Quick-acting insulin (variant) compositions

Abstract

The invention relates to the field of pharmaceutical science and medicine, in particular to a composition of a quick-acting insulin preparation. More specifically, the present invention relates to a composition comprising quick-acting insulin and an amino acid as a stabilizer wherein the amino acid may be selected from lysine and / or a salt thereof, or a combination of lysine and / or a salt thereof and arginine and / or a salt thereof. Compared with the closest prior art, the composition disclosed by the invention shows better chemical and physical stability.

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceutical science and medicine, and specifically to compositions of fast-acting insulin preparations. More specifically, the present invention relates to compositions comprising a fast-acting insulin and an amino acid as a stabilizer, wherein the amino acid can be selected from lysine and / or a salt thereof, or a combination of lysine and / or a salt thereof and arginine and / or a salt thereof. Compared to the closest prior art, the compositions of the present invention exhibit improved chemical and physical stability. Background Art

[0002] Insulin is a peptide hormone that regulates blood glucose levels in mammals by binding to the insulin receptor and initiating a signaling cascade that 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, type 2 diabetes mellitus (T2DM) develops even when sufficient insulin hormone is produced, as a result of reduced sensitivity of tissues to the action of insulin (insulin resistance). The efficiency of insulin in the treatment of diabetes is due to the ability of this hormone to lower blood glucose levels, which has been proven in medical use for decades. However, due to the increasing number of diabetes cases and the need to take into account individual patient characteristics and preferences, there remains a need for new approaches to treat diabetes. [1] .

[0003] Development of rapid-acting and ultra-rapid-acting insulin analogs, such as insulin aspart, with faster onset and shorter duration of action Insulin glulisine and insulin lispro It has become a major advance in the treatment of type 1 and type 2 diabetes. The formulation of is the closest prior art to the composition of the present invention.

[0004] In neutral solution, at pharmaceutical concentrations, insulin and insulin analogs exist as stable zinc-containing hexamers composed of three identical dimer units. The delay in insulin action depends primarily on the time required for the hexamer to dissociate into monomers and dimers that can be absorbed. [2 , 3]The first class of rapid-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 tendency of the molecule to form hexamers, which can be observed in solutions of soluble human insulin. Consequently, 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 [4]. The molecular structure of insulin lispro is identical to that of human insulin, except that at positions 28 and 29 in the B chain of the molecule, the order of lysine and proline is reversed (human insulin: B28ProB29Lys, lispro: B28LysB29Pro). The reversed order of lysine and proline makes the lispro molecule dissociate twice as fast. 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 the lysine residue at position 29 of the B chain is replaced by glutamine, which contributes to the stability of the pharmaceutical preparation in the form of monomers and dimers in solution. [1] .

[0005] The chemical and physical stability of insulin formulations is crucial to ensuring their therapeutic efficacy and the absence of adverse reactions; in this regard, the stability of the formulation must be maintained both during shelf life and during use. Insulin compositions are manufactured, stored, and used in vials, which requires them to maintain stability throughout their shelf life. Stable insulin compositions are particularly suitable for use in delivery devices such as injection pens and continuous infusion systems (pump-assisted), where they are exposed to high temperatures close to body temperature and / or mechanical stress.

[0006] It is known that fast-acting insulin analogs in monomeric or dimeric form have reduced physicochemical stability compared to hexamer [5] , for example, they form high molecular weight impurities; they also have a strong tendency to form aggregates (fibrillation), which manifest as turbidity and precipitation. High molecular weight impurities (dimers, trimers and polymers) and aggregates can reduce the dosage of insulin and also promote irritation or immune response in patients. In addition, insoluble aggregates can clog and damage needles, cannulas and pump tubing in continuous infusion systems. In order to ensure the quality of insulin compositions, it is necessary to improve their chemical and physical stability.

[0007] Pharmaceutical preparations of insulin aspart are commercially available (e.g., (Novo Nordisk A / S), (GEROPHARM OOO)). It has been shown that overall glycemic control can be improved by adding the excipient nicotinamide, which accelerates the absorption of insulin aspart compared to insulin aspart formulations lacking nicotinamide. Specifically, (Lack of niacinamide) compared to (containing nicotinamide) has been shown to provide improved overall glycemic control and better postprandial glycemic control without increasing the overall risk of severe or confirmed hypoglycemia in patients with T1DM and T2DM. The pharmacokinetics of the drug may better mimic the rapid endogenous secretion of insulin after a meal, thereby providing improved postprandial glycemic control. [6] .

[0008] Nicotinamide can accelerate the absorption of insulin (WO91 / 09617), including insulin aspart (WO / 9610417), but it has a negative impact on chemical stability and increases the content of impurities (RU2533217). By introducing arginine, the absorption of insulin can be improved. Chemical stability of drug product (DP) formulations. Arginine can reduce the content of dimers and polymers as well as the content of deamidated insulin during storage (RU2533217). DP formulation is another closest prior art to the present invention.

[0009] Patent RU2533217 discloses (see Example 2) that arginine added to a composition containing insulin aspart and nicotinamide reduces the amount of degradation products formed, 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.

[0010] A small 6-week study (n = 37) of infusion system clogging and malfunction reported no infusion system clogging with either FIASP DP (25 subjects) or insulin aspart (12 subjects). However, unexplained hyperglycemia and early infusion set replacement were more common with FIASP compared with insulin aspart. [7] .

[0011] Therefore, there remains a need for fast-acting insulin compositions with enhanced chemical stability while also having acceptable physical stability. The aforementioned technical problems are particularly relevant to fast-acting insulin compositions, such as insulin aspart, insulin lispro, or insulin glulisine, which are characterized by lower stability compared to hexameric forms of insulin, and compositions containing nicotinamide. Nicotinamide, while capable of accelerating insulin absorption, negatively affects the chemical stability of insulin. Summary of the Invention

[0012] The inventors have surprisingly found that fast-acting insulin compositions containing an amino acid as a stabilizer (the amino acid being lysine and / or a salt thereof, or a combination of lysine and / or a salt thereof and arginine and / or a salt thereof) have improved chemical and physical stability compared to insulin compositions without added amino acids and to insulin compositions containing arginine amino acid as a stabilizer.

[0013] Therefore, the present invention relates to a composition with improved chemical and physical stability comprising a fast-acting insulin and an amino acid, which is lysine or a salt thereof, or a combination of lysine or a salt thereof and arginine or a salt thereof.

[0014] The present invention relates to a composition for lowering blood sugar levels in a subject, the composition comprising fast-acting insulin and an amino acid, wherein the amino acid is lysine or a salt thereof, or a combination of lysine or a salt thereof and arginine or a salt thereof.

[0015] In a preferred embodiment, the insulin is insulin aspart, insulin lispro or insulin glulisine.

[0016] Insulin glulisine compositions optionally comprise methionine.

[0017] The concentration of fast-acting insulin in the compositions of the present invention is in the range of about 0.2 mM to about 2.0 mM (about 33 U / ml to about 333 U / ml), preferably about 0.3 mM to about 1.2 mM (about 50 U / ml to about 200 U / ml), and even more preferably 0.6 mM (100 U / ml).

[0018] In one embodiment of the present invention, the pH of the composition is between 6.5 and 8.5. In another embodiment, the pH of the composition is between 6.6 and 7.4. In one embodiment, the pH is 7, 7.3, 7.4 or 7.5.

[0019] The compositions of the present invention comprising insulin aspart optionally comprise a nicotinic acid compound selected from nicotinamide (niacinamide), nicotinic acid (nicotinic acid) and / or salts thereof and / or any combination thereof; preferably the nicotinic acid compound is nicotinamide. The concentration range of nicotinamide or other nicotinic acid compounds in the compositions of the present invention comprising insulin aspart is from about 1 mM to about 200 mM.

[0020] The concentration of lysine and / or its salt, or the total concentration of lysine or its salt and arginine or its salt ranges from about 1 mM to about 100 mM.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The composition of the present invention can be used to treat or prevent hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetes.

[0025] 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.

[0026] Therefore, the present invention also relates to an injectable insulin preparation comprising the composition of the present invention.

[0027] Terms and Definitions

[0028] As used herein, the term "human insulin" refers to a 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 bridges. Pharmaceutical insulin preparations have been used to treat diabetes for many years, and more recently, not only naturally derived insulin but also insulin derivatives and analogs have come into use.

[0029] 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 deletion and / or replacement of at least one amino acid residue occurring in native insulin and / or the addition of at least one amino acid residue thereto. Mutations in the insulin molecule are represented by the corresponding chain reference (A or B), position, and the three letter code for the amino acid that replaces the native amino acid.

[0030] The term "fast-acting insulin" or "short-acting insulin" refers to insulin analogs and / or insulin derivatives that begin to act within 5-15 minutes and continue to act for 3-4 hours. Examples of fast-acting insulins include, but are not limited to, insulin aspart, insulin lispro, and insulin glulisine.

[0031] The terms "monomeric human insulin analogs" and "monomeric insulin analogs" are well known in the art and generally refer to rapid human insulin analogs. For example, they include insulin lispro Insulin aspart and insulin glulisine

[0032] In injectable formulations, the fast-acting insulin content of the compositions of the present invention 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 U / ml), and most preferably 0.6 mM (100 U / ml). However, for parenteral administration for other purposes, the insulin compound content may be higher.

[0033] In this case, the IU unit corresponds to 6 nmol.

[0034] As used herein, the term "specimen insulin composition" refers to a product comprising a monomeric insulin analog and an amino acid selected from the combination of lysine and / or its salt or lysine and / or its salt and arginine and / or its salt. Preferably, the monomeric insulin analog in the composition is insulin aspart, insulin lispro or insulin glulisine. The insulin aspart composition of the present invention may optionally comprise a nicotinic acid compound selected from nicotinamide (nicotinamide), nicotinic acid (nicotinic acid) and / or its salt and / or any combination. The semi-specimen insulin composition may optionally further comprise other excipients, such as preservatives, chelating agents, isotonic agents, fillers, stabilizers, antioxidants, polymers and surfactants, metal ions, oil carriers and proteins (such as human serum albumin, gelatin or protein), pH adjusting agents, wherein the semi-specimen insulin composition may be used for treating, preventing or alleviating the severity of a disease or illness by administering the insulin preparation to the human body. Therefore, the semi-specimen insulin composition of the present invention may be designated as a "pharmaceutical composition".

[0035] The terms "protein formulation," "protein composition," and "protein" are used interchangeably herein to refer to any pharmaceutical product that contains, as the active substance, a molecule, either alone or in combination with other compounds, which molecule consists of a polypeptide chain composed of amino acid residues linearly linked by peptide bonds. For example, an insulin composition or insulin is a special case and is encompassed by these terms.

[0036] 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.

[0037] The term "niacin compound" includes niacinamide (nicotinamide), niacin (nicotinic acid) and / or salts thereof and / or any combination.

[0038] 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, N,N-bicine, tris(hydroxymethyl)methylglycine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers and each combination thereof is an alternative embodiment of the present invention.

[0039] The fast-acting insulin composition of the present invention may further comprise other ingredients commonly used in insulin preparations, such as a zinc complexing agent.

[0040] Isotonic agents, such as glycerol, mannitol, sodium chloride, glucose, etc., may also be present in the compositions 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.

[0041] The composition of the present invention may contain a pharmaceutically acceptable preservative, such as phenol, m-cresol, or methylparaben.

[0042] 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.

[0043] 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.

[0044] As used herein, the term "surfactant" (surface active compound, SAC) refers to any molecule or ion consisting of a water-soluble (hydrophilic) part, a head, and a fat-soluble (lipophilic) part. SACs are primarily concentrated at interfaces, with the hydrophilic part directed toward the water (hydrophilic phase) and the lipophilic part directed toward the oil or hydrophobic phase (i.e., glass, air, oil, etc.). SACs reduce the surface tension of liquids. They are also known as amphiphilic compounds. The term "detergent" is often synonymous with the term "surfactant." The use of SACs in pharmaceutical formulations is well known to those skilled in the art. Such SACs include, for example, polysorbate 20, polysorbate 80, poloxamer 188, etc.; these substances are described as excipients in current pharmacopoeias (USP, EP).

[0045] As used herein, the term "stability" refers to the chemical and physical stability of a composition containing a monomeric insulin analog.

[0046] As used herein, the term "chemical stability" in relation to protein compositions (protein formulations) and / or proteins relates to changes in the covalent structure of the protein, resulting 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 properties of the native protein, as well as 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, resulting in covalently linked dimeric, oligomeric and polymeric degradation products. [8] . 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 individual 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 inactive, the levels of high molecular weight proteins should be kept low.

[0047] Physical instability of protein compositions (protein preparations) and / or proteins may be caused by the aggregation of protein molecules to form higher-order aggregates or even precipitates. The physical denaturation of insulin is called fibrillation. In the fibrillar state, the elongated peptide chains are arranged in a parallel or antiparallel direction and connected to each other by hydrogen bonds, forming so-called β-structures or β-sheets. Fibrils generally represent the lowest energy state of the protein; only in a strongly alkaline environment can the protein regenerate from this state to its native properly folded state. 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 the acceleration of fibrillation. Fibrillation is believed to be caused by the monomerization of insulin. Compared with human insulin, monomeric insulin analogs that easily dissociate from the hexameric unit into the monomeric form are more likely to form fibrils.

[0048] Physical stability can be assessed by methods well known in the art, for example by measuring turbidity (optical density). Turbidity is caused by aggregation or precipitation of proteins or complexes in the composition.

[0049] 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 enhanced emission at approximately 485 nm. Unbound Thioflavin T is essentially non-fluorescent at these wavelengths.

[0050] A "stable composition" is one in which the extent of protein aggregation and the amount of impurities are within normal ranges and do not exceed these limits over time.

[0051] The term "aqueous preparation" refers to a preparation containing water. The terms "aqueous solution" and "aqueous suspension" refer to a solution or suspension containing water, respectively. Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Example

[0052] Example 1. Preparation of a composition of the present invention comprising insulin aspart

[0053] 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 4-17 listed in Table 1. The formulation of composition 1 corresponds to one of the closest prior art compositions, namely DP. Composition 2 has a formulation similar to Composition 1, except that it lacks L-arginine hydrochloride. Composition 3 has a formulation similar to Composition 1, except that it lacks niacinamide. Composition 4 has a formulation similar to Composition 3, except that it contains added L-lysine hydrochloride in place of L-arginine hydrochloride. Composition 5 has a formulation similar to Composition 3, except that it contains a combination of added L-lysine and L-arginine.

[0054] The method of preparing the composition comprises:

[0055] a) preparing an insulin aspart solution by dissolving insulin aspart in water or a buffer;

[0056] b) preparing a zinc salt solution by dissolving the zinc salt in water or a buffer;

[0057] c) preparing a preservative solution by dissolving the preservative (phenolic compound) in water or a buffer;

[0058] d) preparing an isotonic agent solution by dissolving the isotonic agent in water or a buffer;

[0059] e) preparing a niacinamide solution and / or other niacin compound solution by dissolving niacinamide and / or other niacin compound (if present in the composition) in water or a buffer;

[0060] f) preparing a lysine and / or arginine solution by dissolving an amino acid and / or a salt thereof in water or a buffer;

[0061] g) mixing solution a), solution b), solution c), d), e) (if present) and solution f) according to the composition formula given in Table 1;

[0062] h) adjusting the pH of the mixture g) to 6.6-7.4, followed by sterile filtration;

[0063] i) Aseptically fill the prepared composition into the cartridge.

[0064] The compositions of the present invention may be produced using any other method.

[0065] Table 1. Qualitative and quantitative composition of the claimed compositions comprising insulin aspart

[0066]

[0067]

[0068] Example 2. Physical and chemical stability analysis of the claimed composition comprising insulin aspart

[0069] The chemical stability of compositions 1-17 produced according to Example 1 was determined after sterile cartridges filled with the compositions were thermostated at 40°C for 1 and 2 weeks.

[0070] Using HPLC with UV detection, the Insulin aspart-related impurities were determined on an octadecyl silica gel column at a wavelength of 214 nm and a mobile phase flow rate of 1.0 ml / min.

[0071] Elution was performed in gradient mode using a mobile phase consisting of:

[0072] Mobile phase A (MP A): acetonitrile: pH 3.4 buffer solution (1.4% anhydrous sodium sulfate solution (w / V), 0.13% concentrated orthophosphoric acid solution (V / V), 2 M sodium hydroxide, to pH 3.4 ± 0.05) 10:90 (V / V);

[0073] Mobile phase B (MP B): acetonitrile: water 50:50 (V / V).

[0074] The amounts of B28IsoAsp, deamidated forms (B3iso, A21Asp, B3Asp) and other related impurities were determined using internal normalization by the absorbance areas of the corresponding peaks as a percentage of the sum of the peak areas of the peaks eluting after niacinamide and preservatives.

[0075] In the case of a 10 μm particle size High molecular weight protein (HMWP) levels were determined by size exclusion liquid chromatography using a hydrophilic silica gel column with a pore size of 1.5 mm. 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 in isocratic mode at a flow rate of 0.5 ml / min and detection at a wavelength of 276 nm.

[0076] 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 the aspart monomer (i.e., nicotinamide and preservatives) were excluded from the calculation.

[0077] The physical stability of the protein compositions was assessed by measuring their tendency to fibrillate using the Thioflavin-T (ThT) test. The selected method is described in literature, such as RU 2533217. The fibrillation tendency of the finished pharmaceutical products of Compositions 1, 3-17, and the original Fiasp-DP from Example 1 was determined. Composition 1 is similar to the Fiasp-DP formulation. The compositions were tested immediately after preparation.

[0078] Solution preparation

[0079] Thioflavin stock solution

[0080] 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.

[0081] Thioflavin working solution

[0082] Prepare a 200 μM thioflavin working solution in purified water. Adjust the volume ratio of stock solution to purified water to 1:14.7.

[0083] The solution is ready for use.

[0084] 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 monitoring were performed at 37°C.

[0085] Based on the measurements, for each sample, the signal in relative fluorescence units is plotted in hours. Analysis was performed using a 4-parameter model.

[0086] 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.

[0087] Together with T1 / 2max, the ThT fluorescence curve was used to visually determine the lag time, ie, the time point at which ThT fluorescence differed from the background level.

[0088] Table 2 shows the results of chemical and physical stability studies of compositions 1, 3-17 comprising insulin aspart.

[0089] Table 2. Physical and chemical stability data of compositions 1, 3-17 comprising insulin aspart prepared according to Example 1.

[0090]

[0091]

[0092] *Original DP: Fiasp; **N / A - “not analyzed”.

[0093] Based on chemical and physical stability studies on insulin aspart compositions prepared with phosphate buffer or Tris buffer, it was unexpectedly found that the addition of lysine and / or its salts, and / or a combination of lysine and / or its salts with arginine and / or its salts to the compositions of the present invention can reliably and significantly reduce impurities compared to the prototype after storage at 40°C for 2 weeks, while at the same time improving the physical stability relative to the prototype.

[0094] Nicotinamide, used in a composition without added amino acids (Composition 2), negatively impacted chemical stability during storage. Compared to the impurity levels in Composition 2, which lacked arginine, the arginine in Composition 1, similar to FIASP DP, prevented the chemical degradation that occurs after two weeks of storage at 40°C when HMWP and deamidated insulin decrease—degradation specifically due to the addition of nicotinamide. Replacing the arginine in Composition 1 with an equimolar amount of lysine (Composition 6) reliably reduced impurity levels; increasing the lysine concentration to 100 mM promoted a greater reduction in impurities. Lysine concentrations greater than 100 mM are undesirable due to reduced physical stability of the composition. The combination of lysine and arginine (Composition 10) exhibited the lowest impurity levels.

[0095] Similar results were obtained for the compositions prepared using Tris buffer (compositions 11-17), which showed better chemical stability than the corresponding compositions prepared using phosphate buffer.

[0096] For compositions lacking niacinamide (compositions 3-5), stabilization by lysine or a combination of lysine and arginine was superior to stabilization by arginine.

[0097] Example 3. Preparation of the claimed composition containing insulin glulisine

[0098] In one embodiment, the pharmaceutical composition of the present invention is prepared as an aqueous solution. Table 3 shows the qualitative and quantitative contents of some prepared compositions. The compositions of the present invention include compositions 2-7 listed in Table 3, but are not limited thereto. The formulation of composition 1 corresponds to the original DP, 100 U / ml subcutaneous solution. Compositions 2-7 were similar to the original DP, except that they contained added L-lysine hydrochloride or a combination of L-lysine hydrochloride and L-arginine hydrochloride.

[0099] The method for preparing the composition comprises:

[0100] a) preparing an insulin glulisine solution by dissolving insulin glulisine in water or a buffer;

[0101] b) preparing a preservative solution by dissolving the preservative in water or a buffer;

[0102] c) preparing an isotonic agent solution by dissolving the isotonic agent in water or a buffer;

[0103] d) preparing a surfactant solution by dissolving the surfactant in water or a buffer;

[0104] e) preparing a solution of lysine and / or a combination of lysine and arginine by dissolving the amino acids and / or their salts in water or a buffer;

[0105] f) mixing solution a) with solutions b), c), d) and e) according to the composition formula given in Table 3;

[0106] g) The pH of the mixture f) was adjusted to 7.3 and then sterile filtered.

[0107] h) Aseptically filling the prepared composition into the cartridge.

[0108] The composition of the present invention may be obtained using any other means.

[0109] Table 3. Qualitative and quantitative composition of the claimed composition comprising insulin glulisine

[0110]

[0111]

[0112] Example 4. Physical and chemical stability analysis of the claimed composition containing insulin glulisine

[0113] The chemical stability of compositions 1-7 prepared according to Example 3 was determined after 1 and 2 weeks of thermostating at 40°C in sterile filled cartridges.

[0114] The HPLC with UV detection was used to analyze the The insulin-glulisine related impurities were determined on an octadecyl silica column with a mobile phase flow rate of 1.0 ml / min and a wavelength of 214 nm.

[0115] Elution was performed in gradient mode using a mobile phase consisting of:

[0116] Mobile phase A (MP A): acetonitrile: pH 2.3 buffer solution (0.1 M sodium sulfate solution, orthophosphoric acid qs pH 2.3) 10:90 (V:V);

[0117] Mobile phase B (MP B): acetonitrile: pH 2.3 buffer solution (0.1 M sodium sulfate solution, orthophosphoric acid qs pH 2.3) 50:50 (V:V).

[0118] The amount of A21 desamidated insulin glulisine and other related impurities was determined by the sum of the absorption areas of the corresponding peaks using an internal normalization method, which was measured as a percentage of the sum of the areas of the peaks eluting after m-cresol.

[0119] Use the method that is similar to the HMWP assay (referring to embodiment 2) in the composition that comprises insulin aspart to measure HMWP content in the composition that comprises insulin glulisine.HMWP content uses internal normalization method, by comparing the absorption area sum at all peaks that retention time is shorter with the insulin glulisine monomer peak to determine.The peak (meta-cresol peak) that retention time is longer than insulin glulisine monomer is excluded from calculating.

[0120] The physical stability of the compositions comprising insulin glulisine was assessed similarly to the assessment of the physical stability of the compositions comprising insulin aspart (see Example 2).

[0121] Table 4 shows the results of stability studies of compositions comprising insulin glulisine.

[0122] Table 4. Physical and chemical stability data of compositions 1-7 comprising insulin glulisine prepared according to Example 3.

[0123]

[0124] *Original DP:

[0125] Based on the chemical and physical stability studies on compositions 1-7 containing insulin glulisine, it was unexpectedly found that after storage at 40°C for 2 weeks, the addition of lysine and / or its salts, and / or a combination of lysine and / or its salts with arginine and / or its salts to the compositions of the present invention can significantly and reliably reduce impurities compared to the prototype, while the physical stability is improved relative to the prototype.

[0126] Increasing the lysine concentration to 100 mM helps reduce the amount of impurities. Lysine concentrations greater than 100 mM are undesirable because the physical stability of the composition is reduced. The lowest impurity levels were observed for the combination of lysine and arginine.

[0127] Example 5. Preparation of the claimed composition containing insulin lispro

[0128] In one embodiment, the pharmaceutical compositions of the present invention are prepared as aqueous solutions. Table 5 shows the qualitative and quantitative contents of some prepared compositions. Compositions of the present invention include, but are not limited to, compositions 2-8 listed in Table 5. Composition 1 is formulated similarly to the original Humalog DP, 100 U / ml intravenous / subcutaneous solution. Compositions 2-7 are similar to the original DP formulation with the addition of L-lysine hydrochloride or a combination of L-lysine hydrochloride and L-arginine hydrochloride.

[0129] The method of preparing the composition comprises:

[0130] a) preparing an insulin solution by dissolving insulin in water or a buffer;

[0131] b) preparing a zinc solution by dissolving zinc oxide in a dilute hydrochloric acid solution;

[0132] c) preparing a preservative solution by dissolving the preservative in water or a buffer;

[0133] d) preparing an isotonic agent solution by dissolving the isotonic agent in water or a buffer;

[0134] e) preparing a lysine and / or arginine solution by dissolving an amino acid and / or a salt thereof in water or a buffer;

[0135] f) mixing solution a) with solutions b), c), d), and e) according to the composition formula given in Table 1;

[0136] g) The pH of the mixture e) is adjusted to 7-8 and then sterile filtered.

[0137] h) Aseptically filling the prepared composition into the cartridge.

[0138] The composition of the present invention may be obtained using any other method.

[0139] Table 5. Qualitative and quantitative composition of the claimed composition comprising insulin lispro

[0140]

[0141] Example 6. Physical and chemical stability analysis of the claimed composition containing insulin lispro

[0142] The chemical stability of compositions 1-8 (compositions comprising insulin lispro) prepared according to Example 5 was determined after thermostating at 40°C for 1 and 2 weeks in sterile filled cartridges.

[0143] A21-desamidated insulin lispro and other insulin lispro-related impurities were determined by HPLC with UV detection in a column packed with 3.5 μm octadecyl silica gel. A mobile phase consisting of the following components was used as the eluent:

[0144] Mobile phase A (MPA): sodium perchlorate buffer solution, pH 2.2;

[0145] Mobile phase B (MP B): acetonitrile for chromatography.

[0146] Elution was performed in gradient mode with a mobile phase flow rate of 0.9 ml / min and a detection wavelength of 214 nm.

[0147] The amount of A21-desamidated insulin lispro and other related impurities was determined by summing the absorption areas of the corresponding peaks using internal normalization, which was measured as a percentage of the sum of the areas of the peaks eluting after m-cresol.

[0148] Use the method that is similar to the HMWP mensuration (referring to embodiment 2) that comprises the compositions of insulin aspart or insulin glulisine, measure the HMWP amount that comprises the compositions of insulin lispro.HMWP content adopts interior normalization method, measures by the absorption area sum at all peaks shorter than retention time with the insulin lispro monomer peak.The peak (meta-cresol peak) that retention time is longer than insulin lispro monomer is excluded from calculating.

[0149] The physical stability of the compositions comprising insulin lispro was evaluated similarly to the physical stability of the compositions comprising insulin aspart (see Example 2).

[0150] Table 6 shows the results of chemical and physical stability studies of compositions comprising insulin lispro.

[0151] Based on the chemical and physical stability studies on compositions 1-8 containing insulin lispro, it was unexpectedly found that after storage at 40°C for 2 weeks, the addition of lysine and / or its salts, and / or a combination of lysine and / or its salts with arginine and / or its salts to the compositions of the present invention can significantly and reliably reduce impurities relative to the prototype, while at the same time improving the physical stability relative to the prototype.

[0152] Increasing the lysine concentration to 100 mM helps reduce the amount of impurities. Lysine concentrations greater than 100 mM are undesirable because the physical stability of the composition is reduced. The lowest impurity levels were observed for the combination of lysine and arginine.

[0153] Table 6. Physical and chemical stability data of compositions 1-8 comprising insulin lispro prepared according to Example 5.

[0154]

[0155]

[0156]

[0157] *Original drug "Humalog"

[0158] References

[0159] 1.O.M.Selivanova,S.Yu.Grishin,A.V.Glyakina,et al.Analysis of insulinanalogues and strategy for their further development.Advances in biologicalchemistry.2018,58:313-346。

[0160] 2.Brange J.,Owens D.R.,Kang S.,Volund A.Monomeric Insulins and TheirExperimental and Clinical Implications.Diabetes Care.1990,13(9):923–954.doi:10.2337 / diacare.13.9.923。

[0161] 3.Schlein M.Insulin Formulation Characterization-the ThioflavinTAssays.AAPS Journal.2017,19(2):397-408.

[0162] 4.Directions for medical use of Fiasp DP.URL:http: / / grls.rosminzdrav.ru(accessed on 30Aug 2022)。

[0163] 5.Brange J.Stability of Insulin.Kluwer Academic Publisher.1994,18–23。

[0164] 6.Paquot N.,Scheen A.J.Faster aspart insulin RevueMedicalede Liege.2018,73(4):211-215。

[0165] 7.Muchmore D.B.Pump Users Clamor for Faster Insulin:Is Fast-ActingInsulin Aspart Ready for Them?Journal ofDiabetes Science and Technology.2018,12(1):152–154.doi:10.1177 / 1932296817750166。

[0166] 8.Ahem T.J.,Manning M.C.Stability of Protein Pharmaceuticals.PlenumPress.New York.1992。

Claims

1. A composition for lowering blood glucose levels in a subject, wherein the composition comprises a fast-acting insulin and an amino acid, wherein the amino acid is lysine or a salt thereof, or a combination of lysine or a salt thereof and arginine or a salt thereof.

2. The composition according to claim 1, wherein the fast-acting insulin is selected from the group consisting of insulin aspart (B28Asp), insulin lispro (B28LysB29Pro) and insulin glulisine (3BLys29BGlu).

3. The composition according to claim 2, wherein the fast-acting insulin is insulin aspart (B28Asp).

4. The composition according to claim 1, wherein the amino acid is lysine or a salt thereof. The composition according to claim 1 , wherein the amino acid is a combination of lysine or a salt thereof and arginine or a salt thereof.

6. The composition according to claim 3, wherein the amino acid is lysine or a salt thereof.

7. The composition according to claim 3, wherein the amino acids are a combination of lysine and arginine.

8. The composition according to claim 2, wherein the fast-acting insulin is insulin glulisine (3BLys29BGlu).

9. The composition according to claim 8, wherein the composition further comprises methionine.

10. The composition according to claim 1, wherein the content of insulin ranges from about 0.2 mM (33 U / ml) to about 2 mM (333 U / ml).

11. The composition according to claim 1, wherein the content of insulin ranges from about 0.3 mM (50 U / ml) to about 1.2 mM (200 U / ml).

12. The composition according to claim 10 or 11, wherein the insulin is insulin aspart and the composition further comprises a niacin compound.

13. The composition according to claim 12, wherein the composition comprises niacinamide as the niacin compound.

14. The composition according to claim 13, wherein the composition comprises about 1 mM to about 200 mM nicotinamide.

15. The composition according to claim 10 or 11, wherein the composition comprises about 1 mM to about 100 mM lysine or a salt thereof.

16. The composition according to claim 10 or 11, wherein the composition comprises about 1 mM to about 100 mM of a combination of lysine or a salt thereof and arginine or a salt thereof.

17. The composition according to claim 1, wherein the composition further comprises metal ions, a preservative, an isotonicity agent, a stabilizer, a buffer and an acidity regulator.

18. The composition according to claim 1, for use in treating or preventing hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes.

19. 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 17.

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