EA042403B1 - FAST-ACTING INSULIN COMPOSITION - Google Patents

Description

The present invention is a pharmaceutical composition of insulin for parenteral injection for counteracting prandial and postprandial blood glucose spikes. The proposed composition contains insulin and a polyphosphate compound, and is characterized by faster absorption of insulin from injection sites than the existing industrial compositions of insulin. The proposed composition is suitable for quickly providing insulin activity when insulin is needed, for example, when eating.

Diabetes mellitus is a chronic disorder characterized by hyperglycemia due to impaired insulin secretion, insulin action, or both. Type 1 diabetes mellitus is characterized by little or no secretory ability to produce insulin, and type 1 diabetic patients require insulin to survive. Type 2 diabetes mellitus, characterized by the combined effect of impaired insulin secretion and insulin resistance, results in elevated blood glucose levels. In at least one third of patients with type 2 diabetes, the disease progresses to the absolute need for insulin therapy.

The temporal profile of insulin action is important in regulating postprandial blood glucose levels. In healthy individuals, the pancreas dramatically increases insulin secretion in response to ingested food, resulting in elevated blood insulin levels within minutes. In individuals with type 1 diabetes, and in some individuals with type 2 diabetes, it is necessary to specifically administer insulin. However, injected insulin enters the blood more slowly than endogenously produced insulin, and its slow onset of action can lead to hyperglycemia during the early postprandial period. Too long action can lead to excess insulin levels between meals, resulting in late postprandial hypoglycemia and/or weight gain.

Previous attempts have been made to accelerate the action of insulin preparations. Fast-acting insulin analogs became available in the 1990s and early 2000s. Even with so-called fast-acting insulin analogues such as insulin lispro (HUMALOG®), insulin aspart (NOVOLOG®) and insulin glulisine (APIDRA®), the maximum circulating insulin concentration is not reached until 50-90 minutes after injection. This is not fast enough to match carbohydrate digestion profiles.

Later research was done in an attempt to develop a product with a faster action profile than described above. For example, US 2014/0378383 describes insulin compositions containing a combination of a substituted anionic compound consisting of a saccharide skeleton formed from 1-hexose saccharide units containing partially substituted carboxyl functional groups with a polyanionic compound, and states that such a combination provides the ability to accelerate intake of insulin into the blood. Similarly, US 2015/0231160 describes insulin compositions containing a combination of an oligosaccharide and a polyanionic compound, and states that such a combination allows for a significant reduction before the onset of action of a fast-acting insulin analogue dosage form. These documents describe a number of polyanionic compounds, including polyphosphoric acids such as triphosphate, but do not describe any composition containing triphosphate, but not containing a substituted anionic compound, as described in US 2014/0378373, or an oligosaccharide, as described in US 2015 /0231160, and does not provide any data on the pharmacokinetics or pharmacodynamics of compositions containing triphosphate or any other polyphosphoric acid. US 2014357554 and US 2015273022 describe compositions claimed to have a rapid onset of action of insulin that contain EDTA, citrate and magnesium-containing compounds, one example of which is magnesium pyrophosphate. The magnesium compound is claimed to minimize irritation at the site of injection but does not alter the rate of subcutaneous absorption, and no data are available on formulations containing magnesium pyrophosphate.

There continues to be a need for mealtime insulin formulations that have faster uptake of insulin from the site of injection and a faster onset of action than existing commercial insulin products.

The present invention addresses these needs by providing pharmaceutically acceptable insulin dosage forms that have faster insulin delivery to the blood and a faster onset of action than existing commercial insulin products.

The essence of the invention

According to a first aspect of the present invention, there is provided a pharmaceutical composition comprising insulin and about 10 to about 30 mM triphosphate, provided that said composition contains neither a saccharide multimer nor EDTA.

In some embodiments, the triphosphate concentration is from about 20 mM to about 25 mM, such as about 20 mM.

In some embodiments, the pharmaceutical composition further comprises zinc.

- 1 042403

In some embodiments, the zinc concentration is from about 0.2 to about 5 mM.

In some embodiments, the pharmaceutical composition further comprises a tonicity agent, which may be glycerol.

In some embodiments, the pharmaceutical composition further comprises one or more preservatives, wherein the one or more preservatives may be selected from the group consisting of phenol, meta-cresol, and benzyl alcohol.

In some embodiments, the insulin is selected from the group consisting of human insulin, insulin lispro, insulin aspart, and insulin glulisine.

In some embodiments, the insulin concentration is from about 40 to about 500 IU/ml, for example, from about 100 to about 200 IU/ml.

According to a second aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a person in need thereof an effective dose of the above pharmaceutical composition.

According to a third aspect of the present invention, the use of the above pharmaceutical composition for the treatment of diabetes is provided.

According to a fourth aspect of the present invention, there is provided an insulin delivery device comprising any of the above pharmaceutical compositions.

In some embodiments, the product is a refillable vial.

In some embodiments, the product is a reusable pen.

In some embodiments, the product is a pre-filled disposable pen.

In some embodiments, the product is an auto-injector.

In some embodiments, the product is a CSII pump.

Detailed description of the invention

According to a first aspect of the present invention, there is provided a pharmaceutical composition comprising insulin and a polyphosphate compound selected from the group consisting of pyrophosphate, trimetaphosphate, triphosphate and tetraphosphate, provided that said composition contains neither a saccharide multimer nor EDTA.

In some embodiments, the polyphosphate concentration is from about 5 to about 50 mM. In some embodiments, the polyphosphate concentration is from about 10 mM to about 30 mM. In some embodiments, the polyphosphate concentration is from about 20 mM to about 25 mM. In some embodiments, the polyphosphate concentration is selected from the group consisting of 5, 10, 15, 20, 25, or 30 mM.

In some embodiments, the composition further comprises zinc. In some embodiments, the zinc concentration is from about 0.2 to about 5 mM.

In some embodiments, the composition further comprises a tonicity agent. In some embodiments, the tonicity agent is glycerol.

In some embodiments, the composition further comprises one or more preservatives. In some embodiments, one or more preservatives are selected from the group consisting of phenol, meta-cresol, and benzyl alcohol.

In some embodiments, the insulin is selected from the group consisting of human insulin, insulin lispro, insulin aspart, and insulin glulisine. In some embodiments, the insulin concentration is from about 40 to about 500 IU/mL. In some embodiments, the insulin concentration is from about 100 to about 200 IU/mL.

In accordance with another aspect of the present invention, there is provided a method of treating diabetes comprising administering to a person in need thereof an effective dose of one of the compositions described above.

According to another aspect of the present invention, one of the above compositions is provided for use as a medicine.

According to another aspect of the present invention, one of the above compositions is provided for use in the treatment of diabetes.

According to another aspect of the present invention, there is provided an article of manufacture comprising one of the compositions described above. In some embodiments, the commercial product is a refillable vial. In some embodiments, the commercial product is a pre-filled disposable pen. In some embodiments, the commercial product is a refillable pen. In some embodiments, the industrial product is an autoinjector. In some embodiments, the article of manufacture is a continuous subcutaneous insulin infusion (CSII) pump.

In this context, a saccharide multimer means any compound containing more than one saccharide unit that is linked to each other, including, for example, substituted anionic compounds described in US 2014/0378383 and oligosaccharides described in US 2015/0231160.

In this context, the term does not contain saccharide multimer or EDTA means that the composition does not contain saccharide multimers or EDTA, or contains only a de minimis amount of saccharide multimers or EDTA, which does not affect the time profile of insulin action.

As used herein, insulin means human insulin or a structural variant, mutant, or analog of human insulin that has the functional activity of human insulin. Human insulin analogs include, but are not limited to, insulin lispro, insulin aspart, and insulin glulisine, or other fast-acting insulin analogs. Insulin for industrial products can be obtained using the method of recombinant DNA or chemical synthesis. Recombinant methods are well known and highly preferred. The human insulin molecule (CAS no. 11061-68-0) consists of two amino acid chains, A and B, whose sequences are well understood. The chains are linked to each other by two disulfide bonds: CysA7-CysB7 and CysA20-CysB19. The A chain has an intrachain disulfide bond at the CysA6-CysA11 position.

The A-chain of human insulin has the following amino acid sequence:

Gly Ne Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tut Gin Leu Asn Tut Cys Asn (SEQ ID

NO:1)

The B-chain of human insulin has the following amino acid sequence:

Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg

Gly Phe Phe Tyr Thr Pro Lys Thr (SEQ ID NO: 2).

Insulin lispro (CAS No. 133107-64-9), the active ingredient in HUMALOG®, has been shown to be equally effective as human insulin on a molar basis, but its effect after subcutaneous injection is faster and shorter than that of soluble human insulin. insulin. For insulin lispro, a corresponding kinetic profile was observed with a lower T _max and half-life and a higher C _max compared to human insulin. Insulin lispro is biologically equivalent to insulin in several in vitro assays, including insulin receptor binding in cultured lymphocytes, human placenta, and human liver, as well as glucose transport in adipocytes. HUMALOG® contains m-cresol as a preservative and stabilizer, a tonicity modifier (glycerol), a buffering agent (dibasic sodium phosphate), a stabilizer (zinc oxide) and a carrier pH adjusting agent.

The insulin lispro molecule consists of a human insulin A chain fused to a lispro insulin B chain, the amino acid sequence of which is shown in SEQ ID NO: 3, below:

Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg

Gly Phe Phe Tu Thr Lys Pro Thr (SEQ ID NO: 3).

One unit of insulin lispro is equivalent to 0.0347 mg of insulin lispro.

Insulin aspart (CAS no. 116094-23-6), the active ingredient in NOVOLOG®, is another fast-acting insulin analogue. Its structure consists of the A chain of human insulin and a B chain analog presented in the following amino acid sequence:

Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg

Gly Phe Phe Tyr Thr Asp Lys Thr (SEQ ID NO: 4).

One unit of insulin aspart corresponds to 6 nmol, which corresponds to 0.035 mg of salt-free anhydrous insulin aspart.

Insulin glulisine (CAS No. 207748-29-6), the active ingredient in APIDRA®, is another fast-acting insulin analogue. The insulin glulisine molecule consists of a human insulin A chain and a modified B chain relative to human insulin, as shown in the following amino acid sequence:

Phe Vai Lys Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tut Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr (SEQ ID NO: 5).

One unit of insulin glulisine corresponds to approximately 0.0349 mg of insulin glulisine.

Compositions according to the present invention have insulin concentrations from 0.24 to 3 mm (40-500 IU/ml; 1.4-17.5 mg/ml). Compositions according to the present invention in most cases have specific concentrations of 40, 100, 200, 300, 400 and 500 IU/ml (1.4, 3.5, 7, 10.5, 14 and 17.5 mg/ml). Preferred concentrations are 100 and 200 IU/ml.

Polyphosphates are inorganic multiply charged polyvalent anions consisting of 2 or more phosphate groups covalently linked by P-O-P bonds. They are widely used in detergents, foods, cosmetics, and biomedicine (as chelating agents, buffers, and crosslinkers, among other uses. Many polyphosphates

- 3 042403 are recognized as completely safe by the US Food and Drug Administration for Food Use (GRAS), including, for example, the polyphosphates listed below in table. 1.

Table 1

Examples of GRAS polyphosphates

Gras substance Formula (MM) CAS No. 21 C.F.R. calcium pyrophosphate Ca ₂ P ₂ O ₇ (254.1) 7790-76-3 182.8223 Potassium pyrophosphate K4P2O7 (330.3) 7320-34-5 No Potassium tripolyphosphate K ₅ R ₃ 0yu (453.5) 13845-36-8 No Sodium hydrogen pyrophosphate Na ₂ H ₂ P2O ₇ (221.9) 7758-16-9 182.1087 Sodium pyrophosphate Na ₄ P ₂ O ₇ (265.9) 7722-88-5 182.6760 sodium tetraphosphate Na ₆ P40i3 (469.8) 14986-84-6 No sodium trimetaphosphate Na ₃ P ₃ O9 (305.9) 7785-84-4 No Gras substance Formula (MM) CAS No. 21 C.F.R. Sodium tripolyphosphate Na ₅ P ₃ Oi ₀ (367.9) 7758-29-4 182.1810 182.1610

Triphosphate is used as a crosslinking agent in polymeric nanocarriers, in particular chitosan nanocarriers, for oral, nasal, parenteral, or transdermal delivery of a wide range of medically important payloads such as antigens, anticancer drugs, genetic materials, and proteins, including insulins. . Kouchak, et al., Effect of different molecular weights of chitosan on preparation and characterization of insulin loaded nanoparticles by ion gelation method, 4 Internat'l J. Drug Dev. & Res. 271 (2012). However, contrary to the present invention, such carriers are not intended to accelerate the action profile of the administered insulin.

The polyphosphates presented herein that are suitable for increasing the rate of insulin absorption from the injection site are pyrophosphate (diphosphate [O3-POP-O3] ⁴ , P2O7) and triphosphate ([O3-PO-(PO2)-OP-O3] ⁵ , P3O ₁₀ ). The effect on insulin absorption is presumably due to these polyphosphates, as well as trimetaphosphate (P3O9)- and tetraphosphate (P4O13-6). The particular polyphosphate compound used may be the acid form or various salt forms, in particular alkali metal (eg sodium and potassium) salts. Pyrophosphate, triphosphate, trimetaphosphate and tetraphosphate, as well as their various salts, in particular their salts with alkali metals (eg sodium and potassium) and alkaline earth metals (eg calcium and magnesium) can be used according to the present invention. Among these, triphosphate and salts thereof are preferable. The concentration of polyphosphate in the compositions varies from 5 to 50 mm, in particular, 5, 10, 15, 20, 25, 30, 35, 40 or 50 mm. Some compositions have polyphosphate concentrations ranging from 10 to 30 mM. Some compositions have polyphosphate concentrations ranging from 15 to 25 mM.

Commercial insulin formulations contain approximately 2.4 zinc atoms per six insulin molecules (HUMULIN® R U-500) and some contain approximately 3.0 zinc atoms per six insulin molecules (HUMALOG®, NOVOLOG®). Some embodiments of the present invention contain zinc at a concentration sufficient to provide about 2-4 zinc atoms per six insulin molecules. Other implementation options contain zinc at a concentration of up to about 5 mm. In some embodiments, the zinc concentration ranges from about 0.2 to about 5 mM. In some embodiments, the zinc concentration ranges from about 0.5 to about 2 mM. In some embodiments, the zinc concentration is selected from the group consisting of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.3, and 2 mm.

The proposed compositions are sterile when initially manufactured. When supplied in the form of a refillable vial or cartridge, an antimicrobial preservative compound or mixture of compounds that are compatible with other components of the dosage form is usually added at an effective concentration sufficient to meet regulatory and pharmacopoeial requirements for antimicrobial preservatives. See USP articles. Insulin lispro injection. USP29-NF24; articles of the British Pharmacopoeia, 2008, volume III: Insulin aspart injection; USP articles. insulin assays; and the general sections of the USP. USP29-NF24. Rockville, MD: USP; 2005. Antimicrobial effectiveness testing; cc. 2499-2500. Preferred preservatives are arylic acids and phenolic compounds or mixtures of such compounds. The preservatives most commonly used in insulin products are phenol, m-cresol, and benzyl alcohol. Effective concentrations can be readily determined by the methods mentioned above. For example, modern commercial compositions contain 3.15 mg/ml m-cresol

- 4 042403 (HUMALOG® and APIDRA®)), 1.72 mg/ml m-cresol and 1.50 mg/ml phenol (NOVOLOG®) and 2.5 mg/ml mcresol (HUMULIN® R U-500).

The pH of the insulin compositions of the present invention is typically between 7.0 and 7.8 and is adjusted with physiologically acceptable acids and bases, typically 10% hydrochloric acid and 10% sodium hydroxide. The pH of commercial insulin formulations is typically between 7.2 and 7.6, with 7.4±0.1 being the usual pH requirement.

It is desirable that the composition approximate as closely as possible the tonicity (i.e., osmolality) of the body fluids at the site of injection when the composition is administered, since solutions that are not approximately isotonic with the body fluids can cause a painful burning sensation upon administration. Thus, it is desirable that the compositions be approximately isotonic with the body fluids at the site of injection. If the osmolality of the composition in the absence of a tonic agent is sufficiently lower than the tissue osmolality (for blood, about 300 mOsmol/kg; EP requirement for osmolality: >240 mOsmol/kg), then it is usually necessary to add a tonicity agent to increase the tonicity of the composition to a value of about 300 mOsmol/kg. kg. Typical tonicity agents are glycerol (glycerin) and sodium chloride. The amount of tonicity agent added can be readily determined by standard methods. Remington: The Science and Practice of Pharmacy, David B. Troy and Paul Beringer, eds., Lippincott Williams & Wilkins, 2006, pp. 257-259; Remington: Essentials of Pharmaceutics, Linda Ed Felton, Pharmaceutical Press, 2013, pp. 277-300.

Compositions of the present invention are typically administered subcutaneously, or as multiple injections per day (MDI) from a pre-filled disposable pen, refillable pen, automatic pen, refillable vial, or CSII pump.

Additional embodiments of the present invention include the embodiments described below.

1. A pharmaceutical composition containing insulin and a polyphosphate compound selected from the group consisting of pyrophosphate, triphosphate, trimetaphosphate and tetraphosphate.

2. A pharmaceutical composition according to any of the preceding embodiments, characterized in that said composition contains neither a saccharide multimer nor EDTA.

3. A pharmaceutical composition according to any of the preceding embodiments, wherein the polyphosphate compound is a triphosphate.

4. A pharmaceutical composition according to any one of the preceding embodiments, wherein the polyphosphate compound is a pyrophosphate.

5. A pharmaceutical composition according to any one of the preceding embodiments, characterized in that the concentration of the polyphosphate is from about 5 to about 50 mM.

6. A pharmaceutical composition according to any one of the preceding embodiments, characterized in that the concentration of the polyphosphate is from about 10 to about 30 mM.

7. Pharmaceutical composition according to any of the preceding embodiments, characterized in that the concentration of the polyphosphate is selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40 and 50 mM.

8. Pharmaceutical composition according to any of the preceding embodiments, additionally containing zinc.

9. A pharmaceutical composition according to any one of the preceding embodiments, wherein the zinc concentration is from about 0.2 to about 5 mM.

10. A pharmaceutical composition according to any of the preceding embodiments, characterized in that the zinc concentration is from about 0.5 to about 2 mm.

11. A pharmaceutical composition according to any of the preceding embodiments, characterized in that the zinc concentration is selected from the group consisting of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 2 and 5 mM.

12. A pharmaceutical composition according to any of the preceding embodiments, further comprising a tonicity agent.

13. A pharmaceutical composition according to any of the preceding embodiments, further comprising a tonicity agent which is glycerol.

14. A pharmaceutical composition according to any of the preceding embodiments, further comprising one or more preservatives.

15. A pharmaceutical composition according to any of the preceding embodiments, wherein the one or more preservatives are selected from the group consisting of phenol, meta-cresol and benzyl alcohol.

16. A pharmaceutical composition according to any one of the preceding embodiments, wherein the insulin is selected from the group consisting of human insulin, insulin lispro, insulin aspart, and insulin glulisine.

17. A pharmaceutical composition according to any of the preceding embodiments, different

- 5 042403 Unfortunately, the insulin concentration is from about 40 to about 500 IU/ml.

18. A pharmaceutical composition according to any of the preceding embodiments, characterized in that the insulin concentration is from about 100 to about 200 IU/ml.

19. A method of treating diabetes, comprising administering to a person in need thereof an effective dose of a pharmaceutical composition according to any of the preceding embodiments.

20. A pharmaceutical composition according to any of the preceding embodiments for use as a medicine.

21. A pharmaceutical composition according to any of the preceding embodiments for use in the treatment of diabetes.

22. A pharmaceutical composition according to any of the preceding embodiments, comprising a mixture of two or more polyphosphate compounds selected from the group consisting of pyrophosphate, triphosphate, trimetaphosphate, and tetraphosphate.

23. An industrial product containing any of the pharmaceutical compositions described above.

24. A reusable vial containing any of the pharmaceutical compositions described above.

25. A refillable pen containing any of the pharmaceutical compositions described above.

26. A pre-filled disposable pen containing any of the pharmaceutical compositions described above.

27. An automatic syringe pen containing any of the pharmaceutical compositions described above.

28. A CSII pump containing any of the pharmaceutical compositions described above.

Pharmacokinetic (PK) and pharmacodynamic (PD) studies

Study 1. 25 mM pyrophosphate or 25 mM triphosphate.

Fifteen castrated male Yucatán miniature pigs (aged 17 months and with an average body weight of 40 kg) with diabetes (caused by alloxan) were used, which were pre-installed with a vascular access device. Diabetic animals were kept separately and had free access to drinking water at all times. They were fed twice daily with S-9 total food and administered with appropriate maintenance doses of basal and prandial insulin twice daily to control their diabetic condition.

Dosage forms of experimental drugs (dosage form A and B) were formulated and delivered overnight in the cold to the research center. They were kept in the refrigerator until the time of dosing and then returned to the refrigerator after dosing of all animals was complete. During dosing, the experimental drugs were kept in an insulated box until removed. The HUMALOG® insulin control preparation was administered from a commercial vial.

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table 2

Compositions of experimental and control preparations

Name drug The composition of the drug HUMALOG 3.5 mg/ml insulin lispro 7 mM sodium phosphate 0.3 mm 3.15 mg/ml m-cresol 16 mg/ml glycerol pH 7.4 insulin lispro + 25 mM pyrophosphate (dosage form A) 92 IU/ml insulin lispro (3.2 mg/ml) 7 mM sodium phosphate 0.3 mM zinc 3.15 mg/ml m-cresol 6.33 mg/ml glycerol 25 mM sodium pyrophosphate pH 7.4 insulin lispro + 25 mM triphosphate (dosage form B) 94 IU/ml insulin lispro (3.3 mg/ml) 7 mM sodium phosphate 0.3 mM zinc 3.15 mg/ml m-cresol 4.49 mg/ml glycerol 25 mM sodium triphosphate pH 7.4

The study design was a three-way crossover design. This design allows the administration of each of the three experimental drugs to each individual animal, administering one experimental drug on each day of the study (3 different days at intervals of 7 days). The day before the start of the study, the animals were given half the daily diet and injected with 0.2 U/kg of Humalog Mix 75/25 insulin as the morning maintenance dose. All experimental animals were kept without access to food during the night and did not give them the evening dose of insulin and did not give them food until the administration of the drug on the day of the study.

On the morning of the study day, all animals were placed in restraint slings and vascular access points (provided for blood sampling) were opened and checked for patency. Animals were randomly assigned to experimental groups (3 groups with n=5 per group, total n=15 per experimental treatment).

After two blood samples were taken at baseline (-30 and -20 min), the animals were returned to the pens and given ~300 g of S-9 feed. Twenty minutes after the end of the meal, the animals were injected with the experimental drug subcutaneously in the flank (0 min) using a Terumo insulin syringe (0.5 ml, 1/2 needle). Dose administration included a single injection of 0.2 IU/kg of active insulin. All experimental animals had free access to clean fresh water during the rest of the blood collection period.

Serial blood samples (2.0 ml each) were collected from each animal at the following time points: -30, -20 (then immediately fed), 0 (just before dosing), after 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240 and 360 minutes after SC dosing. The blood samples (anticoagulant: none (serum)) were kept at room temperature for at least 30 minutes, but no more than 2 hours to ensure clotting. The serum was then separated by centrifugation and divided into two aliquots and stored frozen at about -70°C.

- 7 042403

Serum glucose concentrations were determined using a Cobas c311 automated clinical chemistry analyzer (Roche Diagnostics, Indianapolis). Two animals were excluded from the experimental group with Humalog, obtaining n=13 for this experimental drug. One animal was excluded because it did not meet the criteria for a baseline glucose level of >200 mg/dl, and the other animal was not included in the experiment due to port patency problems. Serum glucose concentrations (mg/dL) after administration of dosage forms containing insulin lispro (0.2 IU/kg at time 0) are shown in Table 1 below. 3.

Table 3

Serum glucose concentrations (mg/dL)

Humalog (n=13) Dosage form A (n=15) 25 mM pyrophosphate Dosage form B (n=15) 25 mM triphosphate Time (min.) Average Art. from class. Average Art. from class. Average Art. from class. -thirty 293 46 270 77 292 43 -20 297 47 293 40 299 46 0 322 52 304 48 306 45 5 325 45 312 52 309 48 10 313 51 245 48 238 50 15 285 60 201 47 189 53 thirty 216 80 133 56 108 56 45 165 80 99 63 70 51 60 131 87 70 45 52 54 75 104 80 57 39 44 52 90 83 70 44 35 35 42 105 68 62 40 33 34 36 120 58 52 37 34 35 38 150 49 43 38 38 38 27 180 48 40 47 48 44 33 240 69 44 70 73 74 59 360 143 73 FROM 87 151 106

Serum insulin concentrations were determined by competitive radioimmunoassay (RIA). In the RIA, which measured both endogenous porcine insulin and exogenous insulin, serum insulin was substituted for 125 Kinsulin to bind to guinea pig anti-rat insulin. The antibody complex was precipitated with a goat anti-guinea pig serum reagent. The upper and lower limits of quantitation in RIA were 5000 and 20 pM, respectively, in heat-treated and charcoal-purified serum.

Non-compartmental pharmacokinetic analysis was performed using Phoenix WinNonLin 6.3. For values below the lower limit of quantitation, a value of 20 pM was assigned for calculations. Samples with values above the upper limit of quantitative detection were either diluted and reanalyzed or ignored. As stated above, one animal from the HUMALOG group did not participate in the study due to port patency problems, so n=14 for this experimental formulation.

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Table 4

FC data

Dosage form Tmax (min.) Stach (nM) AUCoo (min*nM) CL/F (ml/min/kg) Humalog (n=14) Average± SO 61.1±11.1 1.30±0.25 141±21.2 12.1±1.97 Median 45.0 1.08 142 8.43 Dosage form A(n=15) 25 mM pyrophosphate Average± SO 32.3±7.6 1.29±0.12 113=1=10.7 10.6±1.12 Median 30.0 1.30 107 11.1 Dosage form B(n=15) 25 mM triphosphate Mean ± SD 14.3±1.88 2.93±0.34 220±31.4 6.88±0.764 Median 15.0 2.53 159 7.56

Abbreviations:

T _max - time at maximum concentration,

C _max - maximum concentration,

AUC _ins . - area under the curve from 0 to infinity,

CL/F -clearance/bioavailability

The PK/PD data demonstrate that pyrophosphate or triphosphate accelerates action and reduces _Tmax compared to HUMALOG. Dosage form And with pyrophosphate shows T _max ∼47% less than HUMALOG (∼33% lower median T _max ) and has a comparable average C _max with HUMALOG. Dosage form B with triphosphate shows T _max ∼77% less than HUMALOG (∼67% lower median T _max ) and ∼125% higher C _max than Humalog. Dosage forms containing 25 mm pyrophosphate or 25 mm triphosphate, provided a shorter T _max and higher C _max compared to HUMALOG.

Study 2. Effect of triphosphate concentration on PK/PD.

Fourteen castrated male Yucatan miniature pigs (aged 14 months and with an average body weight of 35 kg) with diabetes (caused by alloxan) were used, which were pre-installed with a vascular access device. Diabetic animals were kept separately and had free access to drinking water at all times. They were fed twice daily with S-9 total food and administered with appropriate maintenance doses of basal and prandial insulin twice daily to control their diabetic condition.

Experimental preparations were made and brought during the night in the cold. They were kept in the refrigerator until the time of dosing and then returned to the refrigerator after dosing of all animals was completed. During dosing, the experimental drugs were kept in an insulated box until removed. The HUMALOG control preparation was an industrial vial.

- 9 042403

Table 5

Compositions of experimental and control preparations

Name of the drug The composition of the drug HUMALOG 3.5 mg/ml insulin lispro 7 mM sodium phosphate, pH 7.4 0.3 mM Zn molecules 16 mg/ml glycerol 3.15 mg/ml m-cresol Dosage form C: insulin lispro + 5 mM triphosphate 3.5 mg/ml insulin lispro 7 mM sodium phosphate 0.3 mM zinc 13.70 mg/ml glycerol 3.15 mg/ml m-cresol 5 mM sodium triphosphate, pH 7.4 Dosage form D: insulin lispro + 10 mM triphosphate 3.5 mg/ml insulin lispro 7 mM sodium phosphate 0.3 mM zinc 11.40 mg/ml glycerol 3.15 mg/ml m-cresol 10 mM sodium triphosphate, pH 7.4 Dosage form E: insulin lispro + 20 mM triphosphate 3.5 mg/ml insulin lispro 7 mM sodium phosphate 0.3 mM zinc 6.79 mg/ml glycerol 3.15 mg/ml m-cresol 20 mM sodium triphosphate, pH 7.4

The study design was a four-way cross-over design, allowing each individual animal to be administered each of the three experimental drugs and the control drug, administering one experimental drug on each day of the study (4 different days at 7-day intervals).

The day before the start of the study, the animals were given half the daily diet and injected with 0.2 U/kg of Humalog Mix 75/25 insulin as the morning maintenance dose. All experimental animals were kept without access to food during the night and did not give them the evening dose of insulin and did not give them food until the administration of the drug on the day of the study.

On the morning of the study day, all animals were placed in restraint slings and vascular access points (provided for blood sampling) were opened and checked for patency. Animals were randomly assigned to experimental groups (4 groups with n=3-4 per group, total n=14 per experimental treatment). Two pigs were excluded from the study as they were seen by a veterinarian and the total n=14 was reduced to n=12 before any other exclusions. One animal was excluded from the HUMALOG group and two animals were excluded from the insulin lispro + triphosphate 10 mM group because they did not meet the inclusion criteria, so n=11 and n=10, respectively, for these groups.

After two blood samples were taken at baseline (-30 and -20 min), the animals were returned to the pens and given ~300 g of S-9 feed. Twenty minutes after the end of the meal, the animals were injected with the experimental drug subcutaneously in the flank (0 min) using a Terumo insulin syringe (0.5 ml, 1/2 needle). All experimental animals had free access to clean fresh water during the rest of the blood collection period.

Serial blood samples (2.0 ml each) were collected from each animal at the following time points: -30, -20 (then immediately fed), 0 (just before dosing), after 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240 and 360 minutes post SC dosing. Blood samples (anticoagulant: none (serum)) were kept at room temperature for at least 30 minutes, but no more than 2 hours to ensure clotting. The serum was then separated by centrifugation and divided into two aliquots and stored frozen at about -70°C. The next day, aliquots were delivered by dry ice delivery service.

Serum insulin concentrations were determined by competitive radioimmunoassay (RIA) as described above. Data were analyzed by non-compartmental pharmacokinetic analysis using Phoenix WinNonLin 6.3 as described above. Serum glucose concentrations were determined using a Cobas C3I Automated Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Ind.).

The results of measuring glucose in serum (mg / dl) are presented below in table. 6.

Table 6

Serum glucose results (mg/dl) HUMALOG Dosage form C insulin lispro + 5 mM triphosphate Dosage form D insulin lispro + 10 mM triphosphate Dosage form E insulin lispro + 20 mM triphosphate Time (min.) Average st.o. Average st.o. Average st.o. Average st.o. -thirty 287 63 266 49 283 43 285 34 -20 299 65 275 52 295 40 292 37 0 306 65 280 54 294 43 299 43 5 314 64 281 53 294 43 300 49 10 320 61 252 47 266 39 256 52 15 314 59 221 45 232 40 215 56 thirty 305 65 161 50 171 44 146 59 45 277 73 141 57 141 47 114 59 60 246 74 126 64 126 60 99 61 75 219 73 FROM 63 FROM 60 96 63 90 183 76 106 73 102 57 92 63 105 160 71 95 63 99 57 96 63 120 140 72 82 60 94 55 93 60 150 117 72 77 52 81 51 96 58 180 105 80 77 59 72 48 106 71 240 133 93 115 83 99 67 163 101 360 219 93 164 94 163 94 197 92

The PK parameters as a function of triphosphate concentration are presented in Table 1 below. 7.

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Table 7

FC data

Dosage form Tshah (min.) Stach (nM) AiSbesk. (min.*nM) CL/F (ml/min/kg) Humalog N=12 Average ± SO 58.8 + 6.0 0.89 + 0.098 127+12 10.3 + 0.89 Median 60 1.03 119 10.2 Dosage form C: insulin lispro + 5 mM triphosphate N=12 Average ± SO 52.5 + 13.0 1.05 + 0.093 132+25 11.6+1.5 Median 37.5 1.12 120 9.97 Dosage form D: insulin lispro +10 mM triphosphate N=10 Mean ± SD 28.0+10.6 1.23 + 0.15 127+29 12.0+1.5 Median 15.0 1.21 98.6 12.2 Dosage form E: insulin lispro + 20 mM triphosphate N=12 Average ± SO 12.9+2.5 1.67 + 0.14 116+13 11.8+1.2 Median 10.0 1.84 109 11.0

Abbreviations:

Tmax - time at maximum insulin concentration,

Cmax - the maximum concentration of insulin,

AUC _ins . - area under the curve from 0 to infinity,

CL/F - clearance/bioavailability

Triphosphate at a concentration of 5, 10 or 20 mM in dosage forms like HUMALOG accelerates the time of action, reduces Tmax and increases Cmax compared to HUMALOG®, and this occurs in a dose-dependent manner.

Study 3. Effects on various industrial insulins.

Fifteen castrated male Yucatan miniature pigs with diabetes (induced by alloxan) pre-inserted with a vascular access device were used to study the effect of triphosphate on the serum glucose profile and serum insulin action time profile for various commercial insulins. The maintenance and nutrition of animals, as well as the delivery and storage of experimental and control drugs were carried out as described above in studies 1 and 2.

Experimental formulations (dosage forms F, G, and H in the following table) were formulated by adding enough triphosphate to commercial vials of HUMULINR®, NOVOLOG®, and APIDRA® to achieve a triphosphate concentration of 20 mM. It should be noted that the concentrations of other ingredients listed in the following table reflect the concentrations of these ingredients in commercial vials of the indicated products; the reported concentrations have not been adjusted to account for the slight dilution resulting from the addition of triphosphate.

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Table 8

Compositions of experimental and control preparations

Name of the drug The composition of the drug HUMALOG 3.5 mg/ml insulin lispro 7 mM sodium phosphate, pH 7.4 0.3 mM Zn 16 mg/ml glycerol 3.15 mg/ml m-cresol Dosage form F: NOVOLOG + 20 mM triphosphate 100 U/ml insulin aspart 19.6 µg/mL zinc 16 mg/ml glycerol 1.50 mg/ml phenol 1.72 mg/ml m-cresol 1.25 mg/ml anhydrous disodium hydrogen phosphate 0.58 mg/ml sodium chloride 20 mM sodium triphosphate Dosage form G: APIDRA + 20 mM triphosphate 100 U/ml insulin glulisine 3.15 mg/ml m-cresol 6 mg/ml tromethamine, 5 mg/ml sodium chloride 0.01 mg/ml polysorbate 20 20 mM sodium triphosphate Dosage form H: HUMULIN + 20 mM triphosphate 100 U/ml insulin 16 mg/ml glycerol 2.6 mg/ml m-cresol 0.23 mM Zn 20 mM sodium triphosphate

The study was a four-way cross-over design, allowing each individual animal to be administered each of the three experimental drugs and the control drug, administering one experimental drug on each day of the study (4 different days at 7-day intervals). Animals were prepared for the study as described above for studies 1 and 2.

Animals were randomly assigned to experimental groups (4 groups with n=3-4 per group, total n=15 per experimental treatment). One animal was excluded from the HUMULIN R + 20 mM triphosphate and NOVOLOG + 20 mM triphosphate group due to port patency problems, so n=14 for these experimental groups. One animal was excluded from the HUMALOG group due to port patency problems and one animal was excluded from the HUMALOG group because it did not meet the inclusion criteria, and for this group n=13.

Collection of blood samples at baseline, injection of experimental drugs, collection of blood samples, and collection, transport and measurement of blood and serum samples were performed as described above for studies 1 and 2.

Serum glucose concentrations were determined using an AU480 automated clinical chemistry analyzer (Beckman Coulter). The results of measuring glucose in serum (mg / dl) are presented below in table. 9.

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Table 9

Serum glucose results (mg/dL)

HUMALOG Dosage form F: HUMULIN + 20 mM triphosphate Dosage form G: APIDRA + 20 mM triphosphate Dosage form H: NOVOLOG + 20 mM triphosphate Time (min.) Average ST.off. Average ST.off. Average ST.off. Average ST.off. -thirty 343 39 335 62 336 45 343 56 -20 360 38 356 64 350 45 355 58 0 372 43 368 61 366 42 367 48 5 386 37 371 63 378 39 373 51 10 362 39 315 64 307 37 296 50 15 348 39 274 65 276 45 255 50 thirty 274 65 209 73 201 63 177 56 45 239 82 187 85 169 76 139 59 60 190 90 184 101 143 76 118 67 75 173 82 185 110 130 79 FROM 76 90 150 77 175 115 114 67 105 82 105 136 67 164 118 104 64 103 84 120 129 68 164 124 102 68 102 82 150 116 67 145 123 94 68 94 78 180 111 73 134 122 91 65 92 71 240 131 80 120 108 107 65 103 74 360 174 101 116 111 149 86 149 88

Serum insulin concentrations were determined by competitive radioimmunoassay (RIA) as described above for studies 1 and 2. Data were analyzed by non-compartmental pharmacokinetic analysis using Phoenix WinNonLin. Pharmacokinetic parameters as a function of triphosphate concentration are presented in Table 1 below. 10.

Table 10

FC data

Dosage form Tshah (min.) Stach (nM) AiSbesk. (min.*nM) CL/F (ml/min/kg) HUMALOG N=13 Average ± SO 58.8±7.49 1.12±0.176 129±9.27 9.88 ± 0.669 Median 60 1.00 120 9.99 Dosage form F: HUMULIN + 20 mM triphosphate N=14 Average ± SO 22.5 ± 9.55 1.45±0.230 173 ± 14.6 7.48 ± 0.527 Median 7.50 1.12 159 7.54 Dosage form G: APIDRA + 20 mM triphosphate N= 15 Average ± SO 17.7±4.02 1.65±0.196 166 ± 11.7 7.75±0.527 Median 10.0 1.52 153 7.85 Dosage form H: NOVOLOG + 20 mM triphosphate N=14 Average ± SO 27.9 ± 10.8 1.10±0.111 121±12.7 10.9±0.784 Median 12.5 0.980 108 ID

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Abbreviations:

T _max - time at the maximum concentration of insulin,

C _max - the maximum concentration of insulin,

AUC _ins . - area under the curve from 0 to infinity,

CL/F - clearance/bioavailability

PK/PD data demonstrate that the use of triphosphate in formulations of various commercial insulins accelerates the time of action and shortens Tmax, compared with HUMALOG without triphosphate. All dosage forms containing 20 mm triphosphate caused a reduction in Tmax compared with pure HUMALOG. Dosage forms containing APIDRA and HUMULIN with 20 mM triphosphate caused an increase in C _max compared to pure HUMALOG.

Clinical Study

Conducted a clinical study to study the pharmacokinetic and pharmacodynamic action of the compositions according to the present invention. The study was designed as a 5-period crossover study to compare the effects of subcutaneous (SC) doses of 4 dosage forms containing various concentrations of triphosphate with insulin lispro with insulin lispro without triphosphate. Experimental formulations were formulated by adding sufficient triphosphate and magnesium chloride to commercial insulin lispro U200 to achieve the concentrations shown in Table 1 below. eleven.

Table 11

Compositions of experimental and control preparations

Name of the drug The composition of the drug HUMALOG 100 U/ml insulin lispro 7 mM sodium phosphate 0.3 mM Zn 16 mg/ml glycerol 3.15 mg/ml m-cresol Dosage Form I + 10 mM triphosphate Dosage form J + 20 mM triphosphate Dosage form K + 30 mM triphosphate Dosage form L + 30 mM triphosphate + 7.5 mM MgCl ₂

In addition to insulin lispro, the HUMALOG U-200 dosage form, to which triphosphate and MgCl ₂ were added to obtain experimental preparations, also contained 5 mg/ml tromethamine, 16 mg/ml glycerol, 3.15 mg/ml m-cresol and 0.046 mg/ml. ml Zn ²⁺ .

Healthy subjects were included in the study and each subject was randomly assigned a treatment sequence comprising 15 single SC doses of pure insulin lispro and 15 single SC doses of each of the experimental drugs. For each individual subject, a minimum of 3 days should be allowed between doses.

Blood samples were collected at multiple time points to determine serum insulin lispro concentrations as a function of time. Serum insulin lispro concentrations were measured using a reliable enzyme-linked immunosorbent assay specific for insulin lispro. Pharmacokinetic analyzes were performed using standard non-companment assay methods using Phoenix® version 7.0 (or higher) and S-PLUS® software (version 8.2). Serum concentrations of free insulin lispro were used to calculate pharmacokinetic parameters. The results are presented below in table. 12.

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Table 12

FC data

Treatment LS mean values Experimental drug/reference ratio (95% CI) Tn. (MIN.) Humalog 1.78 Dosage Form I 0.12 0.07 (0.04, 0.10) Dosage form J 0.10 0.05 (0.04, 0.08) Dosage form K 0.09 0.05 (0.03, 0.07) Dosage form L 0.08 0.04 (0.03, 0.07) Early 50% t _max (min.) Humalog 19.06 Dosage Form I 6.04 0.32 (0.26, 0.38) Dosage form J 5.04 0.26 (0.23,0.31) Dosage form K 4.73 0.25 (0.21.030) Dosage form L 4.58 0.24 (0.20, 0.29) AUC(0-15mhh.) Humalog 19.87

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(pmol*h/l) Dosage Form I 136.98 6.89 (5.44, 8.74) Dosage form J 155.78 7.84 (6.19, 9.93) Dosage form K 156.21 7.86 (6.21.9.96) Dosage form L 157.25 7.92 (6.27, 10.00) AUC(0-30mhh.) (pmol*h/l) Humalog 120.07 Dosage Form I 371.42 3.09 (2.63, 3.64) Dosage form J 393.84 3.28 (2.79, 3.86) Dosage form K 397.06 3.31 (2.81.3.89) Dosage form L 393.79 3.28 (2.79, 3.85) Late 50% t max (min.) Humalog 168.28 Dosage Form I 86.10 0.51 (0.45,0.58) Dosage form J 82.10 0.49 (0.42, 0.57) Dosage form K 80.80 0.48 (0.44, 0.52) Dosage form L 84.52 0.50 (0.45, 0.56)

Abbreviations:

LS - obtained by the method of least squares, CI - confidence interval,

T _start . - time to the onset of insulin, early 50%,

Tmax - time to early half-maximal drug concentration, AUC( ₀ -15 _min ) - area under the curve from time zero to 15 min, AUC( ₀ - _{30 min} ) - area under the curve from zero time to 30 min, later than 50% Tmax - time to late half-maximal drug concentration

The P-value for all experimental formulations compared to the Humalog control formulation was <0.0001.

The results demonstrate that dosage forms containing triphosphate are characterized by accelerated pharmacokinetic parameters compared to a control sample that does not contain triphosphate.

In addition, a 5-hour euglycemic glucose clamp test was performed at each period to assess the glucodynamic response to each treatment. In this study, glucose infusion rate (GIR) versus time was used as a measure of insulin exposure. All individual GIR versus time profiles were locally weighted scatterplot smoothing (LOESS) in each experimental group and/or period using S-PLUS software version 8.2. The smoothed data for each subject was used to calculate glucodynamic parameters.

Data analysis demonstrates that dosage forms containing triphosphate are characterized by improved pharmacodynamic parameters compared to a control sample that does not contain triphosphate. The studies described above demonstrate that the addition of small amounts of certain polyphosphates, such as pyrophosphate or triphosphate, to insulin formulations can cause a reduction in _Tmax and an increase in _Cmax in the pharmacokinetic profile of insulin.

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Claims (15)

Sequences. A-chain of human insulin Gly Not Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn (SEQ ID NO:1) B-chain of human insulin Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr (SEQ ID NO: 2). B-chain of insulin lispro Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr (SEQ ID NO: 3). B-chain of insulin aspart Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr (SEQ ID NO: 4). B-chain of insulin glulisine Phe Vai Lys Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr (SEQ ID NO: 5). CLAIM 1. Pharmaceutical composition containing insulin and from about 10 to about 30 mm triphosphate, provided that the specified composition does not contain any saccharide multimer or EDTA. 2. Pharmaceutical composition according to claim 1, characterized in that the concentration of triphosphate is from about 20 to about 25 mm. 3. Pharmaceutical composition according to claim 2, characterized in that the triphosphate concentration is about 20 mM. 4. Pharmaceutical composition according to any one of claims 1 to 3, additionally containing zinc. 5. Pharmaceutical composition according to claim 4, characterized in that the zinc concentration is from about 0.2 to about 5 mm. 6. Pharmaceutical composition according to any one of claims 1 to 5, additionally containing a tonicity agent. 7. Pharmaceutical composition according to claim 6, characterized in that the tonicity agent is glycerol. 8. Pharmaceutical composition according to any one of claims 1 to 7, additionally containing one or more preservatives. 9. Pharmaceutical composition according to claim 8, characterized in that one or more preservatives are selected from the group consisting of phenol, meta-cresol and benzyl alcohol. 10. Pharmaceutical composition according to any one of claims 1 to 9, characterized in that the insulin is selected from the group consisting of human insulin, insulin lispro, insulin aspart and insulin glulisine. 11. Pharmaceutical composition according to any one of claims 1 to 10, characterized in that the insulin concentration is from about 40 to about 500 IU/ml. 12. Pharmaceutical composition according to claim 11, characterized in that the concentration of insulin is from about 100 to about 200 IU/ml. 13. A method of treating diabetes, comprising administering to a person in need thereof an effective dose of a pharmaceutical composition according to any one of claims 1-12. 14. The use of a pharmaceutical composition according to any one of claims 1 to 12 for the treatment of diabetes. 15. An insulin delivery device comprising any of the pharmaceutical compositions of claim 112, wherein the device is a refillable vial, a refillable pen, a prefilled disposable pen, an autoinjector, or a continuous subcutaneous insulin infusion (CSII) pump. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2