KR20140030125A - Preparation comprising insulin, nicotinamide and an amino acid - Google Patents

Abstract

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

Description

Formulations comprising insulin, nicotinamide and amino acids {PREPARATION COMPRISING INSULIN, NICOTINAMIDE AND AN AMINO ACID}

The present invention relates to pharmaceutical formulations comprising insulin compounds, nicotine compounds, and amino acids.

Diabetes is a metabolic disorder in which the ability to use glucose is partially or completely lost. About 5% of the population is diabetic, and the disease is approaching the epidemic proportion.

Since the introduction of insulin in the 1920s, the treatment of diabetes has continued to improve. To prevent blood glucose levels from rising, diabetic patients are given several injections, usually with insulin at each meal. Since diabetics have been treated with insulin for decades, they need an insulin preparation that is safe and improves their quality of life. Among commercially available insulin preparations, mention may be made of fast-acting, intermediate-acting, and sustained-release preparations.

Many different pharmaceutical preparations of insulin have been proposed and used in the treatment of diabetes, for example, standard insulin (eg Actrapid ^® ), isophan insulin (called NPH), insulin zinc suspension (eg Semilente) ^® , Lente ^® and Ultralente ^® ), and biphasic isopaninsulin (eg NovoMix ^® ). Human insulin analogues and derivatives designed for specific action profiles, ie fast or sustained release, have also been developed. Such short-acting any of the commercially available insulin preparations containing the insulin analogues It includes NovoRapid ^® (B28Asp human insulin preparations), Humalog ^® (B28LysB29Pro human insulin preparations), and Apidra ^® (B3LysB29Glu human insulin preparations).

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 EP1283051 discloses insulin preparations containing so-called arginine, wherein arginine is used as a buffer. EP1283051 discloses an improvement in the physical stability of so-called insulin preparations.

Insulin pharmaceutical preparations are most often administered by subcutaneous injection. The action profile of insulin is important for the patient, which means the action of insulin on glucose metabolism as a function of time from the time of injection. Of particular importance in this profile are the time of onset, the maximum of action, and the overall duration of action. For bolus insulin various insulin preparations with different action profiles are desired and required by the patient. Some patients may use insulin preparations with very different action profiles the same day. For example, the desired action profile depends on the time of day and the amount and composition of food consumed by the patient.

The chemical stability of the insulin preparation is equally important to the patient, for example, because it uses many pen-type injection devices, such as devices containing Penfill ^® cartridges, in which the insulin preparation is stored until the entire cartridge is empty. This period may take at least 1-2 weeks for devices containing 1.5-3.0 mL cartridges. Covalent chemical changes in insulin structure occur during storage. This can lead to the formation of less active and / or potentially immunogenic molecules such as deamidation products and high molecular weight conversion products (dimers, polymers). In addition, the physical stability of the insulin preparation is important because prolonged storage can ultimately result in the formation of insoluble fibrils that are biologically inactive and potentially immunogenic.

The present invention relates to insulin preparations having an advantageous rate of absorption and advantageous chemical and physical stability. The present invention relates to insulin preparations comprising human insulin and / or its analogs, nicotinamide or nicotinic acid and / or salts thereof, and arginine.

In one embodiment, the present invention provides

Insulin compounds,

Nicotine compounds, and

● relates to insulin preparations containing arginine.

In another embodiment, the invention also relates to a method for the treatment of diabetes in a subject or to reducing blood glucose levels in a subject comprising administering an insulin preparation according to the invention to a subject or mammal.

1 shows the development of the percentage of degradation products in the total insulin content during two weeks storage of the formulations according to the invention at 37 ° C. Letter A represents the NovoRapid ^® criterion and the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. The addition of nicotinamide (Formulations B and D) increased the formation of degradation products compared to the NovoRapid ^® formulation (Formulation A), but the combined addition of nicotinamide, glutamic acid, and arginine (Formulations C and E) mostly resulted in similar degradation patterns. And low HMWP formation.
2 shows the development of percent degradation products in total insulin content during two weeks storage of the formulations according to the invention at 37 ° C. Letter A represents the NovoRapid ^® criterion and the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Formulations F, G, H and I are added with a combination of nicotinamide, glutamic acid, and arginine and the buffer system is different as phosphate or tris buffer and the concentrations of insulin and Zn are 0.6 mM and 0.3 mM or 1.2 mM and 0.6 mM. Has a degradation pattern similar to Formulation A, which is a NovoRapid® formulation.
Figure 3 shows the plasma glucose concentration (mean +/- SEM, N = 8) after subcutaneous injection of the preparations according to the invention into pigs at a dose of 1 nmol / kg at 0 min. Letter A represents the NovoRapid ^® criterion and the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Compared to the NovoRapid ^® formulation (Formulation A), the initial rate of decrease in plasma glucose was faster in the formulation with nicotinamide (Formulation N) and faster in the combination formulation of nicotinamide and arginine (Formulation M).
Figure 4 shows the plasma glucose concentration (mean +/- SEM, N = 7) after subcutaneous injection of the preparations according to the invention to pigs at a dose of 1 nmol / kg at 0 min. Letter A represents the NovoRapid ^® criterion and the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Compared to the NovoRapid® formulation (Formulation A), the initial rate of degradation of plasma glucose is faster in the combination formulation of nicotinamide, arginine, and glutamic acid (Formulation L) and the combination formulation of nicotinamide and arginine (Formulation K).
FIG. 5 shows the plasma insulin aspart concentration (mean +/- SEM, N = 7) after the subcutaneous injection of the preparations according to the invention into pigs at a dose of 1 nmol / kg at 0 min. Letter A represents the NovoRapid ^® criterion and the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Compared to the NovoRapid ^® formulation (Formulation A), the initial stage of the insulin component of the nicotinamide formulation (Formulation J), the combination formulation of nicotinamide and arginine (Formulation K), the combination formulation of nicotinamide, arginine, and glutamic acid (Formulation L) Absorption rate is remarkably fast.
Fig. 6 shows the plasma glucose concentration (mean +/- SEM, N = 8) after subcutaneous injection of the preparations according to the invention into pigs at a dose of 1 nmol / kg at 0 min. The letter A represents the NovoRapid ^® criterion and the number 11 corresponds to the insulin aspart formulations described in Table 3 of Example 1. Compared to the NovoRapid ^® formulation (Formulation A), the initial rate of degradation of plasma glucose is faster in the combination formulation of nicotinamide and arginine (Formulation 11).
7 shows the plasma insulin aspart concentration (mean +/- SEM, N = 8, twice for each pig) after subcutaneous injection of the preparations according to the invention into pigs at a dose of 1 nmol / kg at 0 min. . The letter A represents the NovoRapid ^® criterion and the number 11 corresponds to the insulin aspart formulations described in Table 3 of Example 1. Compared to the NovoRapid ^® formulation (Formulation A), the initial absorption rate of the insulin component of the formulation with nicotinamide and arginine (Formulation 11) is significantly faster.

Absorption following subcutaneous injection of insulin compounds in the insulin formulations of the present invention has been surprisingly found to be faster than the reference insulin formulation. This property is useful in fast-acting insulins, particularly in connection with several injection regimens in which insulin is given before each meal. Since the onset of action is faster, insulin can be conveniently taken closer to mealtime than conventional fast-acting insulin solutions. In addition, a faster loss of insulin will probably reduce the risk of hypoglycemia after eating.

The insulin preparation of the present invention is a fast-acting insulin preparation comprising an insulin compound such as insulin aspart, a nicotine compound such as nicotinamide, and the amino acid arginine. Optionally, the insulin preparation of the present invention may further comprise amino acids. These insulin preparations have a fast absorption profile that more closely mimics normal physiology than existing therapies. In addition, the insulin formulations of the present invention have chemical and physical stability suitable for commercial pharmaceutical formulations.

The insulin preparations of the present invention not only are physically stable but also surprisingly chemically stable fast-acting insulin preparations. Insulin formulations of the invention provide much faster onset of action compared to existing insulin therapies. Such super fast-acting insulin preparations have the advantage of restoring the first stage insulin release, injection convenience, and blocking hepatic glucose production. Insulin formulations of the present invention have a subcutaneous cell layer with an initial absorption rate increase of 1.5 to 5 times as compared to conventional formulations such as NovoRapid ^® as suggested by several PK / PD experiments in pigs. Has an advantageous rate of absorption into plasma. This fast rate of absorption can improve plasma control and convenience and allow for the transition from pre-meal to post-meal dosages. The present invention is based in part on the surprising finding that even though the addition of nicotinamide allows for an increase in the rate of absorption, it also negatively affects chemical stability by significantly increasing the amount of HMWP. Insulin formulations of the present invention have improved chemical stability by the addition of arginine, which is reflected, for example, as the formation of dimers and polymers and des-amido insulin after storage is reduced. In addition, the insulin formulations of the present invention exhibit improved physical stability, which may be useful for use in pumps.

The present invention provides an insulin preparation comprising the insulin compound according to the invention which is present at a concentration of about 0.1 mM to about 10.0 mM, wherein the preparation has a pH of 3 to 8.5. Formulations also include nicotine compounds and arginine. The formulation may further comprise protease inhibitor (s), metal ions, buffer system, preservative (s), tonicity agent (s), chelating agent (s), stabilizers, and surfactants.

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

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

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

The content of B28Asp human insulin in the solution of the present invention may be in the range of 15 to 500 international units (IU) / mL, preferably 50 to 333 IU / mL in the preparation for injection. However, the content of insulin compound may be higher for other parenteral administration.

The unit "IU" in this context corresponds to 6 nmol.

The term “insulin aspart” refers to human insulin analogue B28Asp human insulin.

The term "initiation" refers to the time from the point of injection until the PK curve turns to increase.

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

An "insulin compound" according to the invention herein is understood as human insulin, insulin analogues and / or any combination thereof.

As used herein, the term “human insulin” refers to human hormones whose structure and properties are well known. Human insulin has two polypeptide chains, A-chain and B-chain, connected by disulfide bridges between cysteine residues. The A-chain is 21 amino acid peptides, the B-chain is 30 amino acid peptides, the two chains are linked by three disulfide bridges, the first is between the cysteines at positions 6 and 11 of the A-chain, The second is between the cysteine at position 7 of the A-chain and the cysteine at position 7 of the B-chain, and the third is between the cysteine at position 20 of the A-chain and the cysteine at position 19 of the B-chain. Is in.

This hormone is synthesized as a short chain-precursor proinsulin (preproinsulin) consisting of 24 amino acids of prepeptide followed by 86 amino acids of proinsulin, and is arranged in the form of prepeptide-B-ArgArg-C-LysArg. -A, where C is a linking peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of linking peptides from the A and B chains.

As used herein, “insulin analogue” refers to a polypeptide derived from the primary structure of naturally occurring insulin, eg, the primary structure of human insulin, by mutation. One or more mutations are made by deleting and / or replacing at least one amino acid residue occurring in naturally occurring insulin and / or by adding at least one amino acid residue. The added and / or substituted amino acid residues may be codeable amino acid residues or other naturally occurring amino acid residues.

In one embodiment the insulin analogue is less than 8 variants (substitution, deletion, addition and any combination thereof) relative to the parental insulin, alternatively less than 7 variants relative to the parental insulin, alternatively less than 6 compared to the parental insulin , Less than 5 strains relative to the parental insulin, alternatively less than 4 strains relative to the parental insulin, alternatively less than 3 strains relative to the parental insulin, alternatively less than 2 strains relative to the parental insulin It includes.

Mutations in the insulin molecule are indicated by describing the chain (A or B), position, and the three letter code of the amino acid that substituted the natural amino acid. "desB30" or "B (1-29)" means a native insulin B chain or analog thereof lacking a B30 amino acid residue, wherein B28Asp human insulin is obtained by replacing the amino acid residue at position 28 of the B chain with Asp It means human insulin.

Examples of insulin analogues are wherein the Pro at position 28 of the B chain is mutated to Asp, Lys, Leu, Val or Ala, and / or the Lys at position B29 is mutated to Pro, Glu or Asp. Furthermore, Asn at position B3 can be mutated to Thr, Lys, Gln, Glu or Asp. The amino acid residue at position A21 may be mutated to Gly. The amino acid at position B1 may be mutated to Glu. The amino acid at position B16 may be mutated to Glu or His. Still other examples of insulin analogues are deletion analogs, eg, analogs lacking the B30 amino acid of human insulin (des (B30) human insulin), analogues lacking the B1 amino acid of human insulin (des (B1) human insulin ), des (B28-B30) human insulin, and des (B27) human insulin. Insulin analogues in which the A- and / or B-chains have N-terminal expansion and insulin analogues in which the A and / or B chains have C-terminal expansion, for example, two arginine at the C-terminus of the B chain Those with residues added are also examples of insulin analogues. Still other examples are insulin analogs that include a combination of the mentioned mutations. Wherein the insulin analogue wherein 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 another example of an insulin analogue . Also insulin analogues of human insulin where the amino acid residue at position A21 is Gly and the insulin analogue is further extended by two arginine residues at the C-terminus are also examples of insulin analogues.

Other examples of insulin analogs include DesB30 human insulin; AspB28 human insulin; AspB28, desB30 human insulin; LysB3, GluB29 human insulin; LysB28, ProB29 human insulin; Gly A21, ArgB31, ArgB32 human insulin; GluA14, HisB25 human insulin; HisA14, HisB25 human insulin; GluA14, HisB25, desB30 human insulin; HisA14, HisB25, desB30 human insulin; GluA14, HisB25, des B27, des B28, des B29, des B30 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 "nicotine 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 nicotine compounds and / or their salts 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 acids arginine and / or salts thereof, 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 1-20 mM arginine.

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

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

The term "glutamic acid" or "Glu" includes amino acid glutamic acid and / or salts thereof.

As used herein, the term “pharmaceutical formulation” or “insulin formulation” refers to insulin compounds, ie, human insulin, analogs and / or combinations thereof, and nicotine compounds and amino acids, optionally with preservatives, chelating agents, enteric modifiers, bulking agents. , Products comprising together with other excipients such as stabilizers, antioxidants, polymers and surfactants, metal ions, oily vehicles and proteins (eg, human serum albumin, gelatin or proteins), wherein the insulin preparation is a human It is useful to treat, prevent or reduce the severity of a disease or disorder by administering the insulin preparation to the patient. Thus, insulin preparations are also known in the art as pharmaceutical preparations or pharmaceutical compositions.

Buffers include but are not limited to sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and tris (hydroxymethyl) -aminomethane (tris), bisine, tricin, malic acid, succinate, male Acid, fumaric acid, tartaric acid, aspartic acid, ethylenediamine, or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

Insulin formulations of the invention may further comprise other components common to insulin formulations, 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 0-25OmM, 0-20OmM or 0-10OmM.

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

Insulin formulations of the invention may further comprise a pharmaceutically acceptable preservative. Preservatives may be present in an amount sufficient to achieve a preservative effect. The amount of preservative in the insulin preparation can be determined, for example, from the literature in the art and / or known amount (s) of the preservative in, for example, a commercial product. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of preservatives in pharmaceutical formulations is described, for example, in Remington: The Science and Practice of Pharmacy , 19 ^th edition, 1995.

Preservatives present in the insulin formulations of the present invention can be, for example, phenol, m-cresol and methylparaben, as used in conventional insulin formulations to date.

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

Insulin formulations of the present invention may further comprise a stabilizer. As used herein, the term “stabilizer” refers to a chemical added to a polypeptide containing pharmaceutical formulation to stabilize the peptide, ie, to increase the shelf life and / or shelf life of such formulation. For your convenience, Remington: The Science and Practice of See Pharmacy , 19 ^th edition, 1995.

Insulin formulations of the invention may further comprise a surfactant. As used herein, the term "surfactant" refers to any molecule or ion consisting of the head and the fat-soluble (lipophilic) portion, which is a water-soluble (hydrophilic) portion. The surfactant preferably accumulates at the interface and the hydrophilic portion is oriented towards the water (hydrophilic phase) and the lipophilic portion is oriented towards the oil- or hydrophobic phase (ie glass, air, oil, etc.). The concentration at which the surfactant begins to form micelles is called the critical micelle concentration or CMC. Surfactants also lower the surface tension of the liquid. Surfactants are also referred to as amphipathic compounds. The term "detergent" is generally used as a synonym for surfactant. The use of surfactants in pharmaceutical formulations is well known to those skilled in the art. For your convenience, Remington: The Science and Practice of See Pharmacy , 19 ^th edition, 1995.

In another embodiment, the present invention relates to an insulin preparation comprising an aqueous solution and a buffer of the insulin compound of the present invention, wherein the insulin compound is present at a concentration of at least 0.1 mM, wherein the preparation is at room temperature (˜25 ° C.). At a pH of about 3.0 to about 8.5.

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

In one embodiment, the method of making an insulin formulation of the present invention is:

a) dissolving the insulin compound or mixture of insulin compounds in water or buffer to prepare a solution;

b) dissolving divalent metal ions in water or a buffer to prepare a solution;

c) dissolving the preservative in water or buffer to prepare a solution;

d) dissolving the tonicity agent in water or buffer to prepare a solution;

e) dissolving the surfactant and / or stabilizer in water or buffer to prepare a solution;

f) mixing solution a) with at least one of solutions b), c), d) and e).

.

Finally, the pH of the mixture obtained in f) is sterile filtered after adjusting the desired pH.

In one embodiment, the method of making an insulin formulation of the present invention is:

a) preparing separate stock solutions of preservatives, isotonic agents, metal ion salts, arginine or arginine salts and nicotine compounds;

b) adding the stock solution (s) of the preservative or mixture of preservatives to water or a buffer to prepare a solution;

c) adding a stock solution of an isotonic agent to solution b);

d) dissolving the insulin compound or mixture of insulin compounds in water or buffer to prepare a solution; Adding HCl to acidify this solution;

e) adding a stock solution of a metal ion salt to d);

f) adding and stirring e) to c);

g) adding and stirring stock solution of arginine or arginine salt to f);

h) adding a stock solution of nicotine compound to g) and adjusting the pH to the desired pH with NaOH / HCl

.

In one embodiment, the method of making an insulin formulation of the present invention is:

a) preparing preservatives, isotonic agents, isolated stock solutions of metal ion salts, and mixed stock solutions of arginine or arginine salts and nicotine compounds;

b) adding the stock solution (s) of the preservative or mixture of preservatives to water or a buffer to prepare a solution;

c) adding a stock solution of an isotonic agent to solution b);

d) dissolving the insulin compound or mixture of insulin compounds in water or buffer to prepare a solution; Adding HCl to acidify this solution;

e) adding a stock solution of a metal ion salt to d);

f) adding and stirring e) to c);

g) adding and stirring arginine or a mixed stock solution of arginine salt and nicotine compound to f);

h) adjusting the pH of g) to the desired pH with NaOH / HCl

.

Insulin formulations of the invention can be used for the treatment of diabetes by parenteral administration. It is recommended that the dosage of the insulin formulations of the invention administered to the patient be selected by the physician.

Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by syringe, optionally a pen type syringe. Alternatively, parenteral administration can be performed by an infusion pump. Insulin formulations containing the insulin compounds of the invention as a further option are suitable for transdermal administration, for example, by needleless injection or from patches, optionally iontophoretic patches, or transmucosal administration, eg buccal administration. Can be done.

Insulin preparations according to the invention can be applied at several sites, for example at topical sites, for example at the skin and mucous membranes, at sites where absorption is bypassed (eg, arterial, venous, cardiac administration) and Administration to a patient in need of such treatment may be by administration to a site that includes absorption (eg, skin, subcutaneous, muscle or abdominal administration).

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

The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w of water. Likewise the term "aqueous solution" is defined as a solution comprising at least 50% w / w of water and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w of water.

Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions.

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

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

As used herein, the term "physical stability" of an insulin preparation is a biologically inert state of a protein as a result of exposure to thermo-mechanical stress and / or as a result of interaction with destabilized interfaces and surfaces such as hydrophobic surfaces and interfaces. Refers to the tendency of proteins to form aggregates that are phosphorus and / or insoluble. The physical stability of the aqueous protein formulation is determined by visual inspection and / or after exposure of the formulation filled in a suitable container (eg cartridge or vial) to mechanical / physical stress (eg agitation) at different temperatures for various time periods. Evaluated by turbidity measurement. Visual inspection of the formulation is carried out in a sharply concentrated light under a dark background. The turbidity of an agent is specified by its visual score, for example, ranking the turbidity on a scale of 0 to 3 (an agent that does not exhibit turbidity corresponds to a visual score of 0, and an agent that exhibits visual turbidity under daylight has a visual score). 3). Formulations are classified as physically unstable with regard to protein aggregation if they show visual turbidity under sunlight. Instead the turbidity of the formulation can be assessed by simple turbidity measurements well known to those skilled in the art. In addition, the physical stability of aqueous protein preparations can be assessed using spectroscopic preparations or probes for the conformational state of the protein. The probe is preferably a small molecule that preferentially binds to the non-natural conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is thioflavin T. Thioflavin T is a fluorescent dye widely used for the detection of amyloid fibrils. If fibrils are present and possibly other protein forms are also present, thioflavin T will show a new maximum excitation at about 450 nm and increased emission at about 482 nm when bound to the fibrillar protein form. Unbound thioflavin T is essentially non-fluorescent at this wavelength.

As used herein, the term “chemical stability” of a protein preparation refers to a change in covalent protein structure that results in the formation of chemical degradation products with potential lowered biological potency and / or potential increased immunogenicity compared to native protein structure. Say. Various chemical degradation products can be formed depending on the type and nature of the natural protein and the environment to which the protein is exposed. An increase in the amount of chemical degradation products is seen primarily during the storage and use of protein preparations. Most proteins exhibit a tendency to deamidate, which is the process of hydrolysis of the side chain amide groups in glutamine or asparaginyl residues to form free carboxylic acids, or hydrolysis of asparaginyl residues to form IsoAsp derivatives. . Another degradation pathway involves the formation of high molecular weight products in which two or more protein molecules are covalently bonded to each other through a transamidation and / or disulfide interaction, which leads to the formation of covalently bound dimers, oligomers and polymer degradation products ( Stability of Protein Pharmaceuticals , Ahern . TJ & Manning M. C, Plenum Press, New York 1992 ). As another variant of chemical degradation, oxidation (eg of methionine residues) can be mentioned. Chemical stability of protein preparations can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions (formation of degradation products can often be accelerated, for example, by increasing temperature). The amount of each individual degradation product is often determined by using different chromatography techniques (eg SEC-HPLC and / or RP-HPLC) to separate the degradation products according to molecular size and / or charge. Low levels of HMWP are advantageous because HMWP products are potentially immunogenic and not biologically active.

The term "stabilized formulation" refers to a formulation having increased physical stability, increased chemical stability or increased physicochemical stability. In general, the formulation should be stable during use and storage (if recommended conditions of use and storage) 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 conditions that cause hyperglycemia. The term is used in metabolic disorders in which the pancreas produces insufficient amounts of insulin, or cells in the body do not properly respond to insulin, making the cells unable to absorb glucose. As a result, glucose accumulates in the blood.

Type 1 diabetes is also referred to as insulin-dependent diabetes (IDDM) and pediatric diabetes and is caused by B-cell destruction and generally results in absolute insulin deficiency.

Type 2 diabetes is also referred to as non-insulin-dependent diabetes mellitus (NIDDM) and adult diabetes, and is associated with significant insulin resistance and thus with predominant insulin secretion defects accompanied by relative insulin deficiency and / or insulin resistance.

As used herein, the term “pharmaceutically acceptable” means that it is suitable for regular pharmaceutical use, ie does not cause any serious side effects in the patient.

As used herein, the term "treatment of a disease" refers to managing and caring for a disease, condition or diseased patient, and includes treating, preventing or alleviating a disease. The purpose of treatment is to fight a disease, condition or illness. Treatment includes the elimination or control of a disease, condition or disorder, the alleviation of symptoms or complications associated with the disease, condition or illness, and the administration of an active compound for the prevention of the disease, condition or illness.

In a broad sense, as used herein, the term “severely ill patient” refers to a patient who is at risk of, or at risk of, a single or multiple organ system arrest, which is acutely life-threatening due to a disease or injury, during complications Patients who are present, and who have had surgery on vital vital organs within one week, or who underwent major surgery within one week. As used herein in a more limited sense, the term “severely ill patient” refers to a patient who is at risk of a single or multiple organ system acute or life-threatening due to a disease or injury, or is at risk for a complication, Means patient. As used herein in a more limited sense, the term “severely ill patient” means a patient who is at risk of or is in a single or multiple organ system arrest situation that is acutely life-threatening due to a disease or injury. Similarly these definitions apply to similar expressions, for example, "acute pain of a patient" and "a seriously ill patient." Seriously ill patients are patients who require heart surgery, brain surgery, chest surgery, abdominal surgery, vascular surgery, or transplantation, or have neurological diseases, brain trauma, respiratory failure, abdominal peritonitis, multiple trauma or severe burns, or severe multiple He suffers from neuropathy.

As used herein, the term "assimilation" refers to a set of metabolic pathways that produce molecules from small units. These reactions require energy. One method of classifying metabolic processes, whether at the cellular level, at the organ level, or at the organism level, is classified as 'fairy tales' or 'catabolism' and vice versa. Assimilation is energized by metabolism, large molecules are broken down into smaller parts and then fully used in respiration. Many assimilation processes are energized by adenosine triphosphate (ATP). Assimilation processes tend to "build up" institutions and organizations. These processes cause cell growth and differentiation and increase body size, a process involving the synthesis of complex molecules. Examples of assimilation processes include bone growth and mineralization and an increase in muscle mass. Endocrinology has traditionally classified hormones as anabolic metabolism hormones or catabolic hormones, depending on where they stimulate metabolism. In addition, the balance between assimilation and catabolism is regulated by biorhythms, and processes such as glucose metabolism vary throughout the day to suit the animal's normal activity cycle. Some examples of "hormonal metabolic effects" of these hormones are stimulation of the bone marrow to increase protein synthesis from amino acids, increased appetite, increased bone remodeling and growth, and production of red blood cells. Through numerous mechanisms, anabolic metabolic hormones stimulate the formation of muscle cells, thus causing an increase in the size of skeletal muscle and resulting in increased strength.

In another embodiment, the 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 invention, the insulin preparations according to the invention are hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and other diseases in which anabolic metabolic effects are required for treatment. Or as a medicament for the treatment or prevention of injury, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases.

In another embodiment of the invention, hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and other diseases or injuries requiring anabolic metabolic effects, myocardial infarction, For use as a medicament for the treatment or prevention of stroke, coronary heart disease and other cardiovascular diseases, the method comprises administering an insulin preparation according to the invention to a patient in need of such treatment in an effective amount for such treatment.

Treatment with the insulin preparations according to the invention also includes, for example, antidiabetics, antiobesity agents, appetite control agents, antihypertensive agents, agents for the treatment and / or prevention of complications caused by or associated with diabetes, and obesity In combination with second, third pharmacologically active substances selected from agents for the treatment and / or prevention of complications and diseases associated with or associated with obesity.

In addition, treatment with an insulin preparation according to the present invention may be combined with high obesity surgery, a surgery that affects glucose levels and / or lipid homeostasis, such as gastric band surgery or gastric bypass surgery.

The production of polypeptides such as insulin is well known in the art. Insulin analogs according to the invention can be produced, for example, by classical peptide synthesis, eg, solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques. See, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999. Insulin analogs may also be produced by a method comprising culturing a host cell containing a DNA sequence capable of encoding the insulin analogs by encoding the insulin analogs in a suitable nutrient medium under conditions that allow expression of the insulin analogs. . In the case of insulin analogues comprising non-natural amino acid residues, the recombinant cell must be modified, for example using tRNA mutants, so that the non-natural amino acid can be incorporated into the analogue. Thus, in short, the insulin analogue according to the invention is prepared analogously to 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 WO 2008034881. Two of them are Escherichia coli ) and expression of large fusion proteins in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7 ^th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, III) pp. 729-739), or the use of signal peptides that allow secretion into the periplasmic space (Chan et al. (1981) PNAS 78: 5401-5404). The third method uses Saccharomyces cerevisiae to secrete insulin precursors into the medium (Thim et al. (1986) PNAS 83: 6766-6770). The prior art discloses numerous insulin precursors expressed in E. coli or Saccharomyces cerevisiae . See US 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

Insulin analogs are produced by expressing DNA sequences encoding insulin analogs in suitable host cells by well known techniques such as, for example, disclosed in US Pat. No. 6,500,645. Insulin analogs are expressed directly or as precursor molecules with an N-terminal extension of the B chain or a C-terminal extension of the B chain. N-terminal expansion may have the function of increasing the yield of the product directly expressed, and may consist of up to 15 amino acid residues in length. N-terminal expansion should be cut in vitro after separation from the culture broth, so there will be a cleavage site following B1. N-terminal extensions of the type suitable for the invention are disclosed in US Pat. No. 5,395,922 and EP 765,395. C-terminal expansion may have the function of protecting mature insulin or insulin analog molecules from intracellular proteolytic processes by host cell exoproteases. C-terminal expansion should be either extracellularly cleaved in culture broth by secreted active carboxypeptidase or cleaved in vitro after separation from the culture broth. A method for producing mature insulin and insulin analogues using C-terminal extension of the B chain removed by carboxypeptidase is disclosed in WO 08037735. The target insulin product of this process may be either two-chain human insulin or two-chain human insulin analogues, which may or may not have a short C-terminal extension of the B chain. The target insulin product will not have C-terminal extension of the B chain. The C-terminal expansion should be able to be subsequently removed from the B chain before further purification steps.

In addition, the present invention contemplates the following non-limiting embodiments, which are further described elsewhere herein:

One.

Insulin compounds,

Nicotine compounds, and

● arginine

Insulin preparations comprising a.

2. The insulin preparation according to embodiment 1, wherein the insulin compound is human insulin or an insulin analogue.

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 of any of the preceding embodiments wherein the insulin compound is B3LysB29Glu human insulin.

8. The method of any of the preceding embodiments wherein the insulin compound is 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 , 0.2-1.5mM, 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 an amount from about 0.1 mM to about 10.0 mM.

10. The insulin preparation of any one of the preceding embodiments wherein the insulin compound is present in an amount from about 0.1 mM to about 3.0 mM.

11. The insulin preparation of any of the preceding embodiments wherein the insulin compound is present in an amount from about 0.1 mM to about 2.5 mM.

12. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.1 mM to about 2.0 mM.

13. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.1 mM to about 1.5 mM.

14. The insulin preparation according to any of the preceding embodiments wherein the insulin compound is present in an amount from about 0.2 mM to about 2.5 mM.

15. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.

16. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.2 mM to about 1.5 mM.

17. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.3 mM to about 3.0 mM.

18. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.3 mM to about 2.5 mM.

19. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.3 mM to about 2.0 mM.

20. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.3 mM to about 1.5 mM.

21. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.5 mM to about 1.3 mM.

22. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.3 mM to about 1.2 mM.

23. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount from about 0.6 mM to about 1.2 mM.

24. The insulin preparation of any of the preceding embodiments wherein the insulin compound is present in an amount of about 0.6 mM or about 1.2 mM.

25. The insulin preparation of any one of the preceding embodiments wherein the insulin compound is present in an amount of about 0.3 mM.

26. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount of about 0.6 mM.

27. The insulin preparation of any one of the preceding embodiments wherein the insulin compound is present in an amount of about 0.9 mM.

28. The insulin preparation of any of the preceding embodiments, wherein the insulin compound is present in an amount of about 1.2 mM.

29. The method according to any of the preceding embodiments wherein the nicotine 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. Insulin preparations.

30. The insulin preparation according to any of the preceding embodiments wherein the nicotine compound is selected from nicotinamide and nicotinic acid and / or salts thereof and / or any combination thereof.

31. The insulin preparation of any of the preceding embodiments wherein the nicotine compound is nicotinamide and / or salts thereof.

32. The insulin preparation of any one of the preceding embodiments wherein the nicotine compound is selected from 1-300 mM, 5-200 mM, 40-120 mM, 70-140 mM or 80-130 mM.

33. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 1 mM to about 300 mM.

34. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 8 mM to about 260 mM.

35. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 50 mM to about 250 mM.

36. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 80 mM to about 250 mM.

37. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 80 mM to about 180 mM.

38. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 5 mM to about 200 mM.

39. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 1 mM to about 150 mM.

40. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 5 mM to about 20 mM.

41. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 20 mM to about 120 mM.

42. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 40 mM to about 120 mM.

43. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 20 mM to about 40 mM.

44. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 60 mM to about 80 mM.

45. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 70 mM to about 140 mM.

46. The insulin preparation of any of the preceding embodiments, which comprises a nicotine compound of about 80 mM to about 130 mM.

47. The insulin formulation of any one of the preceding embodiments, wherein the formulation comprises about 8 mM, 30 mM, 100 mM or 130 mM nicotine compound.

48. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 8 mM nicotine compound.

49. The insulin preparation of any of the preceding embodiments, comprising about 30 mM, 100 mM, or 130 mM nicotine compound.

50. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 30 mM nicotine compound.

51. The insulin preparation of any of the preceding embodiments, comprising about 80 mM nicotine compound.

52. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 100 mM nicotine compound.

53. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 120 mM nicotine compound.

54. Insulin formulation according to any of the preceding embodiments, comprising about 130 mM nicotine compound.

55. Insulin preparation according to any of the preceding embodiments, comprising about 150 mM nicotine compound.

56. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 155 mM nicotine compound.

57. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 180 mM nicotine compound.

58. The insulin preparation according to any of the preceding embodiments, comprising about 230 mM nicotine compound.

59. The method according to any of the preceding embodiments, comprising arginine compounds in the range of 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 Insulin preparations characterized by the above-mentioned.

60. The insulin preparation of any of the preceding embodiments, comprising an arginine compound in the range of 1-120 mM, 8-85 mM or 1-40 mM.

61. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises from about 1 mM to about 120 mM arginine.

62. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises from about 1 mM to about 100 mM arginine.

63. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises from about 5 mM to about 120 mM arginine.

64. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises between about 20 mM and about 90 mM Arginine.

65. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises from about 30 mM to about 85 mM arginine.

66. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises from about 8 mM to about 85 mM arginine.

67. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises from about 30 mM to about 60 mM arginine.

68. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises from about 10 mM to about 40 mM arginine.

69. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises from about 10 mM to about 30 mM of arginine.

70. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises from about 10 mM to about 60 mM arginine.

71. The insulin preparation of any of the preceding embodiments, which comprises about 1 mM to about 40 mM arginine.

72. The method according to any of the preceding embodiments, wherein the arginine is 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM Insulin preparations, characterized in that present in the range selected from 55mM or 60mM.

73. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 1 mM arginine.

74. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 2 mM arginine.

75. Insulin preparation according to any of the preceding embodiments, comprising about 3 mM arginine.

76. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 4 mM arginine.

77. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 5 mM arginine.

78. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 6 mM arginine.

79. The insulin preparation of any of the preceding embodiments, comprising about 7 mM arginine.

80. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 8 mM arginine.

81. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises about 9 mM arginine.

82. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 10 mM arginine.

83. The insulin preparation of any of the preceding embodiments, comprising about 11 mM arginine.

84. The insulin formulation of any of the preceding embodiments, wherein the insulin formulation comprises about 12 mM arginine.

85. The insulin preparation of any of the preceding embodiments, comprising about 13 mM arginine.

86. The insulin preparation of any of the preceding embodiments, comprising about 14 mM arginine.

87. The insulin preparation of any of the preceding embodiments, comprising about 15 mM arginine.

88. The insulin preparation of any of the preceding embodiments, comprising about 17 mM arginine.

89. The insulin preparation of any of the preceding embodiments, comprising about 20 mM arginine.

90. The insulin preparation of any of the preceding embodiments, comprising about 22 mM arginine.

91. The insulin preparation of any of the preceding embodiments, wherein the insulin formulation comprises about 25 mM arginine.

92. The insulin preparation according to any of the preceding embodiments, comprising about 30 mM arginine.

93. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 35 mM arginine.

94. The insulin preparation of any of the preceding embodiments, comprising about 40 mM arginine.

95. The insulin preparation of any of the preceding embodiments, comprising about 45 mM arginine.

96. The insulin formulation of any one of the preceding embodiments, wherein the insulin formulation comprises about 50 mM arginine.

97. The insulin preparation of any of the preceding embodiments, comprising about 55 mM arginine.

98. The insulin preparation of any of the preceding embodiments, comprising about 60 mM arginine.

99. The insulin preparation of any of the preceding embodiments, further comprising a buffer (s).

100. The insulin preparation of embodiment 99, wherein said buffer is a phosphate buffer.

101. The insulin formulation of embodiment 100, comprising about 1 mg / mL to about 20 mg / mL of phosphate buffer.

102. The insulin preparation of embodiment 100, comprising about 1 mg / mL to about 15 mg / mL phosphate buffer.

103. The insulin preparation of embodiment 100, comprising about 1 mg / mL to about 10 mg / mL of phosphate buffer.

104. The insulin preparation of embodiment 100, comprising about 3 mg / mL of phosphate buffer.

105. The insulin preparation of embodiment 99, wherein said buffer is Tris.

106. The insulin preparation of embodiment 105, comprising about 2 mM to about 50 mM Tris.

107. The insulin preparation of embodiment 105, comprising about 10 mM to about 40 mM Tris.

108. The insulin preparation of embodiment 105, comprising about 20 mM to about 30 mM Tris.

109. The insulin preparation of embodiment 105, comprising tris of about 10 mM, 20 mM, 30 mM or 40 mM.

110. The insulin preparation of embodiment 105, comprising about 7 mM Tris.

111. The insulin preparation of embodiment 105, comprising about 10 mM Tris.

112. The insulin preparation of embodiment 105, comprising about 20 mM Tris.

113. The insulin preparation of embodiment 105, comprising about 30 mM Tris.

114. The insulin preparation of embodiment 105, comprising about 40 mM Tris.

115. The insulin preparation of any of the preceding embodiments, further comprising a metal ion.

116. The insulin preparation of embodiment 115, wherein the metal ion is zinc.

117. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 0: 6 to about 6: 6.

118. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 0: 6 to about 5: 6.

119. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 1: 6 to about 6: 6.

120. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 2: 6 to about 6: 6.

121. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 2: 6 to about 5: 6.

122. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 2.5: 6 to about 4.5: 6.

123. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3: 6 to about 4: 6.

124. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 2: 6.

125. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 2.5: 6.

126. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3: 6.

127. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.1: 6.

128. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.2: 6.

129. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.3: 6.

130. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.4: 6.

131. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.5: 6.

132. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.6: 6.

133. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.7: 6.

134. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.8: 6.

135. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 3.9: 6.

136. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 4: 6.

137. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 4.5: 6.

138. The insulin preparation of embodiment 116, wherein the zinc: insulin molar ratio is about 5: 6.

139. The insulin preparation of any of the preceding embodiments, further comprising a stabilizer (s).

140. The insulin preparation of embodiment 139, wherein the stabilizer is a non-ionic detergent.

141. The insulin preparation of embodiment 140, wherein the detergent is polysorbate 20 or polysorbate 80.

142. The insulin preparation of embodiment 140, wherein the detergent is polysorbate 20 (Tween 20).

143. The insulin preparation of embodiment 140, wherein the detergent is polysorbate 80.

144. The insulin preparation of embodiment 140, comprising about 5 to 100 ppm, about 10 to about 50 ppm or about 10 to about 20 ppm polysorbate.

145. The insulin preparation of any of the preceding embodiments, further comprising a preservative preparation (s).

146. The insulin preparation of embodiment 145, wherein the preservative is a phenolic compound.

147. The insulin preparation of embodiment 146, wherein said phenolic compound is present in an amount of about 0 to about 6 mg / ml or about 0 to about 4 mg / ml.

148. The insulin preparation of embodiment 146, wherein the phenolic compound is present in an amount of about 5 to about 100 mM.

149. The insulin preparation of embodiment 146, wherein the phenolic compound is present in an amount of about 5 to about 50 mM.

150. The insulin preparation of embodiment 146, wherein the phenolic compound is present in an amount of about 5 to about 30 mM.

151. The insulin preparation of embodiment 146, wherein the phenolic compound is present in an amount of about 16 mM.

152. The insulin preparation of embodiment 146, wherein said phenolic compound is present in an amount of about 14.4 mM.

153. The insulin preparation of embodiment 145, wherein said preservative is m-cresol.

154. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount of about 0.5 to about 4.0 mg / ml or about 0.6 to about 4.0 mg / ml.

155. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount from about 5 to about 100 mM.

156. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount from about 5 to about 50 mM.

157. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount from about 5 to about 30 mM.

158. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount of about 16 mM.

159. The insulin preparation of embodiment 153, wherein m-cresol is present in an amount of about 14.4 mM.

160. The insulin preparation of any of the preceding embodiments, further comprising glycerol in an amount from about 0.5 to about 2.5%.

161. The insulin preparation of any of the preceding embodiments, further comprising glycerol in an amount from about 0.7 to about 2.0%.

162. The insulin preparation of any of the preceding embodiments, further comprising glycerol in an amount from about 1.0 to about 1.5%.

163. The insulin preparation of any of the preceding embodiments, further comprising glycerol in an amount of about 1.25%.

164. The insulin preparation of any of the preceding embodiments, wherein the pH is neutral to weakly basic.

165. The insulin formulation of any of the preceding embodiments, wherein the pH is from about 6.8 to about 8.0.

166. The insulin preparation of any of the preceding embodiments, wherein the pH is about 6.8.

167. The insulin preparation of any of the preceding embodiments, wherein the pH is about 6.9.

168. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.0.

169. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.1.

170. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.2.

171. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.3.

172. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.4.

173. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.5.

174. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.6.

175. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.7.

176. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.8.

177. The insulin preparation of any of the preceding embodiments, wherein the pH is about 7.9.

178. The insulin preparation of any of the preceding embodiments, wherein the pH is about 8.0.

179. The method of any one of the preceding embodiments, wherein the therapeutically active dosage of an insulin agent is administered to a patient in need of such treatment to reduce blood glucose levels in a mammal.

180. The method of any one of embodiments 1-178, comprising administering an insulin formulation to the subject.

181. The method of any one of embodiments 179 to 180, which is parenteral administration.

182. The method according to any of embodiments 1 to 178, wherein hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and other diseases or wounds that require assimilation upon treatment, Insulin preparations used for the treatment and prevention of myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases and for the treatment of severely ill diabetic and non-diabetic patients.

183. The treatment of embodiment 182, which comprises hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns and surgical injuries, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases, or Insulin preparations for use in prophylaxis.

184. The insulin preparation according to any of embodiments 182 to 183, for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.

185.

Insulin compounds,

● nicotine compound

Arginine, and

● buffer

Insulin preparations comprising a.

186. The insulin preparation of embodiment 185, wherein the insulin compound is human insulin or an insulin analog.

187. The insulin preparation of embodiment 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 of embodiment 186, wherein the insulin compound is B28Asp human insulin.

189. The insulin preparation of any of embodiments 185-188, wherein the insulin compound is present from about 0.2 mM to about 2.0 mM.

190. The insulin preparation of any of embodiments 185-189, wherein the insulin compound is present at about 0.6 mM to about 1.2 mM.

191. The method according to any of embodiments 185 to 190, wherein the nicotine 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. Insulin preparations characterized by the above-mentioned.

192. The insulin preparation according to any of embodiments 185 to 191, wherein the nicotine compound is nicotinamide.

193. The insulin preparation of embodiment 192, comprising about 1 mM to about 250 mM nicotine compound.

194. The insulin preparation of embodiment 192, comprising about 1 mM to about 250 mM nicotine compound.

195. The insulin formulation of embodiment 192, comprising about 80 mM to about 230 mM nicotine compound.

196. The insulin preparation of any of embodiments 185-195, comprising about 10 mM to about 60 mM arginine.

197. The insulin preparation of any of embodiments 185-195, comprising about 10 mM to about 30 mM arginine.

198. The insulin preparation of any of embodiments 185-197, comprising about 20 mM arginine.

199. The insulin preparation of any of embodiments 185 to 198, wherein the buffer is a phosphate buffer.

200. The insulin preparation of any of embodiments 185-199, wherein said buffer is Tris.

201. The insulin formulation of any of embodiments 185 to 200, further comprising a preservative agent (s), isotonic agent (s) and / or stabilizer (s).

202. The insulin preparation of any of embodiments 185 to 201, further comprising a metal ion.

203. The insulin preparation of embodiment 202, wherein the metal ion is zinc.

204. The insulin preparation of embodiment 203, wherein the zinc: insulin molar ratio is about 0: 6 to about 3.5: 6.

205. The insulin preparation of embodiment 203, wherein the zinc: insulin molar ratio is about 2: 6 to about 3: 6.

206. The insulin preparation according to any of embodiments 185 to 205, wherein the preparation has a pH of less than 7.4.

207. The insulin formulation of any of embodiments 185 to 205, wherein the formulation has a pH of about 7.4.

208. The insulin formulation of any of embodiments 185 to 205, wherein the formulation has a pH of about 7.1.

209. B28Asp human insulin; Nicotinamide; zinc; Arginine; And a phosphate buffer.

210. The compound of embodiment 209, wherein B28Asp human insulin is present at a concentration range of about 0.6 mM to about 1.2 mM, wherein nicotinamide is present at a concentration range of about 80 mM to about 260 mM, And wherein arginine is present in a concentration range of about 10 mM to about 40 mM, wherein less than about 4 zinc ions are present per 6 B28Asp human insulin molecule, and wherein the agent is about 7.4 or so Insulin preparations having a pH of less than.

211.

a. B28Asp human insulin, wherein B28Asp human insulin has a concentration range of about 0.6 mM to about 1.2 mM;

b. Nicotinamide, wherein nicotinamide has a concentration range of about 80 mM to about 260 mM;

c. Zinc, wherein less than about 4 zinc ions are present per 6 B28Asp human insulin molecule;

d. Arginine, wherein arginine is present in a concentration range of about 10 mM to about 30 mM; And

e. Phosphate buffers;

Consisting essentially of, wherein the formulation is at a pH of about 7.1.

212. The method of any one of the preceding embodiments, wherein the therapeutically active dose of an insulin agent is administered to a mammal in need of such treatment to reduce blood glucose levels in the mammal.

213. The method of any of embodiments 1-211, comprising administering an insulin formulation to the subject under test.

214. The method according to any one of embodiments 1 to 211, wherein hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical injuries and other diseases requiring assimilation upon treatment, or Insulin preparations for use in the treatment and prevention of injury, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases and for the treatment of severely ill diabetes and non-diabetic patients.

215. The insulin preparation according to any one of embodiments 1 to 211 for use in the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.

In another embodiment of the invention relates to the following:

216.

Insulin compounds,

Nicotine compounds, and

● arginine

Insulin preparations comprising a.

217. The insulin preparation of embodiment 216, wherein the insulin compound is human insulin or an insulin analog.

218. The insulin preparation of any of embodiments 216-217, wherein the insulin compound is B28Asp human insulin.

219. The insulin preparation of any of embodiments 216-218, wherein the insulin compound is B28LysB29Pro human insulin.

220. The insulin preparation of any of embodiments 216 to 219, wherein the insulin compound is B3LysB29Glu human insulin.

221. The insulin preparation of any of embodiments 216 to 220, wherein the insulin compound is about 0.2 mM to about 2.0 mM.

222. The insulin preparation of any of embodiments 216 to 221, wherein the insulin compound is about 0.3 mM to about 1.2 mM.

223. The method according to any of embodiments 216 to 222, wherein the nicotine 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. Insulin preparations.

224. The insulin formulation of any of embodiments 216 to 223, comprising about 1 mM to about 150 mM nicotine compound.

225. The insulin preparation of any of embodiments 216 to 224, comprising about 1 mM to about 85 mM arginine.

226. The insulin formulation of any of embodiments 216 to 225, further comprising metal ions, preservative agent (s), tonicity agent (s) and stabilizer (s) and buffer (s).

227. The method of any of embodiments 216 to 226, wherein a therapeutically active dose of an insulin formulation is administered to the patient in need of such treatment.

228. The method of any of embodiments 216 to 226, comprising administering an insulin formulation to the subject under test.

229. The method according to any of embodiments 216 to 226, wherein hyperglycemia, including hyperglycemia due to stress, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and other diseases or wounds that require assimilation upon treatment, Insulin preparations used for the treatment and prevention of myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases and for the treatment of severely 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 indicated by the full text of each reference individually and specifically for reference, and to the same extent as set forth herein. As incorporated herein by reference (to the maximum extent permitted by law).

All headings and subtitles used herein are for convenience only and do not limit the invention in any way.

 All examples and the use of certain examples or exemplary words provided herein (eg, "such as") are intended to better illustrate the invention and do not limit the scope of the invention unless otherwise indicated in the claims. Do not. The language of the specification should not be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and inclusion of patent documents herein is for convenience only and does not reflect any aspect of the validity, patentability, and / or enforceability of such patent documents.

The present invention includes all modifications and equivalents of the contents recited in the claims appended hereto to the extent permitted by applicable law.

Example

Example  One

Preparation of Pharmaceutical Formulations

Pharmaceutical formulations of the invention may be formulated as aqueous solutions. Aqueous media are made isotonic using, for example, sodium chloride or glycerol. The aqueous medium may also contain zinc ions, buffers, and preservatives added as, for example, zinc acetate or zinc chloride. Arginine may be added as Arg, HCl. The pH value of the formulation is adjusted to the desired value, which may be about 3 to about 8.5, about 3 to about 5, or about 6.5 to about 7.5, depending on the isoelectric point, pi, of the corresponding insulin.

Table 1. Composition of Insulin Preparations According to the Invention

Table 2. Compositions of Other Insulin Preparations According to the Invention

Table 3. Compositions of Other Insulin Preparations According to the Invention

Table 4. Compositions of Other Insulin Formulations According to the Invention

Example  2

Analysis of Chemical Stability of Insulin

Size exclusion chromatography

Quantitative analysis of high molecular weight protein (HMWP) and monomeric insulin aspart was performed on Waters insulin (300x7.8mm, part nr wat 201549), 2.5M acetic acid, 4mM L-arginine, and 20% (V / V) at 40 ° C. ) An eluate containing acetonitrile was used at a flow rate of 1 mL / min. Detection was performed using a tuned absorbance detector (Waters 486) at 276 nm. Injection volumes were 40 μL and 600 μM human insulin standards. HMWP and concentration of the formulations were measured at each sampling time point.

Inverse phase  Chromatography ( UPLC )

Determination of impurities related to insulin aspart was performed in an UPLC system, using a BEH RP C8 2.1 × 100 mm column, 1.7 μm particle size. Waters part no 186002878. was used at 40 ° C. with a flow rate of 0.5 mL / min and detected at 220 nm. Elution was performed using a mobile phase constructed as follows.

A. 10% (w / V) acetonitrile, 2.8% (w / w) sodium sulfate, 0.3% (w / w) o-phosphate, pH 3.5.

B. 70% (w / V) acetonitrile, gradient: 0-11 minutes isocratic with A / B 73% / 27%, 11-12 minutes linear change to A / B 52% / 48%, A / B 73 13-15 min linear change to% / 27%, 15-20 min isocratic gradient at A / B 73% / 27%.

The amount of B28iso-aspartate, desamido and other related impurities was determined as the absorbance area measured as a percentage of the total absorbance area determined after elution of the preservative. The RP-UPLC method is identical to the analytical method used for quality control of insulin aspart pharmaceuticals sold by Novo Nordisk.

The addition of arginine reduces the amount of degradation products formed, in particular HMWP and desamido forms, and increasing the concentration of arginine in the range from 10 to 50 mM leads to further reduction in degradation. Physical stability, measured as latency in ThT assay, decreases with the addition of arginine and decreases significantly with increasing arginine concentration. As shown in Table 4 below, the overall performance of 50mM arginine is superior to 50mM glycine, 50mM glutamic acid or 50mM histidine with respect to the reduction of degradation product formation.

The insulin formulations of the present invention provide not only physically stable but also surprisingly chemically stable fast-acting insulin preparations.

Table 5. Physical and Chemical Stability Data for Insulin Formulations 1-9 in Table 2

Table 6. Chemical Stability Data for Insulin Formulations 10-13 of Table 3

Table 7. Physical and Chemical Stability Data for Insulin Formulations 14-20 in Table 4

Example  3

LYD  Pharmacokinetics in the Pig Model PK ) / Pharmacodynamics ( PD A) study and plasma analysis test

LYD Pig PK / PD  Research

PK / PD studies were conducted on LYD hybrids, livestock female pigs weighing 55-110 kg. Pigs were catheterized into the jugular vein through the ear vein at least two days before the start of the study. The last meal before the start of the study was given to the animals about 18 hours before the injection of the test formulation and the animals had free access to water at any time during the fasting and testing periods.

At time 0 the test formulation was injected subcutaneously in the side of the neck. Blood samples were taken at regular time intervals before and after dosing, samples were collected via catheter and sampled in 1.5 mL glass tubes coated with heparin. Blood samples were stored on ice until centrifugation at 3000 rpm, 4 ° C. for 10 minutes to separate plasma, which was performed 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 term storage, glucose at YSI or Konelab 30i, and insulin aspart concentrations at LOCI.

insulin Aspart  Luminescent Oxygen for Quantification Channeling  Immunoassay ( LOCI )

Insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay and applies the proximity of two beads of europium-coated acceptor beads and streptavidin coated donor beads. Acceptor beads were coated with specific antibodies to human insulin and recognized insulin aspart in plasma samples. The second biotinylated antibody specifically binds to insulin aspart with streptavidin coated beads, and they form a sandwich. Illumination of the bead-aggregate-immune complex releases singlet oxygen from the donor beads, which are directed to the acceptor beads and trigger chemiluminescence. Chemiluminescence was measured and the amount of light generated was proportional to the concentration of insulin aspart.

Compared to the commercial product NovoRapid®, the initial rate of degradation of plasma glucose is faster in the formulations of the invention (Figures 3 and 4). Likewise, when compared to NovoRapid®, the initial absorption rate of the insulin component of the formulation of the present invention is much faster (FIG. 5).

Example  4

protein Preparation  To assess physical stability ThT Small fiber  General introduction of analysis

The low physical stability of the peptide can lead to amyloid fibrils formation, which are aligned in the sample and observed as a macromolecular structure like yarn, resulting in the formation of a gel. This was conventionally measured by visual inspection of the sample. However, this kind of measurement is very subjective and observer dependent. Thus, the application of small molecular indicator probes is even more beneficial. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signal when combined with fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309,274-284]. The time course for small fiber formation can be explained by the sigmoidal curve represented by the following equation [Nielsen et al. (2001) Biochemistry 40, 6036-6046]:

식 (1)

Formula (1)

Where F is ThT fluorescence at time t. The constant t ₀ is the time required to reach 50% of the maximum fluorescence. Two important variables that explain the formation of fibrils are the delay time calculated by t ₀ -2τ and the apparent rate constant K _app = 1 / τ.

Formation of partially folded intermediates of peptides is suggested as a general initiation mechanism for small fibrosis. Some of these intermediates can be nucleated to form a template, on which other intermediates can be assembled, and microfibrillation proceeds. The delay time corresponds to the interval until the critical mass of the nucleus is established, and the apparent rate constant is the rate at which the fibrils themselves form.

Sample preparation

Samples were freshly prepared before each analysis. Each sample composition is described in each example. The pH of the sample was adjusted to the appropriate amount of concentrated NaOH and HClO ₄ Or adjusted to the desired value using HCl. Thioflavin T was added to the sample at a final concentration of 1 μM from stock solution in H ₂ O.

200 μL of sample aliquots were placed in a 96-well microtiter plate (Packard Opti-Plate ™ -96, white polystyrene). Generally, four or eight duplicates of each sample (corresponding to one test condition) were placed in one column of wells. The plate was sealed with a Scotch Pad (Qiagen).

Incubation  And fluorescence measurements

Measurements of incubation, shaking, and ThT fluorescence emission at a given temperature were performed in a Fluoroskan Ascent FL fluorescent plate reader or Varioskan plate reader (Thermo Labsystems). The temperature was adjusted to 37 ° C. The orbital shaking was adjusted to 960 rpm with an amplitude of 1 mm in all provided data. Fluorescence measurements were used with excitation through a 444 nm filter and emission measurements were performed through a 485 nm filter.

Each operation was initiated by plate incubation for 10 minutes at assay temperature. Plates were measured every 20 minutes for the desired time. The plate was shaken and heated as described between each measurement.

Data handling

The measurement points were saved in Microsoft Excel format for further processing, drawing and fitting curves using GraphPad Prism. Background release from ThT in the absence of fibrils was negligible. Data points are typically the average of four or eight samples and are shown with standard deviation error bars. Only data obtained from the same experiment (ie, samples of the same plate) are displayed on the same graph and a relative measure of microfiberization between experiments is ensured.

Fit the data set to equation (1). However, because the complete sigmoidal curve was not always obtained during the measurement time, the delay time was visually determined from the ThT fluorescence curve at a time point where the ThT fluorescence was different from the background level.

Measurement of initial and final concentration

Peptide concentrations in each tested formulation were measured both prior to application of the ThT microfibrillation assay (“initial”) and after completion of the ThT microfibrillation assay (“after the ThT assay”). Concentration was determined by reverse phase HPLC method using frraminide standard as reference. After completion, 150 μL from each duplicate sample was transferred to an eppen rope tube before measurement. They were centrifuged at 30000 G for 40 minutes. The supernatant was filtered through a 0.22 μm filter prior to HPLC system application.

                         SEQUENCE LISTING <110> Novo Nordisk A / S   <120> PREPARATION COMPRISING INSULIN, NICOTINAMIDE AND AN AMINO ACID <130> 8301.204-WO <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 35 <212> PRT <213> Homo sapiens <220> <221> linked to chain B preproinsulin <222> (1) <220> <221> C connecting insulin peptide (222) (3) .. (33) <220> <221> linked to chain A preproinsulin &Lt; 222 > (35) <400> 1 Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa             20 25 30 Xaa Lys Arg         35

Claims (32)

Insulin compounds,
Nicotine compounds, and
● arginine
Insulin preparations comprising a. 2. The insulin preparation of claim 1, wherein the insulin compound is human insulin or an insulin analog. 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. The insulin preparation according to any one of claims 1 to 3, wherein the insulin compound is B28Asp human insulin. 5. The insulin preparation of claim 1, wherein the insulin compound is present in an amount of about 0.2 mM to about 2.0 mM. 6. The insulin preparation according to any one of claims 1 to 5, wherein the insulin compound is present in an amount of about 0.6 mM to about 1.2 mM. The nicotine compound according to any one of claims 1 to 6, wherein the nicotine 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. Insulin preparations, characterized in that. 8. The insulin preparation according to any one of claims 1 to 7, wherein the nicotine compound is nicotin amide. 9. The insulin preparation of claim 1, comprising from about 1 mM to about 250 mM nicotine compound. 10. The insulin preparation according to any one of the preceding claims, comprising from about 1 mM to about 250 mM nicotine compound. The insulin preparation of any one of claims 1 to 10 comprising from about 80 mM to about 230 mM nicotine compound. 12. The insulin formulation of any one of claims 1 to 11, comprising from about 10 mM to about 60 mM arginine. 13. The insulin preparation of any one of claims 1 to 12, comprising from about 10 mM to about 30 mM arginine. 14. The insulin preparation according to any one of claims 1 to 13, comprising about 20 mM arginine. The insulin preparation according to any one of the preceding claims further comprising one or more buffer (s). The insulin preparation of claim 15 wherein the buffer is a phosphate buffer. 16. The insulin formulation of claim 15, wherein the buffer is Tris. 18. The insulin preparation of any one of claims 1 to 17, further comprising a preservative (s), isotonic agent (s) and / or stabilizer (s). The insulin preparation according to any one of claims 1 to 18, further comprising a metal ion. 20. The insulin preparation of claim 19, wherein the metal ion is zinc. The insulin preparation of claim 20 wherein the zinc: insulin molar ratio is about 0: 6 to about 3.5: 6. The insulin preparation of claim 20 wherein the zinc: insulin molar ratio is about 2: 6 to about 3: 6. The insulin preparation of claim 1, wherein the preparation has a pH of less than 7.4. 24. The insulin preparation according to any one of claims 1 to 23, wherein the preparation has a pH of 7.4. The insulin preparation of claim 1, wherein the preparation has a pH of about 7.1. B28Asp Insulin preparations comprising human insulin, nicotinamide, zinc, arginine, and phosphate buffer. The method of claim 26, wherein the B28Asp human insulin is present at a concentration ranging from about 0.6 mM to about 1.2 mM, and nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and arginine is present from about 10 mM to about And a concentration of 40 mM, and less than about 4 zinc ions per six B28Asp human insulin molecules, and the formulation having 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 in a concentration ranging from about 80 mM to about 260 mM,
c. Zinc with 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. Phosphate buffer
Insulin formulation, consisting essentially of, wherein the formulation has a pH of about 7.1. 29. A method for reducing blood glucose levels in a mammal by administering to said mammal in need thereof a therapeutically active dosage of an insulin preparation according to any of claims 1 to 28. 29. A method of treating diabetes in a subject comprising administering to the subject an insulin formulation according to any one of claims 1 to 28. 29. The surgical procedure according to any one of claims 1 to 28, comprising hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical injuries and other diseases that require assimilation to the treatment or Insulin preparations for use in the treatment and prevention of injuries, myocardial infarction, stroke, coronary heart disease and other cardiovascular diseases, and for the treatment of severe and non-diabetic patients. The insulin preparation according to claim 31, which is used for the treatment of hyperglycemia type 2 diabetes and type 1 diabetes.

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