CN103328006A

CN103328006A - Preparation comprising insulin, nicotinamide and an amino acid

Info

Publication number: CN103328006A
Application number: CN2011800602960A
Authority: CN
Inventors: H.B.奥森; S.哈维伦德; U.里伯-马德森; J.斯图里斯; H.纳维; M.施莱恩; S.鲁德维格森
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2010-12-14
Filing date: 2011-12-14
Publication date: 2013-09-25
Also published as: KR20140030125A; WO2012080362A1; BR112013014856A2; IL226336A0; JP2014501239A; CA2821613A1; MX2013006174A; RU2013130374A; AU2011343360A1; EP2651446A1

Abstract

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

Description

Comprise insulin, nicotiamide and amino acid whose preparation

Invention field

The present invention relates to comprise insulin compounds, nicotine compound and amino acid whose pharmaceutical preparation.

Background of invention

The metabolic disorder that diabetes are partially or completely lost for the ability of wherein utilizing glucose.Approximately 5% suffer diabetes in everyone, this disease approaches the epidemic diseases ratio.

Since nineteen twenties is introduced insulin, aspect the treatment diabetes, obtained lasting improvement.In order to help to avoid hyperglycemia level, diabetics usually to put into practice the multiple injection treatment, therefore along with every meal gives insulin.Due to diabetics use of exogenous insulin decades, mainly need safety and life-quality improvement insulin preparation.In commercially available insulin preparation, can mention quick-acting, middle effect and durative action preparation.

In the treatment diabetes, proposed and used the insulin medicament preparation of numerous species, for example insulin regular (Actrapid for example ^?), isophane insulin (being called NPH), lente insulin (Semilente for example ^?, Lente ^?and Ultralente ^?) and biphase isophane insulin (NovoMix for example ^?).Also developed human insulin analogue and derivant, be designed for concrete action characteristic, that is, quick-acting or long-acting.Some commercially available insulin preparations that comprise such Semilente Insulin analog comprise NovoRapid ^?(preparation of aspart), Humalog ^?(preparation of B28LysB29Pro insulin human) and Apidra ^?(preparation of B3LysB29Glu insulin human).

International Application No. WO 91/09617 and WO/9610417 (Novo Nordisk A/S) disclose the insulin preparation that contains nicotiamide or nicotinic acid or its salt.European application EP1283051 contains arginic insulin preparation according to declaring to disclose, and wherein arginine is as buffer agent.EP1283051 is according to the improvement of having declared to disclose the physical stability of insulin preparation.

The most common insulin medicament preparation gives by subcutaneous injection.For the patient, importantly the action characteristic of insulin, refer to the function of insulin to the time that is used as the distance injection of glucose metabolism.Especially, in this characteristic, the total duration of time started, maximum and effect is important.In the situation that the bullet insulin, patient's expectation and requirement have the multiple insulin preparation of different action characteristics.On the same day, a patient may use the insulin preparation with very different action characteristics.The action characteristic of expectation for example depends on amount and the composition of the time that the patient has a dinner every day and the meals of eating.

For the patient, it is also important that the chemical stability of insulin preparation, for example, because a large amount of pen-sample injection devices that use for example contain Penfill ^?the device of cartridge case stores insulin preparation in this device, until whole cartridge case becomes empty, for the device that contains the 1.5-3.0ml cartridge case, may use at least 1-2 week.Between the storage life, insulin structure generation covalent chemical changes.This can cause forming may less activity and/or immunogenic molecule, for example deacylated tRNA amine product and higher molecular weight converted product (dimer, polymer) potentially.In addition, it is also important that the physical stability of insulin preparation, because long term storage can finally cause forming insoluble fibril, it is nonactive and immunogenicity potentially in biology.

Summary of the invention

The present invention relates to have favourable absorption rate and favourable chemistry and the insulin preparation of physical stability.The present invention relates to comprise insulin human and/or its analog, nicotiamide or nicotinic acid and/or its salt and arginic insulin preparation.

In one embodiment, the present invention relates to a kind of insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound, and

Arginine.

In another embodiment, the present invention also comprises a kind of experimenter's of being used for the treatment of diabetes or reduces the method for experimenter's blood sugar level, and described method comprises and gives experimenter or mammal insulin preparation of the present invention.

Invention is described

Be surprised to find that, the absorptance after the insulin compounds in subcutaneous injection insulin preparation of the present invention is faster with reference to insulin preparation.This character can be used for Semilente Insulin, particularly associated with the multiple injection scheme often giving before the meal insulin.Owing to starting sooner effect, compared with conventional Semilente Insulin scheme, insulin can be used easily when more approaching having a dinner.In addition, insulin disappears and may reduce the risk of postprandial hypoglycemia sooner.

For example, for example, Insulin Aspart for comprising insulin compounds (insulin aspart), nicotine compound (nicotiamide) and amino acids Arginine of insulin preparation of the present invention.Optionally, insulin preparation of the present invention can comprise other aminoacid.These insulin preparations have quick absorption characteristic, compared with existing treatment, more closely simulate normal physiology.In addition, insulin preparation of the present invention has chemistry and the physical stability that is applicable to business pharmaceutical preparation.

Insulin preparation of the present invention provides Insulin Aspart, and it is not only stable physically, and unexpectedly also chemically stable.With existing insulinize, compare, insulin preparation of the present invention provides and starts even faster effect.The advantage of supper-fast insulin preparation like this has the first stage of recovery insulin to discharge, and injects convenience and stops hepatic glucose production.Insulin preparation of the present invention has favourable absorption rate from the subcutaneous tissue to blood plasma, when the formulation example with conventional as NovoRapid ^?in the time of relatively, as indicated in the some PK/PD experiments in pig, initial absorption speed improves 1.5-5 doubly.This faster absorption rate can improve that glucemia is controlled and conformance, and can allow to be transferred to administration after the meal from administration before the meal.The present invention's part finds, although add nicotiamide can improve absorption rate, by significantly improving the amount of HMWP, also chemical stability is had to disadvantageous effect based on this accident.By adding arginine, insulin preparation of the present invention has improved chemical stability, is reflected in, and for example, after storage, reduces and forms dimer and polymer and Desamido insulin.Insulin preparation of the present invention can have improved physical stability in addition, and it can be used for pump.

The invention provides the insulin preparation that comprises insulin compounds of the present invention, described insulin compounds exists with the about concentration of the about 10.0mM of 0.1 mM-, and the pH of wherein said preparation is 3-8.5.Described preparation also comprises nicotine compound and arginine.Described preparation also can comprise protease inhibitor, metal ion, buffer system, antiseptic, tonicity agents, chelating agen, stabilizing agent and surfactant.

In one embodiment, metal ion is zinc, and wherein zinc adds as zinc acetate or zinc chloride.

In one embodiment, insulin preparation comprises insulin human, its analog or combination, nicotiamide and/or nicotinic acid and/or its salt and arginine and/or its salt.

In one embodiment, insulin preparation of the present invention comprises aspart, nicotiamide and arginic aqueous solution.

In solution of the present invention, the content of aspart can be in 15-500 iu (IU)/ml scope, for injection preparation, preferably in 50-333 IU/ml scope.Yet, other purpose given for parenteral, the content of insulin compounds can be higher.

In the present context, unit " IU " is corresponding to 6 nmol.

Term " insulin aspart " refers to the human insulin analogue aspart.

Term " beginning " refers to from injection until the PK curve starts the time of improving.

Term " absorption rate " refers to the PK slope of a curve.

" insulin compounds " of the present invention is interpreted as insulin human, insulin analog and/or their any combination herein.

Term used herein " insulin human " refers to its structure and the well-known people's hormone of character.Insulin human has two polypeptide chains, and they connect by the disulphide bridges between cysteine residues, that is, and and A-chain and B-chain.The A-chain is 21 amino acid peptides, the B-chain is 30 amino acid peptides, two chains connect by three disulphide bridges: one between 6 of the A-chain and 11 s' cysteine, between 7 cysteine of second 7 cysteine at the A-chain and B-chain, between 19 cysteine of the 3rd 20 cysteine at the A-chain and B-chain.

Hormone is synthetic as list-chain precursor proinsulin (preproinsulin), it then contains 86 amino acid whose proinsulins by 24 amino acid whose propetides with following structure and forms: propetide-B-Arg Arg-C-Lys Arg-A, wherein C is 31 amino acid whose connection peptides.Arg-Arg and Lys-Arg are for cutting the cutting part of connection peptides and A and B chain.

" insulin analog " used herein refers to for example, the polypeptide derived from the primary structure of naturally occurring insulin (insulin human) by sudden change.One or more sudden changes are by lacking and/or being substituted at least one amino acid residue existed in naturally occurring insulin and/or being undertaken by adding at least one amino acid residue.The amino acid residue that adds and/or replace can be amino acid residue or other naturally occurring amino acid residue of codified.

In one embodiment, insulin analog comprises with respect to the parent insulin and is less than 8 modifications (replace, disappearance, add and their any combination), perhaps with respect to the parent insulin, be less than 7 modifications, perhaps with respect to the parent insulin, be less than 6 modifications, perhaps with respect to the parent insulin, be less than 5 modifications, perhaps with respect to the parent insulin, be less than 4 modifications, or be less than 3 modifications with respect to the parent insulin, or be less than 2 modifications with respect to the parent insulin.

Sudden change in insulin molecule is expressed as the amino acid whose three-letter code that shows chain (A or B), position and replacement natural amino acid." desB30 " or " B (1-29) " is natural insulin B chain or its analog of hypodactylia b30 amino acid residue, and " aspart " refers to the insulin human that wherein 28 amino acids residues of B chain are replaced by Asp.

The example of insulin analog is that wherein the Pro of 28 of B chain is suddenlyd change by Asp, Lys, Leu, Val or Ala, and/or the Lys of position B29 is suddenlyd change by Pro, Glu or Asp.In addition, the Asn of position B3 can be suddenlyd change by Thr, Lys, Gln, Glu or Asp.The amino acid residue of position A21 can be suddenlyd change by Gly.The aminoacid of position B1 can be suddenlyd change by Glu.The aminoacid of position B16 can be suddenlyd change by Glu or His.Other example of insulin analog is delation analogs, for example wherein the analog (des (B30) insulin human) of the disappearance of the b30 amino acid in insulin human, wherein insulin analog (des (B1) insulin human), des (B28-B30) insulin human and des (B27) insulin human of the B1 aminoacid deletion in insulin human.Wherein A-chain and/or B-chain have insulin analog that the N-end extends and insulin analog that wherein A-chain and/or B-chain have a C-end extension (for example two arginine residues join the C-end of B-chain) is also the example of insulin analog.The insulin analog that other example is the combination that comprises mentioned sudden change.Wherein the aminoacid of position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His, and the aminoacid of position B25 is other example that His and the insulin analog that optionally also comprises one or more other sudden changes are insulin analog.The insulin analog that wherein amino acid residue of position A21 is Gly and the insulin human that wherein insulin analog is further extended by two arginine residues at the C-end is also the example of insulin analog.

Other example of insulin analog includes but not limited to: the DesB30 insulin human; Aspart; AspB28, the desB30 insulin human; LysB3, the GluB29 insulin human; LysB28, the ProB29 insulin human; GlyA21, ArgB31, ArgB32 insulin human; GluA14, the HisB25 insulin human; HisA14, the HisB25 insulin human; GluA14, HisB25, desB30 insulin human; HisA14, HisB25, desB30 insulin human; GluA14, HisB25, desB27, desB28, desB29, desB30 insulin human; GluA14, HisB25, GluB27, desB30 insulin human; GluA14, HisB16, HisB25, desB30 insulin human; HisA14, HisB16, HisB25, desB30 insulin human; HisA8, GluA14, HisB25, GluB27, desB30 insulin human; HisA8, GluA14, GluB1, GluB16, HisB25, GluB27, desB30 insulin human; And HisA8, GluA14, GluB16, HisB25, desB30 insulin human.

Term " nicotine compound " comprises nicotiamide, nicotinic acid, nicotinic acid, niacin amide and vitamin B3 and/or its salt and/or their any combination.

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

Term " arginine " or " Arg " comprise amino acids Arginine and/or its salt, for example, and arginine monohydrochloride or Ginamate.

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

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

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

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

Term " glutamic acid " or " Glu " comprise aminoacid glutamic acid and/or its salt.

Term used herein " pharmaceutical preparation " or " insulin preparation " refer to the product that comprises insulin compounds, , insulin human, its analog and/or its combination and nicotine compound and aminoacid, optionally together with other excipient, antiseptic for example, chelating agen, tension regulator, extender, stabilizing agent, antioxidant, polymer and surfactant, metal ion, oiliness solvent and protein are (for example, the human serum albumin, gelatin or protein), by giving the people described insulin preparation, described insulin preparation can be used for treatment, the seriousness of prevention or reduction disease or disease.Therefore, insulin preparation in this area also referred to as pharmaceutical preparation or pharmaceutical composition.

Buffer agent can be selected from but be not limited to sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium phosphate and three (hydroxymethyl)-aminomethane (tris), N-bicine N-, Qu Xin, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, ethylenediamine or their mixture.Each of these concrete buffer agents forms alternative embodiment of the present invention.

Insulin preparation of the present invention also can comprise other common composition of insulin preparation, for example zinc chelating agent citrate for example, and phosphate buffer.

Glycerol and/or mannitol and/or sodium chloride can exist corresponding to the amount of the concentration of 0-250mM, 0-200mM or 0-100mM.

Stabilizing agent, surfactant and antiseptic also can be present in insulin preparation of the present invention.

Insulin preparation of the present invention also can comprise pharmaceutically acceptable antiseptic.Antiseptic can be enough to obtain the amount existence of antisepsis.The amount of antiseptic in insulin preparation for example the document in this field and/or in commodity for example the known quantity of antiseptic determine.Each in these concrete antiseptic forms alternative embodiment of the present invention.In pharmaceutical preparation, use antiseptic for example to be described in Remington:The Science and Practice of Pharmacy (pharmaceutical science with put into practice), the 19th edition, 1995.

The antiseptic be present in insulin preparation of the present invention can be conventional up to now insulin preparation, for example phenol, metacresol and methyl parahydroxybenzoate.

Insulin preparation of the present invention also can comprise chelating agen.In pharmaceutical preparation, use chelating agen well-known for the professional.For convenient, with reference to Remington:The Science and Practice of Pharmacy (pharmaceutical science with put into practice), the 19th edition, 1995.

Insulin preparation of the present invention also can comprise stabilizing agent.Term used herein " stabilizing agent " refers to the chemicals joined in the pharmaceutical preparation that contains polypeptide with stabilized peptide,, improves shelf life and/or the service time of said preparation that is.For convenient, with reference to Remington:The Science and Practice of Pharmacy (pharmaceutical science with put into practice), the 19th edition, 1995.

Insulin preparation of the present invention also can comprise surfactant.Term used herein " surfactant " refers to any molecule or the ion that comprises water solublity (hydrophilic) part termination and fat-soluble (lipotropy) segment.Surfactant is preferably in the interface accumulation, and wherein hydrophilic segment is orientated towards water (aqueous favoring), and the lipotropy part is towards oil phase or hydrophobic phase (that is, glass, air, wet goods) orientation.Concentration when surfactant starts to form micelle is called critical micelle concentration or CMC.In addition, the surface tension of decreasing by surfactant liquid.Surfactant is also referred to as amphipathic compound.Usually the synonym that term " detergent " is surfactant.In pharmaceutical preparation, use surfactant well-known for the professional.For convenient, with reference to Remington:The Science and Practice of Pharmacy (pharmaceutical science with put into practice), the 19th edition, 1995.

In other embodiments, the present invention relates to the insulin preparation of a kind of aqueous solution that comprises insulin compounds of the present invention and buffer agent, wherein said insulin compounds exists with 0.1mM or higher concentration, and at the pH of the lower described preparation of room temperature (approximately 25 ℃), is wherein about 3.0-approximately 8.5.

The invention still further relates to the method for the production of insulin preparation of the present invention.

In one embodiment, the method for the preparation of insulin preparation of the present invention comprises:

A) by dissolve the mixture of insulin compounds or insulin compounds in water or buffer agent, prepare solution;

B) by water or buffer agent, dissolving bivalent metal ion, prepare solution;

C) by water or buffer agent, dissolving antiseptic, prepare solution;

D) by solution tension agent in water or buffer agent, prepare solution;

E) by dissolve surfactant and/or stabilizing agent in water or buffer agent, prepare solution;

F) by solution a) with solution b), c), d) and e) in one or more mix;

Finally will be at f) in the pH regulator of mixture to the pH of expectation, then aseptic filtration.

A) prepare the independent liquid storage of antiseptic, tonicity agents, metal cation salt, arginine or arginine salt and nicotine compound;

B) liquid storage by the mixture by antiseptic or antiseptic joins in water or buffer agent, prepares solution;

C) liquid storage of tonicity agents is joined to solution b) in;

D) by dissolve the mixture of insulin compounds or insulin compounds in water or buffer agent, prepare solution; Add HCl with this solution of acidify;

E) liquid storage of metal cation salt is joined to d) in;

F) by e) join c) in, and stir;

G) liquid storage of arginine or arginine salt is joined to f) in, and stir;

H) liquid storage of nicotine compound is joined to g) in, and use NaOH/HCl to regulate the pH of pH to expectation.

A) prepare the independent liquid storage of antiseptic, tonicity agents, metal cation salt, and the mixing liquid storage of arginine or arginine salt and nicotine compound;

C) liquid storage of tonicity agents is joined to solution b) in;

E) liquid storage of metal cation salt is joined to d) in;

F) by e) join c) in, and stir;

G) the mixing liquid storage of arginine or arginine salt and nicotine compound is joined to f) in, and stir;

H) pH use NaOH/HCl adjusting g) is to the pH of expectation.

By parenteral, give, insulin preparation of the present invention can be used for treating diabetes.Recommendation gives the dosage of patient's insulin preparation of the present invention and is selected by the internist.

By syringe (optionally pen-sample syringe), parenteral gives to implement by subcutaneous, intramuscular, intraperitoneal or intravenous injection.Perhaps, parenteral gives to implement by infusion pump.As other option, the insulin preparation that contains insulin compounds of the present invention also can be suitable for percutaneous and give, for example, by Needleless injection or by patch, optionally ionotherapy patch, or through mucous membrane, for example, oral cavity gives.

Insulin preparation of the present invention can need the patient of this treatment at some positions, for example, and at part, for example, skin and mucosal sites; At non-absorbent position, for example, in tremulous pulse, vein, heart, give; With the position relating to absorption, for example, in skin, under skin, give in muscle or in abdominal part.

In one embodiment of the invention, insulin preparation is aqueous formulation, that is, and and the preparation that comprises water.Such preparation is generally solution or suspension.In other embodiments of the present invention, insulin preparation is aqueous solution.

Term " aqueous formulation " is defined as the preparation that comprises at least 50% w/w water.Equally, term " aqueous solution " is defined as the solution that comprises at least 50% w/w water, and term " waterborne suspension " is defined as the suspension that comprises at least 50% w/w water.

Waterborne suspension can contain the reactive compound with the mixed with excipients that is applicable to manufacture waterborne suspension.

In one embodiment, insulin preparation of the present invention fully is suitable for using in pen-sampling device, for by injection, carrying out insulinize.

In one embodiment, insulin preparation of the present invention can be used for pump, for insulin, gives.

" physical stability " of term insulin preparation used herein refer to due to protein be exposed to thermal-mechanical stress and/or with the interaction of going to stable surface and interface (for example hydrophobic surface and interface), the biology of Protein formation protein nonactive and/or insoluble aggregate tendency.By the container suitable (for example, cartridge case or bottle) in the preparation of filling (for example be exposed to machinery/physical stress at different temperature, stir) visual inspection and/or turbidimetry afterwards of various time periods, the physical stability of evaluation aqueous protein formulations.In dark background, implement the visual inspection of preparation in sharp focus light.The turbidity of preparation characterizes with the vision mark of evaluation turbidity degree, for example 0-3 specification (do not show that the preparation of turbidity is corresponding to vision mark 0, and the preparation that shows the vision turbidity under daylight being corresponding to vision mark 3).About protein aggregation, when it shows the vision turbidity under daylight, said preparation is categorized as unstable physically.Perhaps, by the well-known simple turbidimetry of professional, can estimate the turbidity of preparation.The physical stability of aqueous protein formulations also can be estimated by spectrum agent or the probe of the conformation situation with protein.Probe is preferably micromolecule, and it preferably is combined with the non-natural conformer of protein.An example of the micromolecule spectral probe of protein structure is thioflavine T.Thioflavine T is to be widely used in the fluorescent dye that detects the amyloid fibril.Under fibril exists, same and may the other oroteins structure exist under, thioflavine T causes new maximum excitation under about 450 nm, and when when the fibrillar protein form is combined, the emission strengthened under about 482 nm.Unconjugated thioflavine T is essentially non-fluorescence under these wavelength.

" chemical stability " of term protein preparation used herein refers to that changing the covalency protein structure causes forming the chemical degradation product, with the native protein structure, compares, and has the less biological value of possibility and/or may improve immunogenic character.Various chemical degradation products be can form, the type of native protein and the environment of character and protein exposure depended on.During storing and using protein formulation, usually see the amount that improves the chemical degradation product.Most protein is easy to deacylated tRNA amine, and for the amide side chain base in wherein glutaminyl or asparaginyl-residue is hydrolyzed to form free carboxylic acid, or the asparaginyl-residue is hydrolyzed to form the process of IsoAsp derivant.Other degradation pathway comprises when two or more protein molecules interact each other covalent bond by acylamino-exchange interaction and/or disulphide; form high molecular weight product; cause forming covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. T.J. & Manning M.C., Plenum Press, New York 1992).Oxidation (for example oxidation of methionine residue) can be mentioned as another variant of chemical degradation.By for example, being exposed to different environmental condition (by improving temperature, usually can accelerate to form catabolite) afterwards, at various time points, by measuring the amount of chemical degradation product, but the chemical stability of assess proteins preparation.The amount of each single catabolite for example, is measured by using various chromatographic techniques (, SEC-HPLC and/or RP-HPLC) to separate catabolite usually, and this depends on molecular dimension and/or electric charge.Due to HMWP product immunogenicity and be not biologic activity potentially, low-level HMWP is favourable.

Term " preparation of stabilisation " refers to the preparation of the physics and chemistry stability of the chemical stability of physical stability with raising, raising or raising.Usually, between use and storage life, preparation must be stablized (use and the condition of storage of being obedient to recommendation), until reach expiry date.

Term " diabetes (diabetes) " or " diabetes (diabetes mellitus) " comprise type 1 diabetes, type 2 diabetes mellitus, gestational diabetes (at phenolics) and cause other situation of hyperglycemia.This term for pancreas wherein produce the insulin of q.s not or wherein the cell of health can not suitably respond the metabolic disorder that therefore insulin hinders the Cell uptake glucose.Result is that glucose is accumulated in blood.

Type 1 diabetes, also referred to as insulin-dependent diabetes mellitus (IDDM) and teenager-morbidity diabetes, caused by the B-cytoclasis, usually causes absolute insulin defect.

Type 2 diabetes mellitus, also referred to as non-insulin-dependent diabetes mellitus (NIDDM) and adult-morbidity diabetes, associated with leading insulin resistant, therefore for thering is relative insulin defect and/or the leading defect of insulin secretion of insulin resistant.

Term used herein " pharmaceutically acceptable " refers to and is suitable for normal medicinal application,, can in the patient, not cause any serious harmful event that is.

Term used herein " treatment disease " refers to management and looks after and suffered from the patient of disease, the patient's condition or disease, and comprises treatment, prevents or palliate a disease.The purpose for the treatment of is to struggle with disease, the patient's condition or disease.Treatment comprises and gives reactive compound, to eliminate or control disease, the patient's condition or disease and alleviate the symptom relevant to disease, the patient's condition or disease or complication and prevent disease, the patient's condition or disease.

At it on the widest implication, term used herein " critical patient " refers to and maintains or the patient who maintains the risk of the acute single or multiple tract exhaustion be in peril of one's life due to i or I arranged, wait to perform the operation and follow the patient of complication, and experience vitals operation or experienced large operating patient in nearest one week in nearest one week.On stricter meaning, term used herein " critical patient " refers to and maintains or the patient who maintains the risk of the acute single or multiple tract exhaustion be in peril of one's life due to i or I is arranged, or waits to perform the operation and follow the patient of complication.On even stricter meaning, term used herein " critical patient " refers to and maintains or the patient who maintains the risk of the acute single or multiple tract exhaustion be in peril of one's life due to i or I arranged.Similarly, these definition are applicable to similar statement, for example " critically ill patient " and " patient is critical ".The example of critical patient is the patient who needs operation on heart, operation on brain, thoracic operation, abdominal operation, vascular surgery or transplanting, or suffers from neurological disease, large cerebral trauma, respiratory insufficiency, abdominal part peritonitis, multiple trauma or serious burn or the patient of critical polyneuropathy.

Term used herein " anabolism " refers to one group of metabolic pathway by less cell formation molecule.These reaction needed energy.No matter being cell, organ or organism level, is ' anabolism ' or ' catabolism ' by a kind of mode of metabolic process classification, and the two is relative.Anabolism provides power by catabolism, and in catabolism, macromole destroys becomes less part, in breathing, uses up subsequently.Many anabolic processes provide power by adenosine triphosphate (ATP).Anabolic process is tended to " accumulation " Organ and tissue.These processes produce Growth of Cells and differentiation and raising body sizes, for relating to the synthetic process of complicated molecule.The example of anabolic process comprises bone growth and mineralising and raising muscle quality.The endocrinologist is categorized as hormone anabolism or catabolism traditionally, depends on which part metabolism they stimulate.Also by circadian rhythm, regulate the balance between anabolism and catabolism, for example glucose metabolism fluctuation of this process, with coupling animal normal activity cycle in one day.Some examples of " anabolism " of these hormones are reinvented and grow and stimulate bone marrow (it improves the production of blood rbc) for improving from amino acid whose protein synthesis, raising appetite, raising skeleton.By a plurality of mechanism, anabolic hormone stimulates and forms muscle cell, therefore causes and the size that improves skeletal muscle causes improving intensity.

In another embodiment, insulin analog of the present invention is as medicine, for postponing at type 2 diabetes mellitus or the prevent disease progress.

In one embodiment of the invention, provide insulin preparation of the present invention as medicine, be used for the treatment of or prevent hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and need Other diseases or damage, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder of anabolic action in treatment.

In other embodiments of the present invention, the method that is used for the treatment of or prevents following disease is provided: hyperglycemia comprises stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and needs anabolic Other diseases or damage, myocardial infarction, coronary heart disease and other cardiovascular disorder, apoplexy in treatment, the treatment insulin preparation of the present invention that the patient that described method comprises needs this treatment is effective dose for this treatment.

Use the insulin preparation of the present invention treatment also can be with second or more pharmacological active substance coupling, for example, be selected from anti-diabetic medicament, anti-obesity agents, appetite stimulator medicament, antihypertensive agents, be used for the treatment of and/or the medicament of complication that prevent to be caused by diabetes or relevant to diabetes and be used for the treatment of and/or prevent to be caused by obesity or with obesity relevant complication and the medicament of disease.

Use insulin preparation of the present invention treatment also can with the coupling of bariatrics surgical operation, it is affecting glucose level and/or the homeostatic surgical operation of lipid, for example gastric banding or stomach bypass.

The production of polypeptide (for example, insulin) is well-known in the art.Insulin analog of the present invention can for example synthesize to produce by classical peptide; for example; use the solid-phase peptide of t-Boc or Fmoc chemistry to synthesize or other technology of having established; referring to for example; Greene and Wuts; " Protective Groups in Organic synthesizes (blocking group in organic synthesis) ", John Wiley & Sons, 1999.Insulin analog also can be produced by a kind of method, described method comprises the host cell of cultivating the DNA sequence that contains the analog of encoding, this host cell under the condition that allows the expression of insulin analog, can be in suitable Nutrient medium the expression of insulin analog.For the insulin analog that comprises the alpha-non-natural amino acid residue, should modify recombinant cell, make alpha-non-natural amino acid is incorporated in analog, for example, by using the tRNA mutant.Therefore, briefly, prepared by the preparation of insulin analog of the present invention and known insulin analog similarly.

Some methods can be used for producing insulin human and human insulin analogue.Three kinds for example in WO2008034881, are disclosed for produce the main method of insulin in microorganism.Two kinds in these methods relate to escherichia coli, and it expresses large fused protein (people (1981) such as Frank, Peptides:Proceedings of the 7 in Cytoplasm ^thamerican Peptide Chemistry Symposium (Rich & Gross, chief editor), Pierce Chemical Co., Rockford, III. 729-739 page), or use signal peptide to make it possible to be secreted in periplasmic space people (1981) PNAS 78:5401-5404 such as () Chan.The third method utilizes saccharomyces cerevisiae (Saccharomyces cerevisiae) insulin precurosor is secreted into to (people (1986) the PNAS 83:6766-6770 such as Thim) in culture medium.Prior art discloses many insulin precurosors of expressing in escherichia coli or saccharomyces cerevisiae, referring to U.S5, and 962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

By in for example disclosed well-known technology in US 6500645, by the DNA sequence of the insulin analog that the expression coding is discussed in suitable host cell, produce insulin analog.Insulin analog directly or as precursor molecule is expressed, and it has the extension of N-end or have the extension of C-end on the B-chain on the B-chain.The N-end extends the function that can have the yield that improves the product of directly expressing, and can be up to 15 amino acid residue length.From culture broth bouillon after separating, the N-end extends body and excises outward, therefore has the cutting part by B1.The N-end that is applicable to type of the present invention extends and to be disclosed in US 5,395, and 922 and EP 765,395.The C-end extends to have protects ripe insulin or insulin analog molecule to avoid by the function of hydrolysis processing in host cell exoproteinase cell.The pendant carboxylic group peptidase that the C-end extends through secretion extracellular cutting or in external cutting from the culture broth bouillon separates in the culture broth bouillon.Be disclosed in WO 08037735 for the production of the insulin of the maturation that has the extension of C-end on the B-chain and the method for insulin analog, this C-end extends through carboxypeptidase and removes.The target insulin product of described process can be two-chain insulin human or two-chain human insulin analogue, and its short C-end that can have the B-chain extends or may not have the short C-end extension of B-chain.If target insulin product does not have the C-end of B chain, extend, described C-end extension should be able to be cut from the B-chain before being further purified step.

The present invention also comprises that the non-limiting of following embodiment enumerate, and its other place at this paper further describes:

1. an insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound, and

Arginine.

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

3. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds is aspart, B3LysB29Glu insulin human or B28LysB29Pro insulin human.

4. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds is aspart.

5. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds is B3LysB29Glu insulin human or B28LysB29Pro insulin human.

6. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds is the B28LysB29Pro insulin human.

7. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds is the B3LysB29Glu insulin human.

8. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists to be selected from following scope: 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.

9. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 10.0mM of about 0.1mM-.

10. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 3.0mM of about 0.1mM-.

11. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.5mM of about 0.1mM-.

12. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.1mM-.

13. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.5mM of about 0.1mM-.

14. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.5mM of about 0.2mM-.

15. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.2mM-.

16. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.5mM of about 0.2mM-.

17. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 3.0mM of about 0.3mM-.

18. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.5mM of about 0.3mM-.

19. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.3mM-.

20. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.5mM of about 0.3mM-.

21. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.3mM of about 0.5mM-.

22. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.2mM of about 0.3mM-.

23. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of the about 1.2mM of about 0.6mM-.

24. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds with approximately 0.6 or the amount of about 1.2mM exist.

25. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of about 0.3mM.

26. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of about 0.6mM.

27. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of about 0.9mM.

28. the insulin preparation of any one in foregoing embodiments, wherein said insulin compounds exists with the amount of about 1.2mM.

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

30. the insulin preparation of any one in foregoing embodiments, wherein said nicotine compound is selected from nicotiamide and nicotinic acid and/or its salt and/or their any combination.

31. the insulin preparation of any one in foregoing embodiments, wherein said nicotine compound is nicotiamide and/or its salt.

32. the insulin preparation of any one in foregoing embodiments, wherein said nicotine compound exists to be selected from following scope: 1-300mM; 5-200mM; 40-120mM; 70-140mM or 80-130mM.

33. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 300mM of about 1mM-.

34. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 260mM of about 8mM-.

35. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 250mM of about 50mM-.

36. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 250mM of about 80mM-.

37. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 180mM of about 80mM-.

38. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 200mM of about 5mM-.

39. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 150mM of about 1mM-.

40. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 20mM of about 5mM-.

41. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 120mM of about 20mM-.

42. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 120mM of about 40mM-.

43. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 40mM of about 20mM-.

44. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 80mM of about 60mM-.

45. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 140mM of about 70mM-.

46. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises the about 130mM of about 80mM-.

47. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 8mM, 30mM, 100mM or 130mM.

48. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 8mM.

49. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 30mM, 100mM or 130mM.

50. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 30mM.

51. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 80mM.

52. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 100mM.

53. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 120mM.

54. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 130mM.

55. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 150mM.

56. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 155mM.

57. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 180mM.

58. the insulin preparation of any one in foregoing embodiments, the nicotine compound that described preparation comprises about 230mM.

59. the insulin preparation of any one in foregoing embodiments, the arginine compounds that described preparation comprises following scope: 1-100mM, 5-120mM, 8-85mM, 20-90mM, 30-90mM, 30-85mM, 30-60mM or 10-40mM.

60. the insulin preparation of any one in foregoing embodiments, the arginine compounds that described preparation comprises following scope: 1-120mM, 8-85mM or 1-40mM.

61. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 120mM of about 1mM-.

62. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 100mM of about 1mM-.

63. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 120mM of about 5mM-.

64. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 90mM of about 20mM-.

65. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 85mM of about 30mM-.

66. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 85mM of about 8mM-.

67. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 60mM of about 30mM-.

68. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 40mM of about 10mM-.

69. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 30mM of about 10mM-.

70. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 60mM of about 10mM-.

71. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises the about 40mM of about 1mM-.

72. the insulin preparation of any one in foregoing embodiments, wherein arginine exists to be selected from following scope: 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM, 55mM or 60mM.

73. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 1mM.

74. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 2mM.

75. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 3mM.

76. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 4mM.

77. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 5mM.

78. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 6mM.

79. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 7mM.

80. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 8mM.

81. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 9mM.

82. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 10mM.

83. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 11mM.

84. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 12mM.

85. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 13mM.

86. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 14mM.

87. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 15mM.

88. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 17mM.

89. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 20mM.

90. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 22mM.

91. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 25mM.

92. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 30mM.

93. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 35mM.

94. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 40mM.

95. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 45mM.

96. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 50mM.

97. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 55mM.

98. the insulin preparation of any one in foregoing embodiments, the arginine that described preparation comprises about 60mM.

99. the insulin preparation of any one in foregoing embodiments, described preparation also comprises buffer agent.

100. the insulin preparation of embodiment 99, wherein said buffer agent is phosphate buffer.

101. the insulin preparation of embodiment 100, the phosphate buffer that described preparation comprises the about 20mg/mL of about 1mg/mL-.

102. the insulin preparation of embodiment 100, the phosphate buffer that described preparation comprises the about 15mg/mL of about 1mg/mL-.

103. the insulin preparation of embodiment 100, the phosphate buffer that described preparation comprises the about 10mg/mL of about 1mg/mL-.

104. the insulin preparation of embodiment 100, the phosphate buffer that described preparation comprises about 3mg/mL.

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

106. the insulin preparation of embodiment 105, the Tris that described preparation comprises the about 50mM of about 2mM-.

107. the insulin preparation of embodiment 105, the Tris that described preparation comprises the about 40mM of about 10mM-.

108. the insulin preparation of embodiment 105, the Tris that described preparation comprises the about 30mM of about 20mM-.

109. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 10mM, 20mM, 30mM or 40mM.

110. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 7mM.

111. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 10mM.

112. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 20mM.

113. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 30mM.

114. the insulin preparation of embodiment 105, the Tris that described preparation comprises about 40mM.

115. the insulin preparation of any one in foregoing embodiments, described preparation also comprises metal ion.

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

117. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 6:6 of about 0:6-.

118. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 5:6 of about 0:6-.

119. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 6:6 of about 1:6-.

120. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 6:6 of about 2:6-.

121. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 5:6 of about 2:6-.

122. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 4.5:6 of about 2.5:6-.

123. the insulin preparation of embodiment 116, wherein said zinc: the insulin mol ratio is the about 4:6 of about 3:6-.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

139. the insulin preparation of any one in foregoing embodiments, described preparation also comprises stabilizing agent.

140. the insulin preparation of embodiment 139, wherein said stabilizing agent is nonionic detergent.

141. the insulin preparation of embodiment 140, wherein said detergent is polysorbate20 (polysorbas20) or polysorbate80 (Tween 80).

142. the insulin preparation of embodiment 140, wherein said detergent is polysorbate20 (polysorbas20).

143. the insulin preparation of embodiment 140, wherein said detergent is polysorbate80 (Tween 80).

144. the insulin preparation of embodiment 140, the polysorbate that described preparation comprises about 5-100ppm, the about 50ppm of about 10-or the about 20ppm of about 10-.

145. the insulin preparation of any one in foregoing embodiments, described preparation also comprises antiseptic.

146. the insulin preparation of embodiment 145, wherein said antiseptic is phenolic compound.

147. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of the about 6mg/ml of about 0-or the about 4mg/ml of about 0-.

148. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of the about 100mM of about 5-.

149. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of the about 50mM of about 5-.

150. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of the about 30mM of about 5-.

151. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of about 16mM.

152. the insulin preparation of embodiment 146, wherein said phenolic compound exists with the amount of about 14.4mM.

153. the insulin preparation of embodiment 145, wherein said antiseptic is metacresol.

154. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of the about 4.0mg/ml of about 0.5-or the about 4.0mg/ml of about 0.6-.

155. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of the about 100mM of about 5-.

156. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of the about 50mM of about 5-.

157. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of the about 30mM of about 5-.

158. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of about 16mM.

159. the insulin preparation of embodiment 153, wherein metacresol exists with the amount of about 14.4mM.

160. the insulin preparation of any one in foregoing embodiments, described preparation also comprises the approximately glycerol of 2.5% amount of about 0.5-.

161. the insulin preparation of any one in foregoing embodiments, described preparation also comprises the approximately glycerol of 2.0% amount of about 0.7-.

162. the insulin preparation of any one in foregoing embodiments, described preparation also comprises the approximately glycerol of 1.5% amount of about 1.0-.

163. the insulin preparation of any one in foregoing embodiments, described preparation also comprises the approximately glycerol of 1.25% amount.

164. the insulin preparation of any one in foregoing embodiments, wherein said pH is neutral to alkalescence.

165. the insulin preparation of any one in foregoing embodiments, wherein said pH is about 6.8-approximately 8.0.

166. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 6.8.

167. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 6.9.

168. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.0.

169. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.1.

170. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.2.

171. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.3.

172. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.4.

173. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.5.

174. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.6.

175. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.7.

176. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.8.

177. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 7.9.

178. the insulin preparation of any one in foregoing embodiments, wherein said pH is approximately 8.0.

179. a method that reduces blood glucose level mammals, the insulin preparation of any one in the foregoing embodiments of the patient treatment active dose of described method by needing this treatment.

180. a method for the treatment of experimenter's diabetes, described method comprises the insulin preparation that gives any one in experimenter's embodiment 1-178.

181. the method for any one in embodiment 179-180, described method gives for parenteral.

182. the insulin preparation of any one in embodiment 1-178, described preparation is used for the treatment of or prevents hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and need Other diseases or damage, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder of anabolic action and treat critical diabetes and ND in treatment.

183. the insulin preparation of embodiment 182, described preparation is used for the treatment of or prevents hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn and surgical wound, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder.

184. the insulin preparation of embodiment 182-183, described preparation is used for the treatment of hyperglycemia type 2 diabetes mellitus and type 1 diabetes.

185. an insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound

Arginine, and

Buffer agent.

186. the insulin preparation of embodiment 185, wherein said insulin compounds is insulin human or insulin analog.

187. the insulin preparation of any one in embodiment 185 or 186, wherein said insulin compounds is selected from aspart, B3LysB29Glu insulin human and B28LysB29Pro.

188. the insulin preparation of embodiment 186, wherein said insulin compounds is aspart.

189. the insulin preparation of any one in embodiment 185-188, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.2mM-.

190. the insulin preparation of any one in embodiment 185-189, wherein said insulin compounds exists with the amount of the about 1.2mM of about 0.6mM-.

191. the insulin preparation of any one in embodiment 185-190, wherein said nicotine compound is selected from nicotiamide, nicotinic acid, nicotinic acid, niacin amide and vitamin B3 and/or its salt and/or their any combination.

192. the insulin preparation of any one in embodiment 185-191, wherein said nicotine compound is nicotiamide.

193. the insulin preparation of embodiment 192, the nicotine compound that described preparation comprises the about 250mM of about 1mM-.

194. the insulin preparation of embodiment 192, the nicotine compound that described preparation comprises the about 250mM of about 1mM-.

195. the insulin preparation of embodiment 192, the nicotine compound that described preparation comprises the about 230mM of about 80mM-.

196. the insulin preparation of any one in embodiment 185-195, the arginine that described preparation comprises the about 60mM of about 10mM-.

197. the insulin preparation of any one in embodiment 185-196, the arginine that described preparation comprises the about 30mM of about 10mM-.

198. the insulin preparation of any one in embodiment 185-197, described preparation comprises about 20mM arginine.

199. the insulin preparation of any one in embodiment 185-198, wherein said buffer agent is phosphate buffer.

200. the insulin preparation of any one in embodiment 185-199, wherein said buffer agent is Tris.

201. the insulin preparation of any one in embodiment 185-200, described preparation also can comprise antiseptic, isotonic agent and/or stabilizing agent.

202. the insulin preparation of any one in embodiment 185-201, described preparation also comprises metal ion.

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

204. the insulin preparation of embodiment 203, wherein said zinc: the insulin mol ratio is the about 3.5:6 of about 0:6-.

205. the insulin preparation of embodiment 203, wherein said zinc: the insulin mol ratio is the about 3:6 of about 2:6-.

206. the insulin preparation of any one in embodiment 185-205, the pH of wherein said preparation is less than 7.4.

207. the insulin preparation of any one in embodiment 185-205, the pH of wherein said preparation is approximately 7.4.

208. the insulin preparation of any one in embodiment 185-205, the pH of wherein said preparation is approximately 7.1.

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

210. the insulin preparation of embodiment 209, wherein said aspart with about 0.6 mM-approximately the concentration of 1.2 mM scopes exist, and wherein said nicotiamide with about 80 mM-approximately the concentration of 260 mM scopes exist, and wherein said arginine with about 10 mM-approximately the concentration of 40 mM scopes exist, and every 6 aspart molecules wherein, existence is less than approximately 4 zinc ioies, and the pH of wherein said preparation is approximately 7.4 or lower.

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

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

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

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

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

E. phosphate buffer;

The pH of wherein said preparation is approximately 7.1.

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

213. a method for the treatment of experimenter's diabetes, described method comprises the insulin preparation that gives any one in experimenter's embodiment 1-211.

214. the insulin preparation of any one in embodiment 1-211, described preparation is used for the treatment of or prevents hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and need Other diseases or damage, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder of anabolic action and treat critical diabetes and ND in treatment.

215. the insulin preparation of any one in embodiment 1-211, described preparation is used for the treatment of hyperglycemia type 2 diabetes mellitus and type 1 diabetes.

Other embodiment of the present invention relates to following:

216. an insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound, and

Arginine.

217. the insulin preparation of embodiment 216, wherein said insulin compounds is insulin human or insulin analog.

218. the insulin preparation of embodiment 216-217, wherein said insulin compounds is aspart.

219. the insulin preparation of any one in embodiment 216-218, wherein said insulin compounds is the B28LysB29Pro insulin human.

220. the insulin preparation of any one in embodiment 216-219, wherein said insulin compounds is the B3LysB29Glu insulin human.

221. the insulin preparation of any one in embodiment 216-220, wherein said insulin compounds exists with the amount of the about 2.0mM of about 0.2mM-.

222. the insulin preparation of any one in embodiment 216-221, wherein said insulin compounds exists with the amount of the about 1.2mM of about 0.3mM-.

223. the insulin preparation of any one in embodiment 216-222, wherein said nicotine compound is selected from nicotiamide, nicotinic acid, nicotinic acid, niacin amide and vitamin B3 and/or its salt and/or their any combination.

224. the insulin preparation of any one in embodiment 216-223, the nicotine compound that described preparation comprises the about 150mM of about 1mM-.

225. the insulin preparation of any one in embodiment 216-224, the arginine that described preparation comprises the about 85mM of about 1mM-.

226. the insulin preparation of any one in embodiment 216-225, described preparation also comprises metal ion, antiseptic, isotonic agent and stabilizing agent and buffer agent.

227. a method that reduces blood glucose level mammals, the insulin preparation of any one in the embodiment 216-226 of the patient treatment active dose of described method by needing this treatment.

228. a method for the treatment of experimenter's diabetes, described method comprises the insulin preparation that gives any one in experimenter's embodiment 216-226.

229. the insulin preparation of any one in embodiment 216-226, described preparation is used for the treatment of or prevents hyperglycemia to comprise stress induced hyperglycemia, type 2 diabetes mellitus, glucose tolerance attenuating, type 1 diabetes and burn, surgical wound and need Other diseases or damage, myocardial infarction, apoplexy, coronary heart disease and other cardiovascular disorder of anabolic action and treat critical diabetes and ND in treatment.

Further illustrate the present invention by following examples, it is restrictive that described embodiment should not regard as.

All lists of references that this paper quotes (comprising publication, patent application and patent) are attached to herein by reference and in full, just look like the identical limit that each list of references is single and specifically describe, its incorporated herein by reference and description in full (to greatest extent allowed by law) in this article.

All titles used herein and subtitle be only for convenient, and should not regard as and limit by any way the present invention.

Unless in addition requirement, the use of any and all embodiment provided herein or exemplary language (for example, " such as ") only is intended to describe better the present invention, and do not cause and limit the scope of the invention.In description, do not have language should regard the key element of explanation necessary any undesired protection for practice of the present invention as.

Only quoting and mix for convenient of patent document, and do not reflect effectiveness, the patentability and/or anyways enforceable of these patent documents.

Present invention resides in all modifications and the equivalent of the theme of quoting in claims, in the allowed band of applicable law.

Summary of drawings

During Fig. 1 is presented at preparation of the present invention stores 2 weeks under 37 ℃, the development of total insulin content percentage ratio of catabolite.Letter arefer to NovoRapid ^?reference, the remaining word mother is corresponding to the insulin aspart preparation of describing in the table 1 at embodiment 1.With NovoRapid ^?preparation (preparation a) compare, add nicotiamide (preparation bwith d) cause improving the formation catabolite, and combination adds nicotiamide, glutamic acid and arginine (preparation cwith e), there is the mode of almost similarly degrading, the low HMWP that forms.

During Fig. 2 is presented at preparation of the present invention stores 2 weeks under 37 ℃, the development of total insulin content percentage ratio of catabolite.Letter arefer to NovoRapid ^?reference, the remaining word mother is corresponding to the insulin aspart preparation of describing in the table 1 at embodiment 1.Combination adds nicotiamide, glutamic acid and arginine, preparation f, g, hwith i, concentration (0.6mM and 0.3mM or 1.2mM and the 0.6mM) difference of buffer system (phosphate or tris buffer agent) and insulin and Zn, have and NovoRapid ^?preparation (preparation a) mode of similarly degrading.

After Fig. 3 is presented at 0 minute preparation of the present invention to Corii Sus domestica hemostasis 1nmol/kg dosage, and concentration of glucose in blood plasma (mean+/-SEM, N=8).Letter arefer to NovoRapid ^?reference, the remaining word mother is corresponding to the insulin aspart preparation of describing in the table 1 at embodiment 1.With NovoRapid ^?preparation (preparation a) compare, for the preparation (preparation that adds nicotiamide n), the initial rate that plasma glucose reduces is very fast, for nicotiamide and arginic combination (preparation m), the initial rate that plasma glucose reduces is even faster.

After Fig. 4 is presented at 0 minute preparation of the present invention to Corii Sus domestica hemostasis 1nmol/kg dosage, and concentration of glucose in blood plasma (mean+/-SEM, N=7).Letter arefer to NovoRapid ^?reference, the remaining word mother is corresponding to the insulin aspart preparation of describing in the table 1 at embodiment 1.With NovoRapid ^?preparation (preparation a) compare, for the preparation (preparation of the combination with nicotiamide, arginine and glutamic acid l) and there is the preparation (preparation of nicotiamide and arginic combination k), the initial rate that plasma glucose reduces is very fast.

Fig. 5 shows that 0 minute after the preparation of the present invention to Corii Sus domestica hemostasis 1 nmol/kg dosage, and insulin aspart concentration in blood plasma (mean+/-SEM, N=7).Letter arefer to NovoRapid ^?reference, the remaining word mother is corresponding to the insulin aspart preparation of describing in the table 1 at embodiment 1.With NovoRapid ^?preparation (preparation a) compare, there is the preparation (preparation of nicotiamide j), there is the preparation (preparation of nicotiamide and arginic combination k) and there is the preparation (preparation of the combination of nicotiamide, arginine and glutamic acid l) the initial absorption speed of insulin component significantly faster.

After Fig. 6 is presented at 0 minute preparation of the present invention to Corii Sus domestica hemostasis 1nmol/kg dosage, concentration of glucose in blood plasma (every pig is administered twice for mean+/-SEM, N=8).Letter arefer to NovoRapid ^?reference, numeral 11corresponding to the insulin aspart preparation as described in the table 3 of embodiment 1.With NovoRapid ^?preparation (preparation a) compare, for the preparation (preparation with nicotiamide and arginic combination 11), the initial rate that plasma glucose reduces is very fast.

After Fig. 7 is presented at 0 minute preparation of the present invention to Corii Sus domestica hemostasis 1 nmol/kg dosage, insulin aspart concentration in blood plasma (every pig is administered twice for mean+/-SEM, N=8).Letter arefer to NovoRapid ^?reference, numeral 11corresponding to the insulin aspart preparation as described in the table 3 of embodiment 1.With NovoRapid ^?preparation (preparation a) compare, there is nicotiamide and arginic preparation (preparation 11) the initial absorption speed of insulin component significantly faster.

Embodiment

Embodiment 1

The preparation of pharmaceutical preparation

Pharmaceutical preparation of the present invention can be used as the aqueous solution preparation.Such as using sodium chloride or glycerol, aqueous medium is made etc. and to be oozed.In addition, aqueous medium can contain zinc ion (for example adding as zinc acetate or zinc chloride), buffer agent and antiseptic.Arginine can be used as Arg, and HCl adds.The pH value of preparation is adjusted to the value of expectation, and can be at about 3-approximately between 8.5, at about 3-, approximately between 5 or about 6.5-approximately 7.5, depends on isoelectric point, IP, the pI of the insulin of discussion.

The composition of table 1. insulin preparation of the present invention

The composition of table 2. other insulin preparation of the present invention

The composition of table 3. other insulin preparation of the present invention

The composition of table 4. other insulin preparation of the present invention

。

Embodiment 2

The analysis of insulin chemical stability

Size exclusion chromatography (SEC)

At Waters insulin (300 * 7.8mm, dash number wat 201549) the upper quantitative assay of implementing high molecular weight protein (HMWP) and monomer insulin aspart, eluent contains 2.5M acetic acid, 4mM L-arginine and 20 % (V/V) acetonitrile, flow velocity is 1ml/ minute, under 40 ℃.Use adjustable absorption photometric detector (Waters 486) examinations under 276nm.Volume injected is 40 μ l and 600 μ M insulin human reference materials.Measure HMWP and the concentration of preparation at each sample point.

Reverse-phase chromatography (UPLC)

Use BEH RP C8 2.1 * 100mm post, implement the mensuration of the impurity that insulin aspart is relevant on the UPLC system, particle diameter is 1.7 μ m, Waters dash number 186002878, and flow velocity is 0.5ml/ minute, under 40 ℃, detects, under 220nm.Use is implemented eluting by the following mobile phase formed:

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

B. 70 % (w/V) acetonitrile.Gradient: 0-11 minute, used 73%/27% the permanent solvent system of A/B; 11-12 minute, linearity becomes 52%/48% A/B; 13-15 minute, linearity becomes 73%/27% A/B; 15-20 minute, the permanent solvent system of the A/B gradient with 73%/27%.

Measure the absorbance area with percentage ratio measurement of the amount of the different aspartic acid of B28, impurity that deacylated tRNA amine is relevant with other as the total absorbance area of measuring the eluting antiseptic after.The RP-UPLC method is equal to the analytical method of the insulin aspart medicine of selling for quality control Novo Nordisk.

Add arginine to reduce amount, especially HMWP and the deacylated tRNA amine form of the catabolite formed, improve arginic concentration in the 10-50mM scope, cause further reducing degraded.When adding arginine, in ThT measures, as the physical stability recorded time delay, reduce, and, when arginine concentration improves, physical stability significantly reduces.About forming the reduction of catabolite, the arginic overall performance of 50mM is superior to 50 mM glycine, 50mM glutamic acid or 50mM histidine, as shown in table 4 below.

Insulin preparation of the present invention provides Insulin Aspart, and it is not only stable physically, and unexpectedly also chemically stable.

The physics and chemistry stability data of the insulin preparation 1-9 of table 5. table 2

The chemical stability data of the insulin preparation 10-13 of table 6. table 3

The physics and chemistry stability data of the insulin preparation 14-20 of table 7. table 4

Embodiment 3

Pharmacokinetics in the LYD pig model (PK)/pharmacodynamics (PD) research and plasma analysis are measured

PK/PD research in the LYD pig

The domestic female pigs of the LYD crossbreed to body weight between 55-110kg is implemented PK/PD research.Before beginning one's study, by ear vein, pig is inserted to conduit at least 2 days in jugular vein.Last a meal before beginning one's study is before to animal ejection testing preparation approximately 18 hours, during fasting and the institute of test period free, allow animal freely approach water.

When 0 hours, the subcutaneous test formulation that gives in the side of neck.Get blood sample before administration, and after administration with conventional interval from the extraction with duct sample, and sample introduction is to the 1.5ml glass tubing of prior coating heparin.Blood sample is remained in frozen water, until, by centrifugal by separating plasma 10 minutes with 3000rpm under 4 ℃, carried out in first 30 minutes.Plasma sample is stored under 4 ℃ to the short time (2-3 hour) or-18 ℃ of lower long term storages, and analyze glucose and analyze insulin aspart concentration by LOCI on YSI or Konelab 30i.

For the quantitative luminescent oxygen passage immunoassay (LOCI) of insulin aspart

Insulin aspart LOCI is the sandwich immunoassay based on monoclonal antibody, and use two kinds of beadlet (being coated with the receptor beadlet of europium and the donor-beadlet of painting Streptavidin) near.Receptor beadlet coating needle is to the specific antibody of insulin human and be identified in the insulin aspart in plasma sample.Together with the beadlet that the second biotinylated antibody is coated with Streptavidin, with the insulin aspart specific binding, their form sandwich.Irradiate beadlet-aggregation-immune complex and discharge singlet oxygen from the donor beadlet, it enters the receptor beadlet and triggers chemiluminescence through passage.Measure chemiluminescence, and the concentration of the amount of the light produced and insulin aspart is proportional.

With product sold NovoRapid ^?compare the initial rate that the plasma glucose of preparation of the present invention reduces very fast (Fig. 3 and 4).Equally, when with NovoRapid ^?in the time of relatively, the initial absorption speed of the insulin component of preparation of the present invention is fast (Fig. 5) significantly more.

Embodiment 4

The generality introduction of measuring for the ThT fibrillation of the physical stability of assess proteins preparation

The low physical stability of peptide can cause the amyloid fibril to form, and it is observed in sample is abundant orderly line-sample macromolecular structure, finally causes gel formation.This measures by the visual inspection sample traditionally.Yet this kind of measurement is very subjective, and depends on the observer.Therefore, using micromolecule indicator probe has more advantages.Thioflavine T (ThT) is for such probe and when have distinct fluorescent characteristics [people (1989) the Anal. Biochem. such as Naiki when fibril is combined 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284].The time course that fibril forms can be used following formula [people (2001) Biochemistry such as Nielsen 40, 6036-6046], by sigmoid curve, describe:

equation (1)

Herein, F is the ThT fluorescence when time t.Constant t ₀for reaching 50% required time of maximum fluorescence.Two important parameters of fibril formation are described for passing through t ₀delay-time that-2 τ calculate, the apparent speed constant k _app=1/ τ.

Propose to form the general triggering mechanism of the partially folded intermediate of peptide as fibrillation.In those intermediate seldom nucleation to form the template can assemble other intermediate thereon and to carry out fibrillation.Delay-the time, the apparent speed constant was for forming the speed of fibril itself corresponding to the interval of the critical mass of wherein accumulating core.

Sample preparation

The standby sample of new system before each mensuration.Each sample composition is described in each embodiment.Use appropriate concentrated NaOH and HClO ₄or HCl, the value by the pH regulator of sample to expectation.At H ₂in O, thioflavine T is joined to the sample from liquid storage, to ultimate density be 1 μ M.

The sample aliquot of 200 μ l is placed on to 96 hole microtitration plate (Packard OptiPlate ^tM-96, white polystyrene) in.Usually, for each sample, 4 or 8 copy (corresponding to a test condition) are placed in the string hole.By Scotch Pad (Qiagen) sealing for plate.

Hatch and fluorescence measurement

In Fluoroskan Ascent FL fluorescent screen reader or Varioskan plate reader (Thermo Labsystems), carry out under fixed temperature, hatching, vibrate and measure the ThT fluorescence radiation.By thermoregulation to 37 ℃.In all data that present, use the 1mm amplitude that track vibration is adjusted to 960rpm.Use excites by the 444nm light filter and passes through the 485nm light filter and measure emission, carries out fluorescence measurement.

By plate is hatched 10 minutes at the mensuration temperature, start every operation of taking turns.For the time phase of expectation, within every 20 minutes, measure plate.Between each the measurement, as described in by panel vibration and heating.

Date processing

Measurement point is stored in Microsoft Excel form, for further processing and curve plotting, and uses GraphPad Prism to implement matching.Can ignore there not being the background emission from ThT under fibril.Data point is generally the meansigma methods of 4 or 8 samples, and shows with reference material offset error bar.Only appear at the data (that is, the sample on same plate) that obtain in same experiment in same figure, guarantee the relative measurement of the fibrillation between each experiment.

Data set can with equation (1) matching.Yet, owing to always not realizing complete sigmoid curve during Measuring Time, by ThT fluorescence curve visual determination, be different from the time point of background level time delay as ThT fluorescence.

The measurement of initial and ultimate density

Be applied to ThT fibrillation measure before (" initially ") and complete ThT fibrillation after (" ThT measures afterwards ") the two, measure the peptide concentration of the preparation of each test.Use the Pramlintide reference material as a reference, by Reversed phase HPLC method, measure concentration.After completing, before measurement, for each copy, collect 150 μ l, and be transferred to the Eppendorf pipe.By these under 30000 G centrifugal 40 minutes.Supernatant is filtered by 0.22 μ m filter, be applied to subsequently the HPLC system.

Claims

1. an insulin preparation, described preparation comprises:

Insulin compounds,

The nicotine compound, and

Arginine.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

E. phosphate buffer;

The pH of wherein said preparation is approximately 7.1.

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

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

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

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