WO2016077220A1 - Gip/glp-1 co-agonist peptides for human administration - Google Patents

Gip/glp-1 co-agonist peptides for human administration Download PDF

Info

Publication number
WO2016077220A1
WO2016077220A1 PCT/US2015/059719 US2015059719W WO2016077220A1 WO 2016077220 A1 WO2016077220 A1 WO 2016077220A1 US 2015059719 W US2015059719 W US 2015059719W WO 2016077220 A1 WO2016077220 A1 WO 2016077220A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
pharmaceutical composition
histidine
exemplary aspects
seq
Prior art date
Application number
PCT/US2015/059719
Other languages
French (fr)
Inventor
Ulrike ALTENBURGER
Hanns-Christian Mahler
Astrid Pappenberger
Oliver Boris Stauch
Christine Wurth
Original Assignee
Mb2 Llc
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
Application filed by Mb2 Llc filed Critical Mb2 Llc
Publication of WO2016077220A1 publication Critical patent/WO2016077220A1/en

Links

Classifications

    • 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
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • 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
    • A61K9/08—Solutions

Definitions

  • Diabetes mellitus type II i.e., type 2 diabetes
  • Type 2 diabetes is a heterogeneous group of conditions that constitute approximately 90% of diabetes in the United States.
  • Type 2 diabetes is caused by a combination of insulin resistance and diminished insulin secretion.
  • Weight reduction in obese patients is associated with improvement of insulin resistance and amelioration of diabetes symptoms.
  • GLP-1 glucagon-like peptide- 1
  • GIP glucose dependent insulinotropic peptide
  • GLP-1 and its analogs have been shown to be effective as adjunctive therapy for diabetes.
  • GLP-1 therapy has been associated with weight loss.
  • compositions comprising a GIP/GLP-1 co-agonist peptide for human administration.
  • the pharmaceutical composition comprises a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
  • SEQ ID NO: 2 and examples of peptides comprising SEQ ID NO: 2 are set out in the section entitled "Peptide to be administered" .
  • the peptide comprises a fatty acyl group covalently attached to an amino acid of the peptide.
  • the peptide comprises a fatty acyl group covalently attached to the amino acid at position 40 of the peptide, which is a Lys.
  • the peptide comprises or consists of any one of SEQ ID NOs: 3-8.
  • SEQ ID NOs: 3, 4, 5, 6, 7 and 8 are examples of peptide embodiments encompassed within SEQ ID NO: 2, with varying lengths of fatty acyl groups. It is understood that all references herein to "peptide" and uses or doses thereof includes the peptide or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition comprises about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a
  • the pharmaceutical composition is an aqueous solution and, therefore, comprises water.
  • the water is preferably and sterile.
  • the pharmaceutical composition is preferably sterile.
  • the pharmaceutical composition is an aqueous solution which is chemically and physically stable at physiological pH.
  • the pharmaceutical composition is an aqueous solution characterized by low turbidity and appears clear upon visual inspection.
  • the pharmaceutical composition is a sterile aqueous solution, safe for human administration.
  • the pharmaceutical composition complies with or passes the required antimicrobial efficacy tests, e.g., the test for efficacy of antimicrobial preservation of Section 5.1.3 of the European Pharmacopeia, and preferably complies with the Level B criteria.
  • the pharmaceutical composition comprises a preservative.
  • the pharmaceutical composition comprises phenol.
  • the pharmaceutical composition comprises about 5.0 mg/mL to about 6.0 mg/mL phenol.
  • the pharmaceutical composition comprises an aqueous solution and the aqueous solution comprises (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) water, and optionally (iv) a buffer, and optionally (v) a stabilizer, such as trehalose.
  • the pharmaceutical composition is preferably at physiological pH, e.g. a pH of about 6 to about 8, preferably about 7.
  • the pharmaceutical composition comprises a buffer.
  • the pharmaceutical composition comprises Histidine (e.g., L-Histidine), or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition comprises a combination of L-Histidine base and L-Histidine HC1.
  • the pharmaceutical composition comprises a total concentration of about 15 mM to about 25 mM of Histidine as a combination of L-Histidine base/Histidine HC1.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) a total concentration of about 15 mM to about 25 mM Histidine as a combination of L-Histidine base/Histidine HCl, and (iv) water.
  • the pharmaceutical composition comprises a stabilizer.
  • the pharmaceutical composition comprises trehalose.
  • the pharmaceutical composition comprises about 200 mM to about 300 mM trehalose.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) a total concentration of about 15 mM to about 25 mM Histidine as a combination of L- Histidine base/Histidine HCl, and (v) water.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) a total concentration of about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water.
  • unit doses comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, phenol, trehalose, L-Histidine base/Histidine HCl, and water.
  • the unit dose comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water, optionally, wherein the peptide is present at a concentration of about 1.0 mg/mL to about 10 mg/mL.
  • the unit dose comprises, consists essentially of, or consists of (i) about 0.2 mg to about 0.5 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water, optionally, wherein the peptide is present at a concentration of about 1.0 mg/mL to about 10 mg/mL.
  • an aqueous solution comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) a total concentration of about 20 mM Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL.
  • the pharmaceutical composition is suitable for human administration via injection.
  • the pharmaceutical composition is housed in a syringe, a pen cartridge for use in a pen injector or a glass vial for use with a needle and syringe.
  • Such pharmaceutical compositions are used, for example, to improve or achieve glycemic control, reduce glycemic excursions, improve glucose tolerance, reduce glucose intolerance, treat (reduce) hyperglycemia, treat type 2 diabetes, preserve pancreatic beta-cell function, increase pancreatic beta-cell function, treat insulin resistance, exert an insulinotropic effect, treat (reduce) adiposity, normalize body fat distribution, treat (reduce) dyslipidemia, prevent weight gain, reduce weight gain, reduce food intake, reduce appetite, induce weight loss, treat obesity, treat fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and/or treat metabolic syndrome.
  • NASH non-alcoholic steatohepatitis
  • kits for improving glycemic control or treating diabetes, preferably type II diabetes, in an adult human and methods of reducing weight gain or inducing weight loss in an adult human comprise administering to the adult human a pharmaceutical composition as described herein. Additional methods of use of the pharmaceutical compositions are contemplated herein.
  • the methods comprise steps that lead to the manufacture of the pharmaceutical compositions described herein.
  • the methods comprise steps that lead to the manufacture of an API stock solution of a pharmaceutical composition.
  • the method comprises (i) admixing phenol, L-histidine, or a pharmaceutically acceptable salt thereof, and optionally trehalose, with water, to make a buffer, (ii) adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, (iii) admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer of (i) in a second container, and (iv) adding the contents of the second container to the first container, followed by mixing with a Turbula mixer.
  • Figure 1 represents a graph of the mean change from baseline (i.e. predose on Day 1) in fasting plasma glucose (mg/dL) over time and by dose group, for patients receiving the peptide of SEQ ID NO: 6.
  • Figures 2A-2D are graphs of the results of meal tolerance tests for patients receiving the peptide of SEQ ID NO: 6.
  • Figure 2A represents results at baseline;
  • Figure 2B results at Day 1;
  • Figure 2C results at Day 7; and
  • Figure 2D results at Day 14.
  • Figure 3 represents a graph of the change in blood glucose levels (mg/dL) of mice 0 and 7 days after QD injections for 7 days with a vehicle control, liraglutide (an acylated GLP-1 analog), and three peptides of SEQ ID NO: 2, (a peptide of SEQ ID NO: 5, a peptide of SEQ ID NO: 6, and a peptide of SEQ ID NO: 7), at doses of 25 or 125 nmol/kg.
  • liraglutide an acylated GLP-1 analog
  • Figure 4 represents a graph of the blood glucose levels (mg/dL) of mice 0 and 7 days after the first injection with a vehicle control, liraglutide (at 30 nmol/kg/day), or SEQ ID NO: 6 (at 0.3, 1, 3, 10, or 30 nmol/kg/day).
  • compositions comprising a GIP/GLP-1 co-agonist peptide for human administration.
  • the pharmaceutical composition comprises a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
  • SEQ ID NO: 2 and examples of peptides comprising SEQ ID NO: 2 are set out in the section entitled "Peptide to be administered”.
  • the pharmaceutical composition comprises, consists essentially of, or consists of an aqueous solution, and, therefore, the pharmaceutical composition comprises water.
  • the water is preferably water for injection and is sterile.
  • the pharmaceutical composition is an isotonic solution.
  • the pharmaceutical composition is sterile.
  • the pharmaceutical composition (e.g., the pharmaceutical composition comprising, consisting essentially of, or consisting of an aqueous solution) is chemically and physically stable at physiological pH.
  • the pharmaceutical composition comprises or consists of an aqueous solution characterized by low turbidity.
  • the pharmaceutical composition comprises or consists of a sterile aqueous solution, safe for human administration.
  • the pharmaceutical composition comprises water.
  • the water is preferably water for injection and is sterile.
  • the pharmaceutical composition is an isotonic solution.
  • the pharmaceutical composition is sterile.
  • the pharmaceutical composition (e.g., the pharmaceutical composition comprising, consisting essentially of, or consisting of an aqueous solution) is chemically and physically stable at
  • composition complies with or passes the required antimicrobial efficacy tests, e.g., the test for efficacy of antimicrobial preservation of Section 5.1.3 of the European
  • Pharmacopeia e.g. meeting level B criteria.
  • the pharmaceutical composition (e.g., the pharmaceutical composition comprising, consisting essentially of, or consisting of an aqueous solution) has a physiological pH, e.g. about pH 5.5 to about pH 8.5.
  • the pH of the pharmaceutical composition is within a range of about 6.0 to about 8.0.
  • the pH of the pharmaceutical composition is within a range of about 6.2 to about 7.8.
  • the pH of the pharmaceutical composition is within a range of about 6.5 to about 7.5.
  • the pH of the pharmaceutical composition is within a range of about 6.7 to about 7.3.
  • the pH of the pharmaceutical composition is about 7.0 or 7.0 ⁇ 0.2.
  • the pharmaceutical composition is suitable for human administration via injection.
  • the pharmaceutical composition is stored in a container, such as any of those described herein.
  • the pharmaceutical composition is housed in a pen cartridge for use in a pen injector or a glass vial for use with a needle and syringe.
  • the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the peptide as described herein.
  • the pharmaceutical composition is prepared as a pre-formulated solution ready for injection, e.g., once daily injection. Parenteral injection or extended infusion, e.g. over a period of 10 minutes, 15 minutes, 30 minutes, 1 hour or 2 hours is possible. Subcutaneous injection is preferred.
  • the pharmaceutical composition is packaged as part of a kit. Accordingly, the invention provides a kit comprising any of the pharmaceutical
  • compositions comprising the peptide described herein and a device for administering the peptide to a patient, e.g., adult human.
  • the pharmaceutical compositions of the kits are in the form of an aqueous solution that is sterilized.
  • the peptides are used to prepare pre-formulated solutions ready for injection.
  • the pharmaceutical compositions comprise a lyophilized powder.
  • the device for administering the peptide is a syringe needle, pen device, jet injector or other needle-free injector.
  • the device is a disposable device.
  • the device of the kit is an aerosol dispensing device, wherein the peptides are prepackaged within the aerosol device.
  • the kit comprises a syringe and a needle, and in one embodiment the sterile peptides are prepackaged within the syringe.
  • the pharmaceutical composition is stored or housed in one of various containers.
  • the container in some aspects is a vial, tube, bottle, a single or multi- chambered pre-filled syringe, cartridge, infusion pump (external or implantable), jet injector, pre-filled pen device and the like.
  • kits also include instructions for use or for storage.
  • the pharmaceutical composition of the kit is in the form of a lyophilized powder, and the kit comprises instructions for adding an amount of an aqueous solution to the lyophilized powder.
  • the kits are labeled for storage at ambient room temperature or at refrigerated temperature.
  • the peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2, which is set out below:
  • Xaa at position 20 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB); wherein the Lys at position 40 of which is covalently attached to a fatty acyl group; and wherein the C-terminus is amidated;
  • the peptide consists essentially of the amino acid sequence of SEQ ID NO: 2.
  • the peptide consists of the amino acid sequence of SEQ ID NO: 2.
  • the fatty acyl group attached to the Lys at position 40 of the peptide is a 4-carbon to 30-carbon fatty acyl group.
  • the fatty acyl group is a 4- carbon fatty acyl group, 6-carbon fatty acyl group, 8-carbon fatty acyl group, 10-carbon fatty acyl group, 12-carbon fatty acyl group, 14-carbon fatty acyl group, 16-carbon fatty acyl group, 18-carbon fatty acyl group, 20-carbon fatty acyl group, 22-carbon fatty acyl group, 24-carbon fatty acyl group, 26-carbon fatty acyl group, 28-carbon fatty acyl group, or a 30-carbon fatty acyl group.
  • the fatty acyl acyl group is a 8-carbon to 20-carbon fatty acyl group or a 12-carbon to 20-carbon fatty acyl group, or a 14-carbon to 18-carbon fatty acyl group.
  • the fatty acyl group is a 14-carbon fatty acyl group, a 16-carbon fatty acyl group, or an 18-carbon fatty acyl group.
  • fatty acyl group is linear or branched.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to a C12-C20 fatty acyl group (e.g., a 12-carbon fatty acyl group, a 14-carbon fatty acyl group, 16-carbon fatty acyl group, 18-carbon fatty acyl group, 20-carbon fatty acyl group), as described in SEQ ID NO: 3.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 12-carbon (CI 2) fatty acyl group, as described in SEQ ID NO: 4.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 14-carbon (C14) fatty acyl group, as described in SEQ ID NO: 5.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 16-carbon (C16) fatty acyl group, as described in SEQ ID NO: 6.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 18-carbon (CI 8) fatty acyl group, as described in SEQ ID NO: 7.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 20-carbon (C20) fatty acyl group, as described in SEQ ID NO: 8.
  • the method comprises administering to an adult human a peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • the peptide present in the pharmaceutical compositions of the invention is a peptide of SEQ ID NO: 6:
  • Xaa at position 20 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB); wherein the Lys at position 40 of which is covalently attached to a 16-carbon fatty acyl group; and wherein the C -terminus is amidated;
  • the pharmaceutical composition comprises about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2, or a pharmaceutically acceptable salt thereof.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 9.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 8.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 7.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 6.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 5.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 4.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 3.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 2.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 2.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 3.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 4.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 5.0 mg/mL to about 10.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 6.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 7.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 8.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 9.0 mg/mL to about 10.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration within the range of about 2.5 mg/mL to about 7.5 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 4.0 mg/mL to about 6.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration of about 5.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 7.0 mg/mL to about 8.0 mg/mL. In exemplary aspects, the peptide is present in the
  • the pharmaceutical composition at a concentration within the range of about 2.0 mg/mL to about 8.0 mg/mL, e.g., about 5.0 mg/mL to about 7.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration of about 6.0 mg/mL.
  • the peptide is present in the pharmaceutical composition at a concentration of about 1.0 mg/mL, about 1.5 mg/mL, about 2.0 mg/mL, about 2.5 mg/mL, about 3.0 mg/mL, about 3.5 mg/mL, about 4.0 mg/mL, about 4.5 mg/mL, about 5.0 mg/mL, about 5.5 mg/mL, about 6.0 mg/mL, about 6.5 mg/mL, about 7.0 mg/mL, about 8.0 mg/mL, about 8.5 mg/mL, about 9.0 mg/mL, about 9.5 mg/mL, or about 10.0 mg/mL.
  • the peptide is present in the pharmaceutical composition for use at a once daily dosage of from about 0.5 mg to about 3.0 mg. In exemplary aspects, the peptide is present in the pharmaceutical composition for use at a once daily dosage of from about 0.75 mg to about 2.5 mg, about 0.75 mg to about 2.0 mg, or about 1.0 mg to about 2.0 mg, or about 1.8 mg, or about 1.5 mg. Corresponding methods of administering the peptide at this once daily dosage are also provided.
  • the peptide is in the form of a salt, e.g., a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable salt refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the analog, or separately prepared by reacting a free base function with a suitable acid. Many of the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Acid addition salts may be prepared from inorganic and organic acids.
  • Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2 -hydroxy ethansulfonate (isothionate), lactate, maleate, methane sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate,
  • Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.
  • acids which can be employed to form pharmaceutically acceptable acid addition salts include, for example, an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoric acid, and an organic acid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.
  • an inorganic acid e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoric acid
  • organic acid e.g., oxalic acid, maleic acid, succinic acid, and citric acid.
  • Basic addition salts also can be prepared in situ during the final isolation and purification, or by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium,
  • Representative organic amines useful for the formation of base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.
  • basic nitrogen-containing groups can be quaternized with the analog of the present disclosure as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides
  • arylalkyl halides like benzyl and phenethyl bromides and others.
  • the pharmaceutical composition comprises phenol.
  • the pharmaceutical composition comprises about 5.0 mg/mL to about 6.0 mg/mL phenol.
  • the pharmaceutical composition comprises about 5.25 mg/mL to about 5.75 mg/mL phenol.
  • phenol is present in the
  • the pharmaceutical composition comprises an aqueous solution and the aqueous solution comprises (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, and (iii) water.
  • the pharmaceutical composition comprises Histidine, or a pharmaceutically acceptable salt thereof.
  • the Histidine is L-Histidine.
  • the Histidine is present in the pharmaceutical composition at a concentration within the range of about 15 mM to about 25 mM or about 17.5 mM to about 22.5 mM.
  • the pharmaceutical composition comprises a concentration of about 20 mM L-Histidine
  • the Histidine is provided as a combination of L-Histidine base and Histidine HCl.
  • the pharmaceutical composition comprises a combination of L-Histidine base and L-Histidine HCl.
  • the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 15 mM to about 25 mM.
  • the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 17.5 mM to about 22.5 mM.
  • the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 20 mM.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (iv) water.
  • the pharmaceutical composition comprises trehalose.
  • the trehalose is present in the pharmaceutical composition at a concentration within the range of about 200 mM to about 300 mM.
  • the trehalose is present in the pharmaceutical composition at a concentration within the range of about 225 mM to about 275 mM.
  • the pharmaceutical composition comprises about 250 mM trehalose.
  • the pharmaceutical composition consists of a sterile, isotonic solution comprising the peptide, phenol, trehalose, L-Histidine base, Histidine HCl, and water, and the pH of the isotonic solution is about 7.0.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water.
  • the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water.
  • the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the composition, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg.
  • the pharmaceutical composition is for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human or for use in reducing weight gain or inducing weight loss in an adult human.
  • the pharmaceutical composition is for administration once daily in an amount of about 0.5 to 3.0 mg of the peptide.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 6.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water.
  • the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration within about 1.0 to about 10.0 mg/mL.
  • the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the composition, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg.
  • the pharmaceutical composition is for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human or for use in reducing weight gain or inducing weight loss in an adult human.
  • the pharmaceutical composition is for administration once daily in an amount of about 0.5 to 3.0 mg of the peptide.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water.
  • the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the
  • composition as described herein, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg.
  • an aqueous solution comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of phenol, trehalose, Histidine, and water.
  • the aqueous solution is sterile, isotonic and has a pH of about 7.0.
  • the aqueous solution comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.0 mg/mL to about 6.0 mg/mL phenol, (ii) about 200 mM to about 300 mM trehalose, (iii) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water.
  • the peptide is present in the aqueous solution at a concentration of about 1.0 mg/mL to about 10.0 mg/mL.
  • the aqueous solution comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the aqueous solution at a concentration of about 6.0 mg/mL.
  • the pharmaceutical composition described herein is provided as a multi-use dosage form comprising multiple doses of the pharmaceutical composition.
  • multi-use dosage forms comprising multiple doses of a pharmaceutical composition described herein.
  • each dose of the multi-use dosage form comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2- 8, or a pharmaceutically acceptable salt thereof.
  • each dose of the multi-use dosage form comprises about 0.75 to about 2.5 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof.
  • each dose of the multi-use dosage form comprises about 1.0 to about 2.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof. In exemplary embodiments, each dose of the multi-use dosage form comprises about 1.8 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof. In exemplary aspects, the peptide of the multi-use dosage form is any one of the peptides described herein. See, e.g., the section entitled "Peptide to be administered.”
  • the multi-use dosage form provided herein comprises multiple doses of a sterile pharmaceutical composition
  • a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 mg/mL to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HC1, and (v) water.
  • the multi-use dosage form comprises, consists essentially of, or consists of multiple doses of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein each dose of the multi-use dosage form comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of the peptide (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HC1, and (v) water.
  • the peptide is in a solution comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL, optionally, wherein the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL
  • the multi-use dosage form provided herein comprises multiple doses of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (v) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL. In exemplary aspects, the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL.
  • the pharmaceutical composition described herein is packaged in a container suitable for holding a pharmaceutical composition for injection.
  • the pharmaceutical composition is packaged or housed in a vial, tube, bottle, a single or multi-chambered pre-filled syringe, cartridge, infusion pump (external or
  • the container comprises a volume of the pharmaceutical composition which is suitable for a single injection.
  • the container comprises about 0.3 mL to about 0.5 mL.
  • the container comprises a volume of the pharmaceutical composition which is suitable for multiple injections.
  • the container comprises about 3.0 mL to about 5.0 mL.
  • Such containers are contemplated as part of the invention.
  • containers, pre-filled syringes, pen cartridges, and glass vials comprising one of the pharmaceutical compositions described herein.
  • the container, pre-filled syringe, pen cartridge, or glass vial comprises a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a
  • a pharmaceutically acceptable salt thereof (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water, optionally, wherein the volume of the composition is about 0.2 mL to about 3.0 mL.
  • the container, pre-filled syringe, pen cartridge, or glass vial comprises a volume of the pharmaceutical composition which is suitable for multiple injections.
  • the multi-use container, multi- use pre-filled syringe, multi-use pen cartridge, or multi-use glass vial comprises about 3.0 mL to about 5.0 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or about 3.0 mL to about 5.0 mL of
  • the container, syringe, pen cartridge, or glass vial is for single use.
  • the single-use container, single-use pre-filled syringe, single-use pen cartridge, or single-use glass vial comprises about 0.3 mL to about 0.5 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or about 0.3 mL to about 0.5 mL of a sterile pharmaceutical composition comprising,
  • a pen cartridge for use in a pen injector comprising a
  • a glass vial comprising a pharmaceutical composition described herein, or as manufactured by a method described herein, or a multi-use dosage form described herein, or an aqueous solution described herein.
  • a syringe comprising a pharmaceutical composition described herein, or as manufactured by a method described herein, or a multi-use dosage form described herein, or an aqueous solution described herein.
  • the methods comprise steps that lead to the manufacture of the pharmaceutical compositions described herein.
  • the method of manufacturing a pharmaceutical composition as described herein comprises admixing L-Histidine base, Histidine HCl, trehalose, and phenol with water, and then adding the peptide.
  • the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, and the method of making the pharmaceutical composition comprises admixing L-Histidine base, Histidine HC1, trehalose, and phenol with water, and then adding the peptide.
  • the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water, and the method of making the pharmaceutical composition comprises admixing L-Histidine base, Histidine HC1, trehalose, and phenol with water, and then adding the peptide.
  • the methods comprise making a buffer, making an stock solution comprising the peptide, diluting the stock solution to make a bulk solution, filtering the bulk solution to make a sterile bulk solution and then aliquoting the sterile bulk solution into containers.
  • the buffer is made by admixing, per mL of buffer, about 2.811 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water.
  • the buffer is adjusted to a pH of about 7.0 ⁇ 0.5.
  • the buffer is adjusted to a pH of about 7.0 ⁇ 0.2.
  • the pH of the buffer is adjusted using 1 N NaOH or 1 N HC1.
  • the stock solution is made by adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer described above in a second container, and adding the contents of the second container to the first container, followed by mixing with a shaker-mixed, for example, a Turbula mixer.
  • the method comprises (i) admixing phenol, L-histidine, or a pharmaceutically acceptable salt thereof, and optionally, trehalose with water, to make a buffer, (ii) adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, (iii) admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer of (i) in a second container, and (iv) adding the contents of the second container to the first container, followed by mixing with a Turbula mixer.
  • step (i) comprises admixing, per mL of buffer, about 2.81 1 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water.
  • step (iv) produces a stock solution and the final concentration of the peptide in the stock solution is about 50 mg/mL.
  • the method further comprises diluting the stock solution with buffer to a final peptide concentration within the range of about 1.0 mg/mL to about 10.0 mg/mL to make a bulk solution.
  • the method further comprises filtering the bulk solution through 0.22 ⁇ filters, thereby making the bulk solution a sterile bulk solution.
  • the method further comprises aseptically aliquoting the sterile bulk solution into a plurality of pen cartridges or a plurality of glass vials.
  • compositions provided herein are used, for example, to improve or achieve glycemic control, reduce glycemic excursions, improve glucose tolerance, reduce glucose intolerance, treat (reduce) hyperglycemia, treat type 2 diabetes, preserve pancreatic beta-cell function, increase pancreatic beta-cell function, treat insulin resistance, exert an insulinotropic effect, treat (reduce) adiposity, normalize body fat distribution, treat (reduce) dyslipidemia, prevent weight gain, reduce weight gain, reduce food intake, reduce appetite, induce weight loss, treat obesity, treat fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and/or treat metabolic syndrome.
  • NASH non-alcoholic steatohepatitis
  • the methods comprise administering to the adult human a pharmaceutical composition as described herein.
  • the methods comprise administering once daily to the adult human a pharmaceutical composition as described herein.
  • the pharmaceutical composition consists of a sterile, isotonic solution comprising the peptide, phenol, trehalose, L- Histidine base, Histidine HCl, and water, and the pH of the isotonic solution is about 7.0.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water.
  • the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L- Histidine base/Histidine HCl, and water.
  • a once daily dosage of about 0.50 mg to about 3.0 mg (e.g., about 0.75 mg to about 2.5 mg) of the peptide is administered to the adult human.
  • the pharmaceutical composition is administered via injection, preferably subcutaneous injection, e.g., with a pen injector or with a needle and syringe.
  • the pharmaceutical composition comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, once daily at a dosage of about 0.75 mg to about 2.5 mg, or about 1.8 mg.
  • the method comprises administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, at a daily dosage of from about 0.75 mg to about 2.5 mg.
  • these methods comprise administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, at a daily dosage of from about 0.75 mg to about 2.0 mg, or about 1.0 mg to about 2.0 mg.
  • these methods comprise administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, at a daily dosage of about 1.8 mg, or about 1.5 mg.
  • a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, at a daily dosage of about 1.8 mg, or about 1.5 mg.
  • the peptide is
  • methods of improving glycemic control in an adult human comprising administering to the adult human a pharmaceutical composition as described herein in an amount effective to improve glycemic control or treat diabetes, optionally type II diabetes.
  • methods of reducing weight gain or inducing weight loss in an adult human comprising administering to the adult human a pharmaceutical composition as described herein in an amount effective to reduce weight gain or induce weight loss or treat obesity in an adult human.
  • the pharmaceutical composition used in the method of improving glycemic control in an adult human or the method of reducing weight gain or inducing weight loss in an adult human is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of: (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HC1, and (v) water.
  • the pharmaceutical composition used in the method of improving glycemic control in an adult human or the method of reducing weight gain or inducing weight loss in an adult human is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water.
  • a once daily dosage of about 0.5 mg to about 3.0 mg of the peptide is administered to the adult human.
  • the pharmaceutical composition is administered via injection.
  • the term "treat,” as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the methods of treating obesity or hyperglycemia or diabetes of the invention can provide any amount or any level of treatment.
  • the treatment provided by the method of the invention may include treatment of one or more conditions or symptoms or signs of the obesity or hyperglycemia or diabetes, being treated.
  • the treatment provided by the methods of the invention may encompass slowing the progression of the obesity or
  • the methods can treat obesity by virtue of reducing weight gain, inducing weight loss, reducing food intake, and the like.
  • the methods can treat hyperglycemia or diabetes by reducing blood glucose levels, or normalizing blood glucose levels, or improving or achieving glycemic control, or reducing glycemic excursions, or improving glucose tolerance (reducing glucose intolerance) or improving insulin resistance, preserving pancreatic beta-cell function, increasing pancreatic beta-cell function, exerting an insulinotropic effect and the like.
  • the invention also provides methods of reducing weight gain or inducing weight loss, reducing blood glucose levels, normalizing blood glucose levels.
  • the invention furthermore provides methods of normalizing body fat distribution, reducing food intake, reducing appetite, and the like.
  • the methods of the invention reduce complications associated with obesity, including but not limited to vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia, dyslipidemia, nephropathy and/or musculoskeletal diseases. Accordingly, in exemplary aspects, the invention provides methods of preventing or delaying the onset of or reducing the risk of complications associated with obesity.
  • vascular disease coronary artery disease, myocardial infarction, cerebral vascular disease stroke, peripheral vascular disease, ischemia reperfusion, etc.
  • the invention provides a method of preventing vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases.
  • vascular disease coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.
  • hypertension diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases.
  • the invention provides a method of reducing the risk for vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases.
  • vascular disease coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.
  • hypertension diabetes type II
  • hyperlipidemia dyslipidemia and/or musculoskeletal diseases.
  • the methods of the invention treat obesity-induced nephropathy.
  • the method comprise administering to an adult human in need thereof a pharmaceutical composition described herein.
  • Preferably the peptide of the pharmaceutical composition is administered once daily.
  • the adult human being treated has or suffers from vascular disease (e.g., coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia or dyslipidemia.
  • vascular disease e.g., coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.
  • hypertension onset of diabetes type II
  • hyperlipidemia or dyslipidemia e.g., hypertension of hyperlipidemia or dyslipidemia.
  • the adult human being treated has or suffers from other risk factors for cardiovascular disease, such as patients with insulin resistance or diabetes type II, hypertension, hyperlipidemia, or combinations of these risk factors.
  • the adult human in need thereof has or suffers from a drug -induced obesity.
  • the diabetes being treated by the methods of the invention is diabetes mellitus type I, diabetes mellitus type II, or gestational diabetes, any of which may be insulin-dependent or non-insulin-dependent.
  • the treatment provided by the method of the invention may include treatment of one or more conditions or symptoms or signs of diabetes being treated.
  • the methods of the invention cause an increase in insulin level, a decrease in glucose level, an increase in C-peptide level, a decrease in HbAi c level, a decrease in fructosamine level, and combinations thereof.
  • An increase in insulin level would indicate that administration of the peptide effectively is treating diabetes; and a decrease in glucose level would indicate that administration of the peptide is acting to reduce the levels of blood sugar, e.g., treating hyperglycemia.
  • HbAi c levels depend on blood glucose concentration (i.e., the higher the glucose concentration in blood, the higher the level of HbAi c ), but are not influenced by daily fluctuations in the blood glucose concentration. Instead, they represent the average blood glucose level of approximately the past 4 weeks, strongly weighted toward the most recent 2 weeks. A decrease in HbAi c level would indicate that administration of peptide is reducing the average blood glucose level over the long term.
  • C-peptide serves as a linker between the A- and B- chains of insulin and facilitates the efficient assembly, folding, and processing of insulin in the endoplasmic reticulum.
  • High levels of C-peptide generally indicate high levels of endogenous insulin production, while low levels of C-peptide generally indicate low levels of insulin production.
  • An increase in C-peptide level indicates that administration of the peptide is increasing the producing of insulin.
  • Fructosamine can be used to identify the plasma glucose concentration.
  • the higher the fructosamine concentration the higher the average blood glucose level.
  • Normal fructosamine levels may indicate that a patient is either not diabetic or that the patient has good diabetic control.
  • An increase in fructosamine levels indicates that the patient's average glucose over the last 2 to 3 weeks has been elevated.
  • a decrease in fructosamine level would indicate that administration of the peptide is reducing the average blood glucose level.
  • the invention provides methods of improving or achieving glycemic control, comprising administering to an adult human a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, once daily
  • the method comprises administering to an adult human in need thereof a pharmaceutical composition described herein.
  • the peptide of the pharmaceutical composition is administered once daily.
  • the invention provides methods of treating diabetic nephropathy, comprising administering to an adult human a pharmaceutical composition described herein.
  • a pharmaceutical composition described herein Preferably the peptide of the pharmaceutical composition is administered once daily.
  • the invention provides methods of diabetic retinopathy, comprising administering to an adult human a pharmaceutical composition described herein.
  • a pharmaceutical composition described herein Preferably the peptide of the pharmaceutical composition is administered once daily.
  • the invention provides methods of treating metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, Prader-Willi syndrome, and optionally Alzheimer's disease and Parkinson's disease.
  • the pharmaceutical composition is administered alone, e.g., without any other therapeutic agent, without any other anti-diabetic agent and/or any other anti-obesity agent, including any of those listed below.
  • the pharmaceutical composition is administered in combination with an effective amount of one or more second therapeutic agents.
  • the second therapeutic agent is an anti-diabetic agent, an anti- obesity agent, or a mixture thereof.
  • Anti-diabetic agents known in the art or under investigation include insulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglita
  • Anti-obesity agents known in the art or under investigation include, Leptin and Fibroblast Growth Factor 21 (FGF-21), appetite suppressants, such as phenethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide) or Contrave (bupropion and naltrexone); or lipase inhibitors, similar to xenical (Orlistat) or Cetilistat (also known as ATL-962), or GT
  • the method comprises, in some aspects, administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin.
  • the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin to a patient with insufficient glycemic control.
  • the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin to a patient who has been administered the maximum tolerated dose of monotherapy with
  • Metformin or a sulphonylurea yet the patient still exhibits insufficient glycemic control.
  • the methods comprise administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent.
  • the methods comprise administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea,
  • the method comprises administering to a patient with insufficient glycemic control the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea, thiazolidmedione, gliptin, gliflozin.
  • the method comprises administering to a patient who has been treated with dual therapy, yet the patient still exhibits insufficient glycemic control, the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea, thiazolidinedione, gliptin, gliflozin.
  • Thiazolidinediones also known as glitazones, are known in the art and include, e.g., rosiglitazone, pioglitazone, troglitazone, netoglitazone, rivoglitazone, ciglitazone.
  • Gliptins also known as inhibitors of dipeptidyl peptidase 4 or DPP-4 inhibitors, are known in the art, and include but not limited to, sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, alogliptin, anagliptin, berberine, and lupeol.
  • Gliflozins are SGLT inhibitors (i.e., inhibitors of the SGLT2 glucose transporter) and include, but not limited to, dapagliflozin and sergliflozin.
  • the method comprises, in some aspects, administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea.
  • the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea to a patient with insufficient glycemic control.
  • the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea to a patient who has been administered the maximum tolerated dose of monotherapy with Metformin or a sulphonylurea, yet the patient still exhibits insufficient glycemic control.
  • Sulphonylureas are known in the art and include, but not limited to, carbutamide,
  • This examples provides a method of making a peptide of SEQ ID NO: 2.
  • MBHA resin (4-methylbenzhydrylamine polystyrene resin was used during peptide synthesis.
  • MBHA resin 100-180 mesh, 1% DVB cross-linked polystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmoc protected amino acids were purchased from Midwest Biotech.
  • the solid phase peptide syntheses using Boc-protected amino acids were performed on an Applied Biosystem 43 OA Peptide Synthesizer. Fmoc protected amino acid synthesis was performed using the Applied Biosystems Model 433 Peptide Synthesizer.
  • Synthesis of the peptide was performed on the Applied Biosystem Model 430A Peptide Synthesizer. Synthetic peptides were constructed by sequential addition of amino acids to a cartridge containing 2 mmol of Boc protected amino acid. Specifically, the synthesis was carried out using Boc DEPBT-activated single couplings. At the end of the coupling step, the peptidyl -resin was treated with TFA to remove the N-terminal Boc protecting group. It was washed repeatedly with dimethylformamide (DMF) and this repetitive cycle was repeated for the desired number of coupling steps. After the assembly, the sidechain protection, Fmoc, was removed by 20% piperidine treatment and acylation was conducted using DIC. The peptidyl- resin at the end of the entire synthesis was dried by using dichloromethane (DCM), and the peptide was cleaved from the resin with anhydrous HF.
  • DCM dichloromethane
  • peptidyl-resin was treated with anhydrous hydrogen fluoride (HF), and this typically yielded approximately 350 mg (-50% yield) of a crude deprotected-peptide.
  • HF hydrous hydrogen fluoride
  • the peptidyl-resin (30 mg to 200 mg) was placed in the HF reaction vessel for cleavage. 500 ⁇ , of p-cresol was added to the vessel as a carbonium ion scavenger. The vessel was attached to the HF system and submerged in the methanol/dry ice mixture. The vessel was evacuated with a vacuum pump and 10 ml of HF was distilled to the reaction vessel. This reaction mixture of the peptidyl-resin and the HF was stirred for one hour at 0° C, after which a vacuum was established and the HF was quickly evacuated (10-15 min).
  • the vessel was removed carefully and filled with approximately 35 ml of ether to precipitate the peptide and to extract the p-cresol and small molecule organic protecting groups resulting from HF treatment. This mixture was filtered utilizing a teflon filter and repeated twice to remove all excess cresol. This filtrate was discarded. The precipitated peptide dissolves in approximately 20 ml of 10%> acetic acid (aq). This filtrate, which contained the desired peptide, was collected and lyophilized.
  • the extract was diluted twofold with water and loaded onto a 2.2 X 25 cm Vydac C4 preparative reverse phase column and eluted using an acetonitrile gradient on a Waters HPLC system (A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% ACN and a gradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min.
  • HPLC analysis of the purified peptide demonstrated greater than 95% purity and electrospray ionization mass spectral analysis was used to confirm the identity of the peptide.
  • Acylated peptides were prepared as follows. Peptides were synthesized on a solid support resin using either a CS Bio 4886 Peptide Synthesizer or Applied Biosystems 430A Peptide Synthesizer. In situ neutralization chemistry was used as described by Schnolzer et al, Int. J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides, the target amino acid residue to be acylated was substituted with an N ⁇ -FMOC lysine residue. Treatment of the completed N-terminally BOC protected peptide with 20% piperidine in DMF for 30 minutes removed FMOC/formyl groups.
  • Coupling to a free ⁇ -amino Lys residue can be achieved by coupling a ten-fold molar excess of either an FMOC-protected spacer amino acid (ex. FMOC- Glu-OtBu) or acyl chain (ex. CH 3 (CH 2 )i 4 -COOH)and PyBOP or DEPBT coupling reagent in DMF/N,N-diisopropylethylamine (DIEA). Subsequent removal of the spacer amino acid's FMOC group is followed by repetition of coupling with an acyl chain. Final treatment with 100% TFA resulted in removal of any side chain protecting groups and the N-terminal BOC group.
  • FMOC-protected spacer amino acid ex. FMOC- Glu-OtBu
  • acyl chain ex. CH 3 (CH 2 )i 4 -COOH
  • DIEA DMF/N,N-diisopropylethylamine
  • Peptide resins were neutralized with 5% DIEA/DMF, dried, and then cleaved from the support using HF/p-cresol, 95:5, at 0°C for one hour. Following ether extraction, a 5% acetic acid (HO Ac) solution was used to solvate the crude peptide. A sample of the solution was then verified to contain the correct molecular weight peptide by ESI-MS. Correct peptides were purified by RP-HPLC using a linear gradient of 10% acetonitrile (CH3CN)/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm x 250 mm protein column was used for the purification. Acylated peptide analogs generally completed elution by a buffer ratio of 20:80. Portions were pooled together and checked for purity on an analytical RP-HPLC. Pure fractions were lyophilized yielding white, solid peptides.
  • HO Ac 5% acetic acid
  • the mass spectra were obtained using a Sciex API-Ill electrospray quadrapole mass spectrometer with a standard ESI ion source. Ionization conditions that were used are as follows: ESI in the positive-ion mode; ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing and curtain gas used was nitrogen flow rate of .9 L/min. Mass spectra were recorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwell time. The sample (about lmg/mL) was dissolved in 50%> aqueous acetonitrile with 1% acetic acid and introduced by an external syringe pump at the rate of 5 ⁇ / ⁇ .
  • peptides were analyzed in PBS solution by ESI MS, they were first desalted using a ZipTip solid phase extraction tip containing 0.6 ⁇ , C4 resin, according to instructions provided by the manufacturer (Millipore Corporation, Billerica, MA, see the Millipore website of the world wide web at millipore.com/catalogue.nsf/docs/C5737).
  • PBS Phosphate Buffered Saline
  • HPLC high performance liquid chromatography
  • MALDI analysis The crude peptide samples were dissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of the resulting solution was stored in a 1.5 ml HPLC vial which was then sealed and incubated at 37 °C. Aliquots of ⁇ were drawn out at various time intervals, cooled to room temperature and analyzed by HPLC.
  • HPLC analyses were performed using a Beckman System Gold Chromatography system using a UV detector at 214 nm. HPLC analyses were performed on a 150 mm x 4.6 mm C18 Vydac column. The flow rate was 1 ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent B contained 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to 70%B in 15 minutes). The data were collected and analyzed using Peak Simple Chromatography software.
  • a randomized, double-blind, placebo-controlled, multiple ascending dose study was performed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of a peptide of SEQ ID NO: 6 in adult patients with Type 2 diabetes, over 14 days of treatment.
  • the patients were given doses of 0.25 mg, 0.75 mg, 1.5 mg, 2.0 mg, or 2.5 mg of peptide, or placebo, administered once daily by subcutaneous injection.
  • Patients included in the study had been diagnosed with Type 2 diabetes according to WHO criteria at least 3 months prior to screening, were on a stable dose of metformin for at least 2 months prior to screening, and exhibited evidence of insulin secretory capacity (e.g., fasting serum C-peptide levels above 1.5 ng/mL).
  • fasting plasma glucose prior to the study during was ⁇ 240 mg/dL and hemoglobin Ale (HbAlC) level was > 6.5%> and ⁇ 10.5%.
  • Patients were randomized in a 3: 1 ratio to receive either active drug or placebo as daily subcutaneous injection into the abdomen for 14 days.
  • the therapeutic dose range for effects on glucose control in Type 2 diabetic patients ranged from 0.75 mg to 2.5 mg. This dose range is based on efficacy assessments showing that 0.25 mg had effects that were no different from effects seen with placebo. In contrast, the doses ranging from 0.75 mg to 2.5 mg had
  • Fasting plasma glucose was monitored for the patients receiving the peptide of SEQ ID NO: 6. During a 14 day study, fasting plasma glucose is the best measure of efficacy. The mean change from baseline (i.e. predose on Day 1) in fasting plasma glucose over time and by dose group is depicted in FIGURE 1.
  • a meal tolerance test which assesses stimulated glucose responses, was also conducted for the patients receiving the peptide of SEQ ID NO: 6. Results of meal tolerance tests are shown in FIGURES 2A-2D for baseline, Day 1, Day 7 and Day 14.
  • HbAlc which acts as an integrated measure of daily glucose levels, require longer periods of treatment, usually between 6-12 weeks, to see meaningful and consistent effects.
  • HbAlc A small HbAlc reduction (-0.23%) was seen with placebo, probably due to hospitalization and standardized diet. As observed for the fasting plasma glucose and meal tolerance test above, the effect at 0.25 mg (-0.09%>) was no different from placebo. Higher doses produced an approximately 2-fold greater reduction in HbAlc compared to placebo. HbAlc was reduced by 0.41, 0.50, 0.13 and 0.67%> (absolute value) after 2 weeks of treatment with 0.75, 1.5, 2.0 and 2.5 mg, respectively. The small sample size and the less advanced disease of patients in the 2.0 mg dose group may explain the relatively minor HbAlc decrease (-0.13%) seen in that group.
  • the AUC and Cmax pharmacokinetic parameters demonstrate that the ineffective dose of 0.25 mg is well distinguished from the effective dose of 0.75 mg.
  • the AUC for the 0.75 mg dose was 65.1 ng*h/mL (CV: 28.1%) at Day 1, and 124 ng*h/mL (CV: 37.8%) at Day 14, compared to AUC for the 0.25 mg dose of 22.4 ng*h/mL (CV: 27.3%) at Day 1 and 35.5 ng*h/mL (CV: 23.3%) at Day 14.
  • the Cmax for the 0.75 mg dose was 5.59 ng/niL (CV: 40.5%) at Day 1 and 8.46 ng/niL (CV: 35.0%) at Day 14, while Cmax for the 0.25 mg dose was 1.93 ng/niL (CV: 36.9%) at Day 1 and 2.59 ng/niL (CV: 32.2%) at Day 14.
  • the human dose range of from 0.75 mg to 2.5 mg once daily was surprisingly about 10-fold higher than the doses that would have been predicted from animal studies disclosed in Int'l Patent Pub. No. WO/2010/011439.
  • SEQ ID NOS: 5, 6, 7, which include varying lengths of fatty acyl chains, are all encompassed within SEQ ID NO: 2.
  • SEQ ID NOS: 5, 6, 7, liraglutide or a vehicle control were injected QD into mice at a dose of 25 or 125 nmol/kg for 7 days.
  • the blood glucose levels of the mice were measured 0 and 7 days after injection with peptide or vehicle control and are shown in FIGURE 3.
  • the effects of SEQ ID NOS: 5, 6 and 7 on blood glucose levels were dramatic. At 25 nmol/kg, these peptides caused about a 50% decrease in blood glucose levels.
  • DIO diet-induced obese mice
  • Liraglutide is an acylated peptide that is a GLP-1 agonist, like the peptide of SEQ ID NO: 2, that is not significantly different in molecular weight from SEQ ID NO: 2. Based on the rodent data in WO/2010/011439, it would have been predicted that the effective dose of SEQ ID NO: 2 in humans should be about 10-fold less than the effective dose of liraglutide. However, the data from human clinical studies described herein demonstrate that a surprisingly higher dose of peptides of SEQ ID NO: 2 is necessary to achieve a meaningful therapeutic effect compared to placebo.
  • the peptide referenced and used in this example was a peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 was covalently linked to a C 16 fatty acyl group.
  • compositions were subjected to studies to identify formulations that exhibit physical and chemical stability, comply with the required antimicrobial efficacy test(s), and exhibit low turbidity and sub-visible particle counts. Some studies related to pH/buffer, surfactant, and excipient.
  • the chemical stability of the peptide is influenced by pH. Therefore, the stability of the peptide in compositions comprising different buffer types (at a given physiological pH or within a physiological pH range) was tested. Briefly, pharmaceutical compositions comprising 5 mg/mL peptide, 230 mM trehalose, 0.04% (w/v) Polysorbate 20, 10 mM methionine, and 20 mM of one of 5 types of buffer were made. The pH of buffers ranged from 6.0 to 7.5 (pH 6.0, 6.5, 7.0, and 7.5). The pharmaceutical compositions were stored at different temperatures and the chemical degradation of the peptide of each pharmaceutical composition was measured via RP-HPLC at various time points.
  • the peptide referenced and used in this example was a peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 was covalently linked to a C16 fatty acyl group.
  • compositions comprising 0.0 mg/mL, 0.5 mg/mL, or 3 mg/mL peptide were subjected to turbidity studies with different preservatives. From these studies, it was determined that, for some preservatives, increasing the preservative concentration led to an increase in turbidity level. The increase in turbidity level was dependent upon the concentration of the peptide. The lower the peptide concentration, the higher the increase in turbidity level. If the concentration of the preservative is too low, however, the pharmaceutical composition will not pass the test for efficacy of antimicrobial preservation described in Section 5.1.3 of the European Pharmacopeia and also described in Example 7. See the data in Table 2 below. Thus, the selection of preservative, as well as the amount thereof, must be balanced between turbidity levels and efficacy of microbial preservation.
  • compositions comprising benzyl alcohol at 7.5 mg/mL or at 10 mg/mL were tested and were shown to exhibit low turbidity. These compositions, however, did not pass the test for efficacy of antimicrobial preservation described in Section 5.1.3 of the European Pharmacopeia and also described herein at Example 7. Briefly, the efficacy of antimicrobial preservation was tested for a pharmaceutical composition comprising (i) 3 mg/mL peptide, (ii) 20 mM His/His HCl, pH 7.0, (iii) 230 mM trehalose, (iv) 10 mM methionine, and (v) 10 mg/mL benzyl alcohol.
  • a pharmaceutical composition comprising phenol met the Level B Criteria of Acceptance, when tested for efficacy of antimicrobial preservation, in accordance with Section 5.1.3 of the EP Pharmacopeia.
  • This phenol-containing pharmaceutical composition comprised (i) 3 mg/mL peptide, (ii) 20 mM Histidine/Histidine HCl, pH 7.0, (iii) 240 mM trehalose, and (iv) 5 mg/mL phenol.
  • each test composition comprised a total concentration of 20 mM Histidine base and Histidine HCl, pH 7.0, in addition to 250 mM trehalose, and 1 mg/mL, 6 mg/mL, or 10 mg/mL peptide.
  • Each test composition comprised 4.5 mg/mL, 5.0 mg/mL, 5.5 mg/mL, or 6.0 mg/mL phenol.
  • the pharmaceutical compositions were tested for efficacy of antimicrobial preservation, in accordance with Section 5.1.3 of the EP Pharmacopeia (see Example 7). The results of the assay are shown below in TABLE 2.
  • a buffer comprising the components shown in TABLE 3 was made by carrying out the following steps. The amount of each buffer component indicated in TABLE 3 was added to a first container:
  • An API stock solution containing 50 mg/mL peptide (which peptide was the same as that described in Example 5) was made by first adding the amount of peptide (in powder form) into a glass bottle.
  • the glass bottle was made to fit a Turbula mixer.
  • the buffer described above was added into a separate container and additional IN NaOH was added to the buffer.
  • the amount of IN NaOH added to the buffer was 0.55 mL for each g of peptide in the final API stock solution.
  • the NaOH is to account for the pH drop observed when the peptide is dissolved in the buffer.
  • the buffer (containing the additional NaOH) was added to the glass bottle containing the peptide in powder form, and the glass bottle was then placed onto a Turbula mixer.
  • the contents of the glass bottle were mixed using the Turbula mixer. Once the contents of the glass bottle were sufficiently mixed, the solution was then diluted with the same buffer described above to obtain the desired peptide concentration. In one instance, the peptide concentration was 1.5 mg/mL ⁇ 5% and in another instance, the peptide concentration was 7.5 mg/mL ⁇ 5%.
  • the pH of the diluted solution was then adjusted if it was outside of 7.0 ⁇ 0.2.
  • the diluted solution was then sterile filtered using 0.22 ⁇ low protein-binding filters.
  • the sterile solution was then aseptically aliquoted into sterile glass vials closed with Teflon-coated rubber stoppers and alucrimp caps. The filled glass vials were then stored at a temperature between 2 °C and 8 °C.
  • antimicrobial preservatives may be added, particularly to aqueous preparations, to prevent proliferation or to limit microbial contamination which, during normal conditions of storage and use, particularly for multidose containers, could occur in a product and present a hazard to the patient from infection and spoilage of the preparation.
  • Antimicrobial preservatives must not be used as a substitute for good manufacturing practice.
  • the efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation or by the formulation in which it is incorporated or by the container and closure used.
  • antimicrobial activity of the preparation in its final container is investigated over the period of validity to ensure that such activity has not been impaired by storage. Such investigations may be carried out on samples removed from the final container immediately prior to testing.
  • the antimicrobial activity of the preparation as such or, if necessary, with the addition of a suitable preservative or preservatives provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use of the preparation.
  • the efficacy of the antimicrobial activity may be demonstrated by the test described below. The test is not intended to be used for routine control purposes.
  • the test consists of challenging the preparation, wherever possible in its final container, with a prescribed inoculum of suitable micro-organisms, storing the inoculated preparation at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples so removed.
  • the preservative properties of the preparation are adequate if, in the conditions of the test, there is a significant fall or no increase, as appropriate, in the number of micro-organisms in the inoculated preparation after the times and at the temperatures prescribed.
  • a suitable sample from each container typically 1 ml or 1 g, at zero hour and at appropriate intervals according to the type of the product and determine the number of viable micro-organisms by plate count or membrane filtration (2.6.12). Ensure that any residual antimicrobial activity of the product is eliminated by dilution, by filtration or by the use of a specific inactivator. When dilution procedures are used, due allowance is made for the reduced sensitivity in the recovery of small numbers of viable micro-organisms. When a specific inactivator is used, the ability of the system to support the growth of the test organisms is confirmed by the use of appropriate controls. The procedure is validated to verify its ability to demonstrate the required reduction in count of viable micro-organisms.
  • the A criteria express the recommended efficacy to be achieved. In justified cases where the A criteria cannot be attained, for example for reasons of an increased risk of adverse reactions, the B criteria must be satisfied.
  • a pharmaceutical composition comprising
  • composition of embodiment 1, wherein the peptide consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2.
  • composition is about 7.0.
  • a pharmaceutical composition comprising, consisting essentially of, or consisting of:
  • a pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 6.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water.
  • a multi-use dosage form comprising, consisting essentially of, or consisting of multiple doses of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein each dose of the multi-use dosage form comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of the peptide (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water.
  • the peptide is in a solution comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL, optionally, wherein the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL.
  • An aqueous solution comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL.
  • step (i) comprises admixing, per mL of buffer, about 2.811 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water.
  • step (iv) produces a stock solution and the final concentration of the peptide in the stock solution is about 50 mg/mL.
  • a pen cartridge for use in a pen injector comprising the pharmaceutical composition of any one of embodiments 1 to 23, or as manufactured by the method of embodiment 31 , or the multi-use dosage form of embodiment 24 or 25, or the aqueous solution of embodiment 26.
  • a glass vial comprising the pharmaceutical composition of any one of embodiments 1 to 23, or as manufactured by the method of embodiment 31, or the multi-use dosage form of embodiment 24 or 25, or the aqueous solution of embodiment 26.
  • composition of any one of embodiments 1 to 23 for use with another anti-diabetic and/or anti-obesity agent, optionally for improving glycemic control, or treating diabetes, or treating type II diabetes, or for reducing weight gain or inducing weight loss.
  • composition of claim 38 wherein the anti-diabetic agent is selected from the group consisting of insulin, sulfonylureas, such as tolbutamide (Orinase),
  • acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlint
  • a method of improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human comprising administering to the adult human a pharmaceutical composition of any one of embodiments 1 to 23 in an amount effective to improve glycemic control or treating diabetes, optionally type II diabetes.
  • a method of reducing weight gain or inducing weight loss in an adult human comprising administering to the adult human a pharmaceutical composition of any one of embodiments 1 to 23 in an amount effective to reduce weight gain or induce weight loss in an adult human.

Abstract

Provided herein are pharmaceutical compositions comprising a GIP/GLP-1 co-agonist peptide for human administration. In exemplary embodiments, the pharmaceutical composition comprises a peptide comprising the amino acid sequence of SEQ ID NO: 2, phenol, Histidine, trehalose, and water for injection. Provided herein are pharmaceutical compositions comprising a GIP/GLP-1 co-agonist peptide for human administration. In exemplary embodiments, the pharmaceutical composition comprises a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Description

GIP/GLP-1 CO-AGONIST PEPTIDES FOR HUMAN ADMINISTRATION
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0001] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 5,882 byte ASCII (Text) file named "48494_SeqListing.txt," created on November 4, 2014.
BACKGROUND
[0002] Diabetes mellitus type II (i.e., type 2 diabetes) is a heterogeneous group of conditions that constitute approximately 90% of diabetes in the United States. Type 2 diabetes is caused by a combination of insulin resistance and diminished insulin secretion. Weight reduction in obese patients is associated with improvement of insulin resistance and amelioration of diabetes symptoms.
[0003] There are five widely recognized classes of oral anti-diabetic agents, sulfonylureas, biguanides, meglitinides, thiazolidinediones, and alpha-glucosidase inhibitors. Treatment of type 2 diabetes usually involves choosing one or more of these oral agents as initial therapy (see, e.g., Charpentier G. Diabetes Metab. Res. Rev. 18(Supp. 3):S70-S76 (2002)). Despite the use of multiple drugs, however, the all-over control of diabetes remains inadequate. Only 49.8%> of persons with diabetes achieve the National Diabetes Association target HbAic of less than 7%. Instead, 29.7% of persons with diabetes have an HbAic of greater than 8% (see, e.g., Resnick et al, Diabetes Care 29:531-537 (2006)).
[0004] The incretin hormones, glucagon-like peptide- 1 (GLP-1) and glucose dependent insulinotropic peptide (GIP), are naturally-occurring peptide hormones. Both GLP-1 and GIP stimulate insulin synthesis and secretion in a glucose-dependent manner.
[0005] GLP-1 and its analogs have been shown to be effective as adjunctive therapy for diabetes. GLP-1 therapy has been associated with weight loss.
SUMMARY
[0006] Provided herein are pharmaceutical compositions comprising a GIP/GLP-1 co-agonist peptide for human administration. In exemplary embodiments, the pharmaceutical composition comprises a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. SEQ ID NO: 2 and examples of peptides comprising SEQ ID NO: 2 are set out in the section entitled "Peptide to be administered" . In exemplary aspects, the peptide comprises a fatty acyl group covalently attached to an amino acid of the peptide. In exemplary aspects, the peptide comprises a fatty acyl group covalently attached to the amino acid at position 40 of the peptide, which is a Lys. In exemplary aspects, the peptide comprises or consists of any one of SEQ ID NOs: 3-8. SEQ ID NOs: 3, 4, 5, 6, 7 and 8 are examples of peptide embodiments encompassed within SEQ ID NO: 2, with varying lengths of fatty acyl groups. It is understood that all references herein to "peptide" and uses or doses thereof includes the peptide or a pharmaceutically acceptable salt thereof. In exemplary aspects, the pharmaceutical composition comprises about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a
pharmaceutically acceptable salt thereof.
[0007] In exemplary aspects, the pharmaceutical composition is an aqueous solution and, therefore, comprises water. The water is preferably and sterile. Similarly, the pharmaceutical composition is preferably sterile.
[0008] In exemplary aspects, the pharmaceutical composition is an aqueous solution which is chemically and physically stable at physiological pH. In exemplary aspects, the pharmaceutical composition is an aqueous solution characterized by low turbidity and appears clear upon visual inspection. In exemplary aspects, the pharmaceutical composition is a sterile aqueous solution, safe for human administration. In exemplary aspects, the pharmaceutical composition complies with or passes the required antimicrobial efficacy tests, e.g., the test for efficacy of antimicrobial preservation of Section 5.1.3 of the European Pharmacopeia, and preferably complies with the Level B criteria.
[0009] In exemplary aspects, the pharmaceutical composition comprises a preservative. In exemplary aspects, the pharmaceutical composition comprises phenol. In exemplary aspects, the pharmaceutical composition comprises about 5.0 mg/mL to about 6.0 mg/mL phenol. In exemplary aspects, the pharmaceutical composition comprises an aqueous solution and the aqueous solution comprises (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) water, and optionally (iv) a buffer, and optionally (v) a stabilizer, such as trehalose. The pharmaceutical composition is preferably at physiological pH, e.g. a pH of about 6 to about 8, preferably about 7.
[0010] In exemplary aspects, the pharmaceutical composition comprises a buffer. In exemplary aspects, the pharmaceutical composition comprises Histidine (e.g., L-Histidine), or a pharmaceutically acceptable salt thereof. In exemplary aspects, the pharmaceutical composition comprises a combination of L-Histidine base and L-Histidine HC1. In exemplary aspects, the pharmaceutical composition comprises a total concentration of about 15 mM to about 25 mM of Histidine as a combination of L-Histidine base/Histidine HC1. [0011] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) a total concentration of about 15 mM to about 25 mM Histidine as a combination of L-Histidine base/Histidine HCl, and (iv) water.
[0012] In exemplary aspects, the pharmaceutical composition comprises a stabilizer. In exemplary aspects, the pharmaceutical composition comprises trehalose. In exemplary aspects, the pharmaceutical composition comprises about 200 mM to about 300 mM trehalose.
[0013] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) a total concentration of about 15 mM to about 25 mM Histidine as a combination of L- Histidine base/Histidine HCl, and (v) water.
[0014] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) a total concentration of about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water.
[0015] Also provided herein are unit doses comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, phenol, trehalose, L-Histidine base/Histidine HCl, and water. In exemplary aspects, the unit dose comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water, optionally, wherein the peptide is present at a concentration of about 1.0 mg/mL to about 10 mg/mL. In exemplary aspects, the unit dose comprises, consists essentially of, or consists of (i) about 0.2 mg to about 0.5 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water, optionally, wherein the peptide is present at a concentration of about 1.0 mg/mL to about 10 mg/mL.
[0016] Further provided herein is an aqueous solution comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) a total concentration of about 20 mM Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL.
[0017] In exemplary aspects, the pharmaceutical composition is suitable for human administration via injection. In exemplary aspects, the pharmaceutical composition is housed in a syringe, a pen cartridge for use in a pen injector or a glass vial for use with a needle and syringe.
[0018] Such pharmaceutical compositions are used, for example, to improve or achieve glycemic control, reduce glycemic excursions, improve glucose tolerance, reduce glucose intolerance, treat (reduce) hyperglycemia, treat type 2 diabetes, preserve pancreatic beta-cell function, increase pancreatic beta-cell function, treat insulin resistance, exert an insulinotropic effect, treat (reduce) adiposity, normalize body fat distribution, treat (reduce) dyslipidemia, prevent weight gain, reduce weight gain, reduce food intake, reduce appetite, induce weight loss, treat obesity, treat fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and/or treat metabolic syndrome.
[0019] Accordingly, provided herein are methods of improving glycemic control or treating diabetes, preferably type II diabetes, in an adult human and methods of reducing weight gain or inducing weight loss in an adult human. In exemplary embodiments, the methods comprise administering to the adult human a pharmaceutical composition as described herein. Additional methods of use of the pharmaceutical compositions are contemplated herein.
[0020] Further provided herein are methods of manufacture. In exemplary aspects, the methods comprise steps that lead to the manufacture of the pharmaceutical compositions described herein. In exemplary aspects, the methods comprise steps that lead to the manufacture of an API stock solution of a pharmaceutical composition. In exemplary embodiments, the method comprises (i) admixing phenol, L-histidine, or a pharmaceutically acceptable salt thereof, and optionally trehalose, with water, to make a buffer, (ii) adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, (iii) admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer of (i) in a second container, and (iv) adding the contents of the second container to the first container, followed by mixing with a Turbula mixer. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 represents a graph of the mean change from baseline (i.e. predose on Day 1) in fasting plasma glucose (mg/dL) over time and by dose group, for patients receiving the peptide of SEQ ID NO: 6.
[0022] Figures 2A-2D are graphs of the results of meal tolerance tests for patients receiving the peptide of SEQ ID NO: 6. Figure 2A represents results at baseline; Figure 2B, results at Day 1; Figure 2C, results at Day 7; and Figure 2D, results at Day 14.
[0023] Figure 3 represents a graph of the change in blood glucose levels (mg/dL) of mice 0 and 7 days after QD injections for 7 days with a vehicle control, liraglutide (an acylated GLP-1 analog), and three peptides of SEQ ID NO: 2, (a peptide of SEQ ID NO: 5, a peptide of SEQ ID NO: 6, and a peptide of SEQ ID NO: 7), at doses of 25 or 125 nmol/kg.
[0024] Figure 4 represents a graph of the blood glucose levels (mg/dL) of mice 0 and 7 days after the first injection with a vehicle control, liraglutide (at 30 nmol/kg/day), or SEQ ID NO: 6 (at 0.3, 1, 3, 10, or 30 nmol/kg/day).
DETAILED DESCRIPTION
[0025] Pharmaceutical compositions
[0026] Provided herein are pharmaceutical compositions comprising a GIP/GLP-1 co-agonist peptide for human administration. In exemplary embodiments, the pharmaceutical composition comprises a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. SEQ ID NO: 2 and examples of peptides comprising SEQ ID NO: 2 are set out in the section entitled "Peptide to be administered".
[0027] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of an aqueous solution, and, therefore, the pharmaceutical composition comprises water. The water is preferably water for injection and is sterile. In exemplary aspects, the pharmaceutical composition is an isotonic solution. In exemplary aspects, the pharmaceutical composition is sterile. In exemplary aspects, the pharmaceutical composition (e.g., the pharmaceutical composition comprising, consisting essentially of, or consisting of an aqueous solution) is chemically and physically stable at physiological pH. In exemplary aspects, the pharmaceutical composition comprises or consists of an aqueous solution characterized by low turbidity. In exemplary aspects, the pharmaceutical composition comprises or consists of a sterile aqueous solution, safe for human administration. In exemplary aspects, the
pharmaceutical composition complies with or passes the required antimicrobial efficacy tests, e.g., the test for efficacy of antimicrobial preservation of Section 5.1.3 of the European
Pharmacopeia, e.g. meeting level B criteria.
[0028] In exemplary aspects, the pharmaceutical composition (e.g., the pharmaceutical composition comprising, consisting essentially of, or consisting of an aqueous solution) has a physiological pH, e.g. about pH 5.5 to about pH 8.5. In exemplary aspects, the pH of the pharmaceutical composition is within a range of about 6.0 to about 8.0. In exemplary aspects, the pH of the pharmaceutical composition is within a range of about 6.2 to about 7.8. In exemplary aspects, the pH of the pharmaceutical composition is within a range of about 6.5 to about 7.5. In exemplary aspects, the pH of the pharmaceutical composition is within a range of about 6.7 to about 7.3. In exemplary aspects, the pH of the pharmaceutical composition is about 7.0 or 7.0 ± 0.2.
[0029] In exemplary aspects, the pharmaceutical composition is suitable for human administration via injection. In exemplary aspects, the pharmaceutical composition is stored in a container, such as any of those described herein. In exemplary aspects, the pharmaceutical composition is housed in a pen cartridge for use in a pen injector or a glass vial for use with a needle and syringe. In exemplary aspects, the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the peptide as described herein. In some embodiments, the pharmaceutical composition is prepared as a pre-formulated solution ready for injection, e.g., once daily injection. Parenteral injection or extended infusion, e.g. over a period of 10 minutes, 15 minutes, 30 minutes, 1 hour or 2 hours is possible. Subcutaneous injection is preferred.
[0030] Kits
[0031] In exemplary embodiments, the pharmaceutical composition is packaged as part of a kit. Accordingly, the invention provides a kit comprising any of the pharmaceutical
compositions comprising the peptide described herein and a device for administering the peptide to a patient, e.g., adult human. In exemplary embodiments, the pharmaceutical compositions of the kits are in the form of an aqueous solution that is sterilized. In some embodiments, the peptides are used to prepare pre-formulated solutions ready for injection. In other embodiments the pharmaceutical compositions comprise a lyophilized powder.
[0032] In exemplary aspects, the device for administering the peptide is a syringe needle, pen device, jet injector or other needle-free injector. In exemplary aspects, the device is a disposable device. In some embodiments the device of the kit is an aerosol dispensing device, wherein the peptides are prepackaged within the aerosol device. In another embodiment the kit comprises a syringe and a needle, and in one embodiment the sterile peptides are prepackaged within the syringe. [0033] In exemplary aspects, the pharmaceutical composition is stored or housed in one of various containers. The container in some aspects is a vial, tube, bottle, a single or multi- chambered pre-filled syringe, cartridge, infusion pump (external or implantable), jet injector, pre-filled pen device and the like.
[0034] In exemplary aspects, the kits also include instructions for use or for storage. In exemplary aspects, the pharmaceutical composition of the kit is in the form of a lyophilized powder, and the kit comprises instructions for adding an amount of an aqueous solution to the lyophilized powder. In exemplary aspects, the kits are labeled for storage at ambient room temperature or at refrigerated temperature.
[0035] Peptide to be administered
[0036] In relation to the various aspects of the invention, including pharmaceutical compositions, kits, unit doses, methods, and uses, the peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2, which is set out below:
Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser He Tyr Leu Asp Lys Gin Ala Ala Xaa Glu Phe
Val Asn Trp Leu Leu Ala Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser Lys wherein Xaa at position 2 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB);
wherein Xaa at position 20 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB); wherein the Lys at position 40 of which is covalently attached to a fatty acyl group; and wherein the C-terminus is amidated;
(SEQ ID NO: 2).
[0037] Such peptides have been tested and observed to exhibit agonist activity at both the GLP-1 receptor and the GIP receptor.
[0038] In exemplary aspects, the peptide consists essentially of the amino acid sequence of SEQ ID NO: 2.
[0039] In exemplary aspects, the peptide consists of the amino acid sequence of SEQ ID NO: 2.
[0040] It is understood that all references to "peptide" herein include any pharmaceutically acceptable salts of the peptide. Example salts are described below under the section entitled "Salts."
[0041] In exemplary aspects, the fatty acyl group attached to the Lys at position 40 of the peptide is a 4-carbon to 30-carbon fatty acyl group. For example, the fatty acyl group is a 4- carbon fatty acyl group, 6-carbon fatty acyl group, 8-carbon fatty acyl group, 10-carbon fatty acyl group, 12-carbon fatty acyl group, 14-carbon fatty acyl group, 16-carbon fatty acyl group, 18-carbon fatty acyl group, 20-carbon fatty acyl group, 22-carbon fatty acyl group, 24-carbon fatty acyl group, 26-carbon fatty acyl group, 28-carbon fatty acyl group, or a 30-carbon fatty acyl group. In exemplary aspects, the fatty acyl acyl group is a 8-carbon to 20-carbon fatty acyl group or a 12-carbon to 20-carbon fatty acyl group, or a 14-carbon to 18-carbon fatty acyl group. In exemplary aspects, the fatty acyl group is a 14-carbon fatty acyl group, a 16-carbon fatty acyl group, or an 18-carbon fatty acyl group. In exemplary aspects, fatty acyl group is linear or branched.
[0042] Accordingly, in exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to a C12-C20 fatty acyl group (e.g., a 12-carbon fatty acyl group, a 14-carbon fatty acyl group, 16-carbon fatty acyl group, 18-carbon fatty acyl group, 20-carbon fatty acyl group), as described in SEQ ID NO: 3. In exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 12-carbon (CI 2) fatty acyl group, as described in SEQ ID NO: 4. In exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 14-carbon (C14) fatty acyl group, as described in SEQ ID NO: 5. In exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 16-carbon (C16) fatty acyl group, as described in SEQ ID NO: 6. In exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 18-carbon (CI 8) fatty acyl group, as described in SEQ ID NO: 7. In exemplary aspects, the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 of which is covalently attached to 20-carbon (C20) fatty acyl group, as described in SEQ ID NO: 8. Accordingly, with regard to any of the methods provided herein, the method comprises administering to an adult human a peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
[0043] In exemplary aspects, the peptide present in the pharmaceutical compositions of the invention is a peptide of SEQ ID NO: 6:
Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser He Tyr Leu Asp Lys Gin Ala Ala Xaa Glu Phe
Val Asn Trp Leu Leu Ala Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser Lys wherein Xaa at position 2 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB);
wherein Xaa at position 20 is 2-aminoisobutyric acid or alpha-aminoisobutyric acid (AIB); wherein the Lys at position 40 of which is covalently attached to a 16-carbon fatty acyl group; and wherein the C -terminus is amidated;
(SEQ ID NO: 6). [0044] Peptide Concentration
[0045] In exemplary embodiments, the pharmaceutical composition comprises about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2, or a pharmaceutically acceptable salt thereof. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 9.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 8.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 7.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 6.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 5.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 4.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 3.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 1.0 mg/mL to about 2.0 mg/mL.
[0046] In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 2.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 3.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 4.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 5.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 6.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 7.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 8.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 9.0 mg/mL to about 10.0 mg/mL.
[0047] In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 2.5 mg/mL to about 7.5 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 4.0 mg/mL to about 6.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration of about 5.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration within the range of about 7.0 mg/mL to about 8.0 mg/mL. In exemplary aspects, the peptide is present in the
pharmaceutical composition at a concentration within the range of about 2.0 mg/mL to about 8.0 mg/mL, e.g., about 5.0 mg/mL to about 7.0 mg/mL. In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration of about 6.0 mg/mL.
[0048] In exemplary aspects, the peptide is present in the pharmaceutical composition at a concentration of about 1.0 mg/mL, about 1.5 mg/mL, about 2.0 mg/mL, about 2.5 mg/mL, about 3.0 mg/mL, about 3.5 mg/mL, about 4.0 mg/mL, about 4.5 mg/mL, about 5.0 mg/mL, about 5.5 mg/mL, about 6.0 mg/mL, about 6.5 mg/mL, about 7.0 mg/mL, about 8.0 mg/mL, about 8.5 mg/mL, about 9.0 mg/mL, about 9.5 mg/mL, or about 10.0 mg/mL.
[0049] In exemplary aspects, the peptide is present in the pharmaceutical composition for use at a once daily dosage of from about 0.5 mg to about 3.0 mg. In exemplary aspects, the peptide is present in the pharmaceutical composition for use at a once daily dosage of from about 0.75 mg to about 2.5 mg, about 0.75 mg to about 2.0 mg, or about 1.0 mg to about 2.0 mg, or about 1.8 mg, or about 1.5 mg. Corresponding methods of administering the peptide at this once daily dosage are also provided.
[0050] Salts
[0051] In exemplary aspects, the peptide is in the form of a salt, e.g., a pharmaceutically acceptable salt. As used herein the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the analog, or separately prepared by reacting a free base function with a suitable acid. Many of the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
[0052] Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2 -hydroxy ethansulfonate (isothionate), lactate, maleate, methane sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include, for example, an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoric acid, and an organic acid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.
[0053] Basic addition salts also can be prepared in situ during the final isolation and purification, or by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium,
triethylammonium, diethylammonium, and ethylammonium, amongst others. Other
representative organic amines useful for the formation of base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.
[0054] Further, basic nitrogen-containing groups can be quaternized with the analog of the present disclosure as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
[0055] Phenol
[0056] In exemplary aspects, the pharmaceutical composition comprises phenol. In exemplary aspects, the pharmaceutical composition comprises about 5.0 mg/mL to about 6.0 mg/mL phenol. In exemplary aspects, the pharmaceutical composition comprises about 5.25 mg/mL to about 5.75 mg/mL phenol. In exemplary aspects, phenol is present in the
pharmaceutical composition at a concentration of about 5.5 mg/mL. In exemplary aspects, the pharmaceutical composition comprises an aqueous solution and the aqueous solution comprises (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, and (iii) water.
[0057] Histidine
[0058] In exemplary aspects, the pharmaceutical composition comprises Histidine, or a pharmaceutically acceptable salt thereof. In exemplary aspects, the Histidine is L-Histidine. In exemplary aspects, the Histidine is present in the pharmaceutical composition at a concentration within the range of about 15 mM to about 25 mM or about 17.5 mM to about 22.5 mM. In exemplary aspects, the pharmaceutical composition comprises a concentration of about 20 mM L-Histidine
[0059] In exemplary aspects, the Histidine is provided as a combination of L-Histidine base and Histidine HCl. In exemplary aspects, the pharmaceutical composition comprises a combination of L-Histidine base and L-Histidine HCl. In exemplary aspects, the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 15 mM to about 25 mM. In exemplary aspects, the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 17.5 mM to about 22.5 mM. In exemplary aspects, the total concentration of L-Histidine base and L-Histidine HCl in the pharmaceutical composition is about 20 mM.
[0060] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (iv) water.
[0061] Trehalose
[0062] In exemplary aspects, the pharmaceutical composition comprises trehalose. In exemplary aspects, the trehalose is present in the pharmaceutical composition at a concentration within the range of about 200 mM to about 300 mM. In exemplary aspects, the trehalose is present in the pharmaceutical composition at a concentration within the range of about 225 mM to about 275 mM. In exemplary aspects, the pharmaceutical composition comprises about 250 mM trehalose.
[0063] Exemplary Pharmaceutical Compositions and Aqueous Solutions
[0064] In exemplary aspects, the pharmaceutical composition consists of a sterile, isotonic solution comprising the peptide, phenol, trehalose, L-Histidine base, Histidine HCl, and water, and the pH of the isotonic solution is about 7.0. In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water. In exemplary aspects, the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water. In exemplary aspects, the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the composition, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg. In exemplary aspects, the pharmaceutical composition is for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human or for use in reducing weight gain or inducing weight loss in an adult human. In exemplary aspects, the pharmaceutical composition is for administration once daily in an amount of about 0.5 to 3.0 mg of the peptide.
[0065] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water. In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 6.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and water.
[0066] In exemplary aspects, the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration within about 1.0 to about 10.0 mg/mL. In exemplary aspects, the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the composition, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg. In exemplary aspects, the pharmaceutical composition is for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human or for use in reducing weight gain or inducing weight loss in an adult human. In exemplary aspects, the pharmaceutical composition is for administration once daily in an amount of about 0.5 to 3.0 mg of the peptide.
[0067] In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water. In exemplary aspects, the pharmaceutical composition is provided as a multi-use dosage form comprising multiple doses of the
composition, as described herein, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg.
[0068] Also provided herein is an aqueous solution comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of phenol, trehalose, Histidine, and water. In exemplary aspects, the aqueous solution is sterile, isotonic and has a pH of about 7.0. In exemplary aspects, the aqueous solution comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.0 mg/mL to about 6.0 mg/mL phenol, (ii) about 200 mM to about 300 mM trehalose, (iii) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water. In exemplary aspects, the peptide is present in the aqueous solution at a concentration of about 1.0 mg/mL to about 10.0 mg/mL. In exemplary aspects, the aqueous solution comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM total Histidine as a combination of L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL. In exemplary aspects, the peptide is present in the aqueous solution at a concentration of about 6.0 mg/mL.
[0069] Multi-Use Dosage Forms
[0070] In exemplary embodiments, the pharmaceutical composition described herein is provided as a multi-use dosage form comprising multiple doses of the pharmaceutical composition. Accordingly, provided herein are multi-use dosage forms comprising multiple doses of a pharmaceutical composition described herein. In exemplary aspects, each dose of the multi-use dosage form comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2- 8, or a pharmaceutically acceptable salt thereof. In exemplary embodiments, each dose of the multi-use dosage form comprises about 0.75 to about 2.5 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof. In exemplary embodiments, each dose of the multi-use dosage form comprises about 1.0 to about 2.0 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof. In exemplary embodiments, each dose of the multi-use dosage form comprises about 1.8 mg of a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof. In exemplary aspects, the peptide of the multi-use dosage form is any one of the peptides described herein. See, e.g., the section entitled "Peptide to be administered."
[0071] In exemplary aspects, the multi-use dosage form provided herein comprises multiple doses of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 mg/mL to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HC1, and (v) water.
[0072] In exemplary aspects, the multi-use dosage form comprises, consists essentially of, or consists of multiple doses of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein each dose of the multi-use dosage form comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of the peptide (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HC1, and (v) water. In exemplary aspects of the multi-use dosage form, the peptide is in a solution comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL, optionally, wherein the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL
[0073] In exemplary aspects, the multi-use dosage form provided herein comprises multiple doses of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (v) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL. In exemplary aspects, the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL.
[0074] Containers Comprising the Pharmaceutical Composition
[0075] In exemplary embodiments, the pharmaceutical composition described herein is packaged in a container suitable for holding a pharmaceutical composition for injection. In exemplary aspects, the pharmaceutical composition is packaged or housed in a vial, tube, bottle, a single or multi-chambered pre-filled syringe, cartridge, infusion pump (external or
implantable), jet injector, pre-filled pen device and the like. In exemplary aspects, the container comprises a volume of the pharmaceutical composition which is suitable for a single injection. In exemplary aspects, the container comprises about 0.3 mL to about 0.5 mL. In alternative aspects, the container comprises a volume of the pharmaceutical composition which is suitable for multiple injections. In exemplary aspects, the container comprises about 3.0 mL to about 5.0 mL. Such containers are contemplated as part of the invention.
[0076] Provided herein are containers, pre-filled syringes, pen cartridges, and glass vials comprising one of the pharmaceutical compositions described herein. In exemplary aspects, the container, pre-filled syringe, pen cartridge, or glass vial comprises a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a
pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water, optionally, wherein the volume of the composition is about 0.2 mL to about 3.0 mL. In exemplary aspects, the container, pre-filled syringe, pen cartridge, or glass vial comprises a volume of the pharmaceutical composition which is suitable for multiple injections. In exemplary aspects, the multi-use container, multi- use pre-filled syringe, multi-use pen cartridge, or multi-use glass vial comprises about 3.0 mL to about 5.0 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or about 3.0 mL to about 5.0 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water.
[0077] In exemplary aspects, the container, syringe, pen cartridge, or glass vial is for single use. In exemplary aspects, the single-use container, single-use pre-filled syringe, single-use pen cartridge, or single-use glass vial comprises about 0.3 mL to about 0.5 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, or about 0.3 mL to about 0.5 mL of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a
pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water.
[0078] Provided herein is a pen cartridge for use in a pen injector comprising a
pharmaceutical composition described herein, or as manufactured by a method described herein, or a multi-use dosage form described herein, or an aqueous solution described herein. Also provided herein is a glass vial comprising a pharmaceutical composition described herein, or as manufactured by a method described herein, or a multi-use dosage form described herein, or an aqueous solution described herein. Further provided herein is a syringe comprising a pharmaceutical composition described herein, or as manufactured by a method described herein, or a multi-use dosage form described herein, or an aqueous solution described herein.
[0079] Methods of Manufacture
[0080] Further provided herein are methods of manufacture. In exemplary embodiments, the methods comprise steps that lead to the manufacture of the pharmaceutical compositions described herein. In exemplary embodiments, the method of manufacturing a pharmaceutical composition as described herein comprises admixing L-Histidine base, Histidine HCl, trehalose, and phenol with water, and then adding the peptide. In exemplary aspects, the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HCl, and (v) water, and the method of making the pharmaceutical composition comprises admixing L-Histidine base, Histidine HC1, trehalose, and phenol with water, and then adding the peptide. In exemplary aspects, the pharmaceutical composition is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water, and the method of making the pharmaceutical composition comprises admixing L-Histidine base, Histidine HC1, trehalose, and phenol with water, and then adding the peptide.
[0081] In exemplary embodiments, the methods comprise making a buffer, making an stock solution comprising the peptide, diluting the stock solution to make a bulk solution, filtering the bulk solution to make a sterile bulk solution and then aliquoting the sterile bulk solution into containers. In exemplary aspects, the buffer is made by admixing, per mL of buffer, about 2.811 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water. In exemplary aspects, the buffer is adjusted to a pH of about 7.0 ± 0.5. In exemplary aspects, the buffer is adjusted to a pH of about 7.0 ± 0.2. In exemplary aspects, the pH of the buffer is adjusted using 1 N NaOH or 1 N HC1. In exemplary aspects, the stock solution is made by adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer described above in a second container, and adding the contents of the second container to the first container, followed by mixing with a shaker-mixed, for example, a Turbula mixer.
[0082] In exemplary embodiments, the method comprises (i) admixing phenol, L-histidine, or a pharmaceutically acceptable salt thereof, and optionally, trehalose with water, to make a buffer, (ii) adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer, (iii) admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer of (i) in a second container, and (iv) adding the contents of the second container to the first container, followed by mixing with a Turbula mixer. In exemplary aspects, step (i) comprises admixing, per mL of buffer, about 2.81 1 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water. In exemplary aspects, wherein step (iv) produces a stock solution and the final concentration of the peptide in the stock solution is about 50 mg/mL.
[0083] In exemplary aspects, the method further comprises diluting the stock solution with buffer to a final peptide concentration within the range of about 1.0 mg/mL to about 10.0 mg/mL to make a bulk solution. In exemplary aspects, the method further comprises filtering the bulk solution through 0.22 μιη filters, thereby making the bulk solution a sterile bulk solution. In exemplary aspects, the method further comprises aseptically aliquoting the sterile bulk solution into a plurality of pen cartridges or a plurality of glass vials.
[0084] Methods of Use
[0085] The pharmaceutical compositions provided herein are used, for example, to improve or achieve glycemic control, reduce glycemic excursions, improve glucose tolerance, reduce glucose intolerance, treat (reduce) hyperglycemia, treat type 2 diabetes, preserve pancreatic beta-cell function, increase pancreatic beta-cell function, treat insulin resistance, exert an insulinotropic effect, treat (reduce) adiposity, normalize body fat distribution, treat (reduce) dyslipidemia, prevent weight gain, reduce weight gain, reduce food intake, reduce appetite, induce weight loss, treat obesity, treat fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and/or treat metabolic syndrome.
[0086] Thus, provided herein are methods of using the pharmaceutical composition for any one of the indications described herein. In exemplary embodiments, the methods comprise administering to the adult human a pharmaceutical composition as described herein. In exemplary embodiments, the methods comprise administering once daily to the adult human a pharmaceutical composition as described herein. In exemplary aspects, the pharmaceutical composition consists of a sterile, isotonic solution comprising the peptide, phenol, trehalose, L- Histidine base, Histidine HCl, and water, and the pH of the isotonic solution is about 7.0. In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base/Histidine HCl, and (v) water. In exemplary aspects, the pharmaceutical composition comprises, consists essentially of, or consists of (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM total Histidine as a combination of L- Histidine base/Histidine HCl, and water.
[0087] In exemplary aspects, a once daily dosage of about 0.50 mg to about 3.0 mg (e.g., about 0.75 mg to about 2.5 mg) of the peptide is administered to the adult human. In exemplary aspects, the pharmaceutical composition is administered via injection, preferably subcutaneous injection, e.g., with a pen injector or with a needle and syringe. In exemplary aspects, the pharmaceutical composition comprises a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, once daily at a dosage of about 0.75 mg to about 2.5 mg, or about 1.8 mg. In exemplary aspects, the method comprises administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, at a daily dosage of from about 0.75 mg to about 2.5 mg. In exemplary aspects, these methods comprise administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, at a daily dosage of from about 0.75 mg to about 2.0 mg, or about 1.0 mg to about 2.0 mg. In exemplary aspects, these methods comprise administering to an adult human in need thereof a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, at a daily dosage of about 1.8 mg, or about 1.5 mg. Preferably the peptide is
administered once daily.
[0088] Accordingly, provided herein are methods of improving glycemic control in an adult human, comprising administering to the adult human a pharmaceutical composition as described herein in an amount effective to improve glycemic control or treat diabetes, optionally type II diabetes. Also provided herein are methods of reducing weight gain or inducing weight loss in an adult human comprising administering to the adult human a pharmaceutical composition as described herein in an amount effective to reduce weight gain or induce weight loss or treat obesity in an adult human. In exemplary aspects, the pharmaceutical composition used in the method of improving glycemic control in an adult human or the method of reducing weight gain or inducing weight loss in an adult human is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of: (i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.0 mg/mL to about 6.0 mg/mL phenol, (iii) about 200 mM to about 300 mM trehalose, (iv) about 15 mM to about 25 mM total Histidine as a combination of L-Histidine base and Histidine HC1, and (v) water. In exemplary aspects, the pharmaceutical composition used in the method of improving glycemic control in an adult human or the method of reducing weight gain or inducing weight loss in an adult human is a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L- Histidine base/Histidine HC1, and (iv) water. [0089] In exemplary aspects, a once daily dosage of about 0.5 mg to about 3.0 mg of the peptide is administered to the adult human. In exemplary aspects, the pharmaceutical composition is administered via injection.
[0090] Additional methods of use of the pharmaceutical compositions are contemplated herein.
[0091] As used herein, the term "treat," as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating obesity or hyperglycemia or diabetes of the invention can provide any amount or any level of treatment. Furthermore, the treatment provided by the method of the invention may include treatment of one or more conditions or symptoms or signs of the obesity or hyperglycemia or diabetes, being treated. Also, the treatment provided by the methods of the invention may encompass slowing the progression of the obesity or
hyperglycemia or diabetes. For example, the methods can treat obesity by virtue of reducing weight gain, inducing weight loss, reducing food intake, and the like. For example, the methods can treat hyperglycemia or diabetes by reducing blood glucose levels, or normalizing blood glucose levels, or improving or achieving glycemic control, or reducing glycemic excursions, or improving glucose tolerance (reducing glucose intolerance) or improving insulin resistance, preserving pancreatic beta-cell function, increasing pancreatic beta-cell function, exerting an insulinotropic effect and the like. Accordingly, the invention also provides methods of reducing weight gain or inducing weight loss, reducing blood glucose levels, normalizing blood glucose levels. The invention furthermore provides methods of normalizing body fat distribution, reducing food intake, reducing appetite, and the like.
[0092] In exemplary aspects, the methods of the invention reduce complications associated with obesity, including but not limited to vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia, dyslipidemia, nephropathy and/or musculoskeletal diseases. Accordingly, in exemplary aspects, the invention provides methods of preventing or delaying the onset of or reducing the risk of complications associated with obesity. In exemplary aspects, the invention provides a method of preventing vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases. In exemplary aspects, the invention provides a method of delaying the onset of vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases. In exemplary aspects, the invention provides a method of reducing the risk for vascular disease (coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, diabetes type II, hyperlipidemia, dyslipidemia and/or musculoskeletal diseases.
[0093] In exemplary aspects, the methods of the invention treat obesity-induced nephropathy. The method comprise administering to an adult human in need thereof a pharmaceutical composition described herein. Preferably the peptide of the pharmaceutical composition is administered once daily.
[0094] In exemplary aspects, the adult human being treated has or suffers from vascular disease (e.g., coronary artery disease, myocardial infarction, cerebral vascular disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia or dyslipidemia. In exemplary aspects, the adult human being treated has or suffers from other risk factors for cardiovascular disease, such as patients with insulin resistance or diabetes type II, hypertension, hyperlipidemia, or combinations of these risk factors. In exemplary aspects, the adult human in need thereof has or suffers from a drug -induced obesity.
[0095] In exemplary aspects, the diabetes being treated by the methods of the invention is diabetes mellitus type I, diabetes mellitus type II, or gestational diabetes, any of which may be insulin-dependent or non-insulin-dependent.
[0096] Furthermore, the treatment provided by the method of the invention may include treatment of one or more conditions or symptoms or signs of diabetes being treated.
[0097] In exemplary aspects, the methods of the invention cause an increase in insulin level, a decrease in glucose level, an increase in C-peptide level, a decrease in HbAic level, a decrease in fructosamine level, and combinations thereof. An increase in insulin level would indicate that administration of the peptide effectively is treating diabetes; and a decrease in glucose level would indicate that administration of the peptide is acting to reduce the levels of blood sugar, e.g., treating hyperglycemia.
[0098] HbAic levels depend on blood glucose concentration (i.e., the higher the glucose concentration in blood, the higher the level of HbAic), but are not influenced by daily fluctuations in the blood glucose concentration. Instead, they represent the average blood glucose level of approximately the past 4 weeks, strongly weighted toward the most recent 2 weeks. A decrease in HbAic level would indicate that administration of peptide is reducing the average blood glucose level over the long term.
[0099] C-peptide serves as a linker between the A- and B- chains of insulin and facilitates the efficient assembly, folding, and processing of insulin in the endoplasmic reticulum. High levels of C-peptide generally indicate high levels of endogenous insulin production, while low levels of C-peptide generally indicate low levels of insulin production. An increase in C-peptide level indicates that administration of the peptide is increasing the producing of insulin.
[00100] Fructosamine can be used to identify the plasma glucose concentration. In general, the higher the fructosamine concentration, the higher the average blood glucose level. Normal fructosamine levels may indicate that a patient is either not diabetic or that the patient has good diabetic control. An increase in fructosamine levels indicates that the patient's average glucose over the last 2 to 3 weeks has been elevated. A decrease in fructosamine level would indicate that administration of the peptide is reducing the average blood glucose level.
[00101] In related aspects, the invention provides methods of improving or achieving glycemic control, comprising administering to an adult human a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2-8, or a pharmaceutically acceptable salt thereof, once daily In exemplary aspects, the method comprises administering to an adult human in need thereof a pharmaceutical composition described herein. Preferably the peptide of the pharmaceutical composition is administered once daily.
[00102] In related aspects, the invention provides methods of treating diabetic nephropathy, comprising administering to an adult human a pharmaceutical composition described herein. Preferably the peptide of the pharmaceutical composition is administered once daily.
[00103] In related aspects, the invention provides methods of diabetic retinopathy, comprising administering to an adult human a pharmaceutical composition described herein. Preferably the peptide of the pharmaceutical composition is administered once daily.
[00104] In other related aspects, the invention provides methods of treating metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, Prader-Willi syndrome, and optionally Alzheimer's disease and Parkinson's disease.
[00105] It is understood that the corresponding compositions for use in the methods described herein are contemplated.
[00106] Combinations
[00107] In exemplary aspects of the methods of use provided herein, the pharmaceutical composition is administered alone, e.g., without any other therapeutic agent, without any other anti-diabetic agent and/or any other anti-obesity agent, including any of those listed below.
[00108] In alternative aspects, the pharmaceutical composition is administered in combination with an effective amount of one or more second therapeutic agents.
[00109] In exemplary aspects, the second therapeutic agent is an anti-diabetic agent, an anti- obesity agent, or a mixture thereof. Anti-diabetic agents known in the art or under investigation include insulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) inhibitors; glucokinase activators (GKA); glucagon receptor antagonists (GRA); or FBPase (fructose 1,6-bisphosphatase) inhibitors. For example, the peptides disclosed herein can be administered with insulin.
[00110] Anti-obesity agents known in the art or under investigation include, Leptin and Fibroblast Growth Factor 21 (FGF-21), appetite suppressants, such as phenethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide) or Contrave (bupropion and naltrexone); or lipase inhibitors, similar to xenical (Orlistat) or Cetilistat (also known as ATL-962), or GT 389-255.
[00111] With regard to any one of the inventive methods herein, the method comprises, in some aspects, administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin. In exemplary aspects, the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin to a patient with insufficient glycemic control. In exemplary aspects, the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin to a patient who has been administered the maximum tolerated dose of monotherapy with
Metformin or a sulphonylurea, yet the patient still exhibits insufficient glycemic control.
[00112] In exemplary aspects, the methods comprise administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent. In exemplary aspects, the methods comprise administering the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea,
thiazolidmedione, gliptin, gliflozin. In exemplary aspects, the method comprises administering to a patient with insufficient glycemic control the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea, thiazolidmedione, gliptin, gliflozin. In exemplary aspects, the method comprises administering to a patient who has been treated with dual therapy, yet the patient still exhibits insufficient glycemic control, the peptide of any one of SEQ ID NOs: 2-8 with Metformin and a third therapeutic agent selected from the group consisting of: sulphonylurea, thiazolidinedione, gliptin, gliflozin. Thiazolidinediones, also known as glitazones, are known in the art and include, e.g., rosiglitazone, pioglitazone, troglitazone, netoglitazone, rivoglitazone, ciglitazone. Gliptins, also known as inhibitors of dipeptidyl peptidase 4 or DPP-4 inhibitors, are known in the art, and include but not limited to, sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, alogliptin, anagliptin, berberine, and lupeol. Gliflozins are SGLT inhibitors (i.e., inhibitors of the SGLT2 glucose transporter) and include, but not limited to, dapagliflozin and sergliflozin.
[00113] With regard to any one of the inventive methods herein, the method comprises, in some aspects, administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea. In exemplary aspects, the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea to a patient with insufficient glycemic control. In exemplary aspects, the method comprises administering the peptide of any one of SEQ ID NOs: 2-8 with a sulfonylurea to a patient who has been administered the maximum tolerated dose of monotherapy with Metformin or a sulphonylurea, yet the patient still exhibits insufficient glycemic control.
Sulphonylureas are known in the art and include, but not limited to, carbutamide,
acetohexamide, tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (glibenclamide, Diabeta,
Micronase, Glynase), glimepiride (Amaryl), gliclazide (Diamicron), glibornuride, gliquidone, glisoxepide, and glyclopyramide.
[00114] It is understood that all description of therapeutic methods, pharmaceutical compositions, kits and other similar embodiments described herein contemplate that reference to the peptides include all pharmaceutically acceptable salts, esters, conjugates or prodrugs thereof. Specific embodiments of the invention are further described in the following, nonlimiting examples which illustrate various features. The examples should not be construed as limiting the scope of the invention, as many variations of these embodiments may be practiced and are understood in view of the entire disclosure herein.
EXAMPLES
EXAMPLE 1
[00115] This examples provides a method of making a peptide of SEQ ID NO: 2.
[00116] MBHA resin (4-methylbenzhydrylamine polystyrene resin was used during peptide synthesis. MBHA resin, 100-180 mesh, 1% DVB cross-linked polystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmoc protected amino acids were purchased from Midwest Biotech. The solid phase peptide syntheses using Boc-protected amino acids were performed on an Applied Biosystem 43 OA Peptide Synthesizer. Fmoc protected amino acid synthesis was performed using the Applied Biosystems Model 433 Peptide Synthesizer.
[00117] Peptide synthesis (Boc amino acids/ HF cleavage):
[00118] Synthesis of the peptide was performed on the Applied Biosystem Model 430A Peptide Synthesizer. Synthetic peptides were constructed by sequential addition of amino acids to a cartridge containing 2 mmol of Boc protected amino acid. Specifically, the synthesis was carried out using Boc DEPBT-activated single couplings. At the end of the coupling step, the peptidyl -resin was treated with TFA to remove the N-terminal Boc protecting group. It was washed repeatedly with dimethylformamide (DMF) and this repetitive cycle was repeated for the desired number of coupling steps. After the assembly, the sidechain protection, Fmoc, was removed by 20% piperidine treatment and acylation was conducted using DIC. The peptidyl- resin at the end of the entire synthesis was dried by using dichloromethane (DCM), and the peptide was cleaved from the resin with anhydrous HF.
[00119] After removal of the protecting groups and before HF cleavage, cyclization was performed as described previously (see, e.g., International Patent Application Publication No. WO2008/101017).
[00120] HF treatment of the peptidyl-resin
[00121] The peptidyl-resin was treated with anhydrous hydrogen fluoride (HF), and this typically yielded approximately 350 mg (-50% yield) of a crude deprotected-peptide.
Specifically, the peptidyl-resin (30 mg to 200 mg) was placed in the HF reaction vessel for cleavage. 500 μΐ, of p-cresol was added to the vessel as a carbonium ion scavenger. The vessel was attached to the HF system and submerged in the methanol/dry ice mixture. The vessel was evacuated with a vacuum pump and 10 ml of HF was distilled to the reaction vessel. This reaction mixture of the peptidyl-resin and the HF was stirred for one hour at 0° C, after which a vacuum was established and the HF was quickly evacuated (10-15 min). The vessel was removed carefully and filled with approximately 35 ml of ether to precipitate the peptide and to extract the p-cresol and small molecule organic protecting groups resulting from HF treatment. This mixture was filtered utilizing a teflon filter and repeated twice to remove all excess cresol. This filtrate was discarded. The precipitated peptide dissolves in approximately 20 ml of 10%> acetic acid (aq). This filtrate, which contained the desired peptide, was collected and lyophilized.
[00122] An analytical HPLC analysis of the crude solubilized peptide was conducted under the following conditions [4.6 X 30 mm Xterra C8, 1.50 mL/min, 220 nm, A buffer 0.1% trifiuoroacetic acid (TFA)/ 10% acrylonitrile (CAN), B buffer 0.1% TFA/ 100% ACN, gradient 5-95%B over 15 minutes]. The extract was diluted twofold with water and loaded onto a 2.2 X 25 cm Vydac C4 preparative reverse phase column and eluted using an acetonitrile gradient on a Waters HPLC system (A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% ACN and a gradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLC analysis of the purified peptide demonstrated greater than 95% purity and electrospray ionization mass spectral analysis was used to confirm the identity of the peptide.
[00123] Peptide Acylation
[00124] Acylated peptides were prepared as follows. Peptides were synthesized on a solid support resin using either a CS Bio 4886 Peptide Synthesizer or Applied Biosystems 430A Peptide Synthesizer. In situ neutralization chemistry was used as described by Schnolzer et al, Int. J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides, the target amino acid residue to be acylated was substituted with an N ε -FMOC lysine residue. Treatment of the completed N-terminally BOC protected peptide with 20% piperidine in DMF for 30 minutes removed FMOC/formyl groups. Coupling to a free ε-amino Lys residue can be achieved by coupling a ten-fold molar excess of either an FMOC-protected spacer amino acid (ex. FMOC- Glu-OtBu) or acyl chain (ex. CH3(CH2)i4-COOH)and PyBOP or DEPBT coupling reagent in DMF/N,N-diisopropylethylamine (DIEA). Subsequent removal of the spacer amino acid's FMOC group is followed by repetition of coupling with an acyl chain. Final treatment with 100% TFA resulted in removal of any side chain protecting groups and the N-terminal BOC group. Peptide resins were neutralized with 5% DIEA/DMF, dried, and then cleaved from the support using HF/p-cresol, 95:5, at 0°C for one hour. Following ether extraction, a 5% acetic acid (HO Ac) solution was used to solvate the crude peptide. A sample of the solution was then verified to contain the correct molecular weight peptide by ESI-MS. Correct peptides were purified by RP-HPLC using a linear gradient of 10% acetonitrile (CH3CN)/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm x 250 mm protein column was used for the purification. Acylated peptide analogs generally completed elution by a buffer ratio of 20:80. Portions were pooled together and checked for purity on an analytical RP-HPLC. Pure fractions were lyophilized yielding white, solid peptides.
[00125] Analysis using mass spectrometry
[00126] The mass spectra were obtained using a Sciex API-Ill electrospray quadrapole mass spectrometer with a standard ESI ion source. Ionization conditions that were used are as follows: ESI in the positive-ion mode; ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing and curtain gas used was nitrogen flow rate of .9 L/min. Mass spectra were recorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwell time. The sample (about lmg/mL) was dissolved in 50%> aqueous acetonitrile with 1% acetic acid and introduced by an external syringe pump at the rate of 5 μΕ/ηιίη. [00127] When the peptides were analyzed in PBS solution by ESI MS, they were first desalted using a ZipTip solid phase extraction tip containing 0.6 μΐ, C4 resin, according to instructions provided by the manufacturer (Millipore Corporation, Billerica, MA, see the Millipore website of the world wide web at millipore.com/catalogue.nsf/docs/C5737).
[00128] High Performance Liquid Chromatography (HPLC) analysis:
[00129] Preliminary analyses were performed with these crude peptides to get an
approximation of their relative conversion rates in Phosphate Buffered Saline (PBS) buffer (pH, 7.2) using high performance liquid chromatography (HPLC) and MALDI analysis. The crude peptide samples were dissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of the resulting solution was stored in a 1.5 ml HPLC vial which was then sealed and incubated at 37 °C. Aliquots of ΙΟΟμΙ were drawn out at various time intervals, cooled to room temperature and analyzed by HPLC.
[00130] The HPLC analyses were performed using a Beckman System Gold Chromatography system using a UV detector at 214 nm. HPLC analyses were performed on a 150 mm x 4.6 mm C18 Vydac column. The flow rate was 1 ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent B contained 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to 70%B in 15 minutes). The data were collected and analyzed using Peak Simple Chromatography software.
EXAMPLE 2
[00131] A randomized, double-blind, placebo-controlled, multiple ascending dose study was performed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of a peptide of SEQ ID NO: 6 in adult patients with Type 2 diabetes, over 14 days of treatment. The patients were given doses of 0.25 mg, 0.75 mg, 1.5 mg, 2.0 mg, or 2.5 mg of peptide, or placebo, administered once daily by subcutaneous injection.
[00132] Patients included in the study had been diagnosed with Type 2 diabetes according to WHO criteria at least 3 months prior to screening, were on a stable dose of metformin for at least 2 months prior to screening, and exhibited evidence of insulin secretory capacity (e.g., fasting serum C-peptide levels above 1.5 ng/mL). In addition, fasting plasma glucose prior to the study during was < 240 mg/dL and hemoglobin Ale (HbAlC) level was > 6.5%> and < 10.5%. Patients were randomized in a 3: 1 ratio to receive either active drug or placebo as daily subcutaneous injection into the abdomen for 14 days.
[00133] Therapeutic effect
[00134] Based on the data generated from this study, the therapeutic dose range for effects on glucose control in Type 2 diabetic patients ranged from 0.75 mg to 2.5 mg. This dose range is based on efficacy assessments showing that 0.25 mg had effects that were no different from effects seen with placebo. In contrast, the doses ranging from 0.75 mg to 2.5 mg had
meaningful decreases in both fasting plasma glucose and meal tolerance tests, as well as in HbAlc, after 14 days of treatment. All of these parameters indicate improved glycemic control and utility in treating diabetes. Hence, this data supports the use of peptide comprising SEQ ID NO: 6 in the treatment of diabetes by administering daily doses ranging from 0.75 mg to 2.5 mg.
[00135] Fasting plasma glucose
[00136] Fasting plasma glucose was monitored for the patients receiving the peptide of SEQ ID NO: 6. During a 14 day study, fasting plasma glucose is the best measure of efficacy. The mean change from baseline (i.e. predose on Day 1) in fasting plasma glucose over time and by dose group is depicted in FIGURE 1.
[00137] The effect of the 0.25 mg dose of peptide was no different from placebo. Fasting plasma glucose was slightly reduced by 20 mg/dL in both groups, most likely as a consequence of hospitalization and standardized diet. With the higher doses, there was a clear dose dependent reduction of fasting plasma glucose, with the maximum effect on fasting plasma glucose attained after 11 to 14 days of treatment. All of the doses from 0.75 through 2.5 mg produced meaningful decreases compared to placebo (as well as compared to the 0.25 mg dose). Doses of 0.75 mg, 1.5 mg, 2.0 mg and 2.5 mg achieved a fasting plasma glucose reduction of
approximately 39, 36, 42 and 65 mg/dL, respectively.
[00138] Meal tolerance test
[00139] A meal tolerance test, which assesses stimulated glucose responses, was also conducted for the patients receiving the peptide of SEQ ID NO: 6. Results of meal tolerance tests are shown in FIGURES 2A-2D for baseline, Day 1, Day 7 and Day 14.
[00140] The effect of the 0.25 mg dose of peptide was no different from placebo, after 14 days. In contrast, for the 0.75 mg dose and higher doses, there was a positive and dose- dependent decrease of postprandial plasma glucose concentration following the meal tolerance test. Cmax and AUCo_4h of glucose were clearly reduced with higher doses. After 2 weeks of treatment with 0.75, 1.5, 2.0 or 2.5 mg of peptide, the postprandial glucose Cmax was reduced by 20.6, 30.0, 24.8 and 38.1% relative to baseline, respectively, compared to a 5.3 or 8.4% reduction seen for placebo or 0.25 mg, respectively. Similar reductions from baseline were observed for glucose AUC0-4h: 25.4, 33.6, 32.1 and 40.1% reduction was observed for doses of 0.75, 1.5, 2.0 and 2.5 mg, respectively, compared to a reduction of 10.4 or 10.5% seen for placebo or 0.25 mg, respectively. [00141] HbAlc
[00142] Other parameters such a HbAlc, which acts as an integrated measure of daily glucose levels, require longer periods of treatment, usually between 6-12 weeks, to see meaningful and consistent effects.
[00143] A small HbAlc reduction (-0.23%) was seen with placebo, probably due to hospitalization and standardized diet. As observed for the fasting plasma glucose and meal tolerance test above, the effect at 0.25 mg (-0.09%>) was no different from placebo. Higher doses produced an approximately 2-fold greater reduction in HbAlc compared to placebo. HbAlc was reduced by 0.41, 0.50, 0.13 and 0.67%> (absolute value) after 2 weeks of treatment with 0.75, 1.5, 2.0 and 2.5 mg, respectively. The small sample size and the less advanced disease of patients in the 2.0 mg dose group may explain the relatively minor HbAlc decrease (-0.13%) seen in that group.
[00144] Pharmacokinetics
[00145] The pharmacokinetics of the peptide of SEQ ID NO: 6 were non-linear, with greater accumulation at steady state than predicted from single dose data. While the steady state exposures (Cmax and AUC) increased proportionally with doses from 0.25 to 0.75 mg, steady state exposure increased in a greater than dose-proportional manner between 0.75 and 1.5 mg, with mean Cmax and mean AUC increasing by approximately 5.1 and 5.8-fold, respectively, compared to the actual dose increase of only 2-fold. At higher doses, the steady state exposures increased again proportionally with doses between 1.5 and 2.5 mg.
[00146] The AUC and Cmax pharmacokinetic parameters also demonstrate that the ineffective dose of 0.25 mg is well distinguished from the effective dose of 0.75 mg. The AUC for the 0.75 mg dose was 65.1 ng*h/mL (CV: 28.1%) at Day 1, and 124 ng*h/mL (CV: 37.8%) at Day 14, compared to AUC for the 0.25 mg dose of 22.4 ng*h/mL (CV: 27.3%) at Day 1 and 35.5 ng*h/mL (CV: 23.3%) at Day 14. The Cmax for the 0.75 mg dose was 5.59 ng/niL (CV: 40.5%) at Day 1 and 8.46 ng/niL (CV: 35.0%) at Day 14, while Cmax for the 0.25 mg dose was 1.93 ng/niL (CV: 36.9%) at Day 1 and 2.59 ng/niL (CV: 32.2%) at Day 14.
[00147] Tolerability
[00148] No serious adverse events were experienced at any of the doses tested. The reports of side effects showed a tendency towards an increased rate of adverse events with increasing doses. Based on the tolerability profile of the drug, the dose of 2.5 mg is the highest desirable therapeutic dose to be administered. At this dose, 57%> of patients had nausea and/or diarrhea, 49% had headache, 43% had abdominal distension and 29% had constipation. Although most of these events were mild to moderate in severity, it is clear that doses higher than this would produce an adverse event profile that would not be acceptable for patients. [00149] Literature data in a study of 90 Type 2 diabetic patients has shown that treatment with currently marketed GLP-1 analogues or DPP-IV inhibitors has been associated with increased levels of serum lipase and/or amylase, with serum lipase levels increased more than serum amylase levels. For the peptide of SEQ ID NO: 6, abnormal treatment-emergent increases in lipase levels and amylase levels increased in a dose dependent manner. The largest increase of lipase levels was observed at the 2.5 mg dose (Table 1 below).
Table 1 Summary of Treatment Emergent Lipase and Amylase Elevation
Placebo 0.25 mg 0.75 mg 1.5 mg 2 mg 2.5 mg
(n=13) (n=8) (n=9) (n=9) (n=4) (n=7)
Number of patient(s) with treatment emergent elevation of lipase
Any > ULN 1 - 2 4 3 6
> ULN and < 3-fold the ULN 1 - 2 3 2 6
>3- and < 6.5-fold the ULN - 1 1 -
Number of patient(s) with treatment emergent elevation of amylase
> ULN and < 1.5-fold the ULN 2 1 2 -
Number of patient(s) with treatment emergent elevation of both lipase and amylase
Any > ULN - - 1 1 2 -
Lipase ULN=59 IU/L; Amylase ULN=124 IU/L
[00150] Although these increases were considered mild and are known findings with currently marketed GLP-1 analogues, the 2.5 mg dose would be considered the maximum dose that could be used.
[00151] All of the above data suggest that the range of doses for the peptide of SEQ ID NO: 6 that can be used in the treatment of patients with type 2 diabetes is from 0.75 mg to 2.5 mg once daily.
EXAMPLE 3
[00152] The human dose range of from 0.75 mg to 2.5 mg once daily was surprisingly about 10-fold higher than the doses that would have been predicted from animal studies disclosed in Int'l Patent Pub. No. WO/2010/011439.
[00153] The peptides of SEQ ID NOS: 5, 6, 7, which include varying lengths of fatty acyl chains, are all encompassed within SEQ ID NO: 2. In a first study, SEQ ID NOS: 5, 6, 7, liraglutide or a vehicle control were injected QD into mice at a dose of 25 or 125 nmol/kg for 7 days. The blood glucose levels of the mice were measured 0 and 7 days after injection with peptide or vehicle control and are shown in FIGURE 3. The effects of SEQ ID NOS: 5, 6 and 7 on blood glucose levels were dramatic. At 25 nmol/kg, these peptides caused about a 50% decrease in blood glucose levels.
[00154] In a second study, SEQ ID NO: 6 was tested at different doses in diet-induced obese (DIO) mice (N=8 per group; average initial body weight = 48 g). Mice were subcutaneously injected QD for one week with vehicle only, liraglutide (at 30 nmol/kg of body weight) or mt- 261 (at 0.3, 1, 3, 10 or 30 nmol/kg of body weight). Blood glucose levels of the mice were measured 0 and 7 days after the first injection and are shown in FIGURE 4.
[00155] Doses as low as 3 nmol/kg of SEQ ID NO: 6 caused a significant decrease in blood glucose levels. The decrease in blood glucose levels of mice injected with 3 nmol/kg SEQ ID NO: 6 was similar to the decrease of blood glucose levels of mice injected with 30 nmol/kg liraglutide, demonstrating the higher potency of SEQ ID NO: 6 as compared to liraglutide.
[00156] Liraglutide is an acylated peptide that is a GLP-1 agonist, like the peptide of SEQ ID NO: 2, that is not significantly different in molecular weight from SEQ ID NO: 2. Based on the rodent data in WO/2010/011439, it would have been predicted that the effective dose of SEQ ID NO: 2 in humans should be about 10-fold less than the effective dose of liraglutide. However, the data from human clinical studies described herein demonstrate that a surprisingly higher dose of peptides of SEQ ID NO: 2 is necessary to achieve a meaningful therapeutic effect compared to placebo.
EXAMPLE 4
[00157] Unless noted otherwise, the peptide referenced and used in this example was a peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 was covalently linked to a C 16 fatty acyl group.
[00158] Pharmaceutical compositions were subjected to studies to identify formulations that exhibit physical and chemical stability, comply with the required antimicrobial efficacy test(s), and exhibit low turbidity and sub-visible particle counts. Some studies related to pH/buffer, surfactant, and excipient.
[00159] The chemical stability of the peptide is influenced by pH. Therefore, the stability of the peptide in compositions comprising different buffer types (at a given physiological pH or within a physiological pH range) was tested. Briefly, pharmaceutical compositions comprising 5 mg/mL peptide, 230 mM trehalose, 0.04% (w/v) Polysorbate 20, 10 mM methionine, and 20 mM of one of 5 types of buffer were made. The pH of buffers ranged from 6.0 to 7.5 (pH 6.0, 6.5, 7.0, and 7.5). The pharmaceutical compositions were stored at different temperatures and the chemical degradation of the peptide of each pharmaceutical composition was measured via RP-HPLC at various time points. Chemical degradation of the peptide resulted in an increase in pre-peaks. Because the peptide in the Histidine -based buffers exhibited less degradation over all the pH and temperatures tested, Histidine was selected as the buffer of choice over four other buffer types. EXAMPLE 5
[00160] Unless noted otherwise in this example, the peptide referenced and used in this example was a peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the Lys at position 40 was covalently linked to a C16 fatty acyl group.
[00161] Pharmaceutical compositions comprising 0.0 mg/mL, 0.5 mg/mL, or 3 mg/mL peptide were subjected to turbidity studies with different preservatives. From these studies, it was determined that, for some preservatives, increasing the preservative concentration led to an increase in turbidity level. The increase in turbidity level was dependent upon the concentration of the peptide. The lower the peptide concentration, the higher the increase in turbidity level. If the concentration of the preservative is too low, however, the pharmaceutical composition will not pass the test for efficacy of antimicrobial preservation described in Section 5.1.3 of the European Pharmacopeia and also described in Example 7. See the data in Table 2 below. Thus, the selection of preservative, as well as the amount thereof, must be balanced between turbidity levels and efficacy of microbial preservation.
[00162] Pharmaceutical compositions comprising benzyl alcohol at 7.5 mg/mL or at 10 mg/mL were tested and were shown to exhibit low turbidity. These compositions, however, did not pass the test for efficacy of antimicrobial preservation described in Section 5.1.3 of the European Pharmacopeia and also described herein at Example 7. Briefly, the efficacy of antimicrobial preservation was tested for a pharmaceutical composition comprising (i) 3 mg/mL peptide, (ii) 20 mM His/His HCl, pH 7.0, (iii) 230 mM trehalose, (iv) 10 mM methionine, and (v) 10 mg/mL benzyl alcohol. The test for efficacy of antimicrobial preservation was carried out in accordance with Section 5.1.3 of the EP Pharmacopeia (see Example 7). The results of this test demonstrated that this pharmaceutical formulation failed to meet Level B Criteria of Acceptance. Adjustment of the benzyl alcohol concentration did not lead to a composition that could pass both the turbidity and antimicrobial testing.
[00163] In contrast to the benzyl alcohol-containing pharmaceutical compositions, a pharmaceutical composition comprising phenol met the Level B Criteria of Acceptance, when tested for efficacy of antimicrobial preservation, in accordance with Section 5.1.3 of the EP Pharmacopeia. This phenol-containing pharmaceutical composition comprised (i) 3 mg/mL peptide, (ii) 20 mM Histidine/Histidine HCl, pH 7.0, (iii) 240 mM trehalose, and (iv) 5 mg/mL phenol.
[00164] The use of phenol as a preservative was further analyzed by varying the amount of phenol. In this round of studies, each test composition comprised a total concentration of 20 mM Histidine base and Histidine HCl, pH 7.0, in addition to 250 mM trehalose, and 1 mg/mL, 6 mg/mL, or 10 mg/mL peptide. Each test composition comprised 4.5 mg/mL, 5.0 mg/mL, 5.5 mg/mL, or 6.0 mg/mL phenol. The pharmaceutical compositions were tested for efficacy of antimicrobial preservation, in accordance with Section 5.1.3 of the EP Pharmacopeia (see Example 7). The results of the assay are shown below in TABLE 2.
TABLE 2
Figure imgf000035_0001
[00165] These data suggest that 5.0 mg/mL phenol is the lowest concentration needed in order to meet Level B Criteria of Acceptance.
[00166] The study was repeated using phenol concentrations ranging from 3 mg/mL to 6 mg/mL and peptide concentrations ranging from 1 mg/mL to 10 mg/mL (1 mg/mL, 3 mg/mL, 6 mg/mL, and 10 mg/mL). The results of this study confirmed that 5.0 mg/mL phenol is the lowest concentration needed in order to meet Level B Criteria of Acceptance for all peptide concentrations tested.
EXAMPLE 6
[00167] The following provides an exemplary method of making a pharmaceutical composition of the invention. [00168] A buffer comprising the components shown in TABLE 3 was made by carrying out the following steps. The amount of each buffer component indicated in TABLE 3 was added to a first container:
TABLE 3
Figure imgf000036_0001
[00169] Water for injection was added to near final volume. The pH of the solution was adjusted with IN NaOH or IN HC1 to 7.0±0.2. Water for injection was subsequently added to final volume.
[00170] An API stock solution containing 50 mg/mL peptide (which peptide was the same as that described in Example 5) was made by first adding the amount of peptide (in powder form) into a glass bottle. The glass bottle was made to fit a Turbula mixer. The buffer described above was added into a separate container and additional IN NaOH was added to the buffer. The amount of IN NaOH added to the buffer was 0.55 mL for each g of peptide in the final API stock solution. The NaOH is to account for the pH drop observed when the peptide is dissolved in the buffer. The buffer (containing the additional NaOH) was added to the glass bottle containing the peptide in powder form, and the glass bottle was then placed onto a Turbula mixer. The contents of the glass bottle were mixed using the Turbula mixer. Once the contents of the glass bottle were sufficiently mixed, the solution was then diluted with the same buffer described above to obtain the desired peptide concentration. In one instance, the peptide concentration was 1.5 mg/mL ± 5% and in another instance, the peptide concentration was 7.5 mg/mL ± 5%. The pH of the diluted solution was then adjusted if it was outside of 7.0 ± 0.2. The diluted solution was then sterile filtered using 0.22 μιη low protein-binding filters. The sterile solution was then aseptically aliquoted into sterile glass vials closed with Teflon-coated rubber stoppers and alucrimp caps. The filled glass vials were then stored at a temperature between 2 °C and 8 °C. EXAMPLE 7
5.1.3. EFFICACY OF ANTIMICROBIAL
PRESERVATION
[00171] If a pharmaceutical preparation does not itself have adequate antimicrobial activity, antimicrobial preservatives may be added, particularly to aqueous preparations, to prevent proliferation or to limit microbial contamination which, during normal conditions of storage and use, particularly for multidose containers, could occur in a product and present a hazard to the patient from infection and spoilage of the preparation. Antimicrobial preservatives must not be used as a substitute for good manufacturing practice. The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation or by the formulation in which it is incorporated or by the container and closure used. The
antimicrobial activity of the preparation in its final container is investigated over the period of validity to ensure that such activity has not been impaired by storage. Such investigations may be carried out on samples removed from the final container immediately prior to testing. During development of a pharmaceutical preparation, it shall be demonstrated that the antimicrobial activity of the preparation as such or, if necessary, with the addition of a suitable preservative or preservatives provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use of the preparation. The efficacy of the antimicrobial activitymay be demonstrated by the test described below. The test is not intended to be used for routine control purposes.
TEST FOR EFFICACY OF ANTIMICROBIAL PRESERVATION
[00172] The test consists of challenging the preparation, wherever possible in its final container, with a prescribed inoculum of suitable micro-organisms, storing the inoculated preparation at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples so removed. The preservative properties of the preparation are adequate if, in the conditions of the test, there is a significant fall or no increase, as appropriate, in the number of micro-organisms in the inoculated preparation after the times and at the temperatures prescribed. The criteria of acceptance, in terms of decrease in the number of micro-organisms with time, vary for different types of preparations according to the degree of
protection intended (see Tables 5.1.3.-1/2/3). TEST MICROORGANISMS
Figure imgf000038_0001
METHOD
[00173] To count the viable micro-organisms in the inoculated products, use the agar medium used for the initial cultivation of the respective micro-organisms. Inoculate a series of containers of the product to be examined, each with a suspension of one of the test organisms to give an inoculum of lOsto 10e micro-organisms per millilitre or per gram of the preparation. The volume of the suspension of inoculum does not exceed 1 per cent of the volume of the product. Mix thoroughly to ensure homogeneous distribution. Maintain the inoculated product at 20-25 °C, protected from light. Remove a suitable sample from each container, typically 1 ml or 1 g, at zero hour and at appropriate intervals according to the type of the product and determine the number of viable micro-organisms by plate count or membrane filtration (2.6.12). Ensure that any residual antimicrobial activity of the product is eliminated by dilution, by filtration or by the use of a specific inactivator. When dilution procedures are used, due allowance is made for the reduced sensitivity in the recovery of small numbers of viable micro-organisms. When a specific inactivator is used, the ability of the system to support the growth of the test organisms is confirmed by the use of appropriate controls. The procedure is validated to verify its ability to demonstrate the required reduction in count of viable micro-organisms.
CRITERIA OF ACCEPTANCE
[00174] The criteria for evaluation of antimicrobial activity are given in Tables 5.1.3-1/2/3 in terms of the log reduction in the number of viable microorganisms against the value obtained for the inoculum. Table 5.1.3-1. - Parenteral and ophthalmic preparations
Figure imgf000039_0001
*NR - no recover; **NI - no increase
[00175] The A criteria express the recommended efficacy to be achieved. In justified cases where the A criteria cannot be attained, for example for reasons of an increased risk of adverse reactions, the B criteria must be satisfied.
EXAMPLE EMBODIMENTS OF THE INVENTION
1. A pharmaceutical composition comprising
(i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of any of SEQ ID NOs: 2, or a pharmaceutically acceptable salt thereof,
(ii) about 5.0 mg/mL to about 6.0 mg/mL phenol,
(iii) about 15 mM to about 25 mM Histidine,
(iv) water, and
(iv) optionally, trehalose.
2. The pharmaceutical composition of embodiment 1, wherein the peptide consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2.
3. The pharmaceutical composition ofembodiment 1 or 2, wherein the Lys at position 40 of the peptide is covalently attached to a C12-C30 fatty acyl group.
4. The pharmaceutical composition of any one of embodiments 1 to 3, wherein the fatty acyl group is a C14-C18 fatty acyl group.
5. The pharmaceutical composition of embodiment 4, wherein the Lys at position 40 of the peptide is covalently attached to a C16 fatty acyl group. 6. The pharmaceutical composition of any one of embodiments 1 to 5, wherein the peptide is present in the pharmaceutical composition at a concentration within the range of about 2.0 mg/mL to about 8.0 mg/mL or within the range of about 5.0 mg/mL to about 7.0 mg/mL.
7. The pharmaceutical composition of embodiment 6, wherein the peptide is present in the pharmaceutical composition at a concentration of about 6.0 mg/mL.
8. The pharmaceutical composition of any one of embodiments 1 to 7, wherein the phenol is present in the pharmaceutical composition at a concentration within the range of about 5.25 mg/mL to about 5.75 mg/mL.
9. The pharmaceutical composition of embodiment 8, wherein the phenol is present in the pharmaceutical composition at a concentration of about 5.5 mg/mL.
10. The pharmaceutical composition of any one of embodiments 1 to 9, comprising trehalose.
11. The pharmaceutical composition of embodiment 10, wherein the trehalose is present in the pharmaceutical composition at a concentration within the range of about 200 mM to about 300 mM.
12. The pharmaceutical composition of embodiment 11, wherein the trehalose is present in the pharmaceutical composition at a concentration within the range of about 225 mM to about 275 mM.
13. The pharmaceutical composition of embodiment 12, wherein the trehalose is present in the pharmaceutical composition at a concentration of about 250 mM.
14. The pharmaceutical composition of any one of embodiments 1 to 13, wherein the Histidine is provided as a combination of L-Histidine base and Histidine HC1.
15. The pharmaceutical composition of embodiment 14, wherein the combination of L- Histidine base and Histidine HC1 is present in the pharmaceutical composition at a total concentration within the range of about 15 mM to about 25 mM total Histidine.
16. The pharmaceutical composition of embodiment 15, wherein the combination of L- Histidine base and Histidine HC1 is present in the pharmaceutical composition at a total concentration within the range of about 17.5 mM to about 22.5 mM total Histidine.
17. The pharmaceutical composition of embodiment 16, wherein the combination of L- Histidine base and Histidine HC1 is present in the pharmaceutical composition at a total concentration of about 20mM total Histidine. 18. The pharmaceutical composition of any one of embodiments 1 to 17, wherein the pH of the pharmaceutical composition is within a range of about 6.0 and 8.0, optionally, within a range of about 6.5 to about 7.5.
19. The pharmaceutical composition of embodiment 18, wherein the pH of the
pharmaceutical composition is about 7.0.
20. The pharmaceutical composition of any one of embodiments 1 to 19, wherein the pharmaceutical composition lacks Polysorbate 20.
21. The pharmaceutical composition of any one of embodiments 1 to 20, wherein the pharmaceutical composition consists of a sterile, isotonic solution comprising the peptide, phenol, trehalose, L-Histidine base, Histidine HCl, and water, and the pH of the isotonic solution is about 7.0.
22. A pharmaceutical composition comprising, consisting essentially of, or consisting of:
(i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof,
(ii) about 5.0 mg/mL to about 6.0 mg/mL phenol,
(iii) about 200 mM to about 300 mM trehalose,
(iv) about 15 mM to about 25 mM total Histidine as a combination of L- Histidine base/Histidine HCl, and
(v) water.
23. A pharmaceutical composition comprising, consisting essentially of, or consisting of (i) about 6.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water.
24. A multi-use dosage form comprising, consisting essentially of, or consisting of multiple doses of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein each dose of the multi-use dosage form comprises, consists essentially of, or consists of (i) about 0.5 mg to about 3.0 mg of the peptide (ii) about 5.5 mg/mL phenol, (iii) about 250 mM trehalose, (iv) about 20 mM L-Histidine base/Histidine HCl, and (v) water.
25. The multi-use dosage form of embodiment 24, wherein the peptide is in a solution comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration of about 1.0 mg/mL to about 10 mg/mL, optionally, wherein the peptide is present in the solution at a concentration of about 4.0 mg/mL to about 8 mg/mL.
26. An aqueous solution comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, dissolved in a buffer, said buffer comprising, consisting essentially of, or consisting of (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HC1, and (iv) water, wherein the peptide is present in the aqueous solution at a concentration within a range of about 1.0 to about 10.0 mg/mL.
27. A method of manufacturing the pharmaceutical composition of any one of embodiments 1, 22, and 23.
28. The method of embodiment 27, comprising:
(i) admixing phenol, L-Histidine, or a pharmaceutically acceptable salt thereof, and optionally, trehalose, with water, to make a buffer
(ii) adding a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, to a first container suitable for use in a Turbula mixer,
(iii) admixing 0.55 mL 1 N NaOH per g of peptide of the first container with at least a portion of the buffer of (i) in a second container, and
(iv) adding the contents of the second container to the first container, followed by mixing with a Turbula mixer.
29. The method of embodiment 28, wherein step (i) comprises admixing, per mL of buffer, about 2.811 mg L-histidine base, about 0.394 mg Histidine HC1, about 94.575 mg trehalose, and about 5.5 mg phenol, with water.
30. The method of embodiment 28 or 29, wherein step (iv) produces a stock solution and the final concentration of the peptide in the stock solution is about 50 mg/mL.
31. The method of embodiment 30, comprising diluting the stock solution with buffer to a final peptide concentration within the range of about 1.0 mg/mL to about 10.0 mg/mL to make a bulk solution.
32. The method of embodiment 31 , comprising filtering the bulk solution through a sterilizing grade filter, thereby making the bulk solution a sterile bulk solution. 33. The method of embodiment 32, comprising aseptically aliquoting the sterile bulk solution into a plurality of pen cartridges or a plurality of glass vials.
34. A pen cartridge for use in a pen injector comprising the pharmaceutical composition of any one of embodiments 1 to 23, or as manufactured by the method of embodiment 31 , or the multi-use dosage form of embodiment 24 or 25, or the aqueous solution of embodiment 26.
35. A glass vial comprising the pharmaceutical composition of any one of embodiments 1 to 23, or as manufactured by the method of embodiment 31, or the multi-use dosage form of embodiment 24 or 25, or the aqueous solution of embodiment 26.
36. The pharmaceutical composition of any one of embodiments 1 to 23, the multi-use dosage form of embodiment 24 or 25, the aqueous solution of embodiment 26, the pen cartridge of embodiment 34, or the glass vial of embodiment 35, for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human.
37. The pharmaceutical composition of any one of embodiments 1 to 23, the multi-use dosage form of embodiment 24 or 25, the aqueous solution of embodiment 26, the pen cartridge of embodiment 32, or the glass vial of embodiment 35, for use in reducing weight gain or inducing weight loss in an adult human.
38. The pharmaceutical composition of any one of embodiments 1 to 23 for use with another anti-diabetic and/or anti-obesity agent, optionally for improving glycemic control, or treating diabetes, or treating type II diabetes, or for reducing weight gain or inducing weight loss.
39. The pharmaceutical composition of claim 38 wherein the anti-diabetic agent is selected from the group consisting of insulin, sulfonylureas, such as tolbutamide (Orinase),
acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) inhibitors; glucokinase activators (GKA); glucagon receptor antagonists (GRA); or FBPase (fructose 1,6-bisphosphatase) inhibitors; and/or the anti-obesity agent is selected from the group consisting of Leptin and Fibroblast Growth Factor 21 (FGF-21), appetite suppressants, such as phenethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide) or Contrave (bupropion and naltrexone); or lipase inhibitors, similar to xenical (Orlistat) or Cetilistat (also known as ATL- 962), or GT 389-255.
40. A method of improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human, comprising administering to the adult human a pharmaceutical composition of any one of embodiments 1 to 23 in an amount effective to improve glycemic control or treating diabetes, optionally type II diabetes.
41. A method of reducing weight gain or inducing weight loss in an adult human, comprising administering to the adult human a pharmaceutical composition of any one of embodiments 1 to 23 in an amount effective to reduce weight gain or induce weight loss in an adult human.
42. The method of embodiment 40 or 41, wherein a once daily dosage of about 0.5 mg to about 3.0 mg of the peptide is administered to the adult human.
43. The method of any of embodiments 40 to 42, wherein another anti-diabetic and/or anti- obesity agent is administered.
44. The method of any one of embodiments 40 to 43, wherein the pharmaceutical composition is administered via injection.
45. The method of embodiment 44, wherein the pharmaceutical composition is administered via injection with a pen injector or with a needle and syringe.
[00176] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[00177] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following embodiments) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.
[00178] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.
[00179] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise embodimented. No language in the specification should be construed as indicating any non- embodimented element as essential to the practice of the invention.
[00180] Preferred embodiments of this invention are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the embodiments appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A sterile pharmaceutical composition comprising, consisting essentially of, or consisting of:
(i) about 1.0 to about 10.0 mg/mL of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof,
(ii) about 5.0 mg/mL to about 6.0 mg/mL phenol,
(iii) about 200 mM to about 300 mM trehalose,
(iv) about 15 mM to about 25 mM total Histidine as a combination of L- Histidine base and Histidine HCl, and
(v) water.
2. A sterile pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in a solution comprising (i) about 5.5 mg/mL phenol, (ii) about 250 mM trehalose, (iii) about 20 mM L-Histidine base/Histidine HCl, and (iv) water, optionally, wherein the peptide is present in the solution at a concentration within about 1.0 to about 10.0 mg/mL.
3. A method of manufacturing the pharmaceutical composition of claim 1 or 2 comprising admixing, L-Histidine base, Histidine HCl, trehalose, and phenol with water, and then adding the peptide.
4. A container, pre-filled syringe, pen cartridge, or glass vial comprising the composition of claim 1 or 2.
5. A multi-use dosage form comprising multiple doses of the composition of claim 1 or 2, wherein each dose comprises a total amount of the peptide within the range of about 0.5 mg to about 3.0 mg.
6. The multi-use dosage form of claim 5, housed in a container, pre-filled syringe, pen cartridge, or glass vial .
7. The composition of claim 1 or 2 for use in improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human.
8. The composition of claim 1 or 2 for use in reducing weight gain or inducing weight loss in an adult human.
9. The composition of claim 6 or 7 for administration once daily in an amount of about 0.5 to 3.0 mg of the peptide.
10. A method of improving glycemic control or treating diabetes, optionally type II diabetes, in an adult human, comprising administering the composition of claim 1 or 2 in an amount effective to improve glycemic control or treat diabetes, optionally type II diabetes.
11. A method of reducing weight gain or inducing weight loss or treating obesity in an adult human, comprising administering to the adult human the composition of claim 1 or 2 in an amount effective to reduce weight gain or induce weight loss or treat obesity in an adult human.
12. The method of claim 9 or 10 wherein a once daily dosage of about 0.5 mg to about 3.0 mg of the peptide is administered to the adult human.
13. The method of any one of claims 9-11 wherein the pharmaceutical composition is administered via injection.
PCT/US2015/059719 2014-11-10 2015-11-09 Gip/glp-1 co-agonist peptides for human administration WO2016077220A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462077845P 2014-11-10 2014-11-10
US62/077,845 2014-11-10

Publications (1)

Publication Number Publication Date
WO2016077220A1 true WO2016077220A1 (en) 2016-05-19

Family

ID=55954893

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/059719 WO2016077220A1 (en) 2014-11-10 2015-11-09 Gip/glp-1 co-agonist peptides for human administration

Country Status (1)

Country Link
WO (1) WO2016077220A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018181864A1 (en) 2017-03-31 2018-10-04 Takeda Pharmaceutical Company Limited Gip receptor activating peptide
CN112912100A (en) * 2018-10-26 2021-06-04 诺和诺德股份有限公司 Stable semaglutide compositions and uses thereof
CN115151245A (en) * 2019-12-04 2022-10-04 罗斯制药有限责任公司 Stable liquid formulations comprising glucagon-like peptide 1 and uses thereof
US11779648B2 (en) 2020-07-22 2023-10-10 Novo Nordisk A/S Co-agonists at GLP-1 and GIP receptors suitable for oral delivery
US11840560B2 (en) 2022-01-20 2023-12-12 Novo Nordisk A/S Prodrugs and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010011439A2 (en) * 2008-06-17 2010-01-28 Indiana University Research And Technology Corporation Gip-based mixed agonists for treatment of metabolic disorders and obesity
US20130252893A1 (en) * 2010-11-03 2013-09-26 Arecor Ltd. Novel Composition
WO2014049610A2 (en) * 2012-09-26 2014-04-03 Cadila Healthcare Limited Peptides as gip, glp-1 and glucagon receptors triple-agonist
WO2014124096A1 (en) * 2013-02-06 2014-08-14 Perosphere Inc. Stable glucagon formulations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010011439A2 (en) * 2008-06-17 2010-01-28 Indiana University Research And Technology Corporation Gip-based mixed agonists for treatment of metabolic disorders and obesity
US20110166062A1 (en) * 2008-06-17 2011-07-07 Indiana University Research And Technology Corporation Gip-based mixed agonists for treatment of metabolic disorders and obesity
US20130252893A1 (en) * 2010-11-03 2013-09-26 Arecor Ltd. Novel Composition
WO2014049610A2 (en) * 2012-09-26 2014-04-03 Cadila Healthcare Limited Peptides as gip, glp-1 and glucagon receptors triple-agonist
WO2014124096A1 (en) * 2013-02-06 2014-08-14 Perosphere Inc. Stable glucagon formulations

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018181864A1 (en) 2017-03-31 2018-10-04 Takeda Pharmaceutical Company Limited Gip receptor activating peptide
US10435445B2 (en) 2017-03-31 2019-10-08 Takeda Pharmaceutical Company Limited Peptide compound
US11174301B2 (en) 2017-03-31 2021-11-16 Takeda Pharmaceutical Company Limited Peptide compound
CN112912100A (en) * 2018-10-26 2021-06-04 诺和诺德股份有限公司 Stable semaglutide compositions and uses thereof
CN115151245A (en) * 2019-12-04 2022-10-04 罗斯制药有限责任公司 Stable liquid formulations comprising glucagon-like peptide 1 and uses thereof
US11779648B2 (en) 2020-07-22 2023-10-10 Novo Nordisk A/S Co-agonists at GLP-1 and GIP receptors suitable for oral delivery
US11840560B2 (en) 2022-01-20 2023-12-12 Novo Nordisk A/S Prodrugs and uses thereof

Similar Documents

Publication Publication Date Title
AU2015276203B2 (en) Exendin-4 derivatives as selective glucagon receptor agonists
AU2015243611C1 (en) Dual GLP-1 / glucagon receptor agonists derived from exendin-4
JP5019466B2 (en) Glucagon-like peptide-2 analog
EP1478233B1 (en) Method for administering glp-1 molecules
WO2016077220A1 (en) Gip/glp-1 co-agonist peptides for human administration
KR102350489B1 (en) Stable insulin secretagogue peptide hydro-injection pharmaceutical composition
US7781401B2 (en) Glucagon-like peptide-2 analogs
JP6017754B2 (en) Glucagon / GLP-1 receptor co-agonist
US20160175400A1 (en) Methods for treating metabolic disorders and obesity with gip and glp-1 receptor-active glucagon-based peptides
TW200938225A (en) Ester-based peptide prodrugs
EP3129395A1 (en) Exendin-4 derivatives as peptidic dual glp-1 / glucagon receptor agonists
TW200922615A (en) Insulinotropic peptide derivative wherein its N-terminal amino acid is modified
AU2021229621B2 (en) Peptides as selective GIP receptor agonists
WO2015035419A1 (en) Dosages of gip/glp-1 co-agonist peptides for human administration
EP4267104A1 (en) Therapeutic peptide formulations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15859459

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15859459

Country of ref document: EP

Kind code of ref document: A1