EP3307295A1 - Utilisation de glucagon à faible dose - Google Patents

Utilisation de glucagon à faible dose

Info

Publication number
EP3307295A1
EP3307295A1 EP16751037.9A EP16751037A EP3307295A1 EP 3307295 A1 EP3307295 A1 EP 3307295A1 EP 16751037 A EP16751037 A EP 16751037A EP 3307295 A1 EP3307295 A1 EP 3307295A1
Authority
EP
European Patent Office
Prior art keywords
glucagon
formulation
composition
subject
hours
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16751037.9A
Other languages
German (de)
English (en)
Inventor
Yashvinder Sabharwal
Steven Prestrelski
Michael Sandoval
Martin Donovan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xeris Pharmaceuticals Inc
Original Assignee
Xeris Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/136,650 external-priority patent/US9649364B2/en
Application filed by Xeris Pharmaceuticals Inc filed Critical Xeris Pharmaceuticals Inc
Publication of EP3307295A1 publication Critical patent/EP3307295A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention relates to the fields of medicine, health, and nutrition. More particularly, the present invention relates to methods and compositions for treating diabetes and reducing or controlling body weight or altering body composition in a subject.
  • Diabetes is a serious metabolic disease in which the pancreas produces little or no insulin or the insulin that is present is not used effectively. As a result, blood glucose levels are chronically elevated in the diabetic individual.
  • diabetes There are several main types of diabetes, including insulin-dependent (type 1) diabetes and non-insulin-dependent (type 2) diabetes.
  • type 1 diabetes the pancreas does not produce sufficient levels of insulin for maintaining blood glucose at normal physiological levels.
  • an individual having type 1 diabetes must take insulin daily.
  • type 2 diabetes initially the pancreas generally produces enough insulin, but the body is unable to utilize the insulin effectively, a condition known as "insulin resistance.” However, as the disease progresses, insulin production decreases.
  • individuals with an advanced stage of type 2 diabetes are often prescribed insulin in order to maintain blood glucose levels and minimize complications from the disease.
  • Glucagon is a peptide hormone that is secreted by the pancreas and which raises blood glucose levels. Glucagon has the opposite effect as insulin on blood glucose levels, and both glucagon and insulin are part of a feedback system that keeps blood glucose levels stable in the body.
  • Glucagon is currently prescribed as a treatment for severe hypoglycemia, and smaller doses of glucagon are prescribed for treating mild to moderate hypoglycemia in cases of gastroparesis or other situations where ingestion of oral carbohydrates is not possible. Because glucagon is typically given by intramuscular, intravenous, or subcutaneous injection, it is necessary that the glucagon be in an injectable form. Currently available glucagon products must be reconstituted prior to use, a step that requires a sterile diluent to be injected into a vial containing powdered glucagon, because the hormone is highly unstable when dissolved in solution.
  • glucagon When dissolved in a fluid state, glucagon can form amyloid fibrils, or tightly woven chains of proteins made up of individual glucagon peptides. Once glucagon begins to fibrillate, it becomes useless when injected, as the glucagon cannot be absorbed and used by the body. [0006] As such, what is needed in the art are methods for providing a low dose of glucagon to a subject in need thereof, wherein the glucagon remains stable for an extended period of time once it is reconstituted. The present invention addresses this need and others.
  • controlling or reducing body weight is by controlling or reducing caloric intake in a subject.
  • the method comprises: administering to the subject a low dose of a stable glucagon formulation in response to a hunger cue in the subject, wherein the glucagon formulation is stable for at least one week at controlled room temperature.
  • the methods act via controlling or reducing caloric intake in the subject.
  • the method comprises temporal or chronic administration of the glucagon formulation.
  • the subject in need of controlling or reducing body weight is normoglycemic.
  • the subject is diabetic.
  • the subject is a human adult.
  • the glucagon formulation is administered to the subject at a dose of about 50 ⁇ g to about 200 ⁇ g.
  • the subject is a human child.
  • the glucagon formulation is administered to the subject at a dose of about 5 ⁇ g to about 150 ⁇ g.
  • the present invention provides methods for treating mild or moderate hypoglycemia in a subject in need thereof.
  • the method comprises: administering to the subject a first low dose of a stable glucagon formulation in response to a symptom of mild or moderate hypoglycemia in the subject, wherein the glucagon formulation is stable for at least one week at controlled room temperature; thereby treating the mild or moderate hypoglycemia in the subject.
  • the method comprises chronic administration of the glucagon formulation.
  • the subject in need of treating mild or moderate hypoglycemia is diabetic.
  • the subject is a human adult.
  • the glucagon formulation is administered to the subject at a dose of about 50 ⁇ g to about 200 ⁇ g. In some embodiments, the subject is a human child. In some embodiments, wherein the subject is a human child, the glucagon formulation is administered to the subject at a dose of about 5 ⁇ g to about 150 ⁇ g.
  • the method further comprises: monitoring the subject for one or more symptoms of mild or moderate hypoglycemia subsequent to administering the first low dose of the glucagon formulation; and, if about 30 minutes after administering the first low dose of the glucagon formulation the subject exhibits one or more symptoms of mild or moderate hypoglycemia, administering to the subject a second low dose of the glucagon formulation.
  • the steps of monitoring the subject for one or more symptoms of mild or moderate hypoglycemia and administering a subsequent low dose of glucagon can be repeated two, three, four, five, or more times over a defined period of time in order to treat the mild or moderate hypoglycemia in the subject.
  • the symptom of mild or moderate hypoglycemia is a blood glucose concentration of less than about 70 mg/dL.
  • the method further comprises: monitoring the blood glucose level of the subject subsequent to administering the first low dose of the glucagon formulation; and, if about 30 minutes after administering the first low dose of the glucagon formulation the blood glucose level of the subject is less than about 70 mg/dL, administering to the subject a second low dose of the glucagon formulation.
  • the steps of monitoring the blood glucose concentration of the subject and administering a subsequent low dose of glucagon can be repeated two, three, four, five, or more times over a defined period of time in order to treat the mild or moderate hypoglycemia in the subject.
  • the glucagon formulation for administration according to any of the methods of the present invention is stable for at least one month at controlled room temperature. In some embodiments, the glucagon formulation is stable for at least one week at 40 °C. In some embodiments, the glucagon formulation is stable for at least three months at 4 °C.
  • the glucagon formulation for administration according to any of the methods of the present invention is reconstituted with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is an aqueous carrier.
  • the pharmaceutically acceptable carrier is a non- aqueous carrier.
  • the glucagon, glucagon analogue, or a salt form of either thereof can be fully solubilized in an aprotic polar solvent.
  • Therapeutic molecules typically require an optimal or beneficial ionization profile in order to exhibit prolonged stability when solubilized in an aprotic polar solvent system.
  • An optimal or beneficial ionization profile of a therapeutic molecule may be obtained by direct dissolution of the therapeutic agent in an aprotic polar solvent system containing a specified concentration of at least one ionization stabilizing excipient.
  • Compositions for use with the present invention are stable formulations containing glucagon or glucagon analogue solubilized in an aprotic polar solvent system.
  • the glucagon or glucagon analogue does not need to be previously dried from a buffered aqueous solution prior to reconstitution in the aprotic polar solvent system.
  • glucagon or glucagon analogue is directly dissolved (e.g. a powder as received from a commercial manufacturer or supplier) along with an effective amount of an ionization stabilizing excipient for establishing an appropriate ionization of the glucagon or glucagon analogue in the aprotic polar solvent system.
  • an ionization stabilizing excipient for establishing an appropriate ionization of the glucagon or glucagon analogue in the aprotic polar solvent system.
  • the ability to circumvent the need for drying the peptide from a buffered aqueous solution, for example via lyophilization, prior to reconstitution in the aprotic polar solvent system is anticipated to save considerable time and cost throughout the various product development stages.
  • stabilizing excipients e.g., disaccharides such as trehalose and sucrose
  • stabilizing excipients are often added to the formulation primarily to protect against degradation of the active agent during the drying process.
  • Stable solutions of glucagon or glucagon analogue solubilized in non-aqueous aprotic polar solvents can be prepared by adding a specific predetermined amount of a compound, or combination of compounds, that function as an ionization stabilizing excipient.
  • the amount can be determined by titration studies using glucagon or glucagon analogue and the ionization stabilizing excipient.
  • the ionization stabilizing excipient can act as a proton source (e.g., a molecule that can donate a proton to the therapeutic molecule) in the aprotic polar solvent system that may protonate the ionogenic groups on glucagon or glucagon analogue such that the glucagon or glucagon analogue possesses an ionization profile having an improved physical and chemical stability in the aprotic polar solvent system.
  • a proton source e.g., a molecule that can donate a proton to the therapeutic molecule
  • Certain embodiments are directed to a formulation of glucagon or glucagon analogue comprising a therapeutic agent at a concentration of at least, at most, or about 0.1, 1, 10, 50, or 100 mg/mL to 150, 200, 300, 400, or 500 mg/ml or up to the solubility limit of the glucagon or glucagon analogue in the aprotic polar solvent system comprising a concentration of at least one ionization stabilizing excipient that provides physical and chemical stability to the therapeutic agent.
  • the formulation can comprise an ionization stabilizing excipient at a concentration of at least, at most, or about 0.01, 0.1, 0.5, 1, 10, or 50 mM to 10, 50, 75, 100, 500, 1000 mM, or up to the solubility limit of the ionization stabilizing excipient in the aprotic polar solvent system.
  • the ionization stabilizing excipient concentration is between 0.1 mM to 100 mM.
  • the ionization stabilizing excipient may be a suitable mineral acid, such as hydrochloric acid.
  • the ionization stabilizing excipient may be an organic acid, such as an amino acid, amino acid derivative, or the salt of an amino acid or amino acid derivative (examples include glycine, trimethylglycine (betaine), glycine hydrochloride, and trimethylglycine (betaine) hydrochloride).
  • the amino acid can be glycine or the amino acid derivative trimethylglycine.
  • the aprotic solvent system comprises DMSO.
  • the aprotic solvent can be deoxygenated, e.g., deoxygenated DMSO.
  • the therapeutic agent is glucagon or salt thereof.
  • compositions to be used in conjunction with the present invention can be made by: (a) calculating or determining the appropriate ionization stabilizing excipient or proton concentration needed to achieve a stabilizing ionization profile of glucagon or glucagon analogue in an aprotic polar solvent system; (b) mixing at least one ionization stabilizing excipient with the aprotic polar solvent system to attain an appropriate ionization environment that provides the ionization profile determined in step (a); and (c) solubilizing the glucagon or glucagon analogue in the aprotic solvent having an appropriate environment to physically and chemically stabilize the glucagon or glucagon analogue.
  • the dissolution of the glucagon or glucagon analogue and the addition of the ionization stabilizing excipient to the aprotic polar solvent system can be done in any order or concurrently, thus the ionization stabilizing excipient can be mixed first followed by dissolution of the glucagon or glucagon analogue, or the glucagon or glucagon analogue can be dissolved followed by addition of the ionization stabilizing excipient to the solution, or the ionization stabilizing excipient and the glucagon or glucagon analogue can be added or dissolved in an aprotic polar solvent system concurrently.
  • the entire amount of a component (e.g., glucagon or glucagon analogue or an ionization stabilizing excipient) need not to be mixed at a particular point; that is, a portion of the one or more components can be mixed first, second, or concurrently, and another portion mixed at another time, first, second, or concurrently.
  • the ionization stabilizing excipient may be a suitable mineral acid, such as hydrochloric acid.
  • the concentration of the glucagon or glucagon analogue and/or ionization stabilizing excipient added to the solution can be between 0.01, 0.1, 1, 10, 100, 1000 mM to its solubility limit, including all values and ranges there between.
  • the aprotic polar solvent system is deoxygenated.
  • the aprotic polar solvent system comprises, consists essentially of, or consists of DMSO or deoxygenated DMSO.
  • the glucagon formulation for administration according to any of the methods of the present invention comprises: glucagon or a glucagon analog, or a salt thereof, that has been dried with a carbohydrate and a buffer having a pH of about 2.0 to about 3.5, wherein the glucagon is reconstituted with a pharmaceutically acceptable carrier.
  • the buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof.
  • the carbohydrate is selected from trehalose, hydroxyethyl starch (HES), dextran, and mixtures thereof.
  • the glucagon formulation for administration comprises: (a) glucagon or a glucagon analog, or a salt thereof, wherein the glucagon has been dried in a non-volatile buffer, and wherein the dried glucagon has a pH memory that is about equal to the pH of the glucagon in the non- volatile buffer, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dried glucagon maintains the pH memory that is about equal to the pH of the glucagon in the non-volatile buffer when the dried glucagon is reconstituted in the aprotic polar solvent.
  • the non- volatile buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof.
  • the aprotic polar solvent is selected from dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof.
  • the glucagon formulation for administration according to any of the methods of the present invention is administered subcutaneously.
  • the glucagon formulation is administered via the use of a syringe.
  • the glucagon formulation is administered via the use of a pen injection device.
  • the glucagon formulation is administered via the use of an auto- injector device.
  • the glucagon formulation is administered via the use of a needle-free injection device.
  • the glucagon formulation is administered via the use of a multi-dose injection device.
  • kits for administering a low dose of glucagon to a subject in need of controlling or reducing body weight by controlling or reducing caloric intake In still another aspect, the present invention provides kits for administering a low dose of glucagon to a subject in need of treating mild or moderate hypoglycemia.
  • the kit comprises: (a) a stable glucagon formulation, wherein the glucagon formulation is stable for at least one week at controlled room temperature; (b) a multi-dose cartridge or syringe; and (c) a multi-dose injection device capable of accepting the multi-dose cartridge or syringe.
  • the kit comprises a glucagon formulation that is stable for at least one month at controlled room temperature.
  • the glucagon formulation is stable for at least one week at 40 °C.
  • the glucagon formulation is stable for at least three months at 4 °C.
  • the kit comprises a glucagon formulation is reconstituted with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is an aqueous carrier.
  • the pharmaceutically acceptable carrier is a non-aqueous carrier.
  • the kit comprises a glucagon formulation comprising glucagon or a glucagon analogue at a concentration of at least, at most, or about 0.1, 1, 10, 50, or 100 mg/mL to 150, 200, 300, 400, or 500 mg/ml or up to the solubility limit of the glucagon or glucagon analogue in an aprotic polar solvent system comprising a concentration of at least one ionization stabilizing excipient that provides physical and chemical stability to the therapeutic agent.
  • the formulation can comprise an ionization stabilizing excipient at a concentration of at least, at most, or about 0.01, 0.1, 0.5, 1, 10, or 50 mM to 10, 50, 75, 100, 500, 1000 mM, or up to the solubility limit of the ionization stabilizing excipient in the aprotic polar solvent system.
  • the ionization stabilizing excipient concentration is between 0.1 mM to 100 mM.
  • the ionization stabilizing excipient may be a suitable mineral acid, such as hydrochloric acid.
  • the ionization stabilizing excipient may be an organic acid, such as an amino acid, amino acid derivative, or the salt of an amino acid or amino acid derivative (examples include glycine, trimethylglycine (betaine), glycine hydrochloride, and trimethylglycine (betaine) hydrochloride).
  • the amino acid can be glycine or the amino acid derivative trimethylglycine.
  • the aprotic solvent system comprises DMSO.
  • the aprotic solvent can be deoxygenated, e.g., deoxygenated DMSO.
  • the therapeutic agent is glucagon or salt thereof.
  • the kit comprises a glucagon formulation comprising: glucagon or a glucagon analog, or a salt thereof, that has been dried with a carbohydrate and a buffer having a pH of about 2.0 to about 3.5, wherein the glucagon is reconstituted with a pharmaceutically acceptable carrier.
  • the buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof.
  • the carbohydrate is selected from trehalose, hydroxyethyl starch (HES), dextran, and mixtures thereof.
  • the kit comprises a glucagon formulation comprising: (a) glucagon or a glucagon analog, or a salt thereof, wherein the glucagon has been dried in a non-volatile buffer, and wherein the dried glucagon has a pH memory that is about equal to the pH of the glucagon in the non-volatile buffer, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dried glucagon maintains the pH memory that is about equal to the pH of the glucagon in the non-volatile buffer when the dried glucagon is reconstituted in the aprotic polar solvent.
  • the non-volatile buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof.
  • the aprotic polar solvent is selected from dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof.
  • the kit comprises glucagon that is formulated for dosing a human adult, wherein the dose of the glucagon formulation that is administered is from about 50 ⁇ g to about 150 ⁇ g, or from about 100 ⁇ g to about 200 ⁇ g, e.g., about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 ⁇ g.
  • the kit comprises glucagon that is formulated for dosing a human child, wherein the dose of the glucagon formulation that is administered is from about 5 ⁇ g to about 150 ⁇ g,
  • the kit comprises a multi-dose cartridge or syringe that is pre-filled with the glucagon formulation.
  • the multi-dose injection device of a kit of the present invention is a pen injection device.
  • the multi-dose injection device is an auto-injector device.
  • the multi-dose injection device is a pump, e.g., a glucagon infusion pump or a patch pump.
  • the multi-dose injection device is an implantable injection device.
  • the multi-dose injection device is a needle-free injection device.
  • the multi-dose injection device is a variable dose device.
  • the multi-dose injection device is a low volume injection device.
  • the kit further comprises instructions for administering the glucagon formulation. In some embodiments, the kit comprises instructions for using the glucagon formulation for treating hypoglycemia. In some embodiments, the kit comprises instructions for using the glucagon formulation for controlling or reducing weight or caloric intake.
  • FIG. 1 Size exclusion high-performance liquid chromatography performed on reconstituted commercially available glucagon samples after 4-6 hours incubation at refrigerated temperatures.
  • FIG. 2. Reverse phase high-performance liquid chromatography performed on reconstituted commercially available glucagon samples after 4-6 hours incubation at refrigerated temperatures.
  • FIG. 3 Observed Weigh-Gain During Dosing with either vehicle, or glucagon formulation at 0.5, 1.0, or 2.0 mg/kg/day.
  • FIG. 4 Food Consumption During Treatment with Glucagon
  • the present invention relates to the use of low dose glucagon therapy as a method of treating mild or moderate hypoglycemia in a subject.
  • glucagon formulations for use in low dose glucagon therapy must be reconstituted immediately prior to use, and are only stable for a few hours post-reconstitution before the glucagon fibrillates.
  • mild or moderate hypoglycemia is routinely treated by eating or drinking oral carbohydrates.
  • chronic consumption of glucose-rich food over time, can result in weight gain, which in turn can result in or contribute to other serious health conditions.
  • the stable glucagon formulations described herein have the advantage of significantly increased stability over time at ambient or even physiological temperatures. Because the stable glucagon formulations described herein have a significantly longer shelf life than currently available reconstituted glucagon formulations, these stable glucagon formulations can be used for the chronic administration of low dose glucagon therapy in place of ingestion of oral carbohydrates. By administering a stable glucagon formulation as described herein instead of high calorie, glucose-rich food during episodes of mild or moderate hypoglycemia, caloric intake and body weight can be controlled or reduced. Thus, the present invention also relates to the use of low dose glucagon therapy as a method of controlling or reducing caloric intake and/or body weight.
  • This method of replacing a higher- calorie food or drink with a low dose of glucagon, thereby controlling or reducing caloric intake and/or body weight, is useful not only for individuals in need of controlling blood glucose levels (e.g., individuals having a diabetic condition), but for any individual who wants to control or reduce caloric intake.
  • a low dose means a dose of about 0.1 ⁇ g/kg to about 5 ⁇ g/kg. In some embodiments, a low dose of glucagon is a dose that is from about 5 ⁇ g to about 200 ⁇ g.
  • stable formulation means that at least about 70% chemically and physically stable therapeutic agent (e.g., glucagon) remains 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% chemically and physically stable therapeutic agent remains under these storage conditions.
  • Especially preferred stable formulations are those which do not exhibit degradation after sterilizing irradiation (e.g., gamma, beta or electron beam).
  • chemical stability means that with respect to a therapeutic agent (e.g., glucagon), an acceptable percentage of degradation products produced by chemical pathways such as oxidation or hydrolysis is formed.
  • a formulation is considered chemically stable if no more than about 20% breakdown products are formed after one year of storage at the intended storage temperature of the product (e.g., room temperature); or storage of the product at 30°C/60% relative humidity for one year; or storage of the product at 40°C/75% relative humidity for one month, and preferably three months.
  • a chemically stable formulation has 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% breakdown products formed after an extended period of storage at the intended storage temperature of the product.
  • the term "physical stability" means that with respect to a therapeutic agent (e.g., glucagon), an acceptable percentage of aggregates (e.g., dimers, trimers and larger forms that appear as amyloid fibrils) is formed.
  • a formulation is considered physically stable if no more that about 15% aggregates are formed after one year of storage at the intended storage temperature of the product (e.g., room temperature); or storage of the product at 30°C/60% relative humidity for one year; or storage of the product at 40°C/75% relative humidity for one month, and preferably three months.
  • a physically stable formulation has less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% aggregates formed after an extended period of storage at the intended storage temperature of the product.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable solvent, suspending agent, or vehicle for delivering a therapeutic agent (e.g., glucagon) to a human or animal.
  • the carrier may be liquid, semisolid, or solid.
  • a "pharmaceutically acceptable" ingredient, excipient, or component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio.
  • aprotic polar solvent refers to a polar solvent that does not contain acidic hydrogen and does not act as a hydrogen bond donor.
  • aprotic polar solvents include, but are not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylene carbonate.
  • aprotic polar solvent also encompasses mixtures of two or more aprotic polar solvents, e.g., a mixture of two or more of dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylene carbonate.
  • DMSO dimethylsulfoxide
  • DMF dimethylformamide
  • NMP n-methyl pyrrolidone
  • DMA dimethylacetamide
  • propylene carbonate propylene carbonate
  • controlled room temperature refers to a temperature maintained thermostatically that encompasses the usual and customary working environment of 20 °C to 25 °C that allows for brief deviations between 15 °C and 30 °C.
  • administering means oral ("po") administration, administration as a suppository, topical contact, intravenous (“iv”), intraperitoneal (“ip”), intramuscular (“im”), intralesional, intranasal, or subcutaneous (“sc”) administration, or the implantation of a slow- release device e.g., a mini-osmotic pump, to a subject.
  • Administration can be by any route including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • intracutaneous encompasses administration into the epidermal, dermal or subcutaneous skin layer.
  • treat or “treatment” refer to delaying the onset of, retarding or reversing the progress of, or alleviating or preventing either the disease or condition to which the term applies, such as hypoglycemia, or one or more symptoms of such disease or condition.
  • controlling caloric intake or "reducing caloric intake” refer to reducing the net caloric intake of an individual over a defined period of time, for example, reducing the net caloric intake of an individual on a daily basis, or over a period of days, weeks, or months., e.g., over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more months.
  • caloric intake is controlled or reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the number of calories consumed per day by the individual is decreased by at least 1%, 2%, 3%, 4, %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or more as compared to the daily calorie consumption of the individual prior to treatment with the stable glucagon formulation.
  • caloric intake is controlled or reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the net caloric intake of the individual is decreased by at least 1%, 2%, 3%, 4, %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more as compared to the caloric intake of the individual prior to treatment with the stable glucagon formulation.
  • patient refers to a mammal, for example, a human or a non-human mammal, e.g., a primate, dog, cat, bovine, ovine, porcine, equine, mouse, rat, hamster, rabbit, or guinea pig.
  • a mammal for example, a human or a non-human mammal, e.g., a primate, dog, cat, bovine, ovine, porcine, equine, mouse, rat, hamster, rabbit, or guinea pig.
  • solution refers to a process by which a material(s) in a gas, solid, or liquid state becomes a solute(s), a dissolved component(s), of a solvent, forming a solution of the gas, liquid, or solid in the solvent.
  • a therapeutic agent e.g., glucagon
  • an excipient e.g., an ionization stabilizing excipient
  • dissolve refers to a gas, liquid, or solid becoming incorporated into a solvent to form a solution.
  • excipient refers to a natural or synthetic substance formulated alongside the active or therapeutic ingredient (an ingredient that is not the active ingredient) of a medication, included for the purpose of stabilization, bulking, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, enhancing solubility, adjusting tonicity, mitigating injection site discomfort, depressing the freezing point, or enhancing stability.
  • Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life.
  • an "ionization stabilizing excipient” is an excipient that establishes and/or maintains a particular ionization state for a therapeutic agent.
  • the ionization stabilizing excipient can be, or includes, a molecule that donates at least one proton under appropriate conditions or is a proton source.
  • an acid is a molecule that can donate a proton to another molecule, which by accepting the donated proton may thus be classified as a base.
  • the term “proton” refers to the hydrogen ion, hydrogen cation, or H + .
  • the hydrogen ion has no electrons and is composed of a nucleus that typically consists solely of a proton (for the most common hydrogen isotope, protium).
  • a molecule that can donate a proton to the therapeutic agent is considered an acid or proton source, regardless of whether it is completely ionized, mostly ionized, partially ionized, mostly unionized, or completely unionized in the aprotic polar solvent.
  • a "mineral acid” is an acid that is derived from one or more inorganic compounds. Accordingly, mineral acids may also be referred to as "inorganic acids.” Mineral acids may be monoprotic or polyprotic (e.g. diprotic, triprotic, etc.).
  • mineral acids examples include hydrochloric acid (HCl), sulfuric acid (H 2 S0 4 ), and phosphoric acid (H 3 P0 4 ).
  • an "organic acid” is an organic compound with acidic properties (i.e. can function as a proton source).
  • Carboxylic acids are one example of organic acids.
  • Other known examples of organic acids include, but are not limited to, alcohols, thiols, enols, phenols, and sulfonic acids.
  • Organic acids may be monoprotic or polyprotic (e.g. diprotic, triprotic, etc.).
  • Charge profile “charge state,” “ionization state,” and “ionization profile” may be used interchangeably and refer to the ionization state (i.e. due to protonation and/or deprotonation) of the peptide's ionogenic groups.
  • glucagon refers to the glucagon peptide, analogues thereof, and salt forms of either thereof.
  • "Reconstituted,” when referring to a pharmaceutical composition refers to a composition which has been formed by the addition of an appropriate non-aqueous solvent to a solid material comprising the active pharmaceutical ingredient. Pharmaceutical compositions for reconstitution are typically applied where a liquid composition with acceptable shelf-life cannot be produced. An example of a reconstituted pharmaceutical composition is the solution which results when adding a biocompatible aprotic polar solvent (e.g., DMSO) to a freeze dried composition.
  • a biocompatible aprotic polar solvent e.g., DMSO
  • Derivative in relation to a parent peptide, refers to a chemically modified parent peptide or an analogue thereof, wherein at least one substituent is not present in the parent peptide or an analogue thereof.
  • a parent peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, pegylations and the like.
  • mammal or “mammalian” includes murine (e.g., rats, mice) mammals, rabbits, cats, dogs, pigs, and primates (e.g., monkey, apes, humans).
  • the mammal can be murine or human.
  • the patient can be a mammal or a mammalian patient.
  • the present invention provides methods for controlling or reducing caloric intake in a subject in need thereof.
  • the method comprises administering to the subject a low dose of a stable glucagon formulation in response to a hunger cue in the subject; thereby controlling or reducing caloric intake in the subject.
  • the subject in need of controlling or reducing caloric intake is normoglycemic (i.e., has a normal concentration of glucose in the blood) or is non-diabetic.
  • the subject in need of controlling or reducing caloric intake is diabetic.
  • the term "diabetes" includes insulin-dependent type 1 diabetes, non-insulin- dependent type 2 diabetes, insulin-dependent type 2 diabetes, and gestational diabetes.
  • the subject has type 1 diabetes.
  • the subject has type 2 diabetes.
  • the subject is a human adult. In some embodiments, the subject is a human child (i.e., an individual under the age of 18 years).
  • a "hunger cue" refers to any physiological sensation or symptom that is manifested by the body when a subject is motivated to consume food.
  • a hunger cue may include, but is not limited to, hunger pangs, lightheadedness or loss of balance, nausea, headaches, muscle weakness, lack of energy, or inability to concentrate.
  • Control of or reduction in caloric intake can be measured by any known method.
  • control of or reduction in caloric intake is determined by measuring the number of calories consumed in a day (e.g., in a 24-hour period).
  • caloric intake is controlled or reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the number of calories consumed per day by the individual is decreased by at least 1%, 2%, 3%, 4, %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or more as compared to the daily calorie consumption of the individual prior to treatment with the stable glucagon formulation.
  • the daily caloric intake can be measured over a defined period of time, e.g., over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days; over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks; or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more months.
  • control of or reduction in caloric intake is determined by measuring a reduction in body mass, e.g., body-mass index (BMI).
  • BMI body-mass index
  • the reduction in caloric intake (e.g., as measured by measuring a reduction in body mass) can be measured over a defined period of time.
  • the reduction in caloric intake in an individual can be measured over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more months.
  • caloric intake is controlled or reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the net caloric intake of the individual is decreased by at least 1%, 2%, 3%, 4, %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or more as compared to the caloric intake of the individual prior to treatment with the stable glucagon formulation.
  • caloric intake is reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the BMI of the individual is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more as compared to the BMI of the individual prior to treatment with the stable glucagon formulation.
  • caloric intake is reduced in an individual treated with a stable glucagon formulation according to the methods of the present invention when the individual's body weight is decreased by at least about 5 lb, 10 lb, 15 lb, 20 lb, 25 lb or more as compared to the body weight of the individual prior to treatment with the stable glucagon formulation.
  • the present invention provides methods for treating mild or moderate hypoglycemia in a subject in need thereof.
  • the method comprises administering to the subject a low dose of a stable glucagon formulation in response to a symptom of mild or moderate hypoglycemia in the subject; thereby treating the mild or moderate hypoglycemia in the subject.
  • the method comprises chronic administration of a low dose of a stable glucagon formulation for treating mild or moderate hypoglycemia.
  • the subject in need of treatment for mild or moderate hypoglycemia is diabetic.
  • the term "diabetes" includes insulin-dependent type 1 diabetes, insulin-dependent type 2 diabetes, non-insulin-dependent type 2 diabetes, and gestational diabetes.
  • the subject has type 1 diabetes.
  • the subject has type 2 diabetes.
  • the subject is a human adult. In some embodiments, the subject is a human child (i.e., an individual under the age of 18 years). [0073] In some embodiments, a subject is in need of treatment for mild or moderate hypoglycemia when the subject exhibits one or more symptoms of mild or moderate hypoglycemia.
  • Symptoms of mild or moderate hypoglycemia include, but are not limited to, nausea; extreme hunger; cold, clammy, or wet skin or excessive sweating; rapid heartbeat; trembling; numbness or tingling of fingertips or toes; blurred vision; dizziness; headache; poor coordination; fatigue, lethargy, or drowsiness; and a blood glucose concentration of less than about 70 mg/dL.
  • the symptom of mild or moderate hypoglycemia is a blood glucose concentration of less than about 70 mg/dL. Blood glucose concentration can be measured according to any known method, including but not limited to, capillary blood glucose testing.
  • the method of treating mild or moderate hypoglycemia further comprises, subsequent to administering a first low dose of the glucagon formulation, monitoring one or more symptoms of mild or hypoglycemia in the subject for a defined period of time (e.g., for about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes); wherein if the subject exhibits one or more symptoms of mild or moderate hypoglycemia after the defined period of time, the method further comprises administering to the subject a second low dose of the glucagon formulation.
  • a defined period of time e.g., for about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes
  • the method may further comprise monitoring the subject for one or more symptoms of mild or moderate hypoglycemia subsequent to the second or subsequent dose of the glucagon formulation being administered, and wherein the subject continues to exhibit one or more symptoms of mild or moderate hypoglycemia after a defined period of time, administering a further low dose of the glucagon formulation.
  • the method comprises administering two, three, four, five, or more low doses of a glucagon formulation over a defined period of time (e.g., over the course of several hours) in order to treat the mild or moderate hypoglycemia in the subject.
  • the method comprises monitoring one or more symptoms of mild or hypoglycemia in the subject by measuring the blood glucose level of the subject subsequent to administering a low dose of the glucagon formulation for a defined period of time, e.g., for about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. In some embodiments, the method comprises monitoring the blood glucose level of the subject for about 30 minutes; and, if about 30 minutes after administering the low dose of the glucagon formulation the blood glucose level of the subject is less than about 70 mg/dL, administering to the subject a further low dose of the glucagon formulation.
  • the stable glucagon formulation for administration according to the methods of the present invention can be any glucagon formulation that is stable for extended periods of time at controlled room temperature.
  • the glucagon formulation is stable for at least one week at controlled room temperature.
  • the glucagon formulation is stable for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or longer at controlled room temperature.
  • the stable glucagon formulation is stable for at least 1 month, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer at controlled room temperature.
  • the stable glucagon formulation is a formulation that is functional over a range of temperatures. Suitable glucagon formulations for administration according to the methods of the present invention are described infra.
  • the amount of the glucagon formulation that is administered will vary according to several factors, including the formulation of the composition, patient response, the severity of the condition, the subject's weight, and the judgment of the prescribing physician.
  • the dosage can be increased or decreased over time, as required by an individual patient.
  • General guidance for appropriate dosages of all pharmacological agents used in the present methods is provided in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Edition, 2006, supra, and in a Physicians' Desk Reference (PDR), for example, in the 65th (2011) or 66th (2012) Eds., PDR Network, LLC, each of which is hereby incorporated herein by reference.
  • the dose of the glucagon formulation that is administered is from about 5 ⁇ g to about 200 ⁇ g, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 ⁇ g.
  • the subject is a human adult and the dose of the glucagon formulation that is administered is from about 50 ⁇ g to about 200 ⁇ g, e.g., about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 ⁇ g.
  • the subject is a human adult and the dose of the glucagon formulation that is administered is from about 50 ⁇ g to about 150 ⁇ g, or from about 100 ⁇ g to about 200 ⁇ g.
  • the subject is a human adult and the dose of the glucagon formulation that is administered is about 150 ⁇ g.
  • the subject is a human child and the dose of the glucagon formulation that is administered is from about 5 ⁇ g to about 150 ⁇ g, from about 10 ⁇ g to about 150 ⁇ g, from about 15 ⁇ g to about 150 ⁇ g, or from about 20 ⁇ g to about 150 ⁇ g, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, or about 150 ⁇ g.
  • the subject is a human child and the dose of the glucagon formulation that is administered is determined according to the following formula, wherein each "unit" of glucagon formulation is 10 ⁇ g: for a child ⁇ 2 years of age, a dose of about 2 units (i.e., about 20 ⁇ g) is administered; for a child from 2-15 years of age, a dose of about one unit per each year of the child's age is administered (i.e., 2-15 units, i.e., from about 20 ⁇ g to about 150 ⁇ g); and for a child >15 years of age, a dose of about 15 units (i.e., about 150 ⁇ g) is administered.
  • each "unit" of glucagon formulation is 10 ⁇ g: for a child ⁇ 2 years of age, a dose of about 2 units (i.e., about 20 ⁇ g) is administered; for a child from 2-15 years of age, a dose of about one unit per each year of the child's age is administered (i.e
  • the glucagon formulation is administered chronically.
  • "chronic" administration refers to administration of a dose of a glucagon formulation one or more times per day for an extended period of time.
  • a glucagon formulation is chronically administered if a dose of a glucagon formulation is administered one or more times per day for at least 10 days, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, or for at least 150, 200, 250, 300, or 350 days, at least 1 year, or longer.
  • a glucagon formulation is chronically administered if at least 1, 2, 3, 4, 5, or more doses of a glucagon formulation are administered per day for at least 10 days, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 days or longer. In some embodiments, treatment can continue indefinitely.
  • the number of doses of a glucagon formulation that is administered per day may vary.
  • Stable glucagon formulations can be administered by any method, including oral administration, administration as a suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, or subcutaneous administration or by implantation.
  • a stable glucagon formulation for use in the present invention is administered by subcutaneous, intradermal, or intramuscular administration (e.g., by injection or by infusion).
  • the stable glucagon formulation is administered subcutaneously.
  • the stable glucagon formulation is administered by non-parenteral delivery, e.g., by nasal, buccal, or transdermal administration.
  • a stable glucagon formulation is administered by infusion or by injection using any suitable device.
  • a glucagon formulation of the present invention may be administered via a syringe, a pen injection device, an auto-injector device, or a pump device (e.g., a glucagon infusion pump or a patch pump).
  • the injection device is an implantable injection device.
  • the injection device is a needle-free injection device.
  • the injection device is a multi- dose injector pump device or a multi-dose auto-injector device.
  • the multi-dose injection device (e.g., a multi-dose injector pump device or a multi-dose auto- injector device) is a variable dose device.
  • the injection device is a low volume injection device.
  • the formulation is presented in the device in such a fashion that the formulation is readily able to flow out of the needle upon actuation of an injection device, such as an auto- injector, in order to deliver the glucagon formulation.
  • Suitable pen/autoinjector devices include, but are not limited to, those pen/autoinjection devices manufactured by Becton- Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, West Pharmaceuticals, Inc., and the like.
  • Suitable pump devices include, but are not limited to, those pump devices manufactured by Tandem Diabetes Care, Inc., Delsys Pharmaceuticals, Insulet, Inc., Medtronics, Inc., and the like.
  • Suitable needle-free injection devices include, but are not limited to, those devices manufactured by Zogenix, Inc., Bioject Medical Technologies, Inc., Antares Pharma, Inc., and the like.
  • a glucagon formulation for administration by infusion or injection can be formulated into a preparation suitable for injection or infusion by dissolving, suspending, or emulsifying the glucagon in a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be an aqueous carrier or a non-aqueous carrier.
  • the pharmaceutically acceptable carrier is a non-aqueous carrier including, but are not limited to, lipids, aryl benzonates, alkyl benzonates and triacetin.
  • the nonaqueous carrier is triacetin, benzyl benzoate, miglyol, palm oil or mineral oil.
  • the pharmaceutically acceptable carrier is an aqueous carrier (e.g., water or an aqueous buffer).
  • the pharmaceutically acceptable carrier is an aprotic polar solvent, including but not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), or propylene carbonate.
  • the glucagon formulation that is formulated with a pharmaceutically acceptable carrier e.g., an aqueous carrier or a non-aqueous carrier
  • further comprises one or more surfactants e.g., Tween® 20.
  • the glucagon formulation is formulated as a gel.
  • Methods of formulating pharmacologically active agents as gels for administration by injection are described, for example, in WO 2011/075623, the contents of which are incorporated by reference herein in its entirety.
  • the glucagon formulation is formulated as a fast-dissolving solid matrix, for example, as described in WO 2011/031462, the contents of which are incorporated by reference herein in its entirety.
  • the glucagon formulations and related methods of the present invention may also be used in combination with the administration of other therapies such as weight loss/reduced caloric intake type therapies.
  • weight loss/reduced caloric intake type therapies include exercise, administration of drugs, etc.
  • weight loss/reduced caloric intake drugs include bupropion, leptin, lorcaserin hydrochloride, naltrexone, orlistat, phentermine, topiramate, GLP-1 (glucagon-like peptide- 1 (GLP-1), a GLP-1 agonist, exenatide, or analogs thereof), or any combination of said drugs.
  • the glucagon formulation can be administered before the additional weight loss/reduced caloric intake therapy, simultaneously with the additional therapy, or after the additional therapy. If the glucagon formulation and the additional weight loss/reduced caloric intake therapy are administered separate, then a person having ordinary skill in the art would ensure that a significant period of time did not expire between the time of each administration, such that the glucagon formulation and the additional therapy would be able to exert an advantageously combined effect on the subject.
  • Non-limiting examples of such administration times includes administering the stable glucagon formulation within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, or 15 minutes of administration of the additional therapy.
  • the additional therapy can be administered within 24 hours, 12, hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, or 15 minutes of administration of the stable glucagon formulation.
  • the methods of the present invention comprise administering to a subject in need thereof a low dose of a stable glucagon formulation.
  • Suitable stable glucagon formulations for use in the present invention may comprise any glucagon formulation that is stable for at least one week at controlled room temperature.
  • the glucagon formulation is stable for at least 2 weeks, at least 3 weeks, or at least 4 weeks or longer at controlled room temperature, or at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer at controlled room temperature.
  • the glucagon formulation is stable for an extended period of time over a range of temperatures.
  • the formulation is stable for at least 1, 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer at 40 °C.
  • the glucagon formulation is stable for at least 1, 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer at 4 °C.
  • Exemplary stable glucagon formulations for use in the present invention include any of the glucagon formulations described in WO 2012/059762; WO 2012/059764; US 2011/0237510; US 2012/0071817; US 2012/0046225; or US Application No. 13/417,073 ("Stable Formulations for Parenteral Injection of Peptide Drugs"), filed March 9, 2012, the contents of each of which is incorporated by reference herein in its entirety, and/or any of the glucagon formulations described below.
  • a stable glucagon formulation for use in the present invention may comprise glucagon or a glucagon analog or peptidomimetic, or a salt thereof (e.g., glucagon acetate).
  • the glucagon is present in the formulation in an amount ranging from about 0.5 mg/mL to about 100 mg/mL (e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/mL).
  • the glucagon is present in the formulation in an amount ranging from about 0.5 mg/mL to about 60 mg/mL, from about 10 mg/mL to about 50 mg/mL, from about 20 mg/mL to about 50 mg/mL, from about 5 mg/mL to about 15 mg/mL, or from about 0.5 mg/mL to about 2 mg/mL.
  • the glucagon composition is a non-aqueous ionization stabilized composition.
  • formulations may be prepared wherein the therapeutic agent is dissolved in a biocompatible non-aqueous liquid, such as an aprotic polar solvent.
  • aprotic polar solvents to prepare non-aqueous therapeutic formulations to inhibit many common degradation pathways, particularly those involving water, can significantly improve the stability of the solubilized or dissolved therapeutic molecule(s).
  • problems still remain with the compositions and methods disclosed in the prior art. In particular, direct dissolution of a therapeutic molecule in an aprotic polar solvent is not a suitable approach for preparing stable compositions of most therapeutic molecules.
  • the peptide hormone glucagon when solubilized directly in DMSO at a concentration of 5 mg/mL the peptide hormone glucagon will form insoluble aggregates within one day of storage at room temperature.
  • 5 mg/mL corresponds to approximately 0.45% (w/w) of the peptide compound, indicating that at even relatively low concentrations, direct dissolution in an aprotic polar solvent system is by itself incapable of preventing physical aggregation and/or gelation of a therapeutic molecule.
  • therapeutic molecules that may not form insoluble aggregates in an aprotic polar solvent system may nonetheless be prone to chemical degradation when solubilized directly in an aprotic polar solvent system.
  • the drying process is well known to impose several stresses on the therapeutic molecule, and additional excipients (e.g., lyoprotectants such as trehalose and sucrose, and/or surfactants such as polysorbate 80) must be included in the aqueous solution in sufficient amounts to protect the therapeutic molecule, thereby increasing the cost and complexity of the formulation. Further, the drying process (e.g., spray drying, freeze drying) must often be optimized for a given therapeutic molecule, both at the lab-scale during initial research and development where the process is initially developed, and then during the manufacturing- scale as the process is scaled-up and transferred to instruments and facilities capable of producing commercial- scale batches.
  • excipients e.g., lyoprotectants such as trehalose and sucrose, and/or surfactants such as polysorbate 80
  • the solution resides in dissolving an ionization stabilizing excipient(s) directly in the aprotic polar solvent, coupled with dissolution of the peptide molecule or small molecule directly in the aprotic polar solvent solution.
  • an ionization stabilizing excipient directly in the aprotic polar solvent
  • electrostatic repulsion between therapeutic molecules possessing the same charge polarity i.e. negatively or positively charged
  • may be sufficient in magnitude to prevent physical degradation e.g., via short-range hydrophobic interaction between molecules that lead to aggregation).
  • ionization i.e., protonation or deprotonation
  • chemical degradation can be minimized, as, for example, an excess of protonation may promote chemical instability via degradative reactions such as oxidation (for example, oxidation of methionine residues) and fragmentation (for example, cleavage of the peptide backbone).
  • oxidation for example, oxidation of methionine residues
  • fragmentation for example, cleavage of the peptide backbone
  • a therapeutic peptide For a therapeutic peptide, the extent of protonation required for stability, and thus the amount of the ionization stabilizing excipient required in the solution, will depend on, among other things, the primary structure (i.e., amino acid sequence) and the peptide concentration in the solution. [0095] Without wishing to be bound by theory, it is thought that in order to exhibit enhanced or optimal stability and solubility when formulated in an aprotic polar solvent system, a therapeutic molecule may require a specific ionization profile.
  • the ionization profile is the charge state acquired via protonation and/or deprotonation of the therapeutic molecule's ionogenic groups. For example, protonation of the ionogenic amino acid residues (e.g.
  • arginine, lysine comprising a therapeutic peptide will confer an overall positive charge on the molecules in solution.
  • the relatively long-range electrostatic repulsions between positively charged peptide molecules may inhibit the short-range hydrophobic interactions that can result in physical aggregation and/or gelation.
  • therapeutic molecules dissolved in an aprotic polar solvent system may be physically unstable and lead to the formation of soluble and/or insoluble aggregates.
  • an excipient in a sufficient concentration to function as an ionization stabilizing agent that is capable of imparting the ionization profile for improved physical and/or chemical stability to the active agent in the aprotic polar solvent system.
  • An appropriate concentration of the ionization stabilizing excipient(s) to be added to the solution depends on several factors including, but not limited to, the chemical structure of the ionization stabilizing excipient, the chemical structure of the active agent(s), the concentration of the active(s), the solvent system used, the presence of co- solvents, and the presence of additional excipients or formulation components and their respective concentrations.
  • a composition may be prepared by first adding the ionization stabilizing excipient to the aprotic polar solvent system, followed by addition of the therapeutic molecule.
  • the therapeutic molecule may initially be solubilized in the aprotic polar solvent system followed by addition of the ionization stabilizing excipient.
  • the ionization stabilizing excipient and the therapeutic molecule may be solubilized simultaneously in the aprotic polar solvent system.
  • Each molecule that functions as an ionization stabilizing excipient will exhibit a certain tendency to donate protons to the therapeutic molecule(s) in a given solvent system; this tendency to donate protons may be referred to as the relative acidic strength of the molecule.
  • the concentration of a given proton-donating molecule (ionization stabilizing excipient) required to achieve an appropriate or optimal ionization profile for the therapeutic molecules will be inversely proportional to its acidic strength.
  • the aprotic polar solvent can be deoxygenated prior to preparation of the formulation.
  • Many different techniques can be used in the context of the present invention to deoxygenate or remove oxygen from aprotic polar solvents (degasification or deoxygenation).
  • deoxygenation can, but is not limited to, remove oxygen that is dissolved in a liquid aprotic polar solvent either 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 decrease the solubility of dissolved gas, fractional distillation, membrane degasification, substitution by inert gas, using a reducing agent, freeze-pump-thaw cycling, or long time storage in a container with air-locks.
  • the aprotic polar solvents may have less than 0.1 mM of dissolved oxygen, preferably less than 0.05 mM of dissolved oxygen.
  • Methods known to those of skill in the art can be used to determine the amount of dissolved oxygen in any given aprotic polar solvent (e.g., a dissolved oxygen meter or probe device can be used such as the Dissolved Oxygen Probe commercially available by Vernier (Beaverton, Oregon, USA)).
  • a dissolved oxygen meter or probe device can be used such as the Dissolved Oxygen Probe commercially available by Vernier (Beaverton, Oregon, USA)).
  • a stable glucagon formulation for use in the present invention is a formulation that has been dried with a carbohydrate and a buffer having a pH of about 2.0 to about 3.5. Drying the glucagon with the buffer and the carbohydrate helps to protect against chemical degradation of the glucagon and helps to preserve the a-helix of the glucagon.
  • the glucagon when reconstituted e.g., with a pharmaceutically acceptable carrier
  • the glucagon formulation comprises: glucagon or a glucagon analog, or a salt thereof, that has been dried with a carbohydrate and a buffer having a pH of about 2.0 to about 3.5.
  • the glucagon is reconstituted with a pharmaceutically acceptable carrier.
  • Suitable carbohydrates for use in drying the glucagon include sugars and starches.
  • the carbohydrate is selected from the group consisting of trehalose, hydroxyethyl starch (HES), dextran and mixtures thereof.
  • the carbohydrate is trehalose.
  • the carbohydrate is hydroxyethyl starch (HES).
  • the carbohydrate is a mixture of trehalose and hydroxyethyl starch (HES).
  • Suitable buffers for drying the glucagon include a glycine buffer, a citrate buffer, a phosphate buffer and mixture thereof.
  • the buffer is a glycine buffer.
  • the buffer is a citrate buffer.
  • the pharmaceutically acceptable carrier is an aqueous carrier.
  • the aqueous carrier is water.
  • the pharmaceutically acceptable carrier is a non-aqueous carrier.
  • the nonaqueous carrier is selected from the group consisting of lipids, aryl benzonates, alkyl benzonates and triacetin.
  • the stable glucagon formulation further comprises a surfactant that protects the glucagon from physical damage.
  • the surfactant is polysorbate 20 (e.g., Tween® 20) or polysorbate 80.
  • the stable glucagon formulation further comprises at least one non-aqueous protic solvent.
  • non-aqueous protic solvents include, but are not limited to, polyethylene glycol (PEG), propylene glycol (PG), polyvinylpyrrolidone (PVP), methoxypropylene glycol (MPEG), glycerol, glycofurol, and mixtures thereof.
  • the stable glucagon formulation further comprises one or more of an antioxidant, a chelator, and a preservative.
  • Suitable antioxidants include, but are not limited to, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulphate, sulfites, BHT, BHA, ascorbyl palmitate, propyl gallate, and vitamin E.
  • Suitable chelators include, but are not limited to, EDTA, tartaric acid and salts thereof, glycerin, and citric acid and salts thereof.
  • Suitable preservatives include, but are not limited to, benzyl alcohols, methyl parabens and propyl parabens.
  • a stable glucagon formulation for use in the present invention is a formulation that has been prepared by first freeze-drying the glucagon in a nonvolatile buffer to a dry peptide powder.
  • the dried glucagon has a defined "pH memory" of the pH of the glucagon in the non-volatile buffer from which the glucagon was dried.
  • the resulting glucagon powder is dissolved in an aprotic polar solvent, thereby forming a stable formulation having a moisture content of the formulation is less than 5%.
  • the dried glucagon maintains its defined pH memory when reconstituted in the aprotic polar solvent, i.e., the pH of the glucagon when reconstituted in the aprotic polar solvent is about equal to the pH of the glucagon in the non-volatile buffer from which it was dried.
  • the pH of the reconstituted glucagon is about equal to the pH of the glucagon in the non-volatile buffer from which it was dried when the glucagon is reconstituted in an aprotic polar solvent that is within one pH unit of the pH of the glucagon in the non-volatile buffer from which it was dried.
  • the glucagon had a pH of 3.0 in the non- volatile buffer from which it was dried, a pH memory of from 2.0 to 4.0 for the reconstituted glucagon would be within one pH unit, and thus the pH memory of the reconstituted glucagon would be about equal to the pH of the glucagon in the non-volatile buffer.
  • the pH of the reconstituted glucagon is about equal to the pH of the glucagon in the non-volatile buffer from which it was dried when the glucagon is reconstituted in an aprotic polar solvent that is within half of a pH unit of the pH of the glucagon in the nonvolatile buffer from which it was dried.
  • This glucagon formulation is stable for extended periods of time, is ready for use without the need for reconstitution at the time of use, and is functional over a range of temperatures.
  • the glucagon formulation comprises: (a) glucagon or a glucagon analog, or a salt thereof, wherein the glucagon has been dried in a non-volatile buffer, and wherein the dried glucagon has a pH memory that is about equal to the pH of the glucagon in the non-volatile buffer, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dried glucagon maintains the pH memory that is about equal to the pH of the glucagon in the non-volatile buffer when the dried glucagon is reconstituted in the aprotic polar solvent.
  • the concept of "pH memory,” as used with reference to the glucagon formulations as described herein, relates to the resulting charge profile (protonation state) after drying the glucagon from a buffered aqueous solution (e.g., from a non-volatile buffer).
  • a buffered aqueous solution e.g., from a non-volatile buffer.
  • the protonation state, and thus the solubility and stability of the glucagon, in very low or zero moisture nonaqueous solvents are affected by the aqueous pH of the glucagon solution before drying and the drying conditions employed.
  • the pH memory of the dried glucagon will be about equal to the pH of the glucagon in the non-volatile buffer. See, e.g., Enzymatic Reactions in Organic Media, Koskinen, A.M.P., and Klibanov, A.M., eds., Springer (1996).
  • the pH of the buffered aqueous solution (e.g., non-volatile buffer) in which the glucagon is dried can be optimized to yield a pH memory for the glucagon that results in optimal stability, maximum solubility, and minimal degradation when the dried glucagon is subsequently reconstituted in an aprotic polar solvent. Because aprotic polar solvents do not have exchangeable protons, when the dried glucagon is reconstituted into an aprotic polar solvent, the reconstituted formulation will maintain the solubility and stability characteristics of the optimal pH memory.
  • the pH memory can be measured in several ways.
  • pH memory can be measured by reconstituting the dried glucagon into un-buffered water and measuring the pH of the reconstituted glucagon with a pH indicator such as pH paper or a calibrated pH electrode.
  • pH memory can be determined for glucagon that has been reconstituted in an aprotic polar solvent (e.g., DMSO) by adding at least 20% water to the aprotic polar solvent (e.g., DMSO) and measuring the pH with a pH indicator.
  • aprotic polar solvent e.g., DMSO
  • Suitable non-volatile buffers for drying the glucagon include, for example, glycine buffers, citrate buffers, phosphate buffers, and mixtures thereof.
  • the non-volatile buffer is a glycine buffer or a citrate buffer.
  • the nonvolatile buffer is a glycine buffer.
  • the non- volatile buffer is a mixture of glycine buffer and citrate buffer.
  • the non- volatile buffer is a mixture of citrate buffer and phosphate buffer.
  • the stability of the injectable formulation is further enhanced by the inclusion of one or more stabilizing agents or stabilizing excipients in the formulation.
  • the stabilizing agent or stabilizing excipient is added prior to drying the glucagon.
  • the dried glucagon is reconstituted with the stabilizing agent or stabilizing excipient in the aprotic polar solvent.
  • the stabilizing excipient is selected from sugars, starches, sugar alcohols, and mixtures thereof. Examples of suitable sugars for stabilizing excipients include, but are not limited to, trehalose, glucose, sucrose, etc.
  • suitable starches for stabilizing excipients include, but are not limited to, hydroxyethyl starch (HES).
  • suitable sugar alcohols for stabilizing excipients include, but are not limited to, mannitol and sorbitol.
  • the stabilizing excipient is present in the formulation in an amount that is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% (w/v).
  • the dried glucagon powder is dissolved or reconstituted in an aprotic polar solvent.
  • the aprotic polar solvent is selected from dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), propylene carbonate, and mixtures thereof.
  • the aprotic polar solvent is a mixture of two or more of dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylene carbonate.
  • Dimethylsulfoxide (DMSO), ethyl acetate, and n-methyl pyrrolidone (NMP) are particularly preferred aprotic polar solvents, each of which is a biocompatible solvent.
  • the aprotic polar solvent is dimethylsulfoxide (DMSO).
  • the aprotic polar solvent is n-methyl pyrrolidone (NMP).
  • the aprotic polar solvent is a mixture of dimethylsulfoxide (DMSO) and n-methyl pyrrolidone (NMP). In still other embodiments, the aprotic polar solvent is a mixture of dimethylsulfoxide (DMSO) and ethyl acetate.
  • the dried peptide powder is reconstituted in an aprotic polar solvent that is "neat," i.e., that does not contain a co-solvent.
  • the dried peptide powder is reconstituted in a solution that comprises an aprotic polar solvent and that does not contain water as a co-solvent.
  • the glucagon powder is reconstituted in an aprotic polar solvent that further comprises at least one co-solvent that depresses the freezing point of the formulation, wherein the co-solvent is a polar protic solvent.
  • the co-solvent is selected from ethanol, propylene glycol (PG), glycerol, and mixtures thereof.
  • the co-solvent is ethanol or PG.
  • a co- solvent is present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v), e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (w/v).
  • Such glucagon formulations have very little residual moisture and, thus, the glucagon remains stable over extended periods of time. In some embodiments, the stable glucagon formulation has a moisture content that is less than 5%.
  • the moisture content is less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.025%, or less than 0.01%.
  • a stable glucagon formulation suitable for use according to the methods of the present invention may comprise: glucagon or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a glycine buffer, and wherein the dried glucagon has a pH memory that is about 2.0, about 2.5, or about 3.0; and an aprotic polar solvent selected from DMSO, ethyl acetate, NMP, and mixtures thereof; wherein the moisture content of the formulation is less than about 1%.
  • glucagon or a salt thereof e.g., glucagon acetate
  • an aprotic polar solvent selected from DMSO, ethyl acetate, NMP, and mixtures thereof
  • the glucagon formulation may further comprise one or more stabilizing excipients selected from sugars, starches, and mixtures thereof, and/or one or more co-solvents selected from ethanol, propylene glycol, glycerol, and mixtures thereof.
  • a composition is a stable formulation that includes: (a) a peptide or a salt thereof that has been previously dried from an aqueous composition comprising a partially volatile buffer, a volatile buffer, a strong acid, or a strong base, or any combination thereof, wherein the dried peptide or salt thereof has a first ionization profile that corresponds to the peptide's optimal stability and solubility; and an aprotic polar solvent, wherein the dried peptide or salt thereof is reconstituted into an aprotic polar solvent and has a second ionization profile in the aprotic polar solvent, wherein the first and second ionization profiles are substantially the same, such as within 1 pH unit of one another.
  • One non-limiting method for measuring the ionization state of the dry peptide includes reconstituting the dried peptide into un-buffered water and measuring the pH of the reconstituted peptide with a pH indicator such as pH paper or a calibrated pH electrode.
  • One non-limiting method for measuring the ionization state of the peptide that has been reconstituted in the aprotic polar solvent includes adding at least 20% water to the aprotic polar solvent and measuring the pH with a pH indicator.
  • the peptide or salt thereof can have a third ionization profile when the peptide is in the aqueous composition prior to the aforementioned drying step.
  • the third ionization profile can be different from the first or second ionization profiles by at least 1 pH unit (e.g., the aqueous composition can be formulated such that the pH of the aqueous composition compensates for the loss of counter-ions or buffer components or both during drying of said aqueous composition).
  • the third ionization profile can be substantially the same as the first or second ionization profiles, such as within 1 pH unit of one another.
  • One non- limiting method for measuring the ionization state of the peptide in the aqueous composition prior to said drying step is to measure the pH of the aqueous solution with a pH indicator.
  • the aqueous composition is formulated such that the third ionization profile shifts to the first ionization profile during drying of said aqueous composition.
  • the dried peptide can be partially or fully solubilized within the aprotic polar solvent. Full solubilization can be obtained by adding the dried peptide to the aprotic polar solvent up to the solubility limit of said peptide. For partial solubilization, suspensions and pastes can be formed such that a percentage of the peptide is solubilized in the aprotic polar solvent and a percentage is suspended or dispersed within said aprotic polar solvent.
  • the aqueous composition can include a partially volatile buffer, non-limiting examples of which include sodium acetate or ammonium phosphate or any combination thereof.
  • the aqueous composition can include a volatile buffer, non-limiting examples of which include ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, pyridine acetate, pyridine formate, or triethylammonium acetate, or any combination thereof.
  • the aqueous composition can include a strong acid, a non-limiting example of which includes hydrochloric acid.
  • the aqueous composition can include a strong base, non-limiting examples of which include sodium hydroxide, potassium hydroxide, lithium hydroxide, or calcium hydroxide, or any combination thereof.
  • the aqueous composition does not include any buffer or does not include a non-volatile buffer.
  • the aqueous composition can include a mixture of different buffers.
  • the mixture can include a mixture of non-volatile buffers, a mixture of partially volatile buffers, a mixture of volatile buffers, a mixture of nonvolatile and partially volatile buffers, a mixture of non- volatile and volatile buffers, a mixture of partially volatile and volatile buffers, or a mixture of non-volatile, partially volatile, and volatile buffers.
  • the drying step can be performed by lyophilization, spray drying, desiccation, thin-film freezing, spray freeze drying, or any combination thereof.
  • the moisture or water content of the formulation can be less than 15%, 10%, 5%, 1%, or less.
  • Non-limiting examples of aprotic polar solvents includes dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, dimethylformamide (DMF), propylene carbonate, or mixtures thereof.
  • the formulation can further include a co- solvent that depresses the freezing point of the formulation (e.g., ethanol, propylene glycol, glycerol, and mixtures thereof).
  • the formulation can further include a stabilizing excipient (e.g., a sugar, a starch, or mixtures thereof).
  • the peptide in the formulation is glucagon or a salt thereof.
  • the first or second ionization profiles can correspond to the ionization profile of glucagon when solubilized in an aqueous solution having a pH range of about 2 to 3.
  • the third ionization profile can correspond to the ionization profile of glucagon when solubilized in an aqueous solution having a pH range of about 2 to 3 or can correspond to the ionization profile of glucagon when solubilized in an aqueous solution having a pH range of greater than 3, or greater than 3 to 14, or greater than 3 to 10, or greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or any range therein.
  • the third ionization profile can correspond to the ionization profile of glucagon when solubilized in an aqueous solution having a pH range of less than 2, or less than 2 to 0 or less than 2 to 1 or 1 or 0 or any range therein.
  • the first ionization profile can be maintained by reconstituting the dried peptide or salt thereof in an organic solvent system comprising an organic solvent and an organic phase buffer prior to reconstituting said dried peptide or salt thereof into polar aprotic solvent.
  • the dried peptide or salt thereof can be reconstituted into the polar aprotic solvent with mixing the organic solvent system with the polar aprotic solvent.
  • the organic solvent system can be separated from the polar aprotic solvent via separation methods known in the art.
  • the organic solvent system can be substantially anhydrous (e.g., less than 1 wt. %, less than 0.5 wt. %, or less than 0.1 wt. % water) or anhydrous.
  • the glucagon is processed in order to decrease its particle size by any pharmaceutically acceptable manner known to those skilled in the art.
  • Various methods of particle size manipulation and/or reduction can be utilized in order to prepare the glucagon.
  • Such particle size reduction procedures include, but are not limited to, comminuting processes (cutting, chopping, crushing, grinding, milling, micronizing, nanosizing, freeze drying, spray-freeze-drying, trituration, and microfluidization).
  • Spray-drying includes the steps of atomization of a solution containing one or more solid (e.g., therapeutic agent) via a nozzle spinning disk, or other device, followed by evaporation of the solvent from the droplets.
  • a solution containing one or more solid e.g., therapeutic agent
  • the nature of the powder that results is the function of several variables including the initial solute concentration, size distribution of droplets produced and the rate of solute removal.
  • the particles produced may comprise aggregates of primary particles which consist of crystals and/or amorphous solids depending on the rate and conditions of solvent removal.
  • a spray-drying process for preparing ultra-fine powders of biological macromolecules such as proteins, oligopeptides, high molecular weight polysaccharides, and nucleic acids is described in, for example, U.S. Patent 6,051,256. Freeze-drying procedures are well known in the art, and are described, for example, in U.S. Patent 4,608,764 and U.S.
  • Patent 4,848,094 Spray-freeze-drying processes are described, e.g., in U.S. Patent No.
  • Lyophilization techniques are well known to those skilled in the art. Lyophilization is a dehydration technique that takes place while a product is in a frozen state (ice sublimation under a vacuum) and under a vacuum (drying by gentle heating). These conditions stabilize the product, and minimize oxidation and other degradative processes. The conditions of freeze drying permit running the process at low temperatures, therefore, thermally labile products can be preserved. Steps in freeze drying include pretreatment, freezing, primary drying, and secondary drying. Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent, or increasing the surface area.
  • formulation revision i.e., addition of components to increase stability and/or improve processing
  • decreasing a high vapor pressure solvent or increasing the surface area.
  • Methods of pretreatment include: freeze concentration, solution phase concentration, and formulating specifically to preserve product appearance or to provide lyoprotection for reactive products, and are described, e.g., in U.S. Patent 6,199,297.
  • "Standard” lyophilization conditions are described, e.g., in U.S. Patent 5,031,336, and in "Freeze Drying of Pharmaceuticals” (DeLuca, Patrick P., J. Vac. Sci. Technol., Vol. 14, No. 1, January/February 1977); and "The Lyophilization of Pharmaceuticals: A Literature Review” (Williams, N. A., and G. P. Polli, Journal of Parenteral Science and Technology, Vol. 38, No. 2, March/ April 1984).
  • the lyophilization cycle is partially performed above the glass transition temperature (Tg) of the therapeutic agent formulation to induce a collapse of the mass to form a dense cake containing residue moisture. In other embodiments, the lyophilization cycle is carried out below the glass transition temperature in order to avoid a collapse in order to achieve a complete drying of the particles.
  • Another stable glucagon formulation may be prepared by formulating human glucagon for delivery as a pharmaceutically acceptable semi-solid paste suspension for administration via intracutaneous injection.
  • the following can be used to prepare such a formulation: (1) lyophilized powder development followed by biocompatible carrier development, where in the first step the therapeutic agent (i.e. glucagon, or a glucagon analog or a salt thereof (e.g. glucagon acetate)) is prepared as a powder (either alone, or with excipients); and (2) in the second step the powder is mixed with a biocompatible non-aqueous diluent that does not solubilize the powder, such that a semi-solid suspension of the drug particles in the diluent is prepared.
  • the therapeutic agent i.e. glucagon, or a glucagon analog or a salt thereof (e.g. glucagon acetate)
  • the powder is mixed with a biocompatible non-aqueous diluent that does not solubil
  • This semi-solid suspension may be described as a pharmaceutically acceptable paste for administration via intracutaneous injection, where the solid phase (i.e. powder) promotes thermostability and enables very high concentrations of the therapeutic to be achieved, while the liquid phase (i.e. diluent) serves as a carrier to minimize friction through a needle to allow the formulation to be syringeable.
  • the solid phase i.e. powder
  • the liquid phase i.e. diluent
  • the semi-solid formulation can further include a carrier (e.g., one or more polymers) which imparts thixotropic properties to the formulation.
  • a carrier e.g., one or more polymers
  • the therapeutic agent can be homogeneously incorporated into the thixotropic pharmaceutically acceptable carrier, and said formulation is in the form of a paste or slurry.
  • the therapeutic agent is homogeneously contained within a pharmaceutically acceptable diluent.
  • the diluent functions as a carrier for the drug-containing powder and is preferably biocompatible and is a non- solvent to the powder such that the two phases (solid and liquid) are maintained in the formulation.
  • the carrier in certain embodiments fills the spaces between particles in a way that makes them flow while also serving as a lubricant to minimize friction through a needle.
  • the carrier is selected from the group consisting of alkyl benzoates, aryl benzoates, aralkyl benzoates, triacetin, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), alkanes, cyclic alkanes, chlorinated alkanes, fluorinated alkanes, perfluorinated alkanes, polyethylene glycol, and mixtures thereof.
  • DMSO dimethyl sulfoxide
  • NMP N-methyl-2-pyrrolidone
  • alkanes cyclic alkanes
  • chlorinated alkanes fluorinated alkanes
  • perfluorinated alkanes polyethylene glycol, and mixtures thereof.
  • the stability of the injectable formulation can be further enhanced by the inclusion of one or more stabilizing agents or stabilizing excipients in the formulation prior to drying the glucagon to a powder.
  • the stabilizing excipient is selected from sugars, starches, sugar alcohols, amino acids, and mixtures thereof.
  • suitable sugars for stabilizing excipients include, but are not limited to, trehalose, glucose, sucrose, etc.
  • suitable starches for stabilizing excipients include, but are not limited to, hydroxyethyl starch (HES).
  • suitable sugar alcohols for stabilizing excipients include, but are not limited to, mannitol and sorbitol.
  • An examples of an amino acid includes, but are not limited to, glycine.
  • the stabilizing excipient is present in the formulation in an amount that is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% (w/v).
  • a stable semi-solid suspension of glucagon for administration via intracutaneous injection and suitable for use according to the methods of the present invention may comprise: glucagon or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried (e.g. lyophilized) in a glycine buffer, and wherein the dried glucagon has a pH memory that is about 2.0, about 2.5, or about 3.0.
  • the glucagon formulation may further comprise one or more stabilizing excipients selected from sugars, starches, and mixtures thereof.
  • the powder is blended with a biocompatible non-aqueous carrier using, for example, a planetary mixer, and where the percent solids content (i.e. percent solids by weight) of the semi-solid suspension can vary between 1 to 99% dependent upon the physicochemical properties of the powder.
  • glucagon Current injectable formulations of glucagon are composed of 1 mg of glucagon in 49 mg of lactose at pH less than 3.0.
  • the formulation envisioned by the present invention provides an efficacious dose of glucagon in a minimal volume using as little stabilizer as possible to stabilize the glucagon during the freeze-drying powder development process as well as during the shelf life, and may be achieved by freeze-drying the glucagon at as high a concentration as possible.
  • An example of such a formulation would be 1 mg of glucagon in 3 microliters total volume, producing a highly concentrated formulation of approximately 333 mg/mL.
  • the glucagon-containing powder was prepared via lyophilization (freeze-drying), but the powder may be prepared by other methods well known to those skilled in the art, examples of which include spray drying and spray freeze drying, as well as additional particle engineering technologies such as thin film freezing.
  • An additional formulation for weight loss is a semi-solid co-formulation of glucagon (or a glucagon analog, or a salt thereof) and GLP-1 (glucagon-like peptide- 1 (GLP- 1), a GLP-1 agonist, exenatide, or analogs thereof), where both therapeutic agents are prepared into separate powder (each powder containing a single therapeutic agent) that are then mixed together and suspended in biocompatible diluent to produce a pharmaceutically acceptable semi-solid suspension that is administered to the patient via intracutaneous injection.
  • GLP-1 glucagon-like peptide- 1
  • exenatide or analogs thereof
  • the glucagon ((or a glucagon analog, or a salt thereof) can be dried to a powder in a buffering system, that may also include stabilizing excipients, such that it has a pH memory between 1 to 4 and preferably between 2 to 3.
  • the GLP-1 (glucagon-like peptide- 1, a GLP-1 agonist, exenatide, or analogs thereof) can be dried to a powder in a buffering system that may also include stabilizing excipients, such that it has a pH memory between 3 to 6 and preferably between 4 to 5.
  • the buffering systems may be dried, for example, via lyophilization.
  • the two powders can then be mixed together using, for example, an orbital blender, to prepare a uniform blend and suspended in a biocompatible non-aqueous carrier that is a non- solvent for both powders.
  • the powders are blended with the selected biocompatible non-aqueous carrier using, for example, a planetary mixer, and where the percent solids content (i.e. percent solids by weight) of the semi-solid suspension can vary between 1 - 99% dependent upon the physicochemical properties of the powders.
  • the molar ratio of glucagon to GLP-1 may be range between 0.1:1 to 100:1, and preferably between 0.5:1 to 10:1.
  • the injectable formulation is in controlled (slow) release form.
  • the formulation may comprise a pharmaceutically acceptable polymer (e.g. PLGA, PLA) in an amount effective to slow the release of the therapeutic agent(s) from said formulation upon administration via injection into the epidermal, dermal or subcutaneous layer of an animal.
  • a pharmaceutically acceptable polymer e.g. PLGA, PLA
  • the therapeutic agent(s) may be incorporated into liposomes or conjugated to or incorporated with polysaccharides and/or other polymers to provide a controlled release of the therapeutic agent from said formulation upon administration via injection into the epidermal, dermal or subcutaneous layer of an animal.
  • kits for administering a stable glucagon formulation according to the methods of the present invention.
  • the present invention provides kits for controlling or reducing body weight by controlling or reducing caloric intake in a subject in need thereof.
  • the present invention provides kits for treating mild or moderate hypoglycemia in a subject in need thereof.
  • the kit comprises: (a) a stable glucagon formulation, wherein the glucagon formulation is stable for at least one week at controlled room temperature; (b) a multi-dose cartridge or syringe; and (c) a multi-dose injection device capable of accepting the multi-dose cartridge or syringe.
  • a suitable glucagon formulation for use in a kit of the present invention can be any glucagon formulation described herein.
  • the kit comprises a stable glucagon formulation, wherein the glucagon formulation is stable for at least one week at controlled room temperature.
  • the kit comprises a glucagon formulation that is stable for an extended period of time over a range of temperatures, e.g., a glucagon formulation that is stable for at least 1, 2, 3, or 4 weeks or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months at 40°C, or a glucagon formulation that is stable for at least 1, 2, 3, or 4 weeks or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months at 4°C.
  • the kit comprises a stable glucagon formulation wherein the glucagon formulation comprises: glucagon or a glucagon analog, or a salt thereof, that has been dried with a carbohydrate and a buffer having a pH of about 2.0 to about 3.5, wherein the glucagon is reconstituted with a pharmaceutically acceptable carrier.
  • the glucagon formulation comprises glucagon that has been dried with trehalose and/or HES.
  • the glucagon is reconstituted with a non- aqueous carrier selected from the group consisting of lipids, aryl benzonates, alkyl benzonates, and triacetin.
  • the glucagon formulation further comprises a surfactant, including but not limited to Tween® 20.
  • the kit comprises a stable glucagon formulation wherein the glucagon formulation comprises: (a) glucagon or a glucagon analog, or a salt thereof, wherein the glucagon has been dried in a non- volatile buffer, and wherein the dried glucagon has a pH memory that is about equal to the pH of the glucagon in the non-volatile buffer, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dried glucagon maintains the pH memory that is about equal to the pH of the glucagon in the non-volatile buffer when the dried glucagon is reconstituted in the aprotic polar solvent.
  • the non-volatile buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof.
  • the aprotic polar solvent is selected from dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof.
  • the glucagon formulation further comprises a co- solvent that depresses the freezing point of the formulation, and/or a stabilizing excipient.
  • the multi-dose cartridge or syringe is pre-filled with the glucagon formulation.
  • the multi-dose cartridge is pre-filled with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of the glucagon formulation.
  • Glucagon formulation doses can be any dose described herein, for example, a dose of from about 5 ⁇ g to about 200 ⁇ g, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 ⁇ g.
  • the glucagon formulation dose can be varied (e.g., increased or decreased over time), as required by an individual patient.
  • the kit comprises glucagon that is formulated for dosing a human adult, wherein the dose of the glucagon formulation that is administered is from about 50 ⁇ g to about 150 ⁇ g, or from about 100 ⁇ g to about 200 ⁇ g.
  • the kit comprises glucagon that is formulated for dosing a human child, wherein the dose of the glucagon formulation that is administered is from about 5 ⁇ g to about 150 ⁇ g, from about 10 ⁇ g to about 150 ⁇ g, from about 15 ⁇ g to about 150 ⁇ g, or from about 20 ⁇ g to about 150 ⁇ g, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, or about 150 ⁇ g.
  • the multi-dose injection device capable of accepting the multi-dose cartridge or syringe is a pen injection device, an auto-injector device, or a pump (e.g., a glucagon infusion pump or a patch pump).
  • the multi-dose injection device is an implantable injection device.
  • the multi-dose injection device is a needle-free injection device.
  • the multi-dose device e.g., a pen injection device, auto-injector device, or pump
  • the multi-dose injection device is a low volume injection device.
  • Suitable pen/autoinjector devices for use in the kits of the present invention include, but are not limited to, those pen/autoinjection devices manufactured by Becton- Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, West Pharmaceuticals, Inc., and the like.
  • Suitable pump devices include, but are not limited to, those pump devices manufactured by Tandem Diabetes Care, Inc., Delsys Pharmaceuticals, Insulet, Inc., Medtronics, Inc., and the like.
  • Suitable needle-free injection devices include, but are not limited to, those devices manufactured by Zogenix, Inc., Bioject Medical Technologies, Inc., Antares Pharma, Inc., and the like.
  • the kit further comprises instructions, wherein the instructions direct the administration of the glucagon formulation to control or reduce body weight in the subject in need thereof and/or to treat mild or moderate hypoglycemia in the subject in need thereof.
  • High-performance liquid chromatography was performed on reconstituted samples according to standard methods, filling HPLC vial inserts with 90 ⁇ ⁇ of reconstituted sample and incubating for 4-6 hours at refrigerated temperatures, then injecting 10 ⁇ ⁇ of sample for analysis by size exclusion (SE)-HPLC and reverse phase (RP)-HPLC.
  • SE-HPLC analysis indicated that in less than 6 hours, the glucagon main peak purity dropped to only 42.8%. Two post-glucagon peaks were observed and main peak splitting was also observed.
  • RP-HPLC analysis indicated that in less than 6 hours, the glucagon main peak purity dropped to 27-28%.
  • glycine hydrochloride CAS No. 6000-43-7
  • DMSO CAS No. 67-68-5
  • Table 1 Glucagon sample solutions prepared by dissolving both glycine hydrochloride and glucagon powder directly in DMSO.
  • 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 respectively consisting of 0.1% (v/v) TFA (trifluoroacetic acid) in water and 0.1% (v/v) TFA in acetonitrile.
  • a C8 column BioBasicTM-8; ThermoScientific) (4.6 mm I.D. x 250 mm length, 5 micron particle size) was used with a column temperature of 37 °C, a 1.0 mL/min flow rate, sample injection volume and 280-nm detection wavelength.
  • glucagon formulations prepared via drying from a non- volatile buffer and reconstituting in DMSO (the pH memory formulations as described in Prestrelski '644), and direct dissolution of glucagon in DMSO (the method as described in Stevenson '547).
  • the stability of the formulations are presented as glucagon purity and shown in Table 2 below.
  • Glucagon formulations that had previously been lyophilized from a buffered aqueous solution containing 1 mg/mL glucagon, 2 mM glycine and 1% (w/v) trehalose prior to reconstitution to 5-fold the initial concentration with DMSO (i.e., the composition in the aprotic polar solvent system following reconstitution was 5 mg/mL glucagon, 10 mM glycine, and 5% (w/v) trehalose) also exhibited a glucagon purity of approximately 97% following six weeks of storage at 40 °C.
  • compositions prepared by the method of the present invention provide enhanced stability compared to the prior art methods of direct dissolution of the peptide powder in an aprotic polar solvent.
  • formulations of the present invention may provide an alternative pathway for preparing highly-concentrated, stable glucagon formulations in aprotic polar solvent systems without the need for drying the peptide from a buffered aqueous solution prior to dissolution in the aprotic polar solvent system.
  • glucagon solutions were prepared at a concentration of 5 mg/mL by dissolving glucagon powder in DMSO that included different concentrations of added hydrochloric acid, ranging from 0.001 M (1 mM) to 0.01 M (10 mM).
  • DMSO dimethyl methoxysulfoxide
  • 5 N HC1 was utilized to prepare 10 mM and 5.6 mM HCl in DMSO solutions, while 1 N HCl was used to prepare the 3.2 mM, 1.8 mM, and 1.0 mM solutions.
  • the 10 mM HCl in DMSO solution was prepared by adding 20 of 5 N HCl to 9.98 mL of DMSO (neat), while the 1.0 mM HCl in DMSO solution was prepared by adding 10 ⁇ ⁇ of 1 N HCl to 9.99 mL of DMSO (neat). Samples of each formulation were stored in CZ vials and incubated at 40 °C.
  • Sample solutions were prepared by dissolving glucagon powder to a concentration of 5 mg/mL in DMSO which contained various added concentrations of glycine hydrochloride (CAS No. 6000-43-7), betaine hydrochloride (CAS No. 590-46-5), or hydrochloric acid (1 N; CAS No. 7647-01-0).
  • glycine hydrochloride CAS No. 6000-43-7
  • betaine hydrochloride CAS No. 590-46-5
  • hydrochloric acid 1 N; CAS No. 7647-01-0
  • Table 4 Samples of each formulation were stored in CZ vials and incubated at 40 °C. Following 28 days of storage the chemical stability of the glucagon peptide was assessed by RP-HPLC and the purity reported in Table 4.
  • Glucagon was selected as a model peptide due to its tendency to gel (i.e. form insoluble aggregates) when the molecule is insufficiently protonated.
  • a concentration of up to 2 mM glycine hydrochloride was insufficient to prevent the formation of insoluble aggregates in the solution, though this concentration of both betaine hydrochloride and hydrochloric acid was sufficient to prevent the formation of insoluble aggregates following 28 days of storage at 40 °C.
  • Table 4 Stability (provided as % peptide purity) of 5 mg/mL Glucagon-DMSO Solutions Stored at 40 °C for 28 days.
  • Ionization Stabilized Glucagon Compositions at 5 mg/ml The following example demonstrates the stability of a glucagon solution prepared according to the method of the present invention in the presence of added formulation components (e.g. inactive agents, excipients).
  • Sample solutions were prepared by dissolving glucagon powder to a concentration of 5 mg/mL in DMSO which contained about 3.2 mM of added HC1 (from a stock solution of 1 N HC1). To these solutions were added varying concentrations of moisture, as well as 5.5% (w/v) mannitol (CAS No. 69-65-8), and 1% (v/v) benzyl alcohol (CAS No. 100-51-6). The experimental samples examined are listed in Table 5.
  • Table 5 Stability of 5 mg/mL Glucagon-DMSO Solutions stored at room temperature for 180 days. Stability is provided as glucagon purity as assessed by RP-HPLC
  • glucagon formulation was prepared as follows:
  • Pre-lyophilized bulk drug product is prepared in an aqueous solution of 1.0 mg/mL glucagon, 2 mM glycine, and 1% w/w trehalose, at pH 3.0 as follows:
  • the intermediate bulk drug product includes glucagon lyophiles reconstituted in DMSO that are pooled and aseptically filtered, creating a formulation of 5.0 mg/mL glucagon, 10 mM glycine, 5.0% w/w trehalose, and 94.5% w/w DMSO, with "pH memory" 3.0. All materials used in intermediate drug product formulation steps are DMSO compatible.
  • variable depending on pH adjustment required usually ⁇ 5 ml ( ⁇ 0.1 % w/w) per batch
  • the weekly increase in weights for each group of rats is shown in FIG. 3.
  • the data indicates a dose-related decrease in weight-gain where the last 7 points in the 2 mg/kg/day group were statistically lower relative to the vehicle. Statistical significance was evident at each dose at the end of the study. A few time points at the end of the study were also statistically different in the mid-dose group (see Table 7).
  • the decreased weight gain noted in the glucagon-treated groups was not accompanied by any change in food consumption relative to the vehicle group (FIG. 4), indicating that in rats a mechanism other than food consumption, and specific to glucagon, was involved in the weight gain reduction.

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Abstract

La présente invention concerne des procédés pour réguler ou réduire l'apport calorique chez un sujet par administration d'une faible dose d'une formulation de glucagon stable. La présente invention concerne en outre des procédés pour traiter l'hypoglycémie bénigne ou modérée chez un sujet qui en a besoin par l'administration d'une faible dose d'une formulation de glucagon stable. L'invention concerne également des kits pour mettre en pratique les procédés de l'invention.
EP16751037.9A 2015-06-10 2016-06-10 Utilisation de glucagon à faible dose Pending EP3307295A1 (fr)

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MX2021014614A (es) 2019-05-31 2022-01-06 Xeris Pharmaceuticals Inc Composiciones terapeuticas estables en solventes polares aproticos y metodos de elaboracion de las mismas.

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