CN103328006A - Preparation comprising insulin, nicotinamide and an amino acid - Google Patents

Abstract

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

Description

Formulations containing insulin, niacinamide, and amino acids

field of invention

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

Background of the invention

Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partially or completely lost. About 5% of all people suffer from diabetes, a condition approaching epidemic proportions.

Since the introduction of insulin in the 1920s, continuous improvements have been made in the treatment of diabetes. To help avoid high blood sugar levels, diabetics often practice multiple injection therapy, whereby insulin is given with each meal. Since diabetic patients have been treated with insulin for decades, there is a major need for safety and life-quality improved insulin formulations. Among the commercially available insulin preparations, mention may be made of fast-acting, intermediate-acting and long-acting preparations.

In the treatment of diabetes, many kinds of insulin pharmaceutical preparations have been proposed and used, such as regular insulin (such as Actrapid ^® ), low protamine zinc insulin (called NPH), insulin zinc suspension (such as Semilente ^® , Lente ^® and Ultralente ^? ) and biphasic protamine zinc insulin (eg NovoMix ^® ). Analogs and derivatives of human insulin have also been developed, designed for specific properties of action, ie, fast-acting or long-acting. Some commercially available insulin formulations containing such fast-acting insulin analogs include ^NovoRapid® (a formulation of B28Asp human insulin), Humalog® ⁽ a formulation of B28LysB29Pro human insulin), and Apidra® ⁽ a formulation of B3LysB29Glu human insulin).

International applications WO 91/09617 and WO/9610417 (Novo Nordisk A/S) disclose insulin preparations containing nicotinamide or nicotinic acid or salts thereof. European application EP1283051 purportedly discloses insulin formulations containing arginine, wherein arginine is used as a buffer. EP1283051 purportedly discloses improvements in the physical stability of insulin preparations.

The most common pharmaceutical formulations of insulin are given by subcutaneous injection. What is important to the patient is the action profile of the insulin, which refers to the effect of insulin on glucose metabolism as a function of time since injection. In particular, the onset time, maximum value and total duration of action are important in this property. In the case of bolus insulin, patients expect and demand multiple insulin formulations with different action properties. On the same day, a patient may be taking insulin preparations with very different action properties. The desired profile of action depends, for example, on the time of day the patient eats and the amount and composition of the meals eaten.

Equally important to the patient is the chemical stability of the insulin preparation, e.g. due to the extensive use of pen-like injection devices such as those containing ^Penfill® cartridges in which the insulin preparation is stored until the entire cartridge is emptied, for those containing Devices in 1.5-3.0ml cartridges are likely to be used for at least 1-2 weeks. During storage, a covalent chemical change occurs in the insulin structure. This can lead to the formation of potentially less active and/or potentially immunogenic molecules, such as deamidation products and higher molecular weight conversion products (dimers, polymers). Also of importance is the physical stability of insulin formulations, as long-term storage can eventually lead to the formation of insoluble fibrils, which are biologically inactive and potentially immunogenic.

Summary of the invention

The present invention relates to insulin preparations with favorable absorption rates and favorable chemical and physical stability. The present invention relates to insulin preparations comprising human insulin and/or analogs thereof, nicotinamide or nicotinic acid and/or salts thereof and arginine.

In one embodiment, the present invention relates to an insulin preparation comprising:

Insulin compounds,

nicotine compounds, and

· Arginine.

In another embodiment, the present invention also includes a method for treating diabetes in a subject or reducing blood glucose levels in a subject, the method comprising administering the insulin preparation of the present invention to the subject or mammal.

Figure overview

Figure 1 shows the development of the percentage of total insulin content of degradation products during storage of a formulation according to the invention for 2 weeks at 37°C. The letter A refers to the ^NovoRapid® reference, the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Addition of nicotinamide (formulations B and D ) resulted in increased formation of degradation products compared to the ^NovoRapid® formulation (formulation A ), whereas the combined addition of nicotinamide, glutamic acid and arginine (formulations C and E ) had almost similar Degradation mode, lower formation of HMWP.

Figure 2 shows the development of the percentage of total insulin content of degradation products during storage of formulations according to the invention for 2 weeks at 37°C. The letter A refers to the ^NovoRapid® reference, the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Niacinamide, glutamic acid and arginine were added in combination, formulations F , G , H and I , buffer system (phosphate or tris buffer) and concentrations of insulin and Zn (0.6 mM and 0.3 mM or 1.2 mM and 0.6 mM) with a similar degradation pattern as the ^NovoRapid® formulation (Formulation A ).

Figure 3 shows the glucose concentration in plasma (mean +/- SEM, N=8) after subcutaneous injection of a dose of 1 nmol/kg of the formulation of the invention in pigs at 0 min. The letter A refers to the ^NovoRapid® reference, 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 for the formulation with added niacinamide (Formulation N ) and the initial rate of decrease for the combination of niacinamide and arginine (Formulation M ) The rate is even faster.

Figure 4 shows the glucose concentration in plasma (mean +/- SEM, N=7) after subcutaneous injection of a dose of 1 nmol/kg of the formulation of the invention in pigs at 0 min. The letter A refers to the ^NovoRapid® reference, the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. Compared to the ^NovoRapid® formulation (Formulation A ), plasma glucose The initial rate of decrease is faster.

Figure 5 shows insulin aspart concentrations in plasma (mean +/- SEM, N=7) at 0 min after subcutaneous injection of pigs with a dose of 1 nmol/kg of the formulation of the invention. The letter A refers to the ^NovoRapid® reference, the remaining letters correspond to the insulin aspart formulations described in Table 1 of Example 1. The formulation with niacinamide (Formulation J ), the combination of niacinamide and arginine (Formulation K ) and the combination of niacinamide, arginine and glutamic acid compared to the ^NovoRapid® formulation (Formulation A ) The initial rate of absorption of the insulin component of the formulation of (Formulation L ) was significantly faster.

Figure 6 shows the glucose concentration in plasma (mean +/- SEM, N=8, two doses per pig) after subcutaneous injection of a dose of 1 nmol/kg of the formulation of the invention in pigs at 0 min. The letter A refers to the ^NovoRapid® reference, the number 11 corresponds to the insulin aspart formulation as described in Table 3 of Example 1 . The initial rate of decrease in plasma glucose was faster for the formulation with the combination of niacinamide and arginine (Formulation 11 ) compared to the ^NovoRapid® formulation (Formulation A ).

Figure 7 shows insulin aspart concentrations in plasma (mean +/- SEM, N=8, two doses per pig) after subcutaneous injection of pigs with a dose of 1 nmol/kg of the formulation of the invention at 0 min. The letter A refers to the ^NovoRapid® reference, the number 11 corresponds to the insulin aspart formulation as described in Table 3 of Example 1 . The initial rate of absorption of the insulin component of the formulation with niacinamide and arginine (Formulation 11 ) was significantly faster compared to the ^NovoRapid® formulation (Formulation A ).

Description of the invention

It has surprisingly been found that the absorption of insulin compounds in insulin formulations according to the invention is faster after subcutaneous injection than in reference insulin formulations. This property is useful for rapid-acting insulins, especially in connection with multiple injection regimens where insulin is administered before each meal. Due to the faster onset of action, insulin can be conveniently administered closer to the meal than regular rapid-acting insulin regimens. In addition, the faster disappearance of insulin may reduce the risk of postprandial hypoglycemia.

The insulin preparation of the present invention is a rapid-acting insulin preparation comprising an insulin compound (eg insulin aspart), a nicotinic compound (eg niacinamide) and the amino acid arginine. Optionally, the insulin preparations of the invention may contain other amino acids. These insulin formulations have rapid absorption properties and more closely mimic normal physiology than existing treatments. Furthermore, the insulin formulations of the present invention have chemical and physical stability suitable for commercial pharmaceutical formulations.

The insulin formulations of the present invention provide rapid-acting insulin formulations that are not only physically stable, but surprisingly also chemically stable. The insulin formulations of the present invention provide an even faster onset of action compared to existing insulin treatments. The advantages of such an ultra-rapid insulin formulation are restoration of first-phase insulin release, ease of injection and cessation of hepatic glucose production. The insulin formulations of the present invention have a favorable absorption rate from subcutaneous tissue to plasma, when compared to conventional formulations such as ^NovoRapid® , the initial absorption rate is increased 1.5-5 times as shown by several PK/PD experiments in pigs. This faster rate of absorption may improve glycemic control and convenience, and may allow shifting from pre-meal to post-prandial dosing. The present invention is based in part on the unexpected discovery that while the addition of nicotinamide increases the rate of absorption, it also has an adverse effect on chemical stability by significantly increasing the amount of HMWP. By adding arginine, insulin preparations according to the invention have improved chemical stability, reflected in, for example, reduced formation of dimers and polymers and deamidated insulin after storage. Insulin preparations according to the invention may additionally have improved physical stability, which may be used in pumps.

The invention provides insulin preparations comprising an insulin compound of the invention, said insulin compound being present at a concentration of from about 0.1 mM to about 10.0 mM, and wherein said preparation has a pH of 3-8.5. The formulation also includes a nicotine compound and arginine. The formulations may also contain protease inhibitors, metal ions, buffer systems, preservatives, tonicity agents, chelating agents, stabilizers and surfactants.

In one embodiment, the metal ion is zinc, wherein the 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 formulation of the 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 can be in the range of 15-500 international units (IU)/ml, and for injection preparations, it is preferably in the range of 50-333 IU/ml. However, for other purposes of parenteral administration, the level of insulin compound may be higher.

In this context, 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 profile begins to increase.

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

An "insulin compound" of the invention is herein understood to mean human insulin, insulin analogs and/or any combination thereof.

The term "human insulin" as used herein refers to a human hormone whose structure and properties are well known. Human insulin has two polypeptide chains connected by disulfide bridges between cysteine residues, ie, the A-chain and the B-chain. The A-chain is a 21-amino acid peptide, the B-chain is a 30-amino acid peptide, and the two chains are connected by three disulfide bridges: one between the cysteines at positions 6 and 11 of the A-chain, and the second Between cysteine 7 of the A-chain and cysteine 7 of the B-chain, the third is between cysteine 20 of the A-chain and cysteine 19 of the B-chain between.

The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a 24 amino acid propeptide followed by an 86 amino acid proinsulin having the following structure: Propeptide-B-Arg Arg-C-Lys Arg -A, wherein C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are the cleavage sites for cleavage of linker peptide and A and B chains.

As used herein, "insulin analog" refers to a polypeptide derived by mutation from the primary structure of naturally occurring insulin (eg, human insulin). The one or more mutations are made by deletion and/or substitution of at least one amino acid residue present in naturally occurring insulin and/or by addition of at least one amino acid residue. The added and/or substituted amino acid residues may be codable 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 any combination thereof) relative to the parent insulin, or less than 7 modifications relative to the parent insulin, or less than 6 modifications relative to the parent insulin , or less than 5 modifications relative to the parent insulin, or less than 4 modifications relative to the parent insulin, or less than 3 modifications relative to the parent insulin, or less than 2 modifications relative to the parent insulin.

Mutations in the insulin molecule are expressed as a three-letter code indicating the chain (A or B), position, and amino acid replacing the natural amino acid. "desB30" or "B(1-29)" refers to the natural insulin B chain or its analogs lacking the B30 amino acid residue, and "B28Asp human insulin" refers to human insulin in which the 28th amino acid residue of the B chain is replaced by Asp insulin.

An example of an insulin analogue is one in which Pro at position 28 of the B chain is mutated by Asp, Lys, Leu, Val or Ala, and/or Lys at position B29 is mutated by Pro, Glu or Asp. In addition, the Asn at position B3 can be mutated by Thr, Lys, Gln, Glu or Asp. The amino acid residue at position A21 can be mutated by Gly. The amino acid at position B1 can be mutated by Glu. The amino acid at position B16 can be mutated by Glu or His. Other examples of insulin analogs are deletion analogs, such as 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). insulin), des(B28-B30) human insulin and des(B27) human insulin. Insulin analogues wherein the A-chain and/or B-chain have an N-terminal extension and wherein the A-chain and/or B-chain have a C-terminal extension (for example two arginine residues added to the B-chain) C-terminal) insulin analogs are also examples of insulin analogs. Other examples are insulin analogs comprising combinations of the mutations mentioned. Insulin analogues wherein the amino acid at position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His, and the amino acid at position B25 is His and optionally further comprising one or more additional mutations are insulin analogues other instances. Insulin analogs of human insulin in which the amino acid residue at position A21 is Gly and in which the insulin analog is further extended at the C-terminus by two arginine residues are also examples of insulin analogs.

Other examples of insulin analogs include, but are not limited to: DesB30 human insulin; AspB28 human insulin; AspB28, desB30 human insulin; LysB3, GluB29 human insulin; LysB28, ProB29 human insulin; ; HisA14, HisB25 human insulin; GluA14, HisB25, desB30 human insulin; HisA14, HisB25, desB30 human insulin; GluA14, HisB25, desB27, desB28, desB29, desB30 human insulin; , HisB25, desB30 human insulin; HisA14, HisB16, HisB25, desB30 human insulin; HisA8, GluA14, HisB25, GluB27, desB30 human insulin; HisA8, GluA14, GluB1, GluB16, HisB25, GluB27, desB30 human insulin; and HisA8, GluA14, GluB16, HisB25, desB30 human insulin.

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

According to the present invention, the concentration of the nicotine compound and/or salt thereof is in the range of about 1 mM to about 300 mM or about 5 mM to about 200 mM.

The term "arginine" or "Arg" includes the amino acid arginine and/or a salt thereof, eg, arginine hydrochloride or arginine glutamate.

In one embodiment, the insulin preparation comprises 1-100 mM arginine.

In one embodiment, the insulin preparation comprises 1-20 mM arginine.

In one embodiment, the insulin preparation comprises 20-90 mM arginine.

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

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

The term "pharmaceutical preparation" or "insulin preparation" as used herein refers to a product comprising an insulin compound, i.e., human insulin, analogs thereof and/or combinations thereof and a nicotinic compound and an amino acid, optionally together with other excipients , such as preservatives, chelating agents, tonicity regulators, bulking agents, stabilizers, antioxidants, polymers and surfactants, metal ions, oily vehicles, and proteins (e.g., human serum albumin, gelatin, or proteins), by administering Insulin preparations for humans, said insulin preparations being useful for treating, preventing or reducing the severity of a disease or condition. Therefore, insulin preparations are also referred to in the art as pharmaceutical preparations or pharmaceutical compositions.

The buffering agent may be selected from, but not limited to, sodium acetate, sodium carbonate, citrate, monosodium phosphate, disodium phosphate, sodium phosphate, and tris (hydroxymethyl)-aminomethane (tris), N-dihydroxyethyl Glycine, Trixine, Malate, Succinate, Maleic Acid, Fumaric Acid, Tartaric Acid, Aspartic Acid, Ethylenediamine or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

The insulin preparations of the present invention may also contain other ingredients common to insulin preparations, such as zinc complexing agents such as citrate, and phosphate buffers.

Glycerol and/or mannitol and/or sodium chloride may be present in amounts corresponding to concentrations of 0-250 mM, 0-200 mM or 0-100 mM.

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

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

Preservatives present in the insulin preparations according to the invention may be hitherto customary insulin preparations such as phenol, m-cresol and methylparaben.

The insulin preparations of the invention may also comprise chelating agents. The use of chelating agents in pharmaceutical formulations is well known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th ed., 1995.

The insulin preparations of the invention may also contain stabilizers. The term "stabilizer" as used herein refers to a chemical added to a pharmaceutical formulation containing a polypeptide to stabilize the peptide, ie, to increase the shelf life and/or use time of the formulation. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th ed., 1995.

The insulin preparations of the invention may also comprise surfactants. As used herein, the term "surfactant" refers to any molecule or ion comprising a water-soluble (hydrophilic) head and a fat-soluble (lipophilic) segment. Surfactants accumulate preferably at interfaces where the hydrophilic portion is oriented towards the water (hydrophilic phase) and the lipophilic portion is oriented towards the oily or hydrophobic phase (ie, glass, air, oil, etc.). The concentration at which a surfactant begins to form micelles is called the critical micelle concentration or CMC. In addition, surfactants lower the surface tension of liquids. Surfactants are also known as amphiphiles. Often the term "detergent" is synonymous with surfactant. The use of surfactants in pharmaceutical formulations is well known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th ed., 1995.

In other embodiments, the present invention relates to an insulin preparation comprising an aqueous solution of an insulin compound of the present invention and a buffer, wherein said insulin compound is present at a concentration of 0.1 mM or higher, and wherein The pH of the formulations described below is from about 3.0 to about 8.5.

The invention also relates to a process for the production of the insulin preparations according to the invention.

In one embodiment, the method for preparing the insulin formulation of the invention comprises:

a) preparing a solution by dissolving the insulin compound or mixture of insulin compounds in water or a buffer;

b) preparing a solution by dissolving divalent metal ions in water or buffer;

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

d) Prepare a solution by dissolving the tonicity agent in water or buffer;

e) Prepare solutions by dissolving surfactants and/or stabilizers in water or buffers;

f) mixing solution a) with one or more of solutions b), c), d) and e);

Finally the pH of the mixture in f) is adjusted to the desired pH, followed by sterile filtration.

In one embodiment, the method for preparing the insulin formulation of the invention comprises:

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

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

c) adding the stock solution of the tonicity agent to the solution b);

d) preparing a solution by dissolving the insulin compound or mixture of insulin compounds in water or buffer; adding HCl to acidify the solution;

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

f) Add e) to c) and stir;

g) adding the stock solution of arginine or arginine salt into f), and stirring;

h) Add the stock solution of the nicotine compound to g) and adjust the pH to the desired pH using NaOH/HCl.

In one embodiment, the method for preparing the insulin formulation of the invention comprises:

a) preparation of separate stock solutions of preservatives, tonicity agents, metal ion salts, and mixed stock solutions of arginine or arginine salts and nicotine compounds;

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

c) adding the stock solution of the tonicity agent to the solution b);

d) preparing a solution by dissolving the insulin compound or mixture of insulin compounds in water or buffer; adding HCl to acidify the solution;

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

f) Add e) to c) and stir;

g) adding the mixed stock solution of arginine or arginine salt and nicotine compound into f), and stirring;

h) Adjust the pH of g) to the desired pH using NaOH/HCl.

By parenteral administration, the insulin formulations of the present invention can be used in the treatment of diabetes. The dosage of the insulin preparations of the invention recommended for administration to a patient is chosen by the physician.

Parenteral administration can be effected by subcutaneous, intramuscular, intraperitoneal or intravenous injection with the aid of a syringe (optionally a pen-like syringe). Alternatively, parenteral administration can be by means of an infusion pump. As a further option, insulin formulations containing an insulin compound of the invention may also be adapted for transdermal administration, e.g., by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosally, e.g., buccal administration .

The insulin formulations of the present invention can be administered to patients in need of such treatment at several sites, for example, in topical sites, for example, skin and mucosal sites; in non-absorbed sites, for example, in arteries, veins, heart; and in areas involving The site of absorption, eg, in the skin, under the skin, in the muscle or in the abdomen is administered.

In one embodiment of the invention, the insulin formulation is an aqueous formulation, ie a formulation comprising water. Such formulations are generally solutions or suspensions. In other embodiments 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. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w/w water.

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

In one embodiment, the insulin formulation of the invention is well suited for administration in a pen-like device for insulin therapy by injection.

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

As used herein, the term "physical stability" of an insulin formulation refers to the biological inability of the protein to form due to exposure of the protein to thermo-mechanical stress and/or interaction with destabilizing interfaces and surfaces (e.g. hydrophobic surfaces and interfaces). Active and/or propensity for insoluble aggregates. Aqueous properties are assessed by visual inspection and/or turbidity measurements after exposure of formulations filled in suitable containers (e.g., cartridges or vials) to mechanical/physical stress (e.g., agitation) at different temperatures for various periods of time. Physical stability of protein formulations. Visual inspection of formulations was performed in sharply focused light against a dark background. The turbidity of the formulations is characterized by a visual score assessing the degree of turbidity, for example on a scale of 0-3 (formulations showing no turbidity correspond to a visual score of 0, while formulations showing visual turbidity in daylight correspond to a visual score of 3). With respect to protein aggregation, the formulation was classified as physically unstable when it exhibited visual turbidity in daylight. 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 probes of the conformational state of the protein. Probes are preferably small molecules that preferably bind to non-native conformers of proteins. One example of a small molecule spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye widely used to detect amyloid fibrils. In the presence of fibrils, and possibly other protein structures, Thioflavin T causes a new excitation maximum at about 450 nm and, when bound to a fibril protein form, an enhanced emission at about 482 nm. Unconjugated Thioflavin T is essentially non-fluorescent at these wavelengths.

As used herein, the term "chemical stability" of a protein formulation refers to changes in the covalent protein structure that result in the formation of chemical degradation products that may have less biological potency and/or may enhance immunogenicity compared to the native protein structure. nature. 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. During storage and use of protein formulations, increased levels of chemical degradation products are often seen. Most proteins are susceptible to deamidation, a process in which 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 include the formation of high molecular weight products when two or more protein molecules are covalently bound to each other through amidation and/or disulfide interactions, resulting in the formation of covalently bound dimers, oligomers and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. T.J. & Manning M.C., Plenum Press, New York 1992). Oxidation, eg of methionine residues, can be mentioned as another variant of chemical degradation. The chemical stability of protein formulations can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions (eg, by increasing temperature, which generally accelerates the formation of degradation products). The amount of each individual degradation product is typically determined by separating the degradation products using various chromatographic techniques (eg, SEC-HPLC and/or RP-HPLC), depending on molecular size and/or charge. Low levels of HMWP are advantageous since HMWP products are potentially immunogenic and not biologically active.

The term "stabilized formulation" refers to a formulation that has increased physical stability, increased chemical stability, or increased physical and chemical stability. In general, formulations must be stable (subject to recommended use and storage conditions) during 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 for metabolic disorders in which the pancreas produces insufficient amounts of insulin or in which the cells of the body do not respond properly to insulin thus preventing the cells from absorbing glucose. As a result, glucose builds up in the blood.

Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, results from the destruction of B-cells, often resulting in absolute insulin deficiency.

Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and is thus a relative insulin deficiency with insulin resistance and/or a predominant insulin secretion defect.

The term "pharmaceutically acceptable" as used herein means suitable for normal pharmaceutical use, ie, not causing any serious adverse events in patients.

As used herein, the term "treating a disease" refers to the management and care of a patient already suffering from a disease, condition or disorder, and includes treating, preventing or alleviating the disease. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes the administration of an active compound to eliminate or manage the disease, condition or disorder as well as to alleviate symptoms or complications associated with the disease, condition or disorder and to prevent the disease, condition or disorder.

In its broadest sense, the term "critically ill patient" as used herein refers to a patient who sustains or is at risk of sustaining acute life-threatening failure of one or more organ systems due to disease or injury, pending surgery and associated complications patients, and those who have undergone major organ surgery within the last week or have undergone major surgery within the last week. In a stricter sense, the term "critically ill patient" as used herein refers to a patient who maintains or is at risk of sustaining acute life-threatening failure of one or more organ systems due to disease or injury, or who is undergoing surgery and associated complications patient. In an even stricter sense, the term "critically ill patient" as used herein refers to a patient who sustains or is at risk of sustaining acute life-threatening failure of a single or multiple organ system due to disease or injury. Similarly, these definitions apply to similar expressions such as "moribund patient" and "patient critically ill". Examples of critically ill patients are patients requiring heart surgery, brain surgery, chest surgery, abdominal surgery, vascular surgery or transplantation, or patients with neurological disease, brain trauma, respiratory insufficiency, abdominal peritonitis, multiple trauma or severe burns or Patients with critically ill polyneuropathy.

As used herein, the term "anabolism" refers to a set of metabolic pathways that build molecules from smaller units. These reactions require energy. One way of classifying metabolic processes, whether at the cellular, organ or organismal level, is as 'anabolism' or 'catabolism', relative to each other. Anabolism is powered by catabolism, in which large molecules are broken down into smaller parts, which are then used up in respiration. Many anabolic processes are powered by adenosine triphosphate (ATP). Anabolic processes tend to "build up" organs and tissues. These processes result in cell growth and differentiation and increase in body size, processes involving the synthesis of complex molecules. Examples of anabolic processes include bone growth and mineralization and improving muscle mass. Endocrinologists have traditionally classified hormones as anabolic or catabolic, depending on which part of metabolism they stimulate. The balance between anabolism and catabolism is also regulated by circadian rhythms, processes such as glucose metabolism that fluctuate to match the animal's normal cycle of activity throughout the day. Some examples of "anabolism" of these hormones are increasing protein synthesis from amino acids, increasing appetite, increasing bone remodeling and growth, and stimulating bone marrow (which increases production of red blood cells). Through several mechanisms, anabolic hormones stimulate the formation of muscle cells, thus causing an increase in the size of skeletal muscle, resulting in increased strength.

In another embodiment, the insulin analogues of the present invention are used as medicaments for delaying or preventing disease progression in type 2 diabetes.

In one embodiment of the present invention there is provided an insulin preparation of the present invention for use as a medicament for the treatment or prevention of hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgery Wounds and other diseases or injuries requiring anabolic effects in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders.

In other embodiments of the present invention, methods are provided for treating or preventing hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and in the treatment of Other diseases or injuries requiring anabolism, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, said method comprising administering to a patient in need of such treatment a therapeutic insulin formulation of the invention in an amount effective for such treatment.

Treatment with the insulin preparations according to the invention may also be combined with a second or more pharmacologically active substance, e.g. Agents for preventing complications caused by or associated with diabetes and agents for treating and/or preventing complications and conditions caused by or associated with obesity.

Treatment with insulin formulations according to the invention may also be combined with bariatric surgery, which is a surgical procedure affecting glucose levels and/or lipid homeostasis, such as gastric banding or gastric bypass.

The production of polypeptides (eg, insulin) is well known in the art. Insulin analogues of the invention can be produced, for example, by classical peptide synthesis, for example, solid-phase peptide synthesis using t-Boc or Fmoc chemistry or other established techniques, see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis (Protecting Groups in Organic Synthesis)", John Wiley & Sons, 1999. Insulin analogs may also be produced by a method comprising culturing a host cell containing the DNA sequence encoding the analog in a suitable nutrient medium under conditions permissive for expression of the insulin analog Insulin analogs. For insulin analogs comprising unnatural amino acid residues, recombinant cells should be modified such that the unnatural amino acid is incorporated into the analog, for example by using tRNA mutants. Thus, briefly, the insulin analogs of the invention are prepared analogously to the preparation of known insulin analogs.

Several methods are available for the production of human insulin and human insulin analogs. Three main methods for the production of insulin in microorganisms are disclosed eg in WO2008034881. Two of these methods involve E. coli, which express large fusion proteins in the cytoplasm (Frank et al. (1981), Peptides: Proceedings of the ^7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co. , Rockford, III. pp. 729-739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete insulin precursors into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a number of insulin precursors expressed in E. coli or Saccharomyces cerevisiae, see U.S. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

Insulin analogues are produced by expressing in suitable host cells the DNA sequence encoding the insulin analogue in question by well known techniques as disclosed, for example, in US 6,500,645. Insulin analogues are expressed directly or as precursor molecules with either an N-terminal extension on the B-chain or a C-terminal extension on the B-chain. The N-terminal extension may have the function of increasing the yield of the directly expressed product and may be up to 15 amino acid residues in length. After isolation from the culture broth, the N-terminal extension was excised in vitro, thus having a cleavage site next to B1. N-terminal extensions of the type suitable for use in the present invention are disclosed in US 5,395,922 and EP 765,395. The C-terminal extension may have the function of protecting the mature insulin or insulin analog molecule from intracellular hydrolytic processing by host cell exoproteases. The C-terminal extension is cleaved extracellularly in the culture broth or after isolation from the culture broth in vitro by secreted active carboxypeptidases. A method for producing mature insulin and insulin analogues with a C-terminal extension on the B-chain which is removed by carboxypeptidases is disclosed in WO 08037735. The target insulin product of the process may be a two-chain human insulin or a two-chain human insulin analog, which 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, said C-terminal extension should be able to be cleaved from the B-chain prior to further purification steps.

The present invention also includes the following non-limiting enumeration of embodiments, which are further described elsewhere herein:

1. An insulin preparation, said preparation comprising:

Insulin compounds,

nicotine compounds, and

· Arginine.

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

3. The insulin preparation according to any one 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 one of the preceding embodiments, wherein the insulin compound is B28Asp human insulin.

5. The insulin preparation according to any one of the preceding embodiments, wherein the insulin compound is B3LysB29Glu human insulin or B28LysB29Pro human insulin.

6. The insulin preparation according to any one of the preceding embodiments, wherein the insulin compound is B28LysB29Pro human insulin.

7. The insulin preparation according to any one of the preceding embodiments, wherein the insulin compound is B3LysB29Glu human insulin.

8. The insulin preparation according to any one of the preceding embodiments, wherein the insulin compound is present in a range selected from: 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 0.2-2.5mM; 0.2-2.0mM; 0.2-1.5mM; 0.3-3.0mM;

9. The insulin preparation according to any one 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 according to 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 according to any one 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 according to any one 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 according to any one 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 one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one 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 according to any one of the preceding embodiments, wherein said insulin compound is present in an amount of about 0.6 or about 1.2 mM.

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

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

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

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

29. The insulin preparation according to any one of the preceding embodiments, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nicotinic acid, nicotinamide and vitamin B3 and/or salts thereof and/or any combination thereof.

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

31. The insulin preparation according to any one of the preceding embodiments, wherein said nicotinic compound is nicotinamide and/or a salt thereof.

32. The insulin preparation according to any one of the preceding embodiments, wherein the nicotinic compound is present in a range selected from: 1-300 mM; 5-200 mM; 40-120 mM; 70-140 mM or 80-130 mM.

33. The insulin preparation according to any one of the preceding embodiments, comprising the nicotinic compound from about 1 mM to about 300 mM.

34. The insulin preparation according to any one of the preceding embodiments, comprising from about 8 mM to about 260 mM of the nicotinic compound.

35. The insulin preparation according to any one of the preceding embodiments, comprising from about 50 mM to about 250 mM of the nicotinic compound.

36. The insulin preparation according to any one of the preceding embodiments, comprising from about 80 mM to about 250 mM of the nicotinic compound.

37. The insulin preparation according to any one of the preceding embodiments, comprising from about 80 mM to about 180 mM of the nicotinic compound.

38. The insulin preparation according to any one of the preceding embodiments, comprising from about 5 mM to about 200 mM of the nicotinic compound.

39. The insulin preparation according to any one of the preceding embodiments, comprising from about 1 mM to about 150 mM of the nicotinic compound.

40. The insulin preparation according to any one of the preceding embodiments, comprising from about 5 mM to about 20 mM of the nicotinic compound.

41. The insulin preparation according to any one of the preceding embodiments, comprising from about 20 mM to about 120 mM of the nicotinic compound.

42. The insulin preparation according to any one of the preceding embodiments, comprising from about 40 mM to about 120 mM of the nicotinic compound.

43. The insulin preparation according to any one of the preceding embodiments, comprising from about 20 mM to about 40 mM of the nicotinic compound.

44. The insulin preparation according to any one of the preceding embodiments, comprising from about 60 mM to about 80 mM of the nicotinic compound.

45. The insulin preparation according to any one of the preceding embodiments, comprising from about 70 mM to about 140 mM of the nicotinic compound.

46. The insulin preparation according to any one of the preceding embodiments, comprising from about 80 mM to about 130 mM of the nicotinic compound.

47. The insulin preparation according to any one of the preceding embodiments, comprising about 8 mM, 30 mM, 100 mM or 130 mM of the nicotinic compound.

48. The insulin preparation according to any one of the preceding embodiments, comprising about 8 mM of the nicotinic compound.

49. The insulin preparation according to any one of the preceding embodiments, comprising about 30 mM, 100 mM or 130 mM of the nicotinic compound.

50. The insulin preparation according to any one of the preceding embodiments, comprising about 30 mM of a nicotinic compound.

51. The insulin preparation according to any one of the preceding embodiments, comprising about 80 mM of a nicotinic compound.

52. The insulin preparation according to any one of the preceding embodiments, comprising about 100 mM of a nicotinic compound.

53. The insulin preparation according to any one of the preceding embodiments, comprising about 120 mM of the nicotinic compound.

54. The insulin preparation according to any one of the preceding embodiments, comprising about 130 mM of the nicotinic compound.

55. The insulin preparation according to any one of the preceding embodiments, comprising about 150 mM of the nicotinic compound.

56. The insulin preparation according to any one of the preceding embodiments, comprising about 155 mM of the nicotinic compound.

57. The insulin preparation according to any one of the preceding embodiments, comprising about 180 mM of the nicotinic compound.

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

59. The insulin preparation according to any one of the preceding embodiments, said preparation comprising an arginine compound in the following ranges: 1-100 mM, 5-120 mM, 8-85 mM, 20-90 mM, 30-90 mM, 30-85 mM, 30 -60mM or 10-40mM.

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

61. The insulin preparation according to any one of the preceding embodiments, comprising about 1 mM to about 120 mM arginine.

62. The insulin preparation according to any one of the preceding embodiments, comprising about 1 mM to about 100 mM arginine.

63. The insulin preparation according to any one of the preceding embodiments, comprising about 5 mM to about 120 mM arginine.

64. The insulin preparation according to any one of the preceding embodiments, comprising about 20 mM to about 90 mM arginine.

65. The insulin preparation according to any one of the preceding embodiments, comprising about 30 mM to about 85 mM arginine.

66. The insulin preparation according to any one of the preceding embodiments, comprising about 8 mM to about 85 mM arginine.

67. The insulin preparation according to any one of the preceding embodiments, comprising about 30 mM to about 60 mM arginine.

68. The insulin preparation according to any one of the preceding embodiments, comprising about 10 mM to about 40 mM arginine.

69. The insulin preparation according to any one of the preceding embodiments, comprising about 10 mM to about 30 mM arginine.

70. The insulin preparation according to any one of the preceding embodiments, comprising about 10 mM to about 60 mM arginine.

71. The insulin preparation according to any one of the preceding embodiments, comprising arginine from about 1 mM to about 40 mM.

72. The insulin preparation according to any one of the preceding embodiments, wherein arginine is present in a range selected from 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 , 30mM, 35mM or 40mM, 45mM, 50mM, 55mM or 60mM.

73. The insulin preparation according to any one of the preceding embodiments, comprising about 1 mM arginine.

74. The insulin preparation according to any one of the preceding embodiments, comprising about 2 mM arginine.

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

76. The insulin preparation according to any one of the preceding embodiments, comprising about 4 mM arginine.

77. The insulin preparation according to any one of the preceding embodiments, comprising about 5 mM arginine.

78. The insulin preparation according to any one of the preceding embodiments, comprising about 6 mM arginine.

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

80. The insulin preparation according to any one of the preceding embodiments, comprising about 8 mM arginine.

81. The insulin preparation according to any one of the preceding embodiments, comprising about 9 mM arginine.

82. The insulin preparation according to any one of the preceding embodiments, comprising about 10 mM arginine.

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

84. The insulin preparation according to any one of the preceding embodiments, comprising about 12 mM arginine.

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

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

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

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

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

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

91. The insulin preparation according to any one of the preceding embodiments, comprising about 25 mM arginine.

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

93. The insulin preparation according to any one of the preceding embodiments, comprising about 35 mM arginine.

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

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

96. The insulin preparation according to any one of the preceding embodiments, comprising about 50 mM arginine.

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

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

99. The insulin preparation according to any one of the preceding embodiments, further comprising a buffering agent.

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

101. The insulin preparation according to embodiment 100, said preparation comprising from about 1 mg/mL to about 20 mg/mL of phosphate buffer.

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

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

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

105. The insulin preparation according to embodiment 99, wherein said buffering agent is Tris.

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

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

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

109. The insulin preparation according to embodiment 105, said preparation comprising about 10 mM, 20 mM, 30 mM or 40 mM Tris.

110. The insulin preparation according to embodiment 105, said preparation comprising about 7 mM Tris.

111. The insulin preparation according to embodiment 105, said preparation comprising about 10 mM Tris.

112. The insulin preparation according to embodiment 105, said preparation comprising about 20 mM Tris.

113. The insulin preparation according to embodiment 105, said preparation comprising about 30 mM Tris.

114. The insulin preparation according to embodiment 105, said preparation comprising about 40 mM Tris.

115. The insulin preparation according to any one of the preceding embodiments, further comprising metal ions.

116. The insulin preparation according to embodiment 115, wherein said metal ion is zinc.

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

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

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

120. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is from about 2:6 to about 6:6.

121. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is from about 2:6 to about 5:6.

122. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is from about 2.5:6 to about 4.5:6.

123. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is from about 3:6 to about 4:6.

124. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 2:6.

125. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 2.5:6.

126. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3:6.

127. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.1:6.

128. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.2:6.

129. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.3:6.

130. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.4:6.

131. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.5:6.

132. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.6:6.

133. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.7:6.

134. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.8:6.

135. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 3.9:6.

136. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 4:6.

137. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 4.5:6.

138. The insulin preparation according to embodiment 116, wherein said zinc:insulin molar ratio is about 5:6.

139. The insulin preparation according to any one of the preceding embodiments, further comprising a stabilizer.

140. The insulin preparation according to embodiment 139, wherein said stabilizing agent is a non-ionic detergent.

141. The insulin preparation according to embodiment 140, wherein the detergent is polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80).

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

143. The insulin preparation according to embodiment 140, wherein the detergent is polysorbate 80 (Tween 80).

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

145. The insulin preparation according to any one of the preceding embodiments, further comprising a preservative.

146. The insulin preparation according to embodiment 145, wherein said preservative is a phenolic compound.

147. The insulin preparation according to 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 according to embodiment 146, wherein said phenolic compound is present in an amount from about 5 to about 100 mM.

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

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

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

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

153. The insulin preparation according to embodiment 145, wherein the preservative is m-cresol.

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

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

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

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

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

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

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

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

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

164. The insulin preparation according to any one of the preceding embodiments, wherein said pH is neutral to slightly alkaline.

165. The insulin preparation according to any one of the preceding embodiments, wherein said pH is from about 6.8 to about 8.0.

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

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

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

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

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

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

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

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

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

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

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

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

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

179. A method of reducing blood glucose levels in a mammal by administering to a patient in need of such treatment a therapeutically active dose of the insulin preparation of any one of the preceding embodiments.

180. A method of treating diabetes in a subject, the method comprising administering to the subject an insulin formulation according to any one of embodiments 1-178.

181. The method according to any one of embodiments 179-180 for parenteral administration.

182. The insulin preparation according to any one of embodiments 1-178, for use in the treatment or prevention of hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and In the treatment of other diseases or injuries requiring anabolic effects, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and in the treatment of critically ill diabetic and non-diabetic patients.

183. The insulin formulation according to embodiment 182 for use in the treatment or prevention of hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns and surgical wounds, myocardial infarction, stroke, coronary Heart disease and other cardiovascular conditions.

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

185. An insulin preparation comprising:

Insulin compounds,

· Nicotinic compounds

Arginine, and

• Buffers.

186. The insulin preparation according to embodiment 185, wherein said insulin compound is human insulin or an insulin analog.

187. The insulin preparation according to any one of embodiments 185 or 186, wherein the insulin compound is selected from the group consisting of B28Asp human insulin, B3LysB29Glu human insulin and B28LysB29Pro.

188. The insulin preparation according to embodiment 186, wherein said insulin compound is B28Asp human insulin.

189. The insulin preparation according to any one of embodiments 185-188, wherein said insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.

190. The insulin preparation according to any one of embodiments 185-189, wherein said insulin compound is present in an amount from about 0.6 mM to about 1.2 mM.

191. The insulin preparation according to any one of embodiments 185-190, wherein said nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nicotinic acid, nicotinamide and vitamin B3 and/or salts thereof and/or any combination thereof.

192. The insulin preparation according to any one of embodiments 185-191, wherein said nicotinic compound is nicotinamide.

193. The insulin preparation according to embodiment 192, said preparation comprising from about 1 mM to about 250 mM of the nicotinic compound.

194. The insulin preparation according to embodiment 192, said preparation comprising from about 1 mM to about 250 mM of the nicotinic compound.

195. The insulin preparation according to embodiment 192, said preparation comprising from about 80 mM to about 230 mM of the nicotinic compound.

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

197. The insulin preparation according to any one of embodiments 185-196, comprising about 10 mM to about 30 mM arginine.

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

199. The insulin preparation according to any one of embodiments 185-198, wherein said buffer is a phosphate buffer.

200. The insulin preparation according to any one of embodiments 185-199, wherein said buffering agent is Tris.

201. The insulin preparation according to any one of embodiments 185-200, further comprising preservatives, isotonic agents and/or stabilizers.

202. The insulin preparation according to any one of embodiments 185-201, further comprising metal ions.

203. The insulin preparation according to embodiment 202, wherein said metal ion is zinc.

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

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

206. The insulin preparation according to any one of embodiments 185-205, wherein the pH of said preparation is less than 7.4.

207. The insulin preparation according to any one of embodiments 185-205, wherein said preparation has a pH of about 7.4.

208. The insulin preparation according to any one of embodiments 185-205, wherein the pH of said preparation is about 7.1.

209. An insulin preparation comprising: B28Asp human insulin; nicotinamide; zinc; arginine;

210. The insulin preparation of embodiment 209, wherein said B28Asp human insulin is present at a concentration ranging from about 0.6 mM to about 1.2 mM, and wherein said nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, and wherein said The arginine is present at a concentration ranging from about 10 mM to about 40 mM, and wherein there are less than about 4 zinc ions for every 6 B28Asp human insulin molecules, and wherein the pH of the formulation is about 7.4 or lower.

211. An insulin preparation, said preparation consisting essentially of:

a. B28Asp human insulin, wherein said B28Asp human insulin is present at a concentration ranging from about 0.6 mM to about 1.2 mM;

b. Niacinamide, wherein said nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM;

c. Zinc, wherein there are less than about 4 zinc ions for every 6 B28Asp human insulin molecules;

d. Arginine, wherein said arginine is present at a concentration ranging from about 10 mM to about 30 mM; and

e. Phosphate buffer;

wherein the formulation has a pH of about 7.1.

212. A method of lowering blood glucose levels in a mammal by administering to a mammal in need of such treatment a therapeutically active dose of an insulin preparation according to any one of the preceding embodiments.

213. A method of treating diabetes in a subject, the method comprising administering to the subject an insulin formulation according to any one of embodiments 1-211.

214. The insulin formulation according to any one of embodiments 1-211 for use in the treatment or prevention of hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and In the treatment of other diseases or injuries requiring anabolic effects, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and in the treatment of critically ill diabetic and non-diabetic patients.

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

Other embodiments of the invention relate to the following:

216. An insulin preparation comprising:

Insulin compounds,

nicotine compounds, and

· Arginine.

217. The insulin preparation according to embodiment 216, wherein said insulin compound is human insulin or an insulin analog.

218. The insulin formulation according to embodiments 216-217, wherein said insulin compound is B28Asp human insulin.

219. The insulin preparation according to any one of embodiments 216-218, wherein said insulin compound is B28LysB29Pro human insulin.

220. The insulin preparation according to any one of embodiments 216-219, wherein said insulin compound is B3LysB29Glu human insulin.

221. The insulin preparation according to any one of embodiments 216-220, wherein said insulin compound is present in an amount from about 0.2 mM to about 2.0 mM.

222. The insulin preparation according to any one of embodiments 216-221, wherein said insulin compound is present in an amount from about 0.3 mM to about 1.2 mM.

223. The insulin preparation according to any one of embodiments 216-222, wherein said nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, nicotinic acid, nicotinamide and vitamin B3 and/or salts thereof and/or any combination thereof.

224. The insulin preparation according to any one of embodiments 216-223, comprising the nicotinic compound from about 1 mM to about 150 mM.

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

226. The insulin preparation according to any one of embodiments 216-225, further comprising metal ions, preservatives, isotonic and stabilizers and buffers.

227. A method of reducing blood glucose levels in a mammal by administering to a patient in need of such treatment a therapeutically active dose of the insulin preparation of any one of embodiments 216-226.

228. A method of treating diabetes in a subject, the method comprising administering to the subject an insulin formulation according to any one of embodiments 216-226.

229. The insulin preparation according to any one of embodiments 216-226 for use in the treatment or prevention of hyperglycemia including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, surgical wounds and In the treatment of other diseases or injuries requiring anabolic effects, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and in the treatment of critically ill diabetic and non-diabetic patients.

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

All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference in their entirety as if each reference were individually and specifically indicated to the same extent as if each reference were individually and specifically indicated as being incorporated herein by reference and at This article is described in its entirety (to the fullest extent permitted by law).

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

The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Citation and incorporation of patent documents are for convenience only and do not reflect any view of the validity, patentability, and/or permissibility 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 pharmaceutical preparations

The pharmaceutical formulations of the invention can be formulated as aqueous solutions. Aqueous media are made isotonic, for example, with sodium chloride or glycerol. In addition, the aqueous medium may contain zinc ions (for example added as zinc acetate or zinc chloride), buffers and preservatives. Arginine can be added as Arg, HCl. The pH of the formulation is adjusted to the desired value and may be between about 3 to about 8.5, between about 3 to about 5 or about 6.5 to about 7.5, depending on the isoelectric point, pi, of the insulin in question.

Table 1. Composition of insulin preparations of the present invention

Table 2. Composition of other insulin preparations of the present invention

Table 3. Composition of other insulin preparations of the present invention

Table 4. Composition of other insulin preparations of the present invention

。

.

Example 2

Analysis of Insulin Chemical Stability

size exclusion chromatography

The quantitative determination of high molecular weight protein (HMWP) and monomeric insulin aspart was carried out on Waters insulin (300×7.8mm, part number wat 201549), and the eluent contained 2.5M acetic acid, 4mM L-arginine and 20% ( V/V) Acetonitrile at a flow rate of 1 ml/min at 40°C. Detection was performed at 276 nm using an adjustable absorbance detector (Waters 486). Injection volumes were 40 μl and 600 μM human insulin standard. The HMWP and concentration of the formulations were measured at each sampling point.

Reverse Phase Chromatography (UPLC)

Use the BEH RP C8 2.1×100mm column to implement the determination of insulin aspart related impurities on the UPLC system, the particle size is 1.7μm, Waters part number 186002878, the flow rate is 0.5ml/min, detected at 40°C, at 220nm . Elution was performed using a mobile phase consisting of:

A. 10% (w/v) acetonitrile, 2.8% (w/w) sodium sulfate, 0.3% (w/w) o-phosphoric acid, pH 3.5.

B. 70% (w/v) acetonitrile. Gradient: 0-11 minutes, using 73%/27% A/B constant solvent system; 11-12 minutes, linear to 52%/48% A/B; 13-15 minutes, linear to 73%/27 % A/B; 15-20 minutes with a 73%/27% A/B gradient constant solvent system.

The amount of B28 isoaspartate, deamidation 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 equivalent to the analytical method used for quality control of the insulin aspart drug product sold by Novo Nordisk.

Addition of arginine decreased the amount of degradation products formed, especially HMWP and deamidated forms, and increasing the concentration of arginine in the range of 10-50 mM resulted in a further decrease in degradation. Physical stability, measured as lag time in the ThT assay, decreased when arginine was added and decreased significantly when arginine concentration was increased. The overall performance of 50 mM arginine was superior to that of 50 mM glycine, 50 mM glutamic acid or 50 mM histidine with respect to the reduction in the formation of degradation products, as shown in Table 4 below.

The insulin formulations of the present invention provide rapid-acting insulin formulations that are not only physically stable, but surprisingly also chemically stable.

Table 5. Physical and Chemical Stability Data for Insulin Formulations 1-9 of 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 of Table 4

Example 3

Pharmacokinetic (PK)/pharmacodynamic (PD) studies and plasma analysis assays in the LYD porcine model

PK/PD Study in LYD Pigs

A PK/PD study was performed on LYD cross-bred domestic female pigs weighing between 55-110 kg. Pigs were cannulated in the jugular vein via the ear vein for at least 2 days prior to the start of the study. The last meal before the start of the study was approximately 18 hours before the animals were injected with the test formulation, and the animals were given free access to water during the fasting period and at all times during the test period.

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

Luminescence Oxygen Channel Immunoassay (LOCI) for Quantification of Insulin Aspart

Insulin aspart LOCI is a monoclonal antibody-based sandwich immunoassay and is administered in the vicinity of two beads (europium-coated acceptor-beads and streptavidin-coated donor-beads). The acceptor beads were coated with antibodies specific for human insulin and recognized insulin aspart in the plasma samples. Together with the streptavidin-coated beads, a second biotinylated antibody specifically binds to insulin aspart, which constitute the sandwich. Irradiation of the bead-aggregate-immune complex releases singlet oxygen from the donor bead, which passes through the channel into the acceptor bead and triggers chemiluminescence. Chemiluminescence is measured, and the amount of light produced is proportional to the concentration of insulin aspart.

The formulation of the invention had a faster initial rate of lowering of plasma glucose compared to the marketed product ^NovoRapid® (Figures 3 and 4). Also, the initial rate of absorption of the insulin component of the formulation of the invention was significantly faster when compared to ^NovoRapid® (Figure 5).

Example 4

A General Introduction to the ThT Fibrillation Assay for Assessing the Physical Stability of Protein Preparations

The low physical stability of peptides can lead to the formation of amyloid fibrils, which are observed as well-ordered linear-like macromolecular structures in samples, eventually leading to gel formation. This is traditionally measured by visual inspection of the sample. However, this measurement is very subjective and depends on the observer. Therefore, it is much more advantageous to administer small molecule indicator probes. Thioflavin T (ThT) is such a probe and has distinct fluorescence characteristics when bound to fibrils [Naiki et al. (1989) Anal. Biochem. 177 , 244-249; LeVine (1999) Methods. Enzymol. 309 , 274-284]. The time course of fibril formation can be described by a sigmoid curve using the following expression [Nielsen et al. (2001) Biochemistry 40 , 6036-6046]:

   方程式(1)

Equation (1)

Here, F is ThT fluorescence at time t. The constant _t0 is the time required to reach 50% of the maximum fluorescence. Two important parameters describing fibril formation are the delay-time calculated by t ₀ −2τ, the apparent rate constant k _app =1/τ.

Formation of partially folded intermediates of peptides is proposed as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form templates upon which other intermediates can assemble and fibrillate. The delay-time corresponds to the interval in which the critical mass of nuclei accumulates, and the apparent rate constant is the rate at which the fibrils themselves are formed.

Sample Preparation

Samples were freshly prepared before each assay. Each sample composition is described in each example. Using appropriate amounts of concentrated NaOH and _HClO or HCl, adjust the pH of the sample to the desired value. Thioflavin T was added to samples from stock solutions in _H2O to a final concentration of 1 μM.

Aliquots of 200 μl of samples were placed in 96-well microtiter plates (Packard OptiPlate ^™ -96, white polystyrene). Typically, for each sample, 4 or 8 replicates (corresponding to a test condition) are placed in a row of wells. Plates were sealed with Scotch Pad (Qiagen).

Incubation and fluorescence measurement

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

Each run was initiated by incubating the plate for 10 minutes at the assay temperature. Plates were measured every 20 minutes for the desired time period. Between each measurement, the plate was shaken and heated as described.

data processing

The measured points are stored in Microsoft Excel format for further processing and curve drawing, and the fitting is performed using GraphPad Prism. Background emission from ThT was negligible in the absence of fibrils. 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) appear in the same figure, ensuring a relative measurement of fibrillation between experiments.

The data set can be fitted with equation (1). However, since a complete S-shaped curve was not always achieved during the measurement time, the delay time was determined visually from the ThT fluorescence curve as the time point at which ThT fluorescence differed from the background level.

Measurement of initial and final concentrations

The peptide concentration of each tested formulation was measured both before application to the ThT fibrillation assay ("Initial") and after completion of ThT fibrillation ("Post ThT assay"). Concentrations were determined by reverse phase HPLC method using pramlintide standards as reference. Upon completion, 150 μl per replicate were collected and transferred to Eppendorf tubes before measurement. These were centrifuged at 30,000 G for 40 minutes. The supernatant was filtered through a 0.22 μm filter before being applied to the HPLC system.

Claims (32)

1. an insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound, and

Arginine.

2. the insulin preparation of claim 1, wherein said insulin compounds is insulin human or insulin analog.

3. the insulin preparation of any one in claim 1 or 2, wherein said insulin compounds is selected from aspart, B3LysB29Glu insulin human and B28LysB29Pro.

4. the insulin preparation of any one in aforementioned claim, wherein said insulin compounds is aspart.

5. the insulin preparation of any one in aforementioned claim, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.2mM-.

6. the insulin preparation of any one in aforementioned claim, wherein said insulin compounds exists with the amount of the about 1.2mM of about 0.6mM-.

7. the insulin preparation of any one in aforementioned claim, wherein said nicotine compound is selected from nicotiamide, nicotinic acid, nicotinic acid, niacin amide and vitamin B3 and/or its salt and/or their any combination.

8. the insulin preparation of any one in aforementioned claim, wherein said nicotine compound is nicotiamide.

9. the insulin preparation of any one in aforementioned claim, the nicotine compound that described preparation comprises the about 250mM of about 1mM-.

10. the insulin preparation of any one in aforementioned claim, the nicotine compound that described preparation comprises the about 250mM of about 1mM-.

11. the insulin preparation of any one in aforementioned claim, the nicotine compound that described preparation comprises the about 230mM of about 80mM-.

12. the insulin preparation of any one in aforementioned claim, the arginine that described preparation comprises the about 60mM of about 10mM-.

13. the insulin preparation of any one in aforementioned claim, the arginine that described preparation comprises the about 30mM of about 10mM-.

14. the insulin preparation of any one in aforementioned claim, described preparation comprises about 20mM arginine.

15. the insulin preparation of any one in aforementioned claim, described preparation also comprises one or more buffer agents.

16. the insulin preparation of claim 15, wherein said buffer agent is phosphate buffer.

17. the insulin preparation of claim 15, wherein said buffer agent is Tris.

18. the insulin preparation of any one in aforementioned claim, described preparation also can comprise antiseptic, isotonic agent and/or stabilizing agent.

19. the insulin preparation of any one in aforementioned claim, described preparation also comprises metal ion.

20. the insulin preparation of claim 19, wherein said metal ion is zinc.

21. the insulin preparation of claim 20, wherein said zinc: the insulin mol ratio is the about 3.5:6 of about 0:6-.

22. the insulin preparation of claim 20, wherein said zinc: the insulin mol ratio is the about 3:6 of about 2:6-.

23. the insulin preparation of any one in aforementioned claim, the pH of wherein said preparation is less than 7.4.

24. the insulin preparation of any one in aforementioned claim, the pH of wherein said preparation is approximately 7.4.

25. the insulin preparation of any one in aforementioned claim, the pH of wherein said preparation is approximately 7.1.

26. an insulin preparation, described preparation comprises: aspart; Nicotiamide; Zinc; Arginine; And phosphate buffer.

27. the insulin preparation of claim 26, wherein said aspart with about 0.6 mM-approximately the concentration of 1.2 mM scopes exist, and wherein said nicotiamide with about 80 mM-approximately the concentration of 260 mM scopes exist, and wherein said arginine with about 10 mM-approximately the concentration of 40 mM scopes exist, and every 6 aspart molecules wherein, existence is less than approximately 4 zinc ioies, and the pH of wherein said preparation is approximately 7.4 or lower.

28. an insulin preparation, described preparation is comprised of following basically:

A. aspart, wherein said aspart with about 0.6 mM-approximately the concentration of 1.2 mM scopes exist;

B. nicotiamide, wherein said nicotiamide with about 80 mM-approximately the concentration of 260 mM scopes exist;

C. zinc, wherein every 6 aspart molecules, exist and be less than approximately 4 zinc ioies;

D. arginine, wherein said arginine with about 10 mM-approximately the concentration of 30 mM scopes exist; With

E. phosphate buffer;

The pH of wherein said preparation is approximately 7.1.

29. a method that reduces blood glucose level mammals, the insulin preparation of any one in the aforementioned claim of the mammal therapeutic activity dosage of described method by needing this treatment.

30. a method that is used for the treatment of experimenter's diabetes, described method comprises the insulin preparation that gives any one in experimenter's claim 1-28.

31. the insulin preparation of any one in claim 1-28, described preparation is used for the treatment of or prevents hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and need Other diseases or damage, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder of anabolic action and treat critical diabetes and ND in treatment.

32. the insulin preparation of claim 31, described preparation is used for the treatment of hyperglycemia type 2 diabetes mellitus and type 1 diabetes.

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