AU2013203134B2 - 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

1 PREPARATION COMPRISING INSULIN, NICOTINAMIDE AND AN AMINO ACID The present application is a divisional application of Australian Application No. 2010264636, which is incorporated in its entirety herein by reference. 5 FIELD OF THE INVENTION The present invention relates to pharmaceutical preparations comprising an insulin compound, a nicotinic compound and an amino acid. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be 10 considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions. 15 Since the introduction of insulin in the 1920's, continuous improvements have been made in the treatment of diabetes. To help avoid high glycaemia levels, diabetic patients often practice multiple injection therapy, whereby insulin is administered with each meal. As diabetic patients have been treated with insulin for several decades, there is a major need for safe and life-quality improving insulin preparations. Among the 20 commercially available insulin preparations, rapid-acting, intermediate-acting and prolonged-acting preparations can be mentioned. In the treatment of diabetes mellitus, many varieties of pharmaceutical preparations of insulin have been suggested and used, such as regular insulin (such as Actrapid*), isophane insulin (designated NPH), insulin zinc suspensions (such as 25 Semilente*, Lente*, and Ultralente*), and biphasic isophane insulin (such as NovoMix*). Human insulin analogues and derivatives have also been developed, designed for particular profiles of action, i.e. fast action or prolonged action. Some of the commercially available insulin preparations comprising such rapid acting insulin analogues include NovoRapid@ (preparation of B28Asp human insulin), Humalog@ (preparation of 30 B28LysB29Pro human insulin) and Apidra@ (preparation of B3LysB29GIu human insulin). International applications WO 91/09617 and WO/9610417 (Novo Nordisk A/S) disclose insulin preparations containing nicotinamide or nicotinic acid or a salt thereof.

2 Most often pharmaceutical preparations of insulins are administered by subcutaneous injection. Important for the patient is the action profile of the insulin, meaning the action of insulin on glucose metabolism as a function of time from injection. In this profile, inter alia, the time of the onset, the maximum value and the total duration 5 of action are important. In the case of bolus insulins, a variety of insulin preparations with different action profiles are desired and requested by the patients. One patient may, on the same day, use insulin preparations with very different action profiles. The action profile desired for example, depends on the time of the day and the amount and composition of the meal eaten by the patient. 10 Equally important for the patient is the chemical stability of the insulin preparations, for example, due to the abundant use of pen-like injection devices such as devices which contain Penfill* cartridges, in which an insulin preparation is stored until the entire cartridge is empty which may be at least 1 to 2 weeks for devices containing 1.5-3.Oml cartridges. During storage, covalent chemical changes in the insulin structure 15 occur. This may lead to formation of molecules which may be less active and/or potentially immunogenic such as deamidation products and higher molecular weight transformation products (dimers, polymers). Furthermore, also important is the physical stability of the insulin preparations, since long term storage may eventually lead to formation of insoluble fibrils, which are biologically inactive and potentially 20 immunogenic. Summary of the invention The invention relates to insulin preparations with favourable absorption rate and favourable chemical and physical stability. The present invention relates to insulin 25 preparations comprising human insulin and/or analogues thereof, nicotinamide or nicotinic acid and/or salts thereof and arginine. According to a first aspect, the present invention relates to a pharmaceutical formulation comprising: B28Asp human insulin; a nicotinic compound; arginine; a preservative; and a buffer. 30 According to a second aspect, the present invention relates to a pharmaceutical formulation comprising: B28Asp human insulin; nicotinamide; zinc; arginine; a preservative; and a phosphate buffer.

2a According to a third aspect, the present invention relates to an aqueous pharmaceutical formulation consisting essentially of: B28Asp human insulin; a nicotinic compound; arginine; a buffer; zinc; a preservative; and an isotonicity agent. Unless the context clearly requires otherwise, throughout the description and the 5 claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Description of the drawings Figure 1 shows the development in percentage of total insulin content of 10 degradation products during 2 weeks of storage at 37'C of preparations according to the present invention. The letter A refers to a NovoRapid* reference and remaining letters correspond to insulin as 3 part preparations as described in Table 1 of Example 1. Compared to the NovoRapid* preparation (preparation A), addition of nicotinamide (preparations B and D) leads to an in creased formation of degradation products, whereas the combined addition of nicotinamide, glutamic acid and arginine (preparations C and E), has a mostly similar degradation pattern, 5 with lower formation of HMWP. Figure 2 shows the development in percentage of total insulin content of degradation prod ucts during 2 weeks of storage at 37"C of preparations according to this invention. The letter A refers to a NovoRapid* reference and remaining letters correspond to insulin aspart prepa 10 rations as described in Table 1 of Example 1. The combined addition of nicotinamide, glu tamic acid and arginine, preparations F, G, H, and I, differing in buffer system, phosphate or tris buffer, and concentration of insulin and Zn, 0.6mM and 0.3mM or 1.2mM and 0.6mM, has a degradation pattern similar to the NovoRapid* preparation, preparation A. 15 Figure 3 shows the glucose concentration (mean +/- SEM, N=8) in plasma after subcutane ous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this inven tion. The letter A refers to a NovoRapid* reference and remaining letters correspond to insu lin aspart preparations as described in Table 1 of Example 1. Compared to the NovoRapid* preparation (preparation A) the initial rate of plasma glucose lowering is faster for the prepa 20 ration with addition of nicotinamide (preparation N) and even faster for a combination of nicotinamide and arginine (preparation M). Figure 4 shows the glucose concentration in plasma (mean +/- SEM, N=7) after subcutane ous injection in pigs of a 1nmol/kg dose at 0 minutes of preparations according to this inven 25 tion. The letter A refers to a NovoRapid* reference and remaining letters correspond to insu lin aspart preparations as described in Table 1 of Example 1. Compared to the NovoRapid* preparation (preparation A), the initial rate of plasma glucose lowering is faster for a prepara tion with a combination of nicotinamide, arginine and glutamic acid (preparation L) and for a preparation with a combination of nicotinamide and arginine (preparation K). 30 Figure 5 shows the insulin aspart concentration in plasma (mean +/- SEM, N=7) after subcu taneous injection in pigs of a 1 nmol/kg dose at 0 minutes of preparations according to this invention. The letter A refers to a NovoRapid* reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1. Compared to the No 35 voRapid* preparation, (preparation A) , the initial absorption rate of the insulin component of 4 the preparations with nicotinamide (preparation J), the combination of nicotinamide and ar ginine (preparation K), and the combination of nicotinamide, arginine and glutamic acid (preparation L) is markedly faster. 5 DESCRIPTION OF THE INVENTION The absorption after subcutaneous injection of the insulin compound in the insulin prepara tions of the present invention was surprisingly found to be faster than that of the reference insulin preparations. This property is useful for rapid-acting insulins, in particular in connec tion with a multiple injection regimen where insulin is given before each meal. With faster on 10 set of action, the insulin can conveniently be taken closer to the meal than with conventional rapid acting insulin solutions. Furthermore, a faster disappearance of insulin probably dimin ishes the risk of post-meal hypoglycaemia. The insulin preparations of the present invention are rapid-acting insulin prepara tions comprising an insulin compound such as insulin aspart, a nicotinic compound, such as 15 nicotinamide and the amino acid arginine. Optionally, the insulin preparations of the present invention may comprise further amino acids such as glutamic acid. These insulin prepara tions have a rapid absorption profile that mimics normal physiology more closely than exist ing therapies. Furthermore, the insulin preparations of the present invention have chemical and physical stability suitable for commercial pharmaceutical preparations. 20 The insulin preparations of the present invention provide an even faster onset of ac tion compared with existing insulin therapies. Such ultra-fast insulin preparations have the advantage of restoring first phase insulin release, injection convenience and shutting down hepatic glucose production. The insulin preparations of the present invention have a favour able absorption rate from subcutis into plasma with an increase in initial absorption rate rang 25 ing from 1.5 to 5 times, when compared to conventional preparations such as NovoRapid*, as suggested by several PK/PD experiments in pigs. This faster absorption rate may improve glycaemic control and convenience and may allow for a shift from pre-meal to post-meal dos ing. The present invention is based in part, on the surprising discovery that although, the ad dition of nicotinamide allows the increase in absorption rate, it also has a negative effect on 30 chemical stability by significantly increasing the amount of HMWP.The insulin preparations of the present invention have an improved chemical stability by addition of arginine, which is reflected in e.g. a reduction in the formation of timers and polymers and desamido insulins after storage. The insulin preparations of the present invention may furthermore also have improved physical stability, which may be useful for use in pumps.

5 The present invention provides an insulin preparation comprising an insulin com pound according to the present invention which is present in a concentration from about 0.1 mM to about 10.0mM, and wherein said preparation has a pH from 3 to 8.5. The preparation also comprises a nicotinic compound and arginine. The preparation may further comprise 5 protease inhibitor(s), metal ions, a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment the insulin preparations comprise a human insulin, an analogue or combinations thereof, nicotinamide and/or nicotinic acid and/or salts thereof and arginine and/or salts thereof. 10 In one embodiment, the insulin preparations according to the present invention com prise an aqueous solution of B28Asp human insulin, nicotinamide and arginine. The content of B28Asp human insulin in the solutions of this invention may be in the range of 15 to 500 international units (IU)/ml, preferably in the range of 50 to 333 IU/ml, in preparations for injection. However, for other purposes of parenteral administration, the con 15 tent of insulin compound may be higher. There is also described herein an insulin preparation comprising an insulin com pound, a nicotinic compound and glutamic acid. In the present context the unit "'U" corresponds to 6 nmol. The term "insulin aspart" refers to the human insulin analogue B28Asp human insu 20 lin. The term 'onset" refers to the time from injection until the PK curve shifts to an in crease. The term "absorption rate" refers to the slope of the PK curve. An "insulin compound" according to the invention is herein to be understood as hu 25 man insulin, an insulin analogue and/or any combination thereof. The term "human insulin" as used herein means the human hormone whose struc ture and properties are well-known. Human insulin has two polypeptide chains that are con nected by disuiphide bridges between cysteine residues, namely the A-chain and the B chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, 30 the two chains being connected by three disuiphide bridges: one between the cysteines in position 6 and 11 of the A-chain, the second between the cysteine in position 7 of the A chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in po sition 20 of the A-chain and the cysteine in position 19 of the B-chain. The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) 35 consisting of a prepeptide of 24 amino acids followed by proinsulin containing 86 amino acids 6 in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting pep tide from the A and B chains. By "insulin analogue" as used herein is meant a polypeptide derived from the pri 5 mary structure of a naturally occurring insulin, for example that of human insulin, by mutation. One or more mutations are made by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or by adding at least one amino acid residue. The added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues. 10 In one embodiment an insulin analogue comprises less than 8 modifications (substi tutions, deletions, additions and any combination thereof) relative to the parent insulin, alter natively less than 7 modifications relative to the parent insulin, alternatively less than 6 modi fications relative to the parent insulin, alternatively less than 5 modifications relative to the parent insulin, alternatively less than 4 modifications relative to the parent insulin, alterna 15 tively less than 3 modifications relative to the parent insulin, alternatively less than 2 modifi cations relative to the parent insulin. Mutations in the insulin molecule are denoted stating the chain (A or B), the position, and the three letter code for the amino acid substituting the native amino acid. By "desB30" or "B(1-29)" is meant a natural insulin B chain or analogue thereof lacking the B30 amino 20 acid residue, and by B28Asp human insulin is meant human insulin wherein the amino acid residue in position 28 of the B chain has been substituted with Asp. Examples of insulin analogues are such wherein Pro in position 28 of the B chain is mutated with Asp, Lys, Leu, VaI, or Ala andlor Lys at position B29 is mutated with Pro, Glu or Asp. Furthermore, Asn at position B3 may be mutated with Thr, Lys, Gin, Glu or Asp. The 25 amino acid residue in position A21 may be mutated with Gly. The amino acid in position B1 may be mutated with Glu. The amino acid in position B16 may be mutated with Glu or His. Further examples of insulin analogues are the deletion analogues e.g. analogues where the B30 amino acid in human insulin has been deleted (des(B30) human insulin), insulin ana logues wherein the B1 amino acid in human insulin has been deleted (des(Bl) human insu 30 lin), des(B28-B30) human insulin and des(B27) human insulin. Insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension such as with two arginine residues added to the C-terminal of the B-chain are also examples of insulin analogues. Further ex amples are insulin analogues comprising combinations of the mentioned mutations. Insulin 35 analogues wherein the amino acid in position A14 is Asn, GIn, Glu, Arg, Asp, Gly or His, the 7 amino acid in position B25 is His and which optionally further comprises one or more addi tional mutations are further examples of insulin analogues, Insulin analogues of human insu lin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues are also examples of insulin 5 analogues. Further examples of insulin analogues include, but are not limited to: DesB30 human insulin; AspB28 human insulin; AspB28,desB30 human insulin; LysB3,GIuB29 human insulin; LysB28,ProB29 human insulin; GlyA21,ArgB31,ArgB32 human insulin; GluA14,HisB25 hu man insulin; HisA14,HisB25 human insulin: GIuA14,HisB25,desB30 human insulin; HisA14, 10 HisB25,desB30 human insulin; GluA14,HisB25desB27,desB28,desS29,desB30 human insu lin; GIuA14,HisB25,GluB27,desB30 human insulin; GIuA14,HisB16,HisB25desB30 human insulin; HisA14,HisB16,HisB25,desB3O human insulin; HisA8,GluA14,HisB25,GiuB27,desB30 human insulin; HisA8,GluA14,GluBlGluB16,HisB25,GluB27,desB30 human insulin; and Hi 15 sA8,GluA14,GluB16,HisB25,desB30 human insulin. The term "nicotinic compound" includes nicotinamide, nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof. According to the present invention, the concentration of the nicotinic compound and/or salts thereof is in the range from about 1mM to about 300mM or from about 5mM to 20 about 200mM. The term "arginine" or "Arg" includes the amino acid arginine and/or a salt thereof. In one embodiment, the insulin preparation comprises 1 to 100mM of arginine. In one embodiment, the insulin preparation comprises 1 to 20mM of arginine. In one embodiment, the insulin preparation comprises 20 to 90mM of arginine. 25 In one embodiment, the insulin preparation comprises 30 to 85mM of arginine. The term "glutamic acid" or "Glu" includes the aminoacid glutamic acid and/or a salt thereof. In one embodiment, the insulin preparation comprises 1 to 100mM of glutamic acid. 30 In one embodiment, the insulin preparation comprises 20 to 90mM of glutamic acid. In one embodiment, the insulin preparation comprises 30 to 85mM of glutamic acid. The term "pharmaceutical preparation" or "insulin preparation" as used herein means a product comprising an insulin compound, i.e., a human insulin, an analogue thereof and/or combinations thereof and a nicotinic compound and an aminoacid, optionally together 35 with other excipients such as preservatives, cheating agents, tonicity modifiers, bulking 8 agents, stabilizers, antioxidants, polymers and surfactants, metal ions, oleaginous vehicles and proteins (e.g., human serum albumin, gelatin or proteins), said insulin preparation being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said insulin preparation to a person. Thus, an insulin preparation is also 5 known in the art as a pharmaceutical preparation or pharmaceutical composition. The buffer may be selected from the group consisting of, but not limited to, sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. 10 Each one of these specific buffers constitutes an alternative embodiment of the invention. The insulin preparation of the present invention may further comprise other ingredi ents common to insulin preparations, for example zinc complexing agents such as citrate, and phosphate buffers. Glycerol and/or mannitol and/or sodium chloride may be present in an amount cor 15 responding to a concentration of 0 to 250mM, 0 to 200mM or 0 to 100mM. Stabilizers, surfactants and preservatives may also be present in the insulin preparations of this invention. The insulin preparations of the present invention may further comprise a pharmaceutically acceptable preservative. The preservative may be present in an amount 20 sufficient to obtain a preserving effect. The amount of preservative in an insulin preparation may be determined from e.g. literature in the field and/or the known amount(s) of preservative in e.g. commercial products. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical preparations is described, for example in Remington: The Science and 25 Practice of Pharmacy, 19 " edition, 1995. The preservative present in the insulin preparation of this invention may be as in the heretofore conventional insulin preparations, for example phenol, m-cresol and methylpara ben. The insulin preparation of the present invention may further comprise a chelating 30 agent. The use of a chelating agent in pharmaceutical preparations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 1 9* edition, 1995. The insulin preparation of the present invention may further comprise a stabilizer. The term "stabilizer" as used herein refers to chemicals added to polypeptide containing 35 pharmaceutical preparations in order to stabilize the peptide, i.e. to increase the shelf life 9 and/or in-use time of such preparations. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19" edition, 1995. The insulin preparation of the present invention may further comprise a surfactant. The term "surfactant" as used herein refers to any molecules or ions that are comprised of a 5 water-soluble (hydrophilic) part, the head, and a fat-soluble (ipophilic) segment. Surfactants accumulate preferably at interfaces, which the hydrophilic part is orientated towards the wa ter (hydrophilic phase) and the lipophilic part towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore, surfactants lower the surface tension 10 of a liquid. Surfactants are also known as amphipathic compounds. The term "detergent" is a synonym used for surfactants in general. The use of a surfactant in pharmaceutical prepara tions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19" edition, 1995. In a further embodiment the invention relates to an insulin preparation comprising an 15 aqueous solution of an insulin compound of the present invention, and a buffer, wherein said insulin compound is present in a concentration from D.1mM or above, and wherein said preparation has a pH from about 3.0 to about 8.5 at room temperature (~25*C). The present invention also relates to methods for producing the insulin preparations of the invention. 20 In one embodiment, the method for making insulin preparations of the invention comprises: a) preparing a solution by dissolving the insulin compound or a mixture of insulin compounds in water or buffer; b) preparing a solution by dissolving a divalent metal ion in water or buffer; 25 c) preparing a solution by dissolving a preservative in water or buffer; d) preparing a solution by dissolving an isotonicity agent in water or buffer; e) preparing a solution by dissolving a surfactant and/or a stabilizer in water or buffer; f) mixing solution a) and one or more of solutions b), c), d), and e); 30 Finally adjusting the pH of the mixture in f) to the desired pH followed by a sterile fil tration. The insulin preparations of the present invention can be used in the treatment of diabetes by parenteral administration, It is recommended that the dosage of the insulin preparations of this invention which is to be administered to the patient be selected by a phy 35 sician.

10 Parenteral administration may be performed by subcutaneous, intramuscular, in traperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. As a further option, the insulin preparations containing the insulin compound of the invention can 5 also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration, Insulin preparations according to the present invention may be administered to a pa tient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, 10 in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen. In one embodiment of the invention the insulin preparation is an aqueous prepara tion, i.e. preparation comprising water. Such preparation is typically a solution or a suspen sion, In a further embodiment of the invention the insulin preparation is an aqueous solution. 15 The term "aqueous preparation" is defined as a preparation comprising at least 50 %w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension com prising at least 50 %w/w water. Aqueous suspensions may contain the active compounds in admixture with excipients 20 suitable for the manufacture of aqueous suspensions. In one embodiment, the insulin preparations of this invention are well-suited for ap plication in pen-like devices used for insulin therapy by injection. In one embodiment the insulin preparations of the present invention can be used in pumps for insulin administration. 25 The term "physical stability" of the insulin preparation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein preparations is evaluated by means of visual inspec 30 tion and/or turbidity measurements after exposing the preparation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different tempera tures for various time periods. Visual inspection of the preparations is performed in a sharp focused light with a dark background. The turbidity of the preparation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a preparation 35 showing no turbidity corresponds to a visual score 0, and a preparation showing visual tur- 11 bidity in daylight corresponds to visual score 3). A preparation is classified physically unsta ble with respect to protein aggregation, when it shows visual turbidity in daylight. Alterna tively, the turbidity of the preparation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein preparations can 5 also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations 10 as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essen tially non-fluorescent at the wavelengths. The term "chemical stability" of the protein preparation as used herein refers to changes in the covalent protein structure leading to formation of chemical degradation prod 15 ucts with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Increasing amounts of chemical degradation products is often seen during storage and use of the protein preparation. Most proteins are prone to deamidation, a 20 process in which the side chain aide group in glutaminyl or asparaginyl residues is hydro lysed to form a free carboxylic acid or asparaginyl residues to form an IsoAsp derivative. Other degradations pathways involves formation of high molecular weight products where two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and 25 polymer degradation products (Stability of Protein Pharmaceuticals, Ahem. T.J & Manning M.C., Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein preparation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of 30 degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the deg radation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since HMWP products are potentially immu nogenic and not biologically active, low levels of HMWP are advantageous.

12 The term "stabilized preparation" refers to a preparation with increased physical sta bility, increased chemical stability or increased physical and chemical stability. In general, a preparation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached. 5 The term "diabetes" or "diabetes mellitus" includes type I diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other states that cause hyperglycaemia. The term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the cells of the body fail to respond appropriately to insulin thus prevent ing cells from absorbing glucose. As a result, glucose builds up in the blood. 10 Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile onset diabetes, is caused by B-cell destruction, usually leading to absolute insulin deficiency. Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance. 15 The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e., not giving rise to any serious adverse events in patients. The term "treatment of a disease" as used herein means the management and care of a patient having developed the disease, condition or disorder and includes treatment, pre vention or alleviation of the disease. The purpose of treatment is to combat the disease, con 20 dition or disorder. Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complica tions associated with the disease, condition or disorder, and prevention of the disease, condi tion or disorder. In another embodiment, an insulin analogue according to the invention is used as a 25 medicament for delaying or preventing disease progression in type 2 diabetes. In one embodiment of the present invention, the insulin preparation according to the invention is for use as a medicament for the treatment or prevention of hyperglycemia includ ing stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabe tes, and bums, operation wounds and other diseases or injuries where an anabolic effect is 30 needed in the treatment, myocardial infarction, stroke, coronary heart disease and other car diovascular disorders is provided. In a further embodiment of the present invention, a method for the treatment or pre vention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or inju 35 ries where an anabolic effect is needed in the treatment, myocardial infarction, coronary 13 heart disease and other cardiovascular disorders, stroke, the method comprising administer ing to a patient in need of such treatment an effective amount for such treatment of an insulin preparation according to the invention, is provided. The treatment with an insulin preparation according to the present invention may 5 also be combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associ ated with diabetes and agents for the treatment and/or prevention of complications and dis orders resulting from or associated with obesity. 10 The treatment with an insulin preparation according to the present invention may also be combined with bariatric surgery - a surgery that influences the glucose levels and/or lipid homeostasis such as gastric banding or gastric bypass. The production of polypeptides, e.g., insulins, is well known in the art. An insulin analogue according to the invention may for instance be produced by classical peptide syn 15 thesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well estab lished techniques, see e.g. Greene and Wuts, "Protective Groups in Organic Synthesis*, John Wiley & Sons, 1999. The insulin analogue may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and ca pable of expressing the insulin analogue in a suitable nutrient medium under conditions per 20 milling the expression of the insulin analogue. For insulin analogues comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue, for instance by use of tRNA mutants. Hence, briefly, the insulin analogues according to the invention are prepared analogously to the preparation of known insulin analogues. 25 Several methods may be used for the production of human insulin and human insu lin analogues. For example three major methods which are used in the production of insulin in microorganisms are disclosed in W02008034881. Two of these involve Escherichia col, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7thAmerican Peptide Chemistry Symposium (Rich & Gross, 30 eds.), Pierce Chemical Co., Rockford, Il1. pp 729-739), or use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a number of insulin precur sors which are expressed in either E coli or Saccharomyces cerevisiae, vide U.S5,962,267, 35 WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

14 The insulin analogues are produced by expressing a DNA sequence encoding the insulin analogue in question in a suitable host cell by well known technique as disclosed in e.g. US 6500645. The insulin analogue is either expressed directly or as a precursor mole cule which has an N-terminal extension on the B-chain or a C-terminal extension on the B 5 chain. The N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long. The N-terminal exten sion is to be cleaved of in vitro after isolation from the culture broth and will therefore have a cleavage site next to B1. N-terminal extensions of the type suitable in the present invention are disclosed in US 5,395,922, and EP 765,395. The C-terminal extension may have the 10 function of protecting the mature insulin or insulin analogue molecule against intracellular proteolytic processing by host cell exoproteases. The C-terminal extension is to be cleaved of either extra-cellularly in the culture broth by secreted, active carboxypeptidase or in vitro after isolation from the culture broth. A method for producing mature insulin and insulin ana logs with C-terminal extensions on the B-chain that are removed by carboxypetidase are dis 15 closed in WO 08037735. The target insulin product of the process may either be a two-chain human insulin or a two-chain human insulin analogue which may or may not have a short C terminal extension of the B-chain. If the target insulin product will have no C-terminal exten sion of the B-chain, then said C-terminal extension should be capable of subsequently being cleaved off from the B-chain before further purification steps. 20 The present invention also contemplates the following non-limiting list of embodi ments, which are further described elsewhere herein: 1. An insulin preparation comprising: * an insulin compound, 25 . a nicotinic compound, and * arginine. 2. The insulin preparation according to embodiment 1, wherein the insulin compound is hu man insulin or an insulin analog. 3. The insulin preparation according to any of the preceding embodiments, wherein the in 30 sulin compound is B28Asp human insulin. 4. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is B28LysB29Pro human insulin. 5. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is B3LysB29Glu human insulin. 35 6. The insulin preparation according to any of the preceding embodiments, wherein the in- 15 sulin compound is present in a range selected from the following: 0.1-10.0mM; 0.1 3.0mM; 0.1-2,6mM; 0.1-2.0mM; 0.1-1.5mM; 0.2-2.5mM; 0.2-2.0mM; 0.2-1.5mM; 0.3 3.0mM; 0.3-2.5mM; 0.3-2.0mM; 0.3-1.5mM; 0.5-1.3mM and 0.6-1.2mM. 7. The insulin preparation according to any of the preceding embodiments, wherein the in 5 sulin compound is present in the amount from about 0.1mM to about 10.0mM. 8. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.1mM to about 3.0mM. 9. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.1mM to about 2.5mM. 10 10. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amountfrom about 0.1mM to about 2.0mM. 11. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.1mM to about 1.5mM. 12. The insulin preparation according to any of the preceding embodiments, wherein the in 15 sulin compound is present in the amount from about 0.2mM to about 2.5mM. 13. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.2mM to about 2.0mM. 14. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.2mM to about 1.5mM. 20 15. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.3mM to about 3.0mM. 16. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.3mM to about 2.5mM. 17. The insulin preparation according to any of the preceding embodiments, wherein the in 25 sulin compound is present in the amount from about 0.3mM to about 2.0mM. 18. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.3mM to about 1.5mM. 19. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.5mM to about 1.3mM. 30 20. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.3mM to about 1.2mM. 21. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount from about 0.6mM to about 1.2mM. 22. The insulin preparation according to any of the preceding embodiments, wherein the in 35 sulin compound is present in the amount of about 0.6 or about 1.2mM.

16 23. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount of about 0.3mM. 24. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount of about 0.6mM. 5 25. The insulin preparation according to any of the preceding embodiments, wherein the in sulin compound is present in the amount of about 1.2mM. 26. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is selected from the group consisting of nicotinamide, nicotinic acid, niacin, niacin amide and vitamin 83 and/or salts thereof and/or any combination thereof. 10 27. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is selected from nicotinamide and nicotinic acid and/or salts thereof and/or any combination thereof. 28. The insulin preparation according to any of the preceding embodiments, wherein the nicotinic compound is present in a range selected from the following: 1-300mM; 5 15 200mM; 40-120mM, 70-140mM or 80-130mM. 29. The insulin preparation according to any of the preceding embodiments, comprising from about 1mM to about 300mM of the nicotinic compound. 30. The insulin preparation according to any of the preceding embodiments, comprising from about 8mM to about 260mM of the nicotinic compound. 20 31. The insulin preparation according to any of the preceding embodiments, comprising from about 6mM to about 200mM of the nicotinic compound. 32. The insulin preparation according to any of the preceding embodiments, comprising from about 1mM to about 150mM of the nicotinic compound. 33. The insulin preparation according to any of the preceding embodiments, comprising from 25 about 5mM to about 20mM of the nicotinic compound. 34. The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 120mM of the nicotinic compound. 35. The insulin preparation according to any of the preceding embodiments, comprising from about 40mM to about 120mM of the nicotinic compound. 30 36. The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 40mM of the nicotinic compound. 37. The insulin preparation according to any of the preceding embodiments, comprising from about 60mM to about 80mM of the nicotinic compound. 38. The insulin preparation according to any of the preceding embodiments, comprising from 35 about 70mM to about 140mM of the nicotinic compound.

17 39. The insulin preparation according to any of the preceding embodiments, comprising from about 80mM to about 130mM of the nicotinic compound. 40. The insulin preparation according to any of the preceding embodiments, comprising about 8mM, 30mM, 100mM or 130mM of the nicotinic compound. 5 41. The insulin preparation according to any of the preceding embodiments, comprising about 8mM of the nicotinic compound. 42. The insulin preparation according to any of the preceding embodiments, comprising about 30mM, 100mM or 130mM of the nicotinic compound. 43. The insulin preparation according to any of the preceding embodiments, comprising 10 about 30mM of the nicotinic compound. 44. The insulin preparation according to any of the preceding embodiments, comprising about 100mM of the nicotinic compound. 45. The insulin preparation according to any of the preceding embodiments, comprising about 130mM of the nicotinic compound. 15 46. The insulin preparation according to any of the preceding embodiments, comprising about 150mM of the nicotinic compound. 47. The insulin preparation according to any of the preceding embodiments, comprising the following ranges of arginine compound: 1-100mM, 5-120mM, 8-85mM, 20-90mM, 30 90mM, 30-85mM, 30-60mM or 10-40mM. 20 48. The insulin preparation according to any of the preceding embodiments, comprising the following ranges of arginine compound: 1-120mM, 8-85mM or 1-40mM. 49. The insulin preparation according to any of the preceding embodiments, comprising from about 1mM to about 120mM of arginine. 50. The insulin preparation according to any of the preceding embodiments, comprising from 25 about 1mM to about 100mM of arginine. 51. The insulin preparation according to any of the preceding embodiments, comprising from about 5mM to about 120mM of arginine. 52. The insulin preparation according to any of the preceding embodiments, comprising from about 20mM to about 90mM of arginine. 30 53. The insulin preparation according to any of the preceding embodiments, comprising from about 30mM to about 85mM of arginine. 54. The insulin preparation according to any of the preceding embodiments, comprising from about 8mM to about 85mM of arginine. 55. The insulin preparation according to any of the preceding embodiments, comprising from 35 about 30mM to about 60mM of arginine.

18 56. The insulin preparation according to any of the preceding embodiments, comprising from about 10mM to about 40mM of arginine. 57. The insulin preparation according to any of the preceding embodiments, comprising from about 1mM to about 40mM of arginine. 5 58. The insulin preparation according to any of the preceding embodiments, wherein arginine is present in a range selected from the following: 1mM, 2mM, 3mM, 4mM, 5mM, SmM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM, 55mM or 60mM. 59. The insulin preparation according to any of the preceding embodiments, comprising 10 about 1mM of arginine. 60. The insulin preparation according to any of the preceding embodiments, comprising about 2mM of arginine. 61. The insulin preparation according to any of the preceding embodiments, comprising about 3mM of arginine. 15 62. The insulin preparation according to any of the preceding embodiments, comprising about 4mM of arginine. 63. The insulin preparation according to any of the preceding embodiments, comprising about 5mM of arginine. 64. The insulin preparation according to any of the preceding embodiments, comprising 20 about 6mM of arginine. 65. The insulin preparation according to any of the preceding embodiments, comprising about 7mM of arginine. 66. The insulin preparation according to any of the preceding embodiments, comprising about 8mM of arginine. 25 67. The insulin preparation according to any of the preceding embodiments, comprising about 9mM of arginine. 68. The insulin preparation according to any of the preceding embodiments, comprising about 10mM of arginine. 69. The insulin preparation according to any of the preceding embodiments, comprising 30 about 15mM of arginine. 70. The insulin preparation according to any of the preceding embodiments, comprising about 20mM of arginine. 71. The insulin preparation according to any of the preceding embodiments, comprising about 25mM of arginine. 35 72. The insulin preparation according to any of the preceding embodiments, comprising 19 about 30mM of arginine. 73. The insulin preparation according to any of the preceding embodiments, comprising about 35mM of arginine. 74. The insulin preparation according to any of the preceding embodiments, comprising 5 about 40mM of arginine. 75. The insulin preparation according to any of the preceding embodiments, comprising about 45mM of arginine. 76. The insulin preparation according to any of the preceding embodiments, comprising about 50mM of arginine. 10 77. The insulin preparation according to any of the preceding embodiments, comprising about 55mM of arginine. 78. The insulin preparation according to any of the preceding embodiments, comprising about 60mM of arginine. 79. The insulin preparation according to any of the preceding embodiments, further compris 15 ing glutamic acid. 80. The insulin preparation according to embodiment 79, wherein glutamic acid is present in a range selected from the following: : 1-100mM, 20-90mM, 30-90mM, 30-85mM or 30 50mM. 81. The insulin preparation according to embodiment 79, comprising from about 1mM to 20 about 100mM of glutamic acid. 82. The insulin preparation according to embodiment 79, comprising from about 20mM to about 90mM of glutamic acid. 83. The insulin preparation according to embodiment 79, comprising from about 30mM to about 85mM of glutamic acid. 25 84. The insulin preparation according to embodiment 79, comprising from about 30mM to about 50mM of glutamic acid. 85. The insulin preparation according to embodiment 79, comprising about 30mM or 50mM of glutamic acid. 86. The insulin preparation according to embodiment 79, comprising about 30mM of glutamic 30 acid. 87. The insulin preparation according to embodiment 79, comprising about 50mM of glutamic acid. 88. The insulin preparation according to any of the preceding embodiments, which further comprises a metal ion, preservative agent(s), isotonicity agent(s) and stabilizer(s), deter 35 gent(s) and buffer(s).

20 89. The insulin preparation according to embodiment 88, wherein said buffer is Tris. 90. The insulin preparation according to embodiment 89, comprising from about 2mM to about 50mM of Tris. 91. The insulin preparation according to embodiment 89, comprising from about 10mM to 5 about 40mM of Tris. 92. The insulin preparation according to embodiment 89, comprising from about 20mM to about 30mM of Tris. 93. The insulin preparation according to embodiment 89, comprising about 10mM, 20mM, 30mM or 40mM of Tris. 10 94. The insulin preparation according to embodiment 89, comprising about 10mM of Tris. 95. The insulin preparation according to embodiment 89, comprising about 20mM of Tris. 96. The insulin preparation according to embodiment 89, comprising about 30mM of Tris. 97. The insulin preparation according to embodiment 89, comprising about 40mM of Tris. 98. The insulin preparation according to embodiment 89, wherein the metal ion is zinc. 15 99. The insulin preparation according to embodiment 98, wherein less than about 6 zinc ions are present per hexamer of insulin compound. 100. The insulin preparation according to embodiment 98, wherein less than about 4 zinc ions are present per hexamer of insulin compound. 101. The insulin preparation according to embodiment 98, wherein less than about 3 zinc 20 ions are present per hexamer of insulin compound. 102. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is from about 2:6 to about 5:6. 103. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is from about 2.5:6 to about 4.5:6. 25 104. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is from about 3:6 to about 4:6. 105. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is about 2:6. 106. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar 30 ratio is about 2.5:6. 107. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is about 3:6. 108. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is about 3.5:6. 35 109. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar 21 ratio is about 4:6. 110. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar ratio is about 4.5:6. 111. The insulin preparation according to embodiment 98, wherein the zinc:insulin molar 5 ratio is about 5:6. 112. The insulin preparation according to embodiment 88, wherein the stabilizer is a non ionic detergent. 113. The insulin preparation according to embodiment 112, wherein the detergent is poly sorbate 20 (Tween 20) or polysorbate 80 (Tween 80). 10 114. The insulin preparation according to embodiment 112, wherein the detergent is poly sorbate 20 (Tween 20). 115. The insulin preparation according to embodiment 112, wherein the detergent is polysorbate 80 (Tween 80). 116. The insulin preparation according to any of embodiments 112-115, comprising from 15 about 5 to 100ppm, from about 10 to about 50ppm or from about 10 to about 20ppm of polysorbate. 117. The insulin preparation according to embodiment 88, further comprising a phenolic compound. 118. The insulin preparation according to embodiment 117, wherein said phenolic com 20 pound is present in the amount from about 0 to about 6mg/ml or from about 0 to about 4mg/ml. 119. The insulin preparation according to embodiment 88, further comprising m-cresol. 120. The insulin preparation according to embodiment 119, wherein m-cresol is present in the amount from about 0.5 to about 4.0mg/ml or from about 0.6 to about 4.0mg/mi. 25 121. An insulin preparation according to any of the previous embodiments, wherein the pH is neutral to weakly basic. 122. An insulin preparation according to any of the previous embodiments, wherein the pH is from about 7.0 to about 8.0. 123. An insulin preparation according to any of the previous embodiments, wherein the 30 pH is about 7.0. 124. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.1. 125. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.2. 35 126. An insulin preparation according to any of the previous embodiments, wherein the 22 pH is about 7.3. 127. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.4. 128. An insulin preparation according to any of the previous embodiments, wherein the 5 pH is about 7.5. 129. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.6. 130. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.7. 10 131. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.8. 132. An insulin preparation according to any of the previous embodiments, wherein the pH is about 7.9. 133. An insulin preparation according to any of the previous embodiments, wherein the 15 pH is about 8.0. 134. A method of reducing the blood glucose level in mammals by administering to a pa tient in need of such treatment a therapeutically active dose of an insulin preparation ac cording to any of the preceding embodiments. 135. A method for the treatment of diabetes mellitus in a subject comprising administer 20 ing to a subject an insulin preparation according to any of the preceding embodiments. 136. A method according to any of the preceding embodiments, for parenteral administra tion. 137. An insulin preparation according to any of the preceding embodiments, for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 25 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders and treat ment of critically ill diabetic and non-diabetic patients. 30 The invention is further illustrated by the following examples which are not to be construed as limiting. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer- 23 ence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. 5 The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the inven tion. 10 The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter re cited in the claims appended hereto as permitted by applicable law. 15 24 EXAMPLES Example I q Preparation of pharmaceutical preparations The pharmaceutical preparations of the present invention may be formulated as an aqueous 5 solution. The aqueous medium is made isotonic, for example, with sodium chloride or glyc erol. Furthermore, the aqueous medium may contain zinc ions, for example added as zinc acetate or zinc chloride, buffers and preservatives. Arginine may be added as Arg, HCI. The pH value of the preparation is adjusted to the desired value and may be between about 3 to about 8.5, between about 3 and about 5 or about 6.5 and about 7.5 depending on the 10 isoelectric point, pl, of the insulin in question. Table 1. Composition of insulin preparations according to this invention Insulin Zn Phenol i. Nac Phos. Tris Glyce- Arginine, Nicotin- Glutamic pH aspart (mM) (mM) cresol (mM) phate (mM) ml HCI amide acid (mM) (mM) (mM) (%wv) (mM) (mM) (mM) A* 0.6 0.3 16 16 10 7 1.6 7.4 B 0.6 0.3 16 16 2 7 130 7.4 o 0.6 0.3 16 16 2 7 50 80 50 7.4 D 0.6 0.3 16 16 2 7 130 7.4 E 0.6 0.3 16 16 2 7 50 80 50 7.4 F 0.6 0.3 16 16 20 7 30 80 30 7.4 G 0.6 0.3 16 16 20 7 30 80 30 7.4 H 1.2 0.6 16 16 20 7 30 80 30 7.4 H 1.2 0.6 16 16 20 7 30 80 30 7.4 J 0.6 0.3 16 16 10 7 1.3 80 7.4 K 0.6 0.3 16 16 10 7 0.77 30 80 7.4 L 0.6 0.3 16 16 10 7 0.24 30 80 30 7.4 1I 0.6 0.3 16 16 10 7 60 100 7.4 N 0.6 0.3 16 16 10 7 1.13 100 7.4 * Commercially available NovoRapid" 15 25 Table 2. Composition of further insulin preparations according to this invention Preparation [Insulin [Zn2+] [phenol] {Arg] mM [Gly] [Glu] [His] [Nicotin nr. aspart] mM mM mM mM mM amide] mM mM 1 0.6 0.3 32 260 2 0.6 0.3 32 10 260 3 0.6 0.3 32 20 260 4 0.6 0.3 32 30 260 5 0.6 0.3 32 40 260 6 0.6 0.3 32 50 260 7 0.6 0.3 32 50 260 8 0.6 0.3 32 50 260 9 0.6 0.3 32 50 260 Example 2 5 Analysis of insulin chemical stability Size Exclusion Chromatography Quantitative determination of high molecular weight protein (HMWP) and monomer insulin aspart was performed on Waters insulin (300 x 7.8mm, part nr wat 201549) with an eluent containing 2.5M acetic acid, 4mM L-arginine and 20 %(VN) acetonitrile at a flow rate of 10 imIl/min. and 40*C. Detection was performed with a tuneable absorbance detector (Waters 486) at 276nm. Injection volume was 40pl and a OOpM human insulin standard. HMWP and concentration of the preparations were measured at each sampling point. Reverse phase chromatographv (UPLC) 15 Determination of the insulin aspart related impurities were performed on a UPLC system us ing a BEH RP C8 2.1 x 100mm column, particle size of 1,7pm. Waters part no 186002878. with a flow rate of 0,5mi/min., at 40* C detection at 220nm. Elution was performed with a mobile phase consisting of the following: A. 10 % (wN) acetonititrile, 2.8% (w/w) sodium sulphate, 0.3 % (w/w) o-phosphoric 20 acid, pH 3.5.

26 B. 70 % (wN) acetonitrile. Gradient: 0-11 min socratic with 73%/27% of A/B, 11-12 linear change to 52%/48% AIB, 13-15 min. linear change to 73%/27% of A/B, 15-20 min. iso cratic gradient at 73%/27% of A/B. The amount of B28iso-aspartate, desamido and other related impurities were de 5 termined as absorbance area measured in percent of total absorbance area determined after elution of the preservatives. The RP-UPLC method is equivalent to the analytical method used for quality control of Novo Nordisk marketed insulin aspart pharmaceuticals. Addition of arginine reduces the amount of degradation products formed, especially HMWP and des-amido forms, increasing the concentration of arginine in the range 10 to 10 50mM leads to further reduction of degradation. The physical stability measured as lag time in the ThT assay is reduced upon addition of arginine and is increasingly reduced when the arginine concentration is increased. The overall performance of 50mM arginine is superior to 50 mM glycine, 50mM glutamic acid, or 50mM histidine regarding reduction of the formation of degradation products, as is shown in Table 3 below. 15 Table 3. Physical and chemical stability data for insulin preparations 1-9 of Table 2 (Example 1). Preparation nr. Physical stabil- Chemical stability ity, lag time Content of degradation product (%) measured as differ (min) in ThT ence between content after incubation for 2 weeks at assay 37'C and at 4'C B28 lsoAsp des-amido Otherre- HMWP forms lated impuri ties 1 160 1.17 3.67 1.73 1.36 2 80 1.30 305 0.82 0.65 3 80 1.30 2.49 0.64 0.34 4 60 1.31 2.26 0.79 0.20 5 60 1.27 2.27 0.37 0.19 6 40 1.36 1.99 0.47 0.16 7 100 1.26 4.72 2.21 1.11 8 50 1.39 3.41 1.07 0.70 9 0 1.75 6.99 2.22 1.01 27 Example 3 Pharmacokinetic (PK)J Pharmacodynamic (PD) studies in LYD pig model and plasma analysis assay 5 PK/PD studies in LYD pigs The PK/PD studies were performed on domestic female pigs, LYD cross-breed, weighing between 55 and 110kg. The pigs were catheterised into the jugular vein through an ear vein at least 2 days before start of the study. The last meal before the start of the study was served to the animals approx. 18 hours prior to the injection of the test preparation, and the 10 animals had free access to water at all time during the fasting period and the test period. At time 0 hours the test preparation was given subcutaneous on the lateral side of the neck. A blood sample was drawn prior dosing and at regular time intervals after dosing samples were drawn from the catheter and sampled into 1.5ml glass tubes pre-coated with heparin. The blood samples were kept in ice water until separation of plasma by centrifugation for 15 1 0min. 3000rpm at 4*C, which was done within the first 30 minutes. Plasma samples were stored at 44C for short time (2-3 hours) or at -18"C for long term storage and were analysed for glucose on YSI or Konelab 30i and for insulin Aspart concentration by LOC. Luminescent Oxygen Channeling Immunoassay (LOCI) for Insulin Aspart quantification 20 The insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay and ap plies the proximity of two beads, the europium-coated acceptor beads and the streptavidin coated donor-beads. The acceptor beads were coated with a specific antibody against hu man insulin and recognize insulin Aspart in plasma samples. A second biotinylated antibody bind specific to insulin Aspart and together with the streptavidin coated beads, they make up 25 the sandwich. Illumination of the beads-aggregate-immunocomplex releases singlet oxygen from the donor beads which channels into the acceptor beads and triggers chemilumines cence. The chemiluminescence was measured and the amount of light generated is propor tional to the concentration of insulin Aspart. 30 Compared to the marketed product NovoRapid, the initial rate of plasma glucose lowering is faster for the preparations of the present invention (Figures 3 and 4). Likewise, when com pared to NovoRapid*, the initial absorption rate of the insulin component of the preparations of the present invention, is markedly faster (Figure 5).

28 Example 4 General introduction to ThT fibrillation assays for the assessment of physical stability 5 of protein formulations Low physical stability of a peptide may lead to amyloid fibril formation, which is observed as well-ordered, thread-like macromolecular structures in the sample eventually resulting in gel formation. This has traditionally been measured by visual inspection of the sample. However, that kind of measurement is very subjective and depending on the observer. Therefore, the 10 application of a small molecule indicator probe is much more advantageous. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signature when binding to fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284]. The time course for fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001) Biochemistry 40, 6036-6046]: 15 I +e ')/7Eq.(1) Here, F is the ThT fluorescence at the time t. The constant to is the time needed to reach 50% of maximum fluorescence. The two important parameters describing fibril forma 20 tion are the lag-time calculated by to - 2T and the apparent rate constant kapp = 1/T.

|

f5+mit Lag-time = to - 2T to Time 29 Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and the fibrillation proceeds. The lag-time corresponds to the interval in which the critical mass of nucleus is built up and the apparent 5 rate constant is the rate with which the fibril itself is formed. Sample preparation Samples were prepared freshly before each assay. Each sample composition is described in each example. The pH of the sample was adjusted to the desired value using appropriate 10 amounts of concentrated NaOH and HCIO 4 or HCI. Thioflavin T was added to the samples from a stock solution in H20 to a final concentration of I gM. Sample aliquots of 200gi were placed in a 96 well microtiter plate (Packard Opti PlateTM96, white polystyrene). Usually, four or eight replica of each sample (corresponding to one test condition) were placed in one column of wells. The plate was sealed with Scotch 15 Pad (Qiagen). Incubation and fluorescence measurement Incubation at given temperature, shaking and measurement of the ThT fluorescence emis sion were done in a Fluoroskan Ascent FL fluorescence platereader or Varioskan platereader 20 (Thermo Labsystems). The temperature was adjusted to 37 "C. The orbital shaking was ad justed to 960rpm with an amplitude of 1mm in all the presented data. Fluorescence meas urement was done using excitation through a 444nm filter and measurement of emission through a 485nm filter. Each run was initiated by incubating the plate at the assay temperature for 10 min. 25 The plate was measured every 20 minutes for a desired period of time. Between each meas urement, the plate was shaken and heated as described. Data handling The measurement points were saved in Microsoft Excel format for further processing and 30 curve drawing and fitting was performed using GraphPad Prism. The background emission from ThT in the absence of fibrils was negligible. The data points are typically a mean of four or eight samples and shown with standard deviation error bars. Only data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a relative measure of fibrillation between experiments.

30 The data set may be fitted to Eq. (1). However, since full sigmodial curves are not always achieved during the measurement time, lag times were here visually determined from the ThT fluorescence curve as the time point at which the ThT fluorescence is different than the background level. 5 Measurement of initial and final concentrations The peptide concentration in each of the tested formulations were measured both before ap plication in the ThT fibrillation assay ('Initial") and after completion of the ThT fibrillation ("Af ter ThT assay"). Concentrations were determined by reverse HPLC methods using a pramlin 10 tide standard as a reference. Before measurement after completion 150pl was collected from each of the replica and transferred to an Eppendorf tube. These were centrifuged at 30000 G for 40mins. The supernatants were filtered through a 0.22pm filter before application on the HPLC system.

Claims (24)

1. A pharmaceutical formulation comprising: B28Asp human insulin; a nicotinic compound; arginine; a preservative; and a buffer

2. A pharmaceutical formulation comprising: B28Asp human insulin; nicotinamide; zinc; arginine; a preservative; and a phosphate buffer.

3. An aqueous pharmaceutical formulation comprising: B28Asp human insulin; a nicotinic compound; arginine; a buffer; zinc; a preservative; and an isotonicity agent.

4. A pharmaceutical formulation according to claim 1 comprising zinc.

5. A pharmaceutical formulation according to any one of claims 1, 3 or 4 wherein the nicotinic compound is nicotinamide.

6. A pharmaceutical formulation according to any one of claims 1 or 3 to 5, wherein the buffer is a phosphate buffer.

7. A pharmaceutical formulation according to claim 2 or claim 6 wherein the nicotinamide is present at a concentration ranging from about 1 mM to about 300 mM.

8. A pharmaceutical formulation according to claim 7 wherein the nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM.

9. A pharmaceutical formulation according to any one of claims 2 to 8, wherein less than about 4 zinc ions are present per six B28Asp human insulin molecules.

10. A pharmaceutical formulation according to any one of claims 2 to 8, wherein about 3 zinc ions are present per six B28Asp human insulin molecules.

11. A pharmaceutical formulation according to any one of claims 2 to 8, wherein less than about 3 zinc ions are present per six B28Asp human insulin molecules.

12. A pharmaceutical formulation according to any one of claims 2 to 8, wherein about 2.5 zinc ions are present per six B28Asp human insulin molecules. - 32

13. A pharmaceutical formulation according to any one of claims 1 to 12, wherein the arginine is present in a concentration from about 10 mM to about 60 mM.

14. A pharmaceutical formulation according to any one of claims 1 to 12, wherein the arginine is present in a concentration ranging from about 10 mM to about 40 mM.

15. A pharmaceutical formulation according to any one of claims 1 to 12, wherein the arginine is present in a concentration of about 20mM.

16. A pharmaceutical formulation according to any one of claims 1 to 15, wherein the formulation has a pH of about 7.1, about 7.2, about 7.3 or about 7.4.

17. A pharmaceutical formulation according to any one of claims 1 to 15, wherein the formulation has a pH of about 7.1.

18. A pharmaceutical formulation according to any one of claims 1 to 17, wherein the preservative is selected from the group consisting of phenol, cresol, and combinations thereof.

19. A pharmaceutical formulation according to any one of claims 1 to 18, wherein B28Asp human insulin is present in the amount from about 0.3mM to about 1.2mM.

20. A pharmaceutical formulation according to any one of claims 1 to 18, wherein B28Asp human insulin is present in the amount of about 0.6mM.

21. A pharmaceutical formulation according to any one of claims 1 to 20, wherein the buffer is sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate or mixture thereof.

22. A pharmaceutical formulation according to any one of claims 3 or 4, wherein the nicotinic compound is nicotinamide, the buffer is a phosphate buffer, the nicotinamide is present at a concentration ranging from about 80 mM to about 260 mM, less than about 4 zinc ions are present per six B28Asp human insulin molecules, the arginine is present in a concentration of about 20mM, the formulation has a pH of about 7.1, the preservative is selected from the group consisting of phenol, cresol, and combinations thereof, and B28Asp human insulin is present in the amount of about 0.6mM. - 33

23. A pharmaceutical formulation according to claim 22, wherein about 3 zinc ions are present per six B28Asp human insulin molecules.

24. A pharmaceutical formulation according to claim 22, wherein about 2.5 zinc ions are present per six B28Asp human insulin molecules.