Preserved formulations

Abstract

To provide pharmaceutical compositions that are suitable for parenteral administration and contain a protein or peptide and a surfactant.SOLUTION: An aqueous composition comprises: a) a protein or peptide; b) A non-ionic surfactant; c) a phenolic preservative; and d) a solvent modifier; wherein the non-ionic surfactant and the phenolic preservative are present in concentrations above their concentration threshold in the absence of a solvent modifier; and wherein the solvent modifier is present in a concentration sufficient to ensure the solution remains clear.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] The present invention relates to a preserved surfactant-containing pharmaceutical composition suitable for parenteral administration. The composition includes one or more preservatives such as metacresol or phenol, one or more surfactants such as polysorbate 80 (PS80), one or more pharmaceutical active 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 proteolysis in the gastrointestinal tract when administered orally, and in some cases, they need to be formulated with nonionic surfactants to ensure the stability of the proteins throughout storage and use conditions. However, a limitation of such surfactant-containing formulations, which require surfactant concentrations above certain levels, is that they cannot be adequately stored in a form for multiple uses 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 previously recognized. 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 Approach, 107J.PHARM.SCI.10, 2531-2537 (2018). However, no solution to its incompatibility is described.

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

[0004] Formulations of protein or peptide-based pharmaceuticals containing surfactants at concentrations similar to those used in current commercially available formulations of dulaglutide, or preservatives at concentrations sufficient to meet sterility regulatory requirements, have been previously described, though not both. For example, U.S. Patent Application 2009 / 0232807 describes formulations of GLP-1-Fc fusion protein and lists various categories and examples of excipients, including those described in the application 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 “solubilizers” and the preservatives described. U.S. Patent Application 20100196405 describes formulations of dulaglutide, such as formulations containing polysorbate 80 at a concentration of about 0.2% (w / v). However, the application does not describe formulations containing preservatives.

[0005] There remains a need for formulations containing surfactants at concentrations sufficient to stabilize proteins or peptides, and preservatives at concentrations sufficient to meet the antimicrobial requirements for multi-use injectable products.

[0006] In one aspect, the present invention is a) Protein or peptide, b) Nonionic surfactants, c) Phenolic preservatives, d) A solvent modifier, comprising The present invention provides a composition in which a nonionic surfactant and a phenolic preservative are present at concentrations exceeding their respective 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.

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

[0008] In another embodiment, the present invention is a) Protein or peptide, b) Nonionic surfactants, c) Phenolic preservatives, d) A solvent modifier, comprising The present invention provides a manufactured article comprising an aqueous composition in which a nonionic surfactant and a phenolic preservative are present at concentrations exceeding their respective 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 embodiment, the present invention provides a method for preparing a composition comprising a nonionic surfactant and a phenolic preservative in concentrations above their respective thresholds, wherein the composition comprises including a solvent modifier in a concentration sufficient to ensure that the composition remains clear.

[0010] As described above, surfactants are included in many protein or peptide-based pharmaceutical formulations to stabilize protein or peptide APIs. As used herein, the term “protein or peptide-based pharmaceutical” means 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. While peptides and proteins are sometimes distinguished by size, with peptides having 2 to 50 amino acids and proteins having more than 50 amino acids, the difference between the two is irrelevant to the purposes of this invention, as the formulations described herein can equally apply to pharmaceuticals containing one or more APIs that are peptides or proteins. The formulations of this invention may be applicable to a wide variety of protein or peptide-based drugs requiring nonionic surfactants for stability purposes.

[0011] A preferred drug for use in the formulation 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 immunoglobulin Fc portion analog, identified by CAS registry number 923950-08-7, and providing the following chemical name: immunoglobulin G4 (synthetic human Fc fragment), comprising a dimer, a peptide (synthetic 16-amino acid linker) fusion protein, and a 7-37-glucagon-like peptide I [8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) fusion protein. Each monomer of dulaglutide has the amino acid sequence described in SEQ ID NO: 1: [ka]

[0012] The two monomers are joined by a disulfide bond between cysteine ​​residues at positions 55 and 58 to form a dimer. The structure, function, production, and use in the treatment of T2DM of dulaglutide are described in detail in US7,452,966 and U.S. Patent Application No. US20100196405. As used herein, the term “dulaglutide” means 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, relying in whole or in part on data relating 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 to form the present invention include, but are not limited to, those described in the following examples, as well as other Fc fusion proteins, other GLP-1 agonists, gastric suppressor peptide (GIP) receptor agonists, glucagon receptor agonists, peptide YY (PYY) and its variants, growth and differentiation (GDF) factors such as GDF15 and its variants, amyrin receptor agonists, calcitonin receptor agonists, and interleukins and their variants.

[0014] Many proteins and peptides are prone to denaturation and / or aggregation when formulated in aqueous solutions, and to mitigate such problems, surfactants are often added to formulations of such proteins and peptides. Surfactants have hydrophilic and hydrophobic parts and are composed of molecules that tend to aggregate in aqueous solutions to form aggregates known as micelles. When surfactants are included in aqueous solutions of peptide-based or protein-based pharmaceuticals, they help to reduce the surface tension of the solution, preventing the peptides or proteins 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), as well as block copolymers such as poloxamer 188 (CAS number 9003-11-6, marketed under trade name PLURONIC® F-68) and poloxamer 407 (PLURONIC® F127).

[0015] The formulations of the present invention comprise one or more nonionic surfactants. In certain embodiments, the nonionic surfactant is a polysorbate-type surfactant. Polysorbates are fatty acid-esterified ethoxylated sorbitan, and specific polysorbates are identified by the type of fatty acid ester associated with polyoxyethylene sorbitan. For example, polysorbate 20 comprises monolaurate, polysorbate 40 comprises monopalmitate, polysorbate 60 comprises monostearate, and polysorbate 80 comprises monooleate. Polysorbate 20 and polysorbate 80 are surfactants commonly used in pharmaceutical products for parenteral administration and are included as surfactants 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 parenterally administered pharmaceutical products and is included as a 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 those skilled in the art can identify the identity and concentration of the surfactant necessary to provide a 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 composition is sterile when first produced, and if the composition is supplied in vials or cartridges for multiple uses, an antimicrobial preservative compound or mixture of compounds compatible with the other components of the formulation is added in a strength sufficient to meet regulatory requirements as well as pharmacopoeia requirements, such as those published by the European Pharmacopoeia (EP) and the United States Pharmacopeia (USP). European Pharmacopoeia, 9th Edition, Section 5.1.3, Efficacy of Antimicrobial Preservatives, United States Pharmacopeia USP <51> Please refer to the antimicrobial efficacy test (Rockville, MD).

[0018] Preservatives commonly used in pharmaceutical products suitable for parenteral administration for multiple uses include phenolic compounds or mixtures of such compounds. Specific examples include phenol (CAS number 108-95-2, molecular formula C6H5OH, molecular weight 94.11), m-cresol (CAS number 108-39-4, molecular formula C7H8O, molecular weight 108.14), benzyl alcohol (CAS number 100-51-6, molecular formula C7H8O, molecular weight 108.14 g / mol), and phenoxyethanol (CAS number 122-99-6, molecular formula C8H 10Examples include O2 (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 the preservative required to satisfy the regulatory requirements of a multi-use product is not limited but depends on several factors, including 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 phenol preservative is phenol at a concentration selected from the group consisting of 3, 3.5, 4, 4.5, or 5 mg / mL. In preferred embodiments, the phenol preservative is phenol at a concentration of about 4 mg / mL. In certain embodiments, the phenol preservative is m-cresol. In certain embodiments, the phenol preservative is m-cresol present at a concentration of about 0.1 to about 10 mg / mL. In certain embodiments, the phenol preservative is m-cresol present at a concentration of about 2 to about 6 mg / mL. In certain embodiments, the phenol preservative is m-cresol present at a concentration of about 3.5 to about 5.5 mg / mL. In certain embodiments, the phenol preservative is m-cresol present at a concentration of about 3.15 mg / mL. In other embodiments, the phenol 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 phenol preservative is a mixture of phenol and m-cresol, with phenol present at a concentration of approximately 1.5 mg / mL and m-cresol at a concentration of 1.58 mg / mL. In certain embodiments, the phenol preservative is a mixture of phenol and m-cresol, with phenol present at a concentration of approximately 2 mg / mL and m-cresol at a concentration of approximately 1.58 mg / mL. In certain embodiments, the phenol preservative is a mixture of phenol and m-cresol, with phenol present at a concentration of approximately 3.5 mg / mL and m-cresol at a concentration of approximately 0.32 mg / mL. In certain embodiments, the phenol preservative is a mixture of phenol and m-cresol, with phenol present at a concentration of approximately 3.5 mg / mL and m-cresol at a concentration of approximately 0.63 mg / mL. However, these embodiments should not be construed as limiting, as those skilled in the art can use known techniques to select the phenol preservative and its concentration necessary to satisfy regulatory requirements. For example, see the European Pharmacopoeia, 9th edition, section 5.01.03, “Efficacy of antimicrobial preservatives,” and the United States Pharmacopeia USP40-NF35. <51> Please refer to the chapter "Antibacterial Efficacy Testing." For example, see 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 present in a composition at certain concentrations, they interact in a way that leads to phase separation, resulting in the formation of unacceptable, visible turbidity or cloudiness. While we do not wish to be bound by theory, this phenomenon is thought to occur when molecules of phenolic preservatives bond with micelles of nonionic surfactants via cross-linking attractive forces. For example, see 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, CC; Liu, Y. Gelation transitions of colloidal systems with bridging attractions. PHYSICAL REVIEW E 2016, 94, (4), 040601. As a result, multiple surfactant micelles bind together, and consequently precipitate from the solution. Those skilled in the art will understand that micelles are aggregates of surfactant molecules, where the hydrophilic portion of the nonionic surfactant molecule forms an outer surface or shell surrounding the hydrophobic portion, and the hydrophobic portion is protected from the aqueous solvent by the outer surface or shell formed by the hydrophilic portion. The concentration of surfactant at which such micelles are formed is known as the critical micelle concentration, or CMC, and can be determined using the art known in the art.For example, see Kerwin, BAPolysorbates 20 and 80 used in the formulation of protein biotherapeutics: Structure and degradation pathways. JOURNAL OF PHARMACEUTICAL SCIENCES 2008, 97, (8), 2924-2935. In this case, too, although we do not wish to be bound by theory, the use of solvent denaturants described herein is thought to inhibit the crosslinking attraction between preservative molecules and surfactant micelles.

[0020] However, regardless of the details of the mechanism, phase separation occurs when the combined concentration of the surfactant and preservative in a given composition is greater than or equal to a concentration referred to herein as their “concentration threshold,” which means the concentration at which the combination of surfactant and preservative causes phase separation in the absence of a solvent modifier, resulting in the formation of a turbid or milky appearance. There is no universal concentration threshold that can be generally applied to any combination of surfactant and preservative. Instead, the concentration threshold depends on the details of the formulation in question, particularly the identity of the surfactant and preservative.

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

[0022] Furthermore, visually detectable phase separation in some compositions containing a combination of a surfactant and a preservative that exceeds a concentration threshold occurs essentially immediately after combining the surfactant and the preservative. However, in other compositions, the phase separation does not become visually apparent until some time has elapsed after the formulation has been prepared. For example, in formulations containing m-cresol, visually detectable phase separation has been observed to occur almost immediately, while in certain phenol-containing formulations, the formulation does not become visually detectable for up to about 15 minutes after it has been prepared. Therefore, to confirm that a solubilizing agent sufficiently reduces phase separation due to a combination of a phenolic preservative and a surfactant that would otherwise exceed its concentration threshold, it is necessary to examine the appearance of the formulation at least 10 minutes, preferably at least 15 minutes, after the formulation has been prepared.

[0023] As noted above, the concentration threshold for a given combination of surfactant and preservative depends on both the identity and concentration of the surfactant(s) and preservative(s). Certain commercial products contain both a surfactant and a preservative, but the combination of surfactant and preservative in those products is below their concentration threshold, and thus they remain clear and colorless. For example, a formulation of insulin glargine sold under the trade name LANTUS® contains 0.02 mg / mL of polysorbate 20 and 2.7 mg / mL of m-cresol, and a formulation of insulin glulisine sold under the trade name APIDRA® contains 0.01 mg / mL of polysorbate 20 and 3.15 mg / mL of m-cresol. However, since the total concentration of polysorbate 20 and m-cresol is below the concentration threshold for this particular combination in both cases, both of these formulations are clear. In fact, as shown in the examples described below, in formulations containing m-cresol at a concentration of 3.15 m-cresol, phase separation does not occur when polysorbate 20 is included at a concentration of about 2-fold or less of its CMC, but phase separation occurs at a concentration of about 5-fold or more of the CMC.

[0024] As used herein, the term "phase separation" refers to the formation of physical particles that precipitate from a solution. The presence or absence of phase separation in a given composition can be determined visually, i.e., by a turbid or milky white appearance, as opposed to a clear appearance, or by analytical techniques known to those skilled in the art. Similarly, as used herein, the term "clear" refers to a solution that is transparent, having no turbid or milky white appearance and containing no solid particles of a material detectable to the eye. Analytical techniques known to those skilled in the art can be used, but the determination of whether a formulation is clear and particle-free can be made visually.

[0025] The present invention relates to the use of a solvent modifier for reducing the occurrence of phase separation in a composition in which a surfactant(s) and a preservative(s) are included at a concentration above their concentration threshold, otherwise (i.e., in the absence of a solvent modifier). Compounds that can be used as a solvent modifier in the formulations of the present invention include PPG (CAS number 57-55-6, molecular formula C3H8O2, molecular weight 76.095), NMP (CAS number 872-50-4, molecular formula C5H9NO, molecular weight 99.133) and PEG400 (CAS number 25322-68-3, molecular formula C 2n H 4n+2 O n+1 , n = 8.2 - 9.1, molecular weight 380 - 420 g / mol) 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 are, in some cases, excipients commonly used in pharmaceutical formulations and may 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 isotonic purposes and is included in insulin-containing formulations 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 they contain surfactants, but in combination with phenolic preservatives in their formulations, their concentration is below the threshold. Similarly, PPG is also a commonly used pharmaceutical excipient for functions other than as a solvent modifier; for example, VICTOZA® (liraglutide) contains 14 mg / mL of PPG but does not contain nonionic surfactants. PEG400 is also a common excipient, for example, it is found in ATIVAN® (lorazepam), but that product does not contain nonionic surfactants. Finally, although not as commonly used as glycerol or PPG, NMP is used in a product called ELIGARD (leuprolide acetate), which is non-aqueous and does not contain phenolic preservatives or surfactants.

[0027] With respect to the concentration of solvent modifiers required to reduce phase separation in which surfactants and preservatives are present at concentrations exceeding their concentration thresholds, the required concentration of solvent modifiers also depends on several variables, including the following identity and concentration: (a) the specific surfactant(s) and preservative(s) used, (b) the specific solvent modifier(s) used, and (c) other excipients in the formulation, particularly isotonic agents, which will be described in more detail later. 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 PEG400. In certain embodiments of the present invention, the solvent modifier is PEG400 present at a concentration of about 5 to about 150 mg / mL. In certain embodiments, the concentration of PEG400 is about 40 to about 120 mg / mL.In certain embodiments, the concentration of PEG400 is selected from the group consisting of about 40, about 80, about 110, and about 120 mg / mL. However, these concentrations should not be interpreted as limiting, as the selection of an appropriate concentration of the solvent modifier to be used in a given composition can be readily determined by those skilled in the art using known techniques, including visual observation and turbidity analysis, as well as particulate matter analysis as described in the examples below.

[0028] In addition to reducing the incompatibility between surfactants and preservatives, solvent modifiers can have additional functions in certain compositions, particularly as isotonic agents. Since the formulations of the present invention are for parenteral administration, administering a solution that is not nearly isotonic with body fluids can cause a painful stinging sensation; therefore, when administering a composition, it is desirable to match the tonicity (i.e., osmotic pressure) of the body fluid at the injection site as closely as possible. If the osmotic pressure of the composition is sufficiently lower than that of the tissue (approximately 300 mOsmol / kg for blood; the European Pharmacopoeia requirement for osmotic pressure is >240 mOsmol / kg), the tonicity of the composition needs to be increased to approximately 300 mOsmol / kg. Glycerol and PPG are examples of solvent modifiers for use in the formulations of the present invention, but are also commonly used as isotonic agents in parenteral products; therefore, such effects 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 an isotonic agent. For example, in the dulaglutide-containing compositions described in the examples below, glycerol and PPG are added at sufficient concentrations to increase the tonicity of the composition so that it is nearly isotonic with body fluids at the injection site, and to reduce the incompatibility between the surfactant(s) and preservative(s) in the composition.

[0029] Increasing the tonicity of a composition below the osmotic pressure of the tissue can also be achieved by adding additional isotonic agents. However, commonly used isotonic agents include sodium chloride and mannitol, and it has been found that in certain formulations, these agents worsen the interaction between surfactants and preservatives, leading to phase separation, and thus lowering the minimum concentration of surfactants and / or preservatives that reach the concentration threshold, and / or requiring higher concentrations of solvent modifiers to avoid phase separation. In any case, if the addition of an isotonic agent is necessary, the amount of isotonic agent 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 an isotonic agent such as sodium chloride or mannitol is necessary, and if this addition worsens the interaction between the surfactant and the preservative, the amount of solvent modifier that needs to be added to prevent undesirable phase separation can be easily determined by those skilled 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 formulater attempting to prepare a multi-use formulation of a protein or peptide-based pharmaceutical can, in some cases, first determine the identity and concentration of a nonionic surfactant required to provide sufficient stabilization, then the identity and concentration of a 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 needed. If phase separation occurs, the formulater can determine whether a different combination of surfactant and preservative can be used, or turn their attention to determining the identity and concentration of the solvent modifier according to the present invention to prevent such phase separation from occurring with that particular combination. Alternatively, the formulater can instead first determine the identity and concentration of a preservative required to provide antimicrobial activity, then the identity and concentration of a surfactant required to provide sufficient stabilization, and then observe whether phase separation occurs when those excipients are combined. Similar to the previous scenario, if phase separation does not occur, the surfactant-preservative combination is below its concentration threshold, and a solvent modifier is not needed. However, if phase separation occurs and no alternative preservative + surfactant combination can be identified to avoid such phase separation, the formulationer should focus on determining the identity and concentration of the solvent modifier according to the present invention.

[0031] In certain embodiments, the formulations of the present invention include one or more buffers for controlling pH, and the identity and concentration of any buffer(s) used may, in certain cases, be relevant to determining the concentration threshold of a given surfactant-preservative system and / or the solvent modifier required to avoid phase separation of that system. A “buffer” is a substance that resists changes in pH through the action of its acid-base conjugate component. 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 a desired range include, but are not limited to, phosphates, acetates, citrates, or their acids, arginine, TRIS, and histidine buffers, and combinations thereof. “TRIS” refers to 2-amino-2-hydroxymethyl-1,3,-propanediol and any pharmaceutically acceptable salt thereof. The free base and hydrochloride 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. Considering the potential relevance of any buffer to the determination of the concentration threshold and / or solvent modifier, the formulater may wish to determine the required buffer before determining the identity and concentration of the surfactant and / or preservative used as described in the previous paragraph.

[0032] The above description relates to how a prescriber can determine the identity and concentration of surfactants, preservatives, and solvent modifiers contained in a formulation, but does not necessarily relate to how the formulation is ultimately assembled after these identities and concentrations have been determined. There may be some variation in the order in which the components are added, but the solvent modifier is usually added before the full concentrations of both the phenol preservative and the surfactant are 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 phenol preservative, then the protein or peptide, and then the surfactant.

[0033] In addition to the components described above, 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 susceptibility to oxidation or trace metals. Such stabilizers may include antioxidants such as methionine, or chelating agents such as EDTA, respectively.

[0034] Due to their sensitivity to proteolysis in the gastrointestinal tract and poor absorption, proteins and peptides have low oral bioavailability, and therefore most proteins and peptides are administered parenterally. The formulations of the present invention are for parenteral administration and may include administration by 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. Since the formulations of the present invention are suitable for multiple-dose administration, they are usually supplied in a container-closed system such as a vial or cartridge from which multiple doses can be drawn and administered. The formulations of the present invention can be supplied, for example, in a vial from which multiple doses for administration to a patient can be drawn by a syringe. The formulations of the present invention may also be supplied in a cartridge for use in a pen device from which multiple doses can be administered. The formulations of the present invention may also be supplied in a container-closed device 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. Aqueous compositions comprising: proteins or peptides, nonionic surfactants, phenolic preservatives, and solvent denaturants.

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

[0037] A composition according to any of the above embodiments, wherein a nonionic surfactant and a phenolic preservative are present at concentrations exceeding their respective concentration thresholds in the absence of a solvent modifier.

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

[0039] A composition of the above embodiment in which the solution remains clear for at least 15 minutes. A composition of a prior embodiment in which the solution remains clear for at least 24 hours. A composition of a prior embodiment in which the solution remains clear for at least 1 week. A composition of a prior embodiment in which the solution remains clear for at least 1 month. A composition of a prior embodiment in which the solution remains clear for at least 6 months. A composition of a prior embodiment in which the solution remains clear for at least 1 year.

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

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

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

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

[0044] A composition according to any of the above embodiments, wherein a protein or peptide is present in a concentration ranging from about 1 to about 10 mg / mL.

[0045] A composition according to any of the above embodiments, wherein the protein or peptide is selected from the group consisting of GLP-1 receptor agonists, insulin, GIP receptor agonists, glucagon receptor agonists, PYY, GDF, amyrin receptor agonists, calcitonin receptor agonists, and interleukins. A composition according to a prior embodiment, wherein the protein or peptide is an Fc fusion protein.

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

[0047] A composition according to any of the above embodiments, wherein the nonionic surfactant is a polysorbate-type surfactant. A composition according to a prior embodiment, wherein the nonionic surfactant is selected from the group consisting of PS20, PS80, poloxamer 188, and poloxamer 407. A composition according to a prior embodiment, wherein the nonionic surfactant is either PS20 or PS80.

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

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

[0050] A composition according to any of the above embodiments, wherein the nonionic surfactant is poloxamer 188. A composition according to a prior embodiment, wherein the concentration of poloxamer 188 is in the range of about 0.01 to about 2 mg / mL. A composition according to a prior embodiment, wherein the concentration of poloxamer 188 is in the range of about 0.5 to about 1.5 mg / mL.

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

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

[0053] In certain embodiments, the phenolic preservative is phenoxyethanol. The composition of a prior embodiment contains phenoxyethanol at a concentration of about 10 to about 15 mg / mL.

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

[0055] A composition according to any of the above embodiments, wherein the phenol preservative is phenol. A composition according to a prior embodiment, wherein the concentration of phenol is about 1 to about 10 mg / mL. A composition according to a prior embodiment, wherein the concentration of phenol is about 3 to about 6 mg / mL. A composition according to a prior embodiment, wherein the concentration of phenol is at least about 3 mg / mL. A composition according to a prior embodiment, 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. A composition according to a prior embodiment, wherein the concentration of phenol is about 5 mg / mL.

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

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

[0058] A composition according to any of the above embodiments, wherein the phenol preservative is a mixture of phenol and m-cresol, with phenol present at a concentration of about 3.5 to about 4 mg / mL and m-cresol present at a concentration of about 0.32 mg / mL to about 0.63 mg / mL. A composition according to a prior 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] A composition according to any of the above embodiments, wherein the solvent modifier is selected from the group consisting of PPG, NMP, PEG400, and glycerol.

[0060] A composition according to any of the above embodiments, wherein the solvent modifier is glycerol. A composition according to 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. A composition according to a prior embodiment, wherein the concentration of glycerol is about 20 to about 80 mg / mL. A composition according to a prior embodiment, wherein the concentration of glycerol is selected from the group consisting of about 20, about 25, or about 80 mg / mL. A composition according to a prior embodiment, wherein the concentration of glycerol is about 20 mg / mL.

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

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

[0063] A composition according to any of the above embodiments, wherein the solvent modifier is PEG400. A composition according to any of the above embodiments, wherein the solvent modifier is PEG400 and is present at a concentration of about 5 to about 150 mg / mL. A composition according to a prior embodiment, wherein the concentration of PEG400 is about 40 to about 120 mg / mL. A composition according to a prior embodiment, wherein the concentration of PEG400 is selected from the group consisting of about 40, about 80, about 110 and about 120 mg / mL.

[0064] A composition according to any of the above embodiments, wherein the composition further comprises an isotonic agent. A composition according to a prior embodiment, wherein the isotonic agent is selected from the group consisting of NaCl and mannitol.

[0065] A composition according to any of the above embodiments, wherein the composition further comprises a buffer. A composition according to a prior embodiment, wherein the buffer is selected from the group consisting of phosphates, acetates, citrates, or their acids, arginine, TRIS, and histidine. A composition according to a prior embodiment, wherein the buffer is a phosphate. A composition according to a prior embodiment, wherein the concentration of the phosphate is about 10 mM. A composition according to any of the above embodiments, further comprising a buffer whose composition is citrate. A composition according to a prior embodiment, wherein the concentration of citrate is about 10 mM.

[0066] A composition of any of the above embodiments, wherein the pH of the composition is approximately 4 to approximately 8. A composition of a prior embodiment, wherein the pH of the composition is approximately 5.5 to approximately 7.5. A composition of a prior embodiment, wherein the pH of the composition is approximately 6.0 to 7.0. A composition of a prior embodiment, wherein the pH of the composition is approximately 6.5 or approximately 7.

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

[0068] Aqueous compositions suitable for parenteral administration include a solvent modifier selected from the group consisting of dulaglutide, PS80, PPG, and glycerol, and a phenol preservative selected from the group consisting of phenol, m-cresol, and mixtures thereof. A composition of a prior embodiment in which the dulaglutide concentration is selected from the group consisting of 1.5, 3, 6, or 9 mg / mL. A composition of a prior embodiment in which the PS80 concentration is either 0.2 or 0.25 mg / mL. A composition of a prior embodiment in which the solvent modifier is either 15 mg / mL of PPG or 20 mg / mL of glycerol. A composition of a prior embodiment in which the phenol preservative is either 4 mg / mL of phenol or a combination of 3.5 mg / mL of phenol and 0.32 mg / mL of m-cresol. A composition of a prior embodiment further comprising a buffer. A composition of a prior embodiment in which the buffer is citrate. A composition of a prior embodiment in which the citrate concentration is 10 mM. A composition of a prior embodiment in which the pH of the composition is about 6.5.

[0069] A container closure system comprising any of the above compositions. A container closure system according to 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 automatic injector containing any of the above compositions.

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

[0073] A method for preparing any of the above compositions, comprising preparing or obtaining a buffer solution, 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: a nonionic surfactant and a phenolic preservative, both exceeding their respective concentration thresholds; and a solvent modifier in the composition at a concentration sufficient to ensure the composition remains clear.

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

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

[0077] [Figure 1] This shows the turbidity of compositions containing m-cresol and PS80 at various concentrations. [Figure 2] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 3] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 4] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 5] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 6]This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 7] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Figure 8] This shows the turbidity of formulations containing polysorbate 80 at various concentrations. [Examples]

[0078] Concentration threshold of a composition containing 0.2 mg / mL of PS80 Commercial dulaglutide formulations sold under the trade name TRULICITY® contain 0.2 mg / mL of PS80 as a stabilizer. To investigate the effect of adding a phenolic preservative, a placebo solution containing 0.2 mg / mL of PS80 was prepared in 10 mM citrate buffer at pH 6.5. Test substances were prepared by adding a sufficient amount of m-cresol or phenol to a sample of this solution, obtaining formulations containing 0.2 mg / mL of PS80 and either 3.15 mg / mL of m-cresol or 5 mg / mL of phenol. The placebo and test substances were visually inspected. The placebo solution was clear and colorless, while the test substances rapidly became turbid or milky in appearance. Therefore, the respective concentration thresholds of the two preservative-containing solutions were exceeded.

[0079] Concentration threshold of a composition containing m-cresol and PS20 Tests were conducted to determine the concentration thresholds for the combination of PS20 and m-cresol, which are nonionic surfactants and phenolic preservatives used in commercially available insulin glargine (marketed under the trade name LANTUS®) and insulin glulisine (marketed under the trade name APIDRA®), respectively, and contain 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 of 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 present at a concentration of approximately twice or less than that of the CMC, but it did occur at concentrations of approximately five times or more than that of the CMC. Therefore, the combination of m-cresol and polysorbate 20 at a concentration of 3.15 mg / mL, which is five times or more than that of the CMC, exceeds the concentration thresholds for m-cresol and polysorbate 20, while the combination of m-cresol and polysorbate 20 at a concentration of 3.15 mg / mL, which is twice or less than that of the CMC (e.g., 0.02 and 0.01 mg / mL used in LANTUS and APIDRA), falls below the concentration thresholds for m-cresol and polysorbate 20.

[0081] Turbidity of compositions containing m-cresol and PS80 at various concentrations Tests will be conducted to evaluate the relationship between the concentrations of both m-cresol and PS80 and the occurrence of phase separation. Batches of 10 mM citrate buffer, adjusted to pH 6.5, will be prepared and used as a control and buffer matrix for the formulation of the test substance. M-cresol will be added to a portion 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 will be measured and dissolved in separate portions of citrate buffer to prepare two stock solutions, one containing 10 mg / mL of polysorbate 80 and the other containing 40 mg / mL of polysorbate 80. The stock solutions of the surfactant will be gradually added to various amounts of phenol preservative-containing solutions in the amounts shown in Table 2 below to produce formulations containing a wide range of polysorbate 80 concentrations. [Table 2]

[0082] The turbidity of the obtained formulation is measured using a HACH turbidimeter (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 outside of the test tube to conceal any minute defects in the glass tube. Approximately 7 mL of the 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 effects of various concentrations of solvent modifiers, commonly used isotonic agents, preservatives, and surfactants. Tests will be conducted to evaluate the effect of including various concentrations of solvent denaturants and other excipients, commonly used as isotonic 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 of m-cresol and either a solvent denaturant or a commonly used isotonic 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 containing 10 mg / mL of polysorbate 80 and the other containing 40 mg / mL of polysorbate. These stock solutions are then gradually added in the amounts shown in Table 2 to various amounts of solvent denaturing agents or isotonic agents listed in Table 3 to produce formulations containing a wide range of polysorbate 80 concentrations. The turbidity of the resulting formulations is measured as described above.

[0086] The results are shown in Figure 2. As shown in Figure 2, the addition of mannitol and NaCl resulted in a leftward shift in the turbidity data compared to the control, suggesting that in this study, their inclusion leads to the generation of more turbidity at a given PS80 concentration. On the other hand, the addition of PPG, glycerol, and NMP resulted in a rightward shift in the turbidity data compared to the control, suggesting that PEG400 prevents the generation of turbidity, and in this study, their inclusion reduces the generation of turbidity at a given PS80 concentration.

[0087] In another series of tests, a 10 L batch of 10 mM citrate buffer containing 2.723 mg / mL citrate and 0.1422 mg / mL sodium citrate, adjusted to pH 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 are obtained from Eli Lilly (Indianapolis, Indiana). Glycerol, propylene glycol, N-methyl-2-pyrrolidone (NMP), and polyethylene glycol 400 (PEG400) are obtained from Sigma-Aldrich (Milwaukee, Wisconsin). [Table 4]

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

[0089] The contributions of both surfactant and preservative concentrations, as well as the adverse 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 of m-cresol did not become turbid at any of the PS80 concentrations examined, including in the presence of mannitol or NaCl. Therefore, none of the compositions containing 1.58 mg / mL of m-cresol examined in this test reached the concentration threshold. However, as the concentration of m-cresol was increased to 3.15 mg / mL, turbidity was observed as the concentration of polysorbate 80 increased. Finally, the presence of either mannitol or NaCl exacerbated the development of turbidity in a dose-dependent manner.

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

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

[0092] The effect of PEG400 at the point when the concentration threshold is reached at specific PS80 and m-cresol concentrations can be seen in Figure 7. As shown in Figure 7, PEG400 reduces turbidity generation in a dose-dependent manner.

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

[0094] In summary, the data from these tests indicate that the concentration threshold is inherent to the identity and concentration of surfactants and preservatives in the composition, and that the occurrence of turbidity-causing phase separation in such compositions may be reduced in a dose-dependent manner by including solvent modifiers, or worsened in a dose-dependent manner by including certain commonly used isotonic agents.

[0095] The effect of concentration thresholds and solvent denaturants in compositions containing model proteins of various molecular weights. Tests will be conducted to confirm that the interaction between surfactants and preservatives that cause turbidity in the composition, and the ability to reduce this phenomenon by including solvent denaturants, does not depend on the identity of the proteins in the composition. The proteins identified for inclusion in this test will be selected to include a wide range of molecular weights, as shown in Table 5 below. [Table 5]

[0096] Monosodium phosphate 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 is.

[0097] A 2L batch of 10mM phosphate buffer was prepared 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, and this was used as the buffer matrix for the test. Subsequently, protein preparations containing PS80, m-cresol, and / or NMP were prepared and visually inspected. Details of the composition and results are provided in Table 6 below. [Table 6]

[0098] The data in Table 6 shows that for all proteins tested, including BSA at multiple concentrations, the combination of 0.2 mg / mL polysorbate 80 and 3.15 mg / mL m-cresol in the absence of a solvent denaturant caused phase separation and resulted in a turbid appearance, while the inclusion of 81 mg / mL NMP prevented such phase separation.

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

[0100] The test is designed to examine the stability of dulaglutide in these compositions. Prepare a 5 mM, pH=6.5 citrate buffer and use it as is. Transfer an appropriate amount of citrate buffer to a 500 mL volumetric flask. Next, add the calculated amounts of preservative and solvent denaturant to the same flask, mix and dissolve to ensure a homogeneous solution. Using a graduated cylinder, measure 38.5 mL of dulaglutide API and transfer it to the volumetric flask. Mix until the solution is homogeneous. Simultaneously, prepare a 100 mg / mL polysorbate 80 stock solution. Transfer approximately 1000 mg of polysorbate to a glass beaker and dissolve it in 10 mL of buffer. Using a transfer pipette, transfer 1 mL of polysorbate 80 stock solution to the volumetric flask. Next, add an appropriate amount of buffer until the liquid meniscus reaches the 500 mL mark. Mix the solution further to ensure homogeneity and filter it through a 0.22 μm filter. Fill a 3 mL cartridge with the filtered drug. The solution inside the cartridge was visually confirmed to be transparent, suggesting that phase separation due to interaction between the surfactant and preservative did not occur.

[0101] Furthermore, the filled cartridges are stored at 5°C for stability testing. This storage temperature of 5°C is representative of the recommended storage temperature of 2–8°C for dulaglutide drugs. At the pre-specified time, the samples are removed from storage, visually confirmed to be clear and free of particulate matter, and tested using the various methods described below.

[0102] HIAC. The HIAC test is used to measure the content of invisible particulate matter, and USP <787> (Invisible microparticles in therapeutic protein injections) and <788> The tests are performed on the test samples described in (Microparticles in Injection). These are in harmony with European Pharmacopoeia 2.9.19 and Japanese Pharmacopoeia 6.07. At each time point, five aliquots of 0.5 mL of solution are taken from a 3 mL cartridge and pooled, so the measurement(s) reflect an average of five samples. The results are provided in Table 8 below. [Table 8]

[0103] USP <788> To comply with the (particulate matter in injectable solutions) requirement, parenteral products containing therapeutic protein injectables such as dulaglutide must contain no more than 6,000 particulate matter particles larger than 10 μm and no more than 600 particulate matter particles larger than 25 μm per container. As shown in Table 8, all samples examined were well within the FDA limits for parenteral products.

[0104] MFI testing is used to detect non-bubble particulate matter present in injectable and parenteral solutions. This method is an informational stability characterization method and is performed using flow imaging techniques to enumerate and classify particles that are not visible to the naked eye in terms of size, concentration, and morphology. Samples are removed from storage and tested after 12 months. Results are provided in Table 9 below. Particulate matter larger than 5 μm with an aspect ratio (AR) greater than 0.85 may be silicone from the stopper, as opposed to protein particles, and have a very round shape. [Table 9]

[0105] The data in Table 9 is comparable to the data from past dulaglutide drug studies.

[0106] SEC. The monomeric purity of dulaglutide is measured using the size exclusion (SEC) HPLC method. This method separates aggregates and fragmented species from intact monomeric proteins.

[0107] The monomeric purity of dulaglutide pharmaceuticals is measured by size exclusion HPLC. This method uses isocratic separation on a 200 angstrom pore-size silica gel column in combination with UV detection at 214 nm. Since this is close to the maximum absorbance of the pharmaceutical peptide backbone, no correction of the response coefficient is necessary. This method separates the high molecular weight morphology (total aggregates) from the monomeric dulaglutide. This method has been demonstrated to be specific and stable in separating the high molecular weight morphology from dulaglutide monomers. Monomers and aggregates are reported as peak area percentages relative to the total area. Data are provided in Table 10. [Table 10]

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

[0109] RP-HPLC. This method is designed to determine the purity of dulaglutide pharmaceuticals and associated substances / impurities. Associated impurities resulting from glycosylation, N-terminal cleavage, linker cleavage, and oxidation of the Fc region are separated from unmodified dulaglutide using reversed-phase gradient HPLC in conjunction with UV detection at 214 nm, which is close to the maximum absorbance of the pharmaceutical peptide backbone, thus eliminating the need for response coefficient correction. This 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. The restriction digestion method is designed to determine modifications of GLP-1 analogs in dulaglutide pharmaceuticals. Pharmaceutical samples are exposed to mild digestion conditions with trypsin to 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 is used in conjunction with reversed-phase gradient HPLC separation and UV detection at 214 nm, which is close to the maximum absorbance of the pharmaceutical peptide backbone, so no correction of the response coefficient is necessary. This method separates related impurities resulting from N-terminal cleavage, N-terminal modification (Des H / HG, pyrubilization), 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. The results are given in Table 12. [Table 12]

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

[0113] CE-SDS NR (non-reducing) electrophoresis using sodium dodecyl sulfate capillary electrophoresis is used to determine the purity of dulaglutide pharmaceuticals. The dulaglutide molecule is denatured, and molecular variants are separated by size via a proprietary gel matrix electrokinetically loaded into an uncoated capillary. Separation occurs when an electric current is applied to the capillary, and molecular variants are detected at 214 nm UV, which is close to the maximum absorbance of the drug's peptide backbone, thus eliminating the need for response coefficient correction. This method separates high molecular weight and single-chain morphologies from monomeric dulaglutide. This method has been demonstrated to be specific and stable, separating aggregated and single-chain morphologies from dulaglutide monomers.

[0114] [Table 13]

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

[0116] In summary, the above study supports the conclusion that a preserved dulaglutide formulation containing the same PS80 content as that used to provide sufficient stability in commercially available TRULICITY formulations can be prepared without phase separation due to interaction between the preservative and surfactant by the use of a solvent denaturant, and that the protein in such a formulation remains sufficiently stable.

Claims

1. 1. An aqueous composition comprising: a) a protein or peptide; b) a nonionic surfactant; and c) a phenolic preservative; and d) a solvent modifier which is glycerol; the nonionic surfactant and the phenolic preservative are present at concentrations above their concentration thresholds in the absence of a solvent modifier; An aqueous composition wherein the solvent modifier is present in a concentration sufficient to ensure that the solution remains clear.

2. 10. The composition of claim 1, wherein the protein or peptide is present at a concentration ranging from about 0.1 to about 100 mg / mL.

3. 10. The composition of claim 1, wherein the protein or peptide is present at a concentration ranging from about 0.5 to about 50 mg / mL.

4. 10. The composition of claim 1, wherein the protein or peptide is present at a concentration ranging from about 1 to about 10 mg / mL.

5. The composition of any one of claims 1 to 4, wherein the protein or peptide is dulaglutide.

6. 6. The composition of claim 5, 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 selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, and mixtures thereof.

9. 9. The composition of claim 8, wherein the non-ionic surfactant is polysorbate 80 at a concentration of about 0.1 mg / mL to about 0.4 mg / mL.

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

11. The composition of any one of claims 1 to 10, wherein the phenolic preservative is selected from the group consisting of phenol, m-cresol, benzyl alcohol and phenoxyethanol.

12. 12. The composition of claim 11, wherein the phenolic preservative is phenol.

13. 13. The composition of claim 12, wherein the phenol concentration is about 4 mg / mL.

14. A composition according to any one of claims 1 to 10, wherein the phenolic preservative is a mixture of phenol and m-cresol.

15. 15. The composition of claim 14, wherein the phenol concentration is about 3.5 mg / mL and the m-cresol concentration is about 0.32 mg / mL.

16. 16. The composition of any one of claims 1 to 15, wherein the glycerol concentration is about 20 mg / mL.

17. The composition of any one of claims 1 to 16, wherein the composition further comprises a buffer.

18. 18. The composition of claim 17, wherein the buffer is selected from the group consisting of TRIS, phosphate, and citrate.

19. 19. The composition of claim 18, wherein the buffer is citrate.

20. 20. The composition of claim 19, wherein the citrate concentration is 10 mM.

21. The composition of any one of claims 1 to 20, wherein the pH of the composition is about 6 to 8.

22. The composition of any one of claims 1 to 21, wherein the pH of the composition is about 6.

5.

23. a) dulaglutide, b) PS80; and c) a phenolic preservative selected from the group consisting of phenol, m-cresol, and mixtures thereof; d) a solvent modifier that is glycerol.

24. 24. The composition of claim 23, wherein the concentration of PS80 is about 0.2 mg / mL.

25. 25. The composition of claim 24, 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.

26. 26. The composition of claim 25, wherein the phenolic preservative is about 4 mg / mL phenol.

27. 27. The composition of claim 26, wherein the phenolic preservative is a combination of about 3.5 mg / mL phenol and about 0.32 mg / mL m-cresol.

28. 28. The composition of any one of claims 23 to 27, wherein the concentration of glycerol is about 20 mg / mL.

29. 29. The composition of any one of claims 23 to 28, further comprising 10 mM citrate buffer, wherein the pH of the composition is 6.

5.

30. A method for preparing a composition according to any one of claims 1 to 29, comprising the steps of: a) preparing or obtaining a buffer solution; b) adding the solvent modifier; c) adding said phenolic preservative; d) adding said protein or peptide; e) adding said non-ionic surfactant.

31. A method for preparing an aqueous composition containing a nonionic surfactant and a phenolic preservative above their concentration thresholds, the method comprising including a solvent modifier in the composition at a concentration sufficient to ensure that the composition remains clear.

32. The method of claim 31, wherein the composition comprises any of the compositions of claims 1 to 29.

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