Methods for preparing stable pramlintide therapeutic formulations in an aprotic polar solvent

Abstract

Certain embodiments relate to formulations of therapeutic agents and methods of making such formulations, the formulations comprising at least one therapeutic agent dissolved in a non-protic polar solvent system, the system comprising at least one ionization stabilizing excipient, the ionization stabilizing excipient being at a concentration sufficient to impart physical and chemical stability to the therapeutic agent.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680056000.0, filed on September 25, 2016, entitled "Method for preparing a stable glucagon therapeutic agent in a nonprotic polar solvent". Technical Field

[0002] This invention generally relates to therapeutic formulations for parenteral administration. In particular, this invention relates to the preparation of stable therapeutic formulations using aprotic polar solvents, which involves dissolving the therapeutic agent (active ingredient) in an aprotic polar solvent system without requiring the peptide to be dried from a buffered aqueous solution prior to dissolution in the aprotic polar solvent system. In addition to the active ingredient, the formulation may also contain stabilizing excipients, particularly ionizing stabilizing excipients. Background Technology

[0003] Peptides exhibit enhanced stability and solubility in aprotic polar solvents compared to aqueous solutions (see US2014 / 0005135 and US8,697,644); however, due to their lack of storage stability, directly dissolving some peptides in aprotic polar solvents is generally not a feasible method for preparing stable and therapeutic compositions. A particular example is glucagon, a 29-amino acid peptide hormone used to treat hypoglycemia. Glucagon has an isoelectric point of approximately 7.0, and the molecule is substantially insoluble at neutral pH. Therefore, aqueous solutions must be prepared to be acidic or alkaline before the molecule can be dissolved at therapeutically relevant concentrations. However, acidic and alkaline solutions promote glucagon degradation pathways, and it is well known that glucagon molecules tend to fibrillate and form gel-like aggregates in dilute acidic solutions. Therefore, due to the instability of glucagon molecules, currently available therapeutic agents are sold in lyophilized powder form, which must be reconstituted with a diluent before use. In contrast, glucagon molecules can exhibit enhanced stability and solubility in aprotic polar solvents such as dimethyl sulfoxide (DMSO).

[0004] Besides peptides and proteins, aprotic polar solvents can also enhance the solubility and stability of therapeutic small molecule drugs compared to aqueous solutions. For example, the small molecule drug diazepam exhibits extremely low solubility (<2 mg / mL) in water at neutral pH. To improve the solubility of diazepam, the pH of aqueous solutions is prepared to be acidic or alkaline, which in turn increases the rate of hydrolysis and degradation of diazepam. Conversely, diazepam is readily soluble in the aprotic polar solvents dimethyl sulfoxide (DMSO) and N-methylpyrrolidone (NMP), with solubility in DMSO and NMP at least an order of magnitude higher (>50 mg / mL) compared to neutral aqueous solutions. Furthermore, diazepam molecules are stable in DMSO and NMP in the absence of formulation excipients, and under accelerated storage conditions (40°C, 75% RH), diazepam exhibits stability for at least 6 months in aprotic polar solvents (see U.S. Patent No. 9,125,805).

[0005] The preparation of non-aqueous peptide formulations by directly dissolving peptides in aprotic polar solvents has been described in the prior art. For example, McMullen (UK Patent Application 2,119,248A, hereinafter referred to as McMullen'248) describes the preparation of insulin solutions by directly dissolving insulin crystals in DMSO. Stevenson et al. (US Patent No. 5,932,547, hereinafter referred to as Stevenson'547) disclose the preparation of peptide compositions by directly dissolving peptides in aprotic polar solvents such as DMSO or dimethylformamide (DMF). The compositions described by Stevenson'547 are solutions prepared by directly dissolving peptide powder obtained from a manufacturer or supplier in a non-aqueous solvent, without the use of stabilizing excipients added to the formulation to establish acceptable ionization properties to prevent physical and / or chemical degradation of the therapeutic molecule. While directly dissolving therapeutic molecules in aprotic polar solvents such as DMSO relative to water can improve the solubility of the therapeutic molecule, the molecule remains susceptible to various physical and chemical degradation pathways. Therefore, for many therapeutic molecules, it has been found that directly dissolving them in aprotic polar solvent systems is not suitable for preparing stable formulations. For example, a solution prepared by directly dissolving glucagon powder in DMSO at therapeutically relevant concentrations (e.g., 5 mg / mL or approximately 0.45% (w / w)) initially forms a clear, single-phase composition, but eventually forms insoluble aggregates within 24 hours at room temperature. Therefore, directly dissolving some peptides in aprotic polar solvents is not a feasible method for preparing stable therapeutic formulations.

[0006] The formulations of this invention also differ from those described by Prestrelski et al. (US Patent 8,697,644, hereinafter referred to as Prestrelski'644), which discloses the preparation of peptide formulations by drying an active ingredient (e.g., a peptide) from a buffered aqueous solution and then reconstituted the peptide powder in an aprotic polar solvent. According to this method, the ionization properties of the molecule acquired from the buffered aqueous solution used for drying can be retained in the powder and continue to be retained after dissolution in the aprotic polar solvent system. The ability of a peptide to retain its ionization properties from the final aqueous solution in a dry state is referred to as "pH memory," from which the peptide was dried. However, this method requires a drying step, such as freeze-drying or spray drying, prior to reconstitution in the aprotic polar solvent, which necessitates a stabilizing excipient to protect the molecule from stresses encountered during drying (e.g., thermal stress, mechanical stress, interfacial stress). Furthermore, the operating parameters and formulation components required for drying molecules must be frequently optimized for specific therapeutic agents, and the transition from laboratory-scale to large-scale production and processing requires further method development and optimization. Therefore, adding a drying step to the product development pathway significantly increases costs in both time and expense.

[0007] Therefore, there is still a need for a formulation platform that can combine the stability and solubility offered by aprotic polar solvent systems, but remove the requirement for drying from aqueous solutions before reconstructing therapeutic molecules in biocompatible aprotic polar solvent systems, thereby simplifying and / or accelerating the product development pathway. Summary of the Invention

[0008] When therapeutic molecules are dissolved in aprotic polar solvent systems, they typically require optimal or beneficial ionization properties to exhibit prolonged stability. This invention relates to a surprising discovery that optimal or beneficial ionization properties of therapeutic molecules can be obtained by directly dissolving the therapeutic agent in an aprotic polar solvent system containing a specific concentration of at least one ionizing stabilizing excipient. Certain embodiments of the invention relate to a method for preparing stable formulations containing at least one therapeutic molecule dissolved in an aprotic polar solvent system, a method that does not require pre-drying the therapeutic molecule from a buffer aqueous solution before reconstructing it in the aprotic polar solvent system.

[0009] Directly dissolving therapeutic agents in aprotic polar solvents or drying them from aqueous solutions before reconstructing them in aprotic polar solvents can lead to potential stability issues and increased manufacturing complexity. The inventors have found a solution to this problem. The solution involves directly dissolving the therapeutic agent (e.g., a powder obtained from a commercial manufacturer or supplier) along with an effective amount of an appropriately ionized, stabilizing excipient for constructing the therapeutic agent in an aprotic polar solvent system.

[0010] In particular, avoiding the drying of peptides from buffered aqueous solutions, such as by freeze-drying, before reconstruction in aprotic polar solvent systems is expected to save significant time and costs throughout the development of various products. It is well known that the development of drying methods is an expensive and time-consuming process step, which often must be tailored to each therapeutic molecule. Furthermore, scaling up the drying process becomes complex because the initial research and optimization processes are at a laboratory scale, while production uses significantly different equipment and / or instruments compared to laboratory scale. Therefore, if stable therapeutic peptide formulations could be prepared by directly dissolving the active ingredient in aprotic polar solvent systems without such a drying step, scale-up and manufacturing would be facilitated by eliminating this expensive and time-consuming process. Moreover, during the drying process, the therapeutic agent is exposed to multiple stresses, which can degrade the molecule; therefore, stabilizing excipients (e.g., disaccharides such as trehalose and sucrose) are typically added to the formulation to prevent degradation of the active agent during drying. By omitting the drying step, as few additional stabilizing excipients as possible can be used, especially those that are typically included during the drying step to provide stability, thus simplifying the overall formulation.

[0011] Another discovery of the inventors is that stable solutions can be prepared by dissolving therapeutic agents in non-aqueous, aprotic polar solvents (e.g., DMSO), achieved by adding specific amounts or combinations of compounds to the non-aqueous, aprotic polar solvent, said compounds acting as ionization-stabilizing excipients. Not wishing to be bound by theory, it is believed that ionization-stabilizing excipients can act as proton sources in aprotic polar solvent systems (e.g., molecules that can donate protons to therapeutic molecules), protonating ionized groups on therapeutic molecules, thereby giving the therapeutic molecules improved physically and chemically stable ionization properties in aprotic polar solvent systems. In one aspect of the invention, a stable formulation for parenteral injection is disclosed. Alternatively, it can be delivered transdermally, for example, by topical application to the skin.

[0012] Some embodiments relate to formulations of therapeutic agents in which the concentration of the therapeutic agent is at least, at most, or about 0.1 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, or from 100 mg / mL to 150 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, or reaches the solubility limit of the therapeutic agent in a proton-polar solvent system, said a proton-polar solvent system containing at least one ionizing stabilizer excipient at a concentration that provides physical and chemical stability to the therapeutic agent. In some aspects, the therapeutic agent is a peptide. In other aspects, the therapeutic agent is a small molecule. The formulation may contain an ionizing stabilizer excipient at a concentration of at least, at most, or about 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM, or 50 mM to 10 mM, 50 mM, 75 mM, 100 mM, 500 mM, 1000 mM, or reaching the solubility limit of the ionizing stabilizer excipient in aprotic polar solvent systems. In some aspects, the concentration of the ionizing stabilizer excipient is from 0.1 mM to 100 mM. In some embodiments, the ionizing stabilizer excipient may be a suitable inorganic acid, such as hydrochloric acid. In some aspects, the ionizing stabilizer excipient may be an organic acid, such as an amino acid, an amino acid derivative, or a salt of an amino acid or a salt of an amino acid derivative (examples include glycine, trimethylglycine (betaine), glycine hydrochloride, and trimethylglycine (betaine) hydrochloride). On the other hand, the amino acid may be glycine or the amino acid derivative trimethylglycine. In some respects, the peptide has fewer than 150, 100, 75, 50, or 25 amino acids. In other respects, the aprotic polar solvent system comprises DMSO. The aprotic polar solvent may be deoxygenated, such as deoxygenated DMSO. In some embodiments, an ionization-stabilizing excipient may be added to the aprotic polar solvent system first, followed by the addition of the therapeutic molecule to prepare the formulation. Alternatively, the therapeutic molecule may be dissolved in the aprotic polar solvent system first, followed by the addition of the ionization-stabilizing excipient. On the other hand, the ionization-stabilizing excipient and the therapeutic molecule may be dissolved simultaneously in the aprotic polar solvent system. In some respects, the therapeutic agent is glucagon or a salt thereof.

[0013] Other embodiments of the invention relate to a method for stabilizing a therapeutic agent (e.g., a peptide or small molecule), comprising the steps of: (a) calculating or determining a suitable ionization-stabilizing excipient or a required proton concentration to achieve stable ionization properties of the target therapeutic agent (e.g., a peptide or small molecule) in a nonproton polar solvent system; (b) mixing at least one ionization-stabilizing excipient with the nonproton polar solvent system to obtain a suitable ionization environment to provide the ionization properties determined in step (a); and (c) dissolving the target therapeutic agent in the nonproton polar solvent, the nonproton polar solvent having a suitable environment capable of physically and chemically stabilizing the therapeutic agent. In some non-limiting aspects, the therapeutic agent is capable of being chemically or physically stable for at least about 0.25, 0.5, 1, 2, 3, 4, or 5 years at room temperature. In some respects, the dissolution of the therapeutic agent and the addition of the ionizing stabilizer to the aprotic polar solvent system can be carried out in any order or simultaneously. Therefore, the ionizing stabilizer can be mixed first, followed by the dissolution of the therapeutic agent; or the therapeutic agent can be dissolved first, followed by the addition of the ionizing stabilizer to the solution; or the ionizing stabilizer and the therapeutic agent can be added to or dissolved simultaneously in the aprotic polar solvent system. On the other hand, the entire amount of the components (e.g., the therapeutic agent or the ionizing stabilizer) does not need to be mixed at a specific point; that is, a portion of one or more components can be mixed at the first, second, or simultaneous stages, while another portion can be mixed at a different time, at the first, second, or simultaneous stages. In some respects, the therapeutic agent can be a peptide, and the ionizing stabilizer can be a suitable inorganic acid, such as hydrochloric acid, sulfuric acid, and / or nitric acid. In some respects, the peptide is less than 150, 100, 75, 50, or 25 amino acids. The concentration of the therapeutic agent and / or ionizing stabilizer added to the solution can be 0.01, 0.1, 1, 10, 100, 1000 mM or up to its solubility limit, including all values ​​and ranges therein. In some respects, the aprotic polar solvent system is deoxygenated. On the other hand, the aprotic polar solvent system contains DMSO or deoxygenated DMSO, is substantially composed of DMSO or deoxygenated DMSO, or is composed of DMSO or deoxygenated DMSO.

[0014] In another aspect of the invention, a method for treating or preventing conditions, diseases, disorders, etc., is disclosed, comprising administering an effective amount of the formulation of the invention to a subject in need to treat or prevent said condition, disease, disorder, etc. In the method of the invention, any suitable dose of the therapeutic agent (e.g., protein, peptide, or small molecule) can be administered. Of course, the administered dose will vary according to known factors, such as the pharmacodynamic characteristics of a particular compound, salt, or combination thereof; the subject's age, health, or weight; the nature and severity of symptoms; the metabolic characteristics of the drug and the patient; the type of concurrent treatment; the frequency of treatment; or the desired effect. In some aspects, hypoglycemia can be treated by administering a formulation containing an effective amount of glucagon as described herein.

[0015] The stable formulations described herein can be used for parenteral injection of any therapeutic agent (protein, peptide, and / or small molecule) that has limited or poor stability or solubility in an aqueous environment. In some respects, the formulations described herein are provided as injectable formulations. These injectable formulations can be applied to the epidermis, dermis, or subcutaneous layer of an animal. In some respects, the formulations are administered intradermally.

[0016] Therefore, in some embodiments, the therapeutic agent or peptide or its salt is selected from: glucagon, pramlintide, insulin, leuprorelin, LHRH agonists, parathyroid hormone (PTH), amylin, botulinum toxin, plasma peptides, amyloid peptides, incretin peptides, cone snail toxins, gastric inhibitory peptides, insulin-like growth factor, growth hormone-releasing factor, antimicrobial factors, glatiramer, glucagon-like peptide (GLP-1), GLP-1 agonists, exenatide, analogues, and mixtures thereof. In a preferred embodiment, the peptide is glucagon or a glucagon analogue or glucagon mimic peptide. In another embodiment, the peptide is parathyroid hormone. In yet another embodiment, the peptide is leuprorelin. In yet another embodiment, the peptide is glatiramer. In yet another embodiment, the first peptide is pramlintide and the second peptide is insulin. In yet another embodiment, the first peptide is glucagon and the second peptide is exenatide.

[0017] definition

[0018] As used herein, the term "dissolution" refers to a process by which a substance in a gaseous, solid, or liquid state becomes a solute or dissolved component of a solvent, thereby forming a solution of the gaseous, liquid, or solid in the solvent. In some respects, therapeutic agents or excipients, such as ionizing stabilizers, are present or completely dissolved in amounts that meet their solubility limits. The term "dissolved in" refers to the incorporation of a gaseous, liquid, or solid into a solvent to form a solution.

[0019] As used herein, the term "excipient" refers to a natural or synthetic substance formulated together with the active or therapeutic ingredient (not a component of the active ingredient) of a drug into a dosage form. This includes substances used for stabilization, expansion, or enhancement of the therapeutic effect of the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, increasing solubility, modulating tension, relieving injection site discomfort, lowering the freezing point, or increasing stability. In addition to contributing to in vitro stability, such as preventing denaturation or aggregation during shelf life, excipients can also be useful during the manufacturing process, for example, by promoting powder flowability or non-stick properties to facilitate the handling of the relevant active substance.

[0020] In the context of this invention, "small molecule drug" refers to a bioactive compound (and its salts) capable of providing a desired, beneficial, and / or pharmacological effect to a subject. These "small molecule drugs" are organic or inorganic compounds. Therefore, small molecule drugs in the context of this invention are not polymeric compounds. Typically, the molecular weight of said small molecule drug is less than about 1000 Daltons. Some small molecule drugs are "water-sensitive" because they become increasingly unstable in the presence of water. Moreover, salts that can be used with small molecule drugs are known to those skilled in the art, including inorganic acid salts, organic acid salts, inorganic base salts, or organic base salts.

[0021] The term "therapeutic agent" encompasses proteins, peptides, small molecule drugs, and their pharmaceutically acceptable salts. Available salts are known to those skilled in the art and include inorganic acid salts, organic acid salts, inorganic base salts, or organic base salts. The therapeutic agents that can be used in this invention are those proteins, peptides, and small molecule compounds that, when administered to humans or animals, affect a desired, beneficial, and often pharmacological effect, whether used alone or in combination with other pharmaceutical excipients or inert ingredients.

[0022] The terms “peptide” and “peptide compound” refer to an amino acid or amino acid-like (peptide mimic) polymer of up to about 200 amino acid residues linked together by amide (CONH) or other bonds. In some respects, peptides can have up to 150, 100, 80, 60, 40, 20, or 10 amino acids. “Protein” and “protein compound” refer to a polymer of more than 200 amino acid residues linked together by amide bonds. Analogs, derivatives, agonists, antagonists, and pharmaceutically acceptable salts of any peptide or protein compound disclosed herein are included in these terms. The term also includes peptides, proteins, peptide compounds, and protein compounds having D-amino acids, amino acids modified in D- or L-configurations and / or peptide mimic units as part of their structure.

[0023] When referring to peptides or proteins, "analog" and "similar" refer to modified peptides or proteins in which one or more amino acid residues have been substituted with other amino acid residues, or one or more amino acid residues have been deleted from the peptide or protein, or one or more amino acid residues have been added to the peptide or protein, or any combination of these modifications. The addition, deletion, or substitution of amino acid residues can occur at any point or multiple points along the primary structure of the peptide, including at the N-terminus and / or C-terminus of the peptide or protein.

[0024] The term "derivative" in relation to a parent peptide or protein refers to a chemically modified parent peptide or protein or its analogue, wherein at least one substituent is not present in the parent peptide or protein or its analogue. A non-limiting example is a parent peptide or protein that has been covalently modified. Typical modifications include amides, carbohydrates, alkyl groups, acyl groups, esters, polyethylene glycol groups, etc.

[0025] A "single-phase solution" refers to a solution prepared by dissolving a therapeutic agent in a solvent or solvent system (e.g., a mixture of two or more solvents), wherein the therapeutic agent is completely dissolved in the solvent and no particulate matter is visible, such that the solution can be described as optically clear. A single-phase solution can also be referred to as a "single-phase system," and is distinguished from a "two-phase system" in that the latter consists of particulate matter (e.g., powder) suspended in a fluid.

[0026] "Suppress" or "reduce" or any variation of these terms includes any measurable reduction or complete suppression to achieve the desired result.

[0027] "Effective" or "treatment" or "prevention," or any variation of these terms, means sufficient to achieve the desired, anticipated, or intended result.

[0028] When referring to therapeutic agents, "chemical stability" means that the percentage of degradation products resulting from chemical pathways such as oxidation and / or hydrolysis and / or cleavage and / or other chemical degradation pathways is acceptable. Specifically, a formulation is considered chemically stable if, after one year of storage at the intended storage temperature (e.g., room temperature), or one year of storage at 25°C / 60% relative humidity, or one month of storage at 40°C / 75% relative humidity, preferably three months, no more than about 20% of degradation products are formed. In some embodiments, after prolonged storage at the intended storage temperature, the chemically stable formulation forms less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of degradation products.

[0029] When referring to therapeutic agents, "physical stability" refers to the acceptable percentage of aggregates (e.g., dimers, trimers, and larger forms) formed. Specifically, a formulation is considered physically stable if it forms no more than about 15% aggregates after one year of storage at its intended storage temperature (e.g., room temperature); or after one year of storage at 25°C / 60% relative humidity; or after one month, preferably three months, of storage at 40°C / 75% relative humidity. In some embodiments, after prolonged storage at the intended storage temperature, the aggregates of a physically stable formulation are less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0030] "Stable formulation" refers to a formulation in which at least about 65% of the therapeutic agent (e.g., peptide or salt thereof) remains chemically and physically stable after two months of storage at room temperature. Particularly preferred formulations are those in which at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the therapeutic agent remains chemically and physically stable under these storage conditions. Particularly preferred stable formulations do not exhibit degradation after exposure to bactericidal radiation (e.g., gamma, beta, or electron beams).

[0031] As used in this article, “parenteral administration” refers to the administration of a therapeutic agent to a patient via a route other than the digestive tract, and any administration that does not pass through the digestive tract.

[0032] As used herein, “parenteral injection” refers to the administration of a therapeutic agent (e.g., peptides or small molecules) by injection into one or more layers of skin or mucous membranes in an animal, such as a human. Standard parenteral injection involves administration to subcutaneous, intramuscular, or intradermal regions in animals, such as humans. These deeper localizations are targeted relative to superficial dermal sites because the tissue expands more easily, thus accommodating the injection volume required to deliver most therapeutic agents, such as 0.1 to 3.0 cc (mL).

[0033] The term "intradermal" includes applications to the epidermis, dermis, or subcutaneous skin layers.

[0034] As used herein, the term "aprotic polar solvent" refers to a polar solvent that does not contain acidic hydrogen and therefore does not act as a hydrogen bond donor. Aprotic polar solvents include, but are not limited to, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), dimethylacetamide (DMA), and propylene glycol carbonate.

[0035] As used herein, the term "aprotic polar solvent system" refers to a solution in which the solvent is a single aprotic polar solvent (e.g., pure DMSO) or a mixture of two or more aprotic polar solvents (e.g., a mixture of DMSO and NMP).

[0036] As used herein, “residual moisture” may refer to the residual moisture in the pharmaceutical powder prepared by the manufacturer / supplier. Typical powders typically have a residual moisture content of up to 10% (w / w). When these powders are dissolved in aprotic polar solvent systems, the residual moisture in the powder is incorporated into the formulation. Additionally, aprotic polar solvents may also contain a certain level of residual moisture. For example, freshly opened USP-grade DMSO may contain up to 0.1% (w / w) moisture. Residual moisture differs from “added moisture,” where water is intentionally added to the formulation, for example, as a co-solvent or to lower the freezing point of the aprotic polar solvent system. Moisture may also be introduced into the formulation during the addition of ionization-stabilizing excipients (e.g., by adding an inorganic acid (e.g., 1N HCl) from a storage aqueous solution). The total moisture content (%w / w, unless otherwise stated) in the formulation immediately after preparation comprises residual moisture and added moisture.

[0037] As will be understood by those skilled in the art, the terms “about,” “approximately,” or “substantially unchanged” are defined as close to, and in a non-limiting embodiment, the term is defined as less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.5%. Furthermore, “substantially free of water” means having less than 5%, 4%, 3%, 2%, 1%, or less water by weight or volume.

[0038] A pharmaceutically acceptable ingredient, excipient, or component is one that is suitable for use with humans and / or animals and has no undue side effects (such as toxicity, irritation, and allergic reactions) that correspond to a reasonable benefit / risk ratio.

[0039] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable solvent, suspension or carrier used to deliver the pharmaceutical compounds of the present invention to a mammal, such as a human.

[0040] The term "ionizing stabilizer" as used herein refers to an excipient that constructs and / or maintains a specific ionization state of a therapeutic agent. In some aspects, the ionizing stabilizer may be or include a molecule that provides at least one proton or a proton source under suitable conditions. According to the Bronsted-Lowry definition, an acid is a molecule that can donate a proton to another molecule, and thus a molecule that receives the donated proton can be classified as a base. As used herein, and as will be understood by those skilled in the art, the term "proton" refers to a hydrogen ion, a hydrogen cation, or H+ ion. +Hydrogen ions have no electrons and are composed of a nucleus that is usually made up of only protons (for the most common hydrogen isotope, protium). Specifically, a molecule can be considered an acid or proton source as long as it can donate protons to a therapeutic agent, regardless of whether it is fully ionized, mostly ionized, partially ionized, mostly unionized, or completely unionized in a nonproton polar system.

[0041] The term "inorganic acid" as used herein refers to an acid derived from one or more inorganic compounds. Therefore, inorganic acids can also be called "mineral acids." Inorganic acids can be monovalent or polyvalent (e.g., divalent, trivalent, etc.). Examples of inorganic acids include hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid (H2SO4), and phosphoric acid (H3PO4).

[0042] As described herein, an "organic acid" is an organic compound that possesses acidic properties (i.e., can be used as a proton source). Carboxylic acids such as acetic acid or citric acid are examples of organic acids. Other known examples of organic acids include, but are not limited to, alcohols, thiols, enols, phenols, and sulfonic acids. Organic acids can be monobasic or polybasic (e.g., dibasic, tribasic, etc.).

[0043] "Charge characteristics", "charge state", "ionization", "ionization state" and "ionization characteristics" are used interchangeably and refer to the ionization state caused by the protonation and / or deprotonation of the ionic groups of the peptide.

[0044] As used herein, a “co-preparation” is a preparation containing two or more therapeutic agents dissolved in a nonprotic polar solvent system. The therapeutic agents may belong to the same class (e.g., a co-preparation containing two or more therapeutic peptides such as insulin and pramlintide), or the therapeutic agents may belong to different classes (e.g., a co-preparation containing one or more therapeutic small molecules and one or more therapeutic peptide molecules, such as GLP-1 and lissotheline).

[0045] In the claims and / or specification, when an element used with the term "comprising" is not preceded by a quantifier, it may mean "one (kind)," but may also mean "one (kind) or more (kinds)," "at least one (kind)," and "one (kind) or more than one (kind)."

[0046] The words “contain,” “have,” “include,” or “contain” are inclusive or open-ended and do not exclude other, unlisted elements or methods.

[0047] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while specific embodiments of the invention have been pointed out, the detailed description and examples are given by way of example only. Furthermore, it is anticipated that, based on this detailed description, changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. Attached Figure Description

[0048] The following drawings are included as part of and in this specification to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these drawings in conjunction with the detailed description of embodiments given herein.

[0049] Figure 1 (Left figure) shows that when glucagon is dissolved directly in DMSO at 5 mg / mL (as described by Stevenson), insoluble particles form after 24 hours at room temperature. (Right figure) shows a formulation (5 mg / mL) prepared by dissolving glucagon in DMSO containing 5 mM glycine hydrochloride, which remains clear after being stored at 40°C for at least six weeks.

[0050] Figure 2 The effect of HCl concentration on the stability of pramlinpeptide when dissolved in DMSO is shown. Detailed Implementation

[0051] When standard small molecules, peptides, and proteins are prepared into aqueous solutions, these molecules may be sensitive to a variety of physical and chemical degradation pathways. For many of these therapeutic molecules, water-dependent degradation pathways (e.g., hydrolysis, racemization, deamidation) are unavoidable, thus these molecules cannot be adequately stabilized. Therefore, many therapeutic agents cannot be prepared as stable solutions for parenteral injection; instead, they are prepared as powders and reconstituted before use.

[0052] To address the physical and / or chemical instability exhibited by many therapeutic molecules in water, formulations can be prepared by dissolving the therapeutic agent in a biocompatible non-aqueous liquid, such as an aprotic polar solvent. Examples of prior art have been described above, particularly Stevenson '547, which discloses the preparation of compositions by directly dissolving peptide powder in an aprotic polar solvent, and Prestrellski '644, which discloses drying peptide powder from a buffered aqueous solution and then dissolving it in DMSO.

[0053] Using aprotic polar solvents to prepare non-aqueous therapeutic formulations can inhibit many common degradation pathways, particularly those involving water, thereby significantly improving the stability of soluble or dissolved therapeutic molecules. However, the compositions and methods disclosed in the prior art still present problems. In particular, for most therapeutic molecules, direct dissolution in aprotic polar solvents is not a suitable method for preparing stable compositions; the dissolution of leuprorelin described in Stevenson's 547 is an exception. As previously mentioned, and as will be detailed in the examples below, when the peptide hormone glucagon is directly dissolved in DMSO at a concentration of 5 mg / mL, insoluble aggregates form after one day of storage at room temperature. For compositions containing only glucagon and DMSO, 5 mg / mL corresponds to approximately 0.45% (w / w) of the peptide compound, indicating that even at relatively low concentrations, direct dissolution of therapeutic molecules in aprotic polar solvent systems does not itself prevent the physical aggregation and / or gelation of the therapeutic molecules. Furthermore, even if therapeutic molecules do not form insoluble aggregates when directly dissolved in aprotic polar solvent systems, they still tend to undergo chemical degradation.

[0054] Without being bound by theory, it is believed that when therapeutic molecules are formulated in aprotic polar solvent systems, they may require specific ionization properties to exhibit enhanced or optimal stability and solubility. Ionization properties are the charge states obtained through the protonation and / or deprotonation of ionized groups of the therapeutic molecule. For example, protonation of a therapeutic peptide comprising ionized amino acid residues (e.g., arginine, lysine) will impart an overall positive charge to the molecules in solution. The relatively long-range electrostatic repulsion between positively charged peptide molecules can suppress short-range hydrophobic interactions that can lead to physical aggregation and / or gelation. Therefore, without sufficient protonation (i.e., optimal or beneficial ionization properties), therapeutic molecules dissolved in aprotic polar solvent systems may be physically unstable, leading to the formation of soluble and / or insoluble aggregates. Therefore, it may be necessary to contain a sufficient concentration of at least one excipient to act as an ionization stabilizer, which imparts ionization properties to the active agent in the aprotic polar solvent system in order to improve its physical and / or chemical stability. As will be explained in the following sections and illustrated by several examples, the appropriate concentration of the ionizing stabilizer that must be added to the solution depends on several factors, including but not limited to the chemical structure of the ionizing stabilizer, the chemical structure of the active agent, the concentration of the active substance, the solvent system used, the presence of a co-solvent, the presence of additional excipients or formulation components, and their respective concentrations.

[0055] The compositions and methods disclosed in Prestrelski '644 are designed to establish optimal ionization properties of therapeutic molecules before they are dissolved in a nonprotic polar solvent systems. As disclosed in Prestrelski '644, peptide powder from a supplier / manufacturer is initially dissolved in a buffered aqueous solution, wherein the pH of the buffered peptide solution is set to provide optimal stability and solubility for that particular peptide. The peptide is then dried from the buffered aqueous solution (e.g., by freeze-drying or spray drying) into a powder such that the ionization properties of the peptide molecules in the powder are approximately equal to those of the peptide molecules in the aqueous solution used as the drying source. The peptide powder is then dissolved in a nonprotic polar solvent system, where the ionization properties of the peptide molecules are approximately equal to those of the peptide molecules in the powder. Therefore, the ionization properties of the peptide molecules in the nonprotic polar solvent system are approximately equal to those of the peptide molecules in the buffered aqueous solution.

[0056] The formulation method disclosed in Prestrelski '644 (referred to as "pH memory" in the '644 patent) overcomes the stability problems (i.e., physical and chemical degradation) encountered when therapeutic molecules are directly dissolved in aprotic polar solvent systems. However, to optimize the ionization properties of the molecules and impart pH memory before their dissolution in aprotic polar solvents, the therapeutic molecules need to be dried from buffered aqueous solutions, a requirement that imposes significant additional costs, both in terms of time and expense, on the formulation development pathway. In particular, the drying process is known to impose several stresses on the therapeutic molecules and must contain sufficient amounts of additional excipients (e.g., lyophilization protectants, such as trehalose and sucrose, and / or surfactants, such as polysorbate 80) in the aqueous solution to protect the therapeutic molecules, thereby increasing the cost and complexity of the formulation. Moreover, for a given therapeutic molecule, the drying process (e.g., spray drying, freeze drying) often needs to be optimized, either at the laboratory scale, i.e., during the initial research and development phase in the initial development stage, or subsequently at the production scale, i.e., when the process is scaled up and transferred to instruments and facilities capable of producing commercial-scale batches. Therefore, the initial development and optimization of the drying process for a given therapeutic molecule, and the subsequent time and cost associated with repurposing the method and incorporating additional steps into the production process, can be very expensive. Thus, there is a need for a method that can provide therapeutic molecules with appropriate ionization properties in aprotic polar solvent systems, without requiring the drying of molecules in buffered aqueous solutions with pH settings to provide the appropriate ionization properties.

[0057] The inventors provide a solution exhibiting enhanced stability and solubility of numerous therapeutic molecules, based on the suitable or optimal ionization properties of these molecules in an aprotic polar solvent, without the need to dry these therapeutic molecules from aqueous solutions to obtain powder, and then dissolve them in an aprotic polar solvent system. This solution involves the direct dissolution of an ion-stabilizing excipient in an aprotic polar solvent, and the direct dissolution of peptide molecules or small molecules in the aprotic polar solvent solution. Not wishing to be bound by theory, it is believed that by providing a sufficient amount of ion-stabilizing excipient, suitable or optimal ionization properties of the therapeutic molecules can be achieved, and electrostatic repulsion between therapeutic molecules with the same charge polarity (i.e., negatively or positively charged) may be sufficient to prevent physical degradation (e.g., through short-range hydrophobic interactions between molecules that lead to aggregation). This is particularly important for molecules that exhibit a tendency to aggregate in solution, especially as the molecular concentration in solution increases. Furthermore, chemical degradation can be minimized by controlling and optimizing the degree of ionization of therapeutic agents (i.e., protonation or deprotonation), since excessive protonation, for example, can promote chemical instability through degradation reactions such as oxidation (e.g., oxidation of methionine residues) and fragmentation (e.g., cleavage of the peptide backbone). Therefore, for some therapeutic molecules, there may be optimal or beneficial ionization properties obtained through protonation, thereby minimizing physical and / or chemical degradation reactions. For therapeutic peptides, the degree of protonation required for their stability, as well as the amount of ionizing stabilizing excipient required in solution, will depend, among other factors, on the peptide's primary structure (i.e., amino acid sequence) and the peptide concentration in solution.

[0058] Each molecule used as an ionizing stabilizer excipient exhibits a certain tendency to donate protons to the therapeutic molecule in a given solvent system; this tendency to donate protons can be referred to as the relative acidity of the molecule. For a fixed concentration of proton-donating molecules (and for simplicity, assuming only monoproton molecules in this example), molecules with greater acidity will protonate the therapeutic molecule to a much greater degree compared to weak acids. Therefore, the concentration of a given proton-donating molecule (ionizing stabilizer excipient) required to achieve proper or optimal ionization properties of the therapeutic molecule will be inversely proportional to its acidity. These and other non-limiting aspects of the invention are discussed herein.

[0059] In some respects, aprotic polar solvents can be deoxygenated prior to formulation preparation. In the context of this invention, many different techniques can be used to deoxygenate or remove oxygen (degassing or deoxygenation) from aprotic polar solvents. For example, deoxygenation is contemplated to include, but is not limited to, removing oxygen dissolved in a liquid aprotic polar solvent by the liquid alone, by the liquid and other solute molecules (e.g., micelles, cyclodextrins, etc.), or by other solute molecules alone. Non-limiting examples of deoxygenation techniques include placing the aprotic polar solvent under reduced pressure and / or heating the liquid to reduce the solubility of dissolved gases, fractionation, membrane degassing, purging with an inert gas, using a reducing agent, refrigeration pump-thaw cycle, or long-term storage in a container with a gas plug. In one embodiment, aprotic polar solvents are deoxygenated by vacuum degassing. In another embodiment, aprotic polar solvents are deoxygenated by using a deoxygenator. In one example, the deoxygenator is a disc or cascade deoxygenator. In another example, the deoxygenator is a spray deoxygenator. In yet another embodiment, a gas-liquid separation membrane is used to deoxygenate the aprotic polar solvent. In one example, a gas-liquid separation membrane and reduced pressure are used to degas the aprotic polar solvent. In one embodiment, a non-oxygen gas (e.g., N2) is bubbled through the liquid to displace or reduce oxygen in the aprotic polar solvent. In one example, the gas bubbled through the aprotic polar solvent is argon, helium, nitrogen, an inert gas, and / or hydrogen, preferably nitrogen. In another example, a stripping tower is used to bubble the gas through the aprotic polar solvent. In yet another embodiment, one or more reducing agents are used to deoxygenate the aprotic polar solvent. Non-limiting examples of reducing agents include ammonium sulfite, hydrogen, active deoxygenating metals, copper, tin, cadmium, Wood's metal alloys (50% bismuth, 25% lead, 12.5% ​​tin, and 12.5% ​​cadmium), etc. In another embodiment, the aprotic polar solvent is degassed by a refrigeration pump-thawing cycle (e.g., at least 1, 2, 3, or more cycles may be used). In one example, the cryogenic pump-thawing cycle involves freezing the aprotic polar solvent in liquid nitrogen, applying a vacuum, and then thawing the solvent in warm water. In one embodiment, the aprotic polar solvent is deoxidized by long-term storage in a steel, glass, or wooden container. In another embodiment, the aprotic polar solvent is subjected to acoustic treatment, ultrasonic treatment, or stirring during the deoxidation process.

[0060] Once treated or deoxygenated, the aprotic polar solvent may have a dissolved oxygen content of less than 0.1 mM, preferably less than 0.05 mM. The amount of dissolved oxygen in any given aprotic polar solvent can be determined using methods known to those skilled in the art (e.g., a dissolved oxygen meter or probe device, such as a dissolved oxygen probe commercially available from Vernier (Beaverton, Oregon, USA)).

[0061] In some respects, the formulations disclosed in this application can be prepared and / or sealed under an inert gas atmosphere. Common methods include backfilling the primary container closure system (e.g., a bottle) to provide an inert gas (e.g., nitrogen, argon) headspace. Secondary container closure systems (e.g., sealed foil bags) can also be sealed in an inert gas atmosphere.

[0062] I. Therapeutic agents

[0063] Therapeutic agents in the context of this invention include peptide or protein compounds, small molecule drugs, and pharmaceutically acceptable salts thereof. The stability of the same therapeutic agent present in deoxygenated aprotic polar solvents can be further improved compared to a therapeutic agent present in untreated aprotic polar solvents. The increased stability is at least partly attributable to a reduction in oxidative degradation of the therapeutic agent or a reduction in oxidative degradation of the aprotic polar solvent, or both. For the treatment of certain diseases or conditions, those skilled in the art know which therapeutic agents are suitable for treating those diseases or conditions and are able to administer an effective amount of the therapeutic agent in the formulations described herein for the treatment of the disease or condition.

[0064] Non-limiting examples of peptides and proteins (and their salts) that can be used in this invention include, but are not limited to, glucagon, pramlintide, insulin, leuprorelin, luteinizing hormone-releasing hormone (LHRH) agonists, parathyroid hormone (PTH), amylin, angiotensin (1-7), botulinum toxin, plasma peptides, amyloid peptides, gastric inhibitory peptides, insulin-like growth factor, growth hormone-releasing factor, antimicrobial factors, glatiramer, glucagon-like peptide-1 (GLP-1), GLP-1 agonists, exenatide, and analogs thereof, amylin analogs (pramlintide), and mixtures thereof. In some preferred aspects, the therapeutic agents are glucagon, insulin, and / or pramlintide.

[0065] Non-limiting examples of small molecule drugs (and their salts) that can be used in the context of this invention include, but are not limited to, adrenaline, benzodiazepines, catecholamines, triptans, sumatriptan, anthraquinone, small chemotherapeutic molecules (e.g., mitoxantrone), small corticosteroid molecules (e.g., methylprednisolone, beclomethasone dipropionate), small immunosuppressive molecules (e.g., azathioprine, cladribine, cyclophosphamide monohydrate, methotrexate), and small anti-inflammatory molecules (e.g., salicylic acid, acetylsalicylic acid, lisolophrenialine, diflunisal, trisalicylic acid choline magnesium, salicylic acid, benorilate, flufenoxuron, mefenoxuron, meclomethasone). Triflumicacid, diclofenac, fenclofenac, alclofenac, fentimicin, ibuprofen, clobiprofen, ketoprofen, naproxen, fenprofen, fenbufen, sulprofen, indoprofen, tiprofen, benzoxaprofen, pyrrolizumab, tometidine, zometidine, clopinar, indomethacin, sulindac, phenylbutazone, hydroxyphenylbutazone, azaacetone, feprazolam, piroxicam, isoxicam, small molecules used to treat neurological disorders (e.g., cimetidine, ranitidine, famotidine, nizatidine, tacrine, 2linblasti, mepiquat, rivastigmine, cyprofen). Small molecules used to treat cancer (e.g., vincristine, 2-linblastin, paclitaxel, docetaxel, cisplatin, irinotecan, topotecan, gemcitabine, temozolomide, imatinib, bortezomib), statins (e.g., atorvastatin, amlodipine, rosuvastatin, sitagliptin, simvastatin, fluvastatin, pitavastatin, lovastatin, pravastatin, simvastatin), and other taxane derivatives used to treat kidney stones. Small molecule drugs for tuberculosis (e.g., rifampin), small molecule antifungal agents (e.g., fluconazole), small molecule anxiolytics and anticonvulsants (e.g., lorazepam), small molecule anticholinergics (e.g., atropine), small molecule β-agonists (e.g., salbutamol sulfate), small molecule mast cell stabilizers and small molecule agents for treating allergic reactions (e.g., sodium cromoglycate), small molecule anesthetics and small molecule antiarrhythmics (e.g., lidocaine), small molecule antibiotics (e.g., tobramycin, ciprofloxacin), small molecule antimigraines (e.g., sumatriptan), and small molecule antihistamines (e.g., diphenhydramine). In a preferred embodiment, the small molecule is adrenaline.

[0066] In the prevention, diagnosis, mitigation, treatment, or cure of disease, the therapeutic agents of the present invention can be administered subcutaneously. Examples of proteins and protein compounds that can be used in the formulation of the present invention and in the delivery system of the present invention include those proteins that are biologically active or can be used to treat diseases or other pathological conditions.

[0067] Each of the aforementioned peptides, proteins, and small molecule drugs is well-known and available from a variety of manufacturers and sources. Furthermore, the amount of peptide, protein, or small molecule drug in a dosage form can vary depending on currently acceptable levels, the needs of the subject / patient (e.g., age, health, weight, nature and progression of symptoms), etc.

[0068] Therapeutic agents supplied by manufacturers or commercial sources are typically provided in powder form for dissolution in the formulations described herein. Many known techniques can be used to form powder formulations for dissolution.

[0069] One or more peptides can be formulated in any suitable dose in the stable formulation of the present invention. Typically, the peptide (or, in embodiments comprising two or more peptides, each peptide) is present in the formulation at an amount of about 0.5 mg / mL to about 100 mg / mL. In some embodiments, the peptide is present in the formulation at an amount of about 10 mg / mL to about 60 mg / mL. In other embodiments, the peptide is present in the formulation at an amount of about 20 mg / mL to about 50 mg / mL. In other embodiments, the peptide is present in the formulation at an amount of about 5 mg / mL to about 15 mg / mL. In other embodiments, the peptide is present in the formulation at an amount of about 0.5 mg / mL to about 2 mg / mL. In other embodiments, the peptide is present in the formulation at an amount of about 1 mg / mL to about 50 mg / mL. Likewise, it will be apparent to those skilled in the art that the dose of the peptide can vary depending on the peptide used and the disease, disorder, or condition to be treated.

[0070] In some embodiments, the formulations of the present invention further comprise antioxidants. In other embodiments, the formulations further comprise chelating agents. In other embodiments, the formulations of the present invention further comprise preservatives.

[0071] II. Preparations

[0072] The formulations of the present invention comprise therapeutic agents present in a proton-polar solvent system containing at least one ionization-stabilizing excipient. The therapeutic agents may be dissolved (e.g., completely or partially dissolved) or suspended (completely or partially) in the proton-polar solvent system. Furthermore, the formulations may be configured as single-phase solutions, ointments or slurries, gels, emulsions, or suspensions.

[0073] In some embodiments, the therapeutic agent is present in a "pure," i.e., non-aqueous, aprotic polar solvent. In other embodiments, the therapeutic agent is present in a solvent system that is a mixture of two or more aprotic polar solvents (i.e., an aprotic polar solvent system). An example is a mixture of 75 / 25 (% v / v) DMSO and NMP. However, in some embodiments, a co-solvent may be used, which is a mixture of one or more aprotic polar solvents. Non-limiting examples of co-solvents include water, ethanol, propylene glycol (PG), glycerol, and mixtures thereof. In some aspects, water may be specifically excluded or limited as a co-solvent, i.e., the co-solvent is a non-aqueous co-solvent. The amount of co-solvent in the formulation may be from about 0.5% (w / v) to about 50% (w / v), for example, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% (w / v). In some embodiments, the amount of co-solvent in the formulation is about 10% (w / v) to about 50% (w / v), about 10% (w / v) to about 40% (w / v), about 10% (w / v) to about 30% (w / v), about 10% (w / v) to about 25% (w / v), about 15% (w / v) to about 50% (w / v), about 15% (w / v) to about 40% (w / v), about 15% (w / v) to about 30% (w / v), or about 15% (w / v) to about 25% (w / v).

[0074] In addition to ionization-stabilizing excipients, the formulations of the present invention may also contain one or more other excipients. In some embodiments, the other excipients are selected from sugars, starches, sugar alcohols, antioxidants, chelating agents, and preservatives. Examples of suitable sugar excipients include, but are not limited to, trehalose, glucose, sucrose, etc. Examples of suitable starches for stabilizing excipients include, but are not limited to, hydroxyethyl starch (HES). Examples of suitable sugar alcohols (also known as polyols) for stabilizing excipients include, but are not limited to, mannitol and sorbitol. Examples of suitable antioxidants include, but are not limited to, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulfate, sulfites, BHT, BHA, ascorbate palmitate, propyl gallate, N-acetyl-L-cysteine ​​(NAC), and vitamin E. Examples of suitable chelating agents include, but are not limited to, EDTA, disodium EDTA, tartaric acid and its salts, glycerol, and citric acid and its salts. Examples of suitable inorganic salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, calcium sulfate, and magnesium sulfate. Examples of suitable preservatives include, but are not limited to, benzyl alcohol, methylparaben, propylparaben, and mixtures thereof. Additional formulation components include local anesthetics such as lidocaine or procaine. In some embodiments, the amount of additional stabilizing excipients present in the formulation is about 0.1% (w / v) to about 60% (w / v), about 1% (w / v) to about 50% (w / v), about 1% (w / v) to about 40% (w / v), about 1% (w / v) to about 30% (w / v), about 1% (w / v) to about 20% (w / v), about 5% (w / v) to about 60% (w / v), and about 5% (w / v) to about 50% (w / v). / v), about 5% (w / v) to about 40% (w / v), about 5% (w / v) to about 30% (w / v), about 5% to about 20% (w / v), about 10% (w / v) to about 60% (w / v), about 10% (w / v) to about 50% (w / v), about 10% (w / v) to about 40% (w / v), about 10% (w / v) to about 30% (w / v), or about 10% (w / v) to about 20% (w / v). In some embodiments, the amount of additional stabilizing excipient present in the formulation is about, at most, or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (w / v).

[0075] III. Treatment Methods

[0076] On the other hand, the present invention provides a method for treating a disease, symptom, or disorder, wherein the method applies to a subject an effective amount of a therapeutic agent in a stable formulation described herein to treat, alleviate, or prevent said disease, symptom, or disorder.

[0077] In some embodiments, the treatment method of the present invention includes administering a therapeutic agent for hypoglycemia to a subject suffering from hypoglycemia, which treats hypoglycemia by using an effective amount of a stabilizing agent as described herein. In some embodiments, the subject is given a stabilizing agent comprising glucagon. In some aspects, hypoglycemia may be caused by diabetes or non-diabetes-related diseases, conditions, and disorders.

[0078] As described by the working group of the American Diabetes Association and the Endocrine Society (Seaquist et al., (2013), Diabetes Care, Vol. 36, pp. 1384-1395), for hypoglycemia, a single threshold of plasma glucose concentration that can be used to define diabetic hypoglycemia is not usually determined because the glucose threshold for symptoms of hypoglycemia (including other reactions) will shift to a lower plasma glucose concentration after a recent pre-hypoglycemic episode and to a higher plasma glucose concentration in patients with poorly controlled diabetes and infrequent hypoglycemia.

[0079] Nevertheless, defining alarm values ​​can raise awareness among patients and caregivers about the potential hazards associated with hypoglycemia. Patients at risk of hypoglycemia (i.e., those treated with sulfonylureas, meglitinides, or insulin) should be alert to the possibility of hypoglycemia when a sustained glucose concentration monitored by self-monitoring plasma glucose or subcutaneous glucose is ≤70 mg / dL (≤3.9 mmol / L). Because this value is a higher glucose threshold relative to the symptoms of non-diabetic individuals and well-controlled diabetic individuals, there is usually also time to prevent the onset of clinical hypoglycemia and some margin for error in the limited accuracy of monitoring devices at hypoglycemic levels.

[0080] Severe hypoglycemia is an event requiring the active administration of carbohydrates, glucagon, or other corrective measures by another person. Plasma glucose levels may be unavailable during the event, but recovery of neurological function after plasma glucose returns to normal is considered sufficient evidence that the event was induced by low plasma glucose levels. Typically, these events occur when plasma glucose levels are ≤50 mg / dL (2.8 mmol / L). Documented symptomatic hypoglycemia is an event with typical hypoglycemic symptoms accompanied by a measured plasma glucose level ≤70 mg / dL (≤3.9 mmol / L). Asymptomatic hypoglycemia events do not involve typical hypoglycemic symptoms, but a plasma glucose level measurement is ≤70 mg / dL (≤3.9 mmol / L). Probable symptomatic hypoglycemia is an event with typical hypoglycemic symptoms but without a measured plasma glucose level, but presumed to be caused by a plasma glucose level ≤70 mg / dL (≤3.9 mmol / L). Pseudohypoglycemia is an event in which a diabetic person reports any typical symptoms of hypoglycemia and a measured plasma glucose concentration >70 mg / dL (>3.9 mmol / L) but close to this level.

[0081] The indications for treatment by this invention further include hypoglycemia-associated autonomic failure (HAAF). As described by Philip E. Cryer in Perspectives in Diabetes, Mechanisms of Hypoglycemia-Associated Autonomic Failure and Its Component Syndromes in Diabetes, Diabetes, Vol. 54, pp. 3592-3601 (2005), “recent iatrogenic hypoglycemia causes a reversal of glucose deficiency (by reducing the adrenaline response to a given level of late hypoglycemia in a setting of reduced insulin deficiency and increased glucagon deficiency) and unconscious hypoglycemia (for a given level of hypoglycemia, reduced sympathetic adrenaline and the resulting neurogenic symptom response), thus leading to a vicious cycle of hypoglycemia.” HAAF affects patients with advanced type I and type II diabetes. Additionally, this invention can also treat hypoglycemia in patients after islet cell transplantation.

[0082] The formulations of this invention can also be used to treat hyperinsulinemia-induced hypoglycemia, which broadly refers to the condition and effect of hypoglycemia caused by excessive insulin. Administration of exogenous insulin to patients with type 1 diabetes is the most common, severe, but often transient, form of hyperinsulinemia-induced hypoglycemia. This type of hypoglycemia can be defined as iatrogenic hypoglycemia and is a limiting factor in glycemic control in both type 1 and type 2 diabetes. Nocturnal hypoglycemia (nocturnal hypoglycemia) is a common iatrogenic hypoglycemia in patients taking exogenous insulin. However, hyperinsulinemia-induced hypoglycemia can also be caused by endogenous insulin, such as congenital excess insulin, insulinoma (an insulin-secreting tumor), exercise-induced hypoglycemia, and reactive hypoglycemia. Reactive hypoglycemia is a non-diabetic form of hypoglycemia caused by postprandial hypoglycemia, usually occurring within four hours of eating. Reactive hypoglycemia can also be referred to as postprandial hypoglycemia. Symptoms and signs of reactive hypoglycemia may include hunger, weakness, tremors, drowsiness, sweating, confusion, and anxiety. Gastric surgery (such as bariatric surgery) is one possible cause, as food may enter the small intestine too quickly after the procedure. Other causes include enzyme deficiencies that make it difficult for the body to break down food, or increased sensitivity to the hormone adrenaline.

[0083] In some embodiments, the disease, condition, or disorder treated using the stable formulation of the present invention is a diabetic condition. Examples of diabetic conditions include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, hypoglycemia, and metabolic syndrome. In some embodiments, the disease, condition, or disorder is hypoglycemia. In some embodiments, the disease, condition, or disorder is diabetes.

[0084] In some embodiments, the treatment method of the present invention includes treating diabetes by administering to a subject suffering from diabetes a therapeutically effective amount of a therapeutic agent in a stable formulation as described herein. In some embodiments, the subject is administered a stable formulation comprising insulin. In some embodiments, the subject is administered a stable formulation comprising pramlinide. In some embodiments, the subject is administered a stable formulation comprising both insulin and pramlinide. In some embodiments, the subject is administered a stable formulation comprising exenatide. In some embodiments, the subject is administered a stable formulation comprising glucagon and exenatide.

[0085] In some cases, epinephrine can be administered to patients who are experiencing or suspected of having an allergic reaction. Epinephrine can be used as an emergency treatment for type I anaphylaxis, which is caused by a variety of sources, including but not limited to food, drugs and / or other allergens, allergen immunotherapy, diagnostic test substances, insect bites and stings, and idiopathic or exercise-induced anaphylaxis.

[0086] The dosage of the peptide or small molecule drug used to treat diseases, conditions, or disorders (e.g., diabetic conditions, hypoglycemia, or allergic reactions) described herein is in accordance with the dosage and duration of treatment practiced by those skilled in the art. General guidance on appropriate dosages of all pharmacological agents used in the methods of this invention is provided in Goodman and Gilman’s *The Pharmacological Basis of Therapeutics*, 11th edition, 2006, above, and in Physicians' Desk Reference (PDR), such as in 65 (2011) or 66 (2012), PDR Network, LLC, each of which is incorporated herein by reference. The appropriate dosage of the peptide drug used to treat diseases, conditions, or disorders as described herein will vary depending on several factors, including the formulation of the composition, patient response, severity of the condition, subject weight, and the prescribing physician's judgment. An effective dosage of the formulation is capable of delivering a medically effective amount of the peptide drug. The dosage may be increased or decreased over time, as required by the individual patient or as determined by a healthcare professional.

[0087] Determining the effective amount or effective dose is entirely within the competence of those skilled in the art, particularly based on the detailed disclosure provided herein. Typically, formulations delivering these doses may contain one, two, three, four, or more small molecules, peptides, or peptide analogs (collectively referred to as “peptides” unless explicitly excluded), wherein the concentration of each peptide is about 0.1 mg / mL up to the solubility limit of the peptide in the formulation. This concentration is preferably from about 1 mg / mL to about 100 mg / mL. In some aspects, the concentrations are about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL. For medical personnel, the concentrations of small molecules are known and can be established and implemented using the disclosure provided herein, for example, from 0.01 mg / mL to 500 mg / mL, or doses of 5, 10, 25, 50, 75, 100, 200, 500 to 1000 mg, including all values ​​and ranges therein.

[0088] The formulations of the present invention can be administered subcutaneously, intradermally, or intramuscularly (e.g., by injection or infusion). In some embodiments, the formulations are administered subcutaneously. The formulations can also be delivered percutaneously, for example by applying the composition topically to the skin (e.g., applying the composition to the skin or loading the composition onto a skin patch and attaching the skin patch to the skin).

[0089] The formulations disclosed in this invention can be administered via infusion or injection using any suitable device. For example, the formulations of this invention can be placed in a syringe (e.g., a pre-filled syringe), a pen-type injection device, an auto-injector device, or a pump device. In some embodiments, the injection device is a multi-dose infusion pump device or a multi-dose auto-injector device. The formulation is presented in such a way that it readily flows from the needle upon activation of the injection device, such as an auto-injector, to deliver the peptide drug. Suitable pen / auto-injector devices include, but are not limited to, those manufactured by Becton-Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, etc. Suitable pump devices include, but are not limited to, those manufactured by Tandem Diabetes Care, Inc., Delsys Pharmaceuticals, etc.

[0090] In some embodiments, the formulations of the present invention are provided in preparation for administration in vials, cartridges, or pre-filled syringes.

[0091] In some embodiments, the stabilizing agent is used to formulate a drug for treating hypoglycemia. In some embodiments, the stabilizing agent comprises glucagon or a salt thereof (e.g., glucagon acetate). In some embodiments, the stabilizing agent comprises glucagon and exenatide.

[0092] In some embodiments, the stabilizing agent is used to formulate a drug for treating diabetes. In some embodiments, the stabilizing agent comprises insulin. In some embodiments, the stabilizing agent comprises exenatide. In some embodiments, the stabilizing agent comprises pramlintide. In some embodiments, the stabilizing agent comprises both insulin and pramlintide.

[0093] IV Reagent / Container

[0094] Kits are also considered for use in certain aspects of the invention. For example, the kit may include the formulations of the invention. The kit may include a container. For example, in one aspect, the formulation may be contained in a container that is ready to be administered to a subject without having to reconstitute or dilute the formulation. That is, the formulation to be administered may be stored in the container and readily available for use as needed. The container may be a device. The device may be a syringe (e.g., a pre-filled syringe), a pen injection device, an autoinjector device, a device capable of pumping or administering the formulation (e.g., an automatic or non-automatic external pump, an implantable pump, etc.), or an infusion bag. Suitable pen / autoinjector devices include, but are not limited to, those manufactured by Becton-Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, etc. Suitable pump devices include, but are not limited to, those manufactured by Tandem Diabetes Care, Inc., Delsys Pharmaceuticals, etc.

[0095] V. Example

[0096] Large quantities of peptides and small molecule formulations can be prepared using the methods disclosed in this application and methods disclosed in the prior art (e.g., dissolving peptides directly in a non-proton polar solvent system and drying the peptides from a buffer aqueous solution before dissolving them in the non-proton polar solvent system). As shown in the examples below, the compositions prepared by the methods of the present invention exhibit higher physical and chemical stability compared to compositions observed when peptide powders are directly dissolved in a non-proton polar solvent system.

[0097] Some embodiments of the invention disclosed herein will be described in more detail through specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. For example, those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0098] Example 1

[0099] In this embodiment, glycine hydrochloride (CAS No. 6000-43-7) was directly dissolved in DMSO (CAS No. 67-68-5) at concentrations of 5 mM, 10 mM, and 20 mM. Glucagon powder (MW = 3483 g / mol; Bachem AG, product number 4074733) was then added to dissolve it into a peptide concentration of 5 mg / mL, thereby preparing a glucagon solution. The prepared sample solutions are shown in Table 1.

[0100] Table 1: Glucagon sample solutions prepared by directly dissolving glycine hydrochloride and glucagon powder in DMSO.

[0101] glucagon concentration solvent Added excipients 5mg / mL DMSO 5mM glycine hydrochloride 5mg / mL DMSO 10mM glycine hydrochloride 5mg / mL DMSO 20mM glycine hydrochloride

[0102] The reversed-phase high-performance liquid chromatography (RP-HPLC) method used to assess chemical stability was a gradient method, with mobile phases A and B being 0.1% (v / v) aqueous solution of TFA (trifluoroacetic acid) and 0.1% (v / v) acetonitrile solution of TFA, respectively. A C8 column (BioBasic) was used. TM -8; ThermoScientific (inner diameter 4.6 mm × length 250 mm, particle size 5 μm), column temperature 37 ℃, flow rate 1.0 mL / min, sample injection volume 6 μL, detection wavelength 280 nm.

[0103] Sample formulations prepared with glycine hydrochloride at different concentrations were sealed in containers with a 13mm diameter. The solutions were stored in 2-mL CZ bottles (Crystal-Zenith, West Pharmaceuticals, PA, USA) with coated rubber stoppers (butyl rubber stoppers coated with a fluorocarbon film, manufactured by West Pharmaceuticals) at 40°C for 6 weeks. The solutions were compared with a 5 mg / mL glucagon formulation prepared by drying (lyophilizing) from a non-volatile buffer and reconstituted in DMSO (as described in Presrelski '644), or with a 5 mg / mL glucagon formulation prepared by directly dissolving glucagon powder in DMSO (as described in Stevenson '547). The stability of the formulations was assessed by RP-HPLC as described above, and the purity of the glucagon is shown in Table 2.

[0104] Visual observation showed that after storage at 40°C for six weeks (42 days), the sample solution containing glycine hydrochloride as a formulation excipient remained clear and colorless, and no precipitation and / or gelation occurred.

[0105] Table 2: Stability of 5 mg / mL glucagon solution stored at 40 °C (provided in peptide purity).

[0106]

[0107] At room temperature, a 5 mg / mL (approximately 0.45% w / w) glucagon solution prepared by directly dissolving glucagon powder in DMSO exhibited physical aggregation over 24 hours, as shown by the formation of insoluble substances. Figure 1In contrast, a 5 mg / mL glucagon solution prepared by dissolving glucagon powder in DMSO in the presence of 5.0 mM glycine hydrochloride remained clear (i.e., no precipitation) and colorless throughout the incubation period (6 weeks at 40°C). A glucagon formulation pre-lyophilized from a buffer solution (pH 3.0) before being reconstituted to 5 times its initial concentration in DMSO also showed approximately 97% glucagon purity after six weeks of storage at 40°C. This buffer solution contained 1.0 mg / mL glucagon, 2.0 mM glycine, and 1.0% (w / v) trehalose (i.e., after reconstitution, the composition in the aprotic polar solvent system was 5.0 mg / mL glucagon, 10.0 mM glycine, and 5.0% (w / v) trehalose).

[0108] Compared to existing techniques that directly dissolve peptide powder in aprotic polar solvents, the compositions prepared by the method of the present invention exhibit enhanced stability. Furthermore, compared to methods that require drying the peptide in a buffered aqueous solution and then reconstitute the powder in an aprotic polar solvent system, the formulations of the present invention can serve as an alternative approach to provide a method for preparing high-concentration, stable glucagon formulations in aprotic polar solvent systems.

[0109] Example 2

[0110] In this embodiment, a 5 mg / mL glucagon solution was prepared by dissolving glucagon powder (Bachem AG, product number 4074733) in DMSO, wherein the DMSO contained different concentrations of added hydrochloric acid, from 0.001 M (1 mM) to 0.01 M (10 mM). To minimize the amount of water added to the formulation, 10 mM and 5.6 mM HCl DMSO solutions were prepared using 5 N HCl, while 3.2 mM, 1.8 mM, and 1.0 mM solutions were prepared using 1 N HCl. For example, a 10 mM HCl DMSO solution was prepared by adding 20 μL of 5 N HCl to 9.98 mL of (pure) DMSO, while a 1.0 mM HCl DMSO solution was prepared by adding 10 μL of 1 N HCl to 9.99 mL of (pure) DMSO. Samples of each formulation were stored in 2 mL CZ bottles (0.5 mL sample per bottle) and incubated at 40°C.

[0111] The chemical stability of the peptides was assessed by RP-HPLC after 28 and 58 days of storage, and their purity is reported in Table 3. The addition of 1.0 mM HCl was insufficient to prevent the formation of insoluble aggregates in 5 mg / mL glucagon solutions, therefore the chemical stability of these samples was not measured. Conversely, glucagon molecules exhibited relatively rapid chemical degradation when 10 mM HCl was added to the solution. Decreasing the concentration of HCl added to the solution increased the overall stability of the glucagon molecules; solutions with 3.2 mM and 1.8 mM HCl showed the highest stability during the examined time periods.

[0112] Table 3: Stability of 5 mg / mL glucagon-DMSO solution stored at 40 °C (provided in peptide purity).

[0113] glucagon Added hydrochloric acid Day 28 Day 58 5mg / mL 10.0mM 36.9％ 0％ 5mg / mL 5.6mM 90.8％ 85.3％ 5mg / mL 3.2mM 98.0％ 96.8％ 5mg / mL 1.8mM 98.3％ 97.4％ 5mg / mL 1.0mM Insoluble aggregates Insoluble aggregates

[0114] Example 3

[0115] Glucagon powder (Bachem AG, product number 4074733) was dissolved in DMSO to prepare 5 mg / mL sample solutions containing various additive concentrations of glycine hydrochloride (CAS No. 6000-43-7), betaine hydrochloride (CAS No. 590-46-5), or hydrochloric acid (IN; CAS No. 7647-01-0). Table 4 lists the different concentrations of each ionizing stabilizer used to prepare the sample formulations. Samples of each formulation were stored in CZ bottles and incubated at 40°C. After 28 days of storage, the chemical stability of the glucagon peptides was assessed by RP-HPLC, and their purity is reported in Table 4. This example demonstrates that the proton-donating ability (i.e., its “strength”) of the added ionizing stabilizer may affect the concentration required to stabilize the therapeutic molecule. The peptide model was chosen because glucagon molecules tend to gel (i.e., form insoluble aggregates) when insufficiently protonated. After being stored at 40°C for 28 days, glycine hydrochloride at a concentration as high as 2 mM was insufficient to prevent the formation of insoluble aggregates in the solution, but betaine hydrochloride and hydrochloric acid at the same concentration were sufficient to prevent the formation of insoluble aggregates.

[0116] Table 4: Stability of 5 mg / mL glucagon-DMSO solution stored at 40°C for 28 days (provided as peptide purity %).

[0117] Glucagon powder Ionization stabilized excipients Concentration added Peptide purity (%) 5mg / mL glycine hydrochloride 0.5mM Insoluble aggregates 5mg / mL glycine hydrochloride 1.0mM Insoluble aggregates 5mg / mL glycine hydrochloride 2.0mM Insoluble aggregates 5mg / mL glycine hydrochloride 3.0mM 98.5％ 5mg / mL glycine hydrochloride 4.0mM 98.6％ 5mg / mL glycine hydrochloride 5.0mM 99.1％ 5mg / mL Betaine hydrochloride 0.5mM Insoluble aggregates 5mg / mL Betaine hydrochloride 2.0mM 98.6％ 5mg / mL Betaine hydrochloride 5.0mM 98.4％ 5mg / mL HCl 1.0mM Insoluble aggregates 5mg / mL HCl 1.8mM 98.3％ 5mg / mL HCl 3.2mM 98.0％

[0118] Example 4

[0119] The following examples demonstrate the stability of glucagon solutions prepared according to the method of the present invention in the presence of added formulation components (e.g., inactive agents, excipients). Sample solutions with a concentration of 5 mg / mL were prepared by dissolving glucagon powder (Bachem AG, product number 4074733) in DMSO containing approximately 3.2 mM of added HCl (from a stock solution of 1N HCl). Different concentrations of water and 5.5% (w / v) mannitol (CAS No. 69-65-8) and 1% (v / v) benzyl alcohol (CAS No. 100-51-6) were added to these solutions. Table 5 lists the experimental samples tested.

[0120] Samples of each formulation were stored in CZ vials and incubated at room temperature (22-23°C). After 180 days of storage, the chemical stability of the glucagon peptides was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 5. This example demonstrates that additional formulation components (e.g., moisture, inactive agents, excipients) can be included in the formulation and still form stable compositions after storage at room temperature for approximately 6 months.

[0121] Table 5: Stability of 5 mg / mL glucagon-DMSO solution stored at room temperature for 180 days. Stability is provided by the percentage of glucagon purity as assessed by RP-HPLC.

[0122]

[0123] Example 5

[0124] The following examples demonstrate the effect of peptide concentration on the amount of ionized stabilizing excipient required for stabilizing formulations.

[0125] Glucagon powder (Bachem AG, product number 4074733) was dissolved in DMSO to prepare sample solutions with concentrations of 20-50 mg / mL, wherein the DMSO contained various concentrations of added HCl (from a 1N stock solution (CAS No. 7647-01-0)). The experimental samples tested are listed in Table 6. The formulations were stored in 2 mL CZ bottles (0.5 mL sample solution per bottle) and placed in a stabilization chamber at 40°C / 75% RH. The physical stability of the samples was assessed by visual inspection, noting the presence of insoluble particles. Higher peptide concentrations required higher concentrations of ionizing stabilizer (HCl in this example) to prevent the aggregation and formation of insoluble aggregates. The chemical stability of the peptides was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 6 (note that formulations containing insoluble particles were not tested by RP-HPLC).

[0126] Table 6: Chemical and physical stability of glucagon-DMSO solution containing added hydrochloric acid after 84 days of storage at 40°C / 75%RH.

[0127]

[0128] Example 6

[0129] The following examples demonstrate the stability of glucagon solutions prepared according to the method of the present invention using nitric acid, sulfuric acid, phosphoric acid or citric acid as ionization stabilizing excipients.

[0130] A sample solution with a concentration of 5 mg / mL was prepared by dissolving glucagon powder (Bachem AG, product number 4074733) in DMSO containing various concentrations of added HNO3 (1M solution prepared from 70% (w / w) stock solution (CAS No. 7697-37-2) with added nitric acid). Table 7 lists the experimental samples tested. The formulation was stored in a container... The sample solution was sealed in 2 mL CZ bottles (0.5 mL per bottle) with coated rubber stoppers and placed in a stability chamber at 40 °C / 75% RH. After 56 days of storage, the chemical stability of the glucagon peptide was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 7.

[0131] Table 7: Stability of 5 mg / mL glucagon-DMSO solution containing added nitric acid after 56 days of storage at 40 °C / 75% RH. Stability is provided by glucagon purity assessed by RP-HPLC.

[0132] glucagon Added nitric acid glucagon purity % 5mg / mL 1.0mM Insoluble aggregates 5mg / mL 2.0mM 96.2％ 5mg / mL 5.0mM 94.8％ 5mg / mL 7.5mM 86.5％ 5mg / mL 10.0mM 78.6％

[0133] Glucagon powder (Bachem AG, product number 4074733) was dissolved in DMSO to prepare a 5 mg / mL sample solution. The DMSO contained various concentrations of added sulfuric acid (from 1N (0.5M) stock solution (CAS No. 7664-93-9)) and 5% (w / v) trehalose (from dihydrate; CAS No. 6138-23-4). The experimental samples tested are listed in Table 8. The sample preparations were stored in a container... The sample solution was sealed in 2 mL CZ bottles (0.5 mL per bottle) with coated rubber stoppers and placed in a stability chamber at 40 °C / 75% RH. After 84 days of storage, the chemical stability of the glucagon peptide was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 8.

[0134] Table 8: Stability of 5 mg / mL glucagon-DMSO solution containing added sulfuric acid and 5% (w / v) trehalose after 84 days of storage at 40 °C / 75% RH. Stability is provided by glucagon purity assessed by RP-HPLC.

[0135] glucagon Added sulfuric acid glucagon purity % 5mg / mL 2.0mM Insoluble aggregates 5mg / mL 4.0mM 95.3％ 5mg / mL 5.0mM 95.2％ 5mg / mL 6.3mM 94.3％ 5mg / mL 7.9mM 93.3％ 5mg / mL 10.0mM 92.3％ 5mg / mL 12.6mM 87.2％

[0136] A sample solution with a concentration of 5 mg / mL was prepared by dissolving glucagon powder (Bachem AG, product number 4074733) in DMSO containing varying concentrations of added phosphoric acid (the phosphoric acid was added from a 1M solution prepared from an 85% (w / w) stock solution (CAS No. 7664-38-2)). Table 9 lists the experimental samples tested. The sample formulations were stored in a container... The sample solution was placed in 2 mL CZ bottles (0.5 mL per bottle) sealed with coated rubber stoppers and placed in a stability chamber at 40 °C / 75% RH. After 80 days of storage, the chemical stability of the glucagon peptide was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 9.

[0137] Table 9: Stability of 5 mg / mL glucagon-DMSO solution containing added phosphate after 80 days of storage at 40°C / 75% RH. Stability is provided by glucagon purity assessed by RP-HPLC.

[0138] glucagon Added phosphoric acid glucagon purity % 5mg / mL 10mM Insoluble aggregates 5mg / mL 20mM 85.4％ 5mg / mL 40mM 88.8％ 5mg / mL 60mM 89.6％ 5mg / mL 80mM 88.7％ 5mg / mL 100mM 89.1％

[0139] A sample solution with a concentration of 5 mg / mL was prepared by dissolving glucagon powder in DMSO containing various concentrations of added citric acid (CAS No. 77-92-9), which was directly dissolved in pure DMSO. Table 10 lists the experimental samples tested. The sample preparations were stored in a container... The sample solution was placed in 2 mL CZ bottles (0.5 mL per bottle) sealed with coated rubber stoppers and placed in a stability chamber at 40 °C / 75% RH. After 65 days of storage, the chemical stability of the glucagon peptide was assessed by RP-HPLC (according to the method described in Example 1), and the purity of glucagon was reported in Table 10.

[0140] Table 10: Stability of 5 mg / mL glucagon-DMSO solution containing added citric acid after 65 days of storage at 40°C / 75% RH. Stability is provided by glucagon purity assessed by RP-HPLC.

[0141] glucagon <![CDATA[[C6H8O7] added]]> glucagon purity % 5mg / mL 2.5mM 90.4％ 5mg / mL 5.0mM 86.8％ 5mg / mL 10.0mM 82.3％ 5mg / mL 15.0mM 78.1％ 5mg / mL 20.0mM 74.0％

[0142] This example demonstrates that various acids, including organic and inorganic acids, can be used as ionization-stabilizing excipients. The required concentration of a given ionization-stabilizing excipient in a given solvent system will vary depending on various formulation parameters, including API, API concentration, the presence of other formulation components (e.g., water, excipients), and the acid strength of the ionization-stabilizing excipient.

[0143] Example 7

[0144] A 1 mg / mL amylineptide analogue formulation was prepared by dissolving pralamineptide acetate powder (molecular weight = 3949.4; CAS No. 196078-30-5; ChemPep, Inc., Wellington, FL) in DMSO in the presence of 5 mM glycine hydrochloride (CAS No. 6000-43-7) or 5 mM anhydrous citric acid (CAS No. 77-92-9). For comparison, pralamineptide acetate powder was directly dissolved in DMSO at the same concentration (without adding excipients). Samples of each formulation were stored in CZ bottles and incubated at 40°C. The sample solutions remained clear (i.e., free of insoluble aggregates) and colorless throughout the study. The chemical stability of the peptides was assessed by RP-HPLC after 14 and 28 days of storage, according to the method described in Example 1. As shown in Table 11, the stability of the solution containing 5 mM glycine hydrochloride and 5 mM citric acid is enhanced compared to the solution containing only pramlinopeptide and DMSO.

[0145] Table 11: Stability of Pramlinpeptide-DMSO solution stored at 40°C (provided as a percentage of peptide purity).

[0146]

[0147] Example 8

[0148] Amylin peptide acetate powder, an analogue of amylin, was dissolved in DMSO (1 mg / mL), and hydrochloric acid of varying concentrations, from 0.00001 M (0.01 mM) to 0.1 M (100 mM), was added. DMSO solutions of 100 mM and 10 mM HCl were prepared using 5 N HCl, and DMSO solutions of 1 mM, 0.1 mM, and 0.01 mM HCl were prepared using 1 N HCl. For example, for a 100 mM HCl DMSO solution, 10 μL of 1 N HCl was added to 9.99 mL of DMSO. Samples of each formulation were stored in CZ bottles and incubated at 40 °C. After 31 days of storage, the chemical stability of the peptides was assessed by RP-HPLC according to the method described in Example 1. Throughout the study, the sample solutions remained clear (i.e., free of insoluble substances) and colorless. However, as shown in Table 12, the addition of a specific amount of HCl (DMSO solution of 1 mM HCl) provides improved peptide stability and minimizes chemical degradation compared to other sample formulations.

[0149] Table 12: Stability of 1 mg / mL pramlinpeptide-DMSO solution stored at 40 °C (provided as peptide purity %).

[0150] Pramlinpeptide Added hydrochloric acid mM Day 0 Day 31 1mg / mL 100 100％ 0％ 1mg / mL 10 100％ 72.9％ 1mg / mL 1 100％ 100.0％ 1mg / mL 0.1 100％ 72.8％ 1mg / mL 0.01 100％ 43.2％

[0151] Example 9

[0152] To further investigate the range of excipiented HCl that could stabilize the amylinide analog pralaminide in DMSO solution, 5 mg / mL pralaminide formulations were prepared by dissolving pralaminide acetate powder in DMSO in the presence of DMSO hydrochloric acid solutions of varying concentrations, from 0.00032 (0.32 mM) to 0.00316 M (3.16 mM). The HCl concentrations studied are shown in Table 13. 0.5 mL volumes of the solution were stored in 2 mL CZ bottles and placed in an incubator set at 40 °C. Throughout the study, the sample formulations remained clear (i.e., free of insoluble material) and colorless. After 31 days of storage, the stability of the peptide in the formulations (assessed by RP-HPLC according to the method described in Example 1) is shown in Table 13.

[0153] Table 13: Stability of 5 mg / mL pramlinpeptide-DMSO solution stored at 40 °C for 31 days (provided as peptide purity %).

[0154] Pramlinpeptide powder concentration Added hydrochloric acid mM peptide purity 5mg / mL 3.16 69.0％ 5mg / mL 1.78 87.3％ 5mg / mL 1.26 94.9％ 5mg / mL 1.00 97.7％ 5mg / mL 0.79 97.0％ 5mg / mL 0.56 90.5％ 5mg / mL 0.32 59.2％

[0155] The data plotted in Table 13 show the optimal range of ion-stabilizing excipients that can be added to optimize peptide stability (approximately 1.00 mM in this example). Figure 2 ).like Figure 2 As shown (where the X-axis represents the concentration of added HCl (mM), expressed logarithmically), deviations from the optimal concentration of added HCl (by increasing or decreasing the HCl concentration) promote the chemical and / or physical degradation of already dissolved pramlinpeptide molecules.

[0156] Example 10

[0157] The concentration of the ion-stabilizing excipient required to stabilize a given peptide will depend on various formulation parameters, including the amino acid sequence of the peptide and the concentration of the peptide in solution. In this example, two different concentrations of pramlinpeptide acetate DMSO solutions were prepared: 1 mg / mL and 5 mg / mL. An aqueous solution of the ionizing stabilizing excipient HCl (5N and 1N concentrations) was added to the solutions to obtain the final HCl concentrations specified in the left column. The samples were then stored at 40°C for one month. As shown in Table 14, the stability of the pramlinpeptide molecule, as assessed by RP-HPLC according to the method described in Example 1, indicates that increasing the drug concentration fivefold requires a roughly corresponding increase in the concentration of added HCl to stabilize the molecule, as the 1 mg / mL pramlinpeptide solution exhibited maximum stability at 1.00 mM HCl (or between 0.56 mM and 1.78 mM HCl), while the 5 mg / mL solution exhibited maximum stability at approximately 3.16 mM and 5.62 mM HCl.

[0158] Table 14: Purity of pramlinpeptide as assessed by RP-HPLC after 31 days of storage at 40°C.

[0159]

[0160] *For the 1 mg / mL pramlinpeptide solution, samples were not prepared at 5.62 mM and 10.0 mM HCl concentrations because the formulation exhibited decreased stability as the HCl concentration increased from 1.78 mM to 3.16 mM.

[0161] Example 11

[0162] In this embodiment, insulin (recombinant human) powder (CAS No. 11061-68-0) was dissolved in DMSO at a concentration of 3.5 mg / mL. After the insulin powder was dissolved, hydrochloric acid of varying concentrations was added to the sample solution. The concentration of added HCl ranged from 0.010 M (10 mM) to 0.00032 M (0.32 mM). The HCl concentrations studied are shown in Table 15. 0.5 mL aliquots of the insulin solution sample were stored in 2 mL CZ bottles and placed in an incubator set at 40 °C. Visual inspection of the stored solutions showed that they remained clear (i.e., free of insoluble substances) and colorless throughout the incubation period. Following the method described in Example 1, the chemical stability of the peptides in the formulation was assessed by RP-HPLC after 14 days of storage, and the results (provided as peptide purity) are shown in Table 15 below.

[0163] Table 15: Stability of 3.5 mg / mL insulin-DMSO solution stored at 40°C for 2 weeks (provided in peptide purity).

[0164] Insulin powder concentration hydrochloric acid mM Insulin purity 3.5 mg / mL 10.0 70.7％ 3.5 mg / mL 5.6 72.4％ 3.5 mg / mL 3.2 74.7％ 3.5 mg / mL 1.8 88.1％ 3.5 mg / mL 1.0 95.5％ 3.5 mg / mL 0.6 97.3％ 3.5 mg / mL 0.3 94.7％

[0165] Example 12

[0166] The following examples demonstrate that the present invention is applicable to the preparation of co-preparations. Recombinant human insulin powder (CAS No. 11061-68-0) was dissolved in pure DMSO to a final concentration of 3.5 mg / mL, wherein different concentrations of hydrochloric acid were added to the DMSO. As shown in Table 16, the added HCl concentration ranged from 0.010 M (10 mM) to 0.00032 M (0.32 mM). Then, prulaminide acetate powder (CAS No. 196078-30-5) at a concentration of 1.0 mg / mL was added to these solutions. Therefore, each sample solution contained 3.5 mg / mL of insulin powder and 1.0 mg / mL of prulaminide powder dissolved in DMSO, wherein the DMSO contained a specific concentration of added HCl. 0.5 mL aliquots of the co-preparation sample solution were placed in 2 mL CZ bottles and stored at room temperature (22-23°C). The stability of the peptides in the formulation was assessed by RP-HPLC (according to the method described in Example 1) after 52 days of storage, and the results are shown in Table 16. As with the previous examples for formulations containing a single API, the co-formulation also exhibited an optimal concentration of added HCl for providing stability to both peptides.

[0167] Table 16: Stability of co-formulations containing 3.5 mg / mL insulin powder and 1.0 mg / mL pramlinpeptide powder dissolved in DMSO after 52 days of storage at room temperature (22-23°C) (provided in peptide purity).

[0168] Insulin powder concentration Pramlinpeptide powder concentration Added hydrochloric acid Combination purity 3.5 mg / mL 1.0 mg / mL 3.2mM 87.4％ 3.5 mg / mL 1.0 mg / mL 1.8mM 92.9％ 3.5 mg / mL 1.0 mg / mL 1.0mM 98.1％ 3.5 mg / mL 1.0 mg / mL 0.6mM 88.8％ 3.5 mg / mL 1.0 mg / mL 0.3mM 87.5％

[0169] Example 13

[0170] The following examples demonstrate that the present invention is applicable to the preparation of small molecule stable formulations. Epinephrine (from hydrogen tartrate) powder (CAS No. 51-42-3) was dissolved in pure DMSO with an API concentration of 10 mg / mL (approximately 55 mM), the DMSO having been mixed with varying concentrations of hydrochloric acid (from a 1N stock solution). As shown in Table 17, the added HCl concentration ranged from 1 mM to 100 mM. 0.5 mL aliquots of the epinephrine solution were stored in 2 mL (Type I) glass vials and placed in a stabilization chamber at 40°C and 75% relative humidity. These samples were prepared at room temperature and stored at room temperature using… The sample is sealed in a bottle with a coated rubber stopper (note that the sample can also be filled under an inert gas (nitrogen, argon)).

[0171] The stability of the small molecules in the formulation was assessed by RP-HPLC after one month of storage, and the results are shown in Table 17. For HPLC analysis, a 1 L aqueous solution consisting of 0.05 M sodium dihydrogen phosphate, 519 mg sodium 1-octanesulfonate, and 45 mg disodium ethylenediaminetetraacetate was prepared, and the pH was adjusted to 3.8 with H3PO4. The mobile phase consisted of an 85:15 (v / v) mixture of the aforementioned aqueous solution and methanol. A BDS Hypersil C8 column (4.6 mm inner diameter × 150 mm length) was used, with an injection volume of 20 μL and a detection wavelength of 280 nm. Before use, the mobile phase was filtered under vacuum through a 0.45 μm nylon filter, and a 10 mg / mL epinephrine sample solution was diluted 200-fold with the mobile phase (e.g., 5 μL sample volume in 1 mL total volume). Because adrenaline solutions are prone to discoloration, partly because adrenaline molecules are oxidized to adrenaline red (characterized by a pink discoloration of the solution) and / or melanin (characterized by a yellow / brown discoloration of the solution), the color of the sample solution should be visually inspected. As shown in Table 17, approximately an equimolar concentration of HCl (50 mM) is added to the adrenaline solution when sealed under ambient conditions to prevent discoloration.

[0172] Furthermore, adrenaline in aqueous solution readily converts from its biologically active stereoisomer (L-adrenaline) to its inactive form (D-adrenaline). Therefore, the enantiomeric purity in DMSO solution was also determined by chiral RP-HPLC. The mobile phase was a mixture of aqueous solution (0.20 M NaCl, 0.05% glacial acetic acid) and acetonitrile 95:5 (v / v). A chiral column (Shodex ORpak CDBS-453; inner diameter 4.6 mm, length 150 mm) was used at a column temperature of 10 °C, a flow rate of 0.5 mL / min, and a detection wavelength of 280 nm. Before use, the mobile phase was filtered under vacuum through a 0.45 μm nylon filter, and the 10 mg / mL sample solution was diluted 200-fold using the chiral HPLC mobile phase. The enantiomeric purity (provided as a percentage of L-adrenaline to total adrenaline) is listed in Table 17.

[0173] It is noted that, as described in the prior art (e.g., U.S. Patent 9,125,805), when adrenaline tartrate is directly dissolved in DMSO, the solution exhibits significant discoloration. Adding HCl to the formulation inhibits the degree of discoloration until 50 mM HCl (approximately equimolar to the concentration of adrenaline molecules in a 10 mg / mL solution) is added, at which point the solution remains clear and colorless throughout the entire one-month storage period. Adding 75 and 100 mM HCl results in significant discoloration of the solution.

[0174] Table 17: Stability of 10 mg / mL epinephrine solution sealed under ambient atmosphere for 1 month at 40°C and 75% RH

[0175] adrenaline concentration Added hydrochloric acid %purity Solution color Enantiomer purity 10mg / mL 0mM 92.6％ Dark red 100％ 10mg / mL 1mM 94.9％ Deep pink 100％ 10mg / mL 10mM 98.5％ light pink 100％ 10mg / mL 25mM 100.0％ Very light pink 100％ 10mg / mL 50mM 100.0％ colorless 100％ 10mg / mL 75mM 91.6％ brown 97.4％ 10mg / mL 100mM 74.8％ Dark brown 93.1％

[0176] Example 14

[0177] The following examples demonstrate the applicability of the present invention to the preparation of stable small molecule formulations, with sample vials sealed under an inert atmosphere. Epinephrine (from hydrogen tartrate) powder (CAS No. 51-42-3) was dissolved in pure DMSO to a final API concentration of 10 mg / mL (approximately 55 mM), wherein different concentrations of hydrochloric acid (from a 1N stock solution) had been added to the DMSO. As shown in Table 18, the added HCl concentration ranged from 1 mM to 100 mM. 0.5 mL aliquots of the epinephrine solution were stored in 2 mL (Type I) glass vials and placed in a stabilization chamber at 40°C and 75% relative humidity. These samples were prepared under ambient conditions but sealed under an inert gas (argon) because epinephrine is known to readily undergo oxidative degradation.

[0178] The small molecule stability of the formulation sealed under an inert gas atmosphere was assessed by RP-HPLC after one month of storage, and the results are shown in Table 18. Chemical stability was analyzed by HPLC as described in Example 13. The color of the sample solution was visually examined because the epinephrine solution readily undergoes degradation-promoted discoloration, partly due to the oxidation of epinephrine molecules to epinephrine red (characterized by a pink discoloration of the solution) and / or melanin (characterized by a yellow / brown discoloration of the solution). As shown in Table 18, sealing the sample vials under an inert gas atmosphere (argon in this particular example) suppressed the pink discoloration caused by sealing the vials under an ambient atmosphere as described in Example 11 above. However, the addition of excess HCl to the formulation (e.g., significantly higher than an equimolar concentration relative to the small molecule API) resulted in a deep yellow / deep brown discoloration similar to that observed in the solution from Example 10.

[0179] As described in Example 13, the argon-backfilled epinephrine sample was also analyzed by chiral HPLC. Prior to use, the mobile phase was filtered under vacuum through a 0.45 μm nylon filter, and the 10 mg / mL sample solution was diluted 200-fold with the chiral HPLC mobile phase. Enantiomer purity (provided as a percentage of L-epinephrine to total epinephrine) is listed in Table 18.

[0180] When adrenaline (derived from bitartrate) was directly dissolved in DMSO and sealed in a glass vial under an argon atmosphere, the solution still exhibited discoloration, but the discoloration was milder compared to samples sealed under ambient conditions. Adding HCl to the formulation suppressed the degree of discoloration; when 10–50 mM HCl (approximately equimolar to adrenaline molecules) was added, the solution remained clear and colorless throughout the entire one-month storage period. Significant discoloration was observed when 75 and 100 mM HCl were added.

[0181] Table 18: Chemical stability of 10 mg / mL epinephrine solution sealed under argon atmosphere and stored at 40 °C and 75% RH for 1 month.

[0182] adrenaline concentration Added hydrochloric acid purity% Solution color Enantiomer purity 10mg / mL 0mM 100.0％ light pink 100.0％ 10mg / mL 1mM 100.0％ Very light pink 100.0％ 10mg / mL 10mM 100.0％ colorless 100.0％ 10mg / mL 25mM 100.0％ colorless 100.0％ 10mg / mL 50mM 100.0％ colorless 100.0％ 10mg / mL 75mM 93.9％ yellow 97.3％ 10mg / mL 100mM 91.1％ orange color 91.1％

[0183] Example 15

[0184] The following examples demonstrate that the present invention is applicable to the preparation of stable small molecule formulations. Epinephrine (from hydrogen tartrate) powder (CAS No. 51-42-3) was dissolved in pure DMSO at an API concentration of 3 mg / mL (approximately 16 mM), wherein different concentrations of hydrochloric acid (from a 1N stock solution) had been added to the DMSO. As shown in Table 19, the added HCl concentration ranged from 0 mM to 25 mM. 0.5 mL aliquots of the epinephrine solution were stored in 2 mL (Type I) glass vials and placed in a stabilization chamber at 40°C and 75% relative humidity. These samples were prepared under ambient conditions and sealed in vials under ambient atmosphere (note that samples can also be filled under an inert gas (e.g., nitrogen, argon)).

[0185] As described in Example 13, the stability of the small molecules in the formulation was assessed by RP-HPLC after 16 weeks of storage, and the results are shown in Table 19. Prior to analysis, the 3 mg / mL epinephrine sample solution was diluted approximately 30-fold with the mobile phase (e.g., 33 μL sample volume in a 1 mL total volume). As shown in Table 19, the addition of approximately an equimolar concentration of HCl (16.4 mM) to the epinephrine tartrate inhibited discoloration of the sample solution when sealed under ambient conditions for approximately 16 weeks (114 days).

[0186] As described in Example 13, the enantiomeric purity in the DMSO-epinephrine solution was examined by chiral RP-HPLC. Prior to analysis, the 3 mg / mL sample solution was diluted 30-fold with a chiral HPLC mobile phase. The enantiomeric purity (provided as a percentage of L-epinephrine to total epinephrine) is listed in Table 19.

[0187] The addition of HCl to the formulation suppressed the degree of discoloration. The solution remained clear and colorless throughout the 16-week storage period until the addition of 16.4 mM HCl (approximately equimolar to adrenaline molecules). The addition of 20 and 25 mM HCl resulted in slight discoloration.

[0188] Table 19: Chemical stability of 3 mg / mL epinephrine solution sealed under ambient atmosphere and stored at 40 °C and 75% RH for 16 weeks.

[0189] adrenaline concentration Added hydrochloric acid %purity Solution color Enantiomer purity 3mg / mL 0mM 74％ brown 99％ 3mg / mL 1mM 76％ Dark red 99％ 3mg / mL 5mM 96％ Dark yellow 100％ 3mg / mL 10mM 100％ Light brown 100％ 3mg / mL 15mM 100％ colorless 100％ 3mg / mL 20mM 97％ very light yellow 96％ 3mg / mL 25mM 90％ Light brown 92％

[0190] In view of the disclosure of this invention, all compositions and / or methods disclosed and claimed herein can be manufactured and implemented according to the disclosure of this invention without excessive experimentation. While the compositions and methods disclosed herein have been described in some embodiments, it will be apparent to those skilled in the art that variations can be made to the compositions and methods, as well as the steps or order of steps, without departing from the concept, spirit, and scope of this invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for the reagents described herein to obtain the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention described herein.

Claims

1. A stable formulation comprising: (a) Pramlinin peptide or a salt thereof, wherein the pramlinin peptide or a salt thereof is not prepared by drying from a buffer aqueous solution prior to dissolution in an aprotic polar solvent; (b) an ionization-stabilizing excipient, wherein the ionization-stabilizing excipient is an inorganic acid selected from hydrochloric acid, sulfuric acid, or nitric acid, and the concentration of the ionization-stabilizing excipient is from 0.1 mM to less than 100 mM; and (c) A nonprotic polar solvent, namely dimethyl sulfoxide; Wherein (i) the pramlinpeptide or its salt is directly dissolved in the aprotic polar solvent in an amount of 0.1 mg / mL up to the solubility limit of the pramlinpeptide or its salt, and (ii) the ionization-stabilizing excipient is directly dissolved in the aprotic polar solvent in an amount sufficient to stabilize the ionization of the pramlinpeptide or its salt.

2. The stable formulation according to claim 1, further comprising an insulin peptide or a salt thereof, wherein the insulin peptide or salt thereof is dissolved in the aprotic polar solvent in an amount of 0.1 mg / mL up to the solubility limit of the insulin peptide or salt thereof.

3. The stable formulation according to claim 2, wherein the ratio of pramlintide to insulin is 1:3.

5.

4. The stable formulation according to claim 1, wherein the aprotic polar solvent is deoxygenated dimethyl sulfoxide.

5. The stable formulation according to claim 1, wherein the moisture content is less than 10% w / w.

6. The stable formulation according to claim 5, wherein the moisture content is less than 5% w / w.

7. The stable formulation according to claim 5, wherein the moisture content is less than 3% w / w.

8. The stable formulation according to claim 1, further comprising less than 10% w / v of preservative.

9. The stable formulation according to claim 8, further comprising less than 5% w / v of preservative.

10. The stable formulation according to claim 8, further comprising less than 3% w / v of preservative.

11. The stable formulation according to claim 8, wherein the preservative is benzyl alcohol.

12. The stable formulation according to claim 1, further comprising less than 10% w / v disaccharide.

13. The stable formulation according to claim 12, further comprising less than 5% w / v disaccharide.

14. The stable formulation according to claim 12, further comprising less than 3% w / v disaccharide.

15. The stable formulation according to claim 12, wherein the disaccharide is trehalose.

16. Use of the stable formulation according to claim 1 in the preparation of a medicament for treating diabetes in a subject of need, wherein an effective amount of the stable formulation according to claim 1 is administered to the subject of need.

17. The use according to claim 16, wherein the stable formulation is administered via parenteral injection.

18. The use according to claim 17, wherein the injection is an intradermal injection.

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