, the preservatives tested showed minimal impact on formulation stability, with pH and temperature being the primary factors influencing stability. At 5°C, the formulations remained stable with little aggregate growth and chemical degradation, but as storage temperatures increased (25°C, 30°C, and 35°C), degradation accelerated accordingly. Subvisible particulate matter was within specifications for all test groups. Overall, the results from this study show robustness across the formulations tested.

[0068] Chemical stability as a function of pH Studies were designed to investigate the chemical stability of BIF formulations with and without preservatives at pH conditions above their pI.

[0069] Preservative-containing compositions were prepared as shown in Table 25 below. [Table 25]

[0070] Preservative-free compositions were prepared as shown in Table 26 below. [Table 26]

[0071] All solutions were filtered through a 0.22-μm PVDF filter and immediately transferred to sterile glass containers. In a laminar flow hood, the solutions were filled into glass vials. The vials were capped and stored at 5° C. and 30° C. for up to 3 months. At appropriate time points, samples were removed and subjected to testing.

[0072] Chemical stability was assessed by anion exchange chromatography (AEX) and the results are shown in Table 27 below. [Table 27] [Table 28]

[0073] As shown in Tables 27 and 28, at 5° C., growth of the total acidic variants (TAVs) was negligible, while at 30° C., growth occurred in response to pH. The presence of preservatives in these compositions did not substantially affect stability.

[0074] Shelf life and use TAV was deemed to be the most relevant chemical stability indicative assay for BIF and therefore the results shown above in Tables 27 and 28 were used for shelf-life and time-of-use estimation. The accelerating effect of storage temperature (T) at time point (t) was factored using the following formula to scale the time scale to a single arbitrary reference temperature (T Ref ) was collapsed.

number

[0075] The data in Tables 27 and 28 indicate that the preferred drug product pH in such embodiments is about 6.5 or less.

[0076] clinical research A clinical study in healthy participants was designed to compare acute injection site pain intensity associated with matrices containing preservatives and tonicity agents. Each participant received one 0.6 mL SC injection on day 1 of periods 1-5. No active medication was administered. The five solution formulations were as follows: [Table 30]

[0077] Pain at the injection site was assessed and quantified using a 100 mm visual analog scale (VAS), where 0 indicated "no pain" and 100 indicated "worst pain imaginable." Data were tabulated and summarized by treatment and time point.

[0078] A mixed-effects model was used to analyze continuous infusion site pain from VAS pain scores at each time post-injection for each formulation. The model was by post-injection measurement time point and included treatment (solution formulation), injection order within cohort (first, second, third, fourth, or fifth injection of the period), cohort (participants were randomized to injection order group) as fixed factors, and participant as a random effect. The Kenward-Roger method was used to estimate denominator degrees of freedom. Type III tests for least squares (LS) means were used for statistical comparisons. 95% confidence intervals (CIs) of the differences were also reported. Differences in LS means were considered statistically significant if the 95% CI excluded 0.

[0079] All adverse events (AEs) were listed. Treatment-emergent AEs were summarized. Any serious adverse events (SAEs) were listed.

[0080] Infusion site reaction (ISR) questionnaires were collected for spontaneously reported ISRs at prespecified time points. ISR data were listed in frequency tables and summarized by treatment.

[0081] All solution formulations, including the reference formulation, were well tolerated, with most participants reporting low-severity (<10 mm) injection-site pain. For all formulations, across all time points (0–60 min post-injection), 76%–100% of participants reported a VAS pain score of <10 mm, with mean VAS pain scores ranging from 0.2–7.1 mm.

[0082] All solution formulations, including the reference formulation, administered by SC injection were well tolerated by participants. There were no deaths or SAEs. One participant discontinued due to an AE not related to the study intervention as judged by the investigator. The frequency of AEs was low overall. All treatment-emergent adverse events (TEAEs) were mild or moderate in severity as judged by the investigator, and TEAEs were not related to the study intervention. Fewer participants reported ISRs at predefined time points 10 to 60 minutes after injection of the test formulation compared with the reference formulation, and fewer ISRs were spontaneously reported. Mild pain was the most common ISR parameter reported at predefined time points and for spontaneously reported events.

[0083] array SEQ ID NO:1 [Table 31]

Claims

1. 1. An aqueous sterile pharmaceutical composition comprising: a) an insulin-Fc fusion; b) phenol, c) one or more additional preservatives selected from the group consisting of phenoxyethanol and benzyl alcohol; d) a tonicity agent; and e) a surfactant; and f) a buffer solution, having a pH of 6 to 7.5; An aqueous, sterile pharmaceutical composition, wherein said phenol and one or more additional preservatives are present in concentrations that allow an in-use shelf life of at least 12 weeks without unacceptable loss of stability.

2. The composition of claim 1, wherein the concentration of phenol is 1.5 to 4 mg / mL.

3. 2. The composition of claim 1, wherein the concentration of phenol is about 1.8 mg / mL.

4. The composition of claim 1 , wherein the additional preservative is phenoxyethanol.

5. The composition according to claim 4, wherein the concentration of phenoxyethanol is 4 to 14 mg / mL.

6. 5. The composition of claim 4, wherein the concentration of phenoxyethanol is about 4 mg / mL.

7. 5. The composition of claim 4, wherein the concentration of phenoxyethanol is about 8 mg / mL.

8. The composition of claim 1 , wherein the additional preservative is benzyl alcohol.

9. The composition of claim 8, wherein the concentration of benzyl alcohol is 5 to 10 mg / mL.

10. 10. The composition of claim 9, wherein the concentration of benzyl alcohol is about 9 mg / mL.

11. The composition of claim 1, wherein the insulin-Fc fusion is insulin efsitora alfa.

12. The composition of claim 11, wherein the concentration of insulin efsitora alfa is 5 to 30 mg / mL.

13. The composition of claim 12, wherein the concentration of insulin efsitora alfa is selected from the group consisting of about 7.15, 14.3, and 28.6 mg / mL.

14. The composition of claim 1, wherein the pH of the composition is from 6.3 to 6.

8.

15. The composition of claim 1 , wherein the pH of the composition is about 6.

5.

16. 2. The composition of claim 1, wherein the tonicity agent is selected from the group consisting of sodium chloride, mannitol and glycerin.

17. The composition of claim 1 , wherein the tonicity agent is glycerin.

18. The composition of claim 17, wherein the concentration of glycerin is 15 to 35 mg / mL.

19. 20. The composition of claim 18, wherein the concentration of glycerin is about 17 mg / mL.

20. 2. The composition of claim 1, wherein the surfactant is selected from the group consisting of poloxamer 188, polysorbate 20 and polysorbate 80.

21. The composition of claim 20, wherein the surfactant is poloxamer 188.

22. The composition of claim 21, wherein the concentration of poloxamer 188 is 0.01 to 0.5 mg / mL.

23. 23. The composition of claim 22, wherein the concentration of poloxamer 188 is about 0.4 mg / mL.

24. 2. The composition of claim 1, wherein the buffer is selected from the group consisting of phosphate, citrate and TRIS.

25. 25. The composition of claim 24, wherein the buffer is phosphate.

26. The composition of claim 25, wherein the concentration of phosphate is 5 to 10 mM.

27. 1. An aqueous sterile pharmaceutical composition comprising: a) insulin efsitora alfa at a concentration of 5 to 30 mg / mL; b) phenol at a concentration of 1.5 to 4 mg / mL; c) benzyl alcohol at a concentration of 4 to 14 mg / mL; d) glycerin at a concentration of 15 to 35 mg / mL; e) Poloxamer 188 at a concentration of 0.01 to 0.5 mg / mL; f) a concentration of 5 to 10 mM phosphate; An aqueous, sterile pharmaceutical composition having a pH of 6.3 to 6.

8.

28. a) the concentration of insulin efsitora alfa is selected from the group consisting of about 7.15, about 14.3, and about 28.6 mg / mL; b) the concentration of phenol is about 1.8 mg / mL; c) the concentration of benzyl alcohol is about 9 mg / mL; d) the concentration of glycerin is about 17 mg / mL; e) the concentration of poloxamer 188 is about 0.4 mg / mL; 28. The composition of claim 27, wherein f) the concentration of phosphate is selected from the group consisting of about 5 mM and about 10 mM, and wherein the composition has a pH of about 6.

5.

29. The composition of claim 28, wherein the concentration of insulin efsitora alfa is about 14.3 mg / mL and the concentration of phosphate is about 5 mM.

30. The composition of claim 28, wherein the concentration of insulin efsitora alfa is about 28.6 mg / mL and the concentration of phosphate is about 10 mM.

31. The composition of claim 28, wherein the concentration of insulin efsitora alfa is about 7.15 mg / mL.

32. A pharmaceutical composition according to any one of claims 1 to 31 for treating diabetes.

33. An article of manufacture comprising any one of the pharmaceutical compositions according to claims 1 to 31.

34. 34. The article of manufacture of claim 33, which is a multi-use vial.

35. 34. The article of manufacture of claim 33, which is a multi-use pen injection device.

36. 34. The article of manufacture of claim 33 which is a pump device for continuous subcutaneous insulin infusion therapy.