Insulin aspart composition (embodiment)

Abstract

The present invention relates to the field of pharmacy and medicine, i.e. To compositions of quick-acting, injectable insulin formulations. More specifically, the present invention relates to a composition comprising insulin aspart, a nicotinic acid compound, a surfactant selected from poloxamer 188, polysorbate 20, polysorbate 80, or a combination thereof, and an amino acid selected from lysine, arginine, and / or a salt thereof, and / or a combination thereof. The compositions of the present invention are useful for reducing blood glucose levels in subjects, particularly suitable for insulin formulations requiring high stability against thermal and / or physical and mechanical stresses.

Description

Technical Field

[0001] The present invention relates to the fields of pharmacy and medicine, specifically to compositions of fast-acting injectable insulin preparations for lowering blood glucose levels in a subject. More specifically, the present invention relates to compositions comprising insulin aspart, wherein the compositions have excellent chemical and physical stability, particularly when exposed to high temperatures and / or high mechanical energy. Background Art

[0002] The stability of pharmaceutical preparations (PP) is crucial to ensuring therapeutic efficacy and the absence of new adverse reactions and should be maintained throughout the PP's shelf life and during use. Stable PP compositions are particularly important when used in delivery devices exposed to high temperatures and / or mechanical stress. For example, stable insulin compositions are essential for use in injection pens and continuous (pump-assisted) infusion systems. Furthermore, insulin compositions are manufactured, stored, and used in vials, which require the insulin composition to remain stable throughout its shelf life.

[0003] Injection pens are widely used by people with diabetes (so-called diabetics) because they enable them to independently and effectively manage their disease by precisely following their doctor's prescription and promptly injecting the desired dose of medication into the targeted area of ​​their body. These pens are very easy to operate, so users can set the desired insulin dose and subsequently inject the prescribed dose into the targeted injection site on their body without requiring any special medical skills and / or education. An injection pen typically consists of a cartridge containing a predetermined amount of insulin. The cartridge includes a plunger and a mechanism that advances the plunger within the cartridge, allowing medication to be administered. Injection pens can be reusable or disposable. In reusable pens, the user replaces the used cartridge and returns the pen's actuator screw to its original position. In disposable pens, the cartridge is not replaceable; after the cartridge's contents are depleted, the pen is discarded. When an insulin formulation is used in a cartridge or in a replacement cartridge, it should be stored outside the refrigerator until the cartridge is used up (usually within a period of at least 2 weeks) or until the manufacturer's recommended shelf life for the cartridge or replacement cartridge in use (i.e., 1 month) has expired. Because insulin injection pens are typically stored in clothing pockets at temperatures close to body temperature, the insulin composition used in such pens is exposed to physical and thermal stresses and therefore has limited stability.

[0004] For continuous infusion systems, the insulin solution administered by the infusion device is stored in a reservoir (e.g., a syringe, a synthetic polymer chamber, a metal container, etc.). The reservoir and a pumping mechanism operably connected thereto are attached to or implanted in the patient's body. Insulin is pumped from the reservoir subcutaneously, intravenously, or intraperitoneally via a small-diameter catheter; thus, the insulin composition is exposed to body temperature and motion, as well as forced pump-assisted motion, and thus to high thermomechanical stress.

[0005] Existing insulin compositions are primarily composed of monomeric insulin analogs, which do not provide adequate physicochemical stability in injection pens and continuous infusion systems. The main problem with insulin administration via injection pens and infusion systems is that insulin solutions tend to form aggregates, fibrils, or insulin precipitates over time. [1] Aggregates and precipitates can damage components of catheters and pumps, and in the case of injection pens, can damage needles, causing insulin to cease being delivered to the patient and resulting in poor blood sugar control.

[0006] Many factors affect the aggregation and precipitation of insulin in solution. The main factors are as follows:

[0007] (a) Storage conditions at elevated temperatures (e.g., 25-37°C) rather than normal (2-8°C) [2] ;

[0008] (b) Mechanical energy resulting from the motion of an object or a pump / actuator [3] ;

[0009] (c) Long-term interactions of insulin molecules with hydrophobic surfaces (e.g., air interfaces) and plastic or metal pump components [4 , 5] .

[0010] Insulin is a peptide hormone that regulates blood glucose levels in mammals by initiating a signaling cascade that, after binding to the insulin receptor, accelerates glucose uptake and glycogen production. When insulin secretion is impaired, the glucose concentration in the blood increases (chronic hyperglycemia), which is the main diagnostic marker of type 1 diabetes mellitus (T1DM). However, in cases where signaling through the insulin receptor is impaired, even if sufficient insulin hormone is produced, type 2 diabetes mellitus (T2DM) develops as a result of reduced sensitivity of tissues to the action of insulin (insulin resistance). Due to the ability of this hormone to lower blood glucose levels, the efficiency of insulin in the treatment of diabetes has been proven in medical use for decades. However, the need for new approaches to treat diabetes remains a real concern due to the increasing incidence of diabetes and the need to take into account individual patient characteristics and preferences. [6] .

[0011] The development of rapid-acting and ultra-rapid-acting insulin analogs with faster onset and shorter duration of action has been a major advance in the treatment of type 1 and type 2 diabetes. In neutral solutions, at pharmaceutical concentrations, insulin and insulin analogs exist as stable zinc-containing hexamers composed of three identical dimer units. The delay in insulin action is primarily due to the time required for the hexamer to dissociate into monomers and dimers that can be absorbed. [7 , 8]The first fast-acting insulins (such as insulin lispro and insulin aspart) were developed based on amino acid mutations in the human insulin sequence that resulted in reduced self-association of insulin oligomers. For example, in insulin aspart, replacing the proline amino acid at position B28 with aspartic acid reduced the molecule's tendency to form hexamers, which is observed in solutions of soluble human insulin. Accordingly, insulin aspart is absorbed more rapidly from subcutaneous adipose tissue than soluble human insulin and is widely used for postprandial blood glucose control in diabetic patients. [9] .

[0012] It is known that fast-acting insulin analogs in monomeric or dimer form have reduced physicochemical stability compared to hexamer

[10] Insulin is a type of insulin that is produced by the action of a polymer or an adjuvant. ...

[0013] Insulin aspart preparations are commercially available (e.g., (Novo Nordisk A / S), (GEROPHARM OOO)). It has been shown that by supplementing the formulation with the adjuvant nicotinamide, which accelerates the absorption of insulin aspart compared to insulin aspart formulations lacking nicotinamide, overall glycemic control can be improved. Specifically, Fiasp (which contains nicotinamide) was shown to provide improved overall glycemic control and better postprandial glycemic control compared to NovoRapid (which lacks nicotinamide), without increasing the overall risk of severe or confirmed hypoglycemia in patients with T1DM and T2DM. The pharmacokinetics of the fast-acting insulin aspart Fiasp better mimic the rapid endogenous secretion of insulin during meals, thereby providing better postprandial glycemic control compared to insulin aspart.

[11] .

[0014] Nicotinamide can accelerate insulin absorption (WO 91 / 09617), including insulin aspart absorption (WO / 9610417). However, it negatively impacts chemical stability by increasing impurity levels (RU 2533217). The incorporation of arginine improves the chemical stability of the Fiasp pharmaceutical formulation (PP), as evidenced by a reduction in dimer and polymer content, as well as deamidated insulin content, during storage (RU 2533217). FIASP is the closest prior art to the present invention.

[0015] A small 6-week study (n=37) of infusion system clogging and malfunction reported no cases of infusion system clogging with FIASP (25 subjects) or insulin aspart (12 subjects). However, unexplained hyperglycemia and early infusion set replacement were more common with FIASP than with insulin aspart.

[12] .

[0016] Patent RU2533217 discloses (see Example 2) that when included in a composition containing insulin aspart and nicotinamide, arginine reduces the formation of degradation products, in particular high molecular weight proteins and deamidated forms, however, at the same time, it reduces the physical stability measured as the lag time in the thioflavin T assay, and that the physical stability continues to decrease with increasing arginine concentration.

[0017] Our study of the fibrillation propensity of the Thioflavin T assay also showed that FIASP was more prone to fibrillation compared with NovoRapid.

[0018] Therefore, there is still a need for an insulin composition with enhanced stability when used in an injection pen or during continuous pump-assisted infusion (i.e., several days to several months). When used in a vial, the stability of the enhanced insulin composition would also be beneficial. The above-mentioned technical problems particularly relate to compositions of monomeric insulin analogs, including insulin aspart analogs having lower stability than the hexameric form of insulin, and also to compositions comprising nicotinic acid compounds, which can accelerate the absorption of insulin but affect its chemical stability. Summary of the Invention

[0019] We have unexpectedly discovered that incorporating a surfactant (e.g., poloxamer 188, polysorbate 20, polysorbate 80, or a combination thereof) and one or more amino acids (e.g., lysine, arginine, and / or salts thereof, and / or combinations thereof) into a composition comprising insulin aspart and nicotinamide improves the chemical and physical stability of insulin aspart compared to the closest prior art.

[0020] The present invention relates to a composition for lowering blood sugar level of a subject, comprising: insulin aspart (B28Asp), a nicotinic acid compound, one or more amino acids selected from lysine, arginine or salts thereof, and a surfactant.

[0021] In a preferred embodiment, the present invention relates to a composition for lowering blood glucose levels in a subject, the composition comprising: insulin aspart, a niacin compound, an amino acid selected from lysine, arginine and / or salts thereof and / or combinations thereof, and a surfactant selected from poloxamer 188, polysorbate 20, polysorbate 80 and / or combinations thereof.

[0022] The concentration of insulin aspart in the composition of the present invention ranges from about 0.2 mM to about 2.0 mM (about 33 U / ml to about 333 U / ml), preferably from about 0.3 mM to about 1.2 mM (about 50 U / ml to about 200 U / ml), and most preferably is 0.6 mM (100 U / ml).

[0023] In one embodiment of the present invention, the pH of the composition is from 6.5 to 8.5, preferably from 6.6 to 7.4, even more preferably at a pH of 7.

[0024] The compositions of the present invention comprise a niacin compound selected from nicotinamide (nicotinamide), nicotinic acid (nicotinic acid) and / or salts thereof and / or any combination thereof. The concentration of niacinamide or other niacin compound in the compositions of the present invention is from about 1 mM to about 200 mM. The preferred niacin compound is niacinamide.

[0025] The concentration of lysine and / or its salt, the concentration of arginine and / or its salt, or the total concentration of the combination of lysine and / or its salt and arginine and / or its salt ranges from about 1 mM to about 100 mM.

[0026] In the composition comprising a combination of lysine and / or its salt and arginine and / or its salt, the molar ratio of the amino acids is 4000:1 to 1:4000, respectively, and the range includes every integer ratio, such as 100:1, 99:1, 98:1, etc. In a preferred embodiment, the molar ratio of lysine:arginine is 1:1, 4:5, 5:4, 6:4; 5:2 or 2:5.

[0027] The composition of the present invention may further comprise protease inhibitors, metal ions, buffer systems, pH adjusters, preservatives, isotonicity agents, chelating agents, stabilizers and surfactants.

[0028] In one embodiment, the compositions of the invention are aqueous compositions, ie they comprise water and are present as a solution or suspension.

[0029] The composition of the present invention can be used to treat or prevent hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetes.

[0030] In a preferred embodiment, the composition of the present invention is administered parenterally. Parenteral administration can be performed subcutaneously, intramuscularly, intraperitoneally, or intravenously using a syringe, which can be an injection pen. Alternatively, parenteral administration can be performed using an infusion pump.

[0031] Therefore, the present invention also relates to an injectable insulin preparation comprising the composition of the present invention.

[0032] The compositions of the present invention exhibit improved chemical and physical stability compared to the closest prior art, which is advantageous to the consumer, particularly when using an injection pen or insulin pump.

[0033] Terms and Definitions

[0034] As used herein, the term "human insulin" refers to the human hormone with well-known structure and properties. Insulin is a 51-amino acid polypeptide divided into two amino acid chains: chain A, consisting of 21 amino acids, and chain B, consisting of 30 amino acids. These chains are interconnected by two disulfide bonds. For many years, insulin pharmaceutical preparations have been used to treat diabetes, and more recently, not only native insulin but also insulin derivatives and analogs have been used.

[0035] As used herein, the term "insulin analogue" refers to a polypeptide derived from the primary structure of native insulin (e.g., human insulin) by mutation. One or more mutations are obtained by deleting and / or replacing at least one amino acid residue present in native insulin and / or adding at least one amino acid residue thereto. Mutations in the insulin molecule are represented by the corresponding chain reference (A or B), the corresponding position number, and the three-letter code for the amino acid that replaces the native amino acid.

[0036] The term "rapid-acting insulin" or "short-acting insulin" refers to insulin analogs and / or insulin derivatives that have an onset of action within 5-15 minutes and a duration of action of 3-4 hours. Examples of rapid-acting insulins include, but are not limited to, insulin aspart, insulin lispro, and insulin glulisine.

[0037] The terms "monomeric human insulin analogs" and "monomeric insulin analogs" are well known in the art and generally refer to rapid-acting human insulin analogs. For example, they include insulin lispro Insulin aspart and insulin glulisine The molecular structure of insulin lispro is identical to that of human insulin, except for positions 28 and 29 in the B chain of the molecule, where the lysine and proline are arranged in reverse order (human insulin: B28ProB29Lys, lispro: B28LysB29Pro). The reversed lysine / proline arrangement makes the dissociation of the insulin lispro molecule two times faster. In insulin aspart (B28Asp), the proline amino acid residue at position 28 of the B chain is replaced by aspartic acid, reducing the tendency of the molecule to form hexamers observed in human insulin solutions. In the insulin glulisine structure (3BLys29BGlu), the asparagine amino acid residue at position 3 of the B chain of the human insulin molecule is replaced by lysine, and lysine 29 of the B chain is replaced by glutamine, which contributes to the stability of the formulation in solution as both monomers and dimers.

[0038] In an injectable formulation, the content of human insulin B28Asp in the solution may range from about 0.2 mM to about 2.0 mM (about 33 to about 333 international units (IU) / ml), preferably from about 0.3 mM to about 1.2 mM (about 50 to about 200 IU / ml), with a most preferred concentration of 0.6 mM (100 U / ml). However, for parenteral administration for other purposes, the content of the insulin compound may be higher.

[0039] In this case, the IU unit corresponds to 6 nmol.

[0040] As used herein, the term "insulin aspart composition" refers to a product comprising: insulin aspart; a nicotinic acid compound selected from nicotinamide (nicotinamide), nicotinic acid (nicotinic acid) and / or their salts and / or any combination thereof; an amino acid selected from lysine, arginine and / or their salts and / or their combinations; and a surfactant selected from poloxamer 188, polysorbate 20, polysorbate 80, or their combinations. The product may further comprise other excipients, such as preservatives, chelating agents, isotonicity agents, fillers, stabilizers, antioxidants, polymers and surfactants other than those mentioned above, metal ions, oil carriers, and proteins (e.g., human serum albumin, gelatin, or proteins), and pH regulators, wherein the insulin aspart composition can be used to treat, prevent, or alleviate the severity of a disease or condition by administering the composition to a human. Therefore, the composition of the present invention may be referred to as a "pharmaceutical composition" or "pharmaceutical preparation."

[0041] The terms "protein preparation," "protein composition," and "protein" are used interchangeably herein to refer to any pharmaceutical agent comprising, as an active substance, a polypeptide chain molecule consisting of amino acid residues linearly linked by peptide bonds, or a combination of such a polypeptide chain molecule and other compounds. For example, an insulin composition or insulin is a special case and is encompassed by these terms.

[0042] The terms "lysine" and "arginine" refer to amino acids, including D- and L-enantiomers and mixtures thereof. The term also includes any pharmacologically acceptable salts of lysine and arginine. Lysine and arginine readily form salts, such as hydrochloride.

[0043] As used herein, the term "surfactant" (surface active compound, SAC) refers to any molecule or ion consisting of a water-soluble (hydrophilic) portion, a head, and a fat-soluble (lipophilic) portion. Surfactants preferably aggregate at interfaces, with the hydrophilic portion facing the water (hydrophilic phase) and the lipophilic portion facing the oil or hydrophobic phase (i.e., glass, air, oil, etc.). Surfactants reduce the surface tension of liquids. They are also referred to as amphiphilic compounds. The term "detergent" is often synonymous with the term "surfactant." Those skilled in the art are familiar with the use of surfactants in pharmaceutical formulations. Such surfactants include, for example, polysorbate 20, polysorbate 80, poloxamer 188, etc.; these substances are described as excipients in current pharmacopoeias (USP, EP).

[0044] The term "niacin compound" includes niacinamide (nicotinamide), niacin (nicotinic acid) and / or salts thereof and / or any combination thereof.

[0045] The buffer may be selected from the group consisting of, but not limited to, sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, and tris(hydroxymethyl)aminomethane, bis(hydroxyethyl)glycine, tris(hydroxymethyl)glycine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers and each combination thereof constitutes an alternative embodiment of the present invention.

[0046] The composition of the present invention may also contain other ingredients commonly used in insulin preparations, such as zinc complexing agents.

[0047] Isotonic agents, such as glycerol, mannitol, sodium chloride, glucose, etc., may also be present in the composition of the present invention. An isotonic agent is a physiologically acceptable compound that imparts appropriate tonicity to the composition and prevents water from diffusing across cell membranes in contact with the pharmaceutical composition.

[0048] The composition of the present invention may contain a pharmaceutically acceptable preservative. The preservative present in the insulin preparation of the present invention may be phenol, m-cresol, methylparaben, etc.

[0049] The composition of the present invention may further comprise a chelating agent. The use of chelating agents in pharmaceutical formulations is well known to those skilled in the art.

[0050] The composition of the present invention may further comprise a stabilizer. As used herein, the term "stabilizer" refers to a chemical substance added to a pharmaceutical formulation comprising a polypeptide to stabilize the polypeptide, ie, to extend the shelf life and / or usage period of such formulation.

[0051] As used herein, the term "stability" refers to the chemical and physical stability of a composition containing a monomeric insulin analog.

[0052] Physical instability of protein compositions (protein preparations) and / or proteins may be due to the aggregation of protein molecules, which leads to the formation of higher-order polymers or even precipitates. The physical denaturation of insulin is called fibrillation. In the fibril state, the elongated peptide chains are arranged in a parallel or antiparallel orientation and are interconnected by hydrogen bonds, forming so-called β-structures or β-folds. Fibrils generally represent the lowest energy state of a protein; regeneration of the protein from this state to its native, properly folded state is only possible in a strongly alkaline environment. Insulin fibrils are in the form of gels or precipitates. Exposure to thermomechanical stress and / or interactions with phase boundaries and hydrophobic surfaces are factors that promote accelerated fibril formation. Fibrillation is believed to be caused by insulin monomerization. Compared to human insulin, monomeric insulin analogs that easily dissociate from hexameric units into monomeric form are more likely to form fibrils.

[0053] Physical stability can be assessed by methods known in the art, such as by measuring turbidity (optical density). Turbidity is caused by aggregation or precipitation of proteins or complexes in the composition.

[0054] The physical stability of aqueous protein formulations can also be assessed using spectroscopic reagents or probes of protein conformational states. The probes are preferably small molecules that primarily bind to non-native protein conformers. An example of a small molecule probe for spectroscopic protein structure determination is Thioflavin T. Thioflavin T is a fluorescent dye widely used to detect amyloid fibrils. In the presence of fibrils and possibly some other protein conformations, Thioflavin T, upon binding to the fibril form of the protein, produces a new excitation maximum at approximately 445 nm and enhances emission at approximately 485 nm. Unbound Thioflavin T is essentially non-fluorescent at these wavelengths.

[0055] As used herein, the term "chemical stability" in relation to protein compositions (protein formulations) and / or proteins refers to changes in the covalent structure of a protein that result in the formation of chemical degradation products that have potentially lower biological activity and / or potentially enhanced immunogenicity compared to the native protein structure. Depending on the type and nature of the native protein and the environmental factors to which the protein is exposed, a variety of chemical degradation products may be formed. An increase in chemical degradation products is typically observed during storage and use of protein formulations. Most proteins are susceptible to deamidation; other degradation pathways involve the formation of high molecular weight products in which two or more protein molecules are covalently bound to each other by transamidation and / or disulfide bonds, producing covalently linked dimeric, oligomeric and polymeric degradation products

[13] . Another chemical degradation variant worth mentioning is oxidation (e.g. oxidation of methionine residues). The chemical stability of a protein formulation and / or protein can be assessed by measuring the amount of chemical degradation products at different time points after exposure to various environmental conditions (e.g., by increasing the temperature, which generally accelerates the formation of degradation products). The amount of each degradation product is typically determined by separating the degradation products according to their molecular size and / or charge using various chromatographic techniques (e.g., size exclusion liquid chromatography (SEC-HPLC) and / or reverse phase high performance liquid chromatography (RP-HPLC)). Since high molecular weight protein products are potentially immunogenic and biologically inactive, HMWP levels must be kept low.

[0056] A "stable composition" is one in which the extent of protein aggregation and the amount of impurities are within normal limits and do not exceed these limits over time.

[0057] In one embodiment, the composition of the present invention is an aqueous composition, ie a composition comprising water and present in the form of a solution or suspension.

[0058] The term "aqueous composition" refers to a composition comprising water. The terms "aqueous solution" and "aqueous suspension" refer to a solution or suspension, respectively, comprising water. Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Example

[0059] Example 1. Preparation of Compositions 1-9.

[0060] In one embodiment, the pharmaceutical composition of the present invention is prepared as an aqueous solution. Table 1 shows the qualitative and quantitative contents of some prepared compositions. The compositions of the present invention include but are not limited to compositions 2, 3, 6-9 listed in Table 1.

[0061] The method of preparing the composition comprises:

[0062] a) preparing an insulin aspart solution by dissolving insulin aspart in water or a buffer;

[0063] b) preparing a zinc salt solution by dissolving the zinc salt in water or a buffer;

[0064] c) preparing a preservative solution by dissolving the preservative (phenolic compound) in water or a buffer;

[0065] d) preparing an isotonic agent solution by dissolving the isotonic agent in water or a buffer;

[0066] e) preparing a nicotinamide solution and / or other nicotinic acid compound solution by dissolving nicotinamide and / or other nicotinic acid compound in water or a buffer;

[0067] f) preparing a surfactant solution by dissolving the surfactant in water or a buffer;

[0068] g) preparing a lysine and / or arginine solution by dissolving an amino acid and / or its salt in water or a buffer;

[0069] h) mixing solution a) with solutions b), c), d), e), f), and g) according to the composition formulation given in Table 1;

[0070] i) adjusting the pH of the mixture h) to 6.6-7.4 and then sterilizing by filtration;

[0071] j) Aseptically fill the prepared composition into the cartridge.

[0072] The composition of the present invention may be obtained using any other method.

[0073] Table 1. Qualitative and quantitative contents of the claimed compositions

[0074]

[0075] Example 2. Physical and chemical stability analysis of the composition

[0076] The chemical stability of compositions 1-9 prepared as in Example 1 was determined after 1 and 2 weeks of thermostating in sterile filled cartridges at 40°C.

[0077] In the case of a 5μm particle size filled The impurities related to insulin aspart were determined on a column of octadecyl silica gel using HPLC with UV detection and the "quantitation" option at a wavelength of 214 nm and a mobile phase flow rate of 1.0 ml / min.

[0078] Elution was performed in gradient mode using a mobile phase consisting of:

[0079] Mobile phase A (MP A): 10% (w / V) acetonitrile, anhydrous sodium sulfate buffer solution pH 3.4 (1.4% (w / V), 0.13% (V / V) concentrated orthophosphoric acid, 2 M sodium hydroxide to pH 3.4 ± 0.05).

[0080] Mobile phase B (MP B): 50% (w / v) acetonitrile.

[0081] B28IsoAsp, deamidated forms (B3iso, A21Asp, B3Asp) and other related impurities were quantified by the absorbance areas of the corresponding peaks (determined as a percentage of the sum of the peak areas of the peaks eluting after niacinamide and preservatives) using an internal normalization method.

[0082] In the case of a 10 μm particle size High molecular weight proteins (HMWP) were quantified by size exclusion liquid chromatography on a hydrophilic silica gel column. A mixture of acetonitrile: glacial acetic acid: 0.1% arginine solution (20:15:65, v / v / v) was used as the eluent. Elution was performed at a flow rate of 0.5 ml / min and a detection wavelength of 276 nm.

[0083] The HMWP content was determined using internal normalization by summing the absorbance areas of all peaks with shorter retention times than the aspart monomer peak. Peaks with longer retention times than aspart monomer (i.e., nicotinamide and preservatives) were excluded from the calculation.

[0084] The physical stability of the protein compositions was assessed by testing their tendency to fibrillate in the Thioflavin-T (ThT) assay. The selected method is described in the literature (e.g., RU 2533217). Compositions 1-9 of Example 1 and the original Fiasp, a finished drug product, were tested for their tendency to fibrillate. Composition 1 was similar to the Fiasp composition. The compositions were tested immediately after preparation.

[0085] Solution preparation

[0086] Thioflavin stock solution

[0087] Dissolve an exact portion of the dried Thioflavin T reagent in methanol to a concentration of 1 mg / ml (corresponding to 3.14 mM). Vortex the solution thoroughly and store protected from light at +2-8°C for up to 6 months.

[0088] Thioflavin working solution

[0089] Prepare a 200 μM thioflavin working solution in purified water. Adjust the volume ratio of stock solution to purified water to 1:14.7.

[0090] Use the freshly prepared solution immediately.

[0091] Aliquot 100 μl of sample into three wells of a black 96-well plate and use purified water as a control. Add 10 μl of Thioflavin Working Solution to all wells. Do not use the edge wells when filling the plate (see Figure 1 The plate was sealed with a transparent film and placed in a CLARIOstar multimode plate reader (BMG, Germany). Fluorescence signals were measured every 20 minutes for 12 hours at wavelengths of 445 / 485 nm. Between readings, the plate was agitated at 700 rpm. Agitation and detection were performed at 37°C.

[0092] Based on the measurements, the signal for each sample was plotted in relative fluorescence units versus time (hours). A 4-parameter model was used for analysis.

[0093] This graph is used to determine T1 / 2max, the time required for half of the sample to aggregate. When the graph is a straight line at zero, the sample is considered non-aggregating; otherwise, the tendency to aggregate is measured by T1 / 2max.

[0094] Together with T1 / 2max, the ThT fluorescence curve is used to visually determine the lag time, that is, the time point at which ThT fluorescence differs from the background level.

[0095] Table 2. Physical and chemical stability data of insulin compositions 1-9 (Example 1) in Table 1

[0096]

[0097]

[0098] *Original Fiasp

[0099] **Not detected, fibrils missing

[0100] Based on chemical and physical stability studies of insulin aspart compositions prepared with phosphate buffer or Tris buffer, it was unexpectedly discovered that, compared to the prior art, the addition of lysine or arginine and / or their salts, and / or their combination with a surfactant (e.g., poloxamer 188, polysorbate 20, polysorbate 80, or a combination thereof), significantly and reliably reduced the amount of impurities contained in the claimed compositions (compositions 2, 3, 6-9) stored at 40°C for two weeks. Another fundamental advantage of the claimed compositions is their enhanced physical stability, i.e., their lack of tendency to fibrillate. The compositions of the present invention exhibit no fibrillation (ND), however, if the amino acid concentration in the composition exceeds 100 mM, their physical stability deteriorates and a tendency to fibrillate develops.

[0101] The physical stability of compositions 1-9 prepared by the method described in Example 1 was tested in an accelerated test. Three 3 / 16 inch (4.7625 mm) Teflon beads were placed in each 2 ml glass HPLC vial. The vials were filled with samples of the test composition. The sealed vials were continuously shaken at a frequency of 40 Hz (average linear acceleration 20 x g) with an amplitude of 12 mm at 37°C, thereby subjecting the produced compositions to relatively high thermal and mechanical stresses, conditions that are conducive to physical instability. The vials were placed on a vibrating mixer in such a way that their long dimension (from top to bottom) was parallel to the direction of linear acceleration, in other words, they were placed on their sides on the mixer surface. For other insulin compositions, the increase in stability under the accelerated conditions has been shown to be associated with a significant increase in the stability of the compositions during use. The turbidity (optical density at 450 nm) of the test samples and controls was measured regularly using a spectrophotometer. The control samples were prepared using the same method as the test samples, but stored at 2-8°C without shaking. The resulting optical density values ​​were calculated by subtracting the control sample optical density from the test sample optical density. Table 3 lists the average optical density values ​​and standard deviations for a number of samples.

[0102] Table 3. Effect of composition formulation and 37°C mechanical energy exposure time on turbidity (optical density at 450 nm)

[0103]

[0104] Under the experimental conditions described above, the turbidity of the resulting compositions (lacking either the combination of surfactant and amino acid (Composition 1) or the surfactant alone (Composition 5)) reached high levels, becoming unacceptable within 24 hours for Composition 1 and within 72 hours for Composition 5. Over the 336-hour experiment, the optical density of all compositions containing surfactant and amino acid was nearly identical to that of the control. Composition 4 also had an optical density nearly identical to that of the control, but this composition contained significantly higher levels of chemical degradation impurities than the samples containing the surfactant and amino acid combination.

[0105] Based on observations with other finished insulin products, it can be assumed that the unexpectedly significantly improved stability of insulin compositions containing a surfactant and amino acid combination observed in accelerated testing will persist for more than 336 hours in actual use because these compositions are subjected to greater stress in the accelerated testing performed than in use in injection pens and infusion systems.

[0106] References

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Claims

1. A composition for lowering blood glucose levels in a subject, the composition comprising: insulin aspart (B28Asp), a niacin compound, one or more amino acids selected from lysine, arginine, or a salt thereof; and a surfactant.

2. The composition according to claim 1, wherein the composition comprises one or more surfactants selected from the group consisting of poloxamer 188, polysorbate 20 and polysorbate 80.

3. The composition according to claim 1, wherein the content of insulin aspart (B28Asp) is about 0.2 mM (33 U / ml) to about 2 mM (333 U / ml).

4. The composition according to claim 1, wherein the insulin aspart is present in an amount of about 0.3 mM (50 U / ml) to about 1.2 mM (200 U / ml).

5. The composition according to claim 1, wherein the niacin compound is niacinamide.

6. The composition according to claim 1, wherein the composition comprises about 1 mM to about 200 mM nicotinamide.

7. The composition according to claim 1, wherein the composition comprises about 1 mM to about 100 mM lysine or a salt thereof.

8. The composition according to claim 1, wherein the composition comprises about 1 mM to about 100 mM arginine or a salt thereof.

9. The composition according to claim 1, wherein the composition comprises about 1 mM to about 100 mM of a combination of arginine or a salt thereof and lysine or a salt thereof.

10. The composition according to claim 1, wherein the composition comprises one or more surfactants selected from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80 at a concentration of about 0.001 to 0.05% w / v.

11. The composition according to claim 1, wherein the composition further comprises metal ions, preservatives, isotonicity agents and stabilizers, buffers and acidity regulators.

12. The composition according to any one of claims 1 to 11, for use in the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetes.

13. An injectable insulin preparation for lowering blood glucose levels in a subject, the preparation comprising a composition according to any one of claims 1 to 11.

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