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

Abstract

A physically stable compositions in the form of an injectable aqueous solution, the pH of which is from 6.0 to 8.0, including at least: a basal insulin of which the isoelectric point (pI) is from 5.8 to 8.5, and a co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical.

Claims

1. A composition in the form of an injectable aqueous solution, the pH of which is from 6.0 to 8.0, comprising at least: a) one basal insulin the isoelectric point (pI) of which is from 5.8 to 8.5, b) a co-polyamino acid bearing carboxylate charges and hydrophobic radicals Hy, said co-polyamino acid consisting of glutamic or aspartic units, and said hydrophobic radicals Hy being radicals of the following formula I: ##STR00143## in which GpR is a radical of formula II or II′: ##STR00144## GpA is a radical of formula III or III′: ##STR00145## GpC is a radical of formula IV: ##STR00146## Hy comprises more than 30 carbon atoms, the * indicate the sites of attachment of the different groups bound by amide functions; a is a whole number equal to 0 or 1; b is a whole number equal to 0 or 1; p is a whole number equal to 1 or 2, and if p is equal to 1, then a is equal to 0 or 1 and GpA is a radical of formula III′, and if p is equal to 2, then a is equal to 1 and GpA is a radical of formula III; c is a whole number equal to 0 or 1, and, if c is equal to 0, then d is equal to 1 or 2; d is a whole number equal to 0, to 1 or 2; r is a whole number equal to 0 or 1, and if r is equal to 0, then the hydrophobic radical of formula I is bound to the co-polyamino acid via a covalent bond between a carbonyl of the hydrophobic radical and a nitrogen atom in N-terminal position of the co-polyamino acid, thus forming an amide function, and if r is equal to 1, then the hydrophobic radical of formula I is bound to the co-polyamino acid: via a covalent bond between a nitrogen atom of the hydrophobic radical and a carbonyl of the co-polyamino acid, thus forming an amide function, or via a covalent bond between a carbonyl of the hydrophobic radical and a nitrogen atom in N-terminal position of the co-polyamino acid, thus forming an amide function; R is a radical selected from the group consisting of: a linear or branched divalent alkyl radical comprising, if GpR is a radical of formula II, from 2 to 12 carbon atoms, or, if GpR is a radical of formula II′, from 1 to 11 carbon atoms; a linear or branched divalent alkyl radical comprising, if GpR is a radical of formula II, from 2 to 11 carbon atoms, or, if GpR is a radical of formula II′, from 1 to 11 carbon atoms, said alkyl radical bearing one or more —CONH.sub.2 functions, and an un-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms; A is a linear or branched alkyl radical comprising from 1 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 linear or branched monovalent alkyl radical, in which x indicates the number of carbon atoms, and: if p is equal to 1, x is from 11 to 25 (11≦x≦25); if p is equal to 2, x is from 9 to 15 (9≦x≦15), the ratio i between the number of hydrophobic radicals and the number of glutamic or aspartic units being between 0<i≦0.5; when several hydrophobic radicals are borne by a co-polyamino acid, then they are identical or different, the degree of polymerization DP in glutamic or aspartic units is from 5 to 250; the free acid functions being in the form of a salt of an alkaline cation selected from the group consisting of Na.sup.+ and K.sup.+.

2. The composition according to claim 1, wherein that said hydrophobic radicals are selected from the hydrophobic radicals of formula I in which p=1, represented by the following formula V: ##STR00147## GpR, GpA, GpC, r and a have the definitions given above.

3. The composition according to claim 1, wherein that said hydrophobic radicals are selected from the hydrophobic radicals of formula I in which a=1 and p=2, represented by the following formula VI: ##STR00148## in which GpR, GpA, GpC, r and a have the definitions given above.

4. The composition according to claim 1, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of the following formula VII: ##STR00149## in which, D represents, independently, either a —CH.sub.2— group (aspartic unit) or a —CH.sub.2—CH.sub.2— group (glutamic unit), Hy is a hydrophobic radical selected from the hydrophobic radicals of formula I, V or VI, in which r=1 and GpR is a radical of Formula II, R.sub.1 is a hydrophobic radical selected from the hydrophobic radicals of formula I, V or VI in which r=0 or r=1 and GpR is a radical of Formula II′, or a radical selected from the group consisting of H, a C2 to C10 linear acyl group, a C4 to C10 branched acyl group, benzyl, a terminal “amino acid” unit and a pyroglutamate, R.sub.2 is a hydrophobic radical selected from the hydrophobic radicals of formula I, V or VI in which r=1 and GpR is a radical of Formula II, an —NR′R″ radical, R′ and R″ which are identical or different being selected from the group consisting of H, the C2 to C10 linear or branched or cyclic alkyls, benzyl, and said alkyl R′ and R″ together optionally forming one or more saturated, unsaturated and/or aromatic carbon rings and/or optionally comprising heteroatoms selected from the group consisting of O, N and S; X represents a cationic entity selected from the group comprising the alkaline cations; n+m represents the degree of polymerization DP of the co-polyamino acid, that is to say the average number of monomer units per co-polyamino acid chain, and 5≦n+m≦250.

5. The composition according to claim 4, wherein that co-polyamino acid bearing carboxylate charges and hydrophobic charges is selected from the co-polyamino acids of formula VII, in which R.sub.1=R′.sub.1 and R.sub.2=R′.sub.2, of the following formula VIIa: ##STR00150## in which m, n, X, D and Hy have the definitions given above, R′.sub.1 is a radical selected from the group consisting of H, a C2 to C10 linear acyl group, a C4 to C10 branched acyl group, benzyl, a terminal “amino acid” unit and a pyroglutamate, R′.sub.2 is a —NR′R″ radical, R′ and R″ which are identical or different being selected from the group consisting of H, the C2 to C10 linear or branched or cyclic alkyls, benzyl, and said alkyl R′ and R″ together optionally forming one or more saturated, unsaturated and/or aromatic carbon rings and/or optionally comprising heteroatoms selected from the group consisting of O, N and S.

6. The composition according to claim 4, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of formula VII, in which n=0, of the following formula VIIb: ##STR00151## in which m, X, D, R.sub.1 and R.sub.2 have the definitions given above, and at least one R.sub.1 or R.sub.2 is a hydrophobic radical of formula I, V or VI.

7. The composition according to claim 6, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of formula VII, in which n=0, of formula VIIb, and R.sub.1 or R.sub.2 is a hydrophobic radical of formula I, V or VI.

8. The composition according to claim 6, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of formula VIIb, in which R.sub.2 is a hydrophobic radical of formula I, V or VI, in which r=1 and GpR is of formula II.

9. The composition according to claim 4, wherein that R.sub.1 is a radical selected from the group consisting of a C.sub.2 to C.sub.10 linear acyl group, a C.sub.4 to C.sub.10 branched acyl group, benzyl, a terminal “amino acid” unit and a pyroglutamate.

10. The composition according to claim 9, wherein that R.sub.1 is a radical selected from the group consisting of a C.sub.2 to C.sub.10 linear acyl group or a C.sub.4 to C.sub.10 branched acyl group.

11. The composition according to claim 4, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of formula VII, VIIa or VIIb, in which the co-polyamino acid is selected from the co-polyamino acids in which group D is a —CH.sub.2— group (aspartic unit).

12. The composition according to claim 4, wherein that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is selected from the co-polyamino acids of formula VII, VIIa or VIIb, in which the co-polyamino acid is selected from the co-polyamino acids in which group D is a —CH.sub.2—CH.sub.2— group (glutamic unit).

13. The composition according to claim 1, wherein that the basal insulin of which the isoelectric point is from 5.8 to 8.5 is insulin glargine.

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

15. The composition according to claim 1, wherein that the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is at most 60 mg/mL.

16. The composition according to claim 1, wherein that the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is at most 40 mg/mL.

17. The composition according to claim 1, wherein that the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is at most 20 mg/mL.

18. The composition according to claim 1, wherein that the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is at most 10 mg/mL.

Description

[0523] FIG. 1: Median curves (on the abscissa, the post-injection time expressed in hours, and, on the ordinate, the delta insulin concentration expressed in pmol/L) of deviation from the basal insulin level after the administration of composition DB3k of Example DB6 (200 U/mL, 0.5 U/kg of insulin) (empty squares) in comparison after the administration of composition C4 (Lantus®) (100 U/mL, 0.50 U/kg) (filled circles); the administrations being carried out on dogs (n=10), by subcutaneous injection.

[0524] FIG. 2: Mean curves (on the abscissa, the post-injection time expressed in hours, and, on the ordinate, the glucose level expressed in percentage of deviation from the basal level) of glycemia in percentage of deviation from the basal level ±standard deviation from the mean after the administration of composition DB3k of Example DB6 (200 U/mL, 0.5 U/kg of insulin) (empty squares) in comparison after the administration of composition C4 (Lantus®) (100 U/mL, 0.50 U/kg) (ful circles); the administrations having been carried out on dogs (n=10), by subcutaneous injection.

EXAMPLES

[0525] The invention is described in greater detail in reference to the following examples in a non-limiting manner.

Part A

[0526] AA: Synthesis of the Hydrophobic Molecules in which p=1

[0527] The radicals are represented in the following Table 1a by the corresponding hydrophobic molecule before grafting onto the co-polyamino acid.

TABLE-US-00002 TABLE 1a list and structure of hydrophobic molecules synthesized according to the invention. No. Structure of the hydrophobic molecule before grafting onto the co-polyamino acids AA4 [00070] AA7 [00071] AA10 [00072]

Example AA4: Molecule AA4

[0528] Molecule A1: product obtained by the reaction between the palmitoyl chloride and L-proline.

[0529] A solution of palmitoyl chloride (23.0 g, 83.7 mmol) in acetone (167 mL) is added dropwise within 90 minutes to a solution of L-proline (10.6 g, 92.1 mmol) in a 1 N aqueous sodium hydroxide solution (230 mL; 230 mmol). After 14 h of stirring at room temperature, the heterogeneous mixture is cooled to 0° C., then filtered through a sintered filter to yield a white solid which is washed with water (2×100 mL), then with diisopropyl ether (100 mL). The solid is dried at reduced pressure. The solid is then dissolved at reflux in 200 mL of water, then 8 mL of a 37% hydrochloric acid solution are added until obtaining a pH of 1. The opalescent reaction medium is then cooled to 0° C. The precipitate obtained is filtered through a sintered filter, then washed with water (5×50 mL) until filtrates of neutral pH are obtained, then it is dried overnight at 50° C. in an oven under a vacuum. The product is purified by recrystallization in diisopropyl ether. A white solid is obtained.

[0530] Yield: 22.7 g (77%).

[0531] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.19-1.45 (24H); 1.58-1.74 (2H); 1.88-2.14 (3H); 2.15-2.54 (3H); 3.47 (1H); 3.58 (1H); 4.41 (0.1H); 4.61 (0.9H) 6.60-8.60 (1H).

[0532] Molecule A2: product obtained by the reaction between molecule A1 and Boc-1-amino-4,7,10-trioxa-13-tridecaneamine. N,N-diisopropylethylamine (DIPEA) (4.1 g, 31.8 mmol), 1-hydroxybenzotriazole (HOBt) (2.2 g, 16.4 mmol), then N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (3.2 g, 16.6 mmol) are added successively at room temperature to a solution of molecule A1 (4.5 g, 12.7 mmol) in 90 mL of chloroform. After 15 minutes of stirring at room temperature, a solution of Boc-1-amino-4,7,10-trioxa-13-tridecane amine (4.5 g, 14.0 mmol) in 5 mL of chloroform is added. After 18 h of stirring at room temperature, a 0.1 N HCl solution (100 mL), then a saturated NaCl solution (50 mL) are added. The phases are separated, then the organic phase is washed successively with a 0.1 N HCl solution/saturated NaCl (100 mL/50 mL), a saturated NaCl solution (100 mL), a saturated NaHCO.sub.3 solution (100 mL), then a saturated NaCl solution (100 mL). The organic phase is dried over anhydrous sodium sulfate, filtered then concentrated at reduced pressure. A yellow oil is obtained after purification by flash chromatograph (methanol, dichloromethane).

[0533] Yield: 7.7 g (92%).

[0534] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.22-1.37 (24H); 1.44 (9H); 1.59-1.67 (2H); 1.67-2.00 (6H); 2.06-2.45 (4H); 3.18-3.76 (18H); 4.28 (0.2H); 4.52 (0.8H); 4.69-5.04 (1H); 6.77 (0.2H); 7.20 (0.8H).

[0535] Molecule AA4

[0536] At 0° C. a 4 M hydrochloric acid solution in dioxane (29.5 mL, 118 mmol) is added dropwise to a solution of molecule A2 (7.7 g, 11.8 mmol) in 90 mL of dichloromethane. After 3 h 30 of stirring at room temperature, the solution is concentrated at reduced pressure. The residue is purified by flash chromatography (methanol, dichloromethane) to yield a yellow oil. A co-evaporation with diisopropyl ether and drying at 50° C. under a vacuum make it possible to obtain molecule AA4 in hydrochloride salt form.

[0537] Yield: 5.4 g (76%).

[0538] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.08-1.40 (24H); 1.49-1.65 (2H); 1.76-2.39 (10H); 3.07-3.28 (3H); 3.34-3.80 (15H); 4.34 (0.05H); 4.64 (0.95H); 7.35 (0.05H); 7.66-8.58 (3.95H).

[0539] LC/MS (ESI): 556.7; (calculated ([M+H].sup.+): 556.5).

Example AA7: Molecule AA7

[0540] Molecule A3:

[0541] product obtained by reaction between arachidonic acid and L-proline.

[0542] Dicyclohexylcarbodiimide (DCC) (10.45 g, 50.6 mmol) and N-hydroxysuccinimide (NHS) (5.83 g, 50.6 mmol) are added successively to an arachidonic acid solution (15.51 g, 49.63 mmol) in THF (500 mL) at 0° C. After 17 h of stirring at room temperature, the medium is cooled to 0° C. for 20 min, filtered through a sintered filter. L-proline (6 g, 52.11 mmol), DIPEA (60.5 mL) and water (50 mL) are added to the filtrate. After 48 h of stirring at room temperature, the mixture is treated with a 1N aqueous HCl solution to pH 1, and the resulting solid is filtered through a sintered filter, washed with water until the pH of the mother liquors is neutral, then dried under a vacuum to yield a yellowish solid. After purification by chromatography on silica gel (cyclohexane, ethyl acetate), a white solid is obtained.

[0543] Yield: 10.96 g (54%).

[0544] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.28 (34H); 1.66 (2H); 1.95-2.15 (2H); 2.34 (2H); 2.45 (1H); 3.47 (1H); 3.56 (1H); 4.60 (1H).

[0545] LC/MS (ESI): 410.4; (calculated ([M+H].sup.+): 410.6).

[0546] Molecule A4: product obtained by the reaction between molecule A3 and Boc-1-amino-4,7,10-trioxa-13-tridecane.

[0547] DIPEA (9.32 mL) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (9.45 g, 29.4 mmol) at room temperature are added to a solution of molecule A3 (10.96 g, 26.75 mmol) in THF (135 mL). After 30 min of stirring, Boc-1-amino-4,7,10-trioxa-13-tridecane (10.29 g, 32.11 mmol) is added. After stirring at room temperature for 18 h, the solvent is evaporated at reduced pressure, and the residue is diluted with ethyl acetate (400 mL). The organic phase is washed with a saturated NaHCO.sub.3 solution, a 1N aqueous HCl solution, water, an aqueous saturated NaCl solution, then dried over Na.sub.2SO.sub.4, filtered and concentrated under a vacuum. The residue is purified by flash chromatography (cyclohexane, ethyl acetate, methanol) to yield a colorless oil which solidifies. A solid is obtained.

[0548] Yield: 14.2 g (75%).

[0549] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.24 (32H); 1.43 (9H); 1.57-2.00 (8H); 2.10-2.45 (4H); 3.20-3.75 (18H); 4.30 (0.20H); 4.55 (0.80H); 5.03 (1H); 6.75 (0.20H); 7.20 (0.80H).

[0550] LC/MS (ESI): 712.8; (calculated ([M+H].sup.+): 713.1).

Molecule AA7

[0551] A 4N HCl solution in dioxane (25 mL) is added to a solution of molecule A4 (14.25 g, 20.01 mmol) in dichloromethane (100 mL) at 0° C. After 20 h of stirring at 0° C. and 4 h of stirring at 25° C., the medium is concentrated under a vacuum. The residue is dissolved four times in methanol and evaporated at reduced pressure to yield a white solid of molecule AA7 in hydrochloride salt form.

[0552] Yield: 12.7 g (98%).

[0553] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.23 (32H); 1.45 (2H); 1.64 (2H); 1.70-2.05 (6H); 2.10-2.30 (2H); 2.82 (2H); 3.08 (2H); 3.30-3.60 (14H); 4.15-4.30 (1H); 7.73-8.13 (4H).

[0554] LC/MS (ESI): 612.7; (calculated ([M+H].sup.+): 612.9).

Example AA10: Molecule AA10

[0555] Molecule A23:

[0556] product obtained by the reaction between nipecotic acid and arachidic acid. By a method similar to the one used for the preparation of molecule A3, applied to arachidic acid (2.30 g, 7.37 mmol) and to nipecotic acid (1.00 g, 7.74 mmol), a white solid is obtained after filtration of the aqueous phase acidified to pH 1 and washing of the solid with water, then with dichloromethane.

[0557] Yield: 1.65 g (53%)

[0558] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.07-1.88 (37H); 2.10 (1H); 2.28-2.45 (2H); 2.52 (1H); 2.91-3.17 (1.5H); 3.42 (0.5H); 3.72 (0.5H); 3.84 (0.5H); 4.08 (0.5H); 4.56 (0.5H).

[0559] LC/MS (ESI): 424.4; 848.0; (calculated ([M+H].sup.+): 424.4; ([2M+H].sup.+): 847.8).

[0560] Molecule A24:

[0561] product obtained by the reaction between molecule A23 and Boc-1-amino-4,7,10-trioxa-13-tridecaneamine.

[0562] By a method similar to the one used for the preparation of molecule A4, applied to molecule A23 (1.65 g, 3.89 mmol) and to TBTU (1.01 g, 7.79 mmol), a white solid of molecule A24 is obtained. Yield: 1.97 g (70%)

[0563] .sup.1H NMR (CDCl3, ppm): 0.88 (3H); 1.15-2.70 (54H); 3.10-3.46 (6H); 3.46-3.71 (12.6H); 3.92 (0.4H); 4.17 (0.6H); 4.49 (0.4H); 4.80-5.16 (1H); 6.35-6.76 (1H).

[0564] LC/MS (ESI): 726.8; (calculated ([M+H].sup.+): 726.6).

Molecule AA10

[0565] By a method similar to the one used for the preparation of molecule AA4, applied to molecule A24 (1.97 g, 2.71 mmol), a white solid of molecule AA10 is obtained by evaporation of the solvent, trituration in acetone, filtration and washing with acetone, then drying at reduced pressure at 50° C.

[0566] Yield: 1.66 g (92%)

[0567] 1H NMR (DMSO-d6, ppm): 0.86 (3H); 1.09-1.90 (42H); 2.05-2.68 (5H); 2.45-2.68 (1H); 2.78-3.19 (6H); 3.36-3.44 (2H); 3.44-3.60 (10H); 3.69-3.87 (1H); 4.20 (0.4H); 4.35 (0.6H).

[0568] LC/MS (ESI): 626.7; (calculated ([M+H].sup.+): 626.5).

[0569] AB: Synthesis of the Co-Polyamino Acids

[0570] Co-Polyamino Acids with Statistical Grafting (Formula VII or VIIa)

TABLE-US-00003 TABLE 1b list of co-polyamino acids synthesized according to the invention No. co-polyamino acids bearing carboxylate charges and hydrophobic radicals AB6 [00073] i = 0.025, DP (m + n) = 20 [00074] R.sub.1 = H or pyroglutamate AB7 [00075] i = 0.03, DP (m + n) = 21 [00076] R1 = CH.sub.3—C(O)—, H or pyroglutamate AB8 [00077] i = 0.03, DP (m + n) = 24 [00078] R.sub.1 = H or pyroglutamate AB10 [00079] i = 0.08, DP (m + n) = 25 [00080] R.sub.1 = H or pyroglutamate AB21 [00081] i = 0.056, DP (m + n) = 22 [00082] R.sub.1 = H or pyroglutamate

[0571] Co-Polyamino Acids with Defined Grafting (Formula VII or VIIb)

TABLE-US-00004 TABLE 1c list of co-polyamino acids synthesized according to the invention. No. co-polyamino acids bearing carboxylate charges and hydrophobic radicals AB17 [00083] AB18 [00084] AB21′ [00085]

Example AB6: Co-Polyamino Acid AB6—Sodium Poly-L-Glutamate Modified by Molecule AA7 and Having a Number Average Molecular Weight (Mn) of 4000 g/mol

[0572] Co-Polyamino Acid AB6-1:

[0573] poly-L-glutamic acid having a relative number average molecular weight (Mn) of 3500 g/mol originating from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by the hexylamine.

[0574] In a round-bottom flask dried beforehand in the oven, γ-benzyl-L-glutamate N-carboxyanhydride (89.9 g, 341 mmol) is placed for 30 min under a vacuum, then anhydrous DMF (200 mL) is introduced. The mixture is then stirred under argon until the dissolution is complete, cooled to 4° C., then hexylamine (2.05 mL, 15.5 mmol) is introduced rapidly. The mixture is stirred at between 4° C. and room temperature for 2 days. The reaction mixture is then heated at 65° C. for 2 h, cooled to room temperature, then poured dropwise into diisopropyl ether (3 L) under stirring. The white precipitate is recovered by filtration, washed with diisopropyl ether (2×200 mL), then dried under a vacuum at 30° C. to yield a poly(gamma-benzyl-L-glutamic) acid (PBLG).

[0575] A 33% hydrobromic acid (HBr) solution in acetic acid (240 mL, 1.37 mol) is added dropwise to a solution of PBLG (74.8 g) in trifluoroacetic acid (TFA, 340 mL) at 4° C. The mixture is stirred at room temperature for 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether and water under stirring (4 L). After 2 h of stirring, the heterogeneous mixture is left to rest overnight. The white precipitate is recovered by filtration, washed with a 1:1 (v/v) mixture of diisopropylether and water (340 mL), then with water (340 mL).

[0576] The solid obtained is then solubilized in water (1.5 L) by adjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by addition of water until obtaining a final volume of 2.1 L.

[0577] The solution is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated until a final volume of 1.8 L is obtained.

[0578] The aqueous solution is then acidified by adding 37% hydrochloric acid solution until a pH of 2 is reached. After 4 h of stirring, the precipitate obtained is filtered, washed with water (2×340 mL), then dried under a vacuum at 30° C. to yield a poly-L-glutamic acid having a number average molecular weight (Mn) of 3500 g/mol with respect to a polyoxyethylene (PEG) standard.

Co-Polyamino Acid AB6

[0579] The co-polyamino acid AB6-1 having a number average molecular weight (Mn) of 3500 g/mol (10.0 g) is solubilized in DMF (420 mL) at 30° C.-40° C., then maintained at this temperature. In parallel, the hydrochloride salt of molecule AA7 (1.47 g, 2.3 mmol) is suspended in DMF (12 mL), and triethylamine (0.23 g, 2.3 mmol) is added, then the mixture is heated slightly under stirring until the dissolution is complete. NMM (7.6 g, 75 mmol), the solution of AA7, then 2-hydroxypyridine N-oxide (HOPO, 0.84 g, 7.5 mmol) are added successively to the solution of co-polyamino acid in DMF. The reaction medium is then cooled to 0° C., then EDC (1.44 g, 7.5 mmol) is added, and the medium is brought back to room temperature over a period of 2 h. The reaction medium is filtered through a 0.2 mm woven filter and poured dropwise under stirring onto 3.5 L of water containing NaCl at 15% by weight and HCl (pH 2). At the end of the addition, the pH is readjusted to 2 with a 37% HCl solution, and the suspension is allowed to rest overnight. The precipitate is collected by filtration, then rinsed with 100 mL of water. The white solid obtained is solubilized in 500 mL of water by slow addition under stirring of a 1 N aqueous solution NaOH until the pH is 7, then the solution is filtered through a 0.45-μm filter. The clear solution obtained is purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution is filtered through a 0.2-μm filter and stored at 2-8° C.

[0580] Dry extract: 21.6 mg/g.

[0581] DP (estimated based on .sup.1H NMR): 20.

[0582] Based on .sup.1H NMR: i=0.025.

[0583] The calculated average molecular weight of the co-polyamino acid AB6 is 3369 g/mol.

[0584] H PLC-aqueous SEC (calibrant PEG): Mn=4000 g/mol.

Example AB7: Co-Polyamino Acid AB7—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group, Modified by Molecule AA7 and Having a Number Average Molecular Weight (Mn) of 3300 g/mol

[0585] Co-polyamino acid AB7-1:

[0586] poly-L-glutamic acid having a relative number average molecular weight (Mn) of 3600 mol and a DP of 21 originating from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by hexylamine and capped at one of its ends by an acetyl group.

[0587] In a round-bottom flask dried beforehand in the oven, γ-benzyl-L-glutamate N-carboxyanhydride (Glu(OBn)-NCA, 100.0 g, 380 mmol) is placed under a vacuum for 30 minutes, then anhydrous DMF (225 mL) is introduced. The mixture is then stirred under argon until the dissolution is complete, cooled to 4° C., then hexylamine (1.78 g, 17 mmol) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days, then precipitated in diisopropyl ether (3.4 L). The precipitate is recovered by filtration, washed two times with diisopropyl ether (225 mL), then dried to yield a white solid which is dissolved in 450 mL of THF. DIPEA, (31 mL, 176 mmol) then acetic anhydride (17 mL, 176 mmol) are added successively to this solution. After one night of stirring at room temperature, the solution is poured slowly into diisopropyl ether (3 L) under stirring. After 1 h of stirring, the precipitate is filtered, washed two times with diisopropyl ether (250 mL), then dried under a vacuum at 30° C. to yield a poly(gamma-benzyl-L-glutamic) acid capped at one of its ends by an acetyl group.

[0588] A 33% hydrobromic acid (HBr) solution in acetic acid (235 mL) is added dropwise to a solution of the co-polyamino acid referred to above (72 g) in trifluoroacetic acid (TFA, 335 mL) at 4° C. The mixture is stirred at room temperature for 3 h 30, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether and water under stirring (4 L). After 2 h of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed with a 1:1 (v/v) mixture of diisopropyl ether and water (340 mL), then with water (340 mL).

[0589] The solid obtained is then solubilized in water (1.5 L) by adjusting the pH to 7 by addition of a 10N aqueous sodium hydroxide solution, then a 1N aqueous sodium hydroxide solution. After solubilization, the solution is diluted by addition of water until obtaining a final volume of 2.1 L. The solution is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated until a final volume of 1.8 L is obtained.

[0590] The aqueous solution is then acidified by addition of 37% hydrochloric acid solution until a pH of 2 is reached. After 4 h of stirring, the precipitate obtained is filtered, washed with water (330 mL), then dried under a vacuum at 30° C. to yield a poly-L-glutamic acid having a number average molecular weight (Mn) of 3600 g/mol with respect to a polyoxyethylene standard (PEG), and a mean degree of polymerization of 21.

[0591] Co-Polyamino Acid AB7

[0592] By a process similar to the one used for the preparation the co-polyamino acid AB6 applied to the hydrochloride salt of molecule AA7 (1.43 g, 2.2 mmol) and to the co-polyamino acid AB7-1 (10.0 g), a poly-L-glutamic acid sodium salt modified by molecule AA7 is obtained.

[0593] Dry extract: 24.3 mg/g.

[0594] DP (estimated based on .sup.1H NMR): 21.

[0595] Based on .sup.1H NMR: i=0.03.

[0596] The calculated average molecular weight of the co-polyamino acid AB7 is 3677 g/mol.

[0597] HPLC-aqueous SEC (calibrant PEG): Mn=3300 g/mol.

Example AB8: Co-Polyamino Acid AB8—Sodium Poly-L-Glutamate Modified by Molecule AA7 and Having a Number Average Molecular Weight (Mn) of 3600 g/Mol

[0598] Co-Polyamino Acid AB8-1:

[0599] poly-L-glutamic acid having a number average molecular weight (Mn) of 3800 g/mol originating from the polymerization of γ-methyl-L-glutamate N-carboxyanhydride initiated by ammonia.

[0600] By a method similar to the one described in the patent application FR-A-2 801 226 applied to γ-methyl-L-glutamic acid N-carboxyanhydride (25.0 g, 133.6 mmol) and to a 0.5 N ammonia solution in dioxane (12.1 mL, 6.05 mmol), a poly-L-glutamic acid is obtained.

[0601] Co-Polyamino Acid AB8

[0602] By a method similar to the one used for the preparation of the co-polyamino acid AB6 applied to the hydrochloride salt of molecule AA7 (2.1 g, 3.24 mmol) and to the co-polyamino acid AB8-1 (14.3 g), a sodium poly-L-glutamate modified by molecule AA7 is obtained.

[0603] Dry extract: 25.2 mg/g.

[0604] DP (estimated based on .sup.1H NMR): 24.

[0605] Based on .sup.1H NMR: i=0.03.

[0606] The calculated average molecular weight of the co-polyamino acid AB8 is 4099 g/mol.

[0607] HPLC-aqueous SEC (calibrant PEG): Mn=3600 g/mol.

Example AB10: Co-Polyamino Acid AB10—Sodium Poly-L-Glutamate Modified by Molecule AA4 and Having a Number Average Molecular Weight (Mn) of 2600 g/Mol

[0608] By a method similar to the one used for the preparation of the co-polyamino acid AB7 applied to the hydrochloride salt of molecule AA4 and to a poly-L-glutamic acid obtained by a method similar to the one used for the preparation of the co-polyamino acid AB6-1, a sodium poly-L-glutamate modified by molecule AA4 is obtained.

[0609] Dry extract: 18.3 mg/g.

[0610] DP (estimated based on .sup.1H NMR: 25.

[0611] Based on .sup.1H NMR: i=0.08.

[0612] The calculated average molecular weight of the co-polyamino acid AB10 is 4870 g/mol.

[0613] HPLC-aqueous SEC (calibrant PEG): Mn=2600 g/mol.

Example AB21: Co-Polyamino Acid AB21—Sodium Poly-L-Glutamate Modified by Molecule AA7 and Having a Number Average Molecular Weight (Mn) of 3400 g/Mol

[0614] By a method similar to the one used for the preparation of the co-polyamino acid AB6 applied to the hydrochloride salt of molecule AA7 (2.44 g, 2.4 mmol) and to a poly-L-glutamic acid (10 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid AB6-1, a sodium poly-L-glutamate modified by molecule AA7 is obtained.

[0615] Dry extract: 22.7 mg/g.

[0616] DP (estimated based on .sup.1H NMR: 22.

[0617] Based on .sup.1H NMR: i=0.056.

[0618] The calculated average molecular weight of the co-polyamino acid AB21 is 4090 g/mol.

[0619] HPLC-aqueous SEC (calibrant PEG): Mn=3400 g/mol.

Example AB17: Co-Polyamino Acid AB17—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule AA7 and Having a Number Average Molecular Weight (Mn) of 3500 g/Mol

[0620] The hydrochloride salt of molecule AA7 (2.80 g, 4.32 mmol), chloroform (5 mL), molecular sieve 4A (1.3 g) as well as Amberlite IRN 150 ion exchange resin (1.3 g) are introduced successively into an appropriate container. After 1 h of stirring on rollers, the medium is filtered and the resin is rinsed with chloroform. The mixture is evaporated, then co-evaporated with toluene. The residue is solubilized in anhydrous DMF (30 mL) to be used directly in the polymerization reaction.

[0621] γ-Benzyl-L-glutamate N-carboxyanhydride (25.0 g, 94.9 mmol) is placed under a vacuum for 30 min in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (140 mL) is introduced. The mixture is stirred under argon until the solubilization is complete, cooled to 4° C., then the solution of molecule AA7 prepared as described above is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days, then heated at 65° C. for 2 h. The reaction mixture is then cooled to room temperature, then poured dropwise in diisopropyl ether (1.7 L) under stirring. The white precipitate is recovered by filtration, washed two times with diisopropyl ether (140 mL), then dried under a vacuum at 30° C. to obtain a white solid. The solid is diluted in TFA (160 mL), and then a 33% hydrobromic acid (HBr) solution in acetic acid (62 mL, 354 mmol) is added dropwise and at 0° C. The solution is stirred for 2 h at room temperature, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether/water and under stirring (1.9 L). After 2 h of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed successively with a 1:1 (v/v) mixture of diisopropyl ether and water (280 mL), then with water (140 mL). The solid obtained is solubilized in water (530 mL) by adjusting the pH to 7 by adding a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by addition of water until obtaining a final volume of 800 mL. The mixture is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated to approximately 30 g/L theoretical and the pH is adjusted to 7.0. The aqueous solution is filtered through 0.2 μm and stored at 4° C.

[0622] Dry extract: 25.2 mg/g.

[0623] DP (estimated by .sup.1H NMR)=26 therefore i=0.038.

[0624] The calculated average molecular weight of the co-polyamino acid AB17 is 4500 g/mol.

[0625] HPLC-aqueous SEC (calibrant PEG): Mn=3500 g/mol.

Example AB18: Co-Polyamino Acid AB18—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule AA7 Having a Number Average Molecular Weight (Mn) of 3700 g/Mol

[0626] A sodium poly-L-glutamate modified at one of its ends by molecule AA7 is obtained by polymerization of γ-methyl-glutamic acid N-carboxyanhydride (25.0 g, 133.6 mmol) using the hydrochloride salt of molecule AA7 (2.80 g, 4.32 mmol) as initiator and carrying out a deprotection of the methyl esters by using a 37% hydrochloric acid solution according to the method described in patent application FR-A-2 801 226.

[0627] Dry extract: 44.3 mg/g.

[0628] DP (estimated by .sup.1H NMR)=22 therefore i=0.045.

[0629] The calculated average molecular weight of the co-polyamino acid AB18 is 3896 g/mol.

[0630] HPLC-aqueous SEC (calibrant PEG): Mn=3700 g/mol.

Example AB21′: Co-Polyamino Acid AB21′-Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule AA10 and Having a Number Average Molecular Weight (Mn) of 3500 g/Mol

[0631] By a method similar to the one used for the preparation of the co-polyamino acid AB17, applied of the hydrochloride salt of molecule AA10 (0.916 g, 1.38 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (7.19 g, 27.3 mmol), a sodium poly-L-glutamate modified at one of its ends by molecule AA10 is obtained.

[0632] Dry extract: 14.8 mg/g

[0633] DP (estimated by .sup.1H NMR)=25, thus i=0.04

[0634] The calculated average molecular weight of the co-polyamino acid AB21′ is 4364 g/mol.

[0635] HPLC-aqueous-SEC (calibrant PEG): Mn=3500 g/mol.

Part B: Hydrophobic Radicals

[0636] BB: Synthesis of the Hydrophobic Molecules in which p=2

[0637] The radicals are represented in the following Table 1c′ by the corresponding hydrophobic molecule before grafting onto the co-polyamino acid

TABLE-US-00005 TABLE 1c′ list of hydrophobic molecules synthesized according to the invention. No. Structure of the hydrophobic molecule before grafting onto the co-polyamino acid BA1 [00086] BA2 [00087] BA3 [00088] BA4 [00089] BA5 [00090] BA6 [00091] BA7 [00092]

Example BA1: Molecule BA1

[0638] Molecule B1:

[0639] product obtained by the reaction between decanoic acid and L-proline.

[0640] Dicyclohexyl carbodiimide (DCC) (16.29 g, 78.96 mmol) and N-hydroxysuccinimide (NHS) (9.09 g, 78.96 mmol) are added successively to a decanoic acid solution (14.28 g, 82.91 mmol) in THF (520 mL) at 0° C. After 60 h of stirring at room temperature, the mixture is cooled to 0° C. for 20 min, filtered through a sintered filter. L-Proline (10 g, 86.86 mmol), diisopropylethylamine (DIPEA) (68.8 mL) and water (60 mL) are added to the filtrate. After 24 h of stirring at room temperature, the medium is diluted with water (300 mL). The aqueous phase is washed with ethyl acetate (2×250 mL), acidified to pH .sup.˜1 with a 1N aqueous HCl solution, then extracted with dichloromethane (3×150 mL). The combined organic phases are dried over Na.sub.2SO.sub.4, filtered, concentrated under a vacuum, and the residue is purified by chromatography on silica gel (cyclohexane, ethyl acetate).

[0641] Yield: 14.6 g (69%).

[0642] .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.26 (12H); 1.65 (2H); 2.02 (3H); 2.34 (2H); 2.41 (1H); 3.48 (1H); 3.56 (1H); 4.58 (1H)

[0643] LC/MS (ESI): 270.2; (calculated ([M+H].sup.+): 270.4).

[0644] Molecule B2:

[0645] product obtained by the reaction between molecule B1 and L-lysine.

[0646] By a method similar to the one used for the preparation of molecule B1 applied to molecule B1 (14.57 g, 54.07 mmol) and to L-lysine (4.15 g, 28.39 mmol), a yellow oil is obtained.

[0647] Yield: 16.4 g (93%).

[0648] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (6H); 1.26 (24H); 1.35-1.65 (8H); 1.85-2.35 (12H); 2.53 (0.2H); 2.90 (0.8H); 3.45-3.75 (5H); 4.50-4.70 (3H); 7.82 (1H).

[0649] LC/MS (ESI): 649.6; (calculated ([M+H].sup.+): 649.9).

[0650] Molecule B3:

[0651] product obtained by reaction between molecule B2 and Boc-ethylenediamine.

[0652] DIPEA (8.80 mL) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 8.52 g, 26.54 mmol) at room temperature are added to a solution of molecule B2 (16.4 g, 25.27 mmol) in THF (170 mL). After 30 min of stirring, the Boc-ethylenediamine (4.45 g, 27.8 mmol) is added. After stirring at room temperature for 2 h, the solvent is evaporated at reduced pressure and the residue is diluted with ethyl acetate (400 mL). The organic phase is washed with water (250 mL), a saturated aqueous NaHCO.sub.3 solution (250 mL), a 1 N aqueous HCl solution (250 mL), a saturated aqueous NaCl solution (250 mL), and dried over Na.sub.2SO.sub.4. After filtration and concentration under a vacuum, the residue obtained is purified by chromatography on silica gel (ethyl acetate, methanol) to yield a colorless oil.

[0653] Yield: 12.8 g (64%).

[0654] .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.25-1.60 (42H); 1.80-2.05 (4H); 2.15-2.45 (9H); 3.10-3.75 (10H); 4.30 (1H); 4.50 (2H); 5.50 (0.6H); 5.89 (0.2H); 6.15 (0.2H); 7.03 (1H); 7.47 (1H).

[0655] LC/MS (ESI): 791.8; (calculated ([M+H].sup.+): 792.1).

[0656] Molecule BA1

[0657] A 4N HCl solution in dioxane (20.2 mL) is added to a solution of molecule B3 (12.78 g, 16.15 mmol) in dichloromethane (110 mL) at 5° C. After 20 h of stirring at 5° C., the medium is concentrated under a vacuum. The residue obtained is dissolved in methanol and evaporated under a vacuum, this operation being repeated 4 times to yield a white solid of molecule BA1 in hydrochloride salt form.

[0658] Yield: 11.4 g (97%).

[0659] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.25-1.50 (33H); 1.57 (1H); 1.70-2.40 (12H); 2.82 (2H); 3.00 (2H); 3.25-3.70 (6H); 4.05-4.50 (3H); 7.75-8.45 (6H)

[0660] LC/MS (ESI): 691.6; (calculated ([M+H].sup.+): 692.0).

Example BA2: Molecule BA2

[0661] Molecule B4:

[0662] product obtained by the reaction between lauric acid and L-proline.

[0663] By a method similar to the one used for the preparation of molecule B1 applied to lauric acid (31.83 g, 157.9 mmol) and to L-proline (20 g, 173.7 mmol), a yellow oil is obtained.

[0664] Yield: 34.3 g (73%).

[0665] .sup.1H NMR (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).

[0666] LC/MS (ESI): 298.2; (calculated ([M+H].sup.+): 298.4).

[0667] Molecule B5:

[0668] product obtained by the reaction between molecule B4 and L-lysine.

[0669] By a method similar to the one used for the preparation of molecule B1 applied to molecule B4 (33.72 g, 113.36 mmol) and to L-lysine (8.70 g, 59.51 mmol), a white solid is obtained.

[0670] Yield: 26.2 g (66%).

[0671] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (6H); 1.26 (32H); 1.35-1.65 (8H); 1.85-2.35 (15H); 2.87 (1H); 3.40-3.75 (5H); 4.50-4.75 (3H); 7.87 (1H).

[0672] LC/MS (ESI): 705.6; (calculated ([M+H].sup.+): 706.0).

[0673] Molecule B6:

[0674] product obtained by reaction between Boc-ethylenediamine and molecule B5.

[0675] By a method similar to the one used for the preparation of molecule B3 applied to molecule B5 (25.74 g, 36.51 mmol) and to Boc-ethylenediamine (6.43 g, 40.16 mmol), a colorless oil is obtained.

[0676] Yield: 30.9 g (quantitative).

[0677] .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (6H); 1.35-1.65 (50H); 1.85-2.35 (13H); 3.05-3.75 (10H); 4.25-4.65 (3H); 5.50 (0.4H); 5.88 (0.2H); 6.16 (0.2H); 7.08 (1H); 7.26 (1H); 7.49 (0.2H).

[0678] LC/MS (ESI): 847.8; (calculated ([M+H].sup.+): 848.2).

[0679] Molecule BA2

[0680] By a method similar to the one used for the preparation of molecule BA1 applied to molecule B6 (30.9 g, 36.47 mmol), the residue obtained after concentration under a vacuum is dissolved in methanol and evaporated under a vacuum, this operation being repeated 4 times to yield a white solid of molecule BA2 in hydrochloride salt form after drying at reduced pressure.

[0681] Yield: 27.65 g (97%).

[0682] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.10-2.40 (54H); 2.75-3.15 (4H); 3.25-3.60 (6H); 4.05-4.50 (3H); 7.50-8.50 (6H).

[0683] LC/MS (ESI): 747.6; (calculated ([M+H].sup.+): 748.1).

Example BA3: Molecule BA3

[0684] Molecule B7: product obtained by the reaction between myristic acid and L-proline.

[0685] By a method similar to the one used for the preparation of molecule B1 applied to myristic acid (18.93 g, 82.91 mmol) and to L-proline (10 g, 86.86 mmol), a yellowish oil is obtained.

[0686] Yield: 20 g (78%).

[0687] .sup.1H NMR (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)

[0688] LC/MS (ESI): 326.2; (calculated ([M+H].sup.+): 326.6).

[0689] Molecule B8:

[0690] product obtained by the reaction between molecule B7 and L-lysine

[0691] By a method similar to the one used for the preparation of molecule B1 applied to molecule B7 (20.02 g, 61.5 mmol) and to L-lysine (4.72 g, 32.29 mmol), a white solid is obtained.

[0692] Yield: 12.3 g (53%).

[0693] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.26 (40H); 1.35-1.50 (6H); 1.50-2.10 (10H); 2.10-2.25 (4H); 3.01 (2H); 3.31-3.55 (4H); 4.10-4.40 (3H); 7.68 (0.6H); 7.97 (1H); 8.27 (0.4H); 12.50 (1H).

[0694] LC/MS (ESI): 761.8; (calculated ([M+H].sup.+): 762.1).

[0695] Molecule B9:

[0696] product obtained by the reaction between Boc-ethylenediamine and molecule B8.

[0697] By a method similar to the one used for the preparation of molecule B3 applied to molecule B8 (12 g, 15.77 mmol) and to Boc-ethylenediamine (3.03 g, 18.92 mmol), a colorless oil is obtained after purification by chromatography column on silica gel (ethyl acetate, methanol).

[0698] Yield: 12.5 g (88%).

[0699] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.20-1.55 (55H); 1.50-2.25 (14H); 2.95-3.10 (6H); 3.31-3.55 (4H); 4.10-4.40 (3H); 6.74 (1H); 7.60-8.25 (3H).

[0700] LC/MS (ESI): 904.1; (calculated ([M+H].sup.+): 904.3).

Molecule BA3

[0701] After a method similar to the one used for the preparation of molecule BA1 applied to molecule B9 (12.5 g, 13.84 mmol), the residue obtained after concentration under a vacuum is dissolved in methanol and evaporated under a vacuum, this operation being repeated 4 times to yield a white solid of molecule BA3 in hydrochloride salt form after drying at reduced pressure.

[0702] Yield: 9.2 g (79%).

[0703] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.10-1.65 (48H); 1.70-2.35 (12H); 2.85 (2H); 3.01 (2H); 3.25-3.65 (6H); 4.10-4.50 (3H); 7.70-8.40 (6H).

[0704] LC/MS (ESI): 803.9; (calculated ([M+H].sup.+): 804.2).

Example BA4: Molecule BA4

[0705] Molecule B10:

[0706] product obtained by the reaction between molecule B8 and Boc-1-amino-4,7,10-trioxa-13-tridecane.

[0707] By a method similar to the one used for the preparation of molecule B3 applied to molecule B8 (29.80 g, 39.15 mmol) and to Boc-1-amino-4,7,10-trioxa-13-tridecane (15.05 g, 49.96 mmol), a thick colorless oil is obtained.

[0708] Yield: 25.3 g (61%).

[0709] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.25-2.35 (75H); 2.85-3.20 (6H); 3.25-3.65 (16H); 4.10-4.45 (3H); 6.38 (0.1H); 6.72 (0.9H); 7.50-8.25 (3H).

[0710] LC/MS (ESI): 1064.2; (calculated ([M+H].sup.+): 1064.5).

Molecule BA4

[0711] After a method similar to the one used for the preparation of molecule BA1 applied to molecule B10 (25.3 g, 23.8 mmol), the residue obtained after concentration under a vacuum is dissolved in methanol and evaporated under a vacuum, this operation being repeated 4 times to yield a white solid of molecule BA4 in hydrochloride salt form after drying at reduced pressure.

[0712] Yield: 20.02 g (84%).

[0713] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.15-2.35 (66H); 2.80-3.20 (6H); 3.30-3.65 (16H); 4.10-4.45 (3H); 7.55-8.60 (6H).

[0714] LC/MS (ESI): 964.9; (calculated ([M+H].sup.+): 964.6).

Example BA5: Molecule BA5

Molecule B12: Product Obtained by Reaction Between Molecule A1 and L-Lysine.

[0715] By a method similar to the one used for the preparation of molecule B1 applied to molecule A1 (19.10 g, 54.02 mmol) and to L-lysine (4.15 g, 28.36 mmol), an oily residue is obtained after concentration of the reaction medium at reduced pressure. This residue is diluted in water (150 mL), washed with ethyl acetate (2×75 mL), then the aqueous phase is acidified to pH 1 by slow addition of 6 N HCl. The product is extracted 3 times with dichloromethane, the organic phase is dried over Na.sub.2SO.sub.4, then filtered and concentrated at reduced pressure to yield 11.2 g of yellow oily residue. In parallel, the previous organic phase of ethyl acetate is washed with a 2 N aqueous HCl solution (2×75 mL), a saturated aqueous NaCl solution (75 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated to yield 10.2 g of yellow oily residue. A white residue is obtained by recrystallization of each one of these residues in acetone.

[0716] Yield: 11.83 g (54%)

[0717] .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.06-2.44 (70H); 2.78-2.96 (1H); 3.35-3.75 (5H); 4.28-4.43 (0.1H); 4.43-4.52 (0.2H); 4.52-4.61 (1.8H); 4.61-4.75 (0.9H); 7.74-8.02 (2H).

[0718] LC/MS (ESI): 818.0; (calculated ([M+H].sup.+): 818.7).

Molecule B13: Product Obtained by Coupling Between Molecule B12 and Boc-Ethylenediamine.

[0719] DIPEA (3.42 g, 26.43 mmol) is added to a solution of molecule B12 (18.00 g, 22.02 mmol) at room temperature in THF (110 mL). The reaction medium is cooled to 0° C., then HOBt (337 mg, 2.20 mmol, EDC (4.64 g, 24.23 mmol) and then Boc-ethylenediamine (4.23 g, 26.43 mmol) are added successively. The reaction mixture is stirred for 1 h at 0° C., then for 24 h at room temperature and concentrated at reduced pressure. The residue is dissolved in ethyl acetate (250 mL) and dichloromethane (40 mL), the organic phase is washed with a 1 N aqueous HCl solution (2×125 mL), a saturated aqueous NaCl solution (2×125 mL), dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure. A white solid is obtained after recrystallization two times in acetonitrile.

[0720] Yield: 17.5 g (83%)

[0721] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.15-2.29 (79H); 2.92-3.12 (6H); 3.30-3.59 (4H); 4.06-4.13 (0.65H); 4.16-4.29 (2H); 4.38-4.42 (0.35H); 6.71-6.76 (1H); 7.60-7.69 (1.3H); 7.76-7.81 (0.65H); 7.93-7.97 (0.35H); 8.00-8.04 (0.35H); 8.10-8.17 (0.35H).

[0722] LC/MS (ESI): 960.4; (calculated ([M+H].sup.+): 960.8).

Molecule BA5

[0723] By a method similar to the one used for the preparation of molecule BA1 applied to molecule B13 (24.4 g, 25.43 mmol), the residue obtained after concentration under a vacuum is solubilized in dichloromethane (150 mL), the organic phase is washed 2 times with a 2M aqueous sodium hydroxide solution (90 mL). Acetonitrile (120 mL) is added, and the dichloromethane is eliminated by concentration at reduced pressure. The medium is then allowed to rest for 72 h, and a white solid is obtained after filtration and rinsing with acetonitrile, then drying at reduced pressure. This operation is repeated 4 times.

[0724] Yield: 14.28 g (65%)

[0725] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.06-2.32 (70H); 2.53-2.63 (2H); 2.89-3.61 (10H); 4.04-4.43 (3H); 7.55-7.62 (0.65H); 7.65-7.72 (0.65H); 7.80 (0.65H); 7.91 (0.35H); 8.03 (0.35H); 8.14-8.23 (0.35H).

[0726] LC/MS (ESI): 860.0; (calculated ([M+H].sup.+): 860.8).

Example BA6: Molecule BA6

Molecule B14: Product Obtained by Coupling Between Molecule B7 and 2,3-Diaminopropionic Acid

[0727] By a method similar to the one used for the preparation of molecule B1 applied to molecule B7 (80.00 g, 245.78 mmol) and to the 2,3-diaminopropionic acid dihydrochloride (22.84 g, 129.04 mmol), a white solid is obtained after recrystallization in acetonitrile.

[0728] Yield: 69 g (78%)

[0729] .sup.1H NMR (DMSO-d6, ppm): 0.86 (6H); 1.08-1.38 (40H); 1.40-1.55 (4H); 1.68-2.30 (12H); 3.16-3.66 (6H); 4.20-4.39 (3H); 7.67-8.31 (2H); 12.70 (1H).

[0730] LC/MS (ESI): 719.4; 741.5; (calculated ([M+H].sup.+): 719.6; ([M+Na]+): 741.6).

[0731] Molecule B15: product obtained by coupling between B14 and Boc-ethylenediamine

[0732] By a method similar to the one used for the preparation of molecule B13 applied to molecule B14 (32.00 g, 44.50 mmol) in solution in dichloromethane and to Boc-ethylenediamine (8.56 g, 53.40 mmol), a colorless oil is obtained after purification by chromatography on silica gel (ethyl acetate, methanol).

[0733] Yield: 24.5 g (64%)

[0734] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.16-2.42 (65H); 2.89-3.14 (4H); 3.17-3.66 (6H); 4.11-4.43 (3H); 6.77 (1H); 7.38-8.23 (3H).

[0735] LC/MS (ESI): 861.7; (calculated ([M+H].sup.+): 861.7).

Molecule BA6

[0736] By a method similar to the one used for the preparation of molecule BA5 applied to molecule B15 (24.50 g, 28.45 mmol), a white solid is obtained after recrystallization in acetonitrile.

[0737] Yield: 19.7 g (91%)

[0738] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.10-2.40 (58H); 2.51-2.62 (2H); 2.90-3.16 (2H); 3.16-3.67 (6H); 4.04-4.47 (3H); 7.33-8.27 (3H).

[0739] LC/MS (ESI): 761.5; (calculated ([M+H].sup.+): 761.6).

Example BA7: Molecule BA7

[0740] Molecule B16: product obtained by the reaction between N-(tert-butoxycarbonyl-1,6-diaminohexane and molecule B8.

[0741] By a method similar to the one used for the preparation of molecule B13 applied to molecule B8 (10 g, 13.14 mmol) and to N-(tert-butoxycarbonyl-1,6-diaminohexane (3.41 g, 15.77 mmol) in dichloromethane, a white solid is obtained after recrystallization in acetonitrile.

[0742] Yield: 10.7 g (85%)

[0743] .sup.1H NMR (CDCl3, ppm): 0.88 (6H); 1.17-2.40 (79H); 3.00-3.71 (10H); 4.26-4.58 (3H); 4.67 (1H); 6.74 (1H); 7.34-7.49 (2H).

[0744] LC/MS (ESI): 969.9; (calculated ([M+H].sup.+): 959.8).

Molecule BA7

[0745] After a method similar to the one used for the preparation of molecule BA1 applied to molecule B16 (10.5 g, 10.94 mmol), a 2N aqueous NaOH solution is added dropwise to the reaction cooled to 0° C. The aqueous phase is extracted with dichloromethane, then the organic phase is washed 3 times with a 5% aqueous NaCl solution. After drying over Na.sub.2SO.sub.4, the organic phase is filtered, concentrated under a vacuum, and the residue is recrystallized in acetonitrile.

[0746] Yield: 5.4 g (58%).

[0747] .sup.1H NMR (CDCl3, ppm): 0.88 (6H); 1.19-2.40 (72H); 2.67 (2H); 3.03-3.70 (8H); 4.26-4.57 (3H); 6.71 (1H); 7.39-7.49 (2H).

[0748] LC/MS (ESI): 859.8; (calculated ([M+H].sup.+): 859.7).

BB: Synthesis of the Co-Polyamino Acids

[0749] Co-Polyamino Acids with Statistical Grafting (Formulas VII and VIIa)

TABLE-US-00006 TABLE 1d list of co-polyamino acids synthesized according to the invention No. co-polyamino acids bearing carboxylate charges and hydrophobic radicals BB1 [00093] i = 0.05, DP (m + n) = 23 [00094] R.sub.1 = H or pyroglutamate BB2 [00095] i = 0.047, DP (m + n) = 21 [00096] R.sub.1 = H or pyroglutamate BB3 [00097] i = 0.049, DP (m + n) = 34 [00098] R.sub.1 = H or pyroglutamate BB4 [00099] i = 0.04, DP (m + n) = 65 [00100] R1 = H or pyroglutamate BB5 [00101] i = 0.042, DP (m + n) = 23 [00102] R.sub.1 = CH.sub.3—C(O)—, H or pyroglutamate BB6 [00103] i = 0.04, DP (m + n) = 24 [00104] R.sub.1 = CH.sub.3—C(O)—, H or pyroglutamate BB7 [00105] i = 0.042, DP (m + n) = 22 [00106] R.sub.1 = H or pyroglutamate BB8 [00107] i = 0.026, DP (m + n) = 21 [00108] R.sub.1 = H or pyroglutamate BB9 [00109] i = 0.05, DP (m + n) = 26 [00110] R.sub.1 = H or pyroglutamate BB10 [00111] i = 0.029, DP (m + n) = 22 [00112] R1 = H or pyroglutamate BB11 [00113] i = 0.032, DP (m + n) = 22 [00114] R.sub.1 = CH.sub.3—C(O)—, H or pyroglutamate BB12 [00115] i = 0.03, DP (m + n) = 23 [00116] R.sub.1 = CH.sub.3—C(O)—, H or pyroglutamate BB13 [00117] i = 0.08, DP (m + n) = 25 [00118] R.sub.1 = H or pyroglutamate
Co-Polyamino Acids with Defined Grafting (Formulas VII and VIIb)

TABLE-US-00007 TABLE 1e list of co-polyamino acids synthesized according to the invention No. co-polyamino acids bearing carboxylate charges and hydrophobic radicals BB14 [00119] BB15 [00120] BB16 [00121] BB17 [00122] BB18 [00123] BB19 [00124] BB20 [00125] BB21 [00126] BB22 [00127] BB23 [00128] BB24 [00129] BB25 [00130] BB26 [00131] BB42 [00132] BB44 [00133]

Example BB1: Co-Polyamino Acid BB1—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 2400 g/Mol

[0750] Co-Polyamino Acid BB1-1:

[0751] poly-L-glutamic acid having a relative number average molecular weight (Mn) of 3860 g/mol originating from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by hexylamine.

[0752] In a round-bottom flask dried beforehand in the oven, γ-benzyl-L-glutamate N-carboxyanhydride (90.0 g, 342 mmol) is placed under a vacuum for 30 min, then anhydrous DMF (465 mL) is introduced. The mixture is then stirred under argon until the dissolution is complete, cooled to 4° C., then hexylamine (1.8 mL, 14 mmol) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days. The reaction medium is then heated at 65° C. for 4 h, cooled to room temperature, then poured dropwise into cold diisopropyl ether (6 L) under stirring. The white precipitate is recovered by filtration, washed with diisopropyl ether (500 mL, then 250 mL), then dried under a vacuum at 30° C. to yield a poly(γ-benzyl-L-glutamic acid) (PBLG).

[0753] A 33% hydrobromic acid (HBr) solution in acetic acid (135 mL, 0.77 mol) is added dropwise to a solution of PBLG (42.1 g) in trifluoroacetic acid (TFA, 325 mL) at 4° C. The mixture is stirred at room temperature for 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether and water under stirring (1.6 L). After 1 h 30 of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed with a 1:1 (v/v) mixture of diisopropyl ether and water (200 mL).

[0754] The solid obtained is then solubilized in water (1 L) by adjusting the pH to 7 by adding a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. After solubilization, the theoretical concentration is adjusted to 25 g/L theoretical by addition of water until obtaining a final volume of 1.5 L.

[0755] The solution is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm.

[0756] The aqueous solution is then acidified by addition of 37% hydrochloric acid solution until a pH of 2 is reached. After 4 h of stirring, the precipitate obtained is filtered, then dried under a vacuum at 30° C. to yield a poly-L-glutamic acid having a number average molecular weight (Mn) of 3860 g/mol with respect to a standard of polyoxyethylene (PEG).

Co-Polyamino Acid BB1

[0757] The co-polyamino acid BB1-1 (10.0 g) is solubilized in DMF (700 mL) at 30-40° C. then cooled to 0° C. The hydrochloride salt of molecule BA2 (2.95 g, 3.8 mmol) is suspended in DMF (45 mL), and triethylamine (0.39 g, 3.8 mmol) is then added to this suspension, then the mixture is heated slightly under stirring until the dissolution is complete. N-Methylmorpholine (NMM, 7.6 g, 75 mmol) in DMF (14 mL) and ethyl chloroformate (ECF, 8.1 g, 75 mmol) are added to the solution of co-polyamino acid at 0° C. After 10 min at 0° C., the solution of BA2 is added, and the medium is maintained at 30° C. for 1 h. The reaction medium is poured dropwise onto 6 L of water containing 15% by weight of sodium chloride, and HCl (pH 2), then left to rest overnight. The precipitate is collected by filtration, washed with the sodium chloride solution at pH 2 (1 L) and dried under a vacuum for approximately 1 h. The white solid obtained is taken up in water (600 mL), and the pH is adjusted to 7 by slow addition of a 1 N aqueous NaOH solution. The volume is adjusted to 700 mL by addition of water. After filtration through a 0.45-μm filter, the clear solution obtained is purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. After discharging, the solution is filtered through a 0.2-μm filter and stored at 2-8° C.

[0758] Dry extract: 19.7 mg/g.

[0759] DP (estimated based on .sup.1H NMR): 23.

[0760] Based on .sup.1H NMR: i=0.05.

[0761] The calculated average molecular weight of the co-polyamino acid BB1 is 4350 g/mol.

[0762] HPLC-aqueous SEC (calibrant PEG): Mn=2400 g/mol.

Example BB2: Co-Polyamino Acid BB2—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 4900 g/Mol

[0763] A poly-L-glutamic acid having a number average molecular weight (Mn) of 4100 g/mol (5.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1 is solubilized in DMF (205 mL) at 30-40° C., then maintained at this temperature. In parallel, the hydrochloride salt of molecule BA2 (1.44 g, 1.84 mmol) is suspended in DMF (10 mL), and triethylamine (0.19 g, 1.84 mmol) is added, then the mixture is heated slightly under stirring until the dissolution is complete. NMM (3.7 g, 36.7 mmol), the solution of molecule BA2, then 2-hydroxypyridine N-oxide (HOPO, 0.31 g, 2.76 mmol) are added successively to the solution of co-polyamino acid in DMF. The reaction medium is then cooled to 0° C., then EDC (0.53 g, 2.76 mmol) is added and the temperature of the medium is allowed to rise to room temperature over a time period of 3 h. The reaction medium is poured dropwise onto 1.55 L of water containing 15% by weight of NaCl and HCl (pH 2) under stirring. At the end of the addition, the pH is readjusted to 2 with a 1 N HCl solution, and the suspension is allowed to rest overnight. The precipitate is collected by filtration, then rinsed with 100 mL of water. The white solid obtained is solubilized in 200 mL of water by slow addition of a 1 N aqueous NaOH solution until the pH is 7 under stirring, then the solution is filtered through a 0.45-μm filter. The clear solution obtained is purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered through a 0.2-μm filter and stored at 2-8° C.

[0764] Dry extract: 16.3 mg/g.

[0765] DP (estimated based on .sup.1H NMR): 21.

[0766] Based on .sup.1H NMR: i=0.047.

[0767] The calculated average molecular weight of the co-polyamino acid BB2 is 3932 g/mol.

[0768] HPLC-aqueous SEC (calibrant PEG): Mn=4900 g/mol.

Example BB3: Co-Polyamino Acid BB3—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 6400 g/Mol

[0769] Co-polyamino acid BB3-1: poly-L-glutamic acid having a number average molecular weight (Mn) of 17500 g/mol originating from the polymerization of γ-methyl-L-glutamate N-carboxyanhydride initiated by L-leucinamide.

[0770] A poly-L-glutamic acid having a number average weight (Mn) of 17500 g/mol with respect to a methyl polymethacrylate (PMMA) standard is obtained by polymerization of γ-methyl-glutamic acid N-carboxyanhydride using L-leucinamide as initiator and carrying out a deprotection of the methyl esters by using a 37% hydrochloric acid solution according to the method described in patent application FR-A-2 801 226.

[0771] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (3.23 g, 4.1 mmol) and to the co-polyamino acid BB3-1 (11 g), a sodium poly-L-glutamate modified by molecule BA2 is obtained.

[0772] Dry extract: 27.5 mg/g.

[0773] DP (estimated based on .sup.1H NMR): 34.

[0774] Based on .sup.1H NMR: i=0.049

[0775] The calculated average molecular weight of the co-polyamino acid BB3 is 6405 g/mol.

[0776] H PLC-aqueous SEC (calibrant PEG): Mn=6400 g/mol.

Example BB4: Co-Polyamino Acid BB4—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 10500 g/Mol

[0777] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (5 g, 6.35 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=10800 g/mol (21.7 g obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecular BA2 is obtained.

[0778] Dry extract: 28.2 mg/g.

[0779] DP (estimated based on .sup.1H NMR): 65.

[0780] Based on .sup.1H NMR: i=0.04.

[0781] The calculated average molecular weight of the co-polyamino acid BB4 is 11721 g/mol.

[0782] H PLC-aqueous SEC (calibrant PEG): Mn=10500 g/mol.

Example BB5: Co-Polyamino Acid BB5—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 3600 g/mol

[0783] Co-polyamino acid BB5-1: poly-L-glutamic acid having an Mn of 3700 g/mol originating from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by hexylamine and capped at one of its ends by an acetyl group.

[0784] γ-Benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol) is placed under a vacuum for 30 minutes in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (250 mL) is introduced. The mixture is then stirred under argon until the dissolution is complete, cooled to 4° C., then hexylamine (2.3 mL, 17 mmol) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days, then precipitated in diisopropyl ether (3.4 L). The precipitate is recovered by filtration, washed two times with diisopropyl ether (225 mL), then dried to yield a white solid which is dissolved in 450 mL of THF. N,N-Diisopropylethylamine (DIPEA, 31 mL, 176 mmol) then acetic anhydride (17 mL, 176 mmol) are added successively to this solution. After stirring at room temperature overnight the solution is poured slowly into diisopropyl ether (3 L) for a time period of 30 min and under stirring. After 1 h of stirring, the precipitate is filtered, washed two times with diisopropyl ether (200 mL), then dried under a vacuum at 30° C. to yield a poly(γ-benzyl-L-glutamic acid) capped at one of its ends by an acetyl group.

[0785] A 33% hydrobromic acid (HBr) solution in acetic acid (235 mL, 1.34 mol) is added dropwise to a solution of the capped co-polyamino acid (72 g) in trifluoroacetic acid (TFA, 335 mL) at 4° C. The mixture is stirred at room temperature for 3 h 30, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether and water under stirring (4 L). After 2 h of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed with a 1:1 (v/v) mixture of diisopropyl ether and water (340 mL), then with water (340 mL). The solid obtained is then solubilized in water (1.5 L) by adjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by addition of water until obtaining a final volume of 2.1 L. The solution is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated until a final volume of 1.8 L is obtained. The aqueous solution is then acidified by addition of 37% hydrochloric acid solution until a pH of 2 is reached. After 4 h of stirring, the precipitate obtained is filtered, washed with water (330 mL), then dried under a vacuum at 30° C. to yield a poly-L-glutamic acid having a number average molecular weight (Mn) of 3700 g/mol with respect to a standard of polyoxyethylene (PEG).

Co-Polyamino Acid BB5

[0786] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (6.92 g, 8.8 mmol) and to co-polyamino acid BB5-1 (30.0 g), a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by molecule BA2 is obtained.

[0787] Dry extract: 29.4 mg/g.

[0788] DP (estimated based on .sup.1H NMR): 23.

[0789] Based on .sup.1H NMR: i=0.042.

[0790] The calculated average molecular weight of the co-polyamino acid BB5 is 4302 g/mol.

[0791] HPLC-aqueous SEC (calibrant PEG): Mn=3600 g/mol.

Example BB6: Co-Polyamino Acid BB6—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 4100 g/mol

[0792] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (5.8 g, 7.4 mmol) and to poly-L-glutamic acid having a number average molecular weight Mn=3800 g/mol (25 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1 using ammonia instead of hexylamine, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by molecule BA2 is obtained.

[0793] Dry extract: 27.6 mg/g.

[0794] DP (estimated based on .sup.1H NMR): 24.

[0795] Based on .sup.1H NMR: i=0.04.

[0796] The calculated average molecular weight of the co-polyamino acid BB6 is 4387 g/mol.

[0797] HPLC-aqueous SEC (calibrant PEG): Mn=4100 g/mol.

Example BB7: Co-Polyamino Acid BB7—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 4200 g/Mol

[0798] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (7.07 g, 9.0 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=3600 g/mol (30.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecule BA2 is obtained.

[0799] Dry extract: 28.3 mg/g.

[0800] DP (estimated based on .sup.1H NMR): 22.

[0801] Based on .sup.1H NMR: i=0.042.

[0802] The calculated average molecular weight of the co-polyamino acid BB7 is 4039 g/mol.

[0803] HPLC-aqueous SEC (calibrant PEG): Mn=4200 g/mol.

Example BB8: Co-Polyamino Acid BB8—Sodium Poly-L-Glutamate Modified by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 5200 g/Mol

[0804] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA2 (0.85 g, 1.1 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=4100 g/mol (5.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecule BA2 is obtained.

[0805] Dry extract: 28.6 mg/g.

[0806] DP (estimated based on .sup.1H NMR): 21.

[0807] Based on .sup.1H NMR: i=0.026.

[0808] The calculated average molecular weight of the co-polyamino acid BB8 is 3620 g/mol.

[0809] HPLC-aqueous SEC (calibrant PEG): Mn=5200 g/mol.

Example BB9: Co-Polyamino Acid BB9—Sodium Poly-L-Glutamate Modified by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 4700 g/Mol

[0810] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA3 (3.05 g, 3.6 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=4100 g/mol (10.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecule BA3 is obtained.

[0811] Dry extract: 28.6 mg/g.

[0812] DP (estimated based on .sup.1H NMR): 26.

[0813] Based on .sup.1H NMR: i=0.05.

[0814] The calculated average molecular weight of the co-polyamino acid BB9 is 4982 g/mol.

[0815] HPLC-aqueous SEC (calibrant PEG): Mn=4700 g/mol.

Example BB10: Co-Polyamino Acid BB10—Sodium Poly-L-Glutamate Modified by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 4200 g/Mol

[0816] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA3 (1.90 g, 2.3 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=3500 g/mol (10.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecule BA3 is obtained.

[0817] Dry extract: 25.9 mg/g.

[0818] DP (estimated based on .sup.1H NMR): 22.

[0819] Based on .sup.1H NMR: i=0.029.

[0820] The calculated average molecular weight of the co-polyamino acid BB10 is 3872 g/mol.

[0821] HPLC-aqueous SEC (calibrant PEG): Mn=4200 g/mol.

Example BB11: Co-Polyamino Acid BB11—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Molecule BA4 and Having a Number Average Molecular Weight (Mn) of 3900 g/mol

[0822] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA4 (2.21 g, 2.2 mmol) and to a poly-L-glutamic acid having a number average weight Mn=3700 g/mol (10 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by molecular BA4 is obtained.

[0823] Dry extract: 28.1 mg/g.

[0824] DP (estimated based on .sup.1H NMR): 22.

[0825] Based on .sup.1H NMR: i=0.032.

[0826] The calculated average molecular weight of the co-polyamino acid BB11 is 4118 g/mol.

[0827] HPLC-aqueous SEC (calibrant PEG): Mn=3900 g/mol.

Example BB12: Co-Polyamino Acid BB12—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 3900 g/mol

[0828] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule BA3 (1.9 g, 2.3 mmol) and to a poly-L-glutamic acid having a number average weight Mn=3600 g/mol (10 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by molecular BA3 is obtained.

[0829] Dry extract: 26.7 mg/g.

[0830] DP (estimated based on .sup.1H NMR): 23.

[0831] Based on .sup.1H NMR: i=0.03.

[0832] The calculated average molecular weight of the co-polyamino acid BB12 is 4145 g/mol.

[0833] HPLC-aqueous SEC (calibrant PEG): Mn=3900 g/mol.

Example BB13: Co-Polyamino Acid BB13—Sodium Poly-L-Glutamate Modified by Molecule BA1 and Having a Number Average Molecular Weight (Mn) of 2800 g/Mol

[0834] By a method similar to the one used for the preparation of the co-polyamino acid BB1 applied to the hydrochloride salt of molecule BA1 (3.65 g, 5 mmol) and to a poly-L-glutamic acid having a number average molecular weight Mn=3600 g/mol (10 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1, a sodium poly-L-glutamate modified by molecule BA1 is obtained.

[0835] Dry extract: 25.6 mg/g.

[0836] DP (estimated based on .sup.1H NMR): 25.

[0837] Based on .sup.1H NMR: i=0.08.

[0838] The calculated average molecular weight of the co-polyamino acid BB13 is 5253 g/mol.

[0839] HPLC-aqueous SEC (calibrant PEG): Mn=2800 g/mol.

Example BB14: Co-Polyamino Acid BB14—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule BA2 and Having a Number Average Molecular Weight (Mn) of 4020 g/Mol

[0840] The hydrochloride salt of molecule BA2 (2.12 g, 2.70 mmol), chloroform (40 mL), molecular sieve 4 A (1.5 g) as well as Amberlite IRN 150 ion exchange resin (1.5 g) are introduced successively into an appropriate container. After 1 h of stirring on rollers, the medium is filtered and the residue is rinsed with chloroform. The mixture is evaporated, then co-evaporated with toluene. The residue is solubilized in anhydrous DMF (20 mL) to be used directly in the polymerization reaction.

[0841] γ-Benzyl-L-glutamate N-carboxyanhydride (18 g, 68.42 mmol) is placed under a vacuum for 30 min in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (100 mL) is introduced. The mixture is stirred under argon until the solubilization is complete, cooled to 4° C., then the solution of molecule BA2 prepared as described above is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 2 days, then heated at 65° C. for 2 h. The reaction mixture is then cooled to room temperature, then poured dropwise into diisopropyl ether (1.2 L) under stirring. The white precipitate is recovered by filtration, washed two times with diisopropyl ether (100 mL), then dried under a vacuum at 30° C. to obtain a white solid. The solid is diluted in TFA (105 mL), and a 33% hydrobromic acid (HBr) solution in acetic acid (38 mL, 220 mmol) is then added dropwise and at 0° C. The solution is stirred for 2 h at room temperature, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether/water and under stirring (600 mL). After 2 h of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed successively with a 1:1 (v/v) mixture of diisopropyl ether and water (200 mL), then with water (100 mL). The solid obtained is solubilized in water (450 mL) by adjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. The mixture is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated to approximately 30 g/L theoretical and the pH is adjusted to 7.0. The aqueous solution is filtered through a 0.2-μm filter and stored at 4° C.

[0842] Dry extract: 22.3 mg/g.

[0843] DP (estimated by .sup.1H NMR)=29, thus i=0.034.

[0844] The calculated average molecular weight of the co-polyamino acid BB14 is 5089 g/mol.

[0845] HPLC-aqueous SEC (calibrant PEG): Mn=4020 g/mol.

Example BB15: Co-Polyamino Acid BB15—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 3610 g/Mol

[0846] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the hydrochloride salt of the molecule BA3 (3.62 g, 4.32 mmol) and to 25.0 g (94.97 mmol) of γ-benzyl-L-glutamate N-carboxyanhydride, a sodium poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0847] Dry extract: 26.5 mg/g

[0848] DP (estimated by .sup.1H NMR)=24, thus i=0.042.

[0849] The calculated average molecular weight of the co-polyamino acid BB15 is 4390 g/mol.

[0850] HPLC-aqueous SEC (calibrant PEG): Mn=3610 g/mol.

Example BB16: Co-Polyamino Acid BB16—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule BA4 and Having a Number Average Molecular Weight (Mn) of 3300 g/Mol

[0851] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the hydrochloride salt of the molecule BA4 (5.70 g, 5.70 mmol) and to 29.99 g (113.9 mmol) of γ-benzyl-L-glutamate N-carboxyanhydride, a sodium poly-L-glutamate modified at one of its ends by the molecule BA4 is obtained.

[0852] Dry extract: 32.3 mg/g

[0853] DP (estimated by .sup.1H NMR)=23, thus i=0.043.

[0854] The calculated average molecular weight of the co-polyamino acid BB16 is 4399 g/mol.

[0855] HPLC-aqueous SEC (calibrant PEG): Mn=3300 g/mol.

Example BB17: Co-Polyamino Acid BB17—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 10700 g/Mol

[0856] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the hydrochloride salt of the molecule BA3 (2.51 g, 3 mmol) and at 52.7 g (200 mmol) of γ-benzyl-L-glutamate N-carboxyanhydride, a sodium poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0857] Dry extract: 24.5 mg/g

[0858] DP (estimated by .sup.1H NMR)=65, thus i=0.015.

[0859] The calculated average molecular weight of the co-polyamino acid BB17 is 10585 g/mol.

[0860] HPLC-aqueous SEC (calibrant PEG): Mn=10700 g/mol.

Example BB18: Co-Polyamino Acid BB18—Sodium Poly-L-Glutamate Modified at One of its Ends by Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 6600 g/Mol

[0861] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the hydrochloride salt of the molecule BA3 (2.51 g, 3 mmol) and at 31.6 g (120 mmol) of γ-benzyl-L-glutamate N-carboxyanhydride, a sodium poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0862] Dry extract: 27.3 mg/g

[0863] DP (estimated by .sup.1H NMR)=40, thus i=0.025.

[0864] The calculated average molecular weight of the co-polyamino acid BB18 is 6889 g/mol.

[0865] HPLC-aqueous SEC (calibrant PEG): Mn=6600 g/mol.

Example BB19: Co-Polyamino Acid BB19—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 3400 g/Mol)

[0866] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the hydrochloride salt of the molecule BA3 (36.26 g, 43.2 mmol) and of γ-benzyl-L-glutamate N-carboxyanhydride (250.0 g, 949.7 mmol), a sodium poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0867] Dry extract: 22.4 mg/g

[0868] DP (estimated by .sup.1H NMR)=25, thus i=0.04.

[0869] The calculated average molecular weight of the co-polyamino acid BB19 is 4540 g/mol.

[0870] HPLC-aqueous SEC (calibrant PEG): Mn=3400 g/mol.

Example BB20: Co-Polyamino Acid BB20—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 2500 g/Mol

[0871] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the molecule BA3 in free amine form (1.017 g, 12.7 mmol) and of γ-benzyl-L-glutamate N-carboxyanhydride (5.0 g, 19.0 mmol), a sodium-poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0872] Dry extract: 11.2 mg/g

[0873] DP (estimated by .sup.1H NMR)=17, thus i=0.059.

[0874] The calculated average molecular weight of the co-polyamino acid BB20 is 3332 g/mol.

[0875] HPLC-aqueous SEC (calibrant PEG): Mn=2500 g/mol.

Example BB21: Co-Polyamino Acid BB21—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 1100 g/Mol

[0876] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the molecule BA3 in free amine form (3.814 g, 4.75 mmol) and of γ-benzyl-L-glutamate N-carboxyanhydride (10.0 g, 38.0 mmol), a sodium-poly-L-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0877] Dry extract: 16.1 mg/g

[0878] DP (estimated by .sup.1H NMR)=9, thus i=0.11.

[0879] The calculated average molecular weight of the co-polyamino acid BB21 is 2123 g/mol.

[0880] H PLC-aqueous SEC (calibrant PEG): Mn=1100 g/mol.

Example BB22: Co-Polyamino Acid BB22—Sodium Poly-D-Glutamate Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 2900 g/Mol

[0881] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the molecule BA3 in free amine form (2.77 g, 3.45 mmol) and of γ-benzyl-D-glutamate N-carboxyanhydride (20.0 g, 76.0 mmol), a sodium-poly-D-glutamate modified at one of its ends by the molecule BA3 is obtained.

[0882] Dry extract: 15.2 mg/g

[0883] DP (estimated by .sup.1H NMR)=21, thus i=0.048.

[0884] The calculated average molecular weight of the co-polyamino acid BB22 is 3936 g/mol.

[0885] H PLC-aqueous SEC (calibrant PEG): Mn=2900 g/mol.

Example B23: Co-Polyamino Acid BB23—a Random Copolymer of Unit Sodium D- or L-Glutamate, Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 2800 g/mol

[0886] γ-Benzyl-L-glutamate N-carboxyanhydride (20.0 g, 76.00 mmol) and γ-benzyl-D-glutamate N-carboxyanhydride (20.0 g, 76.00 mmol) are placed under a vacuum for 30 min in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (75 mL) is introduced. The mixture is stirred under argon until the solubilization is complete, cooled to 4° C., then a solution of molecule BA3 in free amine form (5.55 g, 6.91 mmol) in chloroform (14.5 mL) is introduced rapidly. The mixture is stirred between 4° C. and room temperature for 18 h, then heated at 65° C. for 2 h. The reaction mixture is then cooled to room temperature, then poured dropwise into diisopropyl ether (1.2 L) under stirring. The white precipitate is recovered by filtration, washed three times with diisopropyl ether (80 mL), then dried under a vacuum at 30° C. until obtaining a white solid. The solid is diluted in TFA (152 mL), and a 33% hydrobromic acid (HBr) solution in acetic acid (106 mL, 220 mmol) is then added dropwise and at 0° C. The solution is stirred for 3 h at room temperature then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether/water and under stirring (1.84 L). The aqueous phase is separated in a dropping funnel, and the pH is adjusted to 7.2 by addition of a 10 N aqueous NaOH solution. After addition of water (250 mL), the mixture is filtered through a 0.45-μm filter then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated to approximately 25 g/L, filtered through a 0.2-μm filter and stored at 4° C.

[0887] Dry extract: 28.2 mg/g.

[0888] DP (estimated by .sup.1H NMR)=21, thus i=0.048.

[0889] The calculated average molecular weight of the co-polyamino acid BB23 is 3936 g/mol.

[0890] HPLC-aqueous SEC (calibrant PEG): Mn=2800 g/mol.

Example BB24: Co-Polyamino Acid BB24—a Block Copolymer of Sodium Poly-D-Glutamate and Sodium Poly-L-Glutamate, Modified at One of its Ends by the Molecule BA3 and Having a Number Average Molecular Weight (Mn) of 2800 g/Mol

[0891] γ-Benzyl-D-glutamate N-carboxyanhydride (13.5 g, 51.3 mmol) is placed under a vacuum for 30 min in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (52 mL) is introduced. The mixture is stirred under argon until the solubilization is complete, cooled to 0° C., then a solution of molecule BA3 in free amine form (3.43 g, 4.27 mmol) in chloroform (8.6 mL) is introduced rapidly. The mixture is stirred at 0° C. for 24 h, then a solution of γ-tert-butyl-L-glutamate N-carboxyanhydride (13.5 g, 58.9 mmol) in DMF (15 mL) is added. The mixture is then stirred between 0° C. and room temperature for 21 h, then heated at 65° C. for 2 h. The reaction mixture is then cooled to room temperature, then poured dropwise into diisopropyl ether (0.8 L) under stirring. The white precipitate is recovered by filtration, washed three times with diisopropyl ether (53 mL), then dried under a vacuum at 30° C. until obtaining a white solid. The solid is diluted in TFA (96 mL), and a 33% hydrobromic acid (HBr) solution in acetic acid (68 mL, 388 mmol) is then added dropwise and at 0° C. The solution is stirred for 2 h at room temperature, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether/water and under stirring (1.2 L). After 2 h of stirring, the heterogeneous mixture is allowed to rest overnight. The white precipitate is recovered by filtration, washed successively with a 1:1 (v/v) mixture of diisopropyl ether and water (100 mL), then with water (100 mL). The solid obtained is solubilized in water (900 ml) by adjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. The mixture is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution of co-polyamino acid is then concentrated to approximately 20 g/L theoretical and the pH is adjusted to 7.0. The aqueous solution is filtered through a 0.2-μm filter and stored at 4° C.

[0892] Dry extract: 23.9 mg/g

[0893] DP (estimated by .sup.1H NMR)=25, thus i=0.04

[0894] The calculated average molecular weight of the co-polyamino acid BB24 is 4541 g/mol.

[0895] HPLC-aqueous SEC (calibrant PEG): Mn=2800 g/mol.

Example BB25: Co-Polyamino Acid BB25—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA5 and Having a Number Average Molecular Weight (Mn) of 2800 g/Mol

[0896] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the molecule BA5 in free amine form (1.70 g, 1.98 mmol) and of γ-benzyl-L-glutamate N-carboxyanhydride (11.46 g, 43.5 mmol), a sodium-L-glutamate modified at one of its ends by the molecule BA5 is obtained.

[0897] Dry extract: 19.8 mg/g

[0898] DP (estimated by .sup.1H NMR)=23, thus i=0.043

[0899] The calculated average molecular weight of the co-polyamino acid BB25 is 4295 g/mol.

[0900] HPLC-aqueous SEC (calibrant PEG): Mn=2800 g/mol.

Example BB26: Co-Polyamino Acid BB26—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA6 and Having a Number Average Molecular Weight (Mn) of 2900 g/Mol

[0901] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to the molecule BA6 in free amine form (3.05 g, 4.01 mmol) and of γ-benzyl-L-glutamate N-carboxyanhydride (22.78 g, 86.5 mmol), a sodium-poly-L-glutamate modified at one of its ends by the molecule BA6 is obtained.

[0902] Dry extract: 16.9 mg/g

[0903] DP (estimated by .sup.1H NMR)=21, thus i=0.048

[0904] The calculated average molecular weight of the co-polyamino acid BB26 is 3894 g/mol.

[0905] H PLC-aqueous SEC (calibrant PEG): Mn=2900 g/mol.

Example BB42: Co-Polyamino Acid BB42—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule B8 and Having a Number Average Molecular Weight (Mn) of 3200 g/Mol

[0906] DCC (0.659 g, 3.19 mmol) and NHS (0.365 g, 3.17 mmol) are added to a solution of molecule B8 (2.366 g, 3.11 mmol) in DMF (19.5 mL). After 16 h of stirring at room temperature, the solution is filtered to be used directly in the following reaction.

[0907] γ-Benzyl-L-glutamate N-carboxyanhydride (18.0 g, 68.4 mmol) is placed under a vacuum for 30 min in a round-bottom flask dried beforehand in the oven, then anhydrous DMF (40 mL) is introduced. The mixture is then stirred under argon until the dissolution is complete, cooled to 0° C., then hexylamine (0.411 mL, 3.11 mmol) is introduced rapidly. After 30 h of stirring at 0° C., the solution of molecule B8 prepared above is added. The solution is stirred between 0° C. and room temperature for 72 h, then poured dropwise into diisopropyl ether (0.9 L) under stirring. The precipitate is recovered by filtration, washed with diisopropyl ether (5 times 100 mL), then dried under a vacuum at 30° C. to yield a white solid. The solid is diluted in TFA (69 mL), then the solution is cooled to 4° C. A 33% HBr solution in acetic acid (48 mL, 0.274 mol) is then added dropwise. The mixture is stirred at room temperature for 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl ether and water under stirring (0.8 L). After 2 h of stirring, the heterogeneous mixture is left to rest overnight. The white precipitate is recovered by filtration, washed with a 1:1 (v/v) mixture of diisopropyl ether and water (70 mL), then with water (70 mL). The solid obtained is then solubilized in water (0.42 L) by adjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a 1 N aqueous sodium hydroxide solution. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by addition of water until obtaining a final volume of 0.63 L. The solution is filtered through a 0.45-μm filter, then purified by ultrafiltration against a 0.9% NaCl solution, then water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered through a 0.2-μm filter and stored at 2-8° C.

[0908] Dry extract: 22.2 mg/g

[0909] DP (estimated based on .sup.1H NMR): 22

[0910] Based on .sup.1H NMR: i=0.045

[0911] The calculated average molecular weight of the co-polyamino acid BB42 is 4160 g/mol.

[0912] HPLC-aqueous SEC (calibrant PEG): Mn=3200 g/mol.

Example BB44: Co-Polyamino Acid BB44—Sodium Poly-L-Glutamate Modified at One of its Ends by the Molecule BA7 and Having a Number Average Molecular Weight (Mn) of 3300 g/Mol

[0913] By a method similar to the one used for the preparation of the co-polyamino acid BB14 applied to molecule BA7 in free amine form (4.45 g, 5.18 mmol) and to 30.0 g (113.96 mmol) of γ-benzyl-L-glutamate N-carboxyanhydride, a sodium poly-L-glutamate modified at one of its ends by molecule BA7 is obtained.

[0914] Dry extract: 29.0 mg/g

[0915] DP (estimated by .sup.1H NMR)=25 thus i=0.04

[0916] The calculated average molecular weight of the co-polyamino acid BB44 is 4597 g/mol.

[0917] HPLC-aqueous SEC (calibrant PEG): Mn=3300 g/mol.

Part CE: Counter-Example Co-Polyamino Acids

CEA: Synthesis of the Counter-Example Hydrophobic Molecules

[0918]

TABLE-US-00008 TABLE 1f Counter-example hydrophobic molecules CEA1 [00134] CEA2 [00135] [00136] CEA4 [00137]

Example CEA1: Molecule CEA1

[0919] Molecule CE1:

[0920] product obtained by the reaction between lauric acid and L-lysine.

[0921] By a method similar to the one used for the preparation of molecule B1 applied to lauric acid (19.58 g, 97.72 mmol) and to L-lysine (7.5 g, 51.3 mmol), a beige solid is obtained.

[0922] Yield: 18.8 g (75%)

[0923] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.12-1.78 (42H); 1.96-2.16 (4H); 2.92-3.07 (2H); 4.06-4.20 (1H); 7.70 (1H); 7.95 (1H); 12.40 (1H)

[0924] LC/MS (ESI): 511.5; (calculated ([M+H].sup.+): 511.8).

[0925] Molecule CE2:

[0926] product obtained by the reaction between molecule CE1 and Boc-ethylenediamine.

[0927] By a method similar to the one used for the preparation of molecule B3 applied to molecule CE1 (18.70 g, 36.61 mmol), the residue obtained after concentration at reduced pressure of the reaction medium is triturated in an acetonitrile/THF mixture (1/1 vol.), filtered and washed with acetonitrile. After drying, a pale yellow solid is obtained.

[0928] Yield: 13.7 g (57%)

[0929] .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.09-1.89 (51H); 2.08-2.31 (4H); 3.13-3.48 (6H); 4.35 (1H); 5.20 (1H); 5.85 (1H); 6.46 (1H); 6.94 (1H). LC/MS (ESI): 653.5; (calculated ([M+H].sup.+): 654.0).

Molecule CEA1

[0930] After a method similar to the one used for the preparation of molecule BA1 applied to molecule CE2 (22.2 g, 34 mmol), the residue obtained after concentration under a vacuum is recrystallized in hot methanol. After cooling to room temperature, the solid is filtered, washed with cold methanol, then acetone, and dried under a vacuum to yield a pale yellow solid.

[0931] Yield: 16.3 g (81%).

[0932] .sup.1H NMR (MeOD, ppm): 0.90 (6H); 1.20-1.85 (42H); 2.15-2.30 (4H); 3.06 (2H); 3.19 (2H); 3.40 (1H); 3.55 (1H); 4.13 (1H)

[0933] LC/MS (ESI): 553.5; (calculated ([M+H].sup.+): 553.9).

Molecule CEA2 and Molecule CEA3

[0934] Molecules CEA2 and CEA3 were synthesized according to the protocol described in the patent U.S. Pat. No. 4,826,818 (Kenji M. et al.).

Part CEB: Synthesis of the Counter-Example Co-Polyamino Acids

[0935]

TABLE-US-00009 TABLE 1g counter-example co-polyamino acids CEB1 [00138] CEB2 [00139] CEB3 [00140] CEB4 [00141] CEBX4 [00142]

Example CEB1: Co-Polyamino Acid CEB1—Sodium Poly-L-Glutamate Modified by Molecule CEA1 and Having a Number Average Molecular Weight of 3800 g/Mol

[0936] A poly-L-glutamic acid having a number average molecular weight (Mn) of 8500 g/mol (2.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB1-1 is solubilized in DMF (27 mL) at 30-40° C., then the solution is cooled to 0° C. To this solution, a solution of EDC (0.59 g, 3.06 mmol) in DMF (6 mL), a solution of HOBt (0.41 g, 3.06 mmol) in DMF (1 mL), and DIPEA (1.76 mL, 18.3 mmol) are added. A solution of molecule CEA1 (0.21 g, 1.84 mmol) in DMF (0.9 mL) is then added, and the mixture is stirred at 25° C. for 3 h. The reaction medium is diluted with 11 mL of water added dropwise, then purified by dialysis against a 0.9% aqueous NaCl solution, then water. The solution obtained is filtered through a 0.2-μm filter and stored at 2-8° C.

[0937] Dry extract: 16.3 mg/g.

[0938] DP (estimated based on .sup.1H NMR): 42.

[0939] Based on .sup.1H NMR: i=0.024.

[0940] The calculated average molecular weight of the co-polyamino acid CEB1 is 6924 g/mol.

[0941] HPLC-aqueous SEC (calibrant PEG): Mn=3800 g/mol.

Example CEB2: Co-Polyamino Acid CEB2—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Cholesteryl Leucinate and Having a Number Average Molecular Weight of 2575 g/mol

[0942] The para-toluenesulfonic acid salt of cholesteryl leucinate is prepared according to the method described in the patent U.S. Pat. No. 4,826,818 (Kenji, M. et al.).

[0943] By a method similar to the one used for the preparation of the co-polyamino acid BB13 applied to the para-toluenesulfonic acid salt of cholesteryl leucinate (1.23 g, 1.8 mmol) and to a poly-L-glutamic acid having a number average weight Mn=3600 g/mol (5 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by cholesteryl leucinate is obtained.

[0944] Dry extract: 15.4 mg/g.

[0945] DP (estimated by .sup.1H NMR): 22.

[0946] Based on .sup.1H NMR: i=0.05.

[0947] The calculated average molecular weight of the co-polyamino acid CEB2 is 3973 g/mol.

[0948] HPLC-aqueous SEC (calibrant PEG): Mn=2575 g/mol.

Example CEB3: Co-Polyamino Acid CEB3—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Dilauryl Aspartate and Having a Number Average Molecular Weight of 5820 g/mol

[0949] The para-toluenesulfonic acid salt of dilauryl aspartate is prepared according to the method described in the patent U.S. Pat. No. 4,826,818 (Kenji, M. et al.).

[0950] By a method similar to the one used for the preparation of the co-polyamino acid BB13 applied to the para-toluenesulfonic acid salt of dilauryl aspartate (0.89 g, 1.9 mmol) and to a poly-L-glutamic acid having a number average weight Mn=8500 g/mol (5 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by dilauryl aspartate is obtained.

[0951] Dry extract: 19.3 mg/g.

[0952] DP (estimated by .sup.1H NMR): 43.

[0953] Based on .sup.1H NMR: i=0.05.

[0954] The calculated average molecular weight of the co-polyamino acid CEB3 is 7565 g/mol.

[0955] HPLC-aqueous SEC (calibrant PEG): Mn=5820 g/mol.

Example CEB4: Co-Polyamino Acid CEB4—Sodium Poly-L-Glutamate Modified by (±)-Alpha-Tocopherol and Having a Number Average Molecular Weight of 5085 g/Mol

[0956] By a method similar to the one described in patent FR 2,840,614 (Ping, C. U. et al.) applied to (±)-alpha-tocopherol (0.5 g, 1.16 mmol) and to a poly-L-glutamic acid (3 g) obtained by a method similar to the one described in the patent FR2985429A1, a sodium poly-L-glutamate modified by (±)-alpha-tocopherol is obtained.

[0957] Dry extract: 10.9 mg/g.

[0958] DP (estimated by .sup.1H NMR): 20.

[0959] Based on .sup.1H NMR: i=0.05.

[0960] The calculated average molecular weight of the co-polyamino acid CEB4 is 3473 g/mol.

[0961] HPLC-aqueous SEC (calibrant PEG): Mn=5085 g/mol.

Example CEBX4: Co-Polyamino Acid CEBX4—Sodium Poly-L-Glutamate Capped at One of its Ends by an Acetyl Group and Modified by Molecule CEA1 and Having a Number Average Molecular Weight (Mn) of 3300 g/mol

[0962] By a method similar to the one used for the preparation of the co-polyamino acid BB2 applied to the hydrochloride salt of molecule CEA1 (0.918 g, 1.56 mmol) and to a poly-L-glutamic acid having a number average weight Mn=3700 g/mol (5.0 g) obtained by a method similar to the one used for the preparation of the co-polyamino acid BB5-1, a sodium poly-L-glutamate capped at one of its ends by an acetyl group and modified by molecule CEA1 is obtained.

[0963] Dry extract: 17.4 mg/g

[0964] DP (estimated based on .sup.1H NMR): 22

[0965] Based on .sup.1H NMR: i=0.042.

[0966] The calculated average molecular weight of the co-polyamino acid CEBX4 is 3941 g/mol.

[0967] HPLC-aqueous SEC (calibrant PEG): Mn=3300 g/mol.

Part C—Insulin Glargine, Prandial Insulin or GLP-1 RA Compositions

Example C1: SOLUTION OF RAPID INSULIN ANALOG (HUMALOG®) AT 100 U/ML

[0968] This solution is a commercial solution of insulin lispro marketed by the company ELI LILLY under the name of Humalog®. This product is a rapid 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 obtain 0.0197 mg of zinc ion/mL), sodium hydroxide and hydrochloric acid for the adjustment of the pH (pH 7-7.8), and water.

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

[0969] 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

[0970] 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

[0971] 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

[0972] 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

[0973] 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 D1: Solution of GLP-1 RA Dulaglutide (Trulicity®) at 3 mg/mL

[0974] 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 D2: Solution of GLP-1 RA Exenatide (Byetta®) at 0.25 mg/mL

[0975] 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.

Example D3: Solution of GLP-1 RA Liraglutide (Victoza®) at 6 mg/mL

[0976] This solution is a solution of liraglutide marketed by the company NOVO NORDISK under the name of Victoza®. The excipients in Victoza® are disodium phosphate dihydrate, propylene glycol (1.42 mg/mL), phenol (5.5 mg/mL), and water.

Example D4: Solution of GLP-1 RA Lixisenatide (Lyxumia®) at 0.1 mg/mL

[0977] This solution is a solution of lixisenatide marketed by the company SANOFI under the name of Lyxumia®. The excipients in Lyxumia® are glycerol, sodium acetate, methionine, meta-cresol (25 mM), hydrochloric acid and sodium hydroxide for the adjustment of the pH, and water.

Example D5: Solution of GLP-1 RA Albiglutide (Tanzeum®) at 60 mg/mL

[0978] Albiglutide is presented in a solid form to be reconstituted, marketed by the company GSK under the name of Tanzeum®. In an injection device, the powder and the volume of water required for the solubilization, which initially are in separate compartments, are mixed by actuating the pen by rotation of the distal end of the device until a characteristic “click” noise is obtained. The solution is ready for the injection after a procedure consisting of mixing steps and resting times described in the user instructions for the product. The excipients in Tanzeum® are mannitol (153 mM), polysorbate 80 (0.01% w/w), sodium phosphate (10 mM), trehalose dihydrate (117 mM).

[0979] I. Determination of the Minimum Ratios for Solubilizing Insulin Glargine

Example G3: Protocol for the Determination of the Minimum Concentration for Solubilizing Insulin Glargine at 50 U/mL at pH 7

[0980] 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 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.

[0981] In a 3-mL vial, 0.5 mL 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 of 0.5 mL 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 composition C.sub.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 1 night. This operation is carried out for different concentrations of C.sub.co-polyamino acid stock/excipients (mg/mL) in a manner so as to vary the concentration of co-polyamino acid C.sub.co-polyamino acid (mg/mL) in steps of at most 0.25 mg/mL. After the night at 40° C., the samples are inspected visually and subjected to a static light scattering measurement at an angle of 173° using a zetasizer (Malvern). The minimum concentration of co-polyamino acid enabling the solubilization of insulin glargine is defined as the lowest concentration at which the mixture of co-polyamino acid/insulin glargine at pH 7 is visually clear and presents a scattered intensity of less than 3000 kcps/s.

TABLE-US-00010 TABLE 2 Minimum ratios for solubilizing insulin glargine. Concentration of co- polyamino acid (mg/mL) at the threshold of [Hy]/[insulin glargine] co-polyamino solubilization of glargine ratio (mol/mol) at the acid 50 U/mL at pH 7 threshold of solubilization BB7 <1.5 <1.5 BB5 <2 <1.5 BB3 <2 <1.5 BB14 <2 <1.5 BB10 <1.5 <1 BB15 <1.5 <1 BB11 <1.5 <1 BB16 <1.5 <1 AB6 <2 <1 AB21 <1.5 <1.5 AB17 <1.5 <1 AB18 <1.5 <1 AB21′ <1.5 <1 BB17 <2 <1 BB18 <1.5 <1 BB20 <1 <1 BB21 <1.5 <1.5 BB22 <1 <1 BB23 <1 <1 BB24 <1 <1 BB25 <1 <1 BB26 <1 <1 BB42 <1 <1

[0982] II. Solubilization/Precipitation

[0983] a) Compositions Comprising Insulin Glargine

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).

[0984] 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.

[0985] 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.

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.

[0986] 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/m L)×C.sub.insulin glargine (U/m 0/50 (U/mL).

[0987] 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/m L.

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

[0988] 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.

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

[0989] 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.

[0990] 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.

[0991] The results are presented in Table 3:

TABLE-US-00011 TABLE 3 Compositions of co-polyamino acid/insulin glargine (200 U/mL) prepared with the co-polyamino acids of the invention; solubilization/precipitation of insulin glargine. Solubi- Concen- lization Precip- Insulin Co- tration of of insulin itation Compo- glargine polyamino co-polyamino insulin of insulin sition (U/mL) acid acid (mg/mL) glargine glargine CA3 200 — — NO na CA3b 200 AB18 6 YES YES CA3c 200 BB5 9 YES YES CA3d 200 BB11 6 YES YES CA3e 200 BB14 10 YES YES CA3f 200 BB15 6 YES YES CA3g 200 BB16 6 YES YES CA3l 200 BB15 5 YES YES CA3m 200 AB21′ 6 YES YES CA3n 200 BB18 6 YES YES CA3o 200 BB17 9 YES YES CA3p 200 BB25 4, 5 YES YES CA3q 200 BB26 5 YES YES CA3t 200 BB20 5 YES YES CA3u 200 BB21 5 YES YES

Example DB3: Preparation of Compositions of Co-Polyamino Acid/Insulin Glargine/Dulaglutide at pH 7.1

[0992] 2 mL of the solution of dulaglutide of Example D1 and 1 mL of water are added to 3 mL of the solution of co-polyamino acid/insulin glargine prepared according to the protocol of Example CA2, in which the concentration of insulin glargine is 400 U/mL, in order to obtain 6 mL of a composition at pH 7. The pH is adjusted to 7.1 with a 0.1 N sodium hydroxide solution. The composition containing 7 mg/mL of co-polyamino acid AB6, 200 U/mL of insulin glargine, and 1 mg/mL of dulaglutide is clear, indicating the good solubility of insulin glargine and dulaglutide in the presence of the co-polyamino acid at pH 7.1. This clear solution is placed at +4° C. According to the protocol of Example DB3, the compositions with different co-polyamino acids are prepared and presented in Table 4 below.

Example DB5

[0993] Preparation of compositions of co-polyamino acid/insulin glargine/dulaglutide at pH 7.2 According to the protocol of Example DB3, the compositions with different co-polyamino acids are prepared at pH 7.2 and presented in Table 4 below.

TABLE-US-00012 TABLE 4 Compositions of co-polyamino acid/insulin glargine (200 U/mL)/dulaglutide (1 mg/mL) at pH 7.2. Insulin Co-polyamino glargine Dulaglutide Co-polyamino Composition acid (U/mL) (mg/mL) acid (mg/mL) DB3h BB10 200 1 6 DB3i BB15 200 1 6

Example DB6

[0994] Preparation of compositions of co-polyamino acid/insulin glargine at pH 7.2 According to the protocol of Example CA2, composition DB3k described in Table 5 below was prepared.

TABLE-US-00013 TABLE 5 Compositions of co-polyamino acid BB15/insulin glargine (200 U/mL) at pH 7.2. DB3k Insulin glargine U/mL 200 BB15 mg/mL 5 m-cresol mM 25 Zn(II) mM 1.12 glycerol mM 120 PS20 mM 0.032 mannitol mM 85 PS80 mM 0.051 pH 7.2

Part D′—Counter Examples

[0995] Compositions of counter-example 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 counter-example co-polyamino acid C.sub.counter-example co-polyamino acid (mg/mL). These compositions are presented in the following Table 6.

TABLE-US-00014 TABLE 6 Compositions of counter-example co-polyamino acid/insulin glargine (200 U/mL). Glargine Concentration of co- insulin polyamino acid Composition (U/mL) Co-polyamino acid (mg/mL) CA3h 200 CEB1 13 CA3i 200 CEB2 6 CA3j 200 CEB3 7 CA3k 200 CEB4 10 CA3w 200 CEBX4 5

[0996] III. Determination of the Quantity of Albumin Required to Obtain the Precipitation

Example G1: Preparation of a Diluted Co-Polyamino Acid/Insulin Glargine 65 U/mL Composition at pH 7.1

[0997] 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 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 co-polyamino acid/insulin glargine 65 U/mL composition at pH 7.1.

[0998] 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.stock co-polyamino acid/excipients of a solution of co-polyamino acid of concentration C.sub.stock co-polyamino acid/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 G2: Precipitation of a Co-Polyamino Acid/Insulin Glargine 65 U/mL Composition at pH 7.1, by Varying the Concentration of Albumin

[0999] 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 C.sub.BSA (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.

[1000] 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.

[1001] 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.

[1002] 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.

[1003] One notes that in the compositions of the invention, the critical BSA quantity is lower.

[1004] The results are reported in the following Table 7:

TABLE-US-00015 TABLE 7 Critical albumin concentration (mg/mL) for 80% of precipitation at 1 h (insulin glargine at 50 U/mL). Critical albumin concentration Concentration of (mg/ml) for 80% precipitation co-polyamino acid at 1 h (insulin glargine at 50 in solution insulin Co-polyamino acid U/mL) glargine at 50 U/mL CEB2 ≧3.9 1.5 CEB3 ≧3.9 1.8 CEB4 ≧3.9 1.5 BB7 <3.9 1.8 BB5 <3.9 2.2 BB14 <3.9 2.5 BB10 <3.9 1.5 BB15 <3.9 2.0 BB11 <3.9 1.5 BB16 <3.9 1.5 AB6 <3.9 1.8 AB21 <3.9 1.5 AB17 <3.9 1.5 AB18 <3.9 1.5 AB21′ <3.9 1.5 BB42 <3.9 1.2 BB18 <3.9 1.5 BB17 <3.9 1.75 BB25 <3.9 1.1 BB20 <3.9 1.25 BB21 <3.9 1.25

[1005] IV. Study of the Stability of the Compositions According to the Invention

Part E: Demonstration of the Physical Stability of the Compositions According to the Invention by the Study of Co-Polyamino Acid/Insulin Glargine 200 U/mL

Example E1: Stability Accelerated at 25° C. Under Dynamic Conditions

[1006] 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 weeks of stability corresponds to the duration after which at least 2 vials present visible particles or are turbid.

[1007] These results are in agreement with the US Pharmacopoeia (USP <790>).

[1008] The results of accelerated stability (obtained with different compositions) are presented in Table 8 below.

TABLE-US-00016 TABLE 8 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). Stability at 25° C. under dynamic Composition Co-polyamino acid conditions (in days) CA3 — * CA3b AB18 11 CA3c BB5 >30 CA3d BB11 19 CA3e BB14 11 CA3f BB15 17 CA3h CEB1 7 CA3i CEB2 >15 CA3j CEB3 7 CA3k CEB4 7 CA3m AB21′ 11 CA3n BB18 >44 CA3o BB17 39 CA3p BB25 18 CA3q BB26 39 CA3t BB20 22 CA3u BB21 28 CA3w CEBX4 2 (* Appearance of a precipitate when the pH of the solution of insulin glargine is adjusted to pH 7).

Example E2: Accelerated Stability at 30° C. Under Static Conditions

[1009] 5 3-mL vials filled with 1 mL of composition are placed vertically in an oven maintained at 30° C. The vials are inspected visually daily 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 weeks of stability corresponds to the duration after which at least 2 vials present visible particles or are turbid.

[1010] These results are in agreement with the US pharmacopoeia (USP <790>).

[1011] The accelerated stability results (obtained with different compositions) are presented in Table 9 below.

TABLE-US-00017 TABLE 9 Results of the stabilities of the compositions of co-polyamino acid/insulin glargine (200 U/mL) at 30° C. under static conditions. Stability at 30° C. under static Composition Co-polyamino acid conditions (in week) CA3 — * CA3f BB 15 >7 CA3k CEB4 Yellow after 2.5 weeks (* Appearance of a precipitate when the pH of the solution is adjusted to pH 7).

[1012] V. Examples of Compositions According to the Invention

Example H1: Preparation of Compositions of Co-Polyamino Acids and of Insulin Glargine at 200 U/mL and pH 7.1

[1013] In a manner similar to Example CA3, compositions of insulin glargine at 150 U/mL and of co-polyamino acids are prepared. The are presented in the following table 9a:

TABLE-US-00018 TABLE 9a Composition of co-polyamino acids and of insulin glargine at 200 U/mL and at pH 7.1. Insulin Concentration of glargine co-polyamino acid Composition (U/mL) Co-polyamino acid (mg/mL) CA3 200 — — CA3b 200 AB18 6 CA3c 200 BB5 9 CA3d 200 BB11 6 CA3e 200 BB14 10 CA3f 200 BB15 6 CA3g 200 BB16 6

Example H2: Preparation of Compositions of Co-Polyamino Acids and of Insulin Glargine at 150 U/mL and at pH 7.1

[1014] In a manner similar to Example H1, compositions of insulin glargine at 150 U/mL and of co-polyamino acids are prepared. They are presented in the following Table 9b:

TABLE-US-00019 TABLE 9b Compositions of co-polyamino acid/insulin glargine (150 U/mL). Insulin Concentration of glargine co-polyamino acid Composition (U/mL) Co-polyamino acid (mg/mL) H2b 150 AB18 4.5 H2c 150 BB5 6.75 H2d 150 BB11 4.5 H2e 150 BB14 7.5 H2f 150 BB15 4.5 H2g 150 BB16 4.5

Example H3: Preparation of Compositions of Co-Polyamino Acid and of Insulin Glargine at 300 U/mL and at pH 7.1

[1015] In a manner similar to Example H1, compositions of insulin glargine at 300 U/mL and of co-polyamino acids are prepared. They are presented in the following Table 10:

TABLE-US-00020 TABLE 10 Compositions of co-polyamino acid/insulin glargine (300 U/mL) Concentration of Insulin co-polyamino acid Composition glargine Co-polyamino acid (mg/mL) H3b 300 AB18 9 H3c 300 BB5 13.5 H3d 300 BB11 9 H3e 300 BB14 15 H3f 300 BB15 9 H3g 300 BB16 9

Part F: Pharmacokinetic and Pharmacodynamic Studies in Dogs

Example F1

[1016] Pharmacokinetic and pharmacodynamic study in dogs of the co-polyamino acid BB15 (5 mg/mL)/insulin glargine (200 U/mL)

[1017] Studies in dogs were carried out for the purpose of evaluating the pharmacokinetics and the pharmacodynamics of the insulin after administration of a composition of co-polyamino acid and of insulin glargine (composition DB3K).

[1018] The hypoglycemic effect and the pharmacokinetic profiles of the insulin of the composition DB3k were compared with those of the injection of the composition C4 (Lantus®) (pH 4) at the same dose.

[1019] Ten animals that had fasted for approximately 18 hours received injections in the neck above the interscapular region, at the dose of 0.5 U/kg of insulin. In the hour preceding the injection, a blood sample is drawn in order to determine the basal insulin level, and 3 samples are collected in order to determine the basal glucose level. Blood samples are then drawn during the 23 h after the administration in order to describe the pharmacokinetics of the insulin. The glycemia is determined for 24 h by means of a glucometer. The insulin levels are determined by an ELISA test.

[1020] The median pharmacokinetic curves of the insulin, expressed in deviation from the basal level, are presented in FIG. 1.

[1021] The mean pharmacodynamic curves of the glucose, expressed in percentage of deviation of the basal level, are represented in FIG. 2.

[1022] The pharmacokinetic profiles show that after administration of the composition DB3k a plateau is reached rapidly and maintained up to 14 h after the administration, then the insulin concentrations decrease slowly. The profile is similar to the one obtained after the injection of composition C4 (Lantus®), indicating that the composition DB3k does not modify the kinetics of the insulin glargine.

[1023] The pharmacodynamic results obtained with the administration of the composition DB3k and the injection of the composition C4 (Lantus®) are presented in FIG. 2. After administration of the composition DB3k, a decrease in glycemia is observed until, approximately 4 h after the administration, a plateau is reached, which is maintained up to approximately 13 h. The glycemia then rises gradually until its basal level is reached approximately 19 h after the administration. The profile is similar to the one obtained after the injection of the composition C4 (Lantus®), indicating that the effect of the insulin glargine is well preserved with the composition DB3k.