Preservative preparation

Abstract

The present invention relates to preserved surfactant-containing pharmaceutical compositions suitable for parenteral administration.

Description

Detailed Description of the Invention

[0001] The present invention relates to preserved surfactant-containing pharmaceutical compositions suitable for parenteral administration, which include one or more preservatives, such as meta-cresol or phenol, one or more surfactants, such as polysorbate 80 (PS80), one or more active pharmaceutical ingredients (APIs), such as dulaglutide, and one or more solvent modifiers, such as propylene glycol (PPG), N-methyl-2-pyrrolidone (NMP), polyethylene glycol (PEG) 400, or glycerol.

[0002] Protein and peptide-based pharmaceuticals typically must be administered parenterally due to the susceptibility of proteins and peptides to proteolytic degradation in the gastrointestinal tract when administered orally, and in some cases must be formulated with nonionic surfactants to ensure protein stability throughout storage and use. However, a limitation of such surfactant-containing formulations requiring surfactant concentrations above a certain level is that they cannot be adequately stored for multiple use because the interaction between the surfactant and the preservative results in the formation of unacceptable visible precipitates. This incompatibility between surfactants and preservatives has been recognized previously. For example, S. Kazmi and A. Mitchell, Interaction of Preservatives with Cetomacrogol, 23J.PHARM.PHARMAC.482-489(1970); J. Blanchard, Effect of Sorbitol on Interaction of Phenolic Preservatives with Polysorbate 80,66J.PHARM.SCI.10,1471-1472(1977);J.Blanchard,Effect of Polyols on Interaction of Paraben Preservatives with Polysorbate 80,69J.PHARM.SCI.2,169-173(1980);R.Torosantucci,Protein-Excipient Interactions Evaluated via Nuclear Magnetic Resonance Studies in Polysorbate-Based Multidose Protein Formulations: Influence on Antimicrobial Efficacy and Potential Study See, for example, J. PHARM. SCI. 10, 2531-2537 (2018). However, no solution to the incompatibility is described.

[0003] Therefore, currently available protein- and peptide-based pharmaceuticals that require specific concentrations of surfactants as stabilizers are sold as unpreserved, single-use formulations. For example, dulaglutide, a glucagon-like peptide 1 (GLP-1) receptor agonist fusion protein sold under the trade name TRULICITY™, requires 0.20 mg / mL of polysorbate 80 for stabilization but does not contain a phenol preservative due to phase separation that occurs when the phenol preservative is added at a concentration sufficient to meet regulatory requirements. For subcutaneous use, see TRULICITY (dulaglutide) Injection, prescribing information highlights (first approved by the US FDA in 2014). Therefore, dulaglutide is currently sold in a device that must be disposed of after a single use, which is associated with disadvantages such as increased cost of goods sold (COPS) and increased physical waste compared to preserved, multi-use products.

[0004] Formulations of protein- or peptide-based pharmaceuticals containing surfactants at concentrations similar to those used in current commercial formulations of dulaglutide, or preservatives at concentrations sufficient to meet regulatory sterility requirements, have previously been described, although not both. For example, U.S. Patent Application No. 2009 / 0232807 describes formulations of GLP-1-Fc fusion proteins and lists various excipient categories and examples, including what the application describes as "solubilizers" such as Tween 80® (also known as polysorbate 80) and preservatives such as m-cresol. However, the application does not provide any examples or embodiments of formulations containing both the described "solubilizers" and the described preservatives. U.S. Patent Application No. 20100196405 describes formulations of dulaglutide, including formulations containing polysorbate 80 at a concentration of approximately 0.2% (w / v). However, the application does not describe formulations containing preservatives.

[0005] There remains a need for formulations that contain a surfactant at a concentration sufficient to stabilize the protein or peptide and a preservative at a concentration sufficient to meet the antimicrobial requirements of a multi-use injectable product.

[0006] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a protein or peptide; b) a nonionic surfactant; and c) a phenolic preservative; d) a solvent modifier; A composition is provided in which the nonionic surfactant and phenolic preservative are present at concentrations above their concentration thresholds in the absence of the solvent modifier, and the solvent modifier is present at a concentration sufficient to ensure that the solution remains clear.

[0007] In another aspect, the present invention provides a method for preparing a clear formulation comprising a nonionic surfactant and a phenolic preservative at concentrations above their concentration thresholds in the absence of a solvent modifier, comprising including a solvent modifier in the composition.

[0008] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a protein or peptide; b) a nonionic surfactant; and c) a phenolic preservative; d) a solvent modifier; An article of manufacture is provided that includes an aqueous composition in which a nonionic surfactant and a phenolic preservative are present at concentrations above their concentration thresholds in the absence of a solvent modifier, and the solvent modifier is present at a concentration sufficient to ensure that the solution remains clear.

[0009] In another aspect, the present invention provides a method for preparing a composition comprising a nonionic surfactant and a phenolic preservative above their concentration thresholds, comprising including a solvent modifier in the composition at a concentration sufficient to ensure that the composition remains clear.

[0010] As noted above, surfactants are included in the formulation of many protein- or peptide-based pharmaceuticals to stabilize the protein or peptide API. As used herein, the term "protein or peptide-based pharmaceutical" refers to a pharmaceutically acceptable composition for use in treating or preventing a disease or condition of interest, the composition comprising at least one API that is a peptide or protein. Peptides and proteins are sometimes distinguished by size, with peptides having 2-50 amino acids and proteins having more than 50 amino acids, but the distinction between the two is irrelevant for purposes of the present invention, as the formulations described herein are equally applicable to pharmaceuticals containing one or more APIs that are peptides or proteins. The formulations of the present invention may be applicable to a wide variety of protein- or peptide-based drugs that require a nonionic surfactant for stability purposes.

[0011] A preferred drug for use in the formulations of the present invention is dulaglutide, a human GLP-1R agonist comprising a dimer of a GLP-1 analog fused at its C-terminus via a peptide linker to the N-terminus of an analog of the Fc portion of an immunoglobulin, identified by CAS Registry Number 923950-08-7 and provided with the following chemical names: Immunoglobulin G4 (synthetic human Fc fragment), including dimers, peptide (synthetic 16-amino acid linker) fusion proteins, 7-37-glucagon-like peptide I [8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) fusion proteins. Each dulaglutide monomer has the amino acid sequence set forth in SEQ ID NO: 1: [ka]

[0012] The two monomers are linked by a disulfide bond between the cysteine ​​residues at positions 55 and 58 to form a dimer. The structure, function, production, and use of dulaglutide in the treatment of T2DM are described in more detail in U.S. Pat. No. 7,452,966 and U.S. Patent Application No. US20100196405. As used herein, the term "dulaglutide" refers to any GLP-1R agonist protein dimer 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 a GLP-1R agonist product that relies in whole or in part on data related to dulaglutide submitted to regulatory authorities by Eli Lilly and Company, regardless of whether the party seeking approval of the protein actually identifies the protein as dulaglutide or uses some other term.

[0013] Other examples of proteins or peptides that can be used in forming the present invention include, but are not limited to, those described in the Examples below, as well as other Fc fusion proteins, other GLP-1 agonists, gastric inhibitory peptide (GIP) receptor agonists, glucagon receptor agonists, peptide YY (PYY) and variants thereof, growth and differentiation (GDF) factors such as GDF15 and variants thereof, amylin receptor agonists, calcitonin receptor agonists, and interleukins and variants thereof.

[0014] Many proteins and peptides are prone to denaturation and / or aggregation when formulated in aqueous solutions, and surfactants are often added to such protein and peptide formulations to alleviate these problems. Surfactants are composed of molecules that have hydrophilic and hydrophobic portions and tend to aggregate in aqueous solutions to form aggregates known as micelles. The inclusion of a surfactant in an aqueous solution of a peptide- or protein-based pharmaceutical reduces the surface tension of the solution, helping to prevent the peptide or protein from coming into contact with any oxygen in the container. Examples of surfactants disclosed for use in parenteral pharmaceutical compositions include polysorbates such as polysorbate 20 (TWEEN® 20) and polysorbate 80 (TWEEN® 80), and block copolymers such as poloxamer 188 (CAS No. 9003-11-6, sold under the trade name PLURONIC® F-68) and poloxamer 407 (PLURONIC® F127).

[0015] The formulations of the present invention include one or more nonionic surfactants. In certain embodiments, the nonionic surfactant is a polysorbate-type surfactant. Polysorbates are ethoxylated sorbitans with fatty acid esters, and specific polysorbates are identified by the type of fatty acid ester relative to polyoxyethylene sorbitan. For example, polysorbate 20 contains monolaurate, polysorbate 40 contains monopalmitate, polysorbate 60 contains monostearate, and polysorbate 80 contains monooleate. Polysorbate 20 and polysorbate 80 are commonly used surfactants in pharmaceutical products for parenteral administration and are included as surfactant(s) in certain preferred embodiments of the present invention. In other embodiments, the nonionic surfactant is a poloxamer. Poloxamers are block copolymers composed of a polyoxopropylene chain and two polyoxyethylene chains, and are generally classified by a number indicating the mass of the polyoxypropylene core and the proportion of polyoxyethylene. Examples include poloxamer 188 and poloxamer 407. In particular, poloxamer 188 is a surfactant commonly used in pharmaceutical products for parenteral administration and is included as the surfactant(s) in certain preferred embodiments of the present invention.

[0016] In certain preferred embodiments, the nonionic surfactant is selected from the group consisting of polysorbate 80, polysorbate 20, and poloxamer 188. In certain embodiments, the nonionic surfactant is polysorbate 80. In certain embodiments, the concentration of polysorbate 80 is about 0.01 mg / mL to about 1 mg / mL. In certain embodiments, the concentration of polysorbate 80 is about 0.05 mg / mL to about 0.5 mg / mL. In certain embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL to about 0.4 mg / mL. In certain preferred embodiments, the concentration of polysorbate 80 is about 0.2 mg / mL to about 3 mg / mL. In certain embodiments, the concentration of polysorbate 80 is selected from the group consisting of about 0.2 mg / mL and about 0.25 mg / mL. In certain embodiments, the concentration of polysorbate 80 is about 0.2 mg / mL. In certain embodiments, the concentration of polysorbate 80 is about 0.25 mg / mL. In certain embodiments, the nonionic surfactant is polysorbate 20. In certain embodiments, the concentration of polysorbate 20 is about 0.01 mg / mL to about 1 mg / mL. In certain embodiments, the concentration of polysorbate 20 is about 0.05 mg / mL to about 0.5 mg / mL. In certain embodiments, the concentration of polysorbate 20 is about 0.1 mg / mL to about 0.4 mg / mL. In certain embodiments, the nonionic surfactant is poloxamer 188. In certain embodiments, the concentration of poloxamer 188 is in the range of about 0.01 to about 2 mg / mL. In certain embodiments, the concentration of poloxamer 188 is in the range of about 0.01 to about 2 mg / mL. In certain embodiments, the concentration of poloxamer 188 is in the range of about 0.5 to about 1.5 mg / mL. However, these embodiments should not be construed as limiting, as one of ordinary skill in the art can identify the identity and concentration of surfactant necessary to provide sufficient stabilizing effect in a given composition.

[0017] The formulations of the present invention also include one or more preservatives added to provide antimicrobial properties. However, the compositions are sterile when initially produced, and if the compositions are provided in multi-use vials or cartridges, an antimicrobial preservative compound or mixture of compounds compatible with the other components of the formulation is typically added in a strength sufficient to meet regulatory requirements and pharmacopoeia antimicrobial preservative requirements, such as those published by the European Pharmacopoeia (EP) and the United States Pharmacopoeia (USP). European Pharmacopoeia, 9th Edition, Section 5.1.3, Effectiveness of Antimicrobial Preservatives, US Pharmacopoeia USP <51> , Antibacterial Efficacy Test (Rockville, MD).

[0018] Commonly used preservatives in pharmaceutical products suitable for multiple-dose parenteral administration include phenolic compounds, or mixtures of such compounds. Specific examples include phenol (CAS No. 108-95-2, molecular formula C6H5OH, molecular weight 94.11), m-cresol (CAS No. 108-39-4, molecular formula C7H8O, molecular weight 108.14), benzyl alcohol (CAS No.: 100-51-6, molecular formula C7H8O, molecular weight 108.14 g / mol), and phenoxyethanol (CAS No.: 122-99-6, molecular formula C8H 10O2, molecular weight 138.17 g / mol). In certain embodiments of the present invention, the phenolic preservative is selected from the group consisting of phenol and m-cresol, and mixtures thereof. The concentration of preservative necessary to meet regulatory requirements for multi-use products depends on several factors, including, but not limited to, the identity of the phenolic preservative used and the pH of the solution. In certain embodiments, the phenolic preservative is phenoxyethanol present at a concentration of about 10 to about 15 mg / mL. In certain embodiments, the phenolic preservative is benzyl alcohol. In certain embodiments, the phenolic preservative is benzyl alcohol present at a concentration of about 10 mg / mL. In certain embodiments, the phenolic preservative is phenol. In certain embodiments, the phenolic preservative is phenol present at a concentration of about 1 to about 10 mg / mL. In certain embodiments, the phenolic preservative is phenol present at a concentration of about 3 to about 6 mg / mL. In certain embodiments, the phenolic preservative is phenol at a concentration of at least about 3 mg / mL. In certain embodiments, the phenolic preservative is phenol at a concentration selected from the group consisting of 3, 3.5, 4, 4.5, or 5 mg / mL. In a preferred embodiment, the phenolic preservative is phenol at a concentration of about 4 mg / mL. In certain embodiments, the phenolic preservative is m-cresol. In certain embodiments, the phenolic preservative is m-cresol present at a concentration of about 0.1 to about 10 mg / mL. In certain embodiments, the phenolic preservative is m-cresol present at a concentration of about 2 to about 6 mg / mL. In certain embodiments, the phenolic preservative is m-cresol present at a concentration of about 3.5 to about 5.5 mg / mL. In certain embodiments, the phenolic preservative is m-cresol present at a concentration of about 3.15 mg / mL. In other embodiments, the phenolic preservative is a mixture of phenol and m-cresol. In certain embodiments, the phenolic preservative is a mixture of phenol and m-cresol, where phenol is present at a concentration of about 1 to about 5 mg / mL and m-cresol is present at a concentration of about 0.1 to about 3.5 mg / mL.In certain embodiments, the phenolic preservative is a mixture of phenol and m-cresol, where the phenol is present at a concentration of about 1.5 mg / mL and the m-cresol is present at a concentration of about 1.58 mg / mL. In certain embodiments, the phenolic preservative is a mixture of phenol and m-cresol, where the phenol is present at a concentration of about 2 mg / mL and the m-cresol is present at a concentration of about 1.58 mg / mL. In certain embodiments, the phenolic preservative is a mixture of phenol and m-cresol, where the phenol is present at a concentration of about 3.5 mg / mL and the m-cresol is present at a concentration of about 0.32 mg / mL. In certain embodiments, the phenolic preservative is a mixture of phenol and m-cresol, where the phenol is present at a concentration of about 3.5 mg / mL and the m-cresol is present at a concentration of about 0.63 mg / mL. However, these embodiments should not be construed as limiting, as one of ordinary skill in the art can select the phenolic preservative and its concentration necessary to meet regulatory requirements using known techniques. For example, the European Pharmacopoeia, 9th Edition, Section 5.01.03 "Effectiveness of Antimicrobial Preservatives" and the United States Pharmacopoeia USP40-NF35, <51> See the chapter "Antimicrobial Efficacy Testing." See, e.g., Meyer, BK, et al., Antimicrobial Preservative Use in Parenteral Products: Past and Present, J. PHARM. SCI., Vol. 96, No. 12 (2007).

[0019] However, when both surfactants and preservatives are included in a composition at certain concentrations, they interact to cause phase separation, resulting in the formation of unacceptable visible turbidity or cloudiness. Without wishing to be bound by theory, it is believed that this phenomenon occurs when molecules of the phenolic preservative bind to micelles of the nonionic surfactant through bridging attractive forces. See, for example, Chen, J., et al., From the depletion attraction to the bridging attraction: The effect of solvent molecules on the effective colloidal interactions, THE JOURNAL OF CHEMICAL PHYSICS 2015, 142, 084904; Jie, C., et al., Size effects of solvent molecules on the phase behavior and effective interaction of colloidal systems with the bridging attraction, JOURNAL OF PHYSICS: CONDENSED MATTER 2016, 28, (45), 455102; Yuan, G.; Luo, J.; Han, C.C.; Liu, Y. Gelation transitions of colloidal systems with bridging attractions, PHYSICAL REVIEW E 2016, 94, (4), 040601. This leads to the association of multiple surfactant micelles, which then precipitate from solution. Those skilled in the art will understand that micelles are aggregates of surfactant molecules in which the hydrophilic portions of the nonionic surfactant molecules form an outer surface or shell that surrounds the hydrophobic portions, shielding the hydrophobic portions from the aqueous solvent by the outer surface or shell formed by the hydrophilic portions. The concentration of surfactant at which such micelles form is known as the critical micelle concentration, or CMC, and can be determined using techniques known in the art.See, e.g., Kerwin, BA Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: Structure and degradation pathways. JOURNAL OF PHARMACEUTICAL SCIENCES 2008, 97, (8), 2924-2935. Again, without wishing to be bound by theory, it is believed that the use of solvent modifiers described herein disrupts the cross-linking attraction between the preservative molecules and the surfactant micelles.

[0020] Regardless of the specifics of the mechanism, however, phase separation occurs when the combined concentrations of surfactant and preservative in a given composition are at or above what are referred to herein as their "concentration thresholds," meaning the concentrations at which the combination of surfactant and preservative, in the absence of a solvent modifier, causes phase separation and results in the formation of a cloudy or milky appearance. There is no universal concentration threshold that is generally applicable to any surfactant and preservative combination. Instead, the concentration threshold depends on the details of the formulation, including, inter alia, the identity of the surfactant(s) and preservative(s).

[0021] The concentration threshold of a given surfactant and preservative combination in any given formulation can be determined by those skilled in the art using known methods, including, inter alia, visual observation, but quantitative analyses such as turbidity analysis, as described in the Examples below, can also be used. See, for example, European Pharmacopoeia 7.0, Section 2.2.1, Degree of Liquid Clarity and Turbidity. Other analyses that may not directly reflect the formation of visible phase separation but may be relevant to the likelihood of a given composition for the eventual occurrence or formation of visible phase separation include size exclusion chromatography (SEC), high accuracy particle counter (HIAC) analysis, and microflow imaging (MFI).

[0022] Furthermore, visually detectable phase separation in some compositions containing surfactant and preservative combinations above a concentration threshold occurs essentially immediately after combining the surfactant and preservative, while in other compositions, phase separation does not become visually apparent until some time has passed after the formulation is prepared. For example, visually detectable phase separation has been observed to occur almost immediately in m-cresol-containing formulations, whereas in certain phenol-containing formulations, the formulation does not become visually detectable until up to approximately 15 minutes after the formulation is prepared. Therefore, to ensure that the solvent modifier sufficiently reduces phase separation due to a phenol preservative and surfactant combination that would otherwise exceed its concentration threshold, the appearance of the formulation should be inspected at least 10 minutes, preferably at least 15 minutes, after the formulation is prepared.

[0023] As noted above, the concentration threshold for a given surfactant and preservative combination depends on both the identity and concentration of the surfactant(s) and preservative(s); certain commercially available products contain both surfactants and preservatives, but remain clear and colorless because the surfactant and preservative combinations in those products are below their concentration thresholds. For example, a formulation of insulin glargine sold under the trade name LANTUS® contains 0.02 mg / mL polysorbate 20 and 2.7 mg / mL m-cresol, and a formulation of insulin glulisine sold under the trade name APIDRA® contains 0.01 mg / mL polysorbate 20 and 3.15 mg / mL m-cresol, but both formulations are clear because the combined concentrations of polysorbate 20 and m-cresol are below the concentration thresholds for this particular combination in both cases. In fact, as shown in the Examples below, in a formulation containing m-cresol at a concentration of 3.15 m-cresol, phase separation does not occur when polysorbate 20 is contained at a concentration of about 2 times or less of its CMC, but phase separation occurs at a concentration of about 5 times or more of the CMC.

[0024] As used herein, the term "phase separation" refers to the formation of physical particles that precipitate from solution.Whether or not phase separation occurs in a given composition can be determined visually, i.e., as indicated by a cloudy or milky appearance, as opposed to a transparent appearance, or by analytical techniques known to those skilled in the art.Similarly, as used herein, the term "transparent" refers to a clear solution that does not have a cloudy or milky appearance and does not contain solid particles of material that can be visually detected.Although analytical techniques known to those skilled in the art can be used, determining whether a formulation is transparent and free of particles can be determined visually.

[0025] The present invention involves the use of solvent modifiers to reduce the occurrence of phase separation in compositions that would otherwise (i.e., in the absence of the solvent modifier) ​​contain surfactant(s) and preservative(s) at concentrations above their threshold concentration. Compounds that can be used as solvent modifiers in the formulations of the present invention include PPG (CAS No. 57-55-6, molecular formula C3H8O2, molecular weight 76.095), NMP (CAS No. 872-50-4, molecular formula C5H9NO, molecular weight 99.133), and PEG400 (CAS No. 25322-68-3, molecular formula C 2n H 4n+2 O n+1 , n = 8.2-9.1, molecular weight 380-420 g / mol) and glycerol (CAS number 56-81-5, molecular formula C3H8O3, molecular weight 92.09382).

[0026] It should be noted that the compounds identified in the preceding paragraph that can be used as solvent modifiers in the formulations of the present invention may, in some cases, be commonly used excipients in pharmaceutical formulations and have functions other than their use as solvent modifiers in the formulations of the present invention. For example, glycerol is a commonly used agent for isotonicity purposes and is included in the formulations of insulin-containing products such as LANTUS® (insulin glargine), APIDRA® (insulin glulisine), HUMALOG® (insulin lispro), NOVOLOG® (insulin aspart), TRESIBA® (insulin degludec), HUMULIN® (human insulin), and TOUJEO® (insulin glargine). However, these insulin-containing products either do not contain any surfactants, or contain surfactants but below their concentration threshold in combination with the phenolic preservative(s) in their formulations. Similarly, PPG is also a commonly used pharmaceutical excipient for functions other than its use as a solvent modifier; for example, VICTOZA® (liraglutide) contains 14 mg / mL PPG but does not contain a non-ionic surfactant. PEG 400 is also a common excipient, found in, for example, ATIVAN® (lorazepam), but that product does not contain a non-ionic surfactant. Finally, although not as commonly used as glycerol or PPG, NMP is used in a product called ELIGARD (leuprolide acetate), but that product is non-aqueous and does not contain a phenolic preservative or surfactant.

[0027] Just as the concentration of solvent modifier required to reduce phase separation when surfactants and preservatives are present at concentrations above their threshold concentrations varies for a given surfactant and preservative combination, the required solvent modifier concentration also depends on several variables, including the identity and concentration of: (a) the specific surfactant(s) and preservative(s) used, (b) the specific solvent modifier(s) used, and (c) other excipients in the formulation, particularly tonicity agents, which are described in more detail below. In certain embodiments of the present invention, the solvent modifier is glycerol. In certain embodiments of the present invention, the solvent modifier is glycerol present at a concentration of about 10 to about 100 mg / mL. In certain embodiments, the concentration of glycerol is about 20 to about 80 mg / mL. In certain embodiments, the concentration of glycerol is selected from the group consisting of about 20, about 25, or about 80 mg / mL. In certain embodiments, the concentration of glycerol is about 20 mg / mL. In certain embodiments of the present invention, the solvent modifier is PPG. In certain embodiments of the present invention, the solvent modifier is PPG present at a concentration of about 10 to about 100 mg / mL. In certain embodiments, the concentration of PPG is about 15 to about 60 mg / mL. In certain embodiments, the concentration of PPG is selected from the group consisting of about 15, about 20, or about 60 mg / mL. In certain embodiments, the concentration of PPG is about 15 mg / mL. In certain embodiments of the present invention, the solvent modifier is NMP. In certain embodiments of the present invention, the solvent modifier is NMP present at a concentration of about 10 mg / mL to about 100 mg / mL. In certain embodiments, the concentration of NMP is about 20 to about 90 mg / mL. In certain embodiments, the concentration of NMP is about 27 to about 80 mg / mL. In certain embodiments, the concentration of NMP is selected from the group consisting of about 27, about 54, and about 80 mg / mL. In certain embodiments of the present invention, the solvent modifier is PEG 400. In certain embodiments of the invention, the solvent modifier is PEG 400 present at a concentration of about 5 to about 150 mg / mL, hi certain embodiments, the concentration of PEG 400 is about 40 to about 120 mg / mL.In certain embodiments, the concentration of PEG 400 is selected from the group consisting of about 40, about 80, about 110, and about 120 mg / mL. However, these concentrations should not be construed as limiting, as the selection of an appropriate concentration of solvent modifier for use in a given composition can be readily determined by one of ordinary skill in the art using known techniques, including visual observation and turbidity, and particulate analysis as described in the Examples below.

[0028] In addition to reducing the incompatibility between surfactants and preservatives, solvent modifiers may have additional functions in certain compositions, particularly as tonicity modifiers. Because the formulations of the present invention are intended for parenteral administration, it is desirable to administer the composition with a tonicity (i.e., osmolality) that closely matches the tonicity of the body fluid at the injection site, since administering a solution that is not nearly isotonic with the body fluid can cause a painful, stinging sensation. If the osmolality of the composition is sufficiently lower than the osmolality of the tissue (for blood, approximately 300 mOsmol / kg; the European Pharmacopoeia requirement for osmolality is >240 mOsmol / kg), the tonicity of the composition should be increased to approximately 300 mOsmol / kg. While glycerol and PPG are examples of solvent modifiers for use in the formulations of the present invention, they are also commonly used as tonicity modifiers in parenteral products, and such an effect can be achieved by adding a sufficient concentration of the solvent modifier. Thus, glycerol and / or PPG can be used in the compositions of the present invention to function as both a solvent modifier and / or a tonicity modifier. For example, in the dulaglutide-containing compositions described in the Examples below, glycerol and PPG are added in concentrations sufficient to increase the tonicity of the compositions so that they are approximately isotonic with body fluids at the injection site and to reduce the incompatibility of the surfactant(s) and preservative(s) in those compositions.

[0029] Increasing the tonicity of the composition to be lower than the osmotic pressure of tissue can also be achieved by adding additional tonicity agents.However, commonly used tonicity agents include sodium chloride and mannitol, and it has been found that in certain formulations, these agents can worsen the interaction between surfactants and preservatives, which leads to phase separation, and therefore may reduce the minimum concentration of surfactants and / or preservatives that reach the concentration threshold, and / or require a higher concentration of solvent modifier to avoid phase separation.In any case, if the addition of tonicity agents is necessary, the amount of tonicity agents to be added can be easily determined using standard techniques.Remington: The Science and Practice of Pharmacy, David B. Troy and Paul Beringer, eds., Lippincott Williams & Wilkins, 2006, pp.257-259;Remington: Essentials of Pharmaceutics, Linda Ed Felton, Pharmaceutical Press, 2013, pp.277-300. Furthermore, if the addition of a tonicity agent such as sodium chloride or mannitol is necessary and would exacerbate the interaction between the surfactant and the preservative, the amount of solvent modifier that needs to be added to prevent undesired phase separation can be readily determined by one of skill in the art using known techniques such as those described in the Examples below.

[0030] As described above, the concentrations of surfactants, preservatives, and solvent modifiers for use in the formulations of the present invention can be determined by those skilled in the art using known techniques, such as those described in the Examples below. For example, a formulator preparing a multi-use formulation of a protein or peptide-based pharmaceutical can, in some cases, first determine the identity and concentration of the nonionic surfactant required to provide sufficient stabilizing effect, then determine the identity and concentration of the preservative required to provide sufficient antimicrobial activity, and observe whether phase separation occurs. If phase separation does not occur, the combination of nonionic surfactant and preservative is below its concentration threshold, and no solvent modifier is required. If phase separation occurs, the formulator can determine whether a different surfactant and preservative combination can be used, or focus on determining the identity and concentration of a solvent modifier according to the present invention that will prevent such phase separation from occurring in that particular combination. Alternatively, the formulator can instead first determine the identity and concentration of the preservative required to provide sufficient antimicrobial activity, then determine the identity and concentration of the surfactant required to provide sufficient stabilizing effect, and then observe whether phase separation occurs when those excipients are combined. As in the previous scenario, if phase separation does not occur, the surfactant and preservative combination is below its concentration threshold and no solvent modifier is required. However, if phase separation does occur and an alternative preservative + surfactant combination that avoids such phase separation cannot be identified, the formulator turns their attention to determining the identity and concentration of a solvent modifier in accordance with the present invention.

[0031] In certain embodiments, the formulations of the present invention contain one or more buffers to control pH. The identity and concentration of any buffer(s) used may be relevant in certain cases to determine the concentration threshold of a given surfactant+preservative system and / or the solvent modifier necessary to avoid phase separation in that system. A "buffer" is a substance that resists changes in pH through the action of its acid-base conjugate components. In certain embodiments, the formulations of the present invention have a pH of about 4.0 to about 8.0, preferably about 5.5 to about 7.5, and more preferably about 6.0 to about 7.0. In certain preferred embodiments, the formulations of the present invention have a pH of about 6.5. In certain preferred embodiments, the formulations of the present invention have a pH of about 7. Suitable buffers for controlling the pH of the compositions of the present invention within the desired range include, but are not limited to, phosphate, acetate, citrate, or their acids, arginine, TRIS, and histidine buffers, and combinations thereof. "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol and any pharmacologically acceptable salts thereof. The free base and hydrochloride salt forms (i.e., TRIS-HCl) are two common forms of TRIS. TRIS is also known in the art as trimethylolaminomethane, tromethamine, and tris(hydroxymethyl)aminomethane. Preferred buffers in the compositions of the present invention are citrate or citric acid, and phosphate. Given the potential relevance of any buffer to determining concentration thresholds and / or solvent modifiers, formulators may wish to determine the required buffer before determining the identity and concentration of surfactants and / or preservatives to be used, as described in the previous paragraph.

[0032] The above description relates to how a formulator can determine the identity and concentration of surfactants, preservatives, and solvent modifiers to be included in a formulation, but does not necessarily relate to how the formulation is ultimately assembled once these identities and concentrations have been determined. While there may be some variability in the order in which components are added, the solvent modifier is typically added before the full concentration of both the phenolic preservative and surfactant has been added, i.e., before phase separation occurs. In certain preferred embodiments, the solvent modifier is the first component added to the formulation, followed by the phenolic preservative, followed by the protein or peptide, followed by the surfactant.

[0033] In addition to the above ingredients, the formulations of the present invention may contain other excipients. For example, certain protein or peptide-based pharmaceuticals may require additional stabilizers due to their sensitivity to oxidation or trace metals. Such stabilizers include antioxidants such as methionine, or chelating agents such as EDTA, respectively.

[0034] Proteins and peptides have low oral bioavailability due to their susceptibility to proteolysis and poor absorption in the gastrointestinal tract; therefore, most proteins and peptides are administered parenterally. The formulations of the present invention are intended for parenteral administration, which may include administration via intravenous (IV), subcutaneous (SC), intramuscular (IM), or intraperitoneal (IP) injection. In a preferred embodiment, the formulations of the present invention are designed for SC injection. Because the formulations of the present invention are suitable for multiple-dose administration, they are typically provided in a container closure system, such as a vial or cartridge, from which multiple doses can be withdrawn and administered. The formulations of the present invention can be provided, for example, in a vial, from which multiple doses can be withdrawn by a syringe for administration to a patient. The formulations of the present invention can also be provided in a cartridge for use in a pen device, from which multiple doses can be administered. The formulations of the present invention can also be provided in a container closure, such as a cartridge for use in an auto-injector or infusion pump capable of delivering multiple doses.

[0035] Additional embodiments of the present invention are described below. An aqueous composition comprising: a protein or peptide, a non-ionic surfactant, a phenolic preservative, and a solvent denaturant.

[0036] The composition of the above embodiment, wherein the composition is sterile.

[0037] The composition of any of the above embodiments, wherein the non-ionic surfactant and phenolic preservative are present at concentrations above their concentration thresholds in the absence of the solvent modifier.

[0038] The composition of any of the above embodiments, wherein the solvent modifier is present in a concentration sufficient to ensure that the solution remains clear.

[0039] The composition of the above embodiment, wherein the solution remains clear for at least 15 minutes. The composition of the preceding embodiment, wherein the solution remains clear for at least 24 hours. The composition of the preceding embodiment, wherein the solution remains clear for at least one week. The composition of the preceding embodiment, wherein the solution remains clear for at least one month. The composition of the preceding embodiment, wherein the solution remains clear for at least six months. The composition of the preceding embodiment, wherein the solution remains clear for at least one year.

[0040] The composition of any of the above embodiments, wherein the solution remains clear throughout its shelf life.

[0041] The composition of any of the above embodiments, wherein the solvent modifier is present at a concentration sufficient to prevent phase separation due to interaction between the nonionic surfactant and the phenolic preservative.

[0042] The composition of any of the above embodiments, wherein the protein or peptide is present at a concentration ranging from about 0.1 to about 100 mg / mL.

[0043] The composition of any of the above embodiments, wherein the protein or peptide is present at a concentration ranging from about 0.5 to about 50 mg / mL.

[0044] The composition of any of the above embodiments, wherein the protein or peptide is present at a concentration in the range of about 1 to about 10 mg / mL.

[0045] The composition of any of the above embodiments, wherein the protein or peptide is selected from the group consisting of a GLP-1 receptor agonist, insulin, a GIP receptor agonist, a glucagon receptor agonist, PYY, GDF, an amylin receptor agonist, a calcitonin receptor agonist, and an interleukin. The composition of the preceding embodiment, wherein the protein or peptide is an Fc fusion protein.

[0046] The composition of any of the above embodiments, wherein the protein or peptide is dulaglutide. The composition of the preceding embodiments, wherein the concentration of dulaglutide is about 1.5 to about 9 mg / mL. The composition of the preceding embodiments, wherein the concentration of dulaglutide is selected from the group consisting of 1.5, 3.0, 6.0, and 9.0 mg / mL.

[0047] The composition of any of the above embodiments, wherein the non-ionic surfactant is a surfactant of the polysorbate type. The composition of the preceding embodiments, wherein the non-ionic surfactant is selected from the group consisting of PS20, PS80, poloxamer 188, and poloxamer 407. The composition of the preceding embodiments, wherein the non-ionic surfactant is either PS20 or PS80.

[0048] The composition of any of the above embodiments, wherein the nonionic surfactant is PS80. The composition of the preceding embodiments, wherein the concentration of PS80 is from about 0.01 mg / mL to about 1 mg / mL. The composition of the preceding embodiments, wherein the concentration of PS80 is from about 0.05 mg / mL to about 0.5 mg / mL. The composition of the preceding embodiments, wherein the concentration of PS80 is from about 0.1 mg / mL to about 0.4 mg / mL. The composition of the preceding embodiments, wherein the concentration of PS80 is from about 0.2 mg / mL to about 0.3 mg / mL. The composition of the preceding embodiments, wherein the concentration of polysorbate 80 is either 0.2 mg / mL or 0.25 mg / mL.

[0049] The composition of any of the above embodiments, wherein the nonionic surfactant is PS20. The composition of the preceding embodiments, wherein the concentration of PS20 is greater than about twice its CMC. The composition of the preceding embodiments, wherein the concentration of polysorbate 20 is from about 0.01 mg / mL to about 1 mg / mL. The composition of the preceding embodiments, wherein the concentration of PS20 is from about 0.05 mg / mL to about 0.5 mg / mL. The composition of the preceding embodiments, wherein the concentration of PS20 is from about 0.1 mg / mL to about 0.4 mg / mL.

[0050] The composition of any of the above embodiments, wherein the nonionic surfactant is poloxamer 188. The composition of the preceding embodiment, wherein the concentration of poloxamer 188 ranges from about 0.01 to about 2 mg / mL. The composition of the preceding embodiment, wherein the concentration of poloxamer 188 ranges from about 0.5 to about 1.5 mg / mL.

[0051] The composition of any of the above embodiments, wherein the phenolic preservative is present in a concentration sufficient to meet regulatory and pharmacopoeial antimicrobial preservative requirements.

[0052] The composition of any of the above embodiments, wherein the phenolic preservative is selected from the group consisting of phenol, m-cresol, benzyl alcohol, and phenoxyethanol. The composition of the preceding embodiments, wherein the phenolic preservative is benzyl alcohol. The composition of the preceding embodiments, wherein the benzyl alcohol is present at a concentration of about 10 mg / mL.

[0053] In certain embodiments, the phenolic preservative is phenoxyethanol. The composition of the preceding embodiments, wherein the phenoxyethanol is present in a concentration of about 10 to about 15 mg / mL.

[0054] The composition of any of the above embodiments, wherein the phenolic preservative is selected from the group consisting of phenol and m-cresol and mixtures thereof.

[0055] The composition of any of the above embodiments, wherein the phenol preservative is phenol. The composition of the preceding embodiments, wherein the concentration of phenol is about 1 to about 10 mg / mL. The composition of the preceding embodiments, wherein the concentration of phenol is about 3 to about 6 mg / mL. The composition of the preceding embodiments, wherein the concentration of phenol is at least about 3 mg / mL. The composition of the preceding embodiments, wherein the phenol preservative is phenol at a concentration selected from the group consisting of 3, 3.5, 4, 4.5, or 5 mg / mL. The composition of the preceding embodiments, wherein the concentration of phenol is about 5 mg / mL.

[0056] The composition of any of the above embodiments, wherein the phenolic preservative is m-cresol. The composition of any of the above embodiments, wherein the phenolic preservative is m-cresol and is present at a concentration of about 0.1 to about 10 mg / mL. The composition of the preceding embodiments, wherein the phenolic preservative is m-cresol and is present at a concentration of about 2 to about 6 mg / mL. The composition of the preceding embodiments, wherein the phenolic preservative is m-cresol and is present at a concentration of about 3.5 to about 5.5 mg / mL.

[0057] The composition of any of the above embodiments, wherein the phenolic preservative is a mixture of phenol and m-cresol. The composition of the preceding embodiments, wherein the phenolic preservative is a mixture of phenol and m-cresol, wherein phenol is present at a concentration of about 1 to about 5 mg / mL and m-cresol is present at a concentration of about 0.1 to about 3.5 mg / mL. The composition of the preceding embodiments, wherein the phenolic preservative is a mixture of phenol and m-cresol, wherein phenol is present at a concentration of about 1.5 to about 2 mg / mL and m-cresol is present at a concentration of 1.58 mg / mL.

[0058] The composition of any of the above embodiments, wherein the phenolic preservative is a mixture of phenol and m-cresol, wherein the phenol is present at a concentration of about 3.5 to about 4 mg / mL and the m-cresol is present at a concentration of about 0.32 mg / mL to about 0.63 mg / mL. The composition of the preceding embodiment, wherein the concentration of phenol is about 3.5 mg / mL and the concentration of m-cresol is about 0.32 mg / mL.

[0059] The composition of any of the above embodiments, wherein the solvent denaturant is selected from the group consisting of PPG, NMP, PEG400 and glycerol.

[0060] The composition of any of the above embodiments, wherein the solvent modifier is glycerol. The composition of any of the above embodiments, wherein the solvent modifier is glycerol and is present at a concentration of about 10 to about 100 mg / mL. The composition of the preceding embodiments, wherein the concentration of glycerol is about 20 to about 80 mg / mL. The composition of the preceding embodiments, wherein the concentration of glycerol is selected from the group consisting of about 20, about 25, or about 80 mg / mL. The composition of the preceding embodiments, wherein the concentration of glycerol is about 20 mg / mL.

[0061] The composition of any of the preceding embodiments, wherein the solvent modifier is PPG. The composition of any of the preceding embodiments, wherein the solvent modifier is PPG and is present at a concentration of about 10 to about 100 mg / mL. The composition of the preceding embodiments, wherein the concentration of PPG is about 15 to about 60 mg / mL. The composition of the preceding embodiments, wherein the concentration of PPG is selected from the group consisting of about 15, about 20, or about 60 mg / mL. The composition of the preceding embodiments, wherein the concentration of PPG is about 15 mg / mL.

[0062] The composition of any of the preceding embodiments, wherein the solvent modifier is NMP. The composition of any of the preceding embodiments, wherein the solvent modifier is NMP and is present at a concentration of about 10 mg / mL to about 100 mg / mL. The composition of the preceding embodiments, wherein the concentration of NMP is about 20 to about 90 mg / mL. The composition of the preceding embodiments, wherein the concentration of NMP is about 27 to about 80 mg / mL. The composition of the preceding embodiments, wherein the concentration of NMP is selected from the group consisting of about 27, about 54, and about 80 mg / mL.

[0063] The composition of any of the above embodiments, wherein the solvent modifier is PEG 400. The composition of any of the above embodiments, wherein the solvent modifier is PEG 400 and is present at a concentration of about 5 to about 150 mg / mL. The composition of the preceding embodiments, wherein the concentration of PEG 400 is about 40 to about 120 mg / mL. The composition of the preceding embodiments, wherein the concentration of PEG 400 is selected from the group consisting of about 40, about 80, about 110, and about 120 mg / mL.

[0064] The composition of any of the above embodiments, wherein the composition further comprises a tonicity agent. The composition of the preceding embodiments, wherein the tonicity agent is selected from the group consisting of NaCl and mannitol.

[0065] The composition of any of the above embodiments, wherein the composition further comprises a buffer. The composition of the preceding embodiment, wherein the buffer is selected from the group consisting of phosphate, acetate, citrate, or acids thereof, arginine, TRIS, and histidine. The composition of the preceding embodiment, wherein the buffer is phosphate. The composition of the preceding embodiment, wherein the concentration of phosphate is about 10 mM. The composition of any of the above embodiments, wherein the composition further comprises a buffer that is citrate. The composition of the preceding embodiment, wherein the concentration of citrate is about 10 mM.

[0066] The composition of any of the above embodiments, wherein the pH of the composition is about 4 to about 8. The composition of the preceding embodiment, wherein the pH of the composition is about 5.5 to about 7.5. The composition of the preceding embodiment, wherein the pH of the composition is about 6.0 to 7.0. The composition of the preceding embodiment, wherein the pH of the composition is about 6.5 or about 7.

[0067] The composition of any of the above embodiments, wherein the composition further comprises an additional stabilizer. The composition of the preceding embodiments, wherein the additional stabilizer is an antioxidant or a chelating agent. The composition of the preceding embodiments, wherein the antioxidant is methionine and the chelating agent is EDTA.

[0068] The aqueous composition suitable for parenteral administration comprises dulaglutide, a solvent modifier selected from the group consisting of PS80, PPG, and glycerol, and a phenolic preservative selected from the group consisting of phenol, m-cresol, and a mixture thereof. The composition of the preceding embodiments, wherein the dulaglutide concentration is selected from the group consisting of 1.5, 3, 6, or 9 mg / mL. The composition of the preceding embodiments, wherein the PS80 concentration is either 0.2 or 0.25 mg / mL. The composition of the preceding embodiments, wherein the solvent modifier is either 15 mg / mL PPG or 20 mg / mL glycerol. The composition of the preceding embodiments, wherein the phenolic preservative is either 4 mg / mL phenol or a combination of 3.5 mg / mL phenol and 0.32 mg / mL m-cresol. The composition of the preceding embodiments, further comprising a buffer. The composition of the preceding embodiments, wherein the buffer is citrate. The composition of the preceding embodiments, wherein the citrate concentration is 10 mM. The composition of the preceding embodiments, wherein the pH of the composition is about 6.5.

[0069] A container closure system comprising any of the above compositions. The container closure system of the previous embodiment, wherein the container closure system is a vial or a cartridge.

[0070] A multi-dose pen device comprising any of the above compositions.

[0071] A multi-dose auto-injector containing any of the above compositions.

[0072] An infusion pump comprising any of the above compositions.

[0073] A method of preparing any of the above compositions comprising preparing or obtaining a buffer, then adding a solvent denaturant, then adding a phenol preservative, then adding a protein or peptide-based API, and then adding a surfactant.

[0074] A method for preparing an aqueous composition suitable for parenteral administration comprising including a non-ionic surfactant and a phenolic preservative above their concentration thresholds and including a solvent modifier in the composition at a concentration sufficient to ensure that the composition remains clear.

[0075] The method of the above embodiment, wherein the composition comprises any of the compositions described above.

[0076] Embodiments of the present invention are further described in the following examples, which should not be construed as limiting. [Brief explanation of the drawings]

[0077] (No corresponding part in the original text) [Example]

[0078] Concentration threshold for compositions containing 0.2 mg / mL PS80 The commercial formulation of dulaglutide, sold under the trade name TRULICITY®, contains 0.2 mg / mL PS80 as a stabilizer. To examine the effect of adding a phenolic preservative, a placebo solution containing 0.2 mg / mL PS80 was prepared in 10 mM citrate buffer at pH 6.5. Test articles were prepared by adding sufficient m-cresol or phenol to a sample of this solution to obtain a formulation containing 0.2 mg / mL and either 3.15 mg / mL m-cresol or 5 mg / mL phenol. The placebo and test articles were visually inspected. The placebo solution was clear and colorless, whereas the test articles rapidly became cloudy or milky in appearance, respectively. Thus, the concentration thresholds for the two preservative-containing solutions were exceeded.

[0079] Concentration thresholds for compositions containing m-cresol and PS20 Tests were conducted to determine the concentration thresholds for the combination of PS20 and m-cresol, a nonionic surfactant and phenolic preservative used in the commercial formulations of insulin glargine (sold under the tradenames LANTUS®) and insulin glulisine (sold under the tradename APIDRA®), containing PS20 at concentrations of 0.02 mg / mL and 0.01 mg / mL and m-cresol at concentrations of 2.7 and 3.15 mg / mL, respectively. Placebo solutions were prepared in 10 mM phosphate buffer at pH 7 containing 3.15 mg / mL m-cresol and various concentrations of PS20 ranging from 1 / 4 to 10 times its CMC. Vials were analyzed by visual inspection. The results are provided in Table 1 below. [Table 1]

[0080] The results indicate that phase separation did not occur in these compositions when polysorbate 20 was included at concentrations approximately two times its CMC or less, but did occur at concentrations approximately five times its CMC or more. Thus, the combination of m-cresol and polysorbate 20 at 3.15 mg / mL, at a concentration five times or more the CMC, is above the m-cresol and polysorbate 20 concentration threshold, whereas the combinations of m-cresol and polysorbate 20 at 3.15 mg / mL at concentrations two times or less the CMC (e.g., 0.02 and 0.01 mg / mL used in LANTUS and APIDRA) are below the m-cresol and polysorbate 20 concentration threshold.

[0081] Turbidity of compositions containing various concentrations of m-cresol and PS80 A study was conducted to evaluate the relationship of both m-cresol and PS80 concentrations to the occurrence of phase separation. Batches of 10 mM citrate buffer, adjusted to pH 6.5, were prepared to serve as controls and buffer matrices for formulation of the test article. M-cresol was added to portions of the buffer matrix to prepare solutions containing m-cresol at concentrations of 1.58 mg / mL, 2.70 mg / mL, or 3.15 mg / mL. Polysorbate 80 was measured and dissolved in separate portions of the citrate buffer to prepare two stock solutions, one with 10 mg / mL polysorbate 80 and the other with 40 mg / mL polysorbate. The surfactant stock solutions were gradually added to various amounts of phenolic preservative-containing solutions, in the amounts shown in Table 2 below, to generate formulations containing a wide range of polysorbate 80 concentrations. [Table 2]

[0082] The turbidity of the resulting formulation is measured using a HACH turbidity meter (model: 2100AN, tag number: K349924). The instrument is calibrated using a turbidity standard before use. A thin coating of silicone oil is applied to the exterior of the test tube to hide minor imperfections in the glass tube. Approximately 7 mL of solution is used for turbidity measurement. The results are provided in Figure 1. As shown in Figure 1, the occurrence and magnitude of turbidity depend on the concentrations of both m-cresol and PS80.

[0083] The effect of various concentrations of solvent denaturants, commonly used tonicity agents, preservatives and surfactants. Studies will be conducted to assess the effect of including various concentrations of solvent modifiers and other excipients commonly used as tonicity agents in protein and peptide-based formulations on the compatibility of preservatives and surfactants in solution.

[0084] In one study, a batch of 10 mM phosphate buffer adjusted to pH 6.5 was used as the buffer matrix. Subsequently, buffers containing 3.15 mg / mL m-cresol and either a solvent modifier or a commonly used tonicity agent were prepared as shown in Table 3. [Table 3]

[0085] Polysorbate 80 is measured and dissolved in phosphate buffer to prepare two stock solutions, one with 10 mg / mL polysorbate 80 and the other with 40 mg / mL polysorbate, which are gradually added in the amounts shown in Table 2 above to various amounts of the solvent denaturant or tonicity agent-containing formulations listed in Table 3 above to produce formulations each containing a range of concentrations of polysorbate 80. The turbidity of the resulting formulations is measured as described above.

[0086] The results are provided in Figure 2. As shown in Figure 2, the addition of mannitol and NaCl each resulted in a leftward shift in the turbidity data compared to the control, suggesting that their inclusion resulted in more turbidity development at a given PS80 concentration in this study, while the addition of PPG, glycerol, and NMP each resulted in a rightward shift in the turbidity data compared to the control, suggesting that PEG400 prevented turbidity development and their inclusion resulted in reduced turbidity development at a given PS80 concentration in this study.

[0087] In another series of tests, a 10 L batch of 10 mM citrate buffer containing 2.723 mg / mL citric acid and 0.1422 mg / mL sodium citrate, adjusted to a pH of 6.5, was prepared and used as the buffer matrix. Subsequently, buffers containing m-cresol and various excipients were prepared, as summarized in Table 4. Citric acid, sodium citrate dihydrate, polysorbate 80, m-cresol, liquefied phenol, mannitol, and sodium chloride were obtained from Eli Lilly (Indianapolis, Indiana). Glycerol, propylene glycol, N-methyl-2-pyrrolidone (NMP), and polyethylene glycol 400 (PEG 400) were obtained from Sigma-Aldrich (Milwaukee, Wisconsin). [Table 4]

[0088] Polysorbate 80 was measured and dissolved in phosphate buffer to prepare two stock solutions, one with 10 mg / mL polysorbate 80 and the other with 40 mg / mL polysorbate. These were gradually added in the amounts shown in Table 2 above to various amounts of the solvent denaturant or tonicity agent-containing formulations listed in Table 4 above to produce formulations containing a wide range of polysorbate 80 concentrations. The turbidity of the resulting formulations was measured as described above. The results are presented in Figures 3-8.

[0089] The contributions of both surfactant and preservative concentrations, as well as the detrimental effects of mannitol and NaCl, can be seen in Figures 3 and 4. As seen in Figures 3 and 4, formulations containing 1.58 mg / mL m-cresol do not become turbid at any of the PS80 concentrations examined, including in the presence of mannitol or NaCl. Thus, a concentration threshold was not reached for any of the compositions containing 1.58 mg / mL m-cresol examined in this study. However, when the m-cresol concentration is increased to 3.15 mg / mL, turbidity development is observed as the concentration of polysorbate 80 increases. Finally, the presence of either mannitol or NaCl exacerbates turbidity development in a dose-dependent manner.

[0090] The effect of glycerol and PPG on turbidity development at specific surfactant and preservative concentrations can be seen in Figure 5. As shown in Figure 5, the inclusion of PPG reduces turbidity development in a dose-dependent manner. On the other hand, glycerol resulted in a leftward shift in the turbidity data compared to the control, suggesting that it did not reduce the turbidity of the compositions examined in this study.

[0091] The effect of NMP can be seen in Figure 6. As shown in Figure 6, NMP reduces the occurrence of turbidity in a dose-dependent manner.

[0092] The effect of PEG400 at a particular PS80 and m-cresol concentration, once a concentration threshold is reached, can be seen in Figure 7. As shown in Figure 7, PEG400 reduces turbidity development in a dose-dependent manner.

[0093] Finally, a comparison of the concentration thresholds for combinations of PS80 with m-cresol or phenol in the presence of either mannitol or NaCl can be seen in Figure 8. As shown in Figure 8, although both preservatives caused turbidity development, phenol was more compatible with PS80 than m-cresol at all concentrations examined, and mannitol had a more detrimental effect than NaCl.

[0094] In summary, the data from these studies indicate that a concentration threshold is inherent to the identity and concentration of surfactants and preservatives in the composition, and that the occurrence of phase separation leading to turbidity in such compositions can be either reduced in a dose-dependent manner by the inclusion of solvent modifiers or exacerbated in a dose-dependent manner by the inclusion of certain commonly used tonicity agents.

[0095] Concentration thresholds and the effect of solvent denaturants in compositions containing model proteins of various molecular weights Tests are conducted to confirm that the interaction of surfactants with preservatives to produce turbidity in the composition, and the ability to reduce that phenomenon by including a solvent modifier, is independent of the identity of the protein in the composition. The proteins identified for inclusion in this test are selected to encompass a wide range of molecular weights, as shown in Table 5 below. [Table 5]

[0096] Sodium phosphate monobasic monohydrate, disodium hydrogen phosphate heptahydrate, PS80, and m-cresol are obtained from Eli Lilly (Indianapolis, Indiana). N-methyl-2-pyrrolidone (NMP), cytochrome C, lysozyme, β-lactoglobulin, and thyroglobulin are obtained from Sigma-Aldrich (Milwaukee, Wisconsin). Bovine serum albumin is obtained from Akron. All materials are used as received.

[0097] Prepare a 2 L batch of 10 mM phosphate buffer by combining 0.7821 mg / mL disodium hydrogen phosphate and 0.62 mg / mL monosodium phosphate in water and adjusting the pH to 7.0 to serve as the buffer matrix for testing. Protein formulations containing PS80, m-cresol, and / or NMP are then prepared and visually inspected. Details of the composition and results are provided in Table 6 below. [Table 6]

[0098] The data in Table 6 show that for all proteins tested, including multiple concentrations of BSA, the combination of 0.2 mg / mL polysorbate 80 and 3.15 mg / mL m-cresol in the absence of a solvent modifier causes phase separation, resulting in a turbid appearance, while the inclusion of 81 mg / mL NMP prevents such phase separation from occurring.

[0099] Stability study of preserved dulaglutide formulations The study was designed to examine the stability of preserved formulations of dulaglutide prepared with solvent modifiers according to the present invention. The currently available commercial formulation of TRULICITY® (dulaglutide) contains 3 mg / mL dulaglutide, 0.2 mg / mL PS80, and 46.4 mg / mL mannitol in 10 mM citrate buffer (pH 6.5). As noted above, previous efforts to preserve this formulation by adding a phenolic preservative resulted in phase separation due to the incompatibility of PS80 with the phenolic preservative. However, by using the solvent modifiers described herein, improved formulations were developed that contain enough preservative to achieve sufficient antimicrobial efficacy and the 0.2 mg / mL PS80 required for stability purposes, but without the phase separation observed with non-solvent modifier-containing formulations. The compositions of these formulations are shown in Table 7 below. [Table 7]

[0100] The study is designed to examine the stability of dulaglutide in these compositions. A 5 mM, pH 6.5 citrate buffer solution is prepared and used as is. The appropriate amount of citrate buffer is transferred to a 500 mL volumetric flask. The calculated amounts of preservative and solvent modifier are then added to the same flask and mixed to dissolve and ensure a homogeneous solution. Using a graduated cylinder, 38.5 mL of dulaglutide drug substance is measured and transferred to a volumetric flask. The solution is mixed until homogeneous. Simultaneously, a 100 mg / mL polysorbate 80 stock solution is prepared. Approximately 1000 mg of polysorbate is transferred to a glass beaker and dissolved in 10 mL of buffer. Using a transfer pipette, 1 mL of polysorbate 80 stock solution is transferred to a volumetric flask. The appropriate amount of buffer is then added until the liquid meniscus reaches the 500 mL mark. The solution is further mixed to ensure homogeneity and then filtered through a 0.22 μm filter. The filtered drug product is then filled into 3 mL cartridges. The solution in the cartridge was visually confirmed to be clear, suggesting that no phase separation had occurred due to interactions between the surfactant and preservative.

[0101] Additionally, filled cartridges are stored at 5°C for stability testing. This storage temperature is representative of the recommended storage temperature of 2-8°C for dulaglutide drug products. At pre-specified time points, samples are removed from storage, visually verified to be clear and free of particulates, and tested by various methods described below.

[0102] HIAC. The HIAC test is used to measure subvisible particulate content and is USP <787> (invisible microparticles in therapeutic protein injections) and <788> Test samples were performed as described in (Microparticles for Injection). These are harmonized with European Pharmacopoeia 2.9.19 and Japanese Pharmacopoeia 6.07. At each time point, five aliquots of 0.5 mL of solution were removed from the 3 mL cartridge and pooled, so the measurement result(s) reflect the average of five samples. Results are provided in Table 8 below. [Table 8]

[0103] USP <788> To be compliant with the Particulates in Injectable Solutions (PFIP), parenteral products containing therapeutic protein injections such as dulaglutide must have no more than 6,000 particulates 10 μm or larger and no more than 600 particulates 25 μm or larger per container. As shown in Table 8, all samples examined were well within the FDA limits for parenteral products.

[0104] MFI. The MFI test is used to detect particulate matter other than air bubbles present in injectable and parenteral solutions. This method is a stability characterization method for informational purposes only, using flow imaging technology to enumerate and classify particles not visible to the naked eye with respect to size, concentration, and morphology. Samples are removed from storage and examined after 12 months. Results are provided in Table 9 below. Particulates 5 μm or larger with an aspect ratio (AR) greater than 0.85 are highly circular in shape and may be silicone from stoppers, as opposed to protein particles. [Table 9]

[0105] The data in Table 9 are comparable to previous data for dulaglutide medications.

[0106] SEC. The monomeric purity of dulaglutide is measured using a size exclusion (SEC) HPLC method, which separates aggregates and fragmented species from intact monomeric protein.

[0107] The monomer purity of dulaglutide drug product is measured by size-exclusion HPLC. This method uses isocratic separation on a 200 angstrom pore size silica gel column with UV detection at 214 nm, which is close to the absorbance maximum of the drug's peptide backbone and therefore does not require response factor correction. This method separates higher molecular weight forms (total aggregates) from monomeric dulaglutide. This method has been demonstrated to be specific and stable, separating higher molecular weight forms from dulaglutide monomer. Monomer and aggregates are reported as peak area percent relative to total area. Data are provided in Table 10. [Table 10]

[0108] The data in Table 10 are within the acceptable range for dulaglutide drug products.

[0109] RP-HPLC. This method is designed to determine the purity and related substances / impurities of dulaglutide drug product. Related impurities resulting from glycosylation, N-terminal truncation, linker cleavage, and oxidation of the Fc region are separated from unmodified dulaglutide using reversed-phase gradient HPLC with UV detection at 214 nm, which is close to the absorbance maximum of the drug's peptide backbone and therefore does not require response factor correction. The method has been demonstrated to be specific and stable, separating degradation products from the main peak. [Table 11]

[0110] The data in Table 11 are within the acceptable range for dulaglutide.

[0111] Restriction Digestion. A restriction digestion method was designed to determine modifications to the GLP-1 analog in dulaglutide drug products. Drug samples are subjected to mild trypsin digestion conditions, which release the GLP-1 analog and linker from the Fc portion of the molecule. The GLP-1 analog is digested into three small peptides. This method uses reversed-phase gradient HPLC separation with UV detection at 214 nm, which is close to the absorbance maximum of the drug's peptide backbone and does not require response factor correction. This method separates related impurities resulting from N-terminal truncations, N-terminal modifications (Des H / HG, pyruvylation), oxidation of tryptophan at position 25, and hydroxylation of lysine at position 28 from the unmodified dulaglutide peptide. This method has been demonstrated to be specific and stable, separating related substances and impurities from their respective unmodified peptides. Results are provided in Table 12. [Table 12]

[0112] The data in Table 11 are within the acceptable range for dulaglutide.

[0113] CE-SDS NR. Capillary electrophoresis with sodium dodecyl sulfate, non-reducing (CE-SDS NR) is used to determine the purity of dulaglutide drug products. Dulaglutide molecules are denatured, and molecular variants are separated by size through a proprietary gel matrix electrokinetically loaded into an uncoated capillary. Separation occurs when a current is applied to the capillary, and molecular variants are detected at 214 nm UV, which is close to the absorbance maximum of the drug's peptide backbone and does not require response factor correction. This method separates high molecular weight and single-chain forms from monomeric dulaglutide. This method has been demonstrated to be specific and stable, separating aggregates and single-chain forms from dulaglutide monomers.

[0114] [Table 13]

[0115] The data in Table 13 are within the acceptable range for dulaglutide drug products.

[0116] In summary, the above studies support the conclusion that a preserved formulation of dulaglutide containing the same PS80 content used to provide sufficient stability in the currently available commercial formulation of TRULICITY can be prepared without phase separation due to preservative-surfactant interactions through the use of a solvent modifier, and that the protein in such a formulation remains sufficiently stable.

Claims

1. 1. An aqueous composition comprising: a) dulaglutide, b) a non-ionic surfactant selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, and mixtures thereof; c) a phenolic preservative selected from the group consisting of phenol, m-cresol, benzyl alcohol, phenoxyethanol, and mixtures thereof; d) a solvent modifier which is propylene glycol; A multi-use aqueous composition that prevents phase separation.

2. 10. The composition of claim 1, wherein the solution remains clear.

3. 10. The composition of claim 1, wherein dulaglutide is present at a concentration ranging from about 0.1 to about 100 mg / mL.

4. 10. The composition of claim 1, wherein dulaglutide is present at a concentration ranging from about 0.5 to about 50 mg / mL.

5. 10. The composition of claim 1, wherein dulaglutide is present at a concentration ranging from about 1 to about 10 mg / mL.

6. 10. The composition of claim 1, wherein the concentration of dulaglutide is from about 1.5 to about 9 mg / mL.

7. 7. The composition of claim 6, wherein the concentration of dulaglutide is selected from the group consisting of about 1.5, about 3.0, about 6.0, and about 9.0 mg / mL.

8. 8. The composition of any one of claims 1 to 7, wherein the non-ionic surfactant is polysorbate 80 at a concentration of about 0.1 mg / mL to about 0.4 mg / mL.

9. 9. The composition of claim 8, wherein the concentration of polysorbate 80 is selected from the group consisting of about 0.2 mg / mL and about 0.25 mg / mL.

10. A composition according to any one of claims 1 to 9, wherein the phenolic preservative is phenol.

11. 11. The composition of claim 10, wherein the phenol concentration is about 4 mg / mL.

12. A composition according to any one of claims 1 to 9, wherein the phenolic preservative is a mixture of phenol and m-cresol.

13. 13. The composition of claim 12, wherein the phenol concentration is about 3.5 mg / mL and the m-cresol concentration is about 0.32 mg / mL.

14. The composition of any one of claims 1 to 13, wherein the propylene glycol concentration is about 15 mg / mL.

15. The composition of any one of claims 1 to 14, wherein the composition further comprises a buffer.

16. 16. The composition of claim 15, wherein the buffer is selected from the group consisting of TRIS, phosphate, and citrate.

17. 17. The composition of claim 16, wherein the buffer is citrate.

18. 18. The composition of claim 17, wherein the citrate concentration is 10 mM.

19. The composition of any one of claims 1 to 18, wherein the pH of the composition is about 6 to 8.

20. The composition of any one of claims 1 to 19, wherein the pH of the composition is about 6.

5.

21. a) dulaglutide, b) Polysorbate 80; and c) a phenolic preservative selected from the group consisting of phenol, m-cresol, and mixtures thereof; d) a solvent modifier that is propylene glycol.

22. 22. The composition of claim 21, wherein the concentration of polysorbate 80 is about 0.2 mg / mL.

23. 23. The composition of claim 22, wherein the dulaglutide concentration is selected from the group consisting of about 1.5 mg / mL, about 3 mg / mL, about 6 mg / mL and about 9 mg / mL.

24. 24. The composition of claim 23, wherein the phenolic preservative is about 4 mg / mL phenol.

25. 24. The composition of claim 23, wherein the phenolic preservative is a combination of about 3.5 mg / mL phenol and about 0.32 mg / mL m-cresol.

26. 26. The composition of any one of claims 21 to 25, wherein the concentration of propylene glycol is about 15 mg / mL.

27. 27. The composition of any one of claims 21 to 26, further comprising 10 mM citrate buffer, wherein the pH of the composition is 6.

5.

28. A method for preparing a composition according to any one of claims 1 to 27, comprising the steps of: a) preparing or obtaining a buffer solution; b) adding the solvent modifier; c) adding said phenolic preservative; d) adding dulaglutide; and e) adding said non-ionic surfactant.

29. 1. A method for preventing phase separation in an aqueous composition comprising dulaglutide, a nonionic surfactant, and a phenolic preservative due to interaction between the nonionic surfactant and the phenolic preservative, comprising: formulating said aqueous composition, wherein a solvent modifier is added before the full concentration of both said nonionic surfactant and said phenolic preservative is added; The method wherein the solvent modifier is propylene glycol.

30. The method of claim 30, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, and mixtures thereof; 30. The method of claim 29, wherein the phenolic preservative is a phenolic preservative selected from the group consisting of phenol, m-cresol, benzyl alcohol, phenoxyethanol, and mixtures thereof.

31. 31. The method of claim 29 or 30, wherein the aqueous composition is for multiple use.

32. A method according to any one of claims 29 to 31, wherein the aqueous composition remains clear.

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