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WO2012080362A1 - Preparation comprising insulin, nicotinamide and an amino acid - Google Patents

Preparation comprising insulin, nicotinamide and an amino acid Download PDF

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Publication number
WO2012080362A1
WO2012080362A1 PCT/EP2011/072809 EP2011072809W WO2012080362A1 WO 2012080362 A1 WO2012080362 A1 WO 2012080362A1 EP 2011072809 W EP2011072809 W EP 2011072809W WO 2012080362 A1 WO2012080362 A1 WO 2012080362A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
preparation according
insulin preparation
arginine
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2011/072809
Other languages
French (fr)
Inventor
Helle Birk Olsen
Svend Havelund
Ulla Ribel-Madsen
Jeppe Sturis
Helle Naver
Morten Schlein
Svend Ludvigsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP2013543772A priority Critical patent/JP2014501239A/en
Priority to AU2011343360A priority patent/AU2011343360A1/en
Priority to BR112013014856A priority patent/BR112013014856A2/en
Priority to KR1020137018191A priority patent/KR20140030125A/en
Priority to RU2013130374/15A priority patent/RU2013130374A/en
Priority to MX2013006174A priority patent/MX2013006174A/en
Priority to CA2821613A priority patent/CA2821613A1/en
Priority to EP11794776.2A priority patent/EP2651446A1/en
Priority to CN2011800602960A priority patent/CN103328006A/en
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of WO2012080362A1 publication Critical patent/WO2012080362A1/en
Priority to IL226336A priority patent/IL226336A0/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/465—Nicotine; Derivatives thereof
    • 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
    • A61K38/28—Insulins
    • 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
    • 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
    • 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
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to pharmaceutical preparations comprising an insulin com- pound, a nicotinic compound and an amino acid.
  • Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions.
  • Some of the commercially available insulin preparations comprising such rapid acting insulin analogues include NovoRapid® (preparation of B28Asp human insulin), Humalog® (preparation of B28LysB29Pro human insulin) and Apidra® (preparation of
  • insulin preparations of insulins are administered by subcutaneous injection.
  • the action profile of the insulin meaning the action of insulin on glucose metabolism as a function of time from injection.
  • the time of the onset, the maximum value and the total duration of action are important.
  • bolus insulins a variety of insulin preparations with different action profiles are desired and requested by the patients.
  • One patient may, on the same day, use insulin preparations with very different action profiles.
  • the action profile desired for example depends on the time of the day and the amount and composition of the meal eaten by the patient.
  • the chemical stability of the insulin preparations for example, due to the abundant use of pen-like injection devices such as devices which contain Penfill ® cartridges, in which an insulin preparation is stored until the entire cartridge is empty which may be at least 1 to 2 weeks for devices containing 1.5-3.0ml cartridges.
  • pen-like injection devices such as devices which contain Penfill ® cartridges
  • an insulin preparation is stored until the entire cartridge is empty which may be at least 1 to 2 weeks for devices containing 1.5-3.0ml cartridges.
  • covalent chemical changes in the insulin structure occur. This may lead to formation of molecules which may be less active and/or potentially immunogenic such as deamidation products and higher molecular weight transformation products (dimers, polymers).
  • dimers, polymers molecular weight transformation products
  • the physical stability of the insulin preparations since long term storage may eventually lead to formation of insoluble fibrils, which are biologically inactive and potentially immunogenic.
  • the invention relates to insulin preparations with favourable absorption rate and favourable chemical and physical stability.
  • the present invention relates to insulin preparations compris- ing human insulin and/or analogues thereof, nicotinamide or nicotinic acid and/or salts thereof and arginine.
  • the present invention relates to an insulin preparation comprising:
  • the present invention also contemplates a method for the treatment of diabetes mellitus in a subject or for reducing the blood glucose level in a subject comprising administering to a subject or mammal an insulin preparation according to the invention.
  • Figure 1 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37 ° C of preparations according to the present invention.
  • the letter A refers to a NovoRapid ® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 .
  • addition of nicotinamide leads to an increased formation of degradation products
  • the combined addition of nicotinamide, glutamic acid and arginine has a mostly similar degradation pattern, with lower formation of HMWP.
  • Figure 2 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37 ° C of preparations according to this invention.
  • the letter A refers to a NovoRapid ® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1.
  • the letter A refers to a NovoRapid ® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 .
  • the initial rate of plasma glucose lowering is faster for the preparation with addition of nicotinamide (preparation N) and even faster for a combination of nicotinamide and arginine (preparation M).
  • the letter A refers to a NovoRapid ® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 .
  • the initial rate of plasma glucose lowering is faster for a preparation with a combination of nicotinamide, arginine and glutamic acid (preparation L) and for a preparation with a combination of nicotinamide and arginine (preparation K).
  • the letter A refers to a NovoRapid ® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1.
  • the initial absorption rate of the insulin component of the preparations with nicotinamide preparation J
  • the combination of nicotinamide and ar- ginine preparation K
  • the combination of nicotinamide, arginine and glutamic acid preparation L
  • the letter A refers to a NovoRapid ® reference and number 11 corresponds to a insulin aspart preparation as described in Table 3 of Example 1 .
  • the initial rate of plasma glucose lowering is faster for a preparation with a combination of nicotinamide and arginine (preparation 11 ).
  • the letter A refers to a NovoRapid ® reference and number 11 corresponds to a insulin aspart preparation as described in Table 3 of Example 1 .
  • the initial absorption rate of the insulin component of the preparations with nicotinamide and arginine is markedly faster.
  • the absorption after subcutaneous injection of the insulin compound in the insulin preparations of the present invention was surprisingly found to be faster than that of the reference insulin preparations.
  • This property is useful for rapid-acting insulins, in particular in connection with a multiple injection regimen where insulin is given before each meal. With faster on- set of action, the insulin can conveniently be taken closer to the meal than with conventional rapid acting insulin solutions. Furthermore, a faster disappearance of insulin probably diminishes the risk of post-meal hypoglycaemia.
  • the insulin preparations of the present invention are rapid-acting insulin preparations comprising an insulin compound such as insulin aspart, a nicotinic compound, such as nicotinamide and the amino acid arginine.
  • the insulin preparations of the present invention may comprise further amino acids.
  • These insulin preparations have a rapid absorption profile that mimics normal physiology more closely than existing therapies.
  • the insulin preparations of the present invention have chemical and physical stability suitable for commercial pharmaceutical preparations.
  • the insulin preparations of the present invention provide fast-acting insulin preparations which are not only physically stable, but surprisingly also chemically stable.
  • the insulin preparations of the present invention provide an even faster onset of action compared with existing insulin therapies.
  • Such ultra-fast insulin preparations have the advantage of restoring first phase insulin release, injection convenience and shutting down hepatic glucose production.
  • the insulin preparations of the present invention have a favourable absorption rate from subcutis into plasma with an increase in initial absorption rate ranging from 1 .5 to 5 times, when compared to conventional preparations such as NovoRapid ® , as suggested by several PK PD experiments in pigs. This faster absorption rate may improve glycaemic control and convenience and may allow for a shift from pre-meal to post-meal dosing.
  • the present invention is based in part, on the surprising discovery that although, the addition of nicotinamide allows the increase in absorption rate, it also has a negative effect on chemical stability by significantly increasing the amount of HMWP.
  • the insulin preparations of the present invention have an improved chemical stability by addition of arginine, which is reflected in e.g. a reduction in the formation of dimers and polymers and desamido insulins after storage.
  • the insulin preparations of the present invention may furthermore also have improved physical stability, which may be useful for use in pumps.
  • the present invention provides an insulin preparation comprising an insulin compound according to the present invention which is present in a concentration from about 0.1 mM to about 10.0mM, and wherein said preparation has a pH from 3 to 8.5.
  • the preparation also comprises a nicotinic compound and arginine.
  • the preparation may further comprise protease inhibitor(s), metal ions, a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.
  • the metal ion is zinc, wherein zinc is added as zinc acetate or zinc chloride.
  • the insulin preparations comprise a human insulin, an analogue or combinations thereof, nicotinamide and/or nicotinic acid and/or salts thereof and arginine and/or salts thereof.
  • the insulin preparations according to the present invention comprise an aqueous solution of B28Asp human insulin, nicotinamide and arginine.
  • the content of B28Asp human insulin in the solutions of this invention may be in the range of 15 to 500 international units (IU)/ml, preferably in the range of 50 to 333 lU/ml, in preparations for injection. However, for other purposes of parenteral administration, the content of insulin compound may be higher.
  • IU international units
  • IU corresponds to 6 nmol.
  • insulin aspart refers to the human insulin analogue B28Asp human insulin.
  • onset refers to the time from injection until the PK curve shifts to an increase.
  • absorption rate refers to the slope of the PK curve.
  • an “insulin compound” according to the invention is herein to be understood as human insulin, an insulin analogue and/or any combination thereof.
  • human insulin as used herein means the human hormone whose structure and properties are well-known. Human insulin has two polypeptide chains that are con- nected by disulphide bridges between cysteine residues, namely the A-chain and the B- chain.
  • the A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 1 1 of the A-chain, the second between the cysteine in position 7 of the A- chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in po- sition 20 of the A-chain and the cysteine in position 19 of the B-chain.
  • the hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acids followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids.
  • Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting pep- tide from the A and B chains.
  • insulin analogue as used herein is meant a polypeptide derived from the primary structure of a naturally occurring insulin, for example that of human insulin, by mutation.
  • One or more mutations are made by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or by adding at least one amino acid res- idue.
  • the added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues.
  • an insulin analogue comprises less than 8 modifications (substitutions, deletions, additions and any combination thereof) relative to the parent insulin, alternatively less than 7 modifications relative to the parent insulin, alternatively less than 6 modi- fications relative to the parent insulin, alternatively less than 5 modifications relative to the parent insulin, alternatively less than 4 modifications relative to the parent insulin, alternatively less than 3 modifications relative to the parent insulin, alternatively less than 2 modifications relative to the parent insulin.
  • mutants in the insulin molecule are denoted stating the chain (A or B), the position, and the three letter code for the amino acid substituting the native amino acid.
  • desB30 or “B(1 -29)” is meant a natural insulin B chain or analogue thereof lacking the B30 amino acid residue, and by B28Asp human insulin is meant human insulin wherein the amino acid residue in position 28 of the B chain has been substituted with Asp.
  • insulin analogues are such wherein Pro in position 28 of the B chain is mutated with Asp, Lys, Leu, Val, or Ala and/or Lys at position B29 is mutated with Pro, Glu or Asp. Furthermore, Asn at position B3 may be mutated with Thr, Lys, Gin, Glu or Asp. The amino acid residue in position A21 may be mutated with Gly. The amino acid in position B1 may be mutated with Glu. The amino acid in position B16 may be mutated with Glu or His. Further examples of insulin analogues are the deletion analogues e.g.
  • insulin analogues where the B30 amino acid in human insulin has been deleted (des(B30) human insulin), insulin analogues wherein the B1 amino acid in human insulin has been deleted (des(B1 ) human insulin), des(B28-B30) human insulin and des(B27) human insulin.
  • Insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension such as with two arginine residues added to the C-terminal of the B-chain are also examples of insulin analogues. Further examples are insulin analogues comprising combinations of the mentioned mutations.
  • Insulin analogues wherein the amino acid in position A14 is Asn, Gin, Glu, Arg, Asp, Gly or His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations are further examples of insulin analogues.
  • Insulin analogues of human insu- lin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues are also examples of insulin analogues.
  • insulin analogues include, but are not limited to: DesB30 human insulin; AspB28 human insulin; AspB28,desB30 human insulin; LysB3,GluB29 human insulin; LysB28,ProB29 human insulin; GlyA21 ,ArgB31 ,ArgB32 human insulin; GluA14,HisB25 human insulin; HisA14,HisB25 human insulin; GluA14,HisB25,desB30 human insulin; HisA14, HisB25,desB30 human insulin; GluA14,HisB25,desB27,desB28,desB29,desB30 human insulin; GluA14,HisB25,GluB27,desB30 human insulin; GluA14,HisB16,HisB25,desB30 human insulin; HisA14,HisB16,HisB25,desB30 human insulin; HisA14,HisB16,HisB25,desB30 human insulin; HisA14,HisB16,HisB25,desB30
  • nicotinic compound includes nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
  • concentration of the nicotinic compound and/or salts thereof is in the range from about 1 mM to about 300mM or from about 5mM to about 200mM.
  • arginine or "Arg” includes the amino acid arginine and/or a salt thereof, e.g. arginine hydrochloride or arginine glutamate.
  • the insulin preparation comprises 1 to 100mM of arginine. In one embodiment, the insulin preparation comprises 1 to 20mM of arginine.
  • the insulin preparation comprises 20 to 90mM of arginine. In one embodiment, the insulin preparation comprises 30 to 85mM of arginine.
  • glutamic acid or "Glu” includes the aminoacid glutamic acid and/or a salt thereof.
  • pharmaceutical preparation or "insulin preparation” as used herein means a product comprising an insulin compound, i.e., a human insulin, an analogue thereof and/or combinations thereof and a nicotinic compound and an aminoacid, optionally together with other excipients such as preservatives, chelating agents, tonicity modifiers, bulking agents, stabilizers, antioxidants, polymers and surfactants, metal ions, oleaginous vehicles and proteins (e.g., human serum albumin, gelatine or proteins), said insulin preparation being useful for treating, preventing or reducing the severity of a disease or disorder by
  • an insulin preparation is also known in the art as a pharmaceutical preparation or pharmaceutical composition.
  • the buffer may be selected from the group consisting of, but not limited to, sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan (tris), bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, ethylendiamine or mixtures thereof.
  • tris tris(hydroxymethyl)-aminomethan
  • the insulin preparation of the present invention may further comprise other ingredients common to insulin preparations, for example zinc complexing agents such as citrate, and phosphate buffers.
  • zinc complexing agents such as citrate, and phosphate buffers.
  • Glycerol and/or mannitol and/or sodium chloride may be present in an amount corresponding to a concentration of 0 to 250mM, 0 to 200mM or 0 to 100mM.
  • Stabilizers, surfactants and preservatives may also be present in the insulin preparations of this invention.
  • the insulin preparations of the present invention may further comprise a
  • the preservative may be present in an amount sufficient to obtain a preserving effect.
  • the amount of preservative in an insulin preparation may be determined from e.g. literature in the field and/or the known amount(s) of
  • preservative in e.g. commercial products. Each one of these specific preservatives constitutes an alternative embodiment of the invention.
  • the use of a preservative in pharmaceutical preparations is described, for example in Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the preservative present in the insulin preparation of this invention may be as in the heretofore conventional insulin preparations, for example phenol, m-cresol and methylpara- ben.
  • the insulin preparation of the present invention may further comprise a chelating agent.
  • a chelating agent in pharmaceutical preparations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the insulin preparation of the present invention may further comprise a stabilizer.
  • stabilizer refers to chemicals added to polypeptide containing pharmaceutical preparations in order to stabilize the peptide, i.e. to increase the shelf life and/or in-use time of such preparations.
  • Remington The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the insulin preparation of the present invention may further comprise a surfactant.
  • surfactant refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, the head, and a fat-soluble (lipophilic) segment. Surfactants accumulate preferably at interfaces, which the hydrophilic part is orientated towards the wa- ter (hydrophilic phase) and the lipophilic part towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore, surfactants lower the surface tension of a liquid. Surfactants are also known as amphipathic compounds.
  • detergent is a synonym used for surfactants in general.
  • the use of a surfactant in pharmaceutical prepara- tions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the invention in a further embodiment relates to an insulin preparation comprising an aqueous solution of an insulin compound of the present invention, and a buffer, wherein said insulin compound is present in a concentration from 0.1 mM or above, and wherein said preparation has a pH from about 3.0 to about 8.5 at room temperature ( ⁇ 25°C).
  • the present invention also relates to methods for producing the insulin preparations of the invention.
  • the method for making insulin preparations of the invention comprises:
  • the method for making insulin preparations of the invention comprises:
  • the method for making insulin preparations of the invention comprises:
  • the insulin preparations of the present invention can be used in the treatment of diabetes by parenteral administration. It is recommended that the dosage of the insulin preparations of this invention which is to be administered to the patient be selected by a physician.
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • the insulin preparations containing the insulin compound of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.
  • Insulin preparations according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • topical sites for example, skin and mucosal sites
  • sites which bypass absorption for example, administration in an artery, in a vein, in the heart
  • absorption for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • the insulin preparation is an aqueous preparation, i.e. preparation comprising water.
  • Such preparation is typically a solution or a suspension.
  • the insulin preparation is an aqueous solution.
  • aqueous preparation is defined as a preparation comprising at least 50 %w/w water.
  • aqueous solution is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.
  • Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • the insulin preparations of this invention are well-suited for application in pen-like devices used for insulin therapy by injection.
  • the insulin preparations of the present invention can be used in pumps for insulin administration.
  • physical stability of the insulin preparation refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • Physical stability of the aqueous protein preparations is evaluated by means of visual inspec- tion and/or turbidity measurements after exposing the preparation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of the preparations is performed in a sharp focused light with a dark background.
  • the turbidity of the preparation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a preparation showing no turbidity corresponds to a visual score 0, and a preparation showing visual turbidity in daylight corresponds to visual score 3).
  • a preparation is classified physically unstable with respect to protein aggregation, when it shows visual turbidity in daylight.
  • the turbidity of the preparation can be evaluated by simple turbidity measurements well-known to the skilled person.
  • Physical stability of the aqueous protein preparations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein.
  • the probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein.
  • Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
  • chemical stability of the protein preparation refers to changes in the covalent protein structure leading to formation of chemical degradation prod- ucts with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure.
  • chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Increasing amounts of chemical degradation products is often seen during storage and use of the protein preparation.
  • Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydro- lysed to form a free carboxylic acid or asparaginyl residues to form an IsoAsp derivative.
  • the amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since HMWP products are potentially immunogenic and not biologically active, low levels of HMWP are advantageous.
  • stabilized preparation refers to a preparation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a preparation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.
  • diabetes or "diabetes mellitus” includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other states that cause hyperglycaemia.
  • the term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the cells of the body fail to respond appropriately to insulin thus preventing cells from absorbing glucose. As a result, glucose builds up in the blood.
  • Type 1 diabetes also called insulin-dependent diabetes mellitus (IDDM) and juvenile- onset diabetes, is caused by B-cell destruction, usually leading to absolute insulin deficiency.
  • IDDM insulin-dependent diabetes mellitus
  • juvenile- onset diabetes is caused by B-cell destruction, usually leading to absolute insulin deficiency.
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
  • NIDDM non-insulin-dependent diabetes mellitus
  • adult-onset diabetes is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
  • pharmaceutically acceptable means suited for normal pharmaceutical applications, i.e., not giving rise to any serious adverse events in patients.
  • treatment of a disease means the management and care of a patient having developed the disease, condition or disorder and includes treatment, prevention or alleviation of the disease.
  • the purpose of treatment is to combat the disease, condition or disorder.
  • Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complica- tions associated with the disease, condition or disorder, and prevention of the disease, condition or disorder.
  • a “critically ill patient”, as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, a patient who is being operated and where complications supervene, and a patient who has been operated in a vital organ within the last week or has been subject to major surgery within the last week.
  • the term a “critically ill patient”, as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to dis- ease or injury, or a patient who is being operated and where complications supervene.
  • a critically ill patient refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury.
  • these definitions apply to similar expressions such as "critical illness in a patient” and a "patient is critically ill”.
  • a criti- cally ill patient is a patient in need of cardiac surgery, cerebral surgery, thoracic surgery, abdominal surgery, vascular surgery, or transplantation, or a patient suffering from neurological diseases, cerebral trauma, respiratory insufficiency, abdominal peritonitis, multiple trauma or severe burns, or critical illness polyneuropathy.
  • anabolic means the set of metabolic pathways that construct molecules from smaller units. These reactions require energy.
  • One way of categorizing metabolic processes, whether at the cellular, organ or organism level is as 'anabolic' or as 'catabolic', which is the opposite.
  • Anabolism is powered by catabolism, where large molecules are broken down into smaller parts and then used up in respiration.
  • Many anabolic processes are powered by adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • Anabolic processes tend toward "building up" organs and tissues. These processes produce growth and differentiation of cells and increase in body size, a process that involves synthesis of complex molecules. Examples of anabolic processes include the growth and mineralization of bone and increases in muscle mass.
  • Endocrinologists have traditionally classified hormones as anabolic or catabolic, depending on which part of metabolism they stimulate.
  • the balance between anabolism and catabolism is also regulated by circadian rhythms, with processes such as glucose metabolism fluctuating to match an animal's normal periods of activity throughout the day.
  • Some examples of the "anabolic effects" of these hormones are increased protein synthesis from amino acids, increased appetite, increased bone
  • anabolic hormones stimulate the formation of muscle cells and hence cause an increase in the size of skeletal muscles, leading to increased strength.
  • an insulin analogue according to the invention is used as a medicament for delaying or preventing disease progression in type 2 diabetes.
  • the insulin preparation according to the invention is for use as a medicament for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other car- diovascular disorders is provided.
  • hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other car- diovascular disorders is provided.
  • a method for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, the method comprising administering to a patient in need of such treatment an effective amount for such treatment of an insulin preparation according to the invention, is provided.
  • the treatment with an insulin preparation according to the present invention may also be combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • a second or more pharmacologically active substances e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • the treatment with an insulin preparation according to the present invention may also be combined with bariatric surgery - a surgery that influences the glucose levels and/or lipid homeostasis such as gastric banding or gastric bypass.
  • polypeptides e.g., insulins
  • An insulin analogue according to the invention may for instance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well estab- lished techniques, see e.g. Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999.
  • the insulin analogue may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the insulin analogue in a suitable nutrient medium under conditions permitting the expression of the insulin analogue.
  • the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue, for instance by use of tRNA mutants.
  • the insulin analogues according to the invention are prepared analogously to the preparation of known insulin analogues.
  • a third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770).
  • the prior art discloses a number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741 188.
  • the insulin analogues are produced by expressing a DNA sequence encoding the insulin analogue in question in a suitable host cell by well known technique as disclosed in e.g. US 6500645.
  • the insulin analogue is either expressed directly or as a precursor molecule which has an N-terminal extension on the B-chain or a C-terminal extension on the B- chain.
  • the N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long.
  • the N-terminal extension is to be cleaved of in vitro after isolation from the culture broth and will therefore have a cleavage site next to B1 .
  • N-terminal extensions of the type suitable in the present invention are disclosed in US 5,395,922, and EP 765,395.
  • the C-terminal extension may have the function of protecting the mature insulin or insulin analogue molecule against intracellular proteolytic processing by host cell exoproteases.
  • the C-terminal extension is to be cleaved of either extra-cellularly in the culture broth by secreted, active carboxypeptidase or in vitro after isolation from the culture broth.
  • a method for producing mature insulin and insulin analogs with C-terminal extensions on the B-chain that are removed by carboxypetidase are disclosed in WO 08037735.
  • the target insulin product of the process may either be a two-chain human insulin or a two-chain human insulin analogue which may or may not have a short C- terminal extension of the B-chain. If the target insulin product will have no C-terminal extension of the B-chain, then said C-terminal extension should be capable of subsequently being cleaved off from the B-chain before further purification steps.
  • An insulin preparation comprising:
  • insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B28Asp human insulin, B3LysB29Glu human insulin or B28LysB29Pro human insulin.
  • insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B3LysB29Glu human insulin or B28LysB29Pro human insulin.
  • nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nia- cin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
  • nicotinic compound is selected from nicotinamide and nicotinic acid and/or salts thereof and/or any combination thereof.
  • nic- otinic compound is nicotinamide and/or salts thereof.
  • the nicotinic compound is present in a range selected from the following: 1 -300mM; 5-200mM; 40-120mM, 70-140mM or 80-130mM.
  • the insulin preparation according to any of the preceding embodiments comprising from about 1 mM to about 300mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 8mM to about 260mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 50mM to about 250mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 80mM to about 250mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 80mM to about 180mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 5mM to about 200mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 1 mM to about 150mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 5mM to about 20mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 20mM to about 120mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 40mM to about 120mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 20mM to about 40mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 60mM to about 80mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 70mM to about 140mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising from about 80mM to about 130mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 8mM, 30mM, 100mM or 130mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 8mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 30mM, 10OmM or 130mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 30mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 80mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 100mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 120mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 130mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 150mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 155mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 180mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising about 230mM of the nicotinic compound.
  • the insulin preparation according to any of the preceding embodiments comprising the following ranges of arginine compound: 1 -100mM, 5-120mM, 8-85mM, 20-90mM, 30- 90mM, 30-85mM, 30-60mM or 10-40mM.
  • the insulin preparation according to any of the preceding embodiments comprising the following ranges of arginine compound: 1 -120mM, 8-85mM or 1 -40mM.
  • the insulin preparation according to any of the preceding embodiments comprising from about 1 mM to about 120mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 1 mM to about 10OmM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 5mM to about 120mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 20mM to about 90mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 30mM to about 85mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 8mM to about 85mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 30mM to about 60mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 10mM to about 40mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 10mM to about 30mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 10mM to about 60mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising from about 1 mM to about 40mM of arginine.
  • arginine is present in a range selected from the following: 1 mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM,
  • the insulin preparation according to any of the preceding embodiments comprising about 1 mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 2mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 3mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 4mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 5mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 6mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 7mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 8mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 9mM of arginine.
  • the insulin preparation according to any of the preceding embodiments comprising about 10mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 1 1 mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 12mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 13mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 14mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 15mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 17mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 20mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 22mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 25mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 30mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 35mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 40mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 45mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 50mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 55mM of arginine.
  • the insulin preparation according o any o preceding embodiments comprising about 60mM of arginine.
  • the insulin preparation according to embodiment 100 comprising from about 1 mg/mL to about 20mg/ml_ of phosphate buffer.
  • the insulin preparation according to embodiment 100 comprising from about 1 mg/mL to about 15mg/ml_ of phosphate buffer.
  • the insulin preparation according to embodiment 100 comprising from about 1 mg/mL to about 10mg/mL of phosphate buffer.
  • the insulin preparation according to embodiment 100 comprising about 3mg/mL of phosphate buffer.
  • the insulin preparation according to embodiment 105 comprising from about 2mM to about 50mM of Tris.
  • the insulin preparation according to embodiment 105 comprising from about 10mM to about 40mM of Tris.
  • the insulin preparation according to embodiment 105 comprising from about 20mM to about 30mM of Tris.
  • the insulin preparation according to embodiment 105 comprising about 10mM, 20mM, 30mM or 40mM of Tris.
  • the insulin preparation according to embodiment 105 comprising about 10mM of Tris.
  • the insulin preparation according to embodiment 105 comprising about 20mM of Tris.
  • the insulin preparation according to embodiment 105 comprising about 30mM of Tris.
  • the insulin preparation according to embodiment 105 comprising about 40mM of Tris.
  • the insulin preparation according to any one of the preceding embodiments which further comprises a stabilizer(s).
  • the insulin preparation according to embodiment 140 comprising from about 5 to 100ppm, from about 10 to about 50ppm or from about 10 to about 20ppm of polysorbate.
  • the insulin preparation according to any of the preceding embodiments further comprising glycerol in the amount from about 0.5 to about 2.5%.
  • the insulin preparation according to any of the preceding embodiments further comprising glycerol in the amount from about 0.7 to about 2.0%.
  • the insulin preparation according to any of the preceding embodiments further comprising glycerol in the amount from about 1.0 to about 1 .5%.
  • the insulin preparation according to any of the preceding embodiments further comprising glycerol in the amount of about 1.25%.
  • a method of reducing the blood glucose level in mammals by administering to a patient in need of such treatment a therapeutically active dose of an insulin preparation ac- cording to any of the preceding embodiments.
  • a method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any of embodiments 1 -178.
  • the insulin preparation according to embodiment 182 for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns and operation wounds, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders.
  • An insulin preparation comprising:
  • nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
  • nicotinic compound is nicotinamide.
  • the insulin preparation according to embodiment 192 comprising from about 1 mM to about 250mM of the nicotinic compound.
  • the insulin preparation according to embodiment 192 comprising from about 1 mM to about 250mM of the nicotinic compound.
  • the insulin preparation according to embodiment 192 comprising from about 80mM to about 230mM of the nicotinic compound.
  • the insulin preparation according to any one of embodiments 185-195 comprising from about 10mM to about 60mM of arginine.
  • the insulin preparation according to any one of embodiments 185-196 comprising from about 10mM to about 30mM of arginine.
  • the insulin preparation according to any one of embodiments 185-200 which may further comprises preservative agent(s), isotonicity agent(s) and/or stabilizer(s).
  • the insulin preparation according to any one of embodiments 185-201 which further comprises a metal ion.
  • An insulin preparation comprising: B28Asp human insulin; nicotinamide; zinc; arginine; and a phosphate buffer.
  • the insulin preparation of embodiment 209 wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1 .2 mM, and wherein the nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and wherein the arginine is present in a concentration ranging from about 10 mM to about 40 mM, and wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules, and wherein the preparation has a pH of about 7.4 or less.
  • An insulin preparation consisting essentially of:
  • B28Asp human insulin wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1 .2 mM;
  • Nicotinamide wherein the nicotinamide is present in a concentration ranging from about 80 mM to abour 260 mM;
  • Zinc wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules;
  • Arginine wherein the the arginine is present in a concentration ranging from about 10 mM to about 30 mM;
  • a phosphate buffer wherein the preparation has a pH of about 7.1.
  • a method of reducing the blood glucose level in mammals by administering to a mammal in need of such treatment a therapeutically active dose of an insulin preparation according to any one of the preceding embodiments.
  • a method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any one of embodiments 1 -21 1.
  • An insulin preparation according to any one of embodiments 1 -21 1 for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treatment of critically ill diabetic and non-diabetic patients.
  • An insulin preparation according to any one of embodiments 1 -21 1 for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.
  • An insulin preparation comprising:
  • the insulin preparation according to embodiment 216, wherein the insulin com- pound is human insulin or an insulin analog.
  • the insulin preparation according to any one of embodiments 216-221 wherein the insulin compound is present in the amount from about 0.3mM to about 1.2mM. 223.
  • the insulin preparation according to any one of embodiments 216-223 comprising from about 1 mM to about 150mM of the nicotinic compound.
  • the insulin preparation according to any one of embodiments 216-224 comprising from about 1 mM to about 85mM of arginine.
  • the insulin preparation according to any one of 216-225 which further comprises a metal ion, preservative agent(s), isotonicity agent(s) and stabilizer(s) and buffer(s).
  • a method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any one of embodiments 216-226. 229.
  • the pharmaceutical preparations of the present invention may be formulated as an aqueous solution.
  • the aqueous medium is made isotonic, for example, with sodium chloride or glycerol.
  • the aqueous medium may contain zinc ions, for example added as zinc acetate or zinc chloride, buffers and preservatives.
  • Arginine may be added as Arg, HCI.
  • the pH value of the preparation is adjusted to the desired value and may be between about 3 to about 8.5, between about 3 and about 5 or about 6.5 and about 7.5 depending on the isoe- lectric point, pi, of the insulin in question.
  • HMWP high molecular weight protein
  • monomer insulin aspart was performed on Waters insulin (300 x 7.8mm, part nr wat 201549) with an eluent containing 2.5M acetic acid, 4mM L-arginine and 20 %(V/V) acetonitrile at a flow rate of 1 ml/min. and 40°C.
  • Detection was performed with a tuneable absorbance detector (Waters 486) at 276nm. Injection volume was 40 ⁇ and a 600 ⁇ human insulin standard.
  • HMWP and concentration of the preparations were measured at each sampling point.
  • Determination of the insulin aspart related impurities were performed on a UPLC system using a BEH RP C8 2.1 x 100mm column, particle size of 1 ,7 ⁇ . Waters part no 186002878. with a flow rate of 0,5ml/min., at 40° C detection at 220nm. Elution was performed with a mobile phase consisting of the following:
  • the amount of B28iso-aspartate, desamido and other related impurities were de- termined as absorbance area measured in percent of total absorbance area determined after elution of the preservatives.
  • the RP-UPLC method is equivalent to the analytical method used for quality control of Novo Nordisk marketed insulin aspart pharmaceuticals.
  • arginine reduces the amount of degradation products formed, especially HMWP and des-amido forms, increasing the concentration of arginine in the range 10 to 50mM leads to further reduction of degradation.
  • the physical stability measured as lag time in the ThT assay is reduced upon addition of arginine and is increasingly reduced when the arginine concentration is increased.
  • the overall performance of 50mM arginine is superior to 50 mM glycine, 50mM glutamic acid, or 50mM histidine regarding reduction of the formation of degradation products, as is shown in Table 4 below.
  • the insulin preparations of the present invention provide fast-acting insulin preparations which are not only physically stable, but surprisingly also chemically stable.
  • PK Pharmacokinetic
  • PD Pharmacodynamic
  • the PK/PD studies were performed on domestic female pigs, LYD cross-breed, weighing between 55 and 1 10kg.
  • the pigs were catheterised into the jugular vein through an ear vein at least 2 days before start of the study.
  • the last meal before the start of the study was served to the animals approx. 18 hours prior to the injection of the test preparation, and the animals had free access to water at all time during the fasting period and the test period.
  • the test preparation was given subcutaneous on the lateral side of the neck.
  • a blood sample was drawn prior dosing and at regular time intervals after dosing samples were drawn from the catheter and sampled into 1 .5ml glass tubes pre-coated with heparin.
  • the blood samples were kept in ice water until separation of plasma by centrifugation for 10min. 3000rpm at 4°C, which was done within the first 30 minutes.
  • Plasma samples were stored at 4°C for short time (2-3 hours) or at -18°C for long term storage and were analysed for glucose on YSI or Konelab 30i and for insulin Aspart concentration by LOCI.
  • LOCI Luminescent Oxygen Channeling Immunoassay
  • the insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay and applies the proximity of two beads, the europium-coated acceptor beads and the streptavidin coated donor-beads.
  • the acceptor beads were coated with a specific antibody against human insulin and recognize insulin Aspart in plasma samples.
  • a second biotinylated antibody bind specific to insulin Aspart and together with the streptavidin coated beads, they make up the sandwich. Illumination of the beads-aggregate-immunocomplex releases singlet oxygen from the donor beads which channels into the acceptor beads and triggers chemilumines- cence. The chemiluminescence was measured and the amount of light generated is proportional to the concentration of insulin Aspart.
  • Thioflavin T is such a probe and has a distinct fluorescence signature when binding to fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284].
  • the time course for fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001 ) Biochemistry 40, 6036-6046]: fr m f t
  • F is the ThT fluorescence at the time t.
  • t 0 is the time needed to reach 50% of maximum fluorescence.
  • Lag-time t 0 - 2 ⁇ Time Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and the fibrillation proceeds.
  • the lag-time corresponds to the interval in which the critical mass of nucleus is built up and the apparent rate constant is the rate with which the fibril itself is formed.
  • Samples were prepared freshly before each assay. Each sample composition is described in each example. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCI0 4 or HCI. Thioflavin T was added to the samples from a stock solution in H 2 0 to a final concentration of 1 ⁇ .
  • ThT fluorescence emis- sion Incubation at given temperature, shaking and measurement of the ThT fluorescence emis- sion were done in a Fluoroskan Ascent FL fluorescence platereader or Varioskan platereader (Thermo Labsystems). The temperature was adjusted to 37 °C. The orbital shaking was adjusted to 960rpm with an amplitude of 1 mm in all the presented data. Fluorescence measurement was done using excitation through a 444nm filter and measurement of emission through a 485nm filter.
  • the plate was measured every 20 minutes for a desired period of time. Between each measurement, the plate was shaken and heated as described.
  • the measurement points were saved in Microsoft Excel format for further processing and curve drawing and fitting was performed using GraphPad Prism.
  • the background emission from ThT in the absence of fibrils was negligible.
  • the data points are typically a mean of four or eight samples and shown with standard deviation error bars. Only data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a relative measure of fibrillation between experiments.
  • the data set may be fitted to Eq. (1 ). However, since full sigmodial curves are not always achieved during the measurement time, lag times were here visually determined from the ThT fluorescence curve as the time point at which the ThT fluorescence is different than the background level.
  • the peptide concentration in each of the tested formulations were measured both before application in the ThT fibrillation assay ("Initial") and after completion of the ThT fibrillation ("After ThT assay”). Concentrations were determined by reverse HPLC methods using a pramlin- tide standard as a reference. Before measurement after completion 150 ⁇ was collected from each of the replica and transferred to an Eppendorf tube. These were centrifuged at 30000 G for 40mins. The supernatants were filtered through a 0.22 ⁇ filter before application on the HPLC system.

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Abstract

Insulin preparations comprising an insulin compound or a mixture of two or more insulin compounds, a nicotinic compound and an amino acid.

Description

PREPARATION COMPRISING INSULIN, NICOTINAMIDE AND AN AMINO ACID
FIELD OF THE INVENTION
The present invention relates to pharmaceutical preparations comprising an insulin com- pound, a nicotinic compound and an amino acid.
BACKGROUND OF THE INVENTION
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions.
Since the introduction of insulin in the 1920's, continuous improvements have been made in the treatment of diabetes. To help avoid high glycaemia levels, diabetic patients often practice multiple injection therapy, whereby insulin is administered with each meal. As diabetic patients have been treated with insulin for several decades, there is a major need for safe and life-quality improving insulin preparations. Among the commercially available insulin preparations, rapid-acting, intermediate-acting and prolonged-acting preparations can be mentioned.
In the treatment of diabetes mellitus, many varieties of pharmaceutical preparations of insulin have been suggested and used, such as regular insulin (such as Actrapid®), isophane insulin (designated NPH), insulin zinc suspensions (such as Semilente®, Lente®, and Ul- tralente®), and biphasic isophane insulin (such as NovoMix®). Human insulin analogues and derivatives have also been developed, designed for particular profiles of action, i.e. fast action or prolonged action. Some of the commercially available insulin preparations comprising such rapid acting insulin analogues include NovoRapid® (preparation of B28Asp human insulin), Humalog® (preparation of B28LysB29Pro human insulin) and Apidra® (preparation of
B3LysB29Glu human insulin).
International applications WO 91/09617 and WO/9610417 (Novo Nordisk A/S) disclose insulin preparations containing nicotinamide or nicotinic acid or a salt thereof. European application EP1283051 allegedly discloses insulin preparation containing arginine, where arginine is used as buffering agent. EP1283051 allegedly discloses improvement of physical stability of insulin preparations.
Most often pharmaceutical preparations of insulins are administered by subcutaneous injection. Important for the patient is the action profile of the insulin, meaning the action of insulin on glucose metabolism as a function of time from injection. In this profile, inter alia, the time of the onset, the maximum value and the total duration of action are important. In the case of bolus insulins, a variety of insulin preparations with different action profiles are desired and requested by the patients. One patient may, on the same day, use insulin preparations with very different action profiles. The action profile desired for example, depends on the time of the day and the amount and composition of the meal eaten by the patient.
Equally important for the patient is the chemical stability of the insulin preparations, for example, due to the abundant use of pen-like injection devices such as devices which contain Penfill® cartridges, in which an insulin preparation is stored until the entire cartridge is empty which may be at least 1 to 2 weeks for devices containing 1.5-3.0ml cartridges. During storage, covalent chemical changes in the insulin structure occur. This may lead to formation of molecules which may be less active and/or potentially immunogenic such as deamidation products and higher molecular weight transformation products (dimers, polymers). Furthermore, also important is the physical stability of the insulin preparations, since long term storage may eventually lead to formation of insoluble fibrils, which are biologically inactive and potentially immunogenic.
SUMMARY OF THE INVENTION
The invention relates to insulin preparations with favourable absorption rate and favourable chemical and physical stability. The present invention relates to insulin preparations compris- ing human insulin and/or analogues thereof, nicotinamide or nicotinic acid and/or salts thereof and arginine.
In one embodiment, the present invention relates to an insulin preparation comprising:
• an insulin compound,
· a nicotinic compound, and
• arginine.
In another embodiment, the present invention also contemplates a method for the treatment of diabetes mellitus in a subject or for reducing the blood glucose level in a subject comprising administering to a subject or mammal an insulin preparation according to the invention.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37°C of preparations according to the present invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 . Compared to the NovoRapid® preparation (preparation A), addition of nicotinamide (preparations B and D) leads to an increased formation of degradation products, whereas the combined addition of nicotinamide, glutamic acid and arginine (preparations C and E), has a mostly similar degradation pattern, with lower formation of HMWP.
Figure 2 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37°C of preparations according to this invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1. The combined addition of nicotinamide, glutamic acid and arginine, preparations F, G, H, and I, differing in buffer system, phosphate or tris buffer, and concentration of insulin and Zn, 0.6mM and 0.3mM or 1 .2mM and 0.6mM, has a degradation pattern similar to the NovoRapid® preparation, preparation A.
Figure 3 shows the glucose concentration (mean +/- SEM, N=8) in plasma after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 . Compared to the NovoRapid® preparation (preparation A) the initial rate of plasma glucose lowering is faster for the preparation with addition of nicotinamide (preparation N) and even faster for a combination of nicotinamide and arginine (preparation M).
Figure 4 shows the glucose concentration in plasma (mean +/- SEM, N=7) after subcutane- ous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1 . Compared to the NovoRapid® preparation (preparation A), the initial rate of plasma glucose lowering is faster for a preparation with a combination of nicotinamide, arginine and glutamic acid (preparation L) and for a preparation with a combination of nicotinamide and arginine (preparation K).
Figure 5 shows the insulin aspart concentration in plasma (mean +/- SEM, N=7) after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1. Compared to the No- voRapid® preparation, (preparation A) , the initial absorption rate of the insulin component of the preparations with nicotinamide (preparation J), the combination of nicotinamide and ar- ginine (preparation K), and the combination of nicotinamide, arginine and glutamic acid (preparation L) is markedly faster.
Figure 6 shows the glucose concentration in plasma (mean +/- SEM, N=8, each pig dosed twice) after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this invention. The letter A refers to a NovoRapid® reference and number 11 corresponds to a insulin aspart preparation as described in Table 3 of Example 1 . Compared to the NovoRapid® preparation (preparation A), the initial rate of plasma glucose lowering is faster for a preparation with a combination of nicotinamide and arginine (preparation 11 ).
Figure 7 shows the insulin aspart concentration in plasma (mean +/- SEM, N=8, each pig dosed twice) after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of prepa- rations according to this invention. The letter A refers to a NovoRapid® reference and number 11 corresponds to a insulin aspart preparation as described in Table 3 of Example 1 . Compared to the NovoRapid® preparation (preparation A), the initial absorption rate of the insulin component of the preparations with nicotinamide and arginine (preparation 11 ) is markedly faster. DESCRIPTION OF THE INVENTION
The absorption after subcutaneous injection of the insulin compound in the insulin preparations of the present invention was surprisingly found to be faster than that of the reference insulin preparations. This property is useful for rapid-acting insulins, in particular in connection with a multiple injection regimen where insulin is given before each meal. With faster on- set of action, the insulin can conveniently be taken closer to the meal than with conventional rapid acting insulin solutions. Furthermore, a faster disappearance of insulin probably diminishes the risk of post-meal hypoglycaemia.
The insulin preparations of the present invention are rapid-acting insulin preparations comprising an insulin compound such as insulin aspart, a nicotinic compound, such as nicotinamide and the amino acid arginine. Optionally, the insulin preparations of the present invention may comprise further amino acids. These insulin preparations have a rapid absorption profile that mimics normal physiology more closely than existing therapies. Furthermore, the insulin preparations of the present invention have chemical and physical stability suitable for commercial pharmaceutical preparations. The insulin preparations of the present invention provide fast-acting insulin preparations which are not only physically stable, but surprisingly also chemically stable. The insulin preparations of the present invention provide an even faster onset of action compared with existing insulin therapies. Such ultra-fast insulin preparations have the advantage of restoring first phase insulin release, injection convenience and shutting down hepatic glucose production. The insulin preparations of the present invention have a favourable absorption rate from subcutis into plasma with an increase in initial absorption rate ranging from 1 .5 to 5 times, when compared to conventional preparations such as NovoRapid®, as suggested by several PK PD experiments in pigs. This faster absorption rate may improve glycaemic control and convenience and may allow for a shift from pre-meal to post-meal dosing. The present invention is based in part, on the surprising discovery that although, the addition of nicotinamide allows the increase in absorption rate, it also has a negative effect on chemical stability by significantly increasing the amount of HMWP. The insulin preparations of the present invention have an improved chemical stability by addition of arginine, which is reflected in e.g. a reduction in the formation of dimers and polymers and desamido insulins after storage. The insulin preparations of the present invention may furthermore also have improved physical stability, which may be useful for use in pumps.
The present invention provides an insulin preparation comprising an insulin compound according to the present invention which is present in a concentration from about 0.1 mM to about 10.0mM, and wherein said preparation has a pH from 3 to 8.5. The preparation also comprises a nicotinic compound and arginine. The preparation may further comprise protease inhibitor(s), metal ions, a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.
In one embodiment the metal ion is zinc, wherein zinc is added as zinc acetate or zinc chloride.
In one embodiment the insulin preparations comprise a human insulin, an analogue or combinations thereof, nicotinamide and/or nicotinic acid and/or salts thereof and arginine and/or salts thereof.
In one embodiment, the insulin preparations according to the present invention comprise an aqueous solution of B28Asp human insulin, nicotinamide and arginine.
The content of B28Asp human insulin in the solutions of this invention may be in the range of 15 to 500 international units (IU)/ml, preferably in the range of 50 to 333 lU/ml, in preparations for injection. However, for other purposes of parenteral administration, the content of insulin compound may be higher.
In the present context the unit "IU" corresponds to 6 nmol. The term "insulin aspart" refers to the human insulin analogue B28Asp human insulin.
The term "onset" refers to the time from injection until the PK curve shifts to an increase.
The term "absorption rate" refers to the slope of the PK curve.
An "insulin compound" according to the invention is herein to be understood as human insulin, an insulin analogue and/or any combination thereof.
The term "human insulin" as used herein means the human hormone whose structure and properties are well-known. Human insulin has two polypeptide chains that are con- nected by disulphide bridges between cysteine residues, namely the A-chain and the B- chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 1 1 of the A-chain, the second between the cysteine in position 7 of the A- chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in po- sition 20 of the A-chain and the cysteine in position 19 of the B-chain.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acids followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting pep- tide from the A and B chains.
By "insulin analogue" as used herein is meant a polypeptide derived from the primary structure of a naturally occurring insulin, for example that of human insulin, by mutation. One or more mutations are made by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or by adding at least one amino acid res- idue. The added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues.
In one embodiment an insulin analogue comprises less than 8 modifications (substitutions, deletions, additions and any combination thereof) relative to the parent insulin, alternatively less than 7 modifications relative to the parent insulin, alternatively less than 6 modi- fications relative to the parent insulin, alternatively less than 5 modifications relative to the parent insulin, alternatively less than 4 modifications relative to the parent insulin, alternatively less than 3 modifications relative to the parent insulin, alternatively less than 2 modifications relative to the parent insulin.
Mutations in the insulin molecule are denoted stating the chain (A or B), the position, and the three letter code for the amino acid substituting the native amino acid. By "desB30" or "B(1 -29)" is meant a natural insulin B chain or analogue thereof lacking the B30 amino acid residue, and by B28Asp human insulin is meant human insulin wherein the amino acid residue in position 28 of the B chain has been substituted with Asp.
Examples of insulin analogues are such wherein Pro in position 28 of the B chain is mutated with Asp, Lys, Leu, Val, or Ala and/or Lys at position B29 is mutated with Pro, Glu or Asp. Furthermore, Asn at position B3 may be mutated with Thr, Lys, Gin, Glu or Asp. The amino acid residue in position A21 may be mutated with Gly. The amino acid in position B1 may be mutated with Glu. The amino acid in position B16 may be mutated with Glu or His. Further examples of insulin analogues are the deletion analogues e.g. analogues where the B30 amino acid in human insulin has been deleted (des(B30) human insulin), insulin analogues wherein the B1 amino acid in human insulin has been deleted (des(B1 ) human insulin), des(B28-B30) human insulin and des(B27) human insulin. Insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension such as with two arginine residues added to the C-terminal of the B-chain are also examples of insulin analogues. Further examples are insulin analogues comprising combinations of the mentioned mutations. Insulin analogues wherein the amino acid in position A14 is Asn, Gin, Glu, Arg, Asp, Gly or His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations are further examples of insulin analogues. Insulin analogues of human insu- lin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues are also examples of insulin analogues.
Further examples of insulin analogues include, but are not limited to: DesB30 human insulin; AspB28 human insulin; AspB28,desB30 human insulin; LysB3,GluB29 human insulin; LysB28,ProB29 human insulin; GlyA21 ,ArgB31 ,ArgB32 human insulin; GluA14,HisB25 human insulin; HisA14,HisB25 human insulin; GluA14,HisB25,desB30 human insulin; HisA14, HisB25,desB30 human insulin; GluA14,HisB25,desB27,desB28,desB29,desB30 human insulin; GluA14,HisB25,GluB27,desB30 human insulin; GluA14,HisB16,HisB25,desB30 human insulin; HisA14,HisB16,HisB25,desB30 human insulin;
HisA8,GluA14,HisB25,GluB27,desB30 human insulin;
HisA8,GluA14,GluB1 ,GluB16,HisB25,GluB27,desB30 human insulin; and
HisA8,GluA14,GluB16,HisB25,desB30 human insulin.
The term "nicotinic compound" includes nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof. According to the present invention, the concentration of the nicotinic compound and/or salts thereof is in the range from about 1 mM to about 300mM or from about 5mM to about 200mM.
The term "arginine" or "Arg" includes the amino acid arginine and/or a salt thereof, e.g. arginine hydrochloride or arginine glutamate.
In one embodiment, the insulin preparation comprises 1 to 100mM of arginine. In one embodiment, the insulin preparation comprises 1 to 20mM of arginine.
In one embodiment, the insulin preparation comprises 20 to 90mM of arginine. In one embodiment, the insulin preparation comprises 30 to 85mM of arginine.
The term "glutamic acid" or "Glu" includes the aminoacid glutamic acid and/or a salt thereof.
The term "pharmaceutical preparation" or "insulin preparation" as used herein means a product comprising an insulin compound, i.e., a human insulin, an analogue thereof and/or combinations thereof and a nicotinic compound and an aminoacid, optionally together with other excipients such as preservatives, chelating agents, tonicity modifiers, bulking agents, stabilizers, antioxidants, polymers and surfactants, metal ions, oleaginous vehicles and proteins (e.g., human serum albumin, gelatine or proteins), said insulin preparation being useful for treating, preventing or reducing the severity of a disease or disorder by
administration of said insulin preparation to a person. Thus, an insulin preparation is also known in the art as a pharmaceutical preparation or pharmaceutical composition.
The buffer may be selected from the group consisting of, but not limited to, sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan (tris), bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, ethylendiamine or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.
The insulin preparation of the present invention may further comprise other ingredients common to insulin preparations, for example zinc complexing agents such as citrate, and phosphate buffers.
Glycerol and/or mannitol and/or sodium chloride may be present in an amount corresponding to a concentration of 0 to 250mM, 0 to 200mM or 0 to 100mM.
Stabilizers, surfactants and preservatives may also be present in the insulin preparations of this invention. The insulin preparations of the present invention may further comprise a
pharmaceutically acceptable preservative. The preservative may be present in an amount sufficient to obtain a preserving effect. The amount of preservative in an insulin preparation may be determined from e.g. literature in the field and/or the known amount(s) of
preservative in e.g. commercial products. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical preparations is described, for example in Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
The preservative present in the insulin preparation of this invention may be as in the heretofore conventional insulin preparations, for example phenol, m-cresol and methylpara- ben.
The insulin preparation of the present invention may further comprise a chelating agent. The use of a chelating agent in pharmaceutical preparations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
The insulin preparation of the present invention may further comprise a stabilizer. The term "stabilizer" as used herein refers to chemicals added to polypeptide containing pharmaceutical preparations in order to stabilize the peptide, i.e. to increase the shelf life and/or in-use time of such preparations. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
The insulin preparation of the present invention may further comprise a surfactant. The term "surfactant" as used herein refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, the head, and a fat-soluble (lipophilic) segment. Surfactants accumulate preferably at interfaces, which the hydrophilic part is orientated towards the wa- ter (hydrophilic phase) and the lipophilic part towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore, surfactants lower the surface tension of a liquid. Surfactants are also known as amphipathic compounds. The term "detergent" is a synonym used for surfactants in general. The use of a surfactant in pharmaceutical prepara- tions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
In a further embodiment the invention relates to an insulin preparation comprising an aqueous solution of an insulin compound of the present invention, and a buffer, wherein said insulin compound is present in a concentration from 0.1 mM or above, and wherein said preparation has a pH from about 3.0 to about 8.5 at room temperature (~25°C). The present invention also relates to methods for producing the insulin preparations of the invention.
In one embodiment, the method for making insulin preparations of the invention comprises:
a) preparing a solution by dissolving the insulin compound or a mixture of insulin compounds in water or buffer;
b) preparing a solution by dissolving a divalent metal ion in water or buffer;
c) preparing a solution by dissolving a preservative in water or buffer;
d) preparing a solution by dissolving a tonicity agent in water or buffer;
e) preparing a solution by dissolving a surfactant and/or a stabilizer in water or buffer;
f) mixing solution a) and one or more of solutions b), c), d), and e);
Finally adjusting the pH of the mixture in f) to the desired pH followed by a sterile filtration.
In one embodiment, the method for making insulin preparations of the invention comprises:
a) preparing separate stock solutions of preservative agents, tonicity agent, metal ion salt, arginine or arginine salt and nicotinic compound;
b) preparing a solution by adding stock solution(s) of preservative or a mixture of preservatives to water or buffer;
c) adding stock solution of tonicity agent to solution b);
d) preparing a solution by dissolving the insulin compound or a mixture of insulin compounds in water or buffer; adding HCI to acidify this solution;
e) adding stock solution of metal ion salt to d);
f) adding e) to c) and stirring;
g) adding stock solution of arginine or arginine salt to f) and stirring;
h) adding stock solution of nicotinic compound to g) and adjusting pH with
NaOH/HCI to desired pH.
In one embodiment, the method for making insulin preparations of the invention comprises:
a) preparing separate stock solutions of preservative agents, tonicity agent, metal ion salt, and a mixed stock solution of Arginine or Arginine salt and nicotinic compound b) preparing a solution by adding stock solution(s) of preservative or a mixture of preservatives to water or buffer;
c) adding stock solution of tonicity agent to solution b);
d) preparing a solution by dissolving the insulin compound or a mixture of insulin compounds in water or buffer; adding HCI to acidify this solution;
e) adding stock solution of metal ion salt to d);
f) adding e) to c) and stirring;
g) adding mixed stock solution of Arginine or Arginine salt and nicotinic compound to f) and stirring;
h) adjusting pH of g) with NaOH/HCI to desired pH.
The insulin preparations of the present invention can be used in the treatment of diabetes by parenteral administration. It is recommended that the dosage of the insulin preparations of this invention which is to be administered to the patient be selected by a physician.
Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. As a further option, the insulin preparations containing the insulin compound of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.
Insulin preparations according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.
In one embodiment of the invention the insulin preparation is an aqueous preparation, i.e. preparation comprising water. Such preparation is typically a solution or a suspension. In a further embodiment of the invention the insulin preparation is an aqueous solution.
The term "aqueous preparation" is defined as a preparation comprising at least 50 %w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.
Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. In one embodiment, the insulin preparations of this invention are well-suited for application in pen-like devices used for insulin therapy by injection.
In one embodiment the insulin preparations of the present invention can be used in pumps for insulin administration.
The term "physical stability" of the insulin preparation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein preparations is evaluated by means of visual inspec- tion and/or turbidity measurements after exposing the preparation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of the preparations is performed in a sharp focused light with a dark background. The turbidity of the preparation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a preparation showing no turbidity corresponds to a visual score 0, and a preparation showing visual turbidity in daylight corresponds to visual score 3). A preparation is classified physically unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the preparation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein preparations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
The term "chemical stability" of the protein preparation as used herein refers to changes in the covalent protein structure leading to formation of chemical degradation prod- ucts with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Increasing amounts of chemical degradation products is often seen during storage and use of the protein preparation. Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydro- lysed to form a free carboxylic acid or asparaginyl residues to form an IsoAsp derivative. Other degradations pathways involves formation of high molecular weight products where two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. T.J. & Manning M.C., Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein preparation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since HMWP products are potentially immunogenic and not biologically active, low levels of HMWP are advantageous.
The term "stabilized preparation" refers to a preparation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a preparation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.
The term "diabetes" or "diabetes mellitus" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other states that cause hyperglycaemia. The term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the cells of the body fail to respond appropriately to insulin thus preventing cells from absorbing glucose. As a result, glucose builds up in the blood.
Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile- onset diabetes, is caused by B-cell destruction, usually leading to absolute insulin deficiency.
Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e., not giving rise to any serious adverse events in patients.
The term "treatment of a disease" as used herein means the management and care of a patient having developed the disease, condition or disorder and includes treatment, prevention or alleviation of the disease. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complica- tions associated with the disease, condition or disorder, and prevention of the disease, condition or disorder.
In its broadest sense, the term a "critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, a patient who is being operated and where complications supervene, and a patient who has been operated in a vital organ within the last week or has been subject to major surgery within the last week. In a more restricted sense, the term a "critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to dis- ease or injury, or a patient who is being operated and where complications supervene. In an even more restricted sense, the term a "critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury. Similarly, these definitions apply to similar expressions such as "critical illness in a patient" and a "patient is critically ill". Examples of a criti- cally ill patient is a patient in need of cardiac surgery, cerebral surgery, thoracic surgery, abdominal surgery, vascular surgery, or transplantation, or a patient suffering from neurological diseases, cerebral trauma, respiratory insufficiency, abdominal peritonitis, multiple trauma or severe burns, or critical illness polyneuropathy.
The term "anabolism" as used herein, means the set of metabolic pathways that construct molecules from smaller units. These reactions require energy. One way of categorizing metabolic processes, whether at the cellular, organ or organism level is as 'anabolic' or as 'catabolic', which is the opposite. Anabolism is powered by catabolism, where large molecules are broken down into smaller parts and then used up in respiration. Many anabolic processes are powered by adenosine triphosphate (ATP). Anabolic processes tend toward "building up" organs and tissues. These processes produce growth and differentiation of cells and increase in body size, a process that involves synthesis of complex molecules. Examples of anabolic processes include the growth and mineralization of bone and increases in muscle mass. Endocrinologists have traditionally classified hormones as anabolic or catabolic, depending on which part of metabolism they stimulate. The balance between anabolism and catabolism is also regulated by circadian rhythms, with processes such as glucose metabolism fluctuating to match an animal's normal periods of activity throughout the day. Some examples of the "anabolic effects" of these hormones are increased protein synthesis from amino acids, increased appetite, increased bone
remodeling and growth, and stimulation of bone marrow, which increases the production of red blood cells. Through a number of mechanisms anabolic hormones stimulate the formation of muscle cells and hence cause an increase in the size of skeletal muscles, leading to increased strength.
In another embodiment, an insulin analogue according to the invention is used as a medicament for delaying or preventing disease progression in type 2 diabetes.
In one embodiment of the present invention, the insulin preparation according to the invention is for use as a medicament for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other car- diovascular disorders is provided.
In a further embodiment of the present invention, a method for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, the method comprising administering to a patient in need of such treatment an effective amount for such treatment of an insulin preparation according to the invention, is provided.
The treatment with an insulin preparation according to the present invention may also be combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
The treatment with an insulin preparation according to the present invention may also be combined with bariatric surgery - a surgery that influences the glucose levels and/or lipid homeostasis such as gastric banding or gastric bypass.
The production of polypeptides, e.g., insulins, is well known in the art. An insulin analogue according to the invention may for instance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well estab- lished techniques, see e.g. Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999. The insulin analogue may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the insulin analogue in a suitable nutrient medium under conditions permitting the expression of the insulin analogue. For insulin analogues comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue, for instance by use of tRNA mutants. Hence, briefly, the insulin analogues according to the invention are prepared analogously to the preparation of known insulin analogues.
Several methods may be used for the production of human insulin and human insu- lin analogues. For example three major methods which are used in the production of insulin in microorganisms are disclosed in WO2008034881 . Two of these involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981 ) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, III. pp 729-739), or use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981 ) PNAS 78:5401 -5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741 188.
The insulin analogues are produced by expressing a DNA sequence encoding the insulin analogue in question in a suitable host cell by well known technique as disclosed in e.g. US 6500645. The insulin analogue is either expressed directly or as a precursor molecule which has an N-terminal extension on the B-chain or a C-terminal extension on the B- chain. The N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long. The N-terminal extension is to be cleaved of in vitro after isolation from the culture broth and will therefore have a cleavage site next to B1 . N-terminal extensions of the type suitable in the present invention are disclosed in US 5,395,922, and EP 765,395. The C-terminal extension may have the function of protecting the mature insulin or insulin analogue molecule against intracellular proteolytic processing by host cell exoproteases. The C-terminal extension is to be cleaved of either extra-cellularly in the culture broth by secreted, active carboxypeptidase or in vitro after isolation from the culture broth. A method for producing mature insulin and insulin analogs with C-terminal extensions on the B-chain that are removed by carboxypetidase are disclosed in WO 08037735. The target insulin product of the process may either be a two-chain human insulin or a two-chain human insulin analogue which may or may not have a short C- terminal extension of the B-chain. If the target insulin product will have no C-terminal extension of the B-chain, then said C-terminal extension should be capable of subsequently being cleaved off from the B-chain before further purification steps.
The present invention also contemplates the following non-limiting list of embodi- ments, which are further described elsewhere herein: 1 . An insulin preparation comprising:
• an insulin compound,
• a nicotinic compound, and
· arginine.
2. The insulin preparation according to embodiment 1 , wherein the insulin compound is human insulin or an insulin analog.
3. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B28Asp human insulin, B3LysB29Glu human insulin or B28LysB29Pro human insulin.
4. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B28Asp human insulin.
5. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B3LysB29Glu human insulin or B28LysB29Pro human insulin.
6. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B28LysB29Pro human insulin.
7. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is B3LysB29Glu human insulin.
8. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in a range selected from the following: 0.1 -10.0mM; 0.1 -
3.0mM; 0.1 -2.5mM; 0.1 -2.0mM; 0.1 -1.5mM; 0.2-2.5mM; 0.2-2.0mM; 0.2-1.5mM; 0.3- 3.0mM; 0.3-2.5mM; 0.3-2.0mM; 0.3-1 .5mM; 0.5-1.3mM and 0.6-1.2mM.
9. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.1 mM to about 10.0mM.
10. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.1 mM to about 3.0mM.
1 1 . The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.1 mM to about 2.5mM.
12. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in the amount from about 0.1 mM to about 2.0mM.
13. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.1 mM to about 1.5mM.
14. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.2mM to about 2.5mM.
15. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in the amount from about 0.2mM to about 2.0mM.
16. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.2mM to about 1 .5mM.
17. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in the amount from about 0.3mM to about 3.0mM.
18. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.3mM to about 2.5mM.
19. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.3mM to about 2.0mM.
20. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.3mM to about 1.5mM.
21 . The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.5mM to about 1.3mM.
22. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in the amount from about 0.3mM to about 1.2mM.
23. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount from about 0.6mM to about 1.2mM.
24. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount of about 0.6 or about 1.2mM.
25. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount of about 0.3mM.
26. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount of about 0.6mM.
27. The insulin preparation according to any of the preceding embodiments, wherein the in- sulin compound is present in the amount of about 0.9mM.
28. The insulin preparation according to any of the preceding embodiments, wherein the insulin compound is present in the amount of about 1.2mM.
29. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nia- cin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
30. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is selected from nicotinamide and nicotinic acid and/or salts thereof and/or any combination thereof.
31 . The insulin preparation according to any of the preceding embodiments, wherein the nic- otinic compound is nicotinamide and/or salts thereof. 32. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is present in a range selected from the following: 1 -300mM; 5-200mM; 40-120mM, 70-140mM or 80-130mM.
33. The insulin preparation according to any of the preceding embodiments, comprising from about 1 mM to about 300mM of the nicotinic compound.
34. The insulin preparation according to any of the preceding embodiments, comprising from about 8mM to about 260mM of the nicotinic compound.
35. The insulin preparation according to any of the preceding embodiments, comprising from about 50mM to about 250mM of the nicotinic compound.
36. The insulin preparation according to any of the preceding embodiments, comprising from about 80mM to about 250mM of the nicotinic compound.
37. The insulin preparation according to any of the preceding embodiments, comprising from about 80mM to about 180mM of the nicotinic compound.
38. The insulin preparation according to any of the preceding embodiments, comprising from about 5mM to about 200mM of the nicotinic compound.
39. The insulin preparation according to any of the preceding embodiments, comprising from about 1 mM to about 150mM of the nicotinic compound.
40. The insulin preparation according to any of the preceding embodiments, comprising from about 5mM to about 20mM of the nicotinic compound.
41 . The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 120mM of the nicotinic compound.
42. The insulin preparation according to any of the preceding embodiments, comprising from about 40mM to about 120mM of the nicotinic compound.
43. The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 40mM of the nicotinic compound.
44. The insulin preparation according to any of the preceding embodiments, comprising from about 60mM to about 80mM of the nicotinic compound.
45. The insulin preparation according to any of the preceding embodiments, comprising from about 70mM to about 140mM of the nicotinic compound.
46. The insulin preparation according to any of the preceding embodiments, comprising from about 80mM to about 130mM of the nicotinic compound.
47. The insulin preparation according to any of the preceding embodiments, comprising about 8mM, 30mM, 100mM or 130mM of the nicotinic compound.
48. The insulin preparation according to any of the preceding embodiments, comprising about 8mM of the nicotinic compound. 49. The insulin preparation according to any of the preceding embodiments, comprising about 30mM, 10OmM or 130mM of the nicotinic compound.
50. The insulin preparation according to any of the preceding embodiments, comprising about 30mM of the nicotinic compound.
51 . The insulin preparation according to any of the preceding embodiments, comprising about 80mM of the nicotinic compound.
52. The insulin preparation according to any of the preceding embodiments, comprising about 100mM of the nicotinic compound.
53. The insulin preparation according to any of the preceding embodiments, comprising about 120mM of the nicotinic compound.
54. The insulin preparation according to any of the preceding embodiments, comprising about 130mM of the nicotinic compound.
55. The insulin preparation according to any of the preceding embodiments, comprising about 150mM of the nicotinic compound.
56. The insulin preparation according to any of the preceding embodiments, comprising about 155mM of the nicotinic compound.
57. The insulin preparation according to any of the preceding embodiments, comprising about 180mM of the nicotinic compound.
58. The insulin preparation according to any of the preceding embodiments, comprising about 230mM of the nicotinic compound.
59. The insulin preparation according to any of the preceding embodiments, comprising the following ranges of arginine compound: 1 -100mM, 5-120mM, 8-85mM, 20-90mM, 30- 90mM, 30-85mM, 30-60mM or 10-40mM.
60. The insulin preparation according to any of the preceding embodiments, comprising the following ranges of arginine compound: 1 -120mM, 8-85mM or 1 -40mM.
61 . The insulin preparation according to any of the preceding embodiments, comprising from about 1 mM to about 120mM of arginine.
62. The insulin preparation according to any of the preceding embodiments, comprising from about 1 mM to about 10OmM of arginine.
63. The insulin preparation according to any of the preceding embodiments, comprising from about 5mM to about 120mM of arginine.
64. The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 90mM of arginine.
65. The insulin preparation according to any of the preceding embodiments, comprising from about 30mM to about 85mM of arginine. 66. The insulin preparation according to any of the preceding embodiments, comprising from about 8mM to about 85mM of arginine.
67. The insulin preparation according to any of the preceding embodiments, comprising from about 30mM to about 60mM of arginine.
68. The insulin preparation according to any of the preceding embodiments, comprising from about 10mM to about 40mM of arginine.
69. The insulin preparation according to any of the preceding embodiments, comprising from about 10mM to about 30mM of arginine.
70. The insulin preparation according to any of the preceding embodiments, comprising from about 10mM to about 60mM of arginine.
71 . The insulin preparation according to any of the preceding embodiments, comprising from about 1 mM to about 40mM of arginine.
72. The insulin preparation according to any of the preceding embodiments, wherein arginine is present in a range selected from the following: 1 mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM,
55mM or 60mM.
73. The insulin preparation according to any of the preceding embodiments, comprising about 1 mM of arginine.
74. The insulin preparation according to any of the preceding embodiments, comprising about 2mM of arginine.
75. The insulin preparation according to any of the preceding embodiments, comprising about 3mM of arginine.
76. The insulin preparation according to any of the preceding embodiments, comprising about 4mM of arginine.
77. The insulin preparation according to any of the preceding embodiments, comprising about 5mM of arginine.
78. The insulin preparation according to any of the preceding embodiments, comprising about 6mM of arginine.
79. The insulin preparation according to any of the preceding embodiments, comprising about 7mM of arginine.
80. The insulin preparation according to any of the preceding embodiments, comprising about 8mM of arginine.
81 . The insulin preparation according to any of the preceding embodiments, comprising about 9mM of arginine.
82. The insulin preparation according to any of the preceding embodiments, comprising about 10mM of arginine.
83. The insulin preparation according o any o preceding embodiments, comprising about 1 1 mM of arginine.
84. The insulin preparation according o any o preceding embodiments, comprising about 12mM of arginine.
85. The insulin preparation according o any o preceding embodiments, comprising about 13mM of arginine.
86. The insulin preparation according o any o preceding embodiments, comprising about 14mM of arginine.
87. The insulin preparation according o any o preceding embodiments, comprising about 15mM of arginine.
88. The insulin preparation according o any o preceding embodiments, comprising about 17mM of arginine.
89. The insulin preparation according o any o preceding embodiments, comprising about 20mM of arginine.
90. The insulin preparation according o any o preceding embodiments, comprising about 22mM of arginine.
91 . The insulin preparation according o any o preceding embodiments, comprising about 25mM of arginine.
92. The insulin preparation according o any o preceding embodiments, comprising about 30mM of arginine.
93. The insulin preparation according o any o preceding embodiments, comprising about 35mM of arginine.
94. The insulin preparation according o any o preceding embodiments, comprising about 40mM of arginine.
95. The insulin preparation according o any o preceding embodiments, comprising about 45mM of arginine.
96. The insulin preparation according o any o preceding embodiments, comprising about 50mM of arginine.
97. The insulin preparation according o any o preceding embodiments, comprising about 55mM of arginine.
98. The insulin preparation according o any o preceding embodiments, comprising about 60mM of arginine.
99. The insulin preparation according to any of he preceding embodiments, which further comprises a buffer(s). 100. The insulin preparation according to embodiment 99, wherein said buffer is a phosphate buffer.
101 . The insulin preparation according to embodiment 100, comprising from about 1 mg/mL to about 20mg/ml_ of phosphate buffer.
102. The insulin preparation according to embodiment 100, comprising from about 1 mg/mL to about 15mg/ml_ of phosphate buffer.
103. The insulin preparation according to embodiment 100, comprising from about 1 mg/mL to about 10mg/mL of phosphate buffer.
104. The insulin preparation according to embodiment 100, comprising about 3mg/mL of phosphate buffer.
105. The insulin preparation according to embodiment 99, wherein said buffer is Tris.
106. The insulin preparation according to embodiment 105, comprising from about 2mM to about 50mM of Tris.
107. The insulin preparation according to embodiment 105, comprising from about 10mM to about 40mM of Tris.
108. The insulin preparation according to embodiment 105, comprising from about 20mM to about 30mM of Tris.
109. The insulin preparation according to embodiment 105, comprising about 10mM, 20mM, 30mM or 40mM of Tris.
1 10. The insulin preparation according to embodiment 105, comprising about 7mM of Tris.
1 1 1 . The insulin preparation according to embodiment 105, comprising about 10mM of Tris.
1 12. The insulin preparation according to embodiment 105, comprising about 20mM of Tris.
1 13. The insulin preparation according to embodiment 105, comprising about 30mM of Tris.
1 14. The insulin preparation according to embodiment 105, comprising about 40mM of Tris.
1 15. The insulin preparation according to any of the preceding embodiments, which further comprises a metal ion.
1 16. The insulin preparation according to embodiment 1 15, wherein the metal ion is zinc.
1 17. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 0:6 to about 6:6.
1 18. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 0:6 to about 5:6.
1 19. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 1 :6 to about 6:6.
120. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 2:6 to about 6:6.
121 . The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 2:6 to about 5:6.
122. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 2.5:6 to about 4.5:6.
123. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is from about 3:6 to about 4:6.
124. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 2:6.
125. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 2.5:6.
126. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3:6.
127. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.1 :6.
128. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.2:6.
129. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.3:6.
130. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.4:6.
131 . The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.5:6.
132. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.6:6.
133. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.7:6.
134. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.8:6.
135. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 3.9:6. 136. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 4:6.
137. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 4.5:6.
138. The insulin preparation according to embodiment 1 16, wherein the zinc:insulin molar ratio is about 5:6.
139. The insulin preparation according to any one of the preceding embodiments, which further comprises a stabilizer(s).
140. The insulin preparation according to embodiment 139, wherein the stabilizer is a non-ionic detergent.
141 . The insulin preparation according to embodiment 140, wherein the detergent is polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80).
142. The insulin preparation according to embodiment 140, wherein the detergent is polysorbate 20 (Tween 20).
143. The insulin preparation according to embodiment 140, wherein the detergent is polysorbate 80 (Tween 80).
144. The insulin preparation according to embodiment 140, comprising from about 5 to 100ppm, from about 10 to about 50ppm or from about 10 to about 20ppm of polysorbate.
145. The insulin preparation according to any one of the preceding embodiments, which further comprises a preservative agent(s).
146. The insulin preparation according to embodiment 145, wherein the preservative is a phenolic compound.
147. The insulin preparation according to embodiment 146, wherein said phenolic compound is present in the amount from about 0 to about 6mg/ml or from about 0 to about 4mg/ml.
148. The insulin preparation according to embodiment 146, wherein said phenolic compound is present in the amount of from about 5 to about 100mM.
149. The insulin preparation according to embodiment 146, wherein said phenolic compound is present in the amount of from about 5 to about 50mM.
150. The insulin preparation according to embodiment 146, wherein said phenolic compound is present in the amount of from about 5 to about 30mM.
151 . The insulin preparation according to embodiment 146, wherein said phenolic compound is present in the amount of about 16mM.
152. The insulin preparation according to embodiment 146, wherein said phenolic com- pound is present in the amount of about 14.4mM. 153. The insulin preparation according to embodiment 145, wherein said preservative is m-cresol.
154. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount from about 0.5 to about 4.0mg/ml or from about 0.6 to about 4.0mg/ml. 155. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount of from about 5 to about 100mM.
156. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount of from about 5 to about 50mM.
157. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount of from about 5 to about 30mM.
158. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount of about 16mM.
159. The insulin preparation according to embodiment 153, wherein m-cresol is present in the amount of about 14.4mM.
160. The insulin preparation according to any of the preceding embodiments, further comprising glycerol in the amount from about 0.5 to about 2.5%.
161 . The insulin preparation according to any of the preceding embodiments, further comprising glycerol in the amount from about 0.7 to about 2.0%.
162. The insulin preparation according to any of the preceding embodiments, further comprising glycerol in the amount from about 1.0 to about 1 .5%.
163. The insulin preparation according to any of the preceding embodiments, further comprising glycerol in the amount of about 1.25%.
164. The insulin preparation according to any of the preceding embodiments, wherein the pH is neutral to weakly basic.
165. The insulin preparation according to any of the preceding embodiments, wherein the pH is from about 6.8 to about 8.0.
166. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 6.8.
167. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 6.9.
168. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.0.
169. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.1 .
170. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.2.
171 . The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.3.
172. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.4.
173. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.5.
174. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.6.
175. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.7.
176. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.8.
177. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 7.9.
178. The insulin preparation according to any of the preceding embodiments, wherein the pH is about 8.0.
179. A method of reducing the blood glucose level in mammals by administering to a patient in need of such treatment a therapeutically active dose of an insulin preparation ac- cording to any of the preceding embodiments.
180. A method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any of embodiments 1 -178.
181 . A method according to any of embodiments 179-180, for parenteral administration.
182. An insulin preparation according to any of embodiments 1 -178, for use in the treat- ment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treatment of critically ill diabetic and non-diabetic patients.
183. The insulin preparation according to embodiment 182, for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns and operation wounds, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders.
184. The insulin preparation according to embodiment 182-183, for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes. 185. An insulin preparation comprising:
• an insulin compound,
• a nicotinic compound
• arginine, and
· a buffer.
186. The insulin preparation according to embodiment 185, wherein the insulin compound is human insulin or an insulin analog.
187. The insulin preparation according to any one of embodiments 185 or 186, wherein the insulin compound is selected from the group consisting of B28Asp human insulin, B3LysB29Glu human insulin and B28LysB29Pro.
188. The insulin preparation according to embodiment 186, wherein the insulin compound is B28Asp human insulin.
189. The insulin preparation according to any one of embodiments 185-188, wherein the insulin compound is present in the amount from about 0.2mM to about 2.0mM.
190. The insulin preparation according to any one of embodiments 185-189, wherein the insulin compound is present in the amount from about 0.6mM to about 1.2mM.
191 . The insulin preparation according to any one of embodiments 185-190, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof. 192. The insulin preparation according to any one of embodiments 185-191 , wherein the nicotinic compound is nicotinamide.
193. The insulin preparation according to embodiment 192, comprising from about 1 mM to about 250mM of the nicotinic compound.
194. The insulin preparation according to embodiment 192, comprising from about 1 mM to about 250mM of the nicotinic compound.
195. The insulin preparation according to embodiment 192, comprising from about 80mM to about 230mM of the nicotinic compound.
196. The insulin preparation according to any one of embodiments 185-195, comprising from about 10mM to about 60mM of arginine.
197. The insulin preparation according to any one of embodiments 185-196, comprising from about 10mM to about 30mM of arginine.
198. The insulin preparation according to any one of embodiments 185-197, comprising about 20mM arginine.
199. The insulin preparation according to any one of embodiments 185-198, wherein said buffer is a phosphate buffer. 200. The insulin preparation according to any one of embodiments 185-199, wherein said buffer is Tris.
201 . The insulin preparation according to any one of embodiments 185-200, which may further comprises preservative agent(s), isotonicity agent(s) and/or stabilizer(s).
202. The insulin preparation according to any one of embodiments 185-201 , which further comprises a metal ion.
203. The insulin preparation according to embodiment 202, wherein the metal ion is zinc.
204. The insulin preparation according to embodiment 203, wherein the zinc:insulin molar ratio is from about 0:6 to about 3.5:6.
205. The insulin preparation according to embodiment 203, wherein the zinc:insulin molar ratio is from about 2:6 to about 3:6.
206. The insulin preparation according to any one of embodiments 185-205, wherein the preparation has a pH of less than 7.4.
207. The insulin preparation according to any one of embodiments 185-205, wherein the preparation has a pH of about 7.4.
208. The insulin preparation according to any one of embodiments 185-205, wherein the preparation has a pH of about 7.1
209. An insulin preparation comprising: B28Asp human insulin; nicotinamide; zinc; arginine; and a phosphate buffer.
210. The insulin preparation of embodiment 209, wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1 .2 mM, and wherein the nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and wherein the arginine is present in a concentration ranging from about 10 mM to about 40 mM, and wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules, and wherein the preparation has a pH of about 7.4 or less.
21 1 . An insulin preparation consisting essentially of:
a. B28Asp human insulin, wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1 .2 mM;
b. Nicotinamide, wherein the nicotinamide is present in a concentration ranging from about 80 mM to abour 260 mM;
c. Zinc, wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules;
d. Arginine, wherein the the arginine is present in a concentration ranging from about 10 mM to about 30 mM; and
e. a phosphate buffer; wherein the preparation has a pH of about 7.1.
212. A method of reducing the blood glucose level in mammals by administering to a mammal in need of such treatment a therapeutically active dose of an insulin preparation according to any one of the preceding embodiments.
213. A method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any one of embodiments 1 -21 1.
214. An insulin preparation according to any one of embodiments 1 -21 1 , for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treatment of critically ill diabetic and non-diabetic patients.
215. An insulin preparation according to any one of embodiments 1 -21 1 , for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.
Further embodiments of the invention relate to the following: 216. An insulin preparation comprising:
• an insulin compound,
• a nicotinic compound, and
• arginine.
217. The insulin preparation according to embodiment 216, wherein the insulin com- pound is human insulin or an insulin analog.
218. The insulin preparation according to embodiments 216-217, wherein the insulin compound is B28Asp human insulin.
219. The insulin preparation according to any one of embodiments 216-218, wherein the insulin compound is B28LysB29Pro human insulin.
220. The insulin preparation according to any one of embodiments 216-219, wherein the insulin compound is B3LysB29Glu human insulin.
221 . The insulin preparation according to any one of embodiments 216-220, wherein the insulin compound is present in the amount from about 0.2mM to about 2.0mM.
222. The insulin preparation according to any one of embodiments 216-221 , wherein the insulin compound is present in the amount from about 0.3mM to about 1.2mM. 223. The insulin preparation according to any one of embodiments 216-222, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
224. The insulin preparation according to any one of embodiments 216-223, comprising from about 1 mM to about 150mM of the nicotinic compound.
225. The insulin preparation according to any one of embodiments 216-224, comprising from about 1 mM to about 85mM of arginine.
226. The insulin preparation according to any one of 216-225, which further comprises a metal ion, preservative agent(s), isotonicity agent(s) and stabilizer(s) and buffer(s).
227. A method of reducing the blood glucose level in mammals by administering to a patient in need of such treatment a therapeutically active dose of an insulin preparation according to any one of embodiments 216-226.
228. A method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any one of embodiments 216-226. 229. An insulin preparation according to any one of embodiments 216-226, for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treat- ment of critically ill diabetic and non-diabetic patients.
The invention is further illustrated by the following examples which are not to be construed as limiting.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and 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 (to the maximum extent permitted by law).
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
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 claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.
This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.
EXAMPLES
Example 1
Preparation of pharmaceutical preparations
The pharmaceutical preparations of the present invention may be formulated as an aqueous solution. The aqueous medium is made isotonic, for example, with sodium chloride or glycerol. Furthermore, the aqueous medium may contain zinc ions, for example added as zinc acetate or zinc chloride, buffers and preservatives. Arginine may be added as Arg, HCI. The pH value of the preparation is adjusted to the desired value and may be between about 3 to about 8.5, between about 3 and about 5 or about 6.5 and about 7.5 depending on the isoe- lectric point, pi, of the insulin in question.
Table 1 . Composition of insulin preparations according to this invention
Figure imgf000034_0001
* Commercially available NovoRapid Table 2. Composition of further insulin preparations according to this invention
Preparation [Insulin [Zn2+] [phenol] [Arg] [Gly] [Glu] [His] [Nicotin pH nr. aspart] mM mM mM mM mM mM amide] mM mM
1 0.6 0.3 32 260 7.4
2 0.6 0.3 32 10 260 7.4
3 0.6 0.3 32 20 260 7.4
4 0.6 0.3 32 30 260 7.4
5 0.6 0.3 32 40 260 7.4
6 0.6 0.3 32 50 260 7.4
7 0.6 0.3 32 50 260 7.4
8 0.6 0.3 32 50 260 7.4
9 0.6 0.3 32 50 260 7.4
Table 3. Composition of further insulin preparations according to this invention
Figure imgf000036_0001
Table 4. Composition of further insulin preparations according to this invention
Figure imgf000036_0002
Example 2
Analysis of insulin chemical stability
Size Exclusion Chromatography
Quantitative determination of high molecular weight protein (HMWP) and monomer insulin aspart was performed on Waters insulin (300 x 7.8mm, part nr wat 201549) with an eluent containing 2.5M acetic acid, 4mM L-arginine and 20 %(V/V) acetonitrile at a flow rate of 1 ml/min. and 40°C. Detection was performed with a tuneable absorbance detector (Waters 486) at 276nm. Injection volume was 40μΙ and a 600μΜ human insulin standard. HMWP and concentration of the preparations were measured at each sampling point.
Reverse phase chromatography (UPLC)
Determination of the insulin aspart related impurities were performed on a UPLC system using a BEH RP C8 2.1 x 100mm column, particle size of 1 ,7μιη. Waters part no 186002878. with a flow rate of 0,5ml/min., at 40° C detection at 220nm. Elution was performed with a mobile phase consisting of the following:
A. 10 % (w/V) acetonititrile, 2.8% (w/w) sodium sulphate, 0.3 % (w/w) o-phosphoric acid, pH 3.5.
B. 70 % (w/V) acetonitrile. Gradient: 0-1 1 min isocratic with 73%/27% of A/B, 1 1 -12 linear change to 52%/48% A B, 13-15 min. linear change to 73%/27% of A/B, 15-20 min. isocratic gradient at 73%/27% of A/B.
The amount of B28iso-aspartate, desamido and other related impurities were de- termined as absorbance area measured in percent of total absorbance area determined after elution of the preservatives. The RP-UPLC method is equivalent to the analytical method used for quality control of Novo Nordisk marketed insulin aspart pharmaceuticals.
Addition of arginine reduces the amount of degradation products formed, especially HMWP and des-amido forms, increasing the concentration of arginine in the range 10 to 50mM leads to further reduction of degradation. The physical stability measured as lag time in the ThT assay is reduced upon addition of arginine and is increasingly reduced when the arginine concentration is increased. The overall performance of 50mM arginine is superior to 50 mM glycine, 50mM glutamic acid, or 50mM histidine regarding reduction of the formation of degradation products, as is shown in Table 4 below.
The insulin preparations of the present invention provide fast-acting insulin preparations which are not only physically stable, but surprisingly also chemically stable.
Table 5. Physical and chemical stability data for insulin preparations 1 -9 of Table 2
Preparation nr. Physical stabilChemical stability
ity, lag time Content of degradation product (%) measured as differ(min) in ThT ence between content after incubation for 2 weeks at assay 37°C and at 4°C
B28 IsoAsp des-amido Other reHMWP
forms lated impuri- ties
160 1.17 3.67 1.73 1.36
80 1.30 3.05 0.82 0.65
80 1.30 2.49 0.64 0.34
60 1.31 2.26 0.79 0.20
60 1.27 2.27 0.37 0.19
40 1.36 1.99 0.47 0.16
100 1.26 4.72 2.21 1.11
50 1.39 3.41 1.07 0.70
0 1.75 6.99 2.22 1.01
Table 6. Chemical stability data for insulin preparations 10-13 of Table 3
Figure imgf000039_0002
Table 7. Physical and chemical stability data for insulin preparations 14-20 of Table 4
Figure imgf000039_0001
Example 3
Pharmacokinetic (PK)/ Pharmacodynamic (PD) studies in LYD pig model and plasma analysis assay PK/PD studies in LYD pigs
The PK/PD studies were performed on domestic female pigs, LYD cross-breed, weighing between 55 and 1 10kg. The pigs were catheterised into the jugular vein through an ear vein at least 2 days before start of the study. The last meal before the start of the study was served to the animals approx. 18 hours prior to the injection of the test preparation, and the animals had free access to water at all time during the fasting period and the test period.
At time 0 hours the test preparation was given subcutaneous on the lateral side of the neck. A blood sample was drawn prior dosing and at regular time intervals after dosing samples were drawn from the catheter and sampled into 1 .5ml glass tubes pre-coated with heparin. The blood samples were kept in ice water until separation of plasma by centrifugation for 10min. 3000rpm at 4°C, which was done within the first 30 minutes. Plasma samples were stored at 4°C for short time (2-3 hours) or at -18°C for long term storage and were analysed for glucose on YSI or Konelab 30i and for insulin Aspart concentration by LOCI.
Luminescent Oxygen Channeling Immunoassay (LOCI) for Insulin Aspart quantification The insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay and applies the proximity of two beads, the europium-coated acceptor beads and the streptavidin coated donor-beads. The acceptor beads were coated with a specific antibody against human insulin and recognize insulin Aspart in plasma samples. A second biotinylated antibody bind specific to insulin Aspart and together with the streptavidin coated beads, they make up the sandwich. Illumination of the beads-aggregate-immunocomplex releases singlet oxygen from the donor beads which channels into the acceptor beads and triggers chemilumines- cence. The chemiluminescence was measured and the amount of light generated is proportional to the concentration of insulin Aspart.
Compared to the marketed product NovoRapid®, the initial rate of plasma glucose lowering is faster for the preparations of the present invention (Figures 3 and 4). Likewise, when com- pared to NovoRapid®, the initial absorption rate of the insulin component of the preparations of the present invention, is markedly faster (Figure 5).
Example 4
General introduction to ThT fibrillation assays for the assessment of physical stability of protein formulations
Low physical stability of a peptide may lead to amyloid fibril formation, which is observed as well-ordered, thread-like macromolecular structures in the sample eventually resulting in gel formation. This has traditionally been measured by visual inspection of the sample. However, that kind of measurement is very subjective and depending on the observer. Therefore, the application of a small molecule indicator probe is much more advantageous. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signature when binding to fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284]. The time course for fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001 ) Biochemistry 40, 6036-6046]: fr m ft
F = fi + mit -
Eq.(1 )
Here, F is the ThT fluorescence at the time t. The constant t0 is the time needed to reach 50% of maximum fluorescence. The two important parameters describing fibril formation are the lag-time calculated by t0 - 2τ and the apparent rate constant kapp = 1/τ.
Figure imgf000041_0001
tn
Lag-time = t0 - 2τ Time Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and the fibrillation proceeds. The lag-time corresponds to the interval in which the critical mass of nucleus is built up and the apparent rate constant is the rate with which the fibril itself is formed.
Sample preparation
Samples were prepared freshly before each assay. Each sample composition is described in each example. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCI04 or HCI. Thioflavin T was added to the samples from a stock solution in H20 to a final concentration of 1 μΜ.
Sample aliquots of 200μΙ were placed in a 96 well microtiter plate (Packard Opti-
Plate™-96, white polystyrene). Usually, four or eight replica of each sample (corresponding to one test condition) were placed in one column of wells. The plate was sealed with Scotch
Pad (Qiagen).
Incubation and fluorescence measurement
Incubation at given temperature, shaking and measurement of the ThT fluorescence emis- sion were done in a Fluoroskan Ascent FL fluorescence platereader or Varioskan platereader (Thermo Labsystems). The temperature was adjusted to 37 °C. The orbital shaking was adjusted to 960rpm with an amplitude of 1 mm in all the presented data. Fluorescence measurement was done using excitation through a 444nm filter and measurement of emission through a 485nm filter.
Each run was initiated by incubating the plate at the assay temperature for 10 min.
The plate was measured every 20 minutes for a desired period of time. Between each measurement, the plate was shaken and heated as described.
Data handling
The measurement points were saved in Microsoft Excel format for further processing and curve drawing and fitting was performed using GraphPad Prism. The background emission from ThT in the absence of fibrils was negligible. The data points are typically a mean of four or eight samples and shown with standard deviation error bars. Only data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a relative measure of fibrillation between experiments. The data set may be fitted to Eq. (1 ). However, since full sigmodial curves are not always achieved during the measurement time, lag times were here visually determined from the ThT fluorescence curve as the time point at which the ThT fluorescence is different than the background level.
Measurement of initial and final concentrations
The peptide concentration in each of the tested formulations were measured both before application in the ThT fibrillation assay ("Initial") and after completion of the ThT fibrillation ("After ThT assay"). Concentrations were determined by reverse HPLC methods using a pramlin- tide standard as a reference. Before measurement after completion 150μΙ was collected from each of the replica and transferred to an Eppendorf tube. These were centrifuged at 30000 G for 40mins. The supernatants were filtered through a 0.22μιη filter before application on the HPLC system.

Claims

1 . An insulin preparation comprising:
• an insulin compound,
« a nicotinic compound, and
• arginine.
2. The insulin preparation according to claim 1 , wherein the insulin compound is human insulin or an insulin analog.
3. The insulin preparation according to any one of claims 1 or 2, wherein the insulin com- pound is selected from the group consisting of B28Asp human insulin, B3LysB29Glu human insulin and B28LysB29Pro.
4. The insulin preparation according to any one of the preceding claims, wherein the insulin compound is B28Asp human insulin.
5. The insulin preparation according to any one of the preceding claims, wherein the insulin compound is present in the amount from about 0.2mM to about 2.0mM.
6. The insulin preparation according to any of the preceding claims, wherein the insulin compound is present in the amount from about 0.6mM to about 1 .2mM.
7. The insulin preparation according to any one of the preceding claims, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nia- cin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
8. The insulin preparation according to any one of the preceding claims wherein the nicotinic compound is nicotinamide.
9. The insulin preparation according to any one of the preceding claims, comprising from about 1 mM to about 250mM of the nicotinic compound.
10. The insulin preparation according to any one of the preceding claims, comprising from about 1 mM to about 250mM of the nicotinic compound.
1 1 . The insulin preparation according to any one of the preceding claims, comprising from about 80mM to about 230mM of the nicotinic compound.
12. The insulin preparation according to any one of the preceding claims, comprising from about 10mM to about 60mM of arginine.
13. The insulin preparation according to any one of the preceding claims, comprising from about 10mM to about 30mM of arginine.
14. The insulin preparation according to any one of the preceding claims, comprising about 20mM arginine.
15. The insulin preparation according to any one of the preceding claims, which further comprises one or more buffer(s).
16. The insulin preparation according to claim 15, wherein said buffer is a phosphate buffer.
17. The insulin preparation according to claim 15, wherein said buffer is Tris.
18. The insulin preparation according to any one of the preceding claims, which may further comprises preservative agent(s), isotonicity agent(s) and/or stabilizer(s).
19. The insulin preparation according to any one of the preceding claims, which further comprises a metal ion.
20. The insulin preparation according to claim 19, wherein the metal ion is zinc.
21 . The insulin preparation according to claim 20, wherein the zinc:insulin molar ratio is from about 0:6 to about 3.5:6.
22. The insulin preparation according to claim 20, wherein the zinc:insulin molar ratio is from about 2:6 to about 3:6.
23. The insulin preparation according to any one of the preceding claims, wherein the prepa- ration has a pH of less than 7.4.
24. The insulin preparation according to any one of the preceding claims, wherein the preparation has a pH of about 7.4.
25. The insulin preparation according to any one of the preceding claims, wherein the preparation has a pH of about 7.1.
26. An insulin preparation comprising: B28Asp human insulin; nicotinamide; zinc; arginine; and a phosphate buffer.
27. The insulin preparation of claim 26, wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1.2 mM, and wherein the nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and wherein the arginine is present in a concentration ranging from about 10 mM to about 40 mM, and wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules, and wherein the preparation has a pH of about 7.4 or less.
28. An insulin preparation consisting essentially of:
a. B28Asp human insulin, wherein the B28Asp human insulin is present in a concentration ranging from about 0.6 mM to about 1 .2 mM;
b. Nicotinamide, wherein the nicotinamide is present in a concentration ranging from about 80 mM to abour 260 mM;
c. Zinc, wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules;
d. Arginine, wherein the the arginine is present in a concentration ranging from about 10 mM to about 30 mM; and
e. a phosphate buffer;
wherein the preparation has a pH of about 7.1 .
29. A method of reducing the blood glucose level in mammals by administering to a mammal in need of such treatment a therapeutically active dose of an insulin preparation according to any one of the preceding claims.
30. A method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any one of claims 1 -28.
31 . An insulin preparation according to any one of claims 1 -28, for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treatment of critically ill diabetic and non-diabetic patients.
32. The insulin preparation according to claim 31 , for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.
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