THERMOSTABLE FORMULATION OF A21G HUMAN INSULIN

Abstract

A composition in the form of an injectable aqueous solution, the pH of which is between 7.2 and 8.0 (7.2<pH<8.0) and which includes at least A21G human insulin, the composition being intended to be used in a method for treating diabetes, wherein it is administered as a bolus before meals.

Claims

1. A composition in the form of an injectable aqueous solution, the pH of which is between 7.2 and 8.0 (7.2≤pH≤8.0) comprising at least A21G human insulin.

2. The composition according to claim 1, wherein the concentration of A21G human insulin is between 40 and 1000 U/mL (40 U/mL ≤concentration of A21G human insulin≤1000 U/mL).

3. The composition according to claim 1, wherein the concentration of A21G human insulin is 100 U/mL.

4. The composition according to any one of claims 1 to 3, claim 1, wherein the concentration of A21G human insulin is 300 U/mL.

5. The composition according to claim 1, wherein the zinc salt concentration is between 50 and 600 μM per 100 U/mL of A21G insulin.

6. The composition according to claim 1, wherein the zinc salt concentration is 230 μM per 100 U/mL of A21G insulin.

7. The composition according to claim 1, wherein the composition further comprises a phenolic preservative.

8. The composition according to claim 7, wherein the concentration of phenolic preservative is between 15 and 100 mM.

9. The composition according to claim 7, wherein the phenolic preservative is phenol.

10. The composition according to claim 7, wherein the phenol concentration is between 30 and 75 mM (30 mM≤phenol concentration≤75 mM).

11. The composition according to claim 10, wherein the phenol concentration is 50 mM.

12. The composition according to claim 1, wherein it further comprises a surfactant.

13. The composition according to claim 12, wherein the surfactant is chosen from polysorbates.

14. The composition according to claim 12, wherein the surfactant is chosen from polysorbate 20 or “Tween® 20”.

15. The composition according to claim 13 or 14, claim 13, wherein the polysorbate concentration is between 4 and 20 μM (4 μM≤polysorbate concentration≤20 μM).

16. The composition according to claim 1, wherein it further comprises arginine.

17. The composition according to claim 1, wherein it comprises trishydroxymethylaminomethane.

18. The composition according to claim 17, wherein it comprises trishydroxymethylaminomethane at a concentration between 2 and 100 mM (2 mM≤trishydroxymethylaminomethane concentration≤100 mM).

19. A method for treating diabetes, comprising administering the composition according to claim 1 as a bolus before meals.

20. A method for treating diabetes, comprising administering the composition according to claim 1 as prandial insulin.

Description

DESCRIPTION OF FIGURES

[0218] FIG. 1 represents the pharmacodynamics of composition B5-1 at t0 (i.e., stored at 4° C.) and at t12 weeks (composition B5-1 “aged” at 40° C. for 12 weeks) and composition B4, commercial formulation of human insulin Humulin®.

[0219] In this figure the x-axis represents the time after injection (in minutes) and the y-axis represents blood glycemia (in mg/dL).

FIG. 1:

[0220] Are shown in FIG. 1 the pharmacodynamic results obtained with composition B5-1 administered at t0 (curve plotted with squares) and at t12 weeks at 40° C. (curve plotted with triangles) in comparison with the results obtained with composition B4 (reference human insulin) (curve plotted in dotted lines with circles).

[0221] The following examples illustrate the invention in a non-limiting manner.

EXAMPLES

A Chemistry

Example A1: Preparation of A21G Human Insulin (A21G Insulin)

[0222] Insulin glargine (5 g; Gan & Lee Pharmaceuticals) was mixed with the enzyme carboxypeptidase B (Reference 08039852001; Sigma-Aldrich) at a 1:500 ratio (w/w) at pH 8.0 (pH adjusted by adding trishydroxymethylaminomethane buffer), the final insulin glargine concentration being about 4 mg/m L.

[0223] The solution was left under gentle stirring at 25° C. for 17 h. The mixture was then purified by liquid chromatography, dialyzed against 0.01 N hydrochloric acid then lyophilized.

[0224] A21G human insulin was obtained with a purity of 98% and an approximately 90% yield of.

[0225] The molar mass of A21G insulin measured by mass spectrometry (Maldi-Tof) is 5752 Da.

B Compositions

Example B2: Composition of Rapid Insulin Analog (Humalog®) at 100 U/mL

[0226] This composition is a commercial solution of insulin lispro marketed by ELI LILLY under the name Humalog®. This product is a rapid insulin analog. The excipients in Humalog® are metacresol (3.15 mg/mL), glycerol (16 mg/mL), disodium phosphate (1.88 mg/mL), zinc oxide (to obtain 0.0197 mg zinc ion/mL), sodium hydroxide and hydrochloric acid for pH adjustment (pH 7-7.8) and water.

Example B3: Composition of Slow Insulin Analog (Lantus®) at 100 U/mL

[0227] This composition is a commercial solution of insulin glargine marketed by SANOFI under the name Lantus®. This product is a slow insulin analog. The excipients in Lantus® are zinc chloride (30 μg/mL), m-cresol (2.7 mg/mL), glycerol (20 mg/mL), polysorbate 20 (16 μM), hydroxide sodium and hydrochloric acid for pH adjustment (pH 4) and water.

Example B4: Composition of Human Insulin (Humulin® R) at 100 IU/mL

[0228] This composition is a commercial human insulin solution from ELI LILLY sold under the name Humulin® R. This product is human insulin. The excipients of Humulin® R are glycerol, metacresol, sodium hydroxide and hydrochloric acid for pH adjustment (pH 7.0-7.8) and water.

Example B5: Preparation of Compositions of A21G Insulin at Concentrations of Between 100 and 300 U/mL and Excipients

[0229] A21G human insulin compositions were prepared according to the following protocol.

[0230] An aqueous solution of A21G human insulin at pH 7.3±0.2 is prepared from the lyophilizate obtained in Example A1 and an amount of sodium hydroxide or hydrochloric acid necessary for pH adjustment. This solution is added to a mixture of concentrated solutions of the excipients (zinc chloride, preservative, osmotic agent, trishydroxymethylaminomethane, arginine). A surfactant solution is then added to the mixture. The pH is measured and adjusted, if necessary, between 7.1±0.1 and 8.0±0.1 by adding concentrated solutions of sodium hydroxide or hydrochloric acid. A clear solution is obtained which does not contain visible particles. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the U.S. Pharmacopoeia (USP <790>). The preservative is either phenol (25 to 50 mM) or meta-cresol (25 mM). The concentrations of the stock solutions are chosen so as to obtain a concentration of trishydroxymethylaminomethane from 0 to 50 mM and/or arginine from 0 to 50 mM in the final composition. The concentration of osmotic agent stock solution, here glycerol, is chosen so as to obtain a glycerol concentration of between 50 and 210 mM in the final composition. The surfactant used may be Tween® 20, at a concentration of at least 8 μM, or Poloxamer® 188, at a concentration of 1.2 mg/mL, in the final composition. The concentration of A21G human insulin, C.sub.IHA21G, is between 100 and 300 U/mL. The zinc chloride concentration (μM) corresponds to 2 to 3 times the concentration of A21G human insulin (U/m L), i.e., C.sub.ZnCl2 (μM)=2-3 C.sub.IHA21G (U/mL).

[0231] These compositions are presented in Table 1 below.

Example B6: Preparation of Compositions of A21G Human Insulin at Concentrations of Between 100 and 300 U/mL (Corresponding to 3.5 and 10.5 mg/mL) and of Excipients at pH 4

[0232] These compositions were produced according to the protocol described in Example B5 from an aqueous solution of A21G human insulin at pH 4.0±0.2, prepared from the lyophilizate obtained in Example A1 and an amount of sodium hydroxide or hydrochloric acid allowing adjustment of the pH. The concentration of trishydroxymethylaminomethane is between 0 and 50 mM in the final composition. The concentrations of the other excipients are similar to those mentioned in Example B5.

[0233] These compositions are presented in Table 1 below.

TABLE-US-00001 TABLE 1 A21G insulin compositions prepared according to protocols B5 (B5-1 to B5-13) and B6 (B6-1 to B6-3) Com- Gly- po- Insulin Tris [Zn] cerol pH sition (mg/mL (mM) Additive Surfactant (μM) (mM) (±0.1) B5-1 3.5 10 Phenol Tween ® 20 230 200 7.4 (50 mM) B5-2 3.5 10 Phenol Poloxamer ® 230 184 7.4 (50 mM) 188 (1.2 mg/mL) B5-3 3.5 — m-cresol Poloxamer ® 300 184 7.4 (25 mM) 188 (1.2 mg/mL) B5-4 3.5 10 Phenol Tween ® 20 230 200 7.0 (50 mM) (8 μM) B5-5 3.5 10 Phenol Tween ® 20 230 200 7.2 (50 mM) (8 μM) B5-6 3.5 10 Phenol Tween ® 20 230 200 7.5 (50 mM) (8 μM) B5-7 3.5 10 Phenol Tween ® 20 230 200 8.0 (50 mM) (8 μM) B5-8 10.5 — m-cresol Poloxamer ® 900 184 7.4 (25 mM) 188 (1.2 mg/mL) B5-9 3.5 — Phenol Tween ® 20 230 184 7.4 (50 mM) (8 μM) Arginine (50 mM)  B5-10 7 10 Phenol Tween ® 20 460 184 7.4 (50 mM) (8 μM)  B5-11 10.5 10 Phenol Tween ® 20 690 184 7.4 (50 mM) (8 μM)  B5-12 3.5 50 Phenol Tween ® 20 230 60 7.4 (50 mM) (8 μM) Arginine (40 mM) B6-1 3.5 — m-cresol Poloxamer ® 300 184 4.0 (25 mM) 188 (1.2 mg/mL) B6-2 3.5 — m-cresol Poloxamer ® — 184 4.0 (25 mM) 188 (1.2 mg/mL) B6-3 10.5 — m-cresol Tween ® 20 — 184 4.0 (25 mM) (8 μM)  B5-13 10.5 10 Phenol Tween ® 20 690 200 7.4 (50 mM) (8 μM)

C Stability

Example C1: Study of Physical Stability at 50° C. of the Compositions Prepared in Comparison with Commercial Insulins at a Concentration of 100 U/mL in Vials

[0234] At least five 3-mL vials filled with 1 mL of composition are placed vertically in an oven maintained at 50° C. The vials are visually inspected at least weekly for the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the U.S. Pharmacopoeia (USP <790>): the vials are subjected to lighting of at least 2000 Lux and are observed against a white background and a black background. The number of weeks of stability corresponds to the time during which at least half of the vials remain clear and contain no visible particles on inspection.

[0235] The physical stability results obtained with different compositions are presented in the table below.

TABLE-US-00002 TABLE 2 Results of physical stability studies at 50° C. of compositions B5-1 to B5-3, B5-9, B5-12, B6-1 to B6-3 and B2 to B4. Insulin concentration Composition Insulin (mg/mL) pH 50° C. B5-1 A21G 3.5 7.4 No particles at 15 weeks B5-2 A21G 3.5 7.4 No particles at 15 weeks B5-3 A21G 3.5 7.4 No particles at 8 weeks B5-9 A21G 3.5 7.4 No particles at 9 weeks B5-12 A21G 3.5 7.4 No particles at 6 weeks B6-1 A21G 3.5 4.0 Particles at 1 week B6-2 A21G 3.5 4.0 Particles at 1 week B6-3 A21G 3.5 4.0 Particles at 4 weeks B2 (Humalog ®) Insulin lispro 3.5 7.4 Particles at 1 week B3 (Lantus ®) Insulin glargine 3.6 4.0 Particles at 1 week B4 Human insulin 3.5 7.4 Particles at 2 (Humulin ® R) weeks

[0236] The human insulin compositions A21G at pH 4.0 (B6-1 to B6-3) and the commercial formulations (B2 to B4) are less stable at 50° C. than compositions containing A21G human insulin at pH 7.4 (B5-1 to B5-3, B5-9, and B5-12). These compositions (B5-1 to B5-3, B5-9, and B5-12) make it possible to obtain a physical stability of at least 6 weeks at 50° C.

Example C2: Chemical Stability Study at 40° C. and 50° C.

[0237] The compositions, presented in the following table, are placed in an oven maintained at 40° C. or 50° C.

[0238] The compositions are analyzed at 4, 8, 12 weeks at 40° C. and at 2 weeks at 50° C. At the end of each time interval, a sample is analyzed by RP-HLPC-UV (214 nm) with a Cis column and a phosphate/acetonitrile buffer elution gradient in the presence of sodium heptanesulphonate to determine by surface percentage the proportion of deamidated products dB3/disoB3 as well as other impurities (designated total impurities). At the end of each time interval, a sample is also analyzed by SE-HPLC-UV (276 nm) with a silica column coated with 80-A pores and a water/acetonitrile/trifluoroacetic acid mobile phase to determine by surface percentage the proportion of high molecular weight products (HMWP).

[0239] The results are given as a percentage in the table below.

TABLE-US-00003 TABLE 3 Results of the chemical stabilities of solutions B5-1 to B5-3, B5-9, and B5-12 and commercial human insulin compositions Humulin ® R Com- po- Insulin Degradation 40° C. 40° C. 40° C. 50° C. sition (mg/mL) products T0 4 W 8 W 12 W 2 W B5-1  A21G dB3/disoB3 0.4 1.4 2.8 4.2 1.8 (3.5) HMWP 0.1 0.3 0.7 0.9 0.3 Total imp.* 0.3 1.9 3.7 5.0 2.2 B5-2  A21G dB3/disoB3 0.1 — — — 2.0 (3.5) HMWP 0.2 0.5 Total imp.* 0.3 2.5 B5-3  A21G dB3/disoB3 0.2 3.0 5.7 8.7 4.2 (3.5) HMWP 0.1 0.4 1.0 1.9 0.5 Total imp.* 0.6 1.4 4.1 5.2 2.3 B5-9  A21G dB3/disoB3 0.1 — — — 1.8 (3.5) HMWP 0.1 0.1 Total imp.* 0.3 2.5 B5-12 A21G dB3/disoB3 0.1 1.4 2.8 4.1 1.6 (3.5) HMWP 0.1 0.2 0.2 0.3 0.1 Total imp.* 0.4 2.3 4.3 5.6 2.5 B4 Human dB3/disoB3 2.5 11.7 — — 6.4 insulin HMWP 0.3 2.1 — — 3.4 (3.5) Total imp.* 1.4 12.8 — — 6.5 *Total imp. means all impurities except dB3/disoB3 and HMWP “—” means not measured

[0240] The commercial composition Humulin® R (B4) is analyzed after 4, 8 and 12 weeks at 40° C. and after 2 weeks at 50° C. At these time intervals, this composition no longer meets the specifications of the European Pharmacopoeia in terms of physical stability (see above), nor in terms of percentage of impurities. Formulations B5-1 to B5-3, B5-9 and B5-12 are much more stable.

Example C3: Effect of pH on the Physical and Chemical Stability of Compositions Comprising A21G Human Insulin

[0241] Formulations B5-4 to B5-7 have a composition identical to that of formulation B5-1, except for the pH.

TABLE-US-00004 TABLE 4 Results of the effect of pH on the physical and chemical stability of solutions B5-4 to B5-7 Degradation products after 4 weeks Composition pH Vial 40° C. at 40° C. (relative to t0) B5-4 7.0 ± Particles observed — 0.1 immediately at t0 B5-5 7.2 ± No particles at 11 dB3/disoB3 1.3% 0.1 weeks HMWP 0.1% Total imp.* 1.8% B5-6 7.5 ± No particles at 11 dB3/disoB3 1.4% 0.1 weeks HMWP 0.2% Total imp.* 1.9% B5-7 8.0 ± No particles at 11 dB3/disoB3 1.6% 0.1 weeks HMWP 0.3% Total imp.* 2.4% *“total impurities” means all the impurities except dB3/disoB3 and HMWP —means not measured

[0242] The compositions have good stability between 7.2 and 8.0. It should be noted that even better stability at 40° C. is obtained in a pH range of between 7.2 and 7.5.

Example C4: Physical and Chemical Stability of A21G Human Insulin at 100 and 300 U/mL (i.e., 3.5 and 10.5 mg/mL), at pH 7.4

[0243]

TABLE-US-00005 TABLE 5 Results of the physical stability at 50° C. of the compositions at 100 and 300 U/mL of A21G insulin (B5-3 and B5-8). A21G insulin Formulation (mg/mL) 50° C. B5-3 3.5 No particles at 8 weeks B5-8 10.5 No particles at 4 weeks Particles at 5 weeks

TABLE-US-00006 TABLE 6 Results of the chemical stabilities at 40 and 50° C. of the compositions at 100 and 300 U/mL of A21G insulin (B5-3 and B5-8). Com- A21G Deg- po- insulin radation 40° C. 40° C. 40° C. 50° C. sition (mg/mL) products T0 4 W 8 W 12 W 2 W B5-3 3.5 dB3/disoB3 0.2 3.0 5.7 8.9 4.2 HMWP 0.1 0.4 1.0 1.9 2.3 Total imp. 0.6 1.4 4.1 5.2 0.5 B5-8 10.5 dB3/disoB3 0.4 1.9 3.8 5.9 2.7 HMWP 0.1 0.2 0.5 0.9 2.0 Total imp. 1.2 1.3 3.1 3.9 0.4 “*” “Total imp.” means all impurities except dB3/disoB3 and HMWP

[0244] The results are expressed as a percentage in the table below.

[0245] The results presented above show that composition B5-8 (U300) has good physical stability and excellent chemical stability.

Example C5: Physical and Chemical Stability of A21G Human Insulin at 100 and 300 U/mL (i.e., 3.5 and 10.5 mg/mL) in Cartridges (3 mL), at 40° C.

[0246] The compositions B5-1 and B5-13 of A21G human insulin are inserted into 3-mL cartridges and placed in an oven at 40° C. under static conditions. Cartridges are visually inspected every two weeks for the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the U.S. Pharmacopoeia (USP <790>) described for solutions in vials. Thus, the cartridges are subjected to lighting of at least 2000 Lux and are observed against a white background and a black background. The number of weeks of stability corresponds to the time during which at least half of the cartridges remain clear and do not contain visible particles on inspection.

[0247] The cartridges are analyzed at 4, 8 and 12 weeks at 40° C. At the end of each time interval, a cartridge is taken and an aliquot is analyzed by RP-HLPC-UV (214 nm) with a C18 column and a phosphate/acetonitrile buffer elution gradient in the presence of sodium heptanesulphonate to determine by surface percentage the proportion of deamidated products dB3/disoB3 as well as other impurities (designated total impurities). At the end of each time interval, an aliquot is also analyzed by SE-HPLC-UV (276 nm) with a silica column coated with 80-A pores and a mobile phase of water/acetonitrile/trifluoroacetic acid to determine by surface percentage the proportion of high molecular weight products (HMWP).

[0248] The results are given as a percentage in the table below.

TABLE-US-00007 TABLE 6a Results of the physical stability at 40°C. of the compositions at 100 and 300 U/mL of A21G insulin (B5-1 and B5-13). A21G insulin Composition (mg/mL) 40° C. B5-1 3.5 No particles at 12 weeks B5-13 10.5 No particles at 10 weeks

TABLE-US-00008 TABLE 6b Results of the chemical stabilities of solutions B5-1 and B5-13. Com- Deg- po- Insulin radation 40° C. 40° C. 40° C. 40° C. sition (mg/mL) products T0 4 W 8 W 11 W 12 W B5-1  A21G dB3/disoB3 0.1 1.4 2.6 — 3.8  (3.5) HMWP 0.2 0.3 0.4 — 0.7 Total imp.* 0.6 1.9 3.1 — 4.5 B5-13 A21G dB3/disoB3 0.3 — — 3.2 — (10.5) HMWP 0.2 — — 0.5 — Total imp.* 0.4 — — 4.0 — *Total imp. means all the impurities except dB3/disoB3 and HMWP

[0249] The results are expressed as a percentage in the table below.

[0250] The results presented above show that the compositions B5-1 (U100) and B5-13 (U300) have excellent physical and chemical stability.

D Pharmacodynamics

Example D1: Protocol for Measuring the Pharmacodynamics of Insulin Solutions

[0251] Domestic pigs of around 50 kg, previously catheterized at the jugular, are fasted 2.5 hours before the start of the experiment. In the hour before the insulin injection, 3 blood samples are taken to determine the basal glucose level.

[0252] The injection of the insulin M1 or human insulin formulations at a dose of 0.2 μl/kg is carried out subcutaneously in the flank of the animal using an insulin pen (Novo, Sanofi or Lilly) equipped with a 31 G needle.

[0253] Blood samples are then taken at the following times: 4, 8, 12, 16, 20, 30, 40, 50, 60, 70, 80, 100, 120, 150 and 180 minutes. After each sample, the catheter is rinsed with a dilute heparin solution. A drop of blood is taken to determine the blood glucose level using a glucometer. The average blood glucose curves, expressed in mg/dL, are then plotted.

Example D2: Pharmacodynamic Results of the Insulin Solution A21G B5-1 at Time Zero (t0, i.e., Stored at 4° C.) and after 12 Weeks at 40° C. and of a Solution of Human Insulin

[0254]

TABLE-US-00009 TABLE 7 Compositions used for pharmacodynamics A21G human insulin Human insulin Composition (U/mL) (U/mL) Number of pigs B5-1 100 — 12 B4 — 100 11

[0255] The pharmacodynamic results obtained with the composition B5-1 are illustrated in FIG. 1. Analysis of these profiles indicates that similar hypoglycemic actions are obtained with composition B5-1 administered at t0 (curve plotted with squares) and t12 weeks at 40° C. (curve plotted with triangles). On the other hand, composition B5-1, administered at t0 or at t12 weeks at 40° C., has a hypoglycemic action similar to that obtained with composition B4 (reference human insulin) (curve plotted in dotted lines with circles).