Injectable solution at pH 7 comprising at least one basal insulin for which the pI is from 5.8 to 8.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals

Abstract

A composition in the form of an injectable aqueous solution, whose pH consists from 6.0 to 8.0, including at least: a basal insulin whose isoelectric point includes from 5.8 to 8.5; a co-polyamino-acid bearing carboxylate charges and hydrophobic radicals Hy, the co-polyamino-acid being constituted of glutamic or aspartic units and said hydrophobic radicals Hy according to the following formula I: ##STR00001##

Claims

1. A composition in the form of an injectable aqueous solution, whose pH is comprised from 6.0 to 8.0, comprising at least: a) a basal insulin whose isoelectric point (pI) is comprised from 5.8 to 8.5; b) a co-polyamino acid bearing carboxylate charges and hydrophobic radicals Hy, the co-polyamino acid being constituted of glutamic or aspartic units and the hydrophobic radicals Hy according to the following formula I: ##STR00047## wherein GpR is a radical according to formulas II or II′:
*—CO—R—CO—*  II
or
*—NH—R—CO—*  II′; GpA is a radical according to formula III or III′: ##STR00048## GpC is a radical according to formula IV: ##STR00049## the -* indicate the binding sites of the different groups, that is the co-polyamino acids, GpR, GpA and GpC, bound by amide functions; b is an integer equal to 0 or to 1; p is an integer equal to 2 or 3 and if p is equal to 2 then GpA is a radical according to formula III and, if p is equal to 3 then GpA is a radical according to formula III′; c is an integer equal to 0 or 1, and if c is 0 then d is 1 or 2; d is an integer of 0, 1 or 2; r is an integer equal to 0 or 1, and if r is equal to 0, then the hydrophobic radical according to formula I is bound to the co-polyamino acid through a covalent bond between a carbonyl of the co-polyamino acid and one of the nitrogen atoms of the radical GpA, thereby forming an amide function from the reaction of an amine function, either a primary amine or a secondary amine of the precursor of GpA and an acid function borne by the precursor of the co-polyamino acid, and if r is equal to 1 or 2, then the hydrophobic radical according to formula I is bound to the co-polyamino acid: through a covalent bond between a nitrogen atom of the radical GpR and a carbonyl of the co-polyamino acid, thus forming an amide function resulting from the reaction of an amine function of the precursor of the radical GpR and an acid function borne by the precursor of the co-polyamino acid or through a covalent bond between a carbonyl of the radical GpR and a nitrogen atom in N-terminal position of the co-polyamino acid, thus forming an amide function resulting from the reaction of an acid function of the precursor of the radical GpR and an amine function in N-terminal position borne by the precursor of the co-polyamino acid; R is a radical chosen from the group consisting of: a divalent alkyl radical, linear or branched, comprising from 1 to 11 carbon atoms; and a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms; A, A′ and A″ are identical or different and linear or branched alkyl radicals comprising from 2 to 6 carbon atoms; B is a linear or branched alkyl radical, optionally comprising an aromatic ring, comprising from 1 to 9 carbon atoms; C.sub.x is a monovalent alkyl radical, linear or branched, in which x indicates the number of carbon atoms and; if p is equal to 2, x is comprised from 9 to 15 (9≤x≤15); if p is equal to 3, x is comprised from 7 to 15 (7≤x≤15), the ratio i between the number of hydrophobic radicals and the number of glutamic or aspartic units being from 0 to 0.5 (0<i≤0.5); when several hydrophobic radicals are carried by a co-polyamino acid they are identical or different; the degree of polymerization DP of glutamic or aspartic units is comprised from 5 to 250; and the free acid functions being in the form of an alkaline cation salt chosen from the group consisting of Na.sup.+ and K.sup.+.

2. The composition according to claim 1, wherein Hy comprises from 34 to 70 carbon atoms.

3. The composition according to claim 1, wherein the hydrophobic radicals according to formula I are chosen among the hydrophobic radicals according to formula I wherein p=2, represented in the following formula VI: ##STR00050## wherein GpR, GpA, GpC and r are as defined above.

4. The composition according to claim 3, wherein in the hydrophobic radicals according to formula VI, the radical GpA is chosen from the group consisting of the radicals IIIa and IIIb: ##STR00051##

5. The composition according to claim 1, wherein the copolyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the copolyamino acids according to the following formula VII: ##STR00052## wherein, D is, independently, either a —CH2- group (aspartic unit) or a —CH2-CH2- group (glutamic unit), Hy is a hydrophobic radical chosen among the hydrophobic radicals according to formula I: ##STR00053## formula VI: ##STR00054## wherein GpR, GpA, GpC and r are as defined above, or formula IX: ##STR00055## wherein GpA is according to formula III′ and GpR, GpC and r are as defined above, R.sub.1 is a hydrophobic radical chosen among the hydrophobic radicals according to formula I, VI or IX wherein r=0 or r=1 and GpR is a radical according to formula II, or R.sub.1 is a radical chosen in the group constituted by a H, a C2 to C10 linear acylated group, a C3 to C10 branched acylated group, a benzyl, a terminal amino acid unit and a pyroglutamate, R.sub.2 is a hydrophobic radical chosen among the hydrophobic radicals according to formula I, VI or IX wherein r=0 or r=1 and GpR is a radical according to formula II′, or R.sub.2 is a —NR′R″, R′ and R″ identical or different being chosen in the group consisting of H, C2 to C10 linear, branched or cyclic alkyls, benzyl and said R′ and R″ alkyls may form together one or several saturated, unsaturated and/or aromatic carbon rings and/or may include heteroatoms, chosen in the group consisting of O, N and S; X is a cationic entity chosen in the group consisting of the alkali metal cations; n+m refers to the degree of polymerization DP of the co-polyamino acid, that is the mean number of monomeric unit in a co-polyamino acid chain and 5≤n+m≤250.

6. The composition according to claim 5, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula VII, wherein R.sub.1=R′.sub.1 and R.sub.2=R′.sub.2, according to the following formula VIIa: ##STR00056## wherein, m, n, X, D and Hy are as defined above, R′.sub.1 is a radical chosen in the group constituted by H, a C2 to C10 linear acylated group, a C3 to C10 branched acylated group, a benzyl, a terminal amino acid unit and a pyroglutamate, R′.sub.2 is a radical —NR′R″, R′ and R″ identical or different being chosen in the group consisting of H, C2 to C10 linear, branched or cyclic alkyl and benzyl and said R′ and R″ alkyls may form together one or several saturated, unsaturated and/or aromatic carbon rings and/or may include heteroatoms, chosen in the group consisting of O, N and S.

7. The composition according to claim 5, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula VII wherein n=0 according to formula VIIb: ##STR00057## wherein m, X, D, R.sub.1 and R.sub.2 are as defined above and at least one of R.sub.1 or R.sub.2 is a hydrophobic radical according to formula I, VI or IX.

8. The composition according to claim 7, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula VIIb wherein R.sub.1 is a hydrophobic radical according to formula I, VI or IX wherein r=1 and GpR is according to formula II.

9. The composition according to claim 7, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula VIIb wherein R.sub.2 is a hydrophobic radical according to formula I, VI or IX wherein r=0 or r=1 and GpR is according to formula II′.

10. The composition according to claim 1, wherein the basal insulin for which the isoelectric point is comprised from 5.8 to 8.5 is insulin glargine.

11. The composition according to claim 1, wherein the basal insulin for which the isoelectric point is comprised from 5.8 to 8.5 is a bio-similar insulin glargine.

12. The composition according to claim 1, wherein the composition comprises from 40 to 500 U/mL of basal insulin for which the isoelectric point is comprised from 5.8 to 8.5.

13. The composition according to claim 1, wherein the mass ratio between the basal insulin, for which the isoelectric point is comprised from 5.8 to 8.5, and the co-polyamino acid, that is co-polyamino acid/basal insulin, is comprised from 0.2 to 8.

14. The composition according to claim 1, wherein the composition further comprises a prandial insulin.

15. The composition according to claim 14, wherein the prandial insulin is a recombinant human insulin.

16. The composition according to claim 14, wherein the prandial insulin is chosen in the group consisting of insulin lispro, insulin glulisine and insulin aspart.

17. The composition according to claim 1, wherein the composition further comprises a gastrointestinal hormone.

18. The composition according to claim 17, wherein the gastrointestinal hormone is one of GLP-1 RA analogues or derivatives chosen in the group consisting of exenatide, liraglutide, lixisenatide, albiglutide or dulaglutide, their analogues or derivatives and their pharmaceutically acceptable salts.

Description

(1) FIG. 1: FIG. 1 depicts the glycemia average curves in percent of deviation with respect to the basal level±standard error of the average after simultaneous and separated administrations of insulin lispro (Humalog®—example C1) (100 IU/mL, 0.17 IU/kg) and of insulin glargine (Lantus®—example C4) (100 IU/mL, 0.50 U/kg) (filled circles) and of composition CB3-1 (266 U/ml, 0.67 U/kg) (empty squares); administrations have been carried out on dogs (n=10), by subcutaneous injection, and on the x-axis is the post-injection time (in hours) and y-axis the glucose level (in % of deviation with respect to the basal level).

EXAMPLES

(2) The invention is described in more details with the following examples in a non-limited manner.

Part A—Synthesis of Hydrophobic Molecules

(3) The structures of hydrophobic molecules are represented in Table 1.

(4) TABLE-US-00002 TABLE 1 List of hydrophobic molecules Hyd. Molecule Structures A1 A2 A4

Example A1: Molecule A1

(5) Molecule 1

(6) Product Obtained by Coupling Between Lauric Acid and L-Proline

(7) To a solution of dodecanoïc acid (19.77 g, 98.70 mmol) in THF (1 L) are successively added dicyclohexyl carbodiimide (DCC) (20.77 g, 100.68 mmol) and N-hydroxysuccinimide (NHS) (11.59 g, 100.68 mmol). After stirring for 24 h at room temperature, the medium is cooled down to 0° C. and filtered on frit. L-proline (12.50 g, 108.57 mmol), diisopropylethylamine (DIPEA) (63.78 g, 493.51 mmol) and water (90 mL) are added to the filtrate. After stirring for 24 h to room temperature, the medium is concentrated under reduced pressure, then dissolved into water (300 mL). The aqueous phase is washed with ethyl acetate (2×400 mL), acidified until pH ˜1 with a 1 N HCl aqueous solution then extracted with dichloromethane (3×250 mL). The combined organic phases are dried over Na2SO4, filtered off, and concentrated under reduced pressure. Après purification by chromatography on silica gel (cyclohexane, ethyl acetate), a colourless oil of molecule 1 is obtained. Yield: 20.53 g (70%)

(8) NMR .sup.1H (CDCl.sub.3, ppm): 0.87 (3H); 1.26 (16H); 1.70 (2H); 1.90-2.10 (3H); 2.35 (2H); 2.49 (1H); 3.48 (1H); 3.56 (1H); 4.60 (1H)

(9) LC/MS (ESI): 298.2; (calculé ([M+H].sup.+): 298.3)

(10) Molecule 2

(11) Product Obtained by Coupling Between Molecule 1 and Spermidine

(12) To a solution of molecule 1 (20.48 g, 68.85 mmol) to 0° C. in dichloromethane (DCM, 340 mL) is added 1,1′-carbonyldiimidazole (CDI) (11.05 g, 68.16 mmol) and the reaction medium is stirred at room temperature over 1.5 h. Spermidine (5.41 mL, 34.42 mmol) is added and the mixture is stirred over 20 h. The solution is introduced into water, the phases are separated then the aqueous phase is extracted twice with dichloromethane. The combined organic phases are dried over Na2SO4, filtered, concentrated under vacuum and the residue is purified by gel chromatography on silica (dichloromethane, 7 N ammonia in methanol). The product obtained which is contaminated with imidazole, is solubilized in dichloromethane and washed successively with a NaHCO.sub.3 saturated aqueous solution (twice) then water (twice). The organic phase is dried over Na2SO4, filtered, and concentrated under vacuum to give a yellowish oil. Yield: 17.0 g (70%)

(13) NMR .sup.1H (DMSO-d6, ppm): 0.85 (6H); 1.10-1.55 (42H); 1.71-2.03 (8H); 2.08-2.29 (4H); 2.43-2.52 (4H); 2.97-3.16 (4H); 3.20-3.57 (5H); 4.16-4.28 (2H); 7.62-8.09 (2H)

(14) LC/MS (ESI): 704.7; (calculated ([M+H].sup.+): 704.6)

(15) Molecule A1

(16) To a solution of de molecule 2 (17.0 g, 24.14 mmol) in dichloromethane (120 mL) is added a 4 N chlorhydric acid solution in dioxane (12.07 mmol, 48.29 mmol) at 0° C. The reaction medium is stirred at room temperature over 1 h, then concentrated under reduced pressure. The residue is solubilized in methanol and concentrated under vacuum. The residue is triturated in acetonitrile, filtered, washed with acetonitrile and dried under reduced pressure at 50° C. The molecule A1 is obtained as a white solid.

(17) Yield: 12.39 g (69%)

(18) NMR .sup.1H (DMSO, ppm): 0.85 (6H); 1.08-1.63 (40H); 1.70-2.33 (14H); 2.78-2.88 (4H); 3.00-3.18 (4H); 3.31-3.59 (4H); 4.12-4.34 (2H); 7.77-8.32 (2H); 8.60-8.90 (2H) LC/MS (ESI): 704.8; (calculated ([M+H].sup.+): 704.6)

Example A2: Molecule A2

(19) Molecule 3

(20) Product Obtained by Coupling Between Myristic Acid and L-Proline

(21) Using a similar process than the one used for preparing molecule 1 applied to myristic acid (18.03 g, 78.96 mmol) and to L-proline (10.00 g, 86.86 mmol), a colourless oil of molecule 3 is obtained.

(22) Yield: 17.66 g (69%)

(23) NMR .sup.1H (CDCl.sub.3, ppm): 0.88 (3H); 1.28 (20H); 1.70 (2H); 1.90-2.10 (3H); 2.36 (2H); 2.51 (1H); 3.47 (1H); 3.56 (1H); 4.61 (1H) LC/MS (ESI): 326.4; (calculated ([M+H].sup.+): 326.3)

(24) Molecule 4

(25) Product Obtained by Coupling Between La Molecule 3 and La Spermidine

(26) To a solution of molecule 3 (17.41 g, 53.50 mmol) at 0° C. in dichloromethane (260 mL) is added 1,1′-carbonyldiimidazole (CDI) (8.67 g, 53.50 mmol) and the reaction medium is stirred at room temperature over 1.5 h. Spermidine (4.20 mL, 26.75 mmol) is added and the mixture is stirred over 20 h. The reaction medium is washed 4 times with a 0.2 M NaHCO.sub.3 aqueous solution, dried over Na.sub.2SO.sub.4, filtered, and concentrated under vacuum to give a yellowish solid.

(27) Yield: 20.2 g (99%)

(28) NMR .sup.1H (DMSO-d6, ppm): 0.85 (6H); 1.09-2.30 (62H); 2.45-2.56 (4H); 2.96-3.56 (8H); 3.98-4.28 (2H); 7.69-8.14 (2H) LC/MS (ESI): 761.0; (calculated ([M+H].sup.+): 760.7)

(29) Molecule A2

(30) Using a similar process than the one used for preparing molecule A1 applied to molecule 4 (20.21 g, 26.59 mmol), a white solid is obtained after recrystallization in acetonitrile.

(31) Yield: 16.3 g (77%)

(32) NMR .sup.1H (DMSO-d6, ppm): 0.86 (6H); 1.08-2.36 (62H); 2.78-2.89 (4H); 3.02-3.18 (4H); 3.34-3.61 (4H); 4.14-4.32 (2H); 7.80-8.31 (2H); 8.60-8.88 (2H)

(33) LC/MS (ESI): 760.9; (calculated ([M+H].sup.+): 760.7)

Example A4: Molecule A4

(34) Molecule 6

(35) Product Obtained by Reacting Norspermidine and Di-Tert-Butyl Dicarbonate

(36) To a solution of norspermidine (99.2 g, 0.756 mol) in THF (1.1 L) to 0° C. is added di-tert-butyl dicarbonate (55.0 g, 0.252 mol) in THF (715 mL) dropwise over 2.5 h. After stirring for 24 h at 0° C., le solvent is evaporated under reduced pressure. The residue is taken up into water (600 mL) and the product is extracted with dichloromethane (7×500 mL). The combined organic phases are washed with a saturated NaCl aqueous solution (500 mL), dried over Na2SO4 and concentrated under reduced pressure. La molecule 6 is obtained as a colourless oil and used without further purification.

(37) Molecule 7

(38) Product Obtained by Coupling Between La Molecule 3 and La Molecule 6

(39) To a solution of molecule 3 (20.68 g, 63.50 mmol) and molecule 6 (7.0 g, 30.30 mmol) in DMF (70 mL) at 0° C. are successively added DIPEA (15.64 g, 121 mmol) and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 25.31 g, 66.60 mmol). The reaction mixture is stirred over 24 h with the temperature being risen until 25° C., then the mixture is introduced into iced water (700 mL). The product is extracted with ethyl acetate (300 mL), the organic phase is washed with a 1 N HCl aqueous solution (300 mL), a saturated sodium bicarbonate aqueous solution (300 mL), a saturated NaCl aqueous solution (500 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. A pale yellow oil of molecule 7 is obtained after three successive purifications by flash chromatography (eluents: DCM/MeOH, AcOEt/3% MeOH in DCM, DCM/MeOH).

(40) Yield: 18.9 g (74%)

(41) NMR .sup.1H (CDCl.sub.3, ppm): 0.87 (6H); 1.15-2.42 (69H); 2.79-3.83 (12H); 4.17-4.31 (0.5H); 4.46-4.65 (0.5H); 4.69-4.83 (0.5H); 5.19-5.30 (0.5H); 5.32-5.42 (0.5H); 5.95-6.08 (0.5H); 7.59-7.76 (0.5H); 7.98-8.14 (0.5H)

(42) LC/MS (ESI): 746.53, 846.60; (calculated ([M+H-Boc].sup.+): 746.65 ([M+H].sup.+): 846.70)

(43) Molecule A4

(44) To a solution of molecule 7 (15.82 g, 18.70 mmol) in dichloromethane (52 mL) at 0° C. is added a 5.7 N HCl solution in dioxane (16.4 mL, 93.50 mmol). After 48 h at 0° C., the reaction mixture is concentrated under reduced pressure. The residue is dissolved into dichloromethane (90 mL), cooled down at 0° C. and washed with an 1 N iced soda solution (2×75 mL) then a saturated NaCl aqueous solution (50 mL). After drying over Na.sub.2SO.sub.4, a spatula of activated charcoal is introduced, the medium is stirred over 30 minutes then filtered over celite. The molecule A4 is obtained after concentration under reduced pressure.

(45) Yield: 12.40 g (89%).

(46) NMR .sup.1H (CDCl.sub.3, ppm): 0.87 (6H); 1.08-2.47 (62H); 2.50-3.92 (12H); 4.16-4.33 (0.5H); 4.40-4.62 (0.5H); 4.70-4.88 (0.5H); 5.25-5.39 (0.5H); 7.53-7.77 (0.5H); 7.98-8.28 (0.5H)

(47) LC/MS (ESI): 746.55; (calculated ([M+H].sup.+): 746.65)

Part B—Synthesis of Grafted Copolyamino Acids with Hydrophobic Molecules

(48) The structures of hydrophobic copolyamino acids are represented in Table 2.

(49) TABLE-US-00003 TABLE 2 list and structures of grafted copolyamino acids with hydrophobic molecules Hy. Copolyamino acid Structure B1 0 B2 B3 B4

Example B1: Copolyamino Acid B1-Sodium Poly-L-Glutamate Modified at One of its Extremities with Molecule A2 with Number-Average Molecular Weight (Mn) of 3650 g/Mol

(50) Molecule 5:

(51) Product Obtained by Coupling Between Molecule A2 and the N-Hydroxysuccinimic Ester of N-Boc-γ-Tert-Butyl-L-Glutamate (BocGlu(OtBu)OSu)

(52) To a solution of BocGlu(OtBu)OSu (1.81 g, 4.52 mmol) in 30 mL of chloroform is added a solution of molecule A2 (3 g, 3.77 mmol) and N,N-diisopropylethylamine (DIPEA, 0.49 g, 3.77 mmol) in 8 mL of chloroform. After stirring for 18 h at room temperature, the organic phase is washed with water (75 mL) then with a saturated NaCl aqueous solution (5×80 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Molecule 5 is obtained after purification by flash chromatography (eluent: AcOEt/MeOH).

(53) Yield: 0.92 g (23%)

(54) NMR .sup.1H (CDCl.sub.3, ppm): 0.88 (6H); 1.15-2.44 (84H); 2.91-3.85 (12H); 4.25-4.75 (3H); 5.16-5.39 (1H); 7.28-7.72 (2H)

(55) LC/MS (ESI): 1046.0; (calculated ([M+H].sup.+): 1045.8)

(56) Molecule 6:

(57) ##STR00046##

(58) To a solution of Molecule 5 (0.92 g, 0.88 mmol) in 10 mL of dichloromethane is added a 4 N HCl solution in dioxane (2.2 mL, 8.76 mmol) and the reaction mixture is stirred over 18 h at room temperature. The medium is concentrated under reduced pressure, The residue is taken up in 10 mL of water, the pH is adjusted to 8.0 with the addition of a 1 N NaOH aqueous solution followed by 10 mL of water. The solution is lyophilized, the white solid obtained is then solubilized in chloroform (10 mL), concentrated under reduced pressure, toluene (10 mL) is added and the product is concentrated under vacuum once more, this process is repeated twice.

(59) Yield: 0,748 g (97%)

(60) NMR .sup.1H (CDCl.sub.3, ppm): 0.87 (6H); 1.05-2.88 (66H); 3.04-3.83 (12H); 4.41-4.83 (3H); 7.74-8.63 (5H)

(61) LC/MS (ESI): 889.8; (calculated ([M+H].sup.+): 889.7)

(62) Copolyamino Acid B1:

(63) In a oven-dried round-bottom flask, γ-benzyl-L-glutamate N-carboxyanhydride (29.3 g, 111 mmol) is solubilized in anhydrous DMF (116 mL). The mixture is stirred under argon until complete solubilization, cooled down to 4° C., then molecule 6 (4.50 g, 5 mmol) in chloroform (15 mL) is rapidly introduced. The mixture is stirred between 4° C. and room temperature over 17 h and heated at 65° C. over 2 h. The reaction mixture is concentrated under reduced pressure until the volume of solvent is half reduced, then poured dropwise in diisopropylether (820 mL) while stirring. After 1 h, the white precipitate is collected by filtration, triturated with diisopropylether until a white powder is obtained then is dried under vacuum at 30° C. to give a white solid. The solid (24.50 g) is dissolved in TFA (95 mL), and a 33% HBr solution in acetic acid (67 mL, 385 mmol) is then added dropwise at 0° C. La solution is stirred over 2.5 h at room temperature then poured dropwise over a 1:1 (v/v) diisopropylether/water mixture while stirring (1.2 L). After stirring for 2 h, the heterogenous mixture is rested overnight. The white precipitate is collected by filtration, then washed twice with diisopropylether (100 mL) and twice with water (100 mL). The solid obtained is solubilized in water (600 mL) by adjustment of the pH to 7.2 using 1 N soda aqueous solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoritically adding water to obtain a final volume of 900 mL. The mixture is filtered on 0.45 μm frit then purified by ultrafiltration against a 0.9% NaCl solution, a 0.1 N aqueous soda solution, a 0.9% NaCl solution, a phosphate buffer solution, a 0.9% NaCl solution, then water until the conductimetry of the permeate is inferior to 50 μS/cm. The copolyamino acid solution is then concentrated to about 25 g/L theoritically, the pH is adjusted to 7.2 and the solution is filtered on 0.2 μm frit. La solution is then filtered on 0.45 μm frit and purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is inferior to 50 μS/cm. The copolyamino acid solution is then concentrated to about 25 g/L theoritically, filtered on 0.2 μm frit and kept at 2-8° C.

(64) Dry extract: 15.5 mg/g

(65) DP (estimated by NMR .sup.1H)=27 that is i=0.037

(66) The calculated number-average molecular weight of copolyamino acid B1 is 4800 g/mol

(67) HPLC-SEC aqueous (calibrant PEG): Mn=3650 g/mol

Example B2: Copolyamino Acid B2: Poly-L-Glutamate De Sodium Capped to One of its Extremities with an Acyl Group and Modified with Molecule A2 Having a Number-Average Molecular Weight (Mn) of 3320 g/Mol

(68) Copolyamino acid B2-1: poly-L-glutamique acid of relative number-average molecular weight (Mn) relative of 3480 g/mol and DP 22 obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated using hexylamine and capped to one of its extremities with an acetyl group.

(69) In a 1 L round-bottom flask is added γ-benzyl-L-glutamate N-carboxyanhydride (Glu(OBzl)-NCA, 189 g, 716 mmol), then DMF (425 mL) is introduced. The mixture is then stirred under argon until complete dissolution, cooled down to 4° C., then hexylamine (3.29 g, 32.55 mmol) is rapidly introduced. The mixture is stirred between 4° C. and room temperature over 19 h then poured slowly on diisopropylether (6.3 L) while stirring. After stirring for 3 h, the precipitate is collected by filtration, washed twice with diisopropylether (420 mL) then dried to give a white solid which is solubilized in 850 mL of THF. To this solution are successively added DIPEA (42 g, 325 mmol) and acetic anhydride (33 g, 325 mmol). After stirring overnight at room temperature, la solution is poured slowly over 1 h in diisopropylether (5 L) while stirring. After stirring for 1.5 h, the precipitate is filtered, washed twice with diisopropylether (420 mL) then dried under vacuum to 30° C. to give a poly(gamma-benzyl-L-glutamic) acid capped to one of its extremities with an acetyl group.

(70) To a solution of the copolyamino acid obtained (72 g) in trifluoroacetic acid (TFA, 320 mL) at 4° C., a 33% bromhydric acid solution (HBr) in acetic acid (225 mL, 1.28 mol) is added dropwise over 1 h. The mixture is stirred at room temperature over 2.5 h, then poured dropwise over 1 h on a 1:1 (v/v) diisopropylether and water mixture while stirring (4 L). After stirring for 2 h, the mixture is rested overnight. The precipitate white is collected by filtration, washed with a 1:1 (v/v) diisopropylether and water mixture (320 mL) then with water (320 mL).

(71) The solid obtained is then solubilized in water (1.4 L) by adjusting the pH to 7.5 using a 1 N aqueous soda solution. After solubilisation, the solution is diluted adding water to give a final volume of 2.1 L. The solution is filtered on 0.45 μm frit then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is inferior to 50 μS/cm. The aqueous solution is then acidified using a 37% chlorhydric acid solution 37% while stirring until a pH of 2 is reached. After stirring for 4 h, the precipitate obtained is filtered, washed with water (320 mL) then dried under vacuum at 30° C. to give a poly-L-glutamic acid of number-average molecular weight (Mn) 3480 g/mol compared to a polyoxyethylene (PEG) standard, and of average polymerization degree 22.

(72) Copolyamino Acid B2:

(73) The copolyamino acid B2-1 of number-average molecular weight (Mn) 3480 g/mol (5.0 g) is solubilized in DMF (60 mL) at 40° C. then kept at this temperature. In parallel, the chlorhydrate salt of molecule A2 (881 mg, 1.11 mmol) is suspended in DMF (5.5 mL) and triethylamine (0.1 g, 1.11 mmol) is added, then the mixture is stirred until complete dissolution. To the copolyamino acid B2-1 in DMF, N-methylmorpholine (NMM, 3.73 g, 37 mmol), the solution of molecule A2 then 2-hydroxypyridine N-oxide (HOPO, 410 mg, 3.69 mmol) are added successively. The reaction medium is then cooled down at 0° C., then N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 707 mg, 3.69 mmol) is added and the medium is risen at room temperature in 4 h. The reaction medium is filtered on 0.2 mm woven frit, poured dropwise on 625 mL of water containing 15% NaCl by weight and HCl (pH 2) under stirring and at 10° C., then the suspension is kept at rest overnight at room temperature. The precipitate is collected by filtration, solubilized in 300 mL of water by slowly adding a 1 N NaOH aqueous solution until pH 7 while stirring, then the solution is filtered on 0.45 μm frit. The clear solution obtained is purified by ultrafiltration against a 0.9% NaCl solution, a carbonate buffer solution, a 0.9% NaCl solution, a phosphate buffer solution, a 0.9% NaCl solution, then water until the conductimetry of the permeate is inferior to 50 μS/cm. The solution is filtered on frit 0.2 μm and kept at 2-8° C.

(74) Dry extract: 18.7 mg/g

(75) DP (estimated according to la NMR .sup.1H): 22

(76) According to NMR .sup.1H: i=0.032

(77) The calculated number-average molecular weight of B2 is 3974 g/mol.

(78) HPLC-SEC aqueous (calibrant PEG): Mn=3320 g/mol

Example B3: Copolyamino Acid B3-Sodium Poly-L-Glutamate Modified with Molecule A1 Having a Number-Average Molecular Weight (Mn) of 3100 g/Mol

(79) Copolyamino acid B3-1: poly-L-glutamic acid of relative number-average molecular weight (Mn) relative of 3390 g/mol and DP 22 obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated using hexylamine.

(80) In a 1 L round-bottom flask is added γ-benzyl-L-glutamate N-carboxyanhydride (Glu(OBzl)-NCA, 200 g, 760 mmol), then DMF (450 mL) is introduced. The mixture is then stirred until complete dissolution, cooled down at 4° C., then hexylamine (3.94 g, 34.53 mmol) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 3 days then heated at 80° C. over 2 h. The solution is poured slowly in diisopropylether (6 L) under stirring. After stirring for 1 h, the precipitate is collected by filtration, washed twice with diisopropylether (900 mL) then dried under reduced pressure.

(81) To a solution of copolyamino acid obtained (78 g) in trifluoroacetic acid (TFA, 360 mL) at 4° C., a 33% bromhydric acid solution (HBr) in acetic acid (250 mL, 1.42 mol) is added dropwise over 40 minutes. The mixture is stirred at room temperature over 2.5 h, then poured dropwise on a 1:1 (v/v) diisopropylether and water mixture under stirring (4.2 L). After stirring for 2 h, the mixture is kept at rest overnight. The white precipitate is collected by filtration, washed with a 1:1 (v/v) diisopropylether and water mixture (340 mL) then with water (350 mL).

(82) The solid obtained is then solubilized in water (1.5 L) by adjusting the pH to 7.2 using a 1 N aqueous soda solution. After solubilization, the solution is diluted by addition of water to give a final volume of 2.4 L. The solution is filtered on 0.45 μm frit then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is inferior to 50 μS/cm. The solution aqueous is then acidified using a 37% chlorhydric acid solution while stirring until a pH of 2 is reached. After 18 h, the precipitate obtained is filtered, washed with water (350 mL) then dried under vacuum to 30° C. to give a poly-L-glutamic acid of number-average molecular weight (Mn) 3390 g/mol compared to a polyoxyethylene (PEG) standard, and of average polymerization degree 22.

(83) Copolyamino Acid B3

(84) Using a similar process than the one used for preparing copolyamino acid B2 and applied to the copolyamino acid B3-1 (5 g) and to molecule A1 (1.11 g, 1.50 mmol), a modified sodium poly-L-glutamate with molecule A1 is obtained.

(85) Dry extract: 22 mg/g

(86) DP (estimated according to la NMR .sup.1H): 22

(87) According to NMR .sup.1H: i=0.041

(88) The calculated number-average molecular weight of B3 is 3986 g/mol

(89) HPLC-SEC aqueous (calibrant PEG): Mn=3100 g/mol

Example B4: Copolyamino Acid B4-Sodium Poly-L-Glutamate Modified with Molecule A4 Having a Number-Average Molecular Weight (Mn) of 3250 g/Mol

(90) In an oven-dried round-bottom flask, γ-benzyl-L-glutamate N-carboxyanhydride (25 g, 95 mmol) is solubilized in anhydrous DMF (50 mL). The mixture is stirred under argon until complete solubilization, cooled down at 4° C., then the molecule A4 (3.22 g, 4.32 mmol) in DMF (10 mL) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days, heated at 65° C. over 2 h, then cooled down at room temperature and then poured dropwise in diisopropylether (850 mL) under stirring. After 1 h, the precipitate is collected by filtration, triturated with diisopropylether until obtention of a powder then dried under vacuum at 30° C. The solid (22 g) is diluted in TFA (89 mL), and a 33% bromhydric acid solution (HBr) in acetic acid (62 mL, 354 mmol) is then added dropwise and at 0° C. The solution is stirred for 3.5 h at room temperature then poured dropwise on a 1:1 (v/v) diisopropylether/eau mixture and under stirring (1.8 L). After stirring for 2 h, the heterogenous mixture is kept at rest overnight. The white precipitate is collected by filtration, then washed twice with diisopropylether (90 mL) and twice with water (90 mL). The solid obtained is solubilized in water (540 mL) by adjusting the pH to 7.2 using a 1 N aqueous soda solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoritically by adding water to give a final volume of 810 mL. The mixture is filtered on 0.45 μm frit then is purified by ultrafiltration against a 0.9% NaCl solution then water until the conductimetry of the permeate is inferior to 50 μS/cm. The copolyamino acid solution is then concentrated to about 25 g/L theoritically and filtered on 0.2 μm then kept at 2-8° C.

(91) Dry extract: 23.6 mg/g

(92) DP (estimated according to la NMR .sup.1H): 22 that is i=0.045

(93) The calculated number-average molecular weight of B4 is 4030 g/mol

(94) HPLC-SEC aqueous (calibrant PEG): Mn=3250 g/mol

Part C—Compositions Consisting of Insulin Glargine, or Insulin Glargine and Prandial Insulin

Example C1: Fast-Acting Insulin Analog Solution (Humalog®) at 100 U/mL

(95) This solution is a commercial solution of insulin lispro, marketed by ELI LILLY under the name Humalog®. This product is a fast-acting insulin analog. The excipients in Humalog® are meta-cresol (3.15 mg/mL), glycerol (16 mg/mL), disodium phosphate (1.88 mg/mL), zinc oxide (to have 0.0197 mg of zinc ion/mL), sodium hydroxide and hydrochloric acid to adjust the pH (pH 7-7.8) and water.

Example C2: Solution of Rapid Insulin Analog (NovoLog®) at 100 U/mL

(96) This solution is a commercial solution of insulin aspart marketed by the company NOVO NORDISK under the name of NovoLog® in the United States of America and Novolog® in Europe. This product is a rapid insulin analog. The excipients of Novolog® are glycerol (16 mg), phenol (1.50 mg/mL), meta-cresol (1.72 mg/mL), zinc (19.6 μg/mL), disodium phosphate dihydrate (1.25 mg/mL), sodium chloride (0.5 mg/mL), sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 7.2-7.6), and water.

Example C3: Solution of Rapid Insulin Analog (Apidra®) at 100 U/mL

(97) This solution is a commercial solution of insulin glulisine marketed by the company SANOFI under the name of Apidra®. This product is a rapid insulin analog. The excipients of Apidra® are meta-cresol (3.15 mg/mL), tromethamine (6 mg/mL), sodium chloride (5 mg/mL), polysorbate 20 (0.01 mg/mL), sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 7.3), and water.

Example C4: Solution of Slow-Acting Insulin Analog (Lantus®) at 100 U/mL

(98) This solution is a commercial solution of insulin glargine marketed by the company SANOFI under the name of Lantus®. This product is a slow-acting insulin analog. The excipients in Lantus® are zinc chloride (30 μg/mL), meta-cresol (2.7 mg/mL), glycerol (85%) (20 mg/mL), sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 4) and water.

Example C5: Solution of Human Insulin (ActRapid®) at 100 IU/mL

(99) This solution is a commercial solution of human insulin from NOVO NORDISK sold under the name of ActRapid®. This product is a human insulin. The excipients of ActRapid® are zinc chloride, glycerol, meta-cresol, sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 6.9-7.8), and water.

Example C6: Solution of Human Insulin (Umuline Rapide®) at 100 IU/mL

(100) This solution is a commercial solution of human insulin from ELI LILLY sold under the name of Umuline Rapide®. This product is a human insulin. The excipients of Umuline Rapide® are glycerol, meta-cresol, sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 7.0-7.8), and water.

Example C7: Solution of GLP-1 RA Dulaglutide (Trulicity®) at 3 mg/mL

(101) This solution is a solution of dulaglutide marketed by the company ELI LILLY under the name of Trulicity®. The excipients in Trulicity® are anhydrous citric acid (0.14 mg/mL), mannitol (46.4 mg/mL), polysorbate 80 (0.20 mg/mL), trisodium citrate dihydrate (2.74 mg/mL), and water.

Example C8: Solution of GLP-1 RA Exenatide (Byetta®) at 0.25 mg/mL

(102) This solution is a solution of exenatide marketed by the company ELI LILLY under the name of Byetta®. The excipients in Byetta® are meta-cresol (20 mM), mannitol, glacial acetic acid, sodium acetate trihydrate, and water.

(103) a) Compositions Comprising Insulin Glargine

(104) Preparation method CA1: Preparation of a diluted composition of co-polyamino acid/insulin glargine 50 U/mL at pH 7.1, according to a method using insulin glargine in liquid form (in solution) and a co-polyamino acid in liquid form (in solution).

(105) Concentrated solutions of m-cresol and of glycerol are added to a stock solution of co-polyamino acid at pH 7.1 in a manner so as to obtain a solution of co-polyamino acid of concentration C.sub.co-polyamino acid stock/excipients (mg/mL). The quantity of excipients added is adjusted in a manner so as to obtain a concentration of m-cresol of 35 mM and of glycerol of 184 mM in the composition of co-polyamino acid/insulin glargine 50 U/mL at pH 7.1.

(106) In a sterile jar, a volume V.sub.insulin glargine of a commercial solution of insulin glargine marketed under the name of Lantus® at a concentration of 100 U/mL is added to a volume V.sub.co-polyamino acid stock/excipients of a solution of co-polyamino acid at concentration C.sub.co-polyamino acid stock/excipients (mg/mL) in a manner so as to obtain a diluted composition of co-polyamino acid C.sub.diluted co-polyamino acid (mg/mL)/insulin glargine 50 U/mL at pH 7.1. Turbidity appears. The pH is adjusted to pH 7.1 by addition of concentrated NaOH, and the solution is placed under static conditions in an oven at 40° C. for 2 h until the solubilization is complete. This visually clear solution is placed at +4° C.

(107) Preparation Method CA2: Preparation of a Concentrated Composition of Co-Polyamino Acid/Insulin Glargine at pH 7.1 with the Aid of a Co-Polyamino Acid, According to a Method for Concentrating a Diluted Composition.

(108) A composition of co-polyamino acid/insulin glargine 50 U/mL at pH 7.1 described in Example CA1 is concentrated by ultrafiltration through a 3 kDa membrane made of regenerated cellulose (Amicon® Ultra-15 marketed by the company Millipore). After this ultrafiltration step, the retentate is clear, and the concentration of insulin glargine in the composition is determined by reverse phase chromatography (RP-HPLC). The concentration of insulin glargine in the composition is then adjusted to the desired value by dilution in a solution of excipients m-cresol/glycerol in a manner so as to obtain a final concentration of m-cresol of 35 mM and an osmolarity of 300 mOsm/kg. The pH is measured and adjusted to pH 7.1 by addition of concentrated NaOH and HCl. This solution at pH 7.1, visually clear, has a concentration of insulin glargine C.sub.insulin glargine (U/mL) and a concentration of co-polyamino acid C.sub.co-polyamino acid (mg/mL)=C.sub.diluted co-polyamino acid (mg/mL) x C.sub.insulin glargine (U/mL)/50 (U/mL).

(109) According to this preparation method CA2, compositions of co-polyamino acid/insulin glargine were prepared, for example, with concentrations of insulin glargine of 200 U/mL and 400 U/mL.

Example CA3: Preparation of Compositions of Co-Polyamino Acid/Insulin Glargine 200 U/mL at pH 7.1

(110) Compositions of co-polyamino acid/insulin glargine 200 U/mL are prepared according to the method described in Example CA2 in a manner so as to obtain a concentration of insulin glargine C.sub.insulin glargine=200 U/mL and a concentration of co-polyamino acid C.sub.co-polyamino acid (mg/mL). These compositions are presented in the following Table 3.

(111) TABLE-US-00004 TABLE 3 Composition of insulin glargine (200 U/mL) in presence of copolyamino acid. Concentration in Insulin Copolyamino copolyamino acid glargine Composition acid (in mg/ml) (U/mL) CA3-1 B1 5 200 CA3-2 B2 6 200 CA3-3 B3 9 200 CA3-4 B4 5 200

(112) b) Compositions Comprising Insulin Glargine and Insulin Lispro

(113) Preparation Method CB1: Preparation of a Diluted Composition of Co-Polyamino Acid/Insulin Glargine 43 (U/mL)/Insulin Lispro 13.5 (U/mL)

(114) A volume V.sub.insulin lispro of a commercial solution of insulin lispro Humalog® at 100 U/mL and water is added to a volume V.sub.co-polyamino acid/diluted insulin glargine of the diluted composition of co-polyamino acid/insulin glargine 50 U/mL at pH 7.1 described in Example CA1, in a manner so as to obtain a composition of co-polyamino acid/insulin glargine 43 (U/mL)/insulin lispro 13.5 (U/mL).

(115) Preparation Method CB2: Preparation of a Concentrated Composition of Co-Polyamino Acid/Insulin Glargine/Insulin Lispro at pH 7.1

(116) A composition of co-polyamino acid/insulin glargine 43 (U/mL)/insulin lispro 13.5 (U/mL) described in Example CB1 is concentrated by ultrafiltration through a 3 kDa membrane made of regenerated cellulose (Amicon® Ultra-15 marketed by the company MILLIPORE). After the completion of this ultrafiltration step, the retentate is clear, and the concentration of insulin glargine in the composition is determined by reverse phase chromatography (RP-HPLC). The concentrations of insulin glargine and insulin lispro in the composition are then adjusted to the desired value by dilution in a solution of excipients m-cresol/glycerol in a manner so as to obtain a final concentration of m-cresol of 35 mM and an osmolarity of 300 mOsm/kg. The pH is measured and adjusted if necessary to pH 7.1 by addition of concentrated NaOH and HCl. This solution at pH 7.1, visually clear, has a concentration of insulin glargine C.sub.insulin glargine (U/mL), a concentration of insulin lispro C.sub.insulin lispro=C.sub.insulin glargine×0.33, and a concentration of co-polyamino acid C.sub.co-polyamino acid (mg/mL)=C.sub.diluted co-polyamino acid (mg/mL)×C.sub.insulin glargine (U/mL)/50 (U/mL).

Example CB3: Preparation of Compositions of Co-Polyamino Acid/Insulin Glargine 200 U/mL/Insulin Lispro 66 U/mL at pH 7.1

(117) Des compositions copolyamino acid/insuline glargine 200 U/mL/insuline lispro 66 U/mL are préviaées selon le procédé décrit in l'Example CB2 de manière to obtenir une concentration en insuline glargine C.sub.insuline glargine=200 U/mL, une concentration en insuline lispro C.sub.insuline lispro=66 U/mL and une concentration en copolyamino acid C.sub.copolyamino acid (mg/mL). Ces compositions are présentées in Compositions of co-polyamino acid/insulin glargine 200 U/mL/insulin lispro 66 U/mL are prepared according to the method described in Example CB2 in a manner so as to obtain a concentration of insulin glargine C.sub.insulin glargine=200 U/mL, a concentration of insulin lispro C.sub.insulin lispro=66 U/mL, and a concentration of co-polyamino acid C.sub.co-polyamino acid (mg/mL). These compositions are presented in Table 4.

(118) TABLE-US-00005 TABLE 4 Composition of insulin glargine (200 U/mL) and of insulin lispro (66 U/mL) in presence of copolyamino acid. Concentration in Insulin Insulin Copolyamino copolyamino acid glargine Lispro Composition acid (in mg/ml) (U/ml) (U/ml) CB3-1 B1 5 200 66 CB3-2 B2 6 200 66 CB3-3 B3 9 200 66 CB3-4 B4 5 200 66

Part D—Results

(119) Demonstration of the physical stability of the compositions according to the invention by the study of co-polyamino acid/insulin glargine 200 U/mL compositions and of co-polyamino acid/insulin glargine 200 U/mL/lispro 66 U/mL compositions.

Example D1: Stability Accelerated at 25° C. Under Dynamic Conditions

(120) 3 3-mL vials filled with 1 mL of composition co-polyamino acid/insulin glargine are placed vertically in an orbital stirrer. The stirrer is placed in an oven at 25° C., and the vials are subjected to stirring at 250 rpm. The vials are inspected visually daily/weekly in order to detect the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20): the vials are subjected to illumination of at least 2000 lux and are observed on a white background and on a black background. The number of days of stability corresponds to the duration after which at least 2 vials present visible particles or are turbid.

(121) These results are in agreement with the US Pharmacopoeia (USP <790>).

(122) The results of accelerated stability (obtained with different compositions) are presented in Table 5 and Table 6.

(123) TABLE-US-00006 TABLE 5 results of the stabilities of the compositions of co-polyamino acid/insulin glargine (200 U/mL)/insulin lispro (66 U/mL) at 25° C. under dynamic conditions (with stirring at 250 rpm). Concentration in Copolyamino copolyamino acid Stability Composition acid (mg/mL) (days) CB3-1 B1 5 15 CB3-3 B3 9 >25

(124) TABLE-US-00007 TABLE 6 results of the stabilities of the compositions of co- polyamino acid/insulin glargine (200 U/mL) at 25° C. under dynamic conditions (with stirring at 250 rpm). Concentration in Copolyamino copolyamino acid Stability Composition acid (mg/mL) (days) CA3-1 B1 5 19 CA3-3 B3 9 >25

Example D2: Solubilization/Precipitation

Example CA4: Precipitation of Insulin Glargine in Compositions of Co-Polyamino Acid/Insulin Glargine at 200 U/mL

(125) 1 mL of solution of co-polyamino acid/insulin glargine prepared in Example CA3 is added to 2 mL of a PBS solution containing 20 mg/mL of BSA (bovine serum albumin). The PBS/BSA mixture simulates the composition of the subcutaneous environment. A precipitate appears.

(126) A centrifugation at 4000 rpm is carried out in order to separate the precipitate from the supernatant. Next, the insulin glargine is assayed in the supernatant by RP-HPLC. The result is that insulin glargine is present in majority proportion in a precipitated form.

(127) The results are presented in Table 7:

(128) TABLE-US-00008 TABLE 7 Compositions copolyamino acid/insulin glargine (200 U/mL); solubilization/precipitation of insulin glargine. Insulin glargine Solubilization Precipitation Copolyamino Concentration en of insulin of insulin Composition acid (mg/ml) (U/mL) glargine glargine CA3-1 B1 5 200 YES YES CA3-2 B2 6 200 YES YES CA3-3 B3 9 200 YES YES CA3-4 B4 5 200 YES YES

Example CB4: Precipitation of Insulin Glargine in the Compositions of Co-Polyamino Acid/Insulin Glargine/Insulin Lispro at 200/66 200/66 U/mL

(129) 1 mL of solution of co-polyamino acid/insulin glargine/insulin lispro prepared in Example CB3 is added to 2 mL of a PBS solution containing 20 mg/mL of BSA (bovine serum albumin). The PBS/BSA mixture simulates the composition of the subcutaneous environment. A precipitate appears.

(130) A centrifugation at 4000 rpm is carried out in order to separate the precipitate from the supernatant. Next, the insulin glargine is assayed in the supernatant by RP-HPLC. The result is that insulin glargine is found in majority proportion in a precipitated form. The results are presented in Table.

(131) TABLE-US-00009 TABLE 8 Compositions copolyamino acid/insulin glargine (200 U/mL)/insulin lispro (66 U/mL); solubilization and precipitation of insulin glargine. Concentration in copolyamino Insulin Insulin Solubilization Precipitation Copolyamino acid glargine Lispro of insulin of insulin Composition acid (mg/ml) U/ml U/ml glargine glargine CB3-1 B1 5 200 66 YES YES CB3-2 B2 6 200 66 YES YES CB3-3 B3 9 200 66 YES YES CB3-4 B4 5 200 66 YES YES

Example D3: Preparation of a Diluted Co-Polyamino Acid/Insulin Glargine 65 U/mL Composition at pH 7.1

(132) Concentrated solutions of m-cresol and of glycerol are added to a stock solution of co-polyamino acid at pH 7 in a manner so as to obtain a solution of co-polyamino acid of concentration Cco-polyamino acid stock/excipients (mg/mL). The quantity of excipients added is adjusted in a manner so as to obtain a concentration of m-cresol of 35 mM and of glycerol of 184 mM in the co-polyamino acid/insulin glargine 65 U/mL composition at pH 7.1.

(133) In a sterile jar, a volume V.sub.insulin glargine of a commercial solution of insulin glargine marketed under the name of Lantus® at a concentration of 100 U/mL is added to a volume V.sub.co-polyamino acid stock/excipients of a solution of co-polyamino acid of concentration C.sub.co-polyamino acid stock/excipients (mg/mL) in a manner so as to obtain a diluted co-polyamino acid composition C.sub.diluted co-polyamino acid (mg/mL)/insulin glargine 65 U/mL at pH 7.1. Turbidity appears. The pH is adjusted to pH 7.1 by addition of concentrated NaOH, and the solution is placed under static conditions in an oven at 40° C. for 2 h until the solubilization is complete. This visually clear solution is placed at +4° C.

Example D4: Precipitation of a Co-Polyamino Acid/Insulin Glargine 65 U/mL Composition at pH 7.1, by Varying the Concentration of Albumin

(134) 0.3 mL of a solution of BSA (bovine serum albumin) in a PBS buffer at pH 7.4 (phosphate buffer saline) and 1 mL of diluted co-polyamino acid/insulin glargine 65 U/mL composition, pH 7.1 are introduced, respectively in a disposable UV cuvette in a manner so as to obtain a mixture containing 50 U/mL insulin glargine, an albumin concentration CBSA (mg/mL) in a PBS buffer. Several solutions of BSA in a PBS buffer of variable concentrations are prepared so as to vary the concentration of albumin in the final mixture from 1 to 12.7 mg/mL (1; 2.9; 3.9; 6.8; 9.7; 12.7 mg/mL) and a concentration of physiological salt via the PBS buffer.

(135) After addition of the solution of BSA in the PBS buffer, the mixture is rapidly homogenized by a few back and forth strokes of a pipette. One hour after the mixing, an absorbance measurement at 500 nm is carried out by means of a JASCO V-530 UV-Vis spectrophotometer.

(136) The absorbance measurement at 500 nm makes it possible to evaluate the turbidity of the mixture originating from the precipitation of the insulin glargine. The turbidity increases as a function of the albumin concentration to reach a plateau reflecting the complete precipitation of the insulin glargine.

(137) The critical albumin concentration allowing a quantitative precipitation is defined as the albumin concentration for which the absorbance value at 500 nm reaches 80% of the absorbance measured at the plateau.

(138) One notes that in the compositions of the invention, the critical BSA quantity is lower.

(139) The results are reported in the following Table.

(140) TABLE-US-00010 TABLE 9 Critical albumin concentration (mg/ml) for 80% of precipitation after 1 h (insuline glargine at 50 U/ml). Critical albumin concentration Concentration of copolyamino (mg/ml) for 80% of precipitation acid in solution insuline Copolyamino after 1 h (insulin glargine to glargine to 50 U/ml acid 50 U/ml) (en mg/ml) B1 <7 1.6 B2 <3 1.9 B3 <1 2.9 B4 <4 1.6

Example D5: Examples of Pharmacodynamic Studies in Dogs

(141) Studies in dogs were carried out for the purpose of evaluating the pharmacodynamics of insulin after administration of the composition of copolyamino acid B1 and insulins (composition CB3-1).

(142) The hypoglycemic effects of this composition were compared to those of simultaneous but separate injections of insulin glargine (Lantus®) (pH 4) and a prandial insulin lispro (Humalog®) in the proportions of 75% of insulin glargine/25% of insulin lispro (dose/dose).

(143) Ten animals that had fasted for approximately 18 hours received injections in the neck above the interscapular region at the dose of 0.67 U/kg. In the hour preceding the insulin injection 3 blood samples were drawn in order to determine the basal level of glucose. The glycemia is determined over 24 h by means of a glucometer.

(144) The mean pharmacokinetic curves of glucose expressed in deviation percentage of the basal level are represented in FIG. 1.

(145) The pharmacodynamic results obtained with the separate and simultaneous administrations of insulin lispro (Humalog®—example C1) and insulin glargine (Lantus®—example C4) in comparison to those obtained with the composition described in Example CB3-1 are presented in FIG. 1. The hypoglycemic activity of the composition described in Example CB3-1 is biphasic. The first rapid phase is defined by a pronounced decrease of glycemia for approximately 60 minutes, which is characteristic of the rapid effect of insulin lispro. This first phase is also visible with the double Lantus®/Humalog® injection, indicating that the composition according to the invention does not modify the rapid character of Humalog®. After approximately 60 minutes, the glycemia rises up to 3 hours, before a second slower phase characterized by a less pronounced hypoglycemic activity lasting up to 18-20 hours post injection. This basal second phase is characteristic of the basal effect of insulin glargine, also visible with the double injection, indicating that the effect is indeed maintained with the composition according to the invention described in Example CB3-1.