JP2014501239A - Formulations containing insulin, nicotinamide and amino acids - Google Patents

Abstract

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

Description

  The present invention relates to a pharmaceutical preparation comprising an insulin compound, a nicotinic acid compound and an amino acid.

  Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partially or completely lost. About 5% of the total population suffers from diabetes, and the disability has become an epidemic.

  Since the introduction of insulin in the 1920s, continuous progress has been made in the treatment of diabetes. To help avoid high blood sugar levels, diabetics often provide frequent injection therapy, whereby insulin is administered with each meal. Because diabetics are treated with insulin for decades, they primarily need insulin formulations that are safe and improve quality of life. Among the commercial insulin preparations, there are rapid-acting, intermediate-acting and sustained-acting preparations.

  In the treatment of diabetes mellitus, regular insulin (such as Actrapid®), isophene insulin (referred to as NPH), insulin zinc suspension (Semilente®, Lente® and Ultralente®) Many types of pharmaceutical formulations of insulin have been proposed and used, such as biphasic isophene insulin (such as NovoMix®). Human insulin analogs and derivatives have also been developed that are designed for a specific profile of action, ie, fast acting or long acting. Among the commercial insulin formulations containing such immediate-acting insulin analogues are NovoRapid® (B28Asp human insulin formulation), Humalog® (B28LysB29Pro human insulin formulation) and Apidra® ( B3LysB29Glu human insulin formulation).

  International applications WO 91/09617 and WO / 9610417 (Novo Nordisk A / S) disclose insulin preparations containing nicotinamide or nicotinic acid or salts thereof. European Patent Application No. 1283051 is said to disclose an insulin formulation containing arginine used as a buffering agent. European Patent Application No. 1283051 is said to disclose improvements in the physical stability of insulin formulations.

  In most cases, pharmaceutical formulations of insulin are administered by subcutaneous injection. Of importance to the patient is the action profile of insulin, which means the effect of insulin on glucose metabolism as a function of time since injection. In this profile, among others, the onset time of action, the local maximum of action and the total duration of action are important. In the case of bolus insulin, various insulin formulations with different action profiles are desirable and desired by the patient. Any patient can use insulin preparations with very different action profiles on the same day. The desired action profile will depend, for example, on the time of day and the amount and composition of the meal consumed by the patient.

  Equally important to the patient is the chemical stability of the insulin formulation because it uses a pen injection device many times, such as a device that contains a Penfill® cartridge, The entire cartridge in this device is stored until empty, which can be at least 1-2 weeks for devices containing 1.5-3.0 ml cartridges. During storage, covalent chemical changes occur in the insulin structure. This can lead to the formation of molecules that can be less active and / or potentially immunogenic, such as deamidation products and higher molecular weight transformation products (dimers, polymers). is there. Also equally important is the physical stability of the insulin formulation. This is because long-term storage can ultimately lead to the formation of insoluble fibers that are biologically inert and potentially immunogenic.

International application WO 91/09617 International application WO / 9610417 European Patent Application No. 1283051 EP 214826 WO 2005/012347 WO2008034881 U.S. Patent No. 5,962,267 WO 95/16708 European Patent No. 0055945 European Patent No. 0163529 European Patent No. 0347845 European Patent No. 0741188 U.S. Patent No. 6,500,645 U.S. Pat.No. 5,395,922 EP 765,395 WO 08037735 PCT WO 2007/074133

Remington: The Science and Practice of Pharmacy, 19th edition, 1995 Stability of Protein Pharmaceuticals, Ahern.T.J. & Manning M.C., Plenum Press, New York 1992 Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999 Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (edited by Rich & Gross), Pierce Chemical Co., Rockford, III. 729-739 Chan et al. (1981) PNAS 78: 5401-5404 Thim et al. (1986) PNAS 83: 6766-6770 Naiki et al. (1989) Anal. Biochem. 177, 244-249 LeVine (1999) Methods. Enzymol. 309, 274-284 Nielsen et al. (2001) Biochemistry 40, 6036-6046 Pharmaceutical Research, 21 (2004) 1498-1504 Pharmaceutical Research, 23 (2006) 49-55

  The present invention relates to insulin formulations having a preferred rate of absorption and preferred chemical and physical stability. The present invention relates to an insulin preparation comprising human insulin and / or an analogue thereof, nicotinamide or nicotinic acid and / or a salt thereof and arginine.

In one embodiment, the present invention provides:
An insulin compound, a nicotinic acid compound, and an insulin preparation containing arginine.

  In another embodiment, the present invention also provides a method for treating diabetes mellitus in a subject or a method for reducing blood glucose levels in a subject comprising administering an insulin formulation according to the present invention to a subject or mammal. Contemplate.

FIG. 5 shows the generation of degradation products during storage for 2 weeks at 37 ° C. according to the invention, as a percentage of the total insulin content. As described in Table 1 of Example 1, the letter A refers to the reference NovoRapid® and the remaining letters correspond to the insulin aspart formulation. Compared to NovoRapid® formulation (Formulation A), adding nicotinamide (Formulations B and D) increases the formation of degradation products, whereas nicotinamide, glutamic acid and arginine are added in combination (Formulations C and E) result in an almost similar degradation pattern with less HMWP formation. FIG. 5 shows the generation of degradation products during storage for 2 weeks at 37 ° C. according to the invention, as a percentage of the total insulin content. As described in Table 1 of Example 1, the letter A refers to the reference NovoRapid® and the remaining letters correspond to the insulin aspart formulation. Nicotinamide, glutamic acid and arginine combined, Formulations F, G, H and I have different buffer systems, phosphate buffer or Tris buffer, and insulin and Zn concentrations of 0.6 and 0.6. Although they are mM and 0.3 mM or 1.2 mM and 0.6 mM, they have a degradation pattern similar to the formulation A, NovoRapid® formulation. FIG. 4 shows plasma glucose concentration (mean +/− SEM, N = 8) after subcutaneous injection of pigs with a 1 nmol / kg dose of the formulation according to the invention at 0 minutes. As described in Table 1 of Example 1, the letter A refers to the reference NovoRapid® and the remaining letters correspond to the insulin aspart formulation. Compared to NovoRapid® formulation (Formulation A), the initial rate of plasma glucose lowering is faster for the formulation with nicotinamide (Formulation N) and for the combination of nicotinamide and arginine (Formulation M) Even faster. FIG. 4 shows plasma glucose concentration (mean +/− SEM, N = 7) after subcutaneous injection of pigs with a 1 nmol / kg dose of the formulation according to the invention at 0 min. As described in Table 1 of Example 1, the letter A refers to the reference NovoRapid® and the remaining letters correspond to the insulin aspart formulation. Compared to NovoRapid® formulation (Formulation A), the initial rate of plasma glucose lowering is a formulation containing a combination of nicotinamide, arginine and glutamic acid (Formulation L) and a formulation containing a combination of nicotinamide and arginine (Formulation For K) it is faster. FIG. 7 shows plasma insulin aspart concentration (mean +/− SEM, N = 7) after subcutaneous injection of pigs with a 1 nmol / kg dose of the formulation according to the invention at 0 minutes. As described in Table 1 of Example 1, the letter A refers to the reference NovoRapid® and the remaining letters correspond to the insulin aspart formulation. Insulin in formulations containing nicotinamide (Formulation J), a combination of nicotinamide and arginine (Formulation K) and a combination of nicotinamide, arginine and glutamic acid (Formulation L) compared to NovoRapid® formulation (Formulation A) The initial absorption rate of the components is significantly faster. FIG. 3 shows plasma glucose concentration (mean +/− SEM, N = 8, administered twice to each pig) after subcutaneous injection of pigs with a 1 nmol / kg dose of the formulation according to the invention at 0 minutes. The letter A refers to the reference NovoRapid®, and as described in Table 1 of Example 1, No. 11 corresponds to an insulin aspart formulation. Compared to the NovoRapid® formulation (Formulation A), the initial rate of plasma glucose lowering is faster for the formulation containing the combination of nicotinamide and arginine (Formulation 11). FIG. 2 shows plasma insulin aspart concentration (mean +/− SEM, N = 8, administered twice to each pig) after subcutaneous injection of 1 nmol / kg dose of the formulation according to the invention into pigs at 0 minutes. . The letter A refers to the reference NovoRapid®, and as described in Table 1 of Example 1, No. 11 corresponds to an insulin aspart formulation. Compared to the NovoRapid® formulation (Formulation A), the initial absorption rate of the insulin component of the formulation containing nicotinamide and arginine (Formulation 11) is significantly faster.

  The absorption after subcutaneous injection of the insulin compound in the insulin formulation of the present invention was surprisingly found to be faster than that of the reference insulin formulation. This property is useful for fast acting insulins, particularly in connection with frequent injection regimens where insulin is given before each meal. Because of the faster onset of action, the instant insulin can be conveniently ingested closer to meals than conventional fast acting insulin solutions. Furthermore, faster disappearance of insulin probably reduces the risk of hypoglycemia after a meal.

  The insulin preparation of the present invention is an immediate-acting insulin preparation containing an insulin compound such as insulin aspart, a nicotinic acid compound such as nicotinamide, and arginine which is an amino acid. Optionally, the insulin formulations of the present invention can also contain other amino acids. These insulin formulations have a rapid absorption profile that more closely mimics normal physiology compared to existing therapies. Furthermore, the insulin preparations of the present invention have chemical and physical stability suitable for commercial pharmaceutical preparations.

  The insulin preparation of the present invention provides a fast-acting insulin preparation that is not only physically stable, but surprisingly also chemically stable. The insulin formulations of the present invention begin to act even faster compared to existing insulin therapies. Such a super fast-acting insulin preparation has the advantages of recovering insulin release, which is the first phase, convenience of injection, and stopping glucose production by the liver. The insulin formulation of the present invention has a favorable rate of absorption from the subcutaneous tissue into the plasma, compared to conventional formulations such as NovoRapid®, as suggested by several PK / PD experiments in pigs. If so, the initial increase in absorption rate ranges from 1.5 to 5 times. This faster absorption rate may improve glycemic control and convenience, and may allow switching from pre-meal to post-meal administration. The invention is based in part on the surprising discovery that the addition of nicotinamide allows for an increase in absorption rate, which also has a negative impact on chemical stability by significantly increasing the amount of HMWP. ing. The insulin formulations of the present invention have improved chemical stability due to the addition of arginine, which is reflected, for example, in reducing the formation of dimer and polymer insulin and deamidated insulin after storage. Yes. Insulin formulations of the present invention may also have improved physical stability that may be useful for use in pumps.

  The present invention provides an insulin formulation comprising an insulin compound according to the present invention present at a concentration of about 0.1 mM to about 10.0 mM and having a pH of 3 to 8.5. The formulation also includes a nicotinic compound and arginine. The formulation further comprises protease inhibitors, metal ions, buffer systems, preservatives, isotonic agents, chelating agents, stabilizers and surfactants.

  In one embodiment, the metal ion is zinc added as zinc acetate or zinc chloride.

  In one embodiment, the insulin formulation comprises human insulin, analogs or combinations thereof, nicotinamide and / or nicotinic acid and / or salt thereof, and arginine and / or salt thereof.

  In one embodiment, an insulin formulation according to the invention comprises an aqueous solution of B28Asp human insulin, nicotinamide and arginine.

  The content of B28Asp human insulin in the solutions of the present invention may be in the range of 15 to 500 international units (IU) / ml, and preferably in the range of 50 to 333 IU / ml in the injectable formulation. However, for other purposes of parenteral administration, the content of insulin compound may be higher.

  In this situation, the unit “IU” corresponds to 6 nmol.

  The term “insulin aspart” refers to the human insulin analog B28Asp human insulin.

  The term “onset” refers to the time from injection until the PK curve starts to increase.

  The term “absorption rate” refers to the slope of the PK curve.

  An “insulin compound” according to the invention is to be understood herein as human insulin, insulin analogues and / or combinations thereof.

  As used herein, the term “human insulin” refers to a human hormone whose structure and properties are well known. Human insulin has two polypeptide chains joined by cysteine residues, ie, disulfide bridges between the A and B chains. The A chain is a 21 amino acid peptide, the B chain is a 30 amino acid peptide, the two chains are joined by three disulfide bridges: the first is a cysteine at position 6 of the A chain And the 11th cysteine, the second is between the 7th cysteine in the A chain and the 7th cysteine in the B chain, and the third is the 20th cysteine in the A chain and the 19th in the B chain. It is between the cysteines.

  The hormone is synthesized as a single chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acids and then the following configuration: prepeptide-B-Arg Arg-C-Lys Arg-A [where C Is a peptide with 31 amino acids attached] to become proinsulin containing 86 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleaving the binding peptide from the A and B chains.

  As used herein, “insulin analog” means a polypeptide derived by mutation from the primary structure of naturally occurring insulin, eg, the primary structure of human insulin. One or more variants are generated by deleting and / or replacing at least one amino acid residue present in naturally occurring insulin and / or by adding at least one amino acid residue Is done. The added and / or substituted amino acid residues can be codeable amino acid residues or other naturally occurring amino acid residues.

  In one embodiment, the insulin analogue comprises less than 8 modifications (substitutions, deletions, additions and combinations thereof) compared to the parent insulin, or the analogue comprises less than 7 modifications compared to the parent insulin. Or the analog contains less than 6 modifications compared to the parent insulin, or the analog contains less than 5 modifications compared to the parent insulin, or the analog compares to the parent insulin Or less than 4 modifications, or the analog contains less than 3 modifications compared to the parent insulin, or the analog contains less than 2 modifications compared to the parent insulin.

  Mutations in the insulin molecule are displayed describing the chain (A or B), position and three letter code for the amino acid replacing the natural amino acid. `` DesB30 '' or `` B (1-29) '' means the natural insulin B chain or analog thereof lacking the B30 amino acid residue, and B28Asp human insulin is the amino acid residue at position 28 of the B chain, It means human insulin substituted with Asp.

  Examples of insulin analogues are: Pro at position 28 of the B chain is mutated at Asp, Lys, Leu, Val or Ala and / or Lys at position B29 is mutated at Pro, Glu or Asp It is an example. Furthermore, Asn at position B3 can be mutated with Thr, Lys, Gln, Glu or Asp. The amino acid residue at position A21 can be mutated with Gly. The amino acid at position B1 can be mutated with Glu. The amino acid at position B16 can be mutated with Glu or His. Further examples of insulin analogs are, for example, analogs in which the B30 amino acid in human insulin is deleted (des (B30) human insulin), insulin analogs in which the B1 amino acid in human insulin is deleted (des (B1) Human insulin), des (B28-B30) human insulin and des (B27) human insulin. C-terminal where the A and / or B chains have an insulin analogue with an N-terminal extension and the A and / or B chains have two arginine residues appended to the C-terminal of the B chain Insulin analogues with an extension are also examples of insulin analogues. A further example is an insulin analogue containing the combination of mutations mentioned. The amino acid at position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His, the amino acid at position B25 is His, and optionally further comprises one or more additional mutations Is a further example. An example of an insulin analogue is that the amino acid residue at position A21 is Gly and the insulin analogue is further extended at the C-terminus with two arginine residues.

  Further examples of insulin analogues include 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, HisB16, HisB16, HisB16 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, GluB14G Including, but not limited to, desB30 human insulin.

  The term “nicotinic acid-based compound” includes nicotinamide, nicotinic acid, niacin, niacinamide and vitamin B3 and / or salts thereof and / or any combination thereof.

  According to the present invention, the concentration of the nicotinic acid compound and / or salt thereof ranges from about 1 mM to about 300 mM or from about 5 mM to about 200 mM.

  The term “arginine” or “Arg” includes the amino acid arginine and / or its salts, such as arginine hydrochloride or arginine glutamate.

  In one embodiment, the insulin formulation comprises 1 to 100 mM arginine.

  In one embodiment, the insulin formulation comprises 1 to 20 mM arginine.

  In one embodiment, the insulin formulation comprises 20 to 90 mM arginine.

  In one embodiment, the insulin formulation comprises 30 to 85 mM arginine.

  The term “glutamic acid” or “Glu” includes the amino acid glutamic acid and / or its salts.

  As used herein, the term “pharmaceutical formulation” or “insulin formulation” includes insulin compounds, ie human insulin, analogs thereof and / or combinations thereof, and nicotinic compounds and amino acids, optionally preservatives. , Chelating agents, tonicity modifiers, fillers, stabilizers, antioxidants, polymers and surfactants, metal ions, oily media and other additions such as proteins (eg, human serum albumin, gelatin or protein) By means of a product containing together with an excipient, said insulin preparation is useful for treating, preventing or reducing the severity of a disease or disorder by administration of said insulin preparation to a person. Accordingly, insulin preparations are also known in the art as pharmaceutical preparations or pharmaceutical compositions.

  Buffers include sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and tris (hydroxymethyl) -aminomethane (Tris), bicine, tricine, malic acid, succinate The acid salt, maleic acid, fumaric acid, tartaric acid, aspartic acid, ethylenediamine or mixtures thereof may be selected, but are not limited to these. Each of these specific buffers constitutes an alternative embodiment of the invention.

  The insulin preparation of the present invention may further comprise other components common to insulin preparations, for example, a zinc complexing agent such as citrate and a phosphate buffer.

  Glycerol and / or mannitol and / or sodium chloride may be present in an amount corresponding to a concentration of 0 to 250 mM, 0 to 200 mM or 0 to 100 mM.

  Stabilizers, surfactants and preservatives may also be present in the insulin formulations of the present invention.

The insulin preparation of the present invention may further contain a pharmaceutically acceptable preservative. The preservative may be present in an amount sufficient to obtain a preservative effect. The amount of preservative in the insulin formulation can be determined, for example, from literature in the art and / or from amounts known for preservatives in, for example, commercial products. Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical formulations, for example, Remington: are described in The Science and Practice of Pharmacy, 19 th edition, 1995.

  The preservative present in the insulin preparations of the present invention may be, for example, phenol, m-cresol and methylparaben, as in previous conventional insulin preparations.

The insulin preparation of the present invention may further contain a chelating agent. The use of chelating agents in pharmaceutical formulations is well known to those skilled in the art. For convenience, Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 see.

The insulin preparation of the present invention may further contain a stabilizer. As used herein, the term “stabilizer” refers to a polypeptide containing a pharmaceutical formulation to stabilize the peptide, ie, to increase the shelf life and / or use time of such a formulation. Refers to chemicals added to For convenience, Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 see.

The insulin preparation of the present invention may further contain a surfactant. As used herein, the term “surfactant” refers to any molecule or ion comprising a water-soluble (hydrophilic) moiety, a head and a fat-soluble (lipophilic) segment. The surfactant preferably accumulates at the interface, with the hydrophilic portion directed toward water (hydrophilic phase) and the lipophilic portion directed toward the oil or hydrophobic phase (i.e., glass, air, oil, etc.). It is done. The concentration at which the surfactant begins to form micelles is known as the critical micelle concentration or CMC. Furthermore, the surfactant lowers the surface tension of the liquid. Surfactants are also known as amphiphilic compounds. The term “detergent” is a synonym generally used for surfactants. The use of surfactants in pharmaceutical formulations is well known to those skilled in the art. For convenience, Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 see.

  In a further embodiment, the present invention is an insulin formulation comprising an aqueous solution of the insulin compound of the present invention and a buffer, wherein the insulin compound is present at a concentration of 0.1 mM or more, and the formulation is at room temperature. It relates to an insulin formulation having a pH of about 3.0 to about 8.5 at (about 25 ° C.).

  The invention also relates to a method for producing the insulin formulation of the invention.

In one embodiment, the method for making an insulin formulation of the invention comprises:
a) preparing a solution by dissolving an insulin compound or a mixture of insulin compounds in water or in a buffer;
b) preparing a solution by dissolving divalent metal ions in water or in a buffer;
c) Prepare a solution by dissolving the preservative in water or buffer.
d) preparing a solution by dissolving an isotonic agent in water or buffer;
e) preparing a solution by dissolving the surfactant and / or stabilizer in water or buffer;
f) mixing solution a) with 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 sterile filtration.

In one embodiment, the method for making an insulin formulation of the invention comprises:
a) preparing separate stock solutions of preservatives, isotonic agents, metal ion salts, arginine or arginine salts and nicotinic compounds;
b) preparing a solution by adding a stock solution of preservatives or a mixture of preservatives to water or buffer;
c) adding a stock solution of tonicity agent to solution b);
d) dissolving the insulin compound or mixture of insulin compounds in water or in a buffer; preparing the solution by adding HCl to acidify the solution;
e) adding a metal ion salt stock solution to d);
f) adding e) to c) and stirring;
g) adding a stock solution of arginine or arginine salt to f) and stirring;
h) Add a stock solution of nicotinic acid-based compound to g) and use NaOH / HCl to adjust to the desired pH.

In one embodiment, the method for making an insulin formulation of the invention comprises:
a) preparing preservatives, tonicity agents, separate stock solutions of metal ion salts and mixed stock solutions of arginine or arginine salts and nicotinic compounds;
b) preparing a solution by adding a stock solution of preservatives or a mixture of preservatives to water or buffer;
c) adding a stock solution of tonicity agent to solution b);
d) dissolving the insulin compound or mixture of insulin compounds in water or in a buffer; preparing the solution by adding HCl to acidify the solution;
e) adding a metal ion salt stock solution to d);
f) adding e) to c) and stirring;
g) adding a mixed stock solution of arginine or arginine salt and nicotinic acid compound to f) and stirring;
h) Adjusting the pH of g) to the desired pH using NaOH / HCl.

  The insulin preparation of the present invention can be used in the treatment of diabetes by parenteral administration. It is recommended that the dosage of the insulin preparation of the present invention to be administered to the patient is selected by the attending physician.

  Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection using a syringe and optionally using a pen-like syringe. Alternatively, parenteral administration can be performed using an infusion pump. As a further option, an insulin formulation containing an insulin compound of the invention may be adapted for transmucosal administration, such as transdermal or buccal administration, eg, by needleless injection or from a patch, optionally from an iontophoretic patch. .

  Insulin preparations according to the invention may be applied to patients in need of such treatment at several sites, for example local sites such as skin and mucosal sites, sites that perform bypass absorption, such as in arteries, intravenously. It can be administered at the site of administration into the heart as well as at sites with absorption, for example intradermal, subcutaneous, intramuscular or intraperitoneal administration.

  In one embodiment of the invention, the insulin formulation is an aqueous formulation, ie a formulation comprising water. Such formulations are typically solutions or suspensions. In a further embodiment of the invention, the insulin formulation is an aqueous solution.

  The term “aqueous formulation” is defined as a formulation comprising at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water. Defined.

  Aqueous suspensions may contain the active compounds in admixture with additives suitable for the manufacture of aqueous suspensions.

  In one embodiment, the insulin formulations of the present invention are well suited for application in pen-like devices used for insulin therapy by injection.

  In one embodiment, the insulin formulation of the invention can be used in a pump for insulin administration.

  As used herein, the term “physical stability” is used as a result of exposure of proteins to thermo-mechanical stresses and / or with unstable interfaces and surfaces, such as hydrophobic surfaces and interfaces. Refers to the tendency of a protein to form biologically inert and / or insoluble protein aggregates as a result of an interaction. The physical stability of an aqueous protein formulation is determined by exposing a formulation filled in a suitable container (e.g., cartridge or vial) to mechanical / physical stress (e.g., agitation) for different periods of time at different temperatures. Assessed using visual inspection and / or turbidity measurement. Visual inspection of the formulation is performed in a focused sharp light on a dark background. The turbidity of the formulation is characterized, for example, by a visual scale that ranks the degree of turbidity on a scale from 0 to 3 (a formulation that does not show turbidity corresponds to a visual scale of 0, A formulation showing visual turbidity under sunlight corresponds to a visual scale of 3). A formulation is classified as physically unstable with respect to protein aggregation when it shows visual turbidity in sunlight. Alternatively, the turbidity of the formulation can be assessed by simple turbidity measurements well known to those skilled in the art. The physical stability of aqueous protein formulations can also be assessed by using spectroscopic agents or protein conformational probes. The probe is preferably a small molecule that selectively binds to a non-natural conformer of the protein. An example of a small molecule spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that is widely used for the detection of amyloid fibrils. In the presence of fiber, perhaps other protein configurations as well, when bound to the fiber protein form, Thioflavin T results in a new excitation maximal at about 450 nm and an enhanced emission at about 482 nm. Unbound thioflavin T is essentially non-fluorescent at those wavelengths.

  The term “chemical stability” of a protein formulation as used herein refers to potentially having lower biological potency and / or increased immunogenicity compared to the native protein structure. Refers to a change in the covalent structure of a protein that results in the formation of a potential chemical degradation product. 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. Increases in the amount of chemical degradation products are often seen during storage and use of protein formulations. Most proteins tend to deamidate, in the process, side chain amide groups in glutaminyl or asparaginyl residues are hydrolyzed to form free carboxylic acids or asparaginyl residues to form IsoAsp derivatives. Other degradation pathways are involved in the formation of high molecular weight products, where two or more protein molecules are covalently linked to each other through transamidation and / or disulfide interactions, and covalently linked dimer, oligomer and polymer degradation. This leads to product formation (Stability of Protein Pharmaceuticals, Ahern. TJ & Manning MC, Plenum Press, New York 1992). Oxidation (for example, of methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of a protein formulation measures the amount of chemical degradation products at various times after exposure to different environmental conditions (degradation product formation can often be accelerated, for example, by increasing temperatures). Can be evaluated. The amount of each individual degradation product is often determined using different chromatographic techniques (e.g. SEC-HPLC and / or RP-HPLC) depending on the molecular size and / or charge. Is measured by separating. Because HMWP products are potentially immunogenic and not biologically active, low levels of HMWP are advantageous.

  The term “stabilized formulation” refers to a formulation having increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation 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 (in pregnancy) and other conditions that cause hyperglycemia. This term is used for metabolic disorders in which the pancreas produces insufficient amounts of insulin or somatic cells cannot respond properly to insulin and the cells cannot absorb glucose. As a result, glucose accumulates in the blood.

  Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by B-cell destruction and usually leads to absolute insulin deficiency.

  Type 2 diabetes, also known as non-insulin dependent diabetes mellitus (NIDDM) and adult type diabetes, is primarily insulin resistant and thus has a relative lack of insulin and / or has insulin resistance , Mainly accompanied by a decrease in insulin secretion.

  The term “pharmaceutically acceptable” as used herein means that it is suitable for standard pharmaceutical applications, ie does not cause any serious adverse events in the patient.

  The term “treatment of a disease” as used herein refers to the management and care of a patient who has developed a disease, condition or disorder, and includes the treatment, prevention or alleviation of a disease. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes administration of the active compound to eliminate or inhibit the disease, condition or disorder and to alleviate symptoms or complications associated with the disease, condition or disorder, and prevention of the disease, condition or disorder.

  In its broadest sense, the term “critically ill patient”, as used herein, is causing a life-threatening single-organ or multi-organ failure, or due to disease or injury, or Patients who are at risk of causing it acutely, patients who have complications during surgery, and those who have surgery in vital organs within 7 days or 7 days Refers to patients undergoing major surgery within. In a more limited sense, the term “severe patient”, as used herein, is suffering from a life-threatening single-organ or multi-organ system failure or acute Refers to patients who are at risk of having a complication, or who have complications during surgery. In a more limited sense, the term “severe patient”, as used herein, is suffering from a single or multiple organ system failure or is acutely caused by a disease or injury. Refers to patients at risk for Similarly, these definitions apply to similar expressions such as “critical illness in a patient” and “patient is critically ill”. Examples of critically ill patients include patients who require cardiac surgery, brain surgery, thoracic surgery, abdominal surgery, vascular surgery or transplant surgery or nervous system diseases, brain trauma, respiratory failure, peritonitis, multiple trauma or severe burns or severe burns A patient suffering from polyneuropathy.

  As used herein, the term “anabolic” refers to a series of metabolic pathways that make up a molecule from smaller units. These reactions require energy. One way to classify metabolic processes is to classify as "anabolic" or as the opposite "catabolic" at the cellular, organ or organism level. Assimilation is powered by catabolism that breaks up macromolecules into smaller parts and uses them up for respiration. Many anabolic processes are powered by adenosine triphosphate (ATP). The assimilation process works in the direction of “building up” organs and tissues. These processes cause cells to grow and differentiate, increasing body size, and certain processes are involved in the synthesis of complex molecules. Examples of anabolic processes include bone growth and petrification and increased muscle mass. Endocrinologists have traditionally classified hormones as anabolic or catabolic, depending on which part of the metabolism the hormone stimulates. The balance between assimilation and catabolism is also regulated by circadian rhythms, and processes such as glucose metabolism vary throughout the day to fit the animal's standard activity period. Some examples of the “anabolic” effects of these hormones are bone marrow stimulation that increases protein synthesis from amino acids, increases appetite, increases bone remodeling and growth, and increases red blood cell production. Through many mechanisms, anabolic hormones stimulate muscle cell formation, thus leading to an increase in skeletal muscle size, resulting in increased strength.

  In another embodiment, the insulin analogue according to the invention is used as a medicament for delaying or preventing the worsening of the disease in type 2 diabetes.

  In one embodiment of the invention, stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia including type 1 diabetes, and other diseases where burns, surgical wounds and anabolic effects are required for treatment Or an insulin formulation according to the present invention is provided for use as a medicament for the treatment or prevention of injury, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases.

  In further embodiments of the invention, stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia including type 1 diabetes, and other diseases where burns, surgical wounds and anabolic effects are required for treatment Or a method for the treatment or prevention of injury, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, in patients in need of such treatment, an effective amount for such treatment Of administering an insulin preparation according to the present invention.

  Treatment with an insulin preparation according to the invention is also caused by or associated with a second or more pharmacologically active substance, such as an antidiabetic agent, an antiobesity agent, an appetite regulating agent, an antihypertensive agent, diabetes Can be combined with agents selected from the agents for the treatment and / or prevention of complications and the agents for the treatment and / or prevention of complications and disorders resulting from or associated with obesity.

  Treatment with an insulin formulation according to the invention can also be combined with bariatric surgery, which is a surgery that affects glucose levels and / or lipid homeostasis, such as gastric banding or gastric bypass.

  The production of polypeptides, such as insulin, is well known in the art. Insulin analogues according to the present invention can be produced by way of example by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, e.g. Greene and Wuts , "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999. Insulin compounds contain a DNA sequence encoding the analog and comprise culturing a host cell capable of expressing the insulin analog in a suitable nutrient medium under conditions that permit expression of the insulin analog. It can also be produced by a method. For insulin analogs containing non-natural amino acid residues, the recombinant cell should be modified so that the non-natural amino acid is incorporated into the analog, for example by use of a tRNA variant. Thus, in short, the insulin analogues according to the invention are prepared in the same way as the preparation of known insulin analogues.

  Several methods can be used for the production of human insulin and human insulin analogs. For example, three main methods used for the production of insulin in microorganisms are disclosed in WO2008034881. Two of these involve Escherichia coli and the expression of giant fusion proteins in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross) ), Pierce Chemical Co., Rockford, III. 729-739) or the use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78: 5401-5404). . The third method utilizes Saccharomyces cerevisiae (Thim et al. (1986) PNAS 83: 6766-6770) to secrete insulin precursors into the medium. The prior art discloses many insulin precursors expressed in E. coli or Saccharomyces cerevisiae. See U.S. Patent No. 5,962,267, WO 95/16708, European Patent No. 0055945, European Patent No. 0163529, European Patent No. 0347845 and European Patent No. 0741188.

  Insulin analogs are produced by expressing the DNA sequence encoding the insulin analog in question in a suitable host cell by well-known techniques such as those disclosed in US Pat. No. 6,500,645. Is done. Insulin analogs are expressed directly or as precursor molecules with an N-terminal extension on the B chain or a C-terminal extension on the B chain. The N-terminal extension has the function of increasing the yield of the directly expressed product and can be up to 15 amino acid residues long. The N-terminal extension will be cleaved in vitro after isolation from the culture broth and therefore has a cleavage site adjacent to B1. In the present invention, suitable types of N-terminal extensions are disclosed in US Pat. No. 5,395,922 and European Patent 765,395. The C-terminal extension may have the function of protecting mature insulin or insulin analog molecules against intracellular proteolytic processing by host cell exoproteases. The C-terminal extension will be cleaved extracellularly by the secreted active carboxypeptidase in the culture broth or isolated in vitro after isolation from the culture broth. A method for producing mature insulin and insulin analogues having a C-terminal extension removed by carboxypetidase on the B chain is disclosed in WO 08037735. The target insulin product of this process can be a double-chain human insulin or a double-chain human insulin analog that may or may not have a short C-terminal extension of the B chain. If the target insulin product does not have a C-terminal extension of the B chain, the C-terminal extension can then be cleaved from the B chain before further purification steps. Should.

The present invention also contemplates a non-limiting list of the following embodiments, which are further described elsewhere herein.
1.
Insulin compounds,
An insulin preparation containing a nicotinic acid compound and arginine.
2. Insulin preparation according to embodiment 1, wherein the insulin compound is human insulin or an insulin analogue.
3. Insulin preparation according to embodiment 1 or 2, wherein the insulin compound is B28Asp human insulin, B3LysB29Glu human insulin or B28LysB29Pro human insulin.
4. The insulin preparation according to any one of embodiments 1 to 3, wherein the insulin compound is B28Asp human insulin.
5. The insulin preparation according to any one of embodiments 1 to 4, wherein the insulin compound is B3LysB29Glu human insulin or B28LysB29Pro human insulin.
6. The insulin preparation according to any one of embodiments 1 to 5, wherein the insulin compound is B28LysB29Pro human insulin.
7. The insulin preparation according to any one of claims 1 to 6, wherein the insulin compound is B3LysB29Glu human insulin.
8.Insulin compounds are: 0.1-10.0 mM; 0.1-3.0 mM; 0.1-2.5 mM; 0.1-2.0 mM; 0.1-1.5 mM; 0.2-2.5 mM; 0.2-2.0 mM; 0.2-1.5 mM; 0.3 Insulin preparation according to any of embodiments 1 to 7, present in a range selected from -3.0 mM; 0.3-2.5 mM; 0.3-2.0 mM; 0.3-1.5 mM; 0.5-1.3 mM and 0.6-1.2 mM .
9. Insulin formulation according to any of embodiments 1-8, wherein the insulin compound is present in an amount from about 0.1 mM to about 10.0 mM.
10. Insulin formulation according to any of embodiments 1 to 9, wherein the insulin compound is present in an amount from about 0.1 mM to about 3.0 mM.
11. The insulin formulation according to any of embodiments 1-10, wherein the insulin compound is present in an amount from about 0.1 mM to about 2.5 mM.
12. Insulin formulation according to any of embodiments 1-11, wherein the insulin compound is present in an amount from about 0.1 mM to about 2.0 mM.
13. Insulin formulation according to any of embodiments 1-12, wherein the insulin compound is present in an amount from about 0.1 mM to about 1.5 mM.
14. The insulin formulation according to any of embodiments 1-13, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.5 mM.
15. Insulin formulation according to any of embodiments 1 to 14, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.
16. Insulin formulation according to any of embodiments 1-15, wherein the insulin compound is present in an amount from about 0.2 mM to about 1.5 mM.
17. Insulin formulation according to any of embodiments 1 to 16, wherein the insulin compound is present in an amount from about 0.3 mM to about 3.0 mM.
18. Insulin formulation according to any of embodiments 1 to 17, wherein the insulin compound is present in an amount from about 0.3 mM to about 2.5 mM.
19. Insulin formulation according to any of embodiments 1-18, wherein the insulin compound is present in an amount from about 0.3 mM to about 2.0 mM.
20. Insulin formulation according to any of embodiments 1 to 19, wherein the insulin compound is present in an amount from about 0.3 mM to about 1.5 mM.
21. Insulin formulation according to any of embodiments 1 to 20, wherein the insulin compound is present in an amount from about 0.5 mM to about 1.3 mM.
22. The insulin formulation according to any of embodiments 1-21, wherein the insulin compound is present in an amount from about 0.3 mM to about 1.2 mM.
23. Insulin formulation according to any of embodiments 1-22, wherein the insulin compound is present in an amount from about 0.6 mM to about 1.2 mM.
24. Insulin formulation according to any of embodiments 1 to 23, wherein the insulin compound is present in an amount of about 0.6 mM, or about 1.2 mM.
25. Insulin preparation according to any of embodiments 1 to 24, wherein the insulin compound is present in an amount of about 0.3 mM.
26. Insulin formulation according to any of embodiments 1 to 25, wherein the insulin compound is present in an amount of about 0.6 mM.
27. Insulin formulation according to any of embodiments 1 to 26, wherein the insulin compound is present in an amount of about 0.9 mM.
28. The insulin formulation according to any of embodiments 1-27, wherein the insulin compound is present in an amount of about 1.2 mM.
29. Any of embodiments 1-28 wherein the nicotinic acid-based compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacinamide and vitamin B3 and / or a salt thereof and / or any combination thereof The insulin preparation described.
30. The insulin formulation according to any of embodiments 1-29, wherein the nicotinic acid-based compound is selected from nicotinamide and nicotinic acid and / or a salt thereof and / or any combination thereof.
31. The insulin preparation according to any one of embodiments 1 to 30, wherein the nicotinic acid-based compound is nicotinamide and / or a salt thereof.
32. The nicotinic acid-based compound according to any of embodiments 1-31, wherein the nicotinic acid-based compound is present in a range selected from the following: 1 to 300 mM; 5 to 200 mM; 40 to 120 mM, 70 to 140 mM, or 80 to 130 mM. Insulin preparation.
33. The insulin formulation according to any of embodiments 1-32, comprising from about 1 mM to about 300 mM nicotinic acid compound.
34. The insulin formulation according to any of embodiments 1-33, comprising from about 8 mM to about 260 mM nicotinic acid compound.
35. The insulin formulation according to any of embodiments 1-34, comprising from about 50 mM to about 250 mM nicotinic acid-based compound.
36. The insulin formulation of any of embodiments 1-35, comprising from about 80 mM to about 250 mM nicotinic acid-based compound.
37. Insulin preparation according to any of embodiments 1-36, comprising from about 80 mM to about 180 mM nicotinic acid compound.
38. The insulin formulation of any of embodiments 1-37, comprising from about 5 mM to about 200 mM nicotinic acid compound.
39. The insulin formulation according to any of embodiments 1-38, comprising from about 1 mM to about 150 mM nicotinic acid compound.
40. Insulin formulation according to any of embodiments 1-39, comprising from about 5 mM to about 20 mM nicotinic acid-based compound.
41. Insulin formulation according to any of embodiments 1 to 40, comprising from about 20 mM to about 120 mM nicotinic acid-based compound.
42. The insulin formulation according to any of embodiments 1-41, comprising from about 40 mM to about 120 mM nicotinic acid-based compound.
43. Insulin formulation according to any of embodiments 1-42, comprising from about 20 mM to about 40 mM nicotinic acid compound.
44. The insulin formulation according to any of embodiments 1-43, comprising from about 60 mM to about 80 mM nicotinic acid compound.
45. The insulin formulation according to any of embodiments 1-44, comprising from about 70 mM to about 140 mM nicotinic acid-based compound.
46. The insulin formulation according to any of embodiments 1-45, comprising from about 80 mM to about 130 mM nicotinic acid-based compound.
47. The insulin formulation according to any of embodiments 1-46, comprising about 8 mM, 30 mM, 100 mM, or 130 mM nicotinic acid compound.
48. Insulin preparation according to any of embodiments 1-47, which comprises about 8 mM nicotinic acid compound.
49. The insulin formulation according to any of embodiments 1-48, comprising about 30 mM, 100 mM or 130 mM nicotinic acid compound.
50. Insulin preparation according to any of embodiments 1 to 49, comprising about 30 mM nicotinic acid compound.
51. The insulin formulation according to any of embodiments 1-50, comprising about 80 mM nicotinic acid-based compound.
52. Insulin preparation according to any of embodiments 1 to 51, comprising about 100 mM nicotinic acid compound.
53. The insulin formulation of any of embodiments 1 to 52, comprising about 120 mM nicotinic acid compound.
54. The insulin formulation according to any of embodiments 1 to 53, comprising about 130 mM nicotinic acid compound.
55. Insulin preparation according to any of embodiments 1 to 54, comprising about 150 mM nicotinic acid compound.
56. The insulin formulation according to any of embodiments 1-55, comprising about 155 mM nicotinic acid-based compound.
57. Insulin preparation according to any of embodiments 1 to 56, comprising about 180 mM nicotinic acid compound.
58. Insulin preparation according to any of embodiments 1 to 57, comprising about 230 mM nicotinic acid compound.
59. Any of embodiments 1 to 58 comprising the following range: 1-100 mM, 5-120 mM, 8-85 mM, 20-90 mM, 30-90 mM, 30-85 mM, 30-60 mM or 10-40 mM arginine compound. An insulin preparation according to claim 1.
60. Insulin preparation according to any of embodiments 1 to 59, comprising the following ranges: 1 to 120 mM, 8 to 85 mM or 1 to 40 mM arginine compound.
61. Insulin formulation according to any of embodiments 1 to 60, comprising from about 1 mM to about 120 mM arginine.
62. Insulin formulation according to any of embodiments 1 to 61, comprising about 1 mM to about 100 mM arginine.
63. Insulin formulation according to any of embodiments 1 to 62, comprising from about 5 mM to about 120 mM arginine.
64. Insulin formulation according to any of embodiments 1 to 63, comprising from about 20 mM to about 90 mM arginine.
65. Insulin formulation according to any of embodiments 1 to 64, comprising from about 30 mM to about 85 mM arginine.
66. The insulin formulation according to any of embodiments 1-65, comprising from about 8 mM to about 85 mM arginine.
67. Insulin formulation according to any of embodiments 1 to 66, comprising from about 30 mM to about 60 mM arginine.
68. Insulin preparation according to any of embodiments 1 to 67, comprising about 10 mM to about 40 mM arginine.
69. The insulin formulation according to any of embodiments 1-68, comprising from about 10 mM to about 30 mM arginine.
70. The insulin formulation according to any of embodiments 1-69, comprising from about 10 mM to about 60 mM arginine.
71. Insulin formulation according to any of embodiments 1 to 70, comprising from about 1 mM to about 40 mM arginine.
72. Arginine is selected from the following: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, 45 mM, 50 mM, 55 mM or 60 mM 72. Insulin preparation according to any of embodiments 1 to 71, present in a range of
73. The insulin formulation of any of embodiments 1 to 72, comprising about 1 mM arginine.
74. The insulin formulation according to any of embodiments 1-73, comprising about 2 mM arginine.
75. The insulin formulation according to any of embodiments 1-74, comprising about 3 mM arginine.
76. Insulin formulation according to any of embodiments 1 to 75, comprising about 4 mM arginine.
77. Insulin formulation according to any of embodiments 1 to 76, comprising about 5 mM arginine.
78. Insulin formulation according to any of embodiments 1 to 77, comprising about 6 mM arginine.
79. Insulin formulation according to any of embodiments 1 to 78, comprising about 7 mM arginine.
80. Insulin formulation according to any of embodiments 1 to 79, comprising about 8 mM arginine.
81. Insulin preparation according to any of embodiments 1-80, comprising about 9 mM arginine.
82. Insulin formulation according to any of embodiments 1 to 81, comprising about 10 mM arginine.
83. Insulin formulation according to any of embodiments 1 to 82, comprising about 11 mM arginine.
84. The insulin formulation according to any of embodiments 1-83, comprising about 12 mM arginine.
85. Insulin formulation according to any of embodiments 1 to 84, comprising about 13 mM arginine.
86. The insulin formulation according to any of embodiments 1-85, comprising about 14 mM arginine.
87. Insulin preparation according to any of embodiments 1 to 86, comprising about 15 mM arginine.
88. Insulin preparation according to any of embodiments 1 to 87, comprising about 17 mM arginine.
89. The insulin formulation according to any of embodiments 1-88, comprising about 20 mM arginine.
90. The insulin formulation according to any of embodiments 1-89, comprising about 22 mM arginine.
91. Insulin formulation according to any of embodiments 1 to 90, comprising about 25 mM arginine.
92. The insulin formulation of any of embodiments 1 to 91, comprising about 30 mM arginine.
93. Insulin formulation according to any of embodiments 1 to 92, comprising about 35 mM arginine.
94. Insulin formulation according to any of embodiments 1 to 93, comprising about 40 mM arginine.
95. Insulin formulation according to any of embodiments 1 to 94, comprising about 45 mM arginine.
96. Insulin formulation according to any of embodiments 1 to 95, comprising about 50 mM arginine.
97. The insulin formulation of any of embodiments 1 to 96, comprising about 55 mM arginine.
98. Insulin preparation according to any of embodiments 1 to 97, comprising about 60 mM arginine.
99. The insulin formulation according to any of embodiments 1-98, further comprising a buffer.
100. The insulin formulation of embodiment 99, wherein the buffer is a phosphate buffer.
101. The insulin formulation of embodiment 100, comprising about 1 mg / mL to about 20 mg / mL phosphate buffer.
102. The insulin formulation of embodiment 100, comprising about 1 mg / mL to about 15 mg / mL phosphate buffer.
103. The insulin formulation of embodiment 100, comprising about 1 mg / mL to about 10 mg / mL phosphate buffer.
104. The insulin formulation of embodiment 100, comprising about 3 mg / mL phosphate buffer.
105. The insulin formulation of embodiment 99, wherein the buffer is Tris.
106. The insulin formulation of embodiment 105, comprising about 2 mM to about 50 mM Tris.
107. The insulin formulation of embodiment 105, comprising from about 10 mM to about 40 mM Tris.
108. The insulin formulation of embodiment 105, comprising about 20 mM to about 30 mM Tris.
109. The insulin formulation of embodiment 105, comprising about 10 mM, 20 mM, 30 mM or 40 mM Tris.
110. The insulin formulation of embodiment 105, comprising about 7 mM Tris.
111. The insulin formulation of embodiment 105, comprising about 10 mM Tris.
112. The insulin formulation of embodiment 105, comprising about 20 mM Tris.
113. The insulin formulation according to embodiment 105, comprising about 30 mM Tris.
114. Insulin preparation according to embodiment 105, comprising about 40 mM Tris.
115. The insulin formulation according to any of embodiments 1-114, further comprising a metal ion.
116. The insulin formulation according to embodiment 115, wherein the metal ion is zinc.
117. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is from about 0: 6 to about 6: 6.
118. Insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is from about 0: 6 to about 5: 6.
119. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is from about 1: 6 to about 6: 6.
120. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is from about 2: 6 to about 6: 6.
121. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is from about 2: 6 to about 5: 6.
122. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is from about 2.5: 6 to about 4.5: 6.
123. The insulin formulation of embodiment 116, wherein the zinc: insulin molar ratio is from about 3: 6 to about 4: 6.
124. The insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 2: 6.
125. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is about 2.5: 6.
126. Insulin preparation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3: 6.
127. The insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.1: 6.
128. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is about 3.2: 6.
129. The insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.3: 6.
130. The insulin formulation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.4: 6.
131. Insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.5: 6.
132. Insulin preparation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.6: 6.
133. Insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.7: 6.
134. The insulin formulation of embodiment 116, wherein the molar ratio of zinc: insulin is about 3.8: 6.
135. Insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 3.9: 6.
136. The insulin formulation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 4: 6.
137. Insulin preparation according to embodiment 116, wherein the molar ratio of zinc: insulin is about 4.5: 6.
138. The insulin formulation of embodiment 116, wherein the zinc: insulin molar ratio is about 5: 6.
139. The insulin formulation according to any one of embodiments 1-138, further comprising a stabilizer.
140. The insulin formulation of embodiment 139, wherein the stabilizer is a nonionic detergent.
141. Insulin formulation according to embodiment 140, wherein the detergent is polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80).
142. Insulin preparation according to embodiment 140, wherein the detergent is polysorbate 20 (Tween 20).
143. Insulin preparation according to embodiment 140, wherein the detergent is polysorbate 80 (Tween 80).
144. The insulin formulation of embodiment 140, comprising from about 5 to 100 ppm, from about 10 to about 50 ppm, or from about 10 to about 20 ppm polysorbate.
145. Insulin formulation according to any one of embodiments 1-144, further comprising one or more preservatives.
146. Insulin preparation according to embodiment 145, wherein the preservative is a phenolic compound.
147. Insulin formulation according to embodiment 146, wherein said phenolic compound is present in an amount from about 0 to about 6 mg / ml or from about 0 to about 4 mg / ml.
148. Insulin formulation according to embodiment 146, wherein said phenolic compound is present in an amount from about 5 to about 100 mM.
149. Insulin formulation according to embodiment 146, wherein said phenolic compound is present in an amount from about 5 to about 50 mM.
150. The insulin formulation of embodiment 146, wherein the phenolic compound is present in an amount from about 5 to about 30 mM.
151. The insulin formulation of embodiment 146, wherein the phenolic compound is present in an amount of about 16 mM.
152. The insulin formulation of embodiment 146, wherein the phenolic compound is present in an amount of about 14.4 mM.
153. Insulin preparation according to embodiment 145, wherein said preservative is m-cresol.
154. The insulin formulation of embodiment 153, wherein m-cresol is present in an amount from about 0.5 to about 4.0 mg / ml, or from about 0.6 to about 4.0 mg / ml.
155. The insulin formulation of embodiment 153, wherein m-cresol is present in an amount from about 5 to about 100 mM.
156. Insulin formulation according to embodiment 153, wherein m-cresol is present in an amount from about 5 to about 50 mM.
157. Insulin formulation according to embodiment 153, wherein m-cresol is present in an amount from about 5 to about 30 mM.
158. Insulin formulation according to embodiment 153, wherein m-cresol is present in an amount of about 16 mM.
159. Insulin formulation according to embodiment 153, wherein m-cresol is present in an amount of about 14.4 mM.
160. Insulin formulation according to any of embodiments 1-159, further comprising glycerol in an amount from about 0.5 to about 2.5%.
161. Insulin formulation according to any of embodiments 1-160, further comprising glycerol in an amount from about 0.7 to about 2.0%.
162. Insulin formulation according to any of embodiments 1-161, further comprising glycerol in an amount from about 1.0 to about 1.5%.
163. Insulin formulation according to any of embodiments 1-162, further comprising glycerol in an amount of about 1.25%.
164. Insulin preparation according to any of embodiments 1 to 163, wherein the pH is neutral to weakly basic.
165. Insulin formulation according to any of embodiments 1-164, wherein the pH is from about 6.8 to about 8.0.
166. Insulin formulation according to any of embodiments 1-165, wherein the pH is about 6.8.
167. Insulin formulation according to any of embodiments 1-166, wherein the pH is about 6.9.
168. Insulin formulation according to any of embodiments 1-167, wherein the pH is about 7.0.
169. Insulin formulation according to any of embodiments 1 to 168, wherein the pH is about 7.1.
170. Insulin formulation according to any of embodiments 1 to 169, wherein the pH is about 7.2.
171. Insulin formulation according to any of embodiments 1-170, wherein the pH is about 7.3.
172. Insulin formulation according to any of embodiments 1 to 171, wherein the pH is about 7.4.
173. Insulin formulation according to any of embodiments 1 to 172, wherein the pH is about 7.5.
174. Insulin formulation according to any of embodiments 1 to 173, wherein the pH is about 7.6.
175. Insulin formulation according to any of embodiments 1-174, wherein the pH is about 7.7.
176. Insulin formulation according to any of embodiments 1-175, wherein the pH is about 7.8.
177. Insulin formulation according to any of embodiments 1 to 176, wherein the pH is about 7.9.
178. Insulin formulation according to any of embodiments 1-177, wherein the pH is about 8.0.
179. A method of reducing blood glucose levels in a mammal by administering to a patient in need of treatment a therapeutically active dose of an insulin formulation according to any of embodiments 1 to 178.
180. A method for treating diabetes mellitus in a subject, comprising administering to the subject an insulin preparation according to any of embodiments 1 to 178.
181. The method of any of embodiments 179 to 180, for parenteral administration.
182. Stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia including type 1 diabetes, and other diseases or injuries that require burns, surgical wounds and anabolic effects to treat, myocardial infarction, 177. Insulin preparation according to any of embodiments 1 to 178, for use in the treatment or prevention of stroke, coronary heart disease and other cardiovascular diseases and the treatment of severely diabetic and non-diabetic patients.
183. Treatment of stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia, including type 1 diabetes, and burns, surgical wounds, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases or 187. Insulin preparation according to embodiment 182 for use in prevention.
184. Hyperglycemia The insulin formulation of embodiment 182 or 183 for use in the treatment of type 2 diabetes and type 1 diabetes.
185.
Insulin compounds,
・ Insulin preparations containing nicotinic acid compounds ・ Arginine and ・ Buffers.
186. Insulin preparation according to embodiment 185, wherein the insulin compound is human insulin or an insulin analogue.
187. Insulin formulation according to embodiment 185 or 186, wherein the insulin compound is selected from the group consisting of B28Asp human insulin, B3LysB29Glu human insulin and B28LysB29Pro.
188. Insulin preparation according to embodiment 186, wherein the insulin compound is B28Asp human insulin.
189. Insulin formulation according to any one of embodiments 185 to 188, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.
190. The insulin formulation of any one of embodiments 185 to 189, wherein the insulin compound is present in an amount from about 0.6 mM to about 1.2 mM.
191. Any one of Embodiments 185 through 190 wherein the nicotinic acid-based compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacinamide and vitamin B3 and / or salts thereof and / or any combination thereof. Insulin preparations described in 1.
192. Insulin formulation according to any one of embodiments 185 to 191, wherein the nicotinic acid-based compound is nicotinamide.
193. The insulin formulation of embodiment 192, comprising from about 1 mM to about 250 mM nicotinic acid-based compound.
194. The insulin formulation of embodiment 192, comprising from about 1 mM to about 250 mM nicotinic acid-based compound.
195. The insulin formulation of embodiment 192, comprising from about 80 mM to about 230 mM nicotinic acid-based compound.
196. Insulin formulation according to any one of embodiments 185 to 195, comprising from about 10 mM to about 60 mM arginine.
197. Insulin formulation according to any one of embodiments 185 to 196, comprising from about 10 mM to about 30 mM arginine.
198. The insulin formulation of any one of embodiments 185 to 197, comprising about 20 mM arginine.
199. Insulin formulation according to any one of embodiments 185 to 198, wherein the buffer is a phosphate buffer.
200. Insulin formulation according to any one of embodiments 185 to 199, wherein the buffer is Tris.
201. Insulin formulation according to any one of embodiments 185 to 200, which may further comprise a preservative, isotonic agent and / or stabilizer.
202. The insulin formulation according to any one of embodiments 185 to 201, further comprising a metal ion.
203. The insulin formulation of embodiment 202, wherein the metal ion is zinc.
204. The insulin formulation of embodiment 203, wherein the molar ratio of zinc: insulin is from about 0: 6 to about 3.5: 6.
205. The insulin formulation of embodiment 203, wherein the molar ratio of zinc: insulin is from about 2: 6 to about 3: 6.
206. The insulin formulation of any one of embodiments 185 to 205, having a pH of less than about 7.4.
207. Insulin formulation according to any one of embodiments 185 to 205, having a pH of about 7.4.
208. Insulin formulation according to any one of embodiments 185 to 205, having a pH of about 7.1.
209. An insulin formulation comprising B28Asp human insulin; nicotinamide; zinc; arginine; and phosphate buffer.
210. B28Asp human insulin is present at a concentration ranging from about 0.6 mM to about 1.2 mM, nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and arginine ranges from about 10 mM to about 40 mM. The insulin formulation of embodiment 209, present in concentration, wherein there are less than about 4 zinc ions per 6 B28Asp human insulin molecules, and having a pH of about 7.4 or less.
211.
a. B28Asp human insulin present at a concentration ranging from about 0.6 mM to about 1.2 mM;
b. Nicotinamide present at a concentration ranging from about 80 mM to about 260 mM;
c. Zinc present at less than about 4 zinc ions per 6 B28Asp human insulin molecules;
d. Arginine present at a concentration ranging from about 10 mM to about 30 mM; and
e. Insulin preparation made essential from phosphate buffer and having a pH of about 7.1.
212. A method for reducing blood glucose levels in a mammal by administering to the mammal in need of treatment a therapeutically active dose of an insulin formulation as described in any one of embodiments 1 to 211.
213. A method for the treatment of diabetes mellitus in a subject comprising administering to the subject the insulin formulation of any one of embodiments 1-21.
214. Stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia, including type 1 diabetes, and other diseases or injuries that require treatment for burns, surgical wounds and anabolic effects, myocardial infarction, Insulin preparation according to any one of embodiments 1 to 211, for use in the treatment or prevention of stroke, coronary heart disease and other cardiovascular disorders and the treatment of severely diabetic and non-diabetic patients.
215. Hyperglycemia The insulin formulation according to any one of embodiments 1-21, for use in the treatment of type 2 diabetes and type 1 diabetes.

Further embodiments of the invention relate to:
216.
Insulin compounds,
An insulin preparation containing a nicotinic acid compound and arginine.
217. Insulin preparation according to embodiment 216, wherein the insulin compound is human insulin or an insulin analogue.
218. Insulin preparation according to embodiment 216 or 217, wherein the insulin compound is B28Asp human insulin.
219. Insulin formulation according to any one of embodiments 216 to 218, wherein the insulin compound is B28LysB29Pro human insulin.
220. The insulin formulation according to any one of embodiments 216 to 219, wherein the insulin compound is B3LysB29Glu human insulin.
221. The insulin formulation of any one of embodiments 216 to 220, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.
222. Insulin formulation according to any one of embodiments 216 to 221, wherein the insulin compound is present in an amount from about 0.3 mM to about 1.2 mM.
223. Any one of Embodiments 216 through 222 wherein the nicotinic acid-based compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacinamide and vitamin B3 and / or salts thereof and / or any combination thereof. Insulin preparations described in 1.
224. Insulin preparation according to any one of embodiments 216 to 223, comprising from about 1 mM to about 150 mM nicotinic acid-based compound.
225. The insulin formulation of any one of embodiments 216 to 224, comprising about 85 mM arginine.
226. Insulin formulation according to any one of embodiments 216 to 225, further comprising metal ions, preservatives, isotonic and stabilizing agents, and buffers.
227. A method for reducing blood glucose levels in a mammal by administering to a patient in need of treatment a therapeutically active dose of an insulin formulation according to any one of embodiments 216 to 226.
228. A method for treating diabetes mellitus in a subject comprising administering to the subject an insulin preparation according to any one of embodiments 216 to 226.
229. Stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia, including type 1 diabetes, and other diseases or injuries that require treatment for burns, surgical wounds and anabolic effects, myocardial infarction, Insulin preparation according to any one of embodiments 216 to 226, for use in the treatment or prevention of stroke, coronary heart disease and other cardiovascular diseases and the treatment of severely diabetic and non-diabetic patients.

  The invention is further illustrated by the following examples, which should not be construed as limiting.

  All references cited in this specification, including publications, patent applications and patents, are to the same extent as if each reference was individually and specifically indicated to be incorporated by reference. And to the same extent as fully described herein (to the maximum extent permitted by law), the entire contents of which are hereby incorporated by reference.

  All headings and subheadings are used herein for convenience only and are not to be construed as limiting the invention in any way.

  The use of any and all examples or exemplary words (e.g., "such as") provided herein are intended only to better clarify the present invention, Unless otherwise specifically claimed, the scope of the present invention is not limited. 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.

(Example)
(Example 1)
Preparation of the pharmaceutical formulation The pharmaceutical formulation of the present invention may be prepared as an aqueous solution. The aqueous medium is made isotonic with, for example, sodium chloride or glycerol. In addition, the aqueous medium may contain zinc ions (eg, added as zinc acetate or zinc chloride), buffers and preservatives. Arginine can be added as Arg, HCl. The pH value of the formulation is adjusted to the desired value and can be between about 3 and about 8.5, between about 3 and about 5 or between about 6.5 and about 7.5, which is the isoelectric point of the insulin in question. , Determined by pI.

(Example 2)
Analysis of Insulin Chemical Stability Size Exclusion Chromatography High molecular weight protein (HMWP) and monomeric insulin aspart quantification were performed on Waters Insulin (300 x 7.8 mm, part number wat201549) on 2.5 M acetic acid, 4 mM L- The eluent containing arginine and 20% (V / V) acetonitrile was used at 40 ° C. at a flow rate of 1 ml / min. Detection was performed at 276 nm using a variable wavelength absorbance detector (Waters 486). The injection volume was 40 μl, which was a 600 μM human insulin standard. HMWP and formulation concentrations were measured at each sampling point.

Reversed phase chromatography (UPLC)
Determination of impurities related to insulin aspart using BEH RP C8, 2.1 x 100 mm column, particle size 1.7 μm, Waters part no 186002878, flow rate 0.5 ml / min, 40 ° C., detection at 220 nm Made on the UPLC system. Elution was performed using a mobile phase consisting of:
A. 10% (w / w) acetonitrile, 2.8% (w / w) sodium sulfate, 0.3% (w / w) o-phosphate, pH 3.5.
B. 70% (w / w) acetonitrile. Gradient: 0-11 minutes, A / B is 73% / 27% constant composition, 11-12 minutes is A / B linear change to 52% / 48%, 13-15 minutes is A / B Is a linear change to 73% / 27% and A / B is 73% / 27% isocratic gradient.

  The amount of B28 iso-aspartic acid, deamidates and other related impurities was determined as the absorbance area measured as a percentage of the total absorbance area, which was determined after elution of the preservative. The RP-UPLC method is equivalent to the analytical method used for quality control of insulin aspart drugs sold by Novo Nordisk.

  Addition of arginine reduces the amount of degradation products formed, especially HMWP and deamidated products. Increasing the concentration of arginine in the range of 10 to 50 mM further reduces degradation. The physical stability, measured as lag time in the ThT assay, decreases with the addition of arginine and decreases increasingly with increasing concentrations of arginine. As shown in Table 4 below, the overall capacity of 50 mM arginine is superior to 50 mM glycine, 50 mM glutamic acid or 50 mM histidine in terms of reducing degradation product formation.

  The insulin preparation of the present invention provides a fast-acting insulin preparation that is not only physically stable, but surprisingly also chemically stable.

(Example 3)
Pharmacokinetic (PK) / pharmacodynamic (PD) studies and plasma analysis assays in the LYD porcine model
PK / PD study in LYD pigs
The PK / PD study was conducted on domestic sows that were LYD hybrids weighing between 55 and 110 kg. The pigs were catheterized via the ear vein and into the jugular vein at least 2 days before the start of the study. The last diet before the start of the study was given to the animals approximately 18 hours before the injection of the test formulation, and the animals had free access to water during the fasting period and all times during the test period.

  At time 0, the test formulation was administered subcutaneously on the side of the neck. Blood samples were taken before dosing, and at regular time intervals after dosing, samples were taken from the catheter and placed in heparin precoated 1.5 ml glass tubes. The blood sample was kept in ice water until the plasma was separated by centrifugation. Centrifugation was performed at 3000 rpm for 10 minutes at 4 ° C. within the first 30 minutes. Plasma samples were stored at 4 ° C for a short time (2-3 hours) or at -18 ° C for long periods, analyzed for glucose by YSI or Konelab 30i and for insulin aspart concentrations by LOCI.

Luminescent Oxygen Channeling Immunoassay (LOCI) Method for Quantification of Insulin Aspart Insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay, europium-coated acceptor beads and streptavidin-coated donor The proximity of two beads, which are beads, is applied. The acceptor beads are coated with a specific antibody against human insulin and recognize insulin aspart in the plasma sample. The second biotinylated antibody specifically binds to insulin aspart, which makes up a sandwich with the beads coated with streptavidin. Illumination of the bead-aggregate-immunocomplex releases singlet oxygen from the donor bead that channels to the acceptor bead, causing chemiluminescence. Chemiluminescence is 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 formulations of the invention (FIGS. 3 and 4). Similarly, compared to NovoRapid®, the initial absorption rate of the insulin component of the formulation of the present invention is significantly faster (FIG. 5).

(Example 4)
General Overview of ThT Fibrosis Assays for Evaluation of Physical Stability of Protein Formulations Low physical stability of peptides can cause amyloid fibril formation, as an ordered filamentous polymer structure in the sample Observed and ultimately leads to the formation of a gel. This is traditionally measured by visual inspection of the sample. However, such measurements are very subjective and depend on the observer. Therefore, the application of small molecule indicator probes is much more advantageous. Thioflavin T (ThT) is such a probe that, when bound to a fiber, shows a distinct fluorescent signature [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol 309, pp. 274-284]. The time course of fiber formation can be described by a sigmoidal curve having the following formula [Nielsen et al. (2001) Biochemistry 40, 6036-6046].

Here, F is ThT fluorescence at time t. The constant t ₀ is the time required to reach 50% of maximum fluorescence. Two important parameters describing fiber formation are the lag time calculated by t ₀ -2τ and the apparent rate constant κ _app = 1 / τ.

  The formation of a partially folded peptide intermediate has been proposed as a general initiation mechanism for fibrosis. These intermediates can form nuclei and form a template on which further intermediates can gather, and fiberization hardly occurs. Lag time corresponds to the interval at which a critical amount of nuclei accumulates, and the apparent rate constant is the rate at which the fiber itself is formed.

Sample Preparation Samples were prepared fresh before each assay. The composition of each sample is described in each example. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HClO ₄ or HCl. Thioflavin T was added to the sample from a stock solution in H ₂ O to a final concentration of 1 μM.

  A 200 μl sample aliquot was placed in a 96-well microtiter plate (Packard OptiPlate ™ -96, white polystyrene). Typically, 4 or 8 replicates (corresponding to one test condition) for each sample were placed in a row of wells. The plate was sealed with Scotch Pad (Qiagen).

Incubation and fluorescence measurements Incubation at a given temperature, shaking and measurement of ThT fluorescence were performed in a Fluoroskan Ascent FL fluorescence plate reader or Varioskan plate reader (Thermo Labsystems). The temperature was adjusted to 37 ° C. Orbital shaking was adjusted to 960 rpm with 1 mm amplitude in all presented data. Fluorescence measurements were made using excitation through a 444 nm filter and emission measurement through a 485 nm filter.

  Each run was initiated by incubating the plate at the assay temperature for 10 minutes. Plates were measured every 20 minutes for the desired period. 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 were performed using GraphPad Prism. Background emission from ThT in the absence of fiber was negligible. Data points are typically the average of 4 or 8 samples and are shown with standard deviation error bars. Only data obtained in the same experiment (ie samples on the same plate) are presented in the same graph, ensuring relative measurements of fibrosis during the experiment.

  The data set can be fitted to equation (1). However, a complete sigmoidal curve is not always obtained during the measurement time, and in this specification, the lag time was visually determined from the ThT fluorescence curve as the time when the ThT fluorescence was different from the background level.

Measurement of Initial and Final Concentrations The peptide concentration in each of the formulations tested was measured both before application to the ThT fibrosis assay (“initial”) and after completion of ThT fibrosis (“after ThT assay”). Concentration was determined by reverse HPLC method using pramlintide standard as reference. Before measurement after completion, 150 μl was taken from each of the replicates and transferred to an Eppendorf tube. These were centrifuged at 30000G for 40 minutes. The supernatant was filtered through a 0.22 μm filter and then applied to the HPLC system.

Claims (32)

Insulin compounds,
A nicotinic acid compound and an insulin preparation containing arginine.   2. The insulin preparation according to claim 1, wherein the insulin compound is human insulin or an insulin analogue.   The insulin preparation according to claim 1 or 2, wherein the insulin compound is selected from the group consisting of B28Asp human insulin, B3LysB29Glu human insulin, and B28LysB29Pro.   4. The insulin preparation according to any one of claims 1 to 3, wherein the insulin compound is B28Asp human insulin.   5. The insulin formulation according to any one of claims 1 to 4, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.   6. Insulin formulation according to any of claims 1 to 5, wherein the insulin compound is present in an amount from about 0.6 mM to about 1.2 mM.   The nicotinic acid-based compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacinamide and vitamin B3 and / or a salt thereof and / or combinations thereof, according to any one of claims 1 to 6. Insulin preparation.   The insulin preparation according to any one of claims 1 to 7, wherein the nicotinic acid compound is nicotinamide.   9. The insulin formulation according to any one of claims 1 to 8, comprising from about 1 mM to about 250 mM nicotinic acid compound.   10. Insulin preparation according to any one of claims 1 to 9, comprising from about 1 mM to about 250 mM nicotinic acid compound.   11. The insulin preparation according to any one of claims 1 to 10, comprising from about 80 mM to about 230 mM nicotinic acid compound.   12. Insulin preparation according to any one of the preceding claims, comprising from about 10 mM to about 60 mM arginine.   13. Insulin preparation according to any one of the preceding claims, comprising from about 10 mM to about 30 mM arginine.   14. Insulin preparation according to any one of the preceding claims, comprising about 20 mM arginine.   15. Insulin preparation according to any one of claims 1 to 14, further comprising one or more buffering agents.   16. The insulin preparation according to claim 15, wherein the buffer is a phosphate buffer.   16. The insulin preparation according to claim 15, wherein the buffer is Tris.   The insulin preparation according to any one of claims 1 to 17, further comprising a preservative, an isotonic agent and / or a stabilizer.   The insulin preparation according to any one of claims 1 to 18, further comprising a metal ion.   The insulin preparation according to claim 19, wherein the metal ion is zinc.   21. The insulin formulation of claim 20, wherein the molar ratio of zinc: insulin is from about 0: 6 to about 3.5: 6.   21. The insulin formulation of claim 20, wherein the molar ratio of zinc: insulin is from about 2: 6 to about 3: 6.   23. Insulin preparation according to any one of claims 1 to 22, having a pH of less than 7.4.   24. Insulin preparation according to any one of claims 1 to 23, having a pH of about 7.4.   25. Insulin preparation according to any one of claims 1 to 24, having a pH of about 7.1.   An insulin formulation comprising B28Asp human insulin, nicotinamide, zinc, arginine, and phosphate buffer.   B28Asp human insulin is present at a concentration ranging from about 0.6 mM to about 1.2 mM, nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and arginine is present at a concentration ranging from about 10 mM to about 40 mM. 27. Insulin formulation according to claim 26, present, wherein less than about 4 zinc ions are present per 6 B28Asp human insulin molecules and have a pH of about 7.4 or less. a. B28Asp human insulin present at a concentration ranging from about 0.6 mM to about 1.2 mM;
b. Nicotinamide present at a concentration ranging from about 80 mM to about 260 mM;
c. Zinc present at less than about 4 zinc ions per 6 B28Asp human insulin molecules;
d. Arginine present at a concentration ranging from about 10 mM to about 30 mM; and
e. Insulin preparation made essential from phosphate buffer and having a pH of about 7.1.   30. A method of reducing blood glucose levels in a mammal by administering to a mammal in need of treatment a therapeutically active dose of an insulin formulation according to any one of claims 1 to 28.   29. A method for treating diabetes mellitus in a subject, comprising administering to the subject the insulin preparation according to any one of claims 1 to 28.   Stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, hyperglycemia, including type 1 diabetes, and other diseases or injuries that require treatment of burns, surgical wounds and anabolic effects, myocardial infarction, stroke, 29. Insulin preparation according to any one of claims 1 to 28 for use in the treatment or prevention of coronary heart disease and other cardiovascular diseases and the treatment of severely diabetic and non-diabetic patients.   32. Insulin preparation according to claim 31, for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.

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