FIELD OF THE INVENTION
The present invention relates to pharmaceutical preparations comprising an insulin com-pound  a nicotinic compound and an amino acid.
BACKGROUND OF THE INVENTION
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches ep-idemic proportions.
Since the introduction of insulin in the 1920""s  continuous improvements have been made in the treatment of diabetes. To help avoid high glycaemia levels  diabetic patients often practice multiple injection therapy  whereby insulin is administered with each meal. As dia-betic patients have been treated with insulin for several decades  there is a major need for safe and life-quality improving insulin preparations. Among the commercially available insulin preparations  rapid-acting  intermediate-acting and prolonged-acting preparations can be mentioned.
In the treatment of diabetes mellitus  many varieties of pharmaceutical preparations of insulin have been suggested and used  such as regular insulin (such as Actrapid®)  iso-phane insulin (designated NPH)  insulin zinc suspensions (such as 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 B28LysB29Pro human insulin) and Apidra® (preparation of B3LysB29Glu human insulin).
International applications WO 91/09617 and WO/9610417 (Novo Nordisk A/S) dis-close insulin preparations containing nicotinamide or nicotinic acid or a salt thereof.
Most often pharmaceutical preparations of insulins are administered by subcutane-ous injection. Important for the patient is the action profile of the insulin  meaning the action of insulin on glucose metabolism as a function of time from injection. In this profile  inter alia  the time of the onset  the maximum value and the total duration of action are important. In the case of bolus insulins  a variety of insulin preparations with different action profiles are desired and requested by the patients. One patient may  on the same day  use insulin preparations with very different action profiles. The action profile desired for example  depends on the time of the day and the amount and composition of the meal eaten by the patient.
Equally important for the patient is the chemical stability of the insulin preparations  for example  due to the abundant use of pen-like injection devices such as devices which contain Penfill® cartridges  in which an insulin preparation is stored until the entire cartridge is empty which may be at least 1 to 2 weeks for devices containing 1.5-3.0ml cartridges. During storage  covalent chemical changes in the insulin structure occur. This may lead to formation of molecules which may be less active and/or potentially immunogenic such as deamidation products and higher molecular weight transformation products (dimers  polymers). Further-more  also important is the physical stability of the insulin preparations  since long term stor-age may eventually lead to formation of insoluble fibrils  which are biologically inactive and potentially immunogenic.

SUMMARY OF THE INVENTION
The invention relates to insulin preparations with favourable absorption rate and favourable chemical and physical stability. The present invention relates to insulin preparations compris-ing human insulin and/or analogues thereof  nicotinamide or nicotinic acid and/or salts thereof and arginine.

In one embodiment  the present invention relates to an insulin preparation comprising:
• an insulin compound 
• a nicotinic compound  and
• arginine.
In another embodiment the insulin preparation may further comprise glutamic acid.
In another embodiment  the present invention also contemplates a method for the treatment of diabetes mellitus in a subject or for reducing the blood glucose level in a subject comprising administering to a subject or mammal an insulin preparation according to the in-vention.

DESCRIPTION OF THE DRAWINGS
Figure 1 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37°C of preparations according to the present invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1. Compared to the NovoRapid® preparation (preparation A)  addition of nicotinamide (preparations B and D) leads to an increased formation of degradation products  whereas the combined addition of nicotinamide  glutamic acid and arginine (preparations C and E)  has a mostly similar degradation pattern  with lower formation of HMWP.

Figure 2 shows the development in percentage of total insulin content of degradation products during 2 weeks of storage at 37°C of preparations according to this invention. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart prepa-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.

Figure 3 shows the glucose concentration (mean +/- SEM  N=8) in plasma after subcutane-ous injection in pigs of a 1nmol/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 insulin aspart preparations as described in Table 1 of Example 1. Compared to the NovoRapid® preparation (preparation A) the initial rate of plasma glucose lowering is faster for the prepa-ration with addition of nicotinamide (preparation N) and even faster for a combination of nico-tinamide 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-tion. The letter A refers to a NovoRapid® reference and remaining letters correspond to insulin aspart preparations as described in Table 1 of Example 1. Compared to the NovoRapid® preparation (preparation A)  the initial rate of plasma glucose lowering is faster for a 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).

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-voRapid® preparation  (preparation A)   the initial absorption rate of the insulin component of the preparations with nicotinamide (preparation J)  the combination of nicotinamide and argi-nine (preparation K)  and the combination of nicotinamide  arginine and glutamic acid (prepa-ration L) is markedly faster.

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 connection with a multiple injection regimen where insulin is given before each meal. With faster onset of action  the insulin can conveniently be taken closer to the meal than with conventional rapid acting insulin solutions. Furthermore  a faster disappearance of insulin probably diminishes the risk of post-meal hypoglycaemia.
The insulin preparations of the present invention are rapid-acting insulin preparations comprising an insulin compound such as insulin aspart  a nicotinic compound  such as nicotinamide and the amino acid arginine. Optionally  the insulin preparations of the present invention may comprise further amino acids such as glutamic acid. These insulin preparations have a rapid absorption profile that mimics normal physiology more closely than existing therapies. Furthermore  the insulin preparations of the present invention have chemical and physical stability suitable for commercial pharmaceutical preparations.
The insulin preparations of the present invention provide 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-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 dosing. The present invention is based in part  on the surprising discovery that although  the addition of nicotinamide allows the increase in absorption rate  it also has a negative effect on chemical stability by significantly increasing the amount of HMWP.The insulin preparations of the present invention have an improved chemical stability by addition of arginine  which is reflected in e.g. a reduction in the formation of dimers and polymers and desamido insulins after storage. The insulin preparations of the present invention may furthermore also have improved physical stability  which may be useful for use in pumps.
The present invention provides an insulin preparation comprising an insulin 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 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.
In one embodiment  the insulin preparations according to the present invention comprise an aqueous solution of B28Asp human insulin  nicotinamide and arginine.
The content of B28Asp human insulin in the solutions of this invention may be in the range of 15 to 500 international units (IU)/ml  preferably in the range of 50 to 333 lU/ml  in preparations for injection. However  for other purposes of parenteral administration  the con-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 "IU" corresponds to 6 nmol.
The term “insulin aspart” refers to the human insulin analogue B28Asp human insulin.
The term “onset” refers to the time from injection until the PK curve shifts to an 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-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 disulphide bridges between cysteine residues  namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide  the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 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 position 20 of the A-chain and the cysteine in position 19 of the B-chain.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acids followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A  in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting pep-tide from the A and B chains.
By "insulin analogue" as used herein is meant a polypeptide derived from the primary structure of a naturally occurring insulin  for example that of human insulin  by mutation. One or more mutations are made by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or by adding at least one amino acid residue. The added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues.
In one embodiment an insulin analogue comprises less than 8 modifications (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  alternatively less than 3 modifications relative to the parent insulin  alternatively less than 2 modifications relative to the parent insulin.
Mutations in the insulin molecule are denoted stating the chain (A or B)  the position  and the three letter code for the amino acid substituting the native amino acid. By “desB30” or “B(1-29)” is meant a natural insulin B chain or analogue thereof lacking the B30 amino acid residue  and by B28Asp human insulin is meant human insulin wherein the amino acid residue in position 28 of the B chain has been substituted with Asp.
Examples of insulin analogues are such wherein Pro in position 28 of the B chain is mutated with Asp  Lys  Leu  Val  or Ala and/or Lys at position B29 is mutated with Pro  Glu or Asp. Furthermore  Asn at position B3 may be mutated with Thr  Lys  Gln  Glu or Asp. The amino acid residue in position A21 may be mutated with Gly. The amino acid in position B1 may be mutated with Glu. The amino acid in position B16 may be mutated with Glu or His. Further examples of insulin analogues are the deletion analogues e.g. analogues where the B30 amino acid in human insulin has been deleted (des(B30) human insulin)  insulin ana-logues wherein the B1 amino acid in human insulin has been deleted (des(B1) human insulin)  des(B28-B30) human insulin and des(B27) human insulin. Insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension such as with two arginine residues added to the C-terminal of the B-chain are also examples of insulin analogues. Further ex-amples are insulin analogues comprising combinations of the mentioned mutations. Insulin analogues wherein the amino acid in position A14 is Asn  Gln  Glu  Arg  Asp  Gly or His  the 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 insulin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues are also examples of insulin analogues.
Further examples of insulin analogues include  but are not limited to: DesB30 human insulin; AspB28 human insulin; AspB28 desB30 human insulin; LysB3 GluB29 human insulin; LysB28 ProB29 human insulin; GlyA21 ArgB31 ArgB32 human insulin; GluA14 HisB25 hu-man insulin; HisA14 HisB25 human insulin; GluA14 HisB25 desB30 human insulin; HisA14  HisB25 desB30 human insulin; GluA14 HisB25 desB27 desB28 desB29 desB30 human insu-lin; GluA14 HisB25 GluB27 desB30 human insulin; GluA14 HisB16 HisB25 desB30 human insulin; HisA14 HisB16 HisB25 desB30 human insulin; HisA8 GluA14 HisB25 GluB27 desB30 human insulin; HisA8 GluA14 GluB1 GluB16 HisB25 GluB27 desB30 human insulin; and HisA8 GluA14 GluB16 HisB25 desB30 human insulin.
The term “nicotinic compound” includes nicotinamide  nicotinic acid  niacin  niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
According to the present invention  the concentration of the nicotinic compound and/or salts thereof is in the range from about 1mM to about 300mM or from about 5mM to about 200mM.
The term “arginine” or “Arg” includes the amino acid arginine and/or a salt thereof.
In one embodiment  the insulin preparation comprises 1 to 100mM of arginine.
In one embodiment  the insulin preparation comprises 1 to 20mM of arginine.
In one embodiment  the insulin preparation comprises 20 to 90mM of arginine.
In one embodiment  the insulin preparation comprises 30 to 85mM of arginine.

The term “glutamic acid” or “Glu” includes the aminoacid glutamic acid and/or a salt thereof.
In one embodiment  the insulin preparation comprises 1 to 100mM of glutamic acid.
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 with other excipients such as preservatives  chelating agents  tonicity modifiers  bulking agents  stabilizers  antioxidants  polymers and surfactants  metal ions  oleaginous vehicles and proteins (e.g.  human serum albumin  gelatine or proteins)  said insulin preparation being useful for treating  preventing or reducing the severity of a disease or disorder by administration of said insulin preparation to a person. Thus  an insulin preparation is also known in the art as a pharmaceutical preparation or pharmaceutical composition.
The buffer may be selected from the group consisting of  but not limited to  sodium acetate  sodium carbonate  citrate  sodium dihydrogen phosphate  disodium hydrogen phosphate  sodium phosphate  and tris(hydroxymethyl)-aminomethan  bicine  tricine  malic acid  succinate  maleic acid  fumaric acid  tartaric acid  aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.
The insulin preparation of the present invention may further comprise other 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-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 sufficient to obtain a preserving effect. The amount of preservative in an insulin preparation may be determined from e.g. literature in the field and/or the known amount(s) of preservative in e.g. commercial products. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical preparations is described  for example in Remington: The Science and Practice of Pharmacy  19th edition  1995.
The preservative present in the insulin preparation of this invention may be as in the heretofore conventional insulin preparations  for example phenol  m-cresol and methylpara-ben.
The insulin preparation of the present invention may further comprise a chelating agent. The use of a chelating agent in pharmaceutical preparations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy  19th edition  1995.
The insulin preparation of the present invention may further comprise a stabilizer. The term “stabilizer” as used herein refers to chemicals added to polypeptide containing pharmaceutical preparations in order to stabilize the peptide  i.e. to increase the shelf life and/or in-use time of such preparations. For convenience reference is made to Remington: The Science and Practice of Pharmacy  19th edition  1995.
The insulin preparation of the present invention may further comprise a surfactant. The term “surfactant” as used herein refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part  the head  and a fat-soluble (lipophilic) segment. Surfactants accumulate preferably at interfaces  which the hydrophilic part is orientated towards the water (hydrophilic phase) and the lipophilic part towards the oil- or hydrophobic phase (i.e. glass  air  oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore  surfactants lower the surface tension of a liquid. Surfactants are also known as amphipathic compounds. The term “detergent” is a synonym used for surfactants in general. The use of a surfactant in pharmaceutical preparations is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy  19th edition  1995.
In a further embodiment the invention relates to an insulin preparation comprising an aqueous solution of an insulin compound of the present invention  and a buffer  wherein said insulin compound is present in a concentration from 0.1mM 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.
In one embodiment  the method for making insulin preparations of the invention comprises:
a) preparing a solution by dissolving the insulin compound or a mixture of insulin compounds in water or buffer;
b) preparing a solution by dissolving a divalent metal ion in water or buffer;
c) preparing a solution by dissolving a preservative in water or buffer;
d) preparing a solution by dissolving an isotonicity agent in water or buffer ;
e) preparing a solution by dissolving a surfactant and/or a stabilizer in water or buff-er;
f) mixing solution a) and one or more of solutions b)  c)  d)  and e);
Finally adjusting the pH of the mixture in f) to the desired pH followed by a sterile fil-tration.
The insulin preparations of the present invention can be used in the treatment of di-abetes by parenteral administration. It is recommended that the dosage of the insulin prepa-rations of this invention which is to be administered to the patient be selected by a physician.
Parenteral administration may be performed by subcutaneous  intramuscular  intra-peritoneal or intravenous injection by means of a syringe  optionally a pen-like syringe. Alter-natively  parenteral administration can be performed by means of an infusion pump. As a fur-ther option  the insulin preparations containing the insulin compound of the invention can also be adapted to transdermal administration  e.g. by needle-free injection or from a patch  optionally an iontophoretic patch  or transmucosal  e.g. buccal  administration.
Insulin preparations according to the present invention may be administered to a 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  in a vein  in the heart  and at sites which involve absorption  for example  administration in the skin  under the skin  in a muscle or in the abdomen.
In one embodiment of the invention the insulin preparation is an aqueous preparation  i.e. preparation comprising water. Such preparation is typically a solution or a suspension. In a further embodiment of the invention the insulin preparation is an aqueous solution.
The term “aqueous preparation” is defined as a preparation comprising at least 50 %w/w water. Likewise  the term “aqueous solution” is defined as a solution comprising at least 50 %w/w water  and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.
Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.
In one embodiment  the insulin preparations of this invention are well-suited for 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.
The term “physical stability” of the insulin preparation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing  such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein preparations is evaluated by means of visual inspec-tion and/or turbidity measurements after exposing the preparation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different 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 showing no turbidity corresponds to a visual score 0  and a preparation showing visual tur-bidity in daylight corresponds to visual score 3). A preparation is classified physically unstable with respect to protein aggregation  when it shows visual turbidity in daylight. Alternatively  the turbidity of the preparation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein preparations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils  and perhaps other protein configurations as well  Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
The term “chemical stability” of the protein preparation as used herein refers to changes in the covalent protein structure leading to formation of chemical degradation prod-ucts with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Increasing amounts of chemical degradation products is often seen during storage and use of the protein preparation. Most proteins are prone to deamidation  a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydro-lysed to form a free carboxylic acid or asparaginyl residues to form an IsoAsp derivative. Other degradations pathways involves formation of high molecular weight products where two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions leading to formation of covalently bound dimer  oligomer and polymer degradation products (Stability of Protein Pharmaceuticals  Ahern. T.J. & Manning M.C.  Plenum Press  New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein preparation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the 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.
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.
The term “diabetes” or “diabetes mellitus” includes type 1 diabetes  type 2 diabetes  gestational diabetes (during pregnancy) and other states that cause hyperglycaemia. The term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin  or in which the cells of the body fail to respond appropriately to insulin thus preventing cells from absorbing glucose. As a result  glucose builds up in the blood.
Type 1 diabetes  also called insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes  is caused by B-cell destruction  usually leading to absolute insulin deficiency.
Type 2 diabetes  also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes  is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
The term “pharmaceutically acceptable” as used herein means suited for normal pharmaceutical applications  i.e.  not giving rise to any serious adverse events in patients.
The term “treatment of a disease” as used herein means the management and care of a patient having developed the disease  condition or disorder and includes treatment  pre-vention or alleviation of the disease. The purpose of treatment is to combat the disease  con-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 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 burns  operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment  myocardial infarction  stroke  coronary heart disease and other car-diovascular disorders is provided.
In a further embodiment of the present invention  a method for the treatment or 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 injuries where an anabolic effect is needed in the treatment  myocardial infarction  coronary heart disease and other cardiovascular disorders  stroke  the method comprising administering to a patient in need of such treatment an effective amount for such treatment of an insulin preparation according to the invention  is provided.
The treatment with an insulin preparation according to the present invention may also be combined with a second or more pharmacologically active substances  e.g. selected from antidiabetic agents  antiobesity agents  appetite regulating agents  antihypertensive agents  agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders re-sulting from or associated with obesity.
The treatment with an insulin preparation according to the present invention may also be combined with bariatric surgery - a surgery that influences the glucose levels and/or lipid homeostasis such as gastric banding or gastric bypass.
The production of polypeptides  e.g.  insulins  is well known in the art. An insulin an-alogue according to the invention may for instance be produced by classical peptide synthe-sis  e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques  see e.g. Greene and Wuts  “Protective Groups in Organic Synthesis”  John Wiley & Sons  1999. The insulin analogue may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the insulin analogue in a suitable nutrient medium under conditions permitting the expression of the insulin analogue. For insulin analogues comprising non-natural amino acid residues  the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue  for instance by use of tRNA mutants. Hence  briefly  the insulin analogues according to the invention are prepared analogously to the preparation of known insulin analogues.
Several methods may be used for the production of human insulin and human insulin analogues. For example three major methods which are used in the production of insulin in microorganisms are disclosed in WO2008034881. Two of these involve Escherichia coli  with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross  eds.)  Pierce Chemical Co.  Rockford  Ill. pp 729-739)  or use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae  vide U.S5 962 267  WO 95/16708  EP 0055945  EP 0163529  EP 0347845 and EP 0741188.
The insulin analogues are produced by expressing a DNA sequence encoding the insulin analogue in question in a suitable host cell by well known technique as disclosed in e.g. US 6500645. The insulin analogue is either expressed directly or as a precursor molecule which has an N-terminal extension on the B-chain or a C-terminal extension on the B-chain. The N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long. The N-terminal 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 function of protecting the mature insulin or insulin analogue molecule against intracellular proteolytic processing by host cell exoproteases. The C-terminal extension is to be cleaved of either extra-cellularly in the culture broth by secreted  active carboxypeptidase or in vitro after isolation from the culture broth. A method for producing mature insulin and insulin analogs with C-terminal extensions on the B-chain that are removed by carboxypetidase are disclosed in WO 08037735. The target insulin product of the process may either be a two-chain human insulin or a two-chain human insulin analogue which may or may not have a short C-terminal extension of the B-chain. If the target insulin product will have no C-terminal extension of the B-chain  then said C-terminal extension should be capable of subsequently being cleaved off from the B-chain before further purification steps.
The present invention also contemplates the following non-limiting list of embodi-ments  which are further described elsewhere herein:

1. An insulin preparation comprising:
• an insulin compound 
• a nicotinic compound  and
• arginine.
2. The insulin preparation according to embodiment 1  wherein the insulin compound is hu-man insulin or an insulin analog.
3. The insulin preparation according to any of the preceding embodiments  wherein the in-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.
6. The insulin preparation according to any of the preceding embodiments  wherein the in-sulin compound is present in a range selected from the following: 0.1-10.0mM; 0.1-3.0mM; 0.1-2.5mM; 0.1-2.0mM; 0.1-1.5mM; 0.2-2.5mM; 0.2-2.0mM; 0.2-1.5mM; 0.3-3.0mM; 0.3-2.5mM; 0.3-2.0mM; 0.3-1.5mM; 0.5-1.3mM and 0.6-1.2mM.
7. 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 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. 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 2.0mM.
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-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.
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-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.
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-sulin compound is present in the amount of about 0.6 or about 1.2mM.
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.
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 nic-otinic compound is selected from the group consisting of nicotinamide  nicotinic acid  nia-cin  niacin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
27. The insulin preparation according to any of the preceding embodiments  wherein the nic-otinic 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 nic-otinic compound is present in a range selected from the following: 1-300mM; 5-200mM; 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.
31. The insulin preparation according to any of the preceding embodiments  comprising from about 5mM 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 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.
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 about 70mM to about 140mM of the nicotinic compound.
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.
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 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.
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.
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 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.
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 about 30mM to about 60mM of arginine.
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.
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  6mM  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 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.
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 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.
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 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.
72. The insulin preparation according to any of the preceding embodiments  comprising 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 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.
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-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 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.
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 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-gent(s) and buffer(s).
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 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.
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.
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 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.
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 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.
109. The insulin preparation according to embodiment 98  wherein the zinc:insulin molar 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 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 pol-ysorbate 20 (Tween 20) or polysorbate 80 (Tween 80).
114. The insulin preparation according to embodiment 112  wherein the detergent is pol-ysorbate 20 (Tween 20).
115. The insulin preparation according to embodiment 112  wherein the detergent is pol-ysorbate 80 (Tween 80).
116. The insulin preparation according to any of embodiments 112-115  comprising from 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-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/ml.
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 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.
126. An insulin preparation according to any of the previous embodiments  wherein the 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 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.
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 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 administering 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 diabetes  impaired glucose tolerance  type 1 diabetes  and burns  operation wounds and other diseases or injuries where an anabolic effect is needed in the treatment  myocardial infarction  stroke  coronary heart disease and other cardiovascular disorders and treat-ment of critically ill diabetic and non-diabetic patients.

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

All references  including publications  patent applications  and patents  cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer-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.
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.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity  patentability  and/or enforceability of such patent documents.
This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

EXAMPLES
Example 1
Preparation of pharmaceutical preparations
The pharmaceutical preparations of the present invention may be formulated as an aqueous solution. The aqueous medium is made isotonic  for example  with sodium chloride or glycer-ol. Furthermore  the aqueous medium may contain zinc ions  for example added as zinc ace-tate or zinc chloride  buffers and preservatives. Arginine may be added as Arg  HCl. 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 isoelectric point  pI  of the insulin in question.

Table 1. Composition of insulin preparations according to this invention
Insulin aspart (mM) Zn (mM) Phenol (mM) m-cresol
(mM) NaCl
(mM) Phos-phate
(mM) Tris
(mM) Glyce-rol
(%w/v) Arginine  HCl
(mM) Nicotin-amide
(mM) Glutamic acid
(mM) pH
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
C 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
I 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
M 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®

Preparation nr. [Insulin aspart] mM [Zn2+] mM [phenol] mM [Arg] mM [Gly] mM [Glu] mM [His] mM [Nicotin amide] 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
Table 2. Composition of further insulin preparations according to this invention

Example 2

Analysis of insulin chemical stability
Size Exclusion Chromatography
Quantitative determination of high molecular weight protein (HMWP) and monomer insulin aspart was performed on Waters insulin (300 x 7.8mm  part nr wat 201549) with an eluent containing 2.5M acetic acid  4mM L-arginine and 20 %(V/V) acetonitrile at a flow rate of 1ml/min. and 40°C. Detection was performed with a tuneable absorbance detector (Waters 486) at 276nm. Injection volume was 40µl and a 600µM human insulin standard. HMWP and concentration of the preparations were measured at each sampling point.

Reverse phase chromatography (UPLC)
Determination of the insulin aspart related impurities were performed on a UPLC system us-ing a BEH RP C8 2.1 x 100mm column  particle size of 1 7µm. Waters part no 186002878. with a flow rate of 0 5ml/min.  at 40° C detection at 220nm. Elution was performed with a mobile phase consisting of the following:
A. 10 % (w/V) acetonititrile  2.8% (w/w) sodium sulphate  0.3 % (w/w) o-phosphoric acid  pH 3.5.
B. 70 % (w/V) acetonitrile. Gradient: 0-11 min isocratic with 73%/27% of A/B  11-12 linear change to 52%/48% A/B  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-termined as absorbance area measured in percent of total absorbance area determined after elution of the preservatives. The RP-UPLC method is equivalent to the analytical method used for quality control of Novo Nordisk marketed insulin aspart pharmaceuticals.
Addition of arginine reduces the amount of degradation products formed  especially HMWP and des-amido forms  increasing the concentration of arginine in the range 10 to 50mM leads to further reduction of degradation. The physical stability measured as lag time in the ThT assay is reduced upon addition of arginine and is increasingly reduced when the ar-ginine 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.

Table 3. Physical and chemical stability data for insulin preparations 1-9 of Table 2 (Example 1).
Preparation nr. Physical stabil-ity  lag time (min) in ThT assay Chemical stability
Content of degradation product (%) measured as differ-ence between content after incubation for 2 weeks at 37°C and at 4°C
B28 IsoAsp des-amido forms Other relat-ed impurities HMWP
1 160 1.17 3.67 1.73 1.36
2 80 1.30 3.05 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

Example 3
Pharmacokinetic (PK)/ Pharmacodynamic (PD) studies in LYD pig model and plasma analysis assay

PK/PD studies in LYD pigs
The PK/PD studies were performed on domestic female pigs  LYD cross-breed  weighing between 55 and 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 animals had free access to water at all time during the fasting period and the test period.
At time 0 hours the test preparation was given subcutaneous on the lateral side of the neck. A blood sample was drawn prior dosing and at regular time intervals after dosing samples were drawn from the catheter and sampled into 1.5ml glass tubes pre-coated with heparin. The blood samples were kept in ice water until separation of plasma by centrifugation for 10min. 3000rpm at 4°C  which was done within the first 30 minutes. Plasma samples were stored at 4°C for short time (2-3 hours) or at -18°C for long term storage and were analysed for glu-cose on YSI or Konelab 30i and for insulin Aspart concentration by LOCI.

Luminescent Oxygen Channeling Immunoassay (LOCI) for Insulin Aspart quantification
The insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay and applies the proximity of two beads  the europium-coated acceptor beads and the streptavidin coated donor-beads. The acceptor beads were coated with a specific antibody against human insulin and recognize insulin Aspart in plasma samples. A second biotinylated antibody bind specific to insulin Aspart and together with the streptavidin coated beads  they make up the sandwich. Illumination of the beads-aggregate-immunocomplex releases singlet oxygen from the donor beads which channels into the acceptor beads and triggers chemiluminescence. The chemiluminescence was measured and the amount of light generated is proportional to the concentration of insulin Aspart.

Compared to the marketed product NovoRapid®  the initial rate of plasma glucose lowering is faster for the preparations of the present invention (Figures 3 and 4). Likewise  when com-pared to NovoRapid®  the initial absorption rate of the insulin component of the preparations of the present invention  is markedly faster (Figure 5).

Example 4
General introduction to ThT fibrillation assays for the assessment of physical stability of protein formulations
Low physical stability of a peptide may lead to amyloid fibril formation  which is observed as well-ordered  thread-like macromolecular structures in the sample eventually resulting in gel formation. This has traditionally been measured by visual inspection of the sample. However  that kind of measurement is very subjective and depending on the observer. Therefore  the application of a small molecule indicator probe is much more advantageous. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signature when binding to fibrils [Naiki et al. (1989) Anal. Biochem. 177  244-249; LeVine (1999) Methods. Enzymol. 309  274-284].
The time course for fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001) Biochemistry 40  6036-6046]:

Eq.(1)

Here  F is the ThT fluorescence at the time t. The constant t0 is the time needed to reach 50% of maximum fluorescence. The two important parameters describing fibril for-mation are the lag-time calculated by t0 – 2? and the apparent rate constant kapp = 1/?.

Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and the fibrillation proceeds. The lag-time corresponds to the interval in which the critical mass of nucleus is built up and the apparent rate constant is the rate with which the fibril itself is formed.

Sample preparation
Samples were prepared freshly before each assay. Each sample composition is described in each example. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HClO4 or HCl. Thioflavin T was added to the samples from a stock solution in H2O to a final concentration of 1 ?M.
Sample aliquots of 200?l were placed in a 96 well microtiter plate (Packard OptiPlate?-96  white polystyrene). Usually  four or eight replica of each sample (correspond-ing to one test condition) were placed in one column of wells. The plate was sealed with Scotch Pad (Qiagen).

Incubation and fluorescence measurement
Incubation at given temperature  shaking and measurement of the ThT fluorescence emission were done in a Fluoroskan Ascent FL fluorescence platereader or Varioskan platereader (Thermo Labsystems). The temperature was adjusted to 37 °C. The orbital shaking was 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. 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 curve drawing and fitting was performed using GraphPad Prism. The background emission from ThT in the absence of fibrils was negligible. The data points are typically a mean of four or eight samples and shown with standard deviation error bars. Only data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a relative measure of fibrillation between experiments.
The data set may be fitted to Eq. (1). However  since full sigmodial curves are not always achieved during the measurement time  lag times were here visually determined from the ThT fluorescence curve as the time point at which the ThT fluorescence is different than the background level.

Measurement of initial and final concentrations
The peptide concentration in each of the tested formulations were measured both before ap-plication in the ThT fibrillation assay (“Initial”) and after completion of the ThT fibrillation (“After ThT assay”). Concentrations were determined by reverse HPLC methods using a pramlintide standard as a reference. Before measurement after completion 150µl 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 0.22µm filter before application on the HPLC system.

CLAIMS

1. An insulin preparation comprising:
• an insulin compound 
• a nicotinic compound  and
• arginine.
2. The insulin preparation according to claim 1  wherein the insulin compound is human in-sulin or an insulin analog.
3. The insulin preparation according to any of the preceding claims  wherein the insulin compound is B28Asp human insulin.
4. The insulin preparation according to any of the preceding claims  wherein the insulin compound is B28LysB29Pro human insulin.
5. The insulin preparation according to any of the preceding claims  wherein the insulin compound is B3LysB29Glu human insulin.
6. The insulin preparation according to any of the preceding claims  wherein the insulin compound is present in the amount from about 0.2mM to about 2.0mM.
7. The insulin preparation according to any of the preceding claims  wherein the insulin compound is present in the amount from about 0.3mM to about 1.2mM.
8. The insulin preparation according to any of the preceding claims  wherein the nicotinic compound is selected from the group consisting of nicotinamide  nicotinic acid  niacin  ni-acin amide and vitamin B3 and/or salts thereof and/or any combination thereof.
9. The insulin preparation according to any of the preceding claims  comprising from about 1mM to about 150mM of the nicotinic compound.
10. The insulin preparation according to any of the preceding claims  comprising from about 1mM to about 85mM of arginine.
11. The insulin preparation according to any of the preceding claims  further comprising glu-tamic acid.
12. The insulin preparation according to any of the preceding claims  which further comprises a metal ion  preservative agent(s)  isotonicity agent(s) and stabilizer(s)  detergent(s) and buffer(s).
13. A method of reducing the blood glucose level in mammals by administering to a patient in need of such treatment a therapeutically active dose of an insulin preparation according to any of the preceding claims.
14. A method for the treatment of diabetes mellitus in a subject comprising administering to a subject an insulin preparation according to any of the preceding claims.
15. An insulin preparation according to any of the preceding claims for use in the treatment or prevention of hyperglycemia including stress induced hyperglycemia  type 2 diabetes  impaired glucose tolerance  type 1 diabetes  and burns  operation wounds and other dis-eases or injuries where an anabolic effect is needed in the treatment  myocardial infarc-tion  stroke  coronary heart disease and other cardiovascular disorders and treatment of critically ill diabetic and non-diabetic patients.