Copositions in the form of an injectable aqueous solution comprising amylin, an amylin receptor agonist or an amylin analog and a co-polyamino acid

Abstract

A composition in the form of an injectable aqueous solution, wherein the pH is comprised from 6.0 to 8.0, includes at least:

a) amylin, an amylin receptor agonist or an amylin analog; and

b) a co-polyamino acid bearing carboxylate charges and hydrophobic radicals Hy, wherein the composition does not comprise a basal insulin wherein the isoelectric point pI is comprised from 5.8 to 8.5. The composition may further include a prandial insulin.

Claims

1. Composition in the form of an injectable aqueous solution, wherein the pH is comprised from 6.0 to 8.0, comprising at least: amylin, an amylin receptor agonist or an amylin analog; a co-polyamino acid bearing carboxylate charges carboxylates and at least one hydrophobic radical -Hy according to formula X, said co-polyamino acid being chosen among the co-polyamino acids according to formula I:
[Q(PLG).sub.k][Hy].sub.j[Hy].sub.jFormula I wherein: j 1; 0jn1 and j+j1 and k2 said co-polyamino acid according to formula I bearing at least one hydrophobic radical -Hy, carboxylate charges and consisting of at least two chains of glutamic or aspartic units PLG bound together by an at least divalent linear or branched radical or spacer Q[-*].sub.k consisting of an alkyl chain comprising one or a plurality of heteroatoms chosen in the group consisting of nitrogen and oxygen atoms and/or bearing one or a plurality of heteroatoms consisting of nitrogen and oxygen radicals and/or radicals bearing one or a plurality of heteroatoms consisting of nitrogen and oxygen atoms and/or carboxyl functions. said radical or spacer Q[-*]k being bound to at least two glutamic or aspartic unit chains PLG by an amide function and, said amide bonds binding said radical or spacer Q[-*]k bound to said at least two chains of glutamic or aspartic units result from the reaction between an amine function and an acid function respectively borne either by the precursor Q of the radical or spacer Q[-*]k or by a glutamic or aspartic unit, said hydrophobic radical -Hy being bound either to a terminal amino acid unit and then j1, or to a carboxyl function borne by one of the chains of the glutamic or aspartic units PLG and then j=n1 and n1 is the mean number of monomeric units bearing a hydrophobic radical -Hy.

2. Composition according to claim 1, characterized in that said radical or spacer Q[-*].sub.k is chosen among the radicals according to formula II:
Q[-*].sub.k=([Q].sub.c)[-*].sub.k Formula II wherein 1q5.

3. Composition according to claim 1, characterized in that said radicals Q, identical or different, are chosen in the group consisting of the radicals according to formulas III to VII hereinafter, to form Q[-*]k wherein 1t8, by a radical according to formula IV:
*F.sub.aCH.sub.2.sub.tF.sub.a*Formula III wherein: at least one of u.sub.1 or U.sub.2 is different to 0, if u.sub.10 then u.sub.10 and if u.sub.20 then u.sub.20, u.sub.1 and u.sub.2 are identical and different, 2u4, 0u.sub.14, 0u.sub.14, 0u.sub.24, 0u.sub.24; by a radical according to formula V: ##STR00119## wherein: v, v and v identical or different, are integers0, v+v+v15, by a radical according to formula VI: ##STR00120## wherein: w.sub.1 is different to 0, 0w.sub.21, w.sub.16 and w.sub.16 and/or w.sub.26 and w.sub.26, where F.sub.x=F.sub.a, F.sub.b, F.sub.c, F.sub.d, F.sub.a, F.sub.b, F.sub.c, F.sub.c et F.sub.d identical or different representing functions NH or CO and F.sub.y representing a trivalent nitrogen atom N, two radicals Q being bound together by a covalent bond between a carbonyl function, F.sub.x=CO, and an amine function F.sub.x=NH or F.sub.y=N, thus forming an amide function.

4. Composition according to claim 1, characterized in that the hydrophobic radical -Hy is chosen among the radicals according to formula X as defined hereinafter:
*GpR.sub.rGpG.sub.gGpA.sub.a(GpL.sub.l(GpH.sub.h-GpC].sub.l].sub.aFormula X wherein GpR is chosen among the radicals according to formulas VII, VII or VII: ##STR00121## GpG and GpH identical or different are chosen among the radicals according to formulas XI or XI: ##STR00122## GpA is chosen among the radicals according to formula VIII ##STR00123## wherein A is chosen among the radicals according to VIII, VIII or VIII ##STR00124## GpL is chosen among the radicals according to formula XII ##STR00125## GpC is a radical according to formula IX: ##STR00126## the * indicate the binding sites of the different groups bound by amide functions; a is an integer equal to 0 or to 1 and a=1 if a=0 and a=1, 2 or 3 if a=1; a is an integer equal to 1, to 2 or to 3; b is an integer equal to 0 or to 1; c is an integer equal to 0 or to 1, and if c is equal to 0 then d is equal to 1 or to 2; d is an integer equal to 0, to 1 or to 2; e is an integer equal to 0 or to 1; g is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; h is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; l is an integer equal to 0 or 1 and l=1 if l=0 and l=2 if l=1; l is an integer equal to 1 or to 2; r is an integer equal to 0, to 1 or to 2, and s is an integer equal to 0 or 1; A, A.sub.1, A.sub.2 and A.sub.3 identical or different are linear or branched alkyl radicals comprising from 1 to 8 carbon atoms and optionally substituted by a radical from a saturated, unsaturated or aromatic ring; B is a radical chosen in the group consisting of a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms or a linear or branched alkyl radical, optionally comprising an aromatic nucleus, comprising from 1 to 9 carbon atoms; C.sub.x is a linear or branched monovalent alkyl radical, optionally comprising a cyclic part, wherein x indicates the number of carbon atoms and: when the hydrophobic radical -Hy bears 1 -GpC, then 9x25, when the hydrophobic radical -Hy bears 2 -GpC, then 9x15, when the hydrophobic radical -Hy bears 3 -GpC, then 7x13, when the hydrophobic radical -Hy bears 4 -GpC, then 7x11, when the hydrophobic radical -Hy bears at least 5 -GpC, then 6x11, G is a branched alkyl radical of 1 to 8 carbon atoms, said alkyl radical bearing one or a plurality of free carboxylic acid function(s), R is a radical chosen in the group consisting of a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms, a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms bearing one or a plurality of functions CONH.sub.2 or a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms: the hydrophobic radical(s) -Hy according to formula X being bound to the PLG: via a covalent bond between a carbonyl of the hydrophobic radical -Hy and a nitrogen atom borne by the PLG thus forming an amide function obtained from the reaction of an amine function borne by the PLG and an acid function borne by the precursor Hy of the hydrophobic radical -Hy, and via a covalent bond between a nitrogen atom of the hydrophobic radical -Hy and a carbonyl borne by the PLG thus forming an amide function obtained from the reaction of an amine function of the precursor Hy of the hydrophobic radical -Hy and an acid function borne by the PLG, the ratio M between the number of hydrophobic radicals and the number of glutamic or aspartic units being between 0 M 0.5; when a plurality of 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 for the PLG chains is comprised from 5 to 250; the free carboxylic acid functions being in the form of alkali cation salt chosen in the group consisting of Na.sup.+ and K.sup.+.

5. Composition according to claim 1, characterized in that the co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy is chosen among the co-polyamino acids according to formula XXXa hereinafter: ##STR00127## wherein, D represents, independently, either a group CH.sub.2 (aspartic acid) or a group CH.sub.2CH.sub.2 (glutamic acid), X represents a cationic entity chosen in the group comprising alkali cations, Ra and Ra, identical or different, are either a hydrophobic radical -Hy, or a radical chosen in the group consisting of an H, a C.sub.2 to C.sub.10 linear acyl group, a C.sub.3 to C.sub.10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, at least one of Ra and R a being a hydrophobic radical -Hy, Q and -Hy are as defined above, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5n+m250.

6. Composition according to claim 1, characterized in that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula XXXa ##STR00128## wherein: D represents, independently, either a group CH.sub.2 (aspartic acid) or a group CH.sub.2CH.sub.2 (glutamic acid), X represents a cationic entity chosen in the group comprising alkali cations, Ra and R a, identical or different, are either a hydrophobic radical -Hy, or a radical chosen in the group consisting of an H, a C.sub.2 to C.sub.10 linear acyl group, a C3 to C.sub.10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, at least one of Ra and R a being a hydrophobic radical -Hy, Q and Hy are as defined above, n.sub.1+m.sub.1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n.sub.2+m.sub.2 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid not bearing a radical -Hy, n.sub.1+n.sub.2=n and m.sub.1+m.sub.2=m, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5n+m250.

7. Composition according to claim 1, characterized in that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula XXXb hereinafter: ##STR00129## wherein, D represents, independently, either a group CH.sub.2 (aspartic acid) or a group CH.sub.2CH.sub.2 (glutamic acid), X represents a cationic entity chosen in the group comprising alkali cations, Rb and Rb, identical or different, are either a hydrophobic radical -Hy, or a radical chosen in the group consisting of an H, a C.sub.2 to C.sub.10 linear acyl group, a C.sub.3 to C.sub.10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, at least one of Rb and Rb is a hydrophobic radical -Hy, Q and Hy are as defined above, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5n+m250.

8. Composition according claim 1, characterized in that the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is chosen among the co-polyamino acids according to formula XXXb hereinafter: ##STR00130## wherein: D represents, independently, either a group CH.sub.2 (aspartic acid) or a group CH.sub.2CH.sub.2 (glutamic acid), X represents a cationic entity chosen in the group comprising alkali cations, Q and Hy are as defined above, Rb and Rb, identical or different, are either a hydrophobic radical -Hy, or a radical chosen in the group consisting of an OH, an amine group, a terminal amino acid unit and a pyroglutamate, at least one of Rb and Rb is a hydrophobic radical -Hy, n1+m1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n2+m2 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid not bearing a radical -Hy, n1+n2=n and m1+m2=m, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5n+m250.

9. Composition according to claim 5, wherein the co-polyamino acid is chosen among the co-polyamino acids wherein the group D is a group CH.sub.2 (aspartic unit).

10. Composition according to claim 5, wherein the co-polyamino acid is chosen among the co-polyamino acids wherein the group D is a group CH.sub.2CH.sub.2 (glutamic unit).

11. Composition according to claim 1, characterized in that the molar ratio of co-polyamino acid/amylin, amylin receptor agonist or amylin analog is greater than or equal to 1.

12. Composition according to claim 1, characterized in that the amylin, amylin receptor agonist or amylin analog is amylin.

13. Composition according to claim 1, characterized in that the amylin, amylin receptor agonist or amylin analog is pramlintide.

14. Composition according to claim 1, characterized in that it further comprises a prandial insulin.

15. Composition according to claim 1, characterized in that the molar ratio of co-polyamino acid/insulin is greater than or equal to 1.

16. Composition according to claim 1, characterized in that said composition has a stability measured by ThT greater than that of a reference composition comprising amylin, an amylin receptor agonist or an amylin agonist but not comprising co-polyamino acid bearing carboxylate charges and hydrophobic radicals -Hy, said ThT monitoring being carried out under accelerated stability conditions: under stirring and at 37 C.

17. Co-polyamino acid bearing carboxylate charges carboxylates and at least one hydrophobic radical according to formula X, said co-polyamino acid being chosen among the co-polyamino acids according to formula I:
[Q(PLG)k][Hy]j[Hy]jFormula I wherein: j1; 0jn1 and j+j1 and k2 said co-polyamino acid according to formula I bearing at least one hydrophobic radical -Hy, carboxylate charges and consisting of at least two chains of glutamic or aspartic units PLG bound together by an at least divalent linear or branched radical or spacer Q[-*].sub.k consisting of an alkyl chain comprising one or a plurality of heteroatoms chosen in the group consisting of nitrogen and oxygen atoms and/or bearing one or a plurality of heteroatoms consisting of nitrogen and oxygen radicals and/or radicals bearing one or a plurality of heteroatoms consisting of nitrogen and oxygen atoms and/or carboxyl functions, said radical or spacer Q[-*]k being bound to at least two glutamic or aspartic unit chains PLG by an amide function and, said amide bonds binding said radical or spacer Q[-*]k bound to said at least two chains of glutamic or aspartic units result from the reaction between an amine function and an acid function respectively borne either by the precursor Q of the radical or spacer Q[-*]k or by a glutamic or aspartic unit, said hydrophobic radical -Hy being bound either to a terminal amino acid unit and then j1, or to a carboxyl function borne by one of the chains of the glutamic or aspartic units PLG and then j=n1 and n1 is the mean number of monomeric units bearing a hydrophobic radical -Hy.

18. Hydrophobic radical precursor Hy chosen among the compounds according to formula X as defined hereinafter:
HGpR.sub.rGpG.sub.g-(GpA).sub.a(GpL.sub.l(GpH.sub.h-GpC].sub.l].sub.aFormula X wherein GpR is chosen among the radicals according to formulas VII, VII or VII: ##STR00131## GpG and GpH identical or different are chosen among the radicals according to formulas XI or XI: ##STR00132## GpA is chosen among the radicals according to formula VIII ##STR00133## wherein A is chosen among the radicals according to VIII, VIII or VIII ##STR00134## -GpL is chosen among the radicals according to formula XII ##STR00135## GpC is a radical according to formula IX: ##STR00136## the * indicate the binding sites of the different groups bound by amide functions; a is an integer equal to 0 or to 1 and a=1 if a=0 and a=1, 2 or 3 if a=1; a is an integer equal to 1, to 2 or to 3; b is an integer equal to 0 or to 1; c is an integer equal to 0 or to 1, and if c is equal to 0 then d is equal to 1 or to 2; d is an integer equal to 0, to 1 or to 2; e is an integer equal to 0 or to 1; g is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; h is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; l is an integer equal to 0 or 1 and l=1 if l=0 and l=2 if l=1; l is an integer equal to 1 or to 2; r is an integer equal to 0, 1 or to 2, and s is an integer equal to 0 or 1; A, A.sub.1, A.sub.2 and A.sub.3 identical or different are linear or branched alkyl radicals comprising from 1 to 8 carbon atoms and optionally substituted by a radical from a saturated, unsaturated or aromatic ring; B is a radical chosen in the group consisting of a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms or a linear or branched alkyl radical, optionally comprising an aromatic nucleus, comprising from 1 to 9 carbon atoms; C.sub.x is a radical chosen in the group consisting of a linear or branched monovalent alkyl radical, optionally comprising a cyclic part, wherein x indicates the number of carbon atoms and 6x25: when the hydrophobic radical -Hy bears 1 -GpC.sub.x then 9x25, when the hydrophobic radical -Hy bears 2 -GpC.sub.x then 9x15, when the hydrophobic radical -Hy bears 3 -GpC.sub.x then 7x13, when the hydrophobic radical -Hy bears 4 -GpC.sub.x then 7x11, when the hydrophobic radical -Hy bears at least 5 -GpC.sub.x then 6x11; G is a linear or branched divalent alkyl radical of 1 to 8 carbon atoms, said alkyl radical bearing one or a plurality of free carboxylic acid function(s), R is a radical chosen in the group consisting of a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms, a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms bearing one or a plurality of functions CONH.sub.2 or a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms, the hydrophobic radical(s) -Hy according to formula X being bound to the PLG: via a covalent bond between a carbonyl of the hydrophobic radical -Hy and a nitrogen atom borne by the PLG thus forming an amide function obtained from the reaction of an amine function borne by the PLG and an acid function borne by the precursor Hy of the hydrophobic radical -Hy and/or via a covalent bond between a nitrogen atom of the hydrophobic radical -Hy and a carbonyl borne by the PLG thus forming an amide function obtained from the reaction of an amine function of the precursor Hy of the hydrophobic radical -Hy and an acid function borne by the PLG; the ratio M between the number of hydrophobic radicals and the number of glutamic or aspartic units being between 0M0.5; when a plurality of 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 for the PLG chains is comprised from 5 to 250; the free acid functions being in the form of alkali cation salt chosen in the group consisting of Na.sup.+ and K.sup.+.

19. A method for for improving the physicochemical stability of the composition according to claim 1, comprising adding ionic species chosen in the group of anions, cations and/or zwitterions to the compositions.

Description

[0970] The examples hereinafter illustrate, in a non-limiting manner, the invention.

Part ASynthesis of Hydrophobic Intermediate Compounds Hyd for Obtaining the Radicals -Hy.

[0971]

TABLE-US-00002 # HYDROPHOBIC INTERMEDIATE COMPOUNDS A1 [00064] A2 [00065] A3 [00066] A4 [00067] A5 [00068] A6 [00069] A7 [00070] A8 [00071] A9 [00072] A10 [00073] A11 [00074] A12 [00075] A14 [00076] A15 [00077] A16 [00078] A17 [00079] A18 [00080] A19 [00081] A21 [00082] A22 [00083] A23 [00084] A26 [00085] A27 [00086]

EXAMPLE A1

Molecule A1

[0972] To a solution of L-proline (300.40 g, 2.61 mol) in 2 N aqueous sodium hydroxide (1.63 L) at 0 C. is added slowly over 1 h myristoyl chloride (322 g, 1.30 mol) in solution in dichloromethane (DCM, 1.63 L). At the end of the addition, the reaction medium is returned to 20 C. in 3 h, then stirred for a further 2 h. The mixture is cooled to 0 C. then a 37% HCl aqueous solution (215 mL) is added in 15 min. The reaction medium is stirred for 3 h from 0 C. to 20 C., then cooled to 3 C. 37% HCl (213 mL) is added in 15 min and the mixture is stirred for 1 h from 0 C. to 20 C. The organic phase is separated, washed with a 10% HCl aqueous solution (3430 mL), an aqueous solution saturated with NaCl (430 mL), dried on Na.sub.2SO.sub.4, filtered on cotton then concentrated at reduced pressure. The residue is solubilized in heptane (1.31 L) at 50 C., then the solution is progressively returned to ambient temperature. After priming crystallization using a glass rod, the medium is once again heated to 40 C. for 30 min then returned to ambient temperature for 4 h. A white solid of molecule A1 is obtained after filtration on a sintered filter, washing with heptane (2350 mL) and drying at reduced pressure.

[0973] Yield: 410 g (97%)

[0974] .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).

[0975] LC/MS (ESI): 326.4; 651.7; (calculated ([M+H].sup.+): 326.3; ([2M+H].sup.+): 651.6).

EXAMPLE A2

Molecule A2

[0976] Molecule 1: Product obtained by reacting decanoyl chloride and L-proline.

[0977] By means of a similar method to that used for the preparation of molecule A1 and applied to decanoyl chloride (75.0 g, 393.27 mmol) and to L-proline (90.55 g, 786.53 mmol), a colorless oil of molecule 1 is obtained after washing the organic phase with a 10% HCl aqueous solution (3125 mL), an aqueous solution saturated with NaCl (125 mL), drying on Na2SO4, filtration on cotton followed by concentration at reduced pressure.

[0978] Yield: 104.64 g (99%)

[0979] .sup.1H NMR CDCl.sub.3, ppm): 0.86 (3H); 1.10-1.51 (12H); 1.56-1.80 (2H); 1.83-2.46 (6H); 3.42-3.66 (2H); 4.37-4.41 (0.1H); 4.53-4.60 (0.9H); 10.12 (1H).

[0980] LC/MS (ESI): 270.1; (calculated ([M+H].sup.+): 270.2).

Molecule A2

[0981] To a solution of molecule 1 (90.0 g, 334.09 mmol) in THF (600 mL) at 0 C. are added successively N-hydroxysuccinimide (NHS, 40.4 g, 350.80 mmol) followed by dicyclohexylcarbodiimide (DCC, 72.38 g, 350.80 mmol) in solution in THF (60 mL). After 16 h of stirring at ambient temperature, the reaction medium is filtered and introduced onto a solution of L-lysine hydrochloride (30.51 g, 167.05 mmol) and N,N-diisopropylethylamine (DIPEA, 97.16 g, 751.71 mmol) in water (66 mL) and the mixture is stirred for 48 h at 20 C. After concentration at reduced pressure, water (360 mL) is added and the mixture obtained is treated by successive addition of ethyl acetate (AcOEt, 500 mL) followed by a 5% Na.sub.2CO.sub.3 aqueous solution (1 L). The aqueous phase is then washed once again with AcOEt (200 mL), acidified by adding a 6 N HCl aqueous solution and the product is extracted with dichloromethane (DCM, 3250 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The white solid obtained after crystallization in AcOEt is solubilized in DCM (400 mL), the organic phase is washed with a 1 N HCl aqueous solution (200 mL) followed by an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under vacuum. A white solid of molecule A2 is obtained after crystallization in AcOEt.

[0982] Yield: 75.90 g (70%)

[0983] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.10-2.04 (42 H); 2.07-2.30 (4H); 2.92-3.08 (2H); 3.28-3.57 (4H); 4.07-4.28 (2H); 4.32-4.40 (1H); 7.66-7.73 (0.6H); 7.96-8.09 (1H); 8.27 (0.4H); 12.51 (1H).

[0984] LC/MS (ESI): 649.5 (calculated ([M+H].sup.+): 649.5).

EXAMPLE A3

Molecule A3

[0985] Molecule 2: Product obtained by reacting lauroyl chloride and L-Proline.

[0986] By means of a similar method to that used for the preparation of molecule A1 and applied to lauroyl chloride (27.42 g, 685.67 mmol) and to L-proline (60.0 g, 247.27 mmol), a white solid of molecule 2 is obtained.

[0987] Yield: 78.35 g (96%)

[0988] .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).

[0989] LC/MS (ESI): 298.1 (calculated ([M+H].sup.+): 298.2).

Molecule A3:

[0990] By means of a similar method to that used for the preparation of molecule A2 applied to molecule 2 (42.49 g, 142.86 mmol) and to L-lysine hydrochloride (13.7 g, 75.0 mmol), a white solid of molecule A3 is obtained after crystallization in acetone.

[0991] Yield: 30.17 g (60%)

[0992] .sup.1H NMR (DMSO-d6, ppm): 0.86 (6H); 1.07-2.05 (50H); 2.08-2.30 (4H); 2.93-3.09 (2H); 3.28-3.57 (4H); 4.08-4.29 (2H); 4.33-4.41 (1H); 7.70 (0.6H);7.97-8.07 (1H); 8.28 (0.4H); 12.52 (1H).

[0993] LC/MS (ESI): 705.6; (calculated ([M+H].sup.+): 705.6).

EXAMPLE A4

Molecule A4

[0994] By means of a similar method to that used for the preparation of molecule A2 applied to molecule A1 (200.0 g, 614.44 mmol) and to L-lysine hydrochloride (56.11 g, 307.22 mmol), a white solid of molecule A4 is obtained after crystallization in ethyl acetate.

[0995] Yield: 176.0 g (95%)

[0996] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.08-1.51 (48H); 1.53-2.04 (10H); 2.08-2.30 (4H); 2.93-3.09 (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).

[0997] LC/MS (ESI): 761.8; (calculated ([M+H].sup.+): 761.6).

EXAMPLE A5

Molecule A5

[0998] Molecule 3: Product obtained by reacting Fmoc-Lys(Fmoc)-OH and 2-Cl-trityl chloride resin.

[0999] To a suspension of Fmoc-Lys(Fmoc)-OH (7.32 g, 12.40 mmol) in DCM (60 mL) at ambient temperature is added DIPEA (4.32 mL, 24.80 mmol). After complete solubilization (10 min), the solution obtained is poured onto 2-Cl-trityl chloride resin (100-200 mesh, 1% DVB, 1.24 mmol/g) (4.00 g, 4.96 mmol) previously washed with DCM, in a reaction vessel suitable for solid substrate peptide synthesis. After 2 h of stirring at ambient temperature, HPLC grade methanol (0.8 mL/g resin, 3.2 mL) is added and the medium is stirred at ambient temperature for 15 min. The resin is filtered, washed successively with DCM (360 mL), DMF (260 mL), DCM (260 mL), isopropanol (160 mL) and DCM (360 mL).

[1000] Molecule 4: Product obtained by reacting molecule 3 and an 80:20 DMF/piperidine mixture.

[1001] Molecule 3, previously washed with DMF, is treated with an 80:20 DMF/piperidine mixture (60 mL). After 30 min of stirring at ambient temperature, the resin is filtered, washed successively with DMF (360 mL), isopropanol (160 mL) and DCM (360 mL).

[1002] Molecule 5: Product Obtained by Reacting Molecule 4 and 8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (Fmoc-O2Oc-OH).

[1003] To a suspension of Fmoc-O2Oc-OH (9.56 g, 24.80 mmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 9.43 g, 24.80 mmol) in a 1:1 DMF/DCM mixture (60 mL) is added DIPEA (8.64 mL, 49.60 mmol). After complete solubilization, the solution obtained is poured onto molecule 4. After 2 h of stirring at ambient temperature, the resin is filtered, washed successively with DMF (360 mL), isopropanol (160 mL) and DCM (360 mL).

[1004] Molecule 6: Product obtained by reacting molecule 5 and an 80:20 DMF/piperidine mixture.

[1005] By Means of a similar method to that used for molecule 4 applied to molecule 5, molecule 6 is obtained.

[1006] Molecule 7: Product obtained by reacting molecule 6 and lauric acid.

[1007] By means of a similar method to that used for molecule 5 applied to molecule 6 and to lauric acid (4.97 g, 24.80 mmol) in DMF (60 mL), molecule 7 is obtained.

[1008] Molecule 8: Product obtained by reacting molecule 7 and an 80:20 dichloromethane/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) mixture.

[1009] Molecule 7 is treated with an 80:60 dichloromethane/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) mixture (60 mL). After 20 min of stirring at ambient temperature, the resin is filtered and washed successively with dichloromethane (260 mL). The solvents are evaporated at reduced pressure. Two co-evaporations are then carried out on the residue with dichloromethane (60 mL) followed by diisopropylether (60 mL). A white solid of molecule 8 is obtained after recrystallization in acetonitrile.

[1010] Yield: 2.63 g (66% in 6 stages)

[1011] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (6H); 1.09-1.66 (40H); 1.77-1.98 (2H); 2.13-2.29 (4H); 3.24-3.75 (18H); 3.95-4.07 (4H); 4.65-4.70 (1H); 6.23-6.37 (1H); 6.39-6.62 (1H); 6.74-6.91 (1H); 7.38-7.54 (1H).

[1012] LC/MS (ESI): 801.6 (calculated ([M+H].sup.+): 801.6).

[1013] Molecule 9: Product obtained by reacting molecule 8 and N-Boc ethylenediamine.

[1014] To a solution of molecule 8 (2.63 g, 3.29 mmol) in chloroform (20 mL) at ambient temperature are added successively N-hydroxybenzotriazole (HOBt, 654 mg, 4.27 mmol) and N-Boc ethylenediamine (BocEDA, 580 mg, 3.62 mmol). The mixture is cooled to 0 C. then (3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC 819 mg, 4.27 mmol) is added. The reaction medium is stirred for 15 min at 0 C. followed by 18 h at ambient temperature. The organic phase is washed with an aqueous solution saturated with NH.sub.4Cl (210 mL), an aqueous solution saturated with NaHCO.sub.3 (210 mL), and an aqueous solution saturated with NaCl (210 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A white solid of molecule 9 is obtained after purification by silica gel chromatography (eluent: dichloromethane, methanol).

[1015] Yield: 2.37 g (76%)

[1016] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (6H); 1.08-1.47 (34H); 1.43 (9H); 1.48-1.70 (7H); 1.78-1.87 (1H); 2.14-2.25 (4H); 3.16-3.71 (22H); 3.92-4.04 (4H); 4.47-4.52 (1H); 5.33 (1H); 6.10 (1H); 6.65-7.01 (1H); 7.11-7.30 (2H); 7.47-7.63 (1H).

Molecule A5

[1017] To a solution of molecule 9 (2.37 g, 2.51 mmol) in dichloromethane (50 mL) at ambient temperature is added a 4 M HCl solution in dioxane (6.3 mL) then the medium is stirred for 2 h at ambient temperature. After concentration at reduced pressure, the residue is solubilized in dichloromethane (50 mL) then washed with a 1 N NaOH aqueous solution (212.5 mL) and an aqueous solution saturated with NaCl (25 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A white solid of molecule A5 is obtained after recrystallization in acetonitrile.

[1018] Yield: 1.57 g (74%)

[1019] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (6H); 1.08-1.43 (34H); 1.48-1.71 (7H); 1.74-1.93 (3H); 2.14-2.25 (4H); 2.79-2.86 (2H); 3.17-3.71 (20H); 3.93-4.05 (4H); 4.47-4.54 (1H); 6.08-6.29 (1H); 6.84-7.01 (1H); 7.15-7.32 (2H); 7.50-7.64 (1H).

[1020] LC/MS (ESI): 843.6 (calculated ([M+H].sup.+): 843.7).

EXAMPLE A6

Molecule A6

[1021] Molecule 10: Product obtained by hydrogenating retinoic acid.

[1022] A solution of retinoic acid (19.0 g, 63.24 mmol) in methanol (450 mL) in the presence of 10% palladium on carbon (1.9 g) is placed in a hydrogen atmosphere (1 atm) at ambient temperature. After placing overnight, the reaction medium is filtered on a sintered filter and the filtrate is then concentrated at reduced pressure. A colorless oil of molecule 10 is obtained.

[1023] Yield: 19.50 g (99%)

[1024] .sup.1H NMR CDCl.sub.3, ppm): 0.45-2.01 (35 H); 2.10-2.17 (1H); 2.33-2.38 (1H); 11.14 (1H).

[1025] LC/MS (ESI): 309.3; (calculated ([MH].sup.): 309.3).

[1026] Molecule 11: Product obtained by coupling Boc-1-amino-4,7,10-trioxa-13-tridecane amine (BocTOTA) and molecule 10.

[1027] By means of a similar method to that used for the preparation of molecule 9 applied to molecule 10 (19.3 g, 62.15 mmol) and to BocTOTA (23.9 g, 74.58 mmol), an orange oil of molecule 11 is obtained.

[1028] Yield: 37.05 g (97%)

[1029] .sup.1H NMR CDCl.sub.3, ppm): 0.43-1.71 (49 H); 2.13-2.17 (1H); 3.17-3.24 (2H); 3.32-3.39 (2H); 3.51-3.66 (12H); 4.77 (0.1H); 4.94 (0.9H); 6.13 (0.9H); 6.29 (0.1H).

[1030] LC/MS (ESI): 613.5; (calculated ([M+H].sup.+): 613.5).

Molecule A6

[1031] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 11 (34.9 g, 56.94 mmol), an orange oil of molecule A6 is obtained.

[1032] Yield: 28.5 g (97%)

[1033] .sup.1H NMR CDCl.sub.3, ppm): 0.41-1.96 (42 H); 2.13 (1H); 2.78 (2H); 3.31-3.36 (2H); 3.53 (4H); 3.55-3.58 (4H); 3.60-3.63 (4H); 6.43 (1H).

[1034] LC/MS (ESI): 513.5; (calculated ([M+H].sup.+): 513.5).

EXAMPLE A7

Molecule A7

[1035] Molecule 12: Product Obtained by Reacting Molecule 4 and Fmoc-Glu(OtBu)-OH.

[1036] To a suspension of Fmoc-Glu(OtBu)-OH (10.55 g, 24.80 mmol) and HATU (9.43 g, 24.80 mmol) in a 1:1 DMF/dichloromethane mixture (60 mL) is added DIPEA (8.64 mL, 49.60 mmol). After complete solubilization, the solution obtained is poured onto molecule 4. After 2 h of stirring at ambient temperature, the resin is filtered, washed successively with DMF (360 mL), isopropanol (160 mL) and dichloromethane (360 mL).

[1037] Molecule 13: Product obtained by reacting Molecule 12 and a 50:50 DMF/morpholine mixture.

[1038] Molecule 12, previously washed with DMF, is treated with a 50:50 DMF/morpholine mixture (60 mL). After 1 h 15 of stirring at ambient temperature, the resin is filtered, washed successively with DMF (360 mL), isopropanol (160 mL) and dichloromethane (360 mL).

[1039] Molecule 14: Product obtained by reacting molecule A1 and molecule 13.

[1040] By means of a similar method to that used for molecule 12 applied to molecule 13 and to molecule A1 (8.07 g, 24.80 mmol) in DMF (60 mL), molecule 14 is obtained.

Molecule A7

[1041] By means of a similar method to that used for the preparation of molecule 8 and applied to molecule 14, a white solid of molecule A7 is obtained after purification by silica gel chromatography (eluent: DCM, methanol).

[1042] Yield: 2.92 g (52% in 6 stages)

[1043] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.07-2.32 (88H); 2.95-3.09 (2H); 3.28-3.60 (4H); 4.06-4.19 (1.7H); 4.21-4.38 (2.6H); 4.40-4.46 (0.7H); 7.56-7.63 (0.7H); 7.78-8.09 (2.6H); 8.22-8.31 (0.7H); 12.64 (1H).

[1044] LC/MS (ESI): 1131.8 (calculated ([M+H].sup.+): 1131.8).

EXAMPLE A8

Molecule A8

[1045] Molecule 15: Product obtained by reacting decanoic acid and L-leucine.

[1046] By means of a similar method to that used for the preparation of molecule A2 applied to decanoic acid (8.77 g, 50.94 mmol) and to L-leucine (7.00 g, 53.36 mmol), a white solid of molecule 15 is obtained.

[1047] Yield: 9.17 g (66%)

[1048] .sup.1H NMR (DMSO-d6, ppm): 0.82-0.89 (9H); 1.18-1.65 (17H); 2.04-2.14 (2H); 4.19-4.23 (1H); 7.98 (1H); 12.40 (1H).

[1049] LC/MS (ESI): 286.2 (calculated ([M+H].sup.+): 286.2).

[1050] Molecule 16: Product obtained by reacting molecule 15 and L-lysine methyl ester.

[1051] To a solution of molecule 15 (9.16 g, 32.11 mmol) in THF (160 mL) are added successively triethylamine (8.12 g, 80.27 mmol) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and the medium is stirred for 30 min at ambient temperature. L-lysine methyl ester dihydrochloride (3.93 g, 16.86 mmol) is added and the reaction medium is stirred for 3 h then concentrated at reduced pressure. The residue is diluted with AcOEt (200 mL), the organic phase is filtered and washed with a 1 N HCl aqueous solution then with water, dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A white solid of molecule 16 is obtained after triturating the residue in acetonitrile.

[1052] Yield: 7.33 g (66%)

[1053] .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.06-1.72 (38H); 2.03-2.16 (4H); 2.91-3.07 (2H); 3.60 (1.15H); 3.61 (1.85H); 4.13-4.28 (2H); 4.33-4.44 (1H); 7.79-7.92 (3H); 8.13-8.26 (1H).

[1054] LC/MS (ESI) 695.7 (calculated ([M+H].sup.+): 695.6).

[1055] Molecule 17: Product obtained by saponifying molecule 16.

[1056] To a solution of molecule 16 (7.33 g, 10.55 mmol) in a THF/methanol/water mixture (105 mL) is added LiOH (505.13 mg, 21.09 mmol) at 0 C. then the medium is stirred for 20 h at ambient temperature and concentrated at reduced pressure. The aqueous phase is acidified with a 1 N HCl solution to pH 1 and the solid formed is filtered, washed with water and dried at reduced pressure to arrive at a white solid of molecule 17.

[1057] Yield: 7.09 g (99%)

[1058] .sup.1H NMR (DMSO-d6, ppm): 0.80-0.89 (18H); 1.18-1.73 (40H); 2.03-2.16 (4H); 2.91-3.05 (2H); 4.03-4.13 (1H); 4.21-4.27 (1H); 4.31-4.40 (1H); 7.79-8.02 (4H).

[1059] LC/MS (ESI): 681.7 (calculated ([M+H].sup.+): 681.6).

[1060] Molecule 18: Product obtained by reacting molecule 17 and N-Boc ethylenediamine.

[1061] By means of a similar method to that used for the preparation of molecule 16 applied to molecule 17 (7.09 g, 10.41 mmol) and to N-Boc ethylenediamine (1.83 g, 11.45 mmol), a white solid of molecule 18 is obtained after trituration in acetonitrile.

[1062] Yield: 6.64 g (77%)

[1063] .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.15-1.73 (49H); 2.03-2.18 (4H); 2.92-3.13 (6H); 4.05-4.30 (3H); 6.71-6.83 (1H); 7.69-8.23 (5H).

[1064] LC/MS (ESI): 824.0 (calculated ([M+H].sup.+): 823.7).

Molecule A8

[1065] By means of a similar method to that used for molecule A5 applied to molecule 18 (3.00 g, 3.64 mmol) without basic washing, a beige solid of molecule A8 in hydrochloride salt form is obtained after co-evaporating the residue 4 times in methanol.

[1066] Yield: 2.66 g (96%)

[1067] .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.15-1.76 (40H); 2.03-2.19 (4H); 1.78-2.89 (2H); 2.91-3.07 (2H); 3.22-3.37 (2H); 4.08-4.14 (1H); 4.17-4.28 (2H); 7.81-8.36 (8H).

[1068] LC/MS (ESI): 723.7 (calculated ([M+H].sup.+): 723.6).

EXAMPLE A9

Molecule A9

[1069] Molecule 19: 13-Methyltetradecanoic acid.

[1070] In a dry three-neck round-bottom flask under argon, magnesium (5.50 g, 226.3 mmol) chips are introduced. The magnesium is covered with anhydrous THF (25 mL) and a few drops of 1-bromo-2-methylpropane are added at ambient temperature to initiate the reaction. After observing an exotherm and slight turbidity of the medium, the remaining 1-bromo-2-methylpropane (28.42 g, 207 mmol) diluted in THF (60 mL) is added dropwise in 1 h whereas the temperature of the medium remains stable from 65 to 70 C. The reaction medium is then reflux heated for 2 h.

[1071] In a three-neck round-bottom flask under argon, to a solution of CuCl (280 mg, 2.83 mmol) dissolved in N-methylpyrrolidone (NMP) previously distilled at 0 C. is added dropwise a solution of 11-bromoundecanoic acid (25 g, 94.27 mmol) dissolved in THF (60 mL). To this solution is then added dropwise the slightly warm organomagnesium solution diluted in THF (50 mL) so as to maintain the temperature of the medium below 25 C. The mixture is then stirred at ambient temperature for 16 h. The medium is cooled to 0 C. and the reaction is stopped by slowly adding a 1 N HCl aqueous solution to pH 1 (300 mL) and the medium is extracted with hexane (100 mL) and with ethyl acetate (275 mL). After washing the organic phase with a 1 N HCl aqueous solution (100 mL), water (100 mL) and drying on Na.sub.2SO.sub.4, the solution is filtered and concentrated under vacuum to produce a brown solid. After purification by flash chromatography (cyclohexane, ethyl acetate), a white solid is obtained.

[1072] Yield: 18.1 g (79%)

[1073] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (6H); 1.11-1.18 (2H); 1.20-1.38 (16H); 1.51 (1H); 1.63 (2H); 2.35 (2H).

[1074] Molecule 20: Product obtained by reacting molecule 19 and L-leucine.

[1075] To a solution of molecule 19 (18.05 g, 74.46 mmol) in THF (745 mL) at ambient temperature are added successively DCC (14.63 g, 70.92 mmol) and NHS (8.16 g, 70.92 mmol). After 40 h of stirring at ambient temperature, the medium is cooled to 0 C. for 20 min, filtered on a sintered filter. L-leucine (9.77 g, 74.46 mmol), DIPEA (86 mL) and water (150 mL) are added to the filtrate. After 20 h of stirring at ambient temperature, the medium is diluted with a saturated aqueous solution of NaHCO.sub.3 (200 mL). The aqueous phase is washed with ethyl acetate (2200 mL) and acidified with a 2 N HCl solution to pH 1. The precipitate is filtered, rinsed with plenty of water and vacuum-dried at 50 C. Three times, the solid is triturated in pentane, sonicated then filtered to produce a white solid.

[1076] Yield: 18.8 g (75%)

[1077] .sup.1H NMR CDCl.sub.3, ppm): 0.86 (6H); 0.96 (6H); 1.12-1.18 (2H); 1.20-1.78 (22H); 2.24 (2H); 4.58-4.63 (1H); 5.89 (1H).

[1078] LC/MS (ESI): 356.2; (calculated ([M+H].sup.+): 356.6).

[1079] Molecule 21: Product obtained by reacting molecule 20 and Boc-tri(ethyleneglycol)diamine.

[1080] To a solution of molecule 20 (16.7 g, 46.97 mmol) in THF (235 mL) are added successively DIPEA (20.3 mL) and TBTU at ambient temperature. After 20 min of stirring, Boc-tri(ethyleneglycol)diamine (14 g, 56.36 mmol) is added. After stirring at ambient temperature for 5 h, the mixture is concentrated under vacuum. The residue is taken up with ethyl acetate (500 mL), washed with a saturated aqueous solution of NaHCO.sub.3 (3200 mL), a 1 N HCl aqueous solution (3200 mL) and an aqueous solution saturated with NaCl (3200 mL). After drying on Na.sub.2SO.sub.4, filtration and concentration under vacuum, the residue is purified by flash chromatography (cyclohexane, ethyl acetate, methanol) to produce a colorless oil.

[1081] Yield: 23.5 g (85%)

[1082] .sup.1H NMR CDCl.sub.3, ppm): 0.86 (6H); 0.93 (6H); 1.10-1.17 (2H); 1.19-1.08 (31H); 2.18 (2H); 3.23-3.65 (12H); 4.41-4.56 (1H); 5.12-5.47 (1H); 5.99-6.11 (0.75H); 6.48-6.65 (1H); 7.30-7.40 (0.25H).

Molecule A9

[1083] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 21 (23.46 g, 40.04 mmol) without basic washing, the residue obtained after concentration under vacuum is triturated in an acetonitrile/acetone mixture. The supernatant is removed and the pasty residue is vacuum-dried. The residue is triturated in acetone (150 mL) and the white solid of molecule A9 in hydrochloride salt form is filtered, rinsed with acetone then vacuum-dried.

[1084] Yield: 13.0 g (64%)

[1085] .sup.1H NMR (DMSO-d6, ppm): 0.79-0.90 (12H); 1.09-1.61 (24H); 2.03-2.17 (2H); 2.92-2.98 (2H); 3.15-3.23 (2H); 3.40 (2H); 3.50-3.58 (4H); 3.61 (2H); 4.30-4.23 (1H); 7.88-8.14 (5H).

[1086] LC/MS (ESI): 486.4; (calculated ([MCl].sup.+): 486.8).

EXAMPLE A10

Molecule A10

[1087] Molecule 22: Product obtained by reacting octanoyl chloride and L-proline.

[1088] By means of a similar method to that used for the preparation of molecule A1 and applied to octanoyl chloride (150.0 g, 0.922 mol) and to L-proline (212.3 g, 1.844 mol), a colorless oil of molecule 22 is obtained after washing the organic phase with a 10% HCl aqueous solution (3300 mL), an aqueous solution saturated with NaCl (300 mL), drying on Na.sub.2SO.sub.4, filtration on cotton, concentration at reduced pressure, then the residue is purified by flash chromatography (eluent: DCM, MeOH)

[1089] Yield: 134 g (60%)

[1090] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.10-1.52 (8H); 1.57-1.74 (2H); 1.79-2.52 (6H); 3.37-3.67 (2H); 4.37-4.42 (0.07H); 4.53-5.63 (0.93H); 9.83 (1H).

[1091] LC/MS (ESI): 242.1; (calculated ([M+H].sup.+): 242.2).

[1092] Molecule 23: Product obtained by coupling molecule 22 and L-leucine.

[1093] To a solution of molecule 22 (132 g, 0.547 mol) in THF (924 mL) cooled to a temperature below 5 C. are added successively NHS (66.1 g, 0.574 mol) and DCC (118.5 g, 0.574 mol). After 21 h of stirring, the precipitate is removed by precipitation and the filtrate is added in 30 min to a solution of L-lysine (41.98 g, 0.287 mol) in a mixture of deionized water (82 mL) and DIPEA (476 mL, 2.735 mol) at 15 C. After 23 h of stirring at ambient temperature, the reaction medium is concentrated at reduced pressure to produce an oily residue which is diluted in water (1.3 L). The aqueous phase is washed twice with AcOEt (20.5 L), cooled to a temperature below 10 C., acidified by adding a 6 N HCl solution (120 mL) to attain a pH of 1 then extracted three times with DCM (30,6 L). The organic phases are combined, washed with a saturated NaCl solution (0.6 L), dried on Na.sub.2SO.sub.4 then concentrated at reduced pressure. The foam obtained is taken up with acetone (240 mL) under reflux for 2 h. After leaving overnight at 10 C., pentane (240 mL) is added dropwise. After 1 h of stirring, the precipitate is retrieved by filtering under vacuum, washed with a 1:1 mixture of pentane and acetone (150mL) then vacuum-dried.

[1094] Yield: 83.9 g (52%)

[1095] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (6H); 1.06-1.78 (25H); 1.80-2.41 (13H); 2.80-3.72 (6H); 4.30-4.39 (0.15H); 4.46-4.70 (2.85H); 7.84 (1H); 7.93 (1H).

[1096] LC/MS (ESI): 593.5; (calculated ([M+H].sup.+): 593.4).

[1097] Molecule 24: Product obtained by coupling molecule 23 and L-lysine methyl ester (LysOMe).

[1098] To molecule 23 (76.26 g, 0.129 mol) are successively added HOPO (3.57 g, 32.1 mmol), LysOMe dihydrochloride (15.0 g, 64.3 mmol) and EDC (34.53 g, 0.18 mol). Then DMF (600 mL) previously cooled to 5 C. is added. After dissolution, triethylamine (43.9 mL, 0.315 mol) is added dropwise while maintaining the temperature below 5 C. for 2 h after addition. After leaving overnight at ambient temperature, the reaction medium is poured onto a mixture of water/ice (2 kg) and DCM (0.5 L). After 15 min of stirring, the phases are separated. The aqueous phase is extracted twice with DCM (20.4 L). The organic phases are combined, washed with a 1 N HCl solution (0.5 L) then with a saturated NaCl solution (0.5 L), dried on Na.sub.2SO.sub.4, concentrated at reduced pressure, then the residue is purified by flash chromatography (eluent: DCM, MeOH).

[1099] Yield: 56.7 g (67%)

[1100] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (12H); 1.10-2.40 (82H); 2.86-3.72 (17H); 4.16-4.60 (7H); 6.83-8.01 (6H).

Molecule A10

[1101] A solution of molecule 24 (4.0 g, 3.05 mmol) in ethylenediamine (30 mL) is heated at 50 C. overnight. The reaction medium is then diluted with methyl-tetrahydrofuran then the organic phase is washed 4 times with a saturated NaCl solution (430 mL) then 2 times with water (250 mL) before being dried on Na.sub.2SO.sub.4 and concentrated at reduced pressure. The residue is solubilized in acetonitrile under reflux for 30 min then the solution is cooled to ambient temperature under stirring overnight. The white precipitate is then retrieved by filtering under vacuum, washed with cold acetonitrile (220 mL) then dried under vacuum.

[1102] Yield: 3.0 g (74%)

[1103] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (12H); 1.09-2.37 (84H); 2.74-4.56 (25H); 6.85-8.00 (7H). LC/MS (ESI): 1338.0 (calculated ([M+H].sup.+): 1338.0).

EXAMPLE A11

Molecule A11

[1104] Molecule 25: Product obtained by reacting molecule 13 and lauric acid.

[1105] By means of a similar method to that used for molecule 5 applied to molecule 13 (28 mmol) and lauric acid (28.04 g, 140 mmol) in DMF (330 mL), molecule 25 is obtained.

Molecule A11

[1106] By means of a similar method to that used for molecule 8 applied to molecule 25, a white solid of molecule A11 is obtained after recrystallization in acetonitrile.

[1107] Yield: 13.9 g (56% in 6 stages)

[1108] .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.05-1.61 (60H); 1.62-1.75 (2H); 1.78-1.91 (2H); 2.04-2.27 (8H); 2.96-3.06 (2H); 4.08-4.13 (1H); 4.17-4.22 (1H); 4.27-4.34 (1H); 7.82 (1H); 7.86 (1H); 7.90 (1H); 8.03 (1H); 12.54 (1H).

[1109] LC/MS (ESI+): 881.7 (calculated ([M+H].sup.+): 881.7).

EXAMPLE A12

Molecule A12

[1110] Molecule 26: Product obtained by reacting molecule 13 and Fmoc-Glu(OtBu)-OH.

[1111] By means of a similar method to that used for molecule 5 applied to molecule 13 (9.92 mmol) and to Fmoc-Glu(OtBu)-OH (21.10 g, 49.60 mmol) in N-methyl-2-pyrrolidone (NMP, 120 mL), molecule 26 is obtained.

[1112] Molecule 27: Product obtained by reacting molecule 26 and an 80:20 NMP/piperidine mixture.

[1113] By means of a similar method to that used for molecule 4 applied to molecule 26, using NMP instead of DMF, molecule 27 is obtained.

[1114] Molecule 28: Product obtained by reacting molecule 27 and Fmoc-Glu(OtBu)-OH.

[1115] By means of a similar method to that used for molecule 26 applied to molecule 27 and to Fmoc-Glu(OtBu)-OH (21.10 g, 49.60 mmol), molecule 28 is obtained.

[1116] Molecule 29: Product obtained by reacting molecule 28 and an 80:20 NMP/piperidine mixture.

[1117] By means of a similar method to that used for molecule 27 applied to molecule 28, molecule 29 is obtained.

Molecule 30: Product obtained by reacting molecule 29 and molecule A1.

[1118] By means of a similar method to that used for molecule 26 applied to molecule 29 (4.96 mmol) and to molecule A1 (8.07 g, 24.80 mmol), molecule 30 is obtained.

Molecule A12

[1119] By means of a similar method to that used for molecule 8 applied to molecule 30, a white solid of molecule A12 is obtained after purification by flash chromatography (DCM, MeOH).

[1120] Yield: 4.6 g (50% in 10 stages)

[1121] .sup.1H NMR (CD.sub.3OD, ppm): 0.90 (6H); 1.22-2.53 (140H); 3.12-3.25 (2H); 3.43-3.80 (4H); 4.17-4.54 (9H).

[1122] LC/MS (ESI+): 1894.5 (calculated ([M+Na].sup.+): 1894.2).

EXAMPLE A14

Molecule A14

[1123] Molecule 33: Product obtained by reacting N--Boc-L-Lysine and palmitoyl chloride

[1124] By means of a similar method to that used for the preparation of molecule A1 applied to N--Boc-L-Lysine (53.76 g, 218.28 mmol) and to palmitoyl chloride (50.00 g, 181.90 mmol), a white solid of molecule 33 is obtained after recrystallizing 2 times in acetonitrile and purification by flash chromatography (eluent: dichloromethane, methanol).

[1125] Yield: 49.10 g (70%)

[1126] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.09-1.66 (32H); 1.37 (9H); 2.01 (2H); 2.93-3.06 (2H); 3.78-3.85 (1H); 6.61-6.68 (0.2H); 6.96-6.98 (0.8H); 7.66-7.75 (1H); 12.38 (1H).

[1127] LC/MS (ESI): 385.1 (calculated ([MBoc+H].sup.+): 385.3).

Molecule 34: Product obtained by reacting molecule 33 and methyl iodide.

[1128] To a solution of molecule 33 (23.40 g, 48.28 mmol) in DMF (200 mL) at ambient temperature are added K.sub.2CO.sub.3 (10.01 g, 72.41 mmol) followed by methyl iodide (5.96 mL, 98.55 mmol). The medium is stirred for 48 h. Water (350 mL) is added and the suspension is stirred for 15 min. The latter is then filtered on a sintered filter and the solid obtained is rinsed with water (2250 mL) and vacuum-dried. The solid is then solubilized in DCM (300 mL). The solution is washed with water (200 mL) then with an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A white solid of molecule 34 is obtained after recrystallization in acetonitrile.

[1129] Yield: 19.22 g (80%)

[1130] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.06-2.23 (34H); 1.43 (9H); 3.09-3.33 (2H); 3.72 (3H); 3.94-4.35 (1H); 4.69-5.23 (1H); 5.33-5.75 (1H).

[1131] LC/MS (ESI): 543.3 (calculated ([MH+HCOOH].sup.): 543.4).

[1132] Molecule 35: Product obtained by hydrolyzing molecule 34 with hydrochloric acid

[1133] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 34 in solution in a 1:1 DCM/methanol mixture (385 mL), a white solid of molecule 35 is obtained after concentration at reduced pressure and co-evaporation with DCM followed by methanol.

[1134] Yield: 16.73 g (99%)

[1135] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.08-1.50 (30H); 1.67-1.84 (2H); 2.03 (2H); 2.94-3.13 (2H); 3.74 (3H); 3.92-4.01 (1H); 7.77-7.87 (1H); 8.25-8.73 (3H).

[1136] LC/MS (ESI): 399.2 (calculated ([M+H].sup.+): 399.4).

Molecule A14

[1137] To a suspension of molecule 35 (14.70 g, 33.79 mmol) in a mixture of methyl-THF (338 mL) and DMF (30 mL) are added successively DIPEA (17.70 mL, 101.40 mmol) followed by a solution of succinic anhydride (5.07 g, 50.68 mmol) in THF (60 mL). The medium is stirred for 4 h at ambient temperature. Methyl-THF (100 mL) is added and the organic phase is washed with a 5% HCl aqueous solution (300 mL). The aqueous phase is extracted with methyl-DCM (2150 mL). The combined organic phases are washed with water (2150 mL) then with an aqueous solution saturated with NaCl (150 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. The crude product is purified by flash chromatography (eluent: DCM, methanol) then solubilized in methyl-THF. The purified product is then suspended in water. The suspension is stirred by sonication for 20 min followed by magnetic stirring for 30 min. A white solid of molecule A14 is obtained after filtration and drying at reduced pressure.

[1138] Yield: 12.99 g (77%)

[1139] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.08-1.71 (32H); 2.02 (2H); 2.29-2.45 (4H); 2.94-3.04 (2H); 3.61 (3H); 4.14-4.22 (1H); 7.70 (1H); 8.20 (1H); 12.04 (1H).

[1140] LC/MS (ESI): 499.3 (calculated ([M+H].sup.+): 499.4).

EXAMPLE A15

Molecule A15

[1141] Molecule 36: Product obtained by coupling L-proline and palmitoyl chloride

[1142] By means of a similar method to that used for the preparation of molecule A1 applied to L-proline (38.05 g, 906.00 mmol) and to palmitoyl chloride (14.01 g, 350.16 mmol), a white solid of molecule 36 is obtained.

[1143] Yield: 47.39 g (96%)

[1144] .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).

[1145] LC/MS (ESI): 354.5 (calculated ([M+H].sup.+): 354.3).

[1146] Molecule 37: Product obtained by reacting molecule 36 and N-Bocethylenediamine.

[1147] By means of a similar method to that used for molecule 9 applied to molecule 36 (75.1 g, 212.4 mmol), a white solid of molecule 37 is obtained after trituration in diisopropylether (3400 mL) and vacuum-drying at 40 C.

[1148] Yield: 90.4 g (86%).

[1149] .sup.1H NMR CDCl.sub.3, ppm): 0.88 (3H); 1.20-1.37 (24H); 1.44 (9H); 1.54-1.70 (2H); 1.79-1.92 (1H); 1.92-2.04 (1H); 2.03-2.17 (1H); 2.17-2.44 (3H); 3.14-3.36 (4H); 3.43 (1H); 3.56 (1H); 4.29 (0.1 H); 4.51 (0.9 H); 4.82 (0.1H); 5.02 (0.9H); 6.84 (0.1H); 7.22 (0.9H).

[1150] Molecule 38: Product obtained by hydrolyzing molecule 37 with hydrochloric acid

[1151] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 37 (38.17 g, 76.99 mmol), a white solid of molecule 38 is obtained.

[1152] .sup.1H NMR CDCl.sub.3, ppm): 0.88 (3H); 1.07-1.40 (24H); 1.49-1.63 (2H); 1.77-2.18 (4H); 2.18-2.45 (2H); 3.14-3.32 (2H); 3.42-3.63 (2H); 3.63-3.84 (2H); 4.37 (0.1H); 4.48 (0.9H); 6.81-8.81 (4H).

[1153] LC/MS (ESI): 396.5; (calculated ([M+H].sup.+): 396.4).

Molecule A15

[1154] By means of a similar method to that used for the preparation of molecule A14 applied to molecule 38 (10.00 g, 253.00 mmol), a white solid of molecule A15 is obtained.

[1155] Yield: 10.00 g (80%)

[1156] .sup.1H NMR (DMSO, ppm): 0.85 (3H); 1.07-1.51 (26H); 1.69-2.02 (4H); 2.08-2.53 (6H); 3.01-3.18 (4H); 3.39-3.58 (2H); 4.13-4.18 (0.7H); 4.23-4.27 (0.3H); 7.70-7.78 (1.4H); 7.81-7.86 (0.3H); 8.00-8.04 (0.3H); 12.08 (1H).

[1157] LC/MS (ESI): 496.3 (calculated ([M+H].sup.+): 496.4).

EXAMPLE A16

Molecule A16

[1158] Molecule 39: Product obtained by reacting molecule 36 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

[1159] By means of a similar method to that used for the preparation of molecule 9 applied to molecule 36 (17.00 g, 48.08 mmol) and to Boc-1-amino-4,7,10-trioxa-13-tridecane amine (18.49 g, 57.70 mmol), a pale yellow oil of molecule 39 is obtained.

[1160] Yield: 31.11 g (98%)

[1161] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.17-1.31 (24H); 1.37 (9H); 1.41-1.51 (2H); 1.54-1.67 (4H); 1.69-2.02 (4H); 2.08-2.29 (2H); 2.91-3.00 (2H); 3.01-3.17 (2H); 3.31-3.58 (14H); 4.20 (0.65H); 4.26 (0.35H); 6.29-6.82 (1H); 7.68 (0.65H); 8.02 (0.35H).

[1162] LC/MS (ESI): 656.4 (calculated ([M+H].sup.+): 656.5).

[1163] Molecule 40: Product obtained by hydrolyzing molecule 39 with hydrochloric acid

[1164] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 39 (31.11 g, 47.43 mmol), a yellow wax of molecule 40 is obtained.

[1165] Yield: 27 g (97%)

[1166] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.18-1.31 (24H); 1.40-1.51 (2H); 1.55-1.67 (2H); 1.70-2.04 (6H); 2.09-2.30 (2H); 2.78-2.89 (2H); 2.99-3.18 (2H); 3.33-3.58 (14H); 4.19 (0.65H); 4.27 (0.35H); 7.55-8.14 (4H).

[1167] LC/MS (ESI): 556.3 (calculated ([M+H].sup.+): 556.5).

Molecule A16

[1168] Molecule 40 (26.40 g, 44.50 mmol) in hydrochloride form is solubilized in a mixture of DCM (350 mL) and an aqueous solution of NaHCO.sub.3 (350 mL). The organic phase is separated and the aqueous phase is extracted with DCM (2150 mL). The organic phases are combined dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure to produce a colorless oil. By means of a similar method to that used for the preparation of molecule A14, a yellow resin of molecule A16 is obtained after purification by flash chromatography (eluent: DCM, methanol).

[1169] Yield: 19.93 g (68%)

[1170] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.18-1.30 (24H); 1.40-1.51 (2H); 1.55-1.67 (4H); 1.70-2.02 (4H); 2.07-2.45 (6H); 2.99-3.18 (4H); 3.33-3.57 (14H); 4.19 (0.65H); 4.26 (0.35H); 7.68 (0.65H); 7.78 (1H); 8.02 (0.35H); 12.03 (1H).

[1171] LC/MS (ESI): 656.3 (calculated ([M+H].sup.+): 656.5).

EXAMPLE A17

Molecule A17

[1172] Molecule 41: Product obtained by solid phase peptide synthesis (SPPS)

[1173] Molecule 41 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl resin

[1174] To a solution of 4,7,10-trioxa-1,13-tridecanediamine (TOTA, 76.73 mL, 350 mmol) in DCM (350 mL) is added DIPEA (60.96 mL, 350 mmol). This solution is then poured onto the 2-chlorotrityl resin (47.30 g, 0.74 mmol/g) previously washed with DCM in a reaction vessel suitable for SPPS. After 1.5 h of stirring at ambient temperature, methanol (26 mL) is added and the medium is stirred for 15 min. The resin is filtered, washed successively with DCM (3350 mL), DMF (2350 mL), DCM (2350 mL), isopropanol (1350 mL) and DCM (3350 mL). The -methyl ester of N-Fmoc-L-glutamic acid (1,5 eq) followed by molecule 36 (1.5 eq) are coupled using the coupling agent 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 1.5 equivalents) and DIPEA (3 equivalents) in a 1:1 DCM/DMF mixture. A 1:1 DMF/morpholine mixture is used for the cleavage step of the Fmoc protecting group. The resin is washed with DCM, DMF and methanol after each coupling and deprotection step. The cleavage of the product from the resin is carried out using a 1:1 TFA/DCM mixture. The solvents are then evaporated under vacuum; the residue is solubilized in DCM (500 mL) and the organic phase is washed with a 5% Na.sub.2CO.sub.3 aqueous solution (500 mL). After drying on Na.sub.2SO.sub.4, the organic phase is filtered, concentrated under vacuum and a yellow oil of molecule 41 is obtained after drying at reduced pressure.

[1175] Yield: 15.95 g (65%)

[1176] 1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.16-1.31 (24H); 1.38-1.68 (6H); 1.68-2.37 (12H); 2.58 (2H); 3.01-3.17 (2H); 3.31-3.55 (14H); 3.58 (3H); 4.09-4.18 (0.7H); 4.18-4.29 (1H); 4.36-4.43 (0.3H); 7.62 (0.7H); 7.86 (0.7H); 7.98 (0.3H); 8.23 (0.3H).

[1177] LC/MS (ESI): 699.4 (calculated ([M+H]+): 699.5).

Molecule A17

[1178] By means of a similar method to that used for the preparation of molecule A14 applied to molecule 41 (14.05 g, 20.10 mmol), a yellow resin of molecule A17 is obtained after purification by flash chromatography (eluent: DCM, methanol).

[1179] Yield: 7.70 g (48%)

[1180] 1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.17-1.31 (24H); 1.38-1.54 (2H); 1.54-1.68 (4H); 1.68-2.21 (7H); 2.21-2.36 (5H); 2.36-2.44 (2H); 3.01-3.16 (4H); 3.34-3.55 (14H); 3.57 (3H); 4.10-4.18 (0.7H); 4.18-4.30 (1H); 4.40 (0.3H); 7.60 (0.7H); 7.78 (1H); 7.85 (0.7H); 7.95 (0.3H); 8.22 (0.3H); 12.06 (1H).

[1181] LC/MS (ESI): 799.5 (calculated ([M+H]+): 799.5).

EXAMPLE A18

Molecule A18

[1182] Molecule 42: Product obtained by reacting molecule A1 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

[1183] By means of a similar method to that used for the preparation of molecule 18 applied to molecule A1 (44.80 g, 137.64 mmol) and to Boc-l-amino-4,7,10-trioxa-13-tridecane amine (52.92 g, 165.16 mmol), an orange oil of molecule 42 is obtained.

[1184] Yield: 85.63 g (99%)

[1185] 1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.08-1.56 (20H); 1.43 (9H); 1.58-1.67 (2H); 1.70-2.00 (6H); 2.04-2.41 (4H); 3.16-3.77 (18H); 4.26-4.29 (0.2H); 4.50-4.54 (0.8H); 4.68-5.10 (1H); 6.74 (0.2H); 7.19 (0.8H).

[1186] LC/MS (ESI): 628.4; (calculated ([M+H].sup.+): 628.5).

[1187] Molecule 43: Product obtained by hydrolyzing molecule 42 with hydrochloric acid

[1188] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 42 (43.40 g, 69.12 mmol), a white solid of molecule 43 in hydrochloride salt form is obtained after trituration in diethylether, solubilizing the residue in water and freeze-drying.

[1189] Yield: 38.70 g (98%)

[1190] 1H NMR (DMSO, ppm): 0.85 (3H); 1.07-1.38 (20H); 1.41-1.52 (2H); 1.55-1.66 (2H); 1.70-2.02 (6H); 2.08-2.30 (2H); 2.78-2.87 (2H); 3.00-3.16 (2H); 3.29-3.66 (14H); 4.16-4.22 (0.65 H); 4.25-4.30 (0.35H); 7.74 (0.65H); 7.86 (3H); 8.10 (0.35H).

[1191] LC/MS (ESI): 528.4; (calculated ([M+H].sup.+): 528.4).

Molecule A18

[1192] By means of a similar method to that used for the preparation of molecule A14 applied to molecule 43 (13.09 g, 24.8 mmol), a yellow resin of molecule A18 is obtained after purification by flash chromatography (eluent: DCM, methanol).

[1193] Yield: 8.53 g (55%)

[1194] 1H NMR (DMSO-d6, ppm): 0.86 (3H); 1.10-1.39 (20H); 1.42-1.51 (2H); 1.57-1.67 (4H); 1.71-2.03 (4H); 2.09-2.32 (4H); 2.42 (2H); 3.01-3.17 (4H); 3.36-3.57 (14H); 4.18-4.21 (0.65H); 4.24-4.28 (0.35H); 7.69 (0.65H); 7.80 (1H); 8.03 (0.35H); 12.04 (1H).

[1195] LC/MS (ESI): 628.5 (calculated ([M+H].sup.+): 628.5).

EXAMPLE A19

Molecule A19

[1196] Molecule 44: Product obtained by SPPS

[1197] By means of a similar SPPS method to that used for the preparation of molecule 41 and applied to TOTA, to N-Fmoc-L-Leucine, N-Fmoc-L-proline and to myristic acid, an orange oil of molecule 44 is obtained.

[1198] Yield: 19.87 g (69%)

[1199] .sup.1H NMR CDCl.sub.3, ppm): 0.72-1.06 (9H); 1.09-1.42 (20H); 1.42-2.40 (17H); 2.80 (2H); 3.22-3.81 (16H); 4.25-4.61 (2H); 6.56-7.23 (2H).

[1200] LC/MS (ESI): 641.5; (calculated ([M+H].sup.+): 641.5).

Molecule A19

[1201] After a similar method to that used for the preparation of molecule A14 applied to molecule 44 (13.09 g, 204.42 mmol), 4.81 g of the product obtained by purification by flash chromatography (eluent: DCM, methanol) is solubilized in a mixture of DCM (50 mL) and THF (5.5 mL) then washed with an aqueous solution saturated with NaCl (50 mL), a 0.1 N HCl aqueous solution (50 mL) and an aqueous solution saturated with NaCl (50 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A yellow oil of molecule A19 is obtained.

[1202] Yield: 4.20 g

[1203] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.72-1.02 (9H); 1.08-1.34 (20H); 1.34-2.23 (14H); 2.23-2.35 (3H); 2.42 (2H); 3.01-3.17 (4H); 3.17-3.66 (14H); 4.15-4.44 (2H); 7.53-8.23 (3H); 12.06 (1H).

[1204] LC/MS (ESI): 741.5; (calculated ([M+H].sup.+): 741.5).

EXAMPLE A21

Molecule A21

[1205] Molecule 46: Product obtained by SPPS

[1206] By means of a similar SPPS method to that used for the preparation of molecule 41 and applied to TOTA, to N-Fmoc-L-phenylalanine and to molecule A1, an orange oil of molecule 46 is obtained and used without purification.

[1207] Yield: 15.07 g (72%)

[1208] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.08-1.42 (20H); 1.42-1.62 (2H); 1.62-1.99 (7H); 1.99-2.26 (3H); 2.72 (2H); 2.86 (2H); 2.94-3.72 (18H); 4.20-4.72 (2H); 6.63-7.37 (7H).

[1209] LC/MS (ESI): 675.65; (calculated ([M+H].sup.+): 675.5).

Molecule A21

[1210] By means of a similar method to that used for the preparation of molecule A19 applied to molecule 46 (13.79 g, 20.43 mmol), a white solid of molecule A21 is obtained.

[1211] Yield: 7.56 g (48%)

[1212] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.86 (3H); 1.02-1.42 (21H); 1.42-2.20 (10H); 2.23-2.38 (3H); 2.42 (2H); 2.78-3.18 (6H); 3.23-3.59 (14H); 4.12-4.58 (2H); 7.10-7.30 (5H); 7.53-8.33 (3H); 12.08 (1H).

[1213] LC/MS (ESI): 775.5; (calculated ([M+H].sup.+): 775.5).

EXAMPLE A22

Molecule A22

[1214] By means of a similar method to that used for the preparation of molecule A14 applied to molecule A6 (22.15 g, 43.19 mmol), a yellow oil of molecule A22 is obtained.

[1215] Yield: 25.19 g (95%)

[1216] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.42-1.51 (33H); 1.51-2.05 (8H); 2.29 (2H); 2.41 (2H); 3.07 (4H); 3.38 (4H); 3.43-3.54 (8H); 7.72 (1H); 7.79 (1H); 12.03 (1H).

[1217] LC/MS (ESI): 613.5 (calculated ([M+H].sup.+): 613.5).

EXAMPLE A23

Molecule A23

[1218] Molecule 47: Product obtained by hydrogenating phytol.

[1219] To a solution of phytol (30.00 g, 101.20 mmol) in THF (450 mL) in argon is added platinum dioxide (PtO.sub.2, 1.15 g, 6.61 mmol). The medium is placed under 1 bar of dihydrogen then stirred for 4 h at ambient temperature. After filtering on celite by rinsing with THF, a black oil of molecule 47 is obtained after concentration at reduced pressure.

[1220] Yield: 29.00 g (96%)

[1221] .sup.1H NMR CDCl.sub.3, ppm): 0.84 (6H); 0.86 (6H); 0.89 (3H); 1.00-1.46 (22H); 1.46-1.68 (3H); 3.61-3.73 (2H).

[1222] Molecule 48: Product obtained by oxidizing molecule 47

[1223] To a solution of molecule 47 (29.0 g, 97.13 mmol) in a dichloroethane/water mixture (485 mL/388 mL) are added successively tetrabutylammonium bromide (16.90 g, 52.45 mmol), acetic acid (150 mL, 2.62 mol) followed by KMnO.sub.4 (46.05 g, 291.40 mmol) in small fractions while maintaining the temperature from 16 to 19 C. The reaction medium is then stirred for 4.5 h under reflux, cooled to 10 C. then acidified to pH 1 with a 6 N HCl solution (20 mL). Na.sub.2SO.sub.3 (53.90 g) is added progressively while maintaining the temperature at 10 C. and the medium is stirred until completely discolored. Water (200 mL) is added, the phases are separated and the aqueous phase is extracted with DCM (2400 mL). The combined organic phases are washed with a 10% HCl aqueous solution (20 mL), water (2200 mL), an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A yellow oil of molecule 48 is obtained after purification by flash chromatography (eluent: cyclohexane, AcOEt).

[1224] Yield: 28.70 g (94%)

[1225] .sup.1H NMR CDCl.sub.3, ppm): 0.84 (6H); 0.86 (6H); 0.97 (3H); 1.00-1.41 (20H); 1.52 (1H); 1.96 (1H); 2.14 (1H); 2.35 (1H); 11.31 (1H).

[1226] LC/MS (ESI): 311.1 (calculated ([MH].sup.): 311.3).

[1227] Molecule 49: Product obtained by coupling molecule 48 and methyl L-prolinate.

[1228] By means of a similar method to that used for the preparation of molecule 9 applied to molecule 48 (18.00 g, 57.59 mmol) and to methyl L-prolinate hydrochloride (14.31 g, 86.39 mmol) in DCM (380 mL), a yellow oil of molecule 49 is obtained after washing the organic phase with an aqueous solution saturated with NaHCO.sub.3 (2150 mL), a 10% HCL aqueous solution (2150 mL), an aqueous solution saturated with NaCl (2150 mL), followed by drying on Na.sub.2SO.sub.4, filtration and concentration at reduced pressure.

[1229] Yield: 23.20 g (95%)

[1230] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (20H); 1.43-1.56 (1H); 1.70-1.96 (4H); 1.96-2.32 (3H); 3.33-3.56 (2H); 3.59 (0.6H); 3.67 (2.4H); 4.27 (0.8H); 4.57 (0.2H).

[1231] LC/MS (ESI): 424.4 (calculated ([M+H].sup.+): 424.4).

[1232] Molecule 50: Product obtained by saponifying molecule 49.

[1233] By means of a similar method to that used for the preparation of molecule 17 applied to molecule 49 (21.05 g, 49.68 mmol), a yellow oil of molecule 50 is obtained.

[1234] Yield: 20.40 g (99%)

[1235] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.77-0.91 (15H); 0.97-1.43 (20H); 1.43-1.56 (1H); 1.67-1.96 (4H); 1.96-2.29 (3H); 3.26-3.56 (2H); 4.20 (0.8H); 4.41 (0.2H).

[1236] LC/MS (ESI): 410.3 (calculated ([M+H].sup.+): 410.4).

[1237] Molecule 51: Product obtained by coupling molecule 50 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

[1238] By means of a similar method to that used for the preparation of molecule 9 applied to molecule 50 (8.95 g, 21.85 mmol) and to TOTA (8.40 g, 26.21 mmol), a colorless oil of molecule 51 is obtained after purification by flash chromatography (eluent: DCM, AcOEt, methanol).

[1239] Yield: 10.08 g (65%)

[1240] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (29H); 1.43-1.55 (1H); 1.55-1.66 (4H); 1.71-2.30 (7H); 2.95 (2H); 3.00-3.19 (2H); 3.34-3.58 (14H); 4.17-4.29 (1H); 6.30-6.79 (1H); 7.67 (0.65H); 8.00 (0.35H).

[1241] LC/MS (ESI): 712.6 (calculated ([M+H].sup.+): 712.6).

[1242] Molecule 52: Product obtained by hydrolyzing molecule 42 with hydrochloric acid

[1243] By means of a similar method to that used for the preparation of molecule A5 applied to molecule 51 (10.08 g, 14.16 mmol), the residue obtained after concentration at reduced pressure is solubilized in DCM (200 mL). The organic phase is washed with a 2 N NaOH aqueous solution (2100 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A colorless oil of molecule 52 in neutral amine form is obtained.

[1244] Yield: 8.23 g (95%)

[1245] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (20H); 1.43-1.69 (6H); 1.69-2.30 (8H); 2.56 (2H); 2.99-3.19 (2H); 3.31-3.58 (14H); 4.15-4.29 (1H); 7.70 (0.65H); 8.04 (0.35H).

[1246] LC/MS (ESI): 612.5 (calculated ([M+H]+): 612.5).

Molecule A23

[1247] By means of a similar method to that used for the preparation of molecule A14 applied to molecule 52 (15.40 g, 25.17 mmol), a yellow oil of molecule A23 is obtained.

[1248] Yield: 15.19 g (85%)

[1249] .sup.1H NMR (DMSO-d.sub.6, ppm): 0.76-0.91 (15H); 0.98-2.26 (32H); 2.29 (2H); 2.41 (2H); 2.98-3.18 (4H); 3.32-3.63 (14H); 4.15-4.29 (1H); 7.68 (0.7H); 7.78 (1H); 8.01 (0.3H); 12.02 (1H).

[1250] LC/MS (ESI): 712.5 (calculated ([M+H].sup.+): 712.5).

EXAMPLE A26

Molecule A26

[1251] Molecule 55: Product obtained by SPPS

[1252] Molecule 55 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl chloride (CTC) resin (47.56 g, 0.74 mmol/g).

[1253] The grafting of the first amino acid Fmoc-Glu(OtBu)-OH (2.5 equivalents) is performed in DCM (10 V), in the presence of DIPEA (5.0 equivalents). The unreacted sites are capped with methanol (0.8 mL/g resin) at the end of the reaction.

[1254] The protected amino acids Fmoc-Glu(OtBu)-OH (1.5 equivalents (2)) and molecule A1 (1.5 equivalents) are coupled in DMF (10 V), in the presence of HATU (1.0 equivalent with respect to the acid) and DIPEA (2.0 equivalents with respect to the acid).

[1255] The protecting groups Fmoc are removed using an 80:20 DMF/piperidine solution (10 V).

[1256] The product is cleaved from the resin using an 80:20 DCM/HFIP solution (10 V).

[1257] After concentration at reduced pressure, two co-evaporations are performed on the residue with dichloromethane followed by diisopropylether. The product is purified by silica gel chromatography (dichloromethane, methanol). A colorless gum of molecule 55 is obtained.

[1258] Yield: 21.4 g (69% in 8 stages)

[1259] .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.16-1.30 (20H); 1.34-1.41 (27H); 1.41-1.53 (2H); 1.67-2.33 (18H); 3.26-3.60 (2H); 4.09-4.44 (4H); 7.73 (0.65H); 7.85 (0.65H); 7.93-8.04 (1H); 8.17 (0.35H); 8.27 (0.35H); 12.64 (1H).

[1260] LC/MS (ESI+): 881.7 (calculated ([M+H].sup.+): 881.6).

[1261] Molecule 56: Product obtained by reacting molecule 55 and 2-phthalimido ethylamine.

[1262] By means of a similar method to that used for the preparation of molecule 9 applied to molecule 55 (21.38 g, 24.26 mmol) and to 2-phthalimido ethylamine hydrochloride (HCl.PhthalEDA, 6.60 g, 29.12 mmol) in DCM and in the presence of DIPEA (5.07 mL, 29.12 mmol), a beige foam of molecule 56 is obtained without purification.

[1263] Yield: 25.56 g (100%)

[1264] .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.17-1.30 (20H); 1.34-1.41 (27H); 1.41-1.52 (2H); 1.56-2.32 (18H); 3.18-3.69 (6H); 4.01-4.43 (4H); 7.64-8.30 (8H).

[1265] LC/MS (ESI): 1053.8; (calculated ([M+H].sup.+): 1053.6).

Molecule A26

[1266] Molecule 56 (25.56 g, 24.26 mmol) is solubilized in a solution of 40% methylamine in MeOH (242.5 mL, 2.38 mol) at 4 C. then the mixture is stirred at ambient temperature for 5 h. Silica is added to the reaction medium then the latter is concentrated at reduced pressure. The residue is purified by silica gel chromatography (solid deposition, dichloromethane, methanol, NH.sub.3) to produce molecule A26 in the form a pale yellow gum. This product is solubilized in DCM (250 mL) then the solution is washed with a 10% HCl aqueous solution. The aqueous phase is extracted with DCM (100 mL). The combined organic phases are dried on Na.sub.2SO.sub.4, filtered then concentrated at reduced pressure to produce the hydrochloride of molecule A26 in the form of a white solid.

[1267] Yield: 13.5 g (58%)

[1268] .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.18-1.30 (20H); 1.34-1.42 (27H); 1.42-1.53 (2H); 1.66-2.02 (9H); 2.02-2.39 (9H); 2.79-2.91 (2H); 3.25-3.64 (4H); 4.08-4.46 (4H); 7.68-8.37 (7H).

[1269] LC/MS (ESI): 923.8; (calculated ([M+H].sup.+): 923.6).

EXAMPLE A27

Molecule A27

[1270] Molecule A27 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl chloride (CTC) resin (24.00 g, 1.37 mmol/g).

[1271] The grafting of the first amino acid Fmoc-6-aminohexanoic acid (1.5 equivalents) is performed in DCM (10 V), in the presence of DIPEA (2.5 equivalents). The unreacted sites are capped with methanol (0.8 mL/g resin) at the end of the reaction.

[1272] The protected amino acid Fmoc-Glu-OMe (1.5 equivalents) and palmitic acid (1.5 equivalents) are coupled in DMF (10 V), in the presence of HATU (1.0 equivalent with respect to the acid) and DIPEA (1.5 equivalents with respect to the acid).

[1273] The protecting groups Fmoc are removed using an 80:20 DMF/piperidine solution (10 V).

[1274] The product is cleaved from the resin using an 80:20 DCM/HFIP solution (10 V).

[1275] After concentration at reduced pressure, two co-evaporations are performed on the residue with dichloromethane followed by toluene. The product is purified by recrystallization in ethyl acetate. A white solid of molecule A27 is obtained.

[1276] Yield: 11.54 g (68% in 6 stages)

[1277] .sup.1H NMR CDCl.sub.3, ppm): 0.88 (3H); 1.19-1.35 (24H); 1.35-1.44 (2H); 1.50-1.70 (6H); 1.91-2.01 (1H); 2.14-2.40 (7H); 3.14-3.34 (2H); 3.75 (3H); 4.51-4.59 (1H); 6.53 (1H); 6.70 (1H).

[1278] LC/MS (ESI+): 513.4 (calculated ([M+H].sup.+): 513.4).

Part BHydrophobic Co-Polyamino Acid Synthesis

[1279]

TABLE-US-00003 CO-POLYAMINO ACIDS BEARING CARBOXYLATE CHARGES AND # HYDROPHOBIC RADICALS B1 [00087] B2 [00088] B3 [00089] B4 [00090] B5 [00091] B6 [00092] B9 [00093] [00094] B13 [00095] [00096] B14 [00097] [00098] B15 [00099] B16 [00100] [00101] B17 [00102] B18 [00103] B19 [00104] B20 [00105] B21 [00106] B23 [00107] B24 [00108] B25 [00109] B26 [00110] B27 [00111] B28 [00112] B29 [00113] B30 [00114] B31 [00115]

EXAMPLE B1

Co-Polyamino Acid B1Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A1 and having a Number Average Molar Mass (Mn) of 3600 g/mol

[1280] Co-polyamino acid B1-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine and modified at the extremities thereof by molecule A1.

[1281] In a previously oven-dried flask, -benzyl-L-glutamate N-carboxyanhydride (34.74 g, 132 mmol) is solubilized in anhydrous DMF (78 mL). The mixture is then stirred until complete dissolution, cooled to 0 C., then ethylene diamine (0.205 g, 3.41 mmol) is introduced rapidly and the medium is stirred at 0 C.

[1282] In parallel, molecule A1 (2.26 g, 6.94 mmol) is solubilized in DMF (44 mL), then NHS (0.82 g, 7.12 mmol) and DCC (1.47 g, 7.12 mmol) are added successively. After stirring overnight at ambient temperature, the heterogeneous mixture is filtered on a sintered filter. The filtrate is then added to the polymer solution kept at 0 C. After 24 h, the solution is placed at ambient temperature. After 6 h of stirring, the reaction medium is poured onto diisopropylether (IPE, 1.8 L). The precipitate is filtered on a sintered filter, washed with IPE (330 mL) and dried at 30 C. at reduced pressure.

Co-Polyamino Acid B1

[1283] Co-polyamino acid B1-1 is diluted in trifluoroacetic acid (TFA, 132 mL), then the solution is cooled to 4 C. A 33% HBr solution in acetic acid (92.5 mL, 0.528 mol) is then added dropwise. The mixture is stirred at ambient temperature for 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropylether and water under stirring (0.8 L). After 2 h of stirring, the heterogeneous mixture is left to stand overnight. The white precipitate is retrieved by filtration, washed with IPE (266 mL) then with water (266 mL). The solid obtained is then solubilized in water (690 mL) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by adding water (310 mL), the solution is filtered on a 0.45 m filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1284] Dry extract: 24.3 mg/g

[1285] DP (estimated as per .sup.1H NMR): 40

[1286] As per .sup.1H NMR: i=0.050

[1287] The calculated average molar mass of co-polyamino acid B1 is 6719 g/mol.

[1288] HPLC-Organic SEC (PEG calibrator): Mn=3600 g/mol.

EXAMPLE B2

Co-Polyamino Acid B2Sodium Poly-L-Glutamate Modified at the Extremities thereof by Stearic Acid and having a Number Average Molar Mass (Mn) of 3400 g/mol

[1289] Co-polyamino acid B2-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by hexamethylenediamine.

[1290] In a previously oven-dried flask, -benzyl-L-glutamate N-carboxyanhydride (30.0 g, 114 mmol) is solubilized in anhydrous DMF (67 mL). The mixture is then stirred until complete dissolution, cooled to 0 C., then hexamethylenediamine (0.442 g, 3.8 mmol) is introduced rapidly. After 23 h of stirring at 0 C., a 4 M HCl solution in dioxane (4.7 mL, 18.8 mmol) is added then the reaction medium is poured in 5 min onto a mixture of methanol (94 mL) and IPE (375 mL). The precipitate is filtered on a sintered filter, washed with IPE (270 mL) and dried at 30 C. at reduced pressure.

[1291] Co-polyamino acid B2-2: poly-L-benzylglutamate modified at the extremities thereof by stearic acid.

[1292] To a solution of stearic acid (0.851 g, 2.99 mmol) in DMF (20 mL) at 0 C. are added successively HATU (1.484 g, 3.89 mmol) and DIPEA (1.166 g, 9.02 mmol). The solution is then introduced onto a solution of co-polyamino acid B2-1 (10.0 g) and triethylamine (TEA, 0.309 g, 3.04 mmol) in DMF (110 mL) at 0 C., and the medium is stirred for 18 h from 0 C. to ambient temperature. Dichloromethane (390 mL) is added, the organic phase is washed with 0.1 N HCl aqueous solution (3190 mL), an aqueous solution saturated with NaHCO.sub.3 (2190 mL), an aqueous solution saturated with NaCl (2190 mL) followed by water (190 mL). The medium is then poured onto IPE (1.4 L). The precipitate is filtered on a sintered filter, washed with IPE (2100 mL) and dried at 30 C. at reduced pressure.

Co-Polyamino Acid B2

[1293] By means of a similar method to that used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B2-2 (8.80 g, 36.5 mmol), a sodium poly-L-glutamate modified at the extremities thereof with stearic acid is obtained.

[1294] Dry extract: 17.9 mg/g

[1295] DP (estimated as per .sup.1H NMR): 30

[1296] As per .sup.1H NMR: i=0.0657

[1297] The calculated average molar mass of co-polyamino acid B2 is 5174 g/mol.

[1298] HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol.

EXAMPLE B3

Co-Polyamino Acid B3Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A2 and having a Number Average Molar Mass (Mn) of 3000 g/mol

[1299] Co-polyamino acid B3-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1300] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.765 g, 12.73 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (80.0 g, 304 mmol), co-polyamino acid B3-1 is obtained.

[1301] Co-polyamino acid B3-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A2.

[1302] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B3-1 (30.0 g, 5.56 mmol) and to molecule A2 (7.94 g, 12.24 mmol), a poly-L-benzylglutamate modified at the extremities thereof with molecule A2 is obtained.

Co-Polyamino Acid B3

[1303] To a solution of co-polyamino acid B3-2 (36.6 g, 133.5 mmol) in N,N-dimethylacetamide (DMAc, 146 mL) is added 5% palladium on alumina (7.3 g), then the solution is placed at 60 C. at 10 bar hydrogen. After leaving overnight, the reaction medium is filtered on a sintered filter then on a 0.2 m PTFE filter. The filtrate is then placed under stirring before adding water (1.4 L) previously acidified to pH 2 with a 1 N HCl solution (14 mL) dropwise. After placing overnight, the precipitate is filtered on a sintered filter, washed with water (4110 mL) and dried at 30 C. at reduced pressure.

[1304] The solid obtained is then solubilized in water (1.09 L) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide (121 mL). After solubilization, the solution is basified by adding 1 N sodium hydroxide (26 mL) up to a pH of 12. After 2 h, the solution is neutralized by adding 1 N HCl solution (28 mL). The theoretical concentration is adjusted to 12 g/L theoretical by adding water (650 mL) and ethanol (1040 mL) then the solution is filtered on an R53SLP carbon filter (3M) at a rate of 12 mL/min, then on a 0.2 m PES filter. The solution is then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1305] Dry extract: 21.6 mg/g

[1306] DP (estimated as per .sup.1H NMR): 24

[1307] As per .sup.1H NMR: i=0.0808

[1308] The calculated average molar mass of co-polyamino acid B3 is 4948 g/mol.

[1309] HPLC-Organic SEC (PEG calibrator): Mn=3000 g/mol.

EXAMPLE B4

Co-Polyamino Acid B4Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A3 and having a Number Average Molar Mass (Mn) of 2500 g/mol

[1310] Co-polyamino acid B4-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1311] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (1.644 g, 27.35 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B4-1 is obtained.

[1312] Co-polyamino acid B4-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A3.

[1313] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B4-1 (10.0 g, 3.12 mmol) and to molecule A3 (4.412 g, 6.26 mmol), a poly-L-benzylglutamate modified at the extremities thereof with molecule A3 is obtained.

Co-Polyamino Acid B4

[1314] By means of a similar method to that used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B4-2 (12.0 g, 37.3 mmol), a sodium poly-L-glutamate modified at the extremities thereof with molecule A3 is obtained.

[1315] Dry extract: 21.7 mg/g

[1316] DP (estimated as per .sup.1H NMR): 14

[1317] As per .sup.1H NMR: i=0.134

[1318] The calculated average molar mass of co-polyamino acid B4 is 3464 g/mol.

[1319] HPLC-Organic SEC (PEG calibrator): Mn=2500 g/mol.

EXAMPLE B5

Co-Polyamino Acid B5Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A3 and having a Number Average Molar Mass (Mn) of 2800 g/mol

[1320] Co-polyamino acid B5-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1321] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.95 g, 15.83 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B5-1 is obtained.

[1322] Co-polyamino acid B5-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A3.

[1323] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B5-1 (20.0 g, 3.71 mmol) and to molecule A3 (5.233 g, 7.42 mmol), a poly-L-benzylglutamate modified at the extremities thereof with molecule A3 is obtained.

Co-Polyamino Acid B5

[1324] By means of a similar method to that used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B5-2 (15.6 g, 55.93 mmol), a sodium poly-L-glutamate modified at the extremities thereof with molecule A3 is obtained.

[1325] Dry extract: 27.4 mg/g

[1326] DP (estimated as per .sup.1H NMR): 24

[1327] As per .sup.1H NMR: i=0.077

[1328] The calculated average molar mass of co-polyamino acid B5 is 4956 g/mol.

[1329] HPLC-Organic SEC (PEG calibrator): Mn=2800 g/mol.

EXAMPLE B6

Co-Polyamino Acid B6: Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A4 and having a Number Average Molar Mass (Mn) of 2900 g mol

[1330] Co-polyamino acid B6-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1331] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.951 g, 15.83 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B6-1 is obtained.

[1332] Co-polyamino acid B6-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A4.

[1333] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B6-1 (20.0 g, 3.71 mmol) and to molecule A4 (6.649 g, 8.74 mmol), a poly-L-benzylglutamate modified at the extremities thereof with molecule A4 is obtained.

Co-Polyamino Acid B6

[1334] By means of a similar method to that used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B6-2 (19.7 g, 69.47 mmol), a sodium poly-L-glutamate modified at the extremities thereof with molecule A4 is obtained.

[1335] Dry extract: 28.7 mg/g

[1336] DP (estimated as per .sup.1H NMR): 24

[1337] As per .sup.1H NMR: i=0.0812

[1338] The calculated average molar mass of co-polyamino acid B6 is 5135 g/mol.

[1339] HPLC-Organic SEC (PEG calibrator): Mn=2900 g/mol.

EXAMPLE B9

Co-Polyamino Acid B9Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A7 wherein the Side Chains are Deprotected and having a Number Average Molar Mass (Mn) of 3200 g/mol

[1340] Co-polyamino acid B9-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1341] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.96 g, 15.94 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B9-1 is obtained.

[1342] Co-polyamino acid B9-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A7.

[1343] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B9-1 (25.0 g, 4.64 mmol) and to molecule A7 (10.49 g, 9.27 mmol), a poly-L-benzylglutamate modified at the extremities thereof with molecule A7 is obtained.

[1344] Co-polyamino acid B9-3: poly-L-benzylglutamate modified at the extremities thereof by molecule A7 wherein the side chains are deprotected.

[1345] Co-polyamino acid B9-2 (18.6 g) is solubilized in TFA (100 mL). After 2 h under stirring, the reaction medium is concentrated at reduced pressure.

Co-Polyamino Acid B9

[1346] By means of a similar method to that used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B9-3 (18.0 g, 59.0 mmol), a sodium poly-L-glutamate modified at the extremities thereof with molecule A7 is obtained.

[1347] Dry extract: 21.8 mg/g

[1348] DP (estimated as per .sup.1H NMR): 24

[1349] As per .sup.1H NMR: i=0.0833

[1350] The calculated average molar mass of co-polyamino acid B9 is 5776 g/mol.

[1351] HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol.

EXAMPLE B13

Co-Polyamino Acid B13Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A11 wherein the esters are deprotected and having a number average molar mass (Mn) of 3200 g/mol

[1352] Co-polyamino acid B13-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1353] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (4.76 g, 15.94 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1900 mmol), co-polyamino acid B13-1 is obtained.

[1354] Co-polyamino acid B13-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A11.

[1355] To a solution of co-polyamino acid B13-1 (12.0 g) in DMF (40 mL) at 0 C. are successively added a solution of molecule A11 (5.88 g, 6.67 mmol) in DMF (20 mL), N-2-hydroxypyridine oxide (HOPO, 0.82 g, 7.34 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) (1.66 g, 8.68 mmol), followed by DIPEA (0.97 mL, 5.56 mmol). The reaction medium is stirred at 0 C. for 16 h and at 20 C. for 2 h. Dichloromethane (150 mL) is added and the organic phase is washed with a 0.1 N HCl aqueous solution (675 mL), dried on Na.sub.2SO.sub.4 then filtered. The organic phase is then poured onto IPE (600 mL), then left to stand for 18 h. The white precipitate is retrieved by filtration, washed with IPE (2150 mL) then dried at reduced pressure at 30 C.

[1356] Co-polyamino acid B13-3: poly-L-benzylglutamate modified at the extremities thereof by molecule A11 wherein the esters are deprotected

[1357] Co-polyamino acid B13-2 is solubilized in TFA (60 mL), and the solution is stirred for 2 h at ambient temperature then is poured dropwise onto diisopropylether under stirring (600 mL). After 18 h, the white precipitate is retrieved by filtration, triturated with IPE and dried at reduced pressure.

Co-Polyamino Acid B13

[1358] By means of a similar method to that used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B13-3 (14.5 g), a sodium poly-L-glutamate modified at the extremities thereof with molecule A11 wherein the esters are deprotected is obtained.

[1359] Dry extract: 18.0 mg/g

[1360] DP (estimated as per .sup.1H NMR): 24

[1361] As per .sup.1H NMR: i=0.079

[1362] The calculated average molar mass of co-polyamino acid B13 is 5194 g/mol.

[1363] HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

EXAMPLE B14

Co-Polyamino Acid B14Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A12 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3700 g/mol

[1364] Co-polyamino acid B14-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1365] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (4.76 g, 15.94 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1900 mmol), co-polyamino acid B14-1 is obtained.

[1366] Co-polyamino acid B14-2: poly-L-benzylglutamate modified at the extremities thereof by molecule A12

[1367] By means of a similar method to that used for the preparation of co-polyamino acid B2-2 applied to molecule A12 (2.67 g, 1.43 mmol) and to co-polyamino acid B14-1 (3.5 g), a poly-L-benzylglutamate modified at the two extremities thereof with molecule A2 is obtained.

[1368] Co-polyamino acid B14-3: poly-L-benzylglutamate modified at the extremities thereof by molecule A12 wherein the esters are deprotected

[1369] By means of a similar method to that used for the preparation of co-polyamino acid B13-3 applied to co-polyamino acid B14-2, a poly-L-benzylglutamate modified at the two extremities thereof with molecule A12 wherein the esters are deprotected is obtained.

Co-Polyamino Acid B14

[1370] By means of a similar method to that used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B14-3 (1.97 g), in a hydrogen atmosphere (1 atm, 48 h, 65 C.), a sodium poly-L-glutamate modified at the two extremities thereof with molecule A12 wherein the esters are deprotected is obtained.

[1371] Dry extract: 13.2 mg/g

[1372] DP (estimated as per .sup.1H NMR): 24 0

[1373] As per .sup.1H NMR: i=0.072

[1374] The calculated average molar mass of co-polyamino acid B14 is 6537 g/mol.

[1375] HPLC-Organic SEC (PEG calibrator): Mn=3700 g/mol

EXAMPLE B15

Co-Polyamino Acid B15Butyltetracarboxylic Acid Substituted with Molecule A13 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2700 g/mol

[1376] ##STR00116##

[1377] Molecule 31: Product obtained by polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine.

[1378] A solution of BocEDA (12.00 g, 74.9 mmol) in DMF (12 mL) is prepared. In a reaction vessel, -benzyl-L-glutamate N-carboxyanhydride (78.87 g, 300.0 mmol) is solubilized in DMF (165 mL) at 25 C. The mixture is then stirred until complete dissolution, cooled to 10 C., then the BocEDA solution is introduced rapidly. The reaction medium is stirred at 0 C. for 4 h then a solution of HCl in 1,4-dioxane (3.33 M, 19.8 mL, 65.34 mmol) is added. The reaction medium is stirred at ambient temperature then the solution is poured onto a solution of MeOH/IPE (245 mL/990 mL) cooled by an ice bath. After 62 h of stirring at ambient temperature, the white precipitate is filtered on a sintered filter, washed with IPE (2160 mL) and dried at 30 C. at reduced pressure.

[1379] .sup.1H NMR (DMSO-d6, ppm): 1.35 (9H); 1.70-2.10 (10H); 2.26-2.65 (10H); 2.85-3.18 (4H); 3.85 (1H); 4.14-4.42 (4H); 4.87-5.24 (10H); 6.34-6.86 (1H); 7.11-7.56 (25H); 7.90-8.44 (7H); 8.69 (1H).

[1380] DP (estimated as per .sup.1H NMR): 5.0

[1381] The calculated average molar mass of molecule 31 in hydrochloride salt form is 1292.9 g/mol.

[1382] Molecule 32: Product obtained by coupling molecule 31 and molecule A1.

[1383] Molecule 31 (10.0 g, 7.73 mmol) is solubilized in a mixture of DCM (90 mL) and DIPEA (1.585 g, 9.32 mmol) at 0 C. To this solution are added successively HOPO (1.242 g, 11.18 mmol), molecule A1 (3.335 g, 10.25 mmol) and EDC (2.141 g, 11.17 mmol). After placing under stirring overnight, the reaction medium is washed twice with a 0.1 N HCl solution (2100 mL), twice with a 5% Na.sub.2CO.sub.3 aqueous solution (2100 mL) followed by a saturated NaCl solution (100 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. The residue is solubilized in DCM (30 mL) and the solution is poured onto isopropyl alcohol (600 mL) under stirring at 0 C. The precipitate formed is retrieved by vacuum filtration then vacuum-dried at 30 C.

[1384] Yield: 7.58 g (62%)

[1385] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.06-2.76 (58.6H); 3.06-4.45 (12.4H); 4.88-5.25 (10.8H); 5.72-8.40 (34.4H).

[1386] DP (estimated as per .sup.1H NMR): 5.4

[1387] The calculated average molar mass of molecule 32 in hydrochloride salt form is 1651.6 g/mol.

Molecule A13

[1388] After solubilizing molecule 32 (5.93 g, 3.59 mmol) in DCM (40 mL), the solution is cooled to 0 C. and TFA (40 mL) is added. The reaction medium is stirred at 0 C. for 3 h then is dry concentrated at reduced pressure at ambient temperature. The residue is taken up with DCM (120 mL) and washed with an aqueous carbonate buffer solution at pH 10.4 (3240 mL) then by a 0.1 N HCl aqueous solution (2240 mL). The organic solution is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. A white solid of molecule A13 in hydrochloride salt form is obtained.

[1389] Yield: 5.17 g (91%)

[1390] .sup.1H NMR (TFA-d, ppm): 0.87 (3H); 1.06-1.46 (20H); 1.46-1.68 (2H); 1.68-2.81 (28H); 3.13-4.59 (12.5H); 4.83-5.25 (11H); 7.02-9.13 (37H)

[1391] DP (estimated as per .sup.1H NMR): 5.5

[1392] The calculated average molar mass of molecule A13 in hydrochloride salt form is 1609.8 g/mol.

[1393] Co-polyamino acid B15-1: Molecule A13 (3.47 g, 2.16 mmol) is solubilized in DCM (17 mL) then is added successively at 0 C. butyltetracarboxylic acid (BTCA, 115 mg, 0.49 mmol), HOPO (275 mg, 2.48 mmol), DIPEA (377 L, 2.16 mmol) followed by EDC (473 mg, 2.47 mmol). After placing under stirring overnight at 0 C., the reaction medium is poured onto MeOH (220 mL) under stirring at 0 C. After leaving overnight, the white precipitate is retrieved by vacuum filtration, triturated with cold MeOH then vacuum-dried at 30 C.

Co-Polyamino Acid B15

[1394] A solution of co-polyamino acid B15-1 (2.33 g, 0.362 mmol) in DMAc (33 mL) is placed in a hydrogen atmosphere (1 atm) in the presence of 5% palladium on alumina (465 mg) then the solution is heated to 60 C. After leaving overnight, the solution is cooled, filtered on celite then the filtrate is poured onto a 15% NaCl solution at pH 2 (500 mL). After leaving overnight, the precipitate is filtered on a sintered filter then washed twice with a 15% NaCl solution (28 mL). The solid obtained is then solubilized in water (70 mL) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide. After solubilization, the solution is filtered on a 0.45 m filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1395] Dry extract: 25.8 mg/g

[1396] .sup.1H NMR (D.sub.2O, ppm): 0.90 (10.2H); 1.18-1.46 (68H); 1.53-1.9 (6.8H); 1.86-3.04 (101.2H); 3.17-3.80 (20.4H); 4.19-4.68 (22.1H)

[1397] DP (estimated as per .sup.1H NMR): 5.5

[1398] As per .sup.1H NMR: i=3.4

[1399] The calculated average molar mass of co-polyamino acid B15 is 4261.3 g/mol.

[1400] HPLC-Organic SEC (PEG calibrator): Mn=2700 g/mol.

EXAMPLE B16

Co-Polyamino Acid B16Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A14 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3200 g/mol

[1401] Co-polyamino acid B16-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by 1-amino-4,7,10-trioxa-13-tridecane amine (TOTA).

[1402] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to TOTA (13.96 g, 63.37 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (400.0 g, 1519 mmol), co-polyamino acid B16-1 is obtained.

Co-Polyamino Acid B16

[1403] To a solution of molecule A14 (6.74 g, 13.5 mmol) in DMAc (38 mL) are successively added HOPO (1.65 g, 14.8 mmol), and EDC (3.36 g, 17.6 mmol).

[1404] To a solution of co-polyamino acid B16-1 (30.0 g) in DMAc (113 mL) at ambient temperature are successively added DIPEA (1.90 mL, 13.5 mmol) followed by the solution of molecule A14 previously prepared.

[1405] After 24 h of stirring at ambient temperature, DMAc (82 mL) is added and the solution is placed at 60 C. under 10 bar hydrogen in the presence of 5% palladium on alumina (7.0 g). After 17 h of reaction, the reaction medium is filtered on a sintered filter then on a 0.2 m PTFE filter.

[1406] The filtrate is placed under stirring, then a 300 g/L sodium carbonate solution (46 mL) followed by acetone (275 mL) are then added successively dropwise. After 3 h, the precipitate is filtered on a sintered filter, washed with acetone (370 mL) and dried at reduced pressure.

[1407] After solubilizing the solid obtained in water (1.3 L) then diluting with ethanol (0.7 L), the solution is basified by adding 10 N sodium hydroxide (13 mL) until a pH of 13 is obtained. After 3 h of stirring at ambient temperature, the solution is neutralized by adding 1 N HCl solution (190 mL) then the solution is filtered on an R53SLP carbon filter (3M), then on a 0.2 m PES filter. The solution is then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1408] Dry extract: 21.4 mg/g

[1409] DP (estimated as per .sup.1H NMR): 24

[1410] As per .sup.1H NMR: i=0.078

[1411] The calculated average molar mass of co-polyamino acid B16 is 4761 g/mol.

[1412] HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol.

EXAMPLE B17

Co-Polyaminoacid B17Sodium Poly-L-Glutamate Modified at the Two Extremities thereof by Molecule A15 and having a Number Average Molar Mass (Mn) of 3200 g/mol

[1413] Co-polyamino acid B17-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1414] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylenediamine (4.77 g, 79.37 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1899 mmol), co-polyamino acid B17-1 is obtained.

Co-Polyamino Acid B17

[1415] By means of a similar method to that used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B17-1 (15.0 g) and to molecule A 15 (3.45 g) with a saponification step at pH 12 for 50 min, co-polyamino acid B17 is obtained.

[1416] Dry extract: 20.3 mg/g

[1417] DP (estimated as per .sup.1H NMR): 24

[1418] As per .sup.1H NMR: i=0.048

[1419] The calculated average molar mass of co-polyamino acid B17 is 4237 g/mol.

[1420] HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

EXAMPLE B18

Co-Polyaminoacid B18Sodium Poly-L-Glutamate Modified at the Two Extremities thereof by Molecule A16 and having a Number Average Molar Mass (Mn) of 3150 g/mol

[1421] Co-polyamino acid B18-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

[1422] By means of a similar method to that used for the preparation of co-polyamino acid B2-1 applied to ethylenediamine (4.74 g, 78.89 mmol) and to -benzyl-L-glutamate N-carboxyanhydride (498.4 g, 1893 mmol), co-polyamino acid B18-1 is obtained.

Co-Polyamino Acid B18

[1423] By means of a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B18-1 (14.0 g) and to molecule A 16 (4.26 g), co-polyamino acid B18 is obtained.

[1424] Dry extract: 9.7 mg/g

[1425] DP (estimated as per .sup.1H NMR): 24

[1426] As per .sup.1H NMR: i=0.075

[1427] The calculated average molar mass of co-polyamino acid B18 is 4839 g/mol.

[1428] HPLC-Organic SEC (PEG calibrator): Mn=3150 g/mol

EXAMPLE B19

Co-Polyamino Acid B19Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A17 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3400 g/mol

[1429] By means of a similar method to that used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B18-1 (20.39 g) and to molecule A17 (7.553 g), co-polyamino acid B19 is obtained.

[1430] Dry extract: 18.6 mg/g

[1431] DP (estimated as per .sup.1H NMR): 24

[1432] As per .sup.1H NMR: i=0.066

[1433] The calculated average molar mass of co-polyamino acid B19 is 4936 g/mol.

[1434] HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol

EXAMPLE B20

Co-Polyaminoacid B20Sodium Poly-L-Glutamate Modified at the Two Extremities thereof by Molecule A18 and having a Number Average Molar Mass (Mn) of 3200 g/mol

[1435] By means of a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (12.45 g) and to molecule A 18 (3.56 g), co-polyamino acid B20 is obtained.

[1436] Dry extract: 16.8 mg/g

[1437] DP (estimated as per .sup.1H NMR): 24

[1438] As per .sup.1H NMR: i=0.075

[1439] The calculated average molar mass of co-polyamino acid B20 is 4784 g/mol. HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

EXAMPLE B21

Co-Polyaminoacid B21Sodium Poly-L-Glutamate Modified at the Two Extremities thereof by Molecule A19 and having a Number Average Molar Mass (Mn) of 3600 g/mol

[1440] By means of a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (12.16 g) and to molecule A 19 (4.16 g), co-polyamino acid B21 is obtained.

[1441] Dry extract: 26.4 mg/g

[1442] DP (estimated as per .sup.1H NMR): 24

[1443] As per .sup.1H NMR: i=0.077

[1444] The calculated average molar mass of co-polyamino acid B21 is 5023 g/mol.

[1445] HPLC-Organic SEC (PEG calibrator): Mn=3600 g/mol

EXAMPLE B23

Co-Polyaminoacid B23Sodium Poly-L-Glutamate Modified at the two Extremities thereof by Molecule A21 and having a Number Average Molar Mass (Mn) of 3350 g/mol

[1446] By means of a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (18.68 g) and to molecule A21 (7.03 g), co-polyamino acid B23 is obtained.

[1447] Dry extract: 23.2 mg/g

[1448] DP (estimated as per .sup.1H NMR): 24

[1449] As per .sup.1H NMR: i=0.080

[1450] The calculated average molar mass of co-polyamino acid B23 is 5140 g/mol.

[1451] HPLC-Organic SEC (PEG calibrator): Mn=3350 g/mol

EXAMPLE B24

Co-Polyaminoacid B24Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A1 and having a Number Average Molar Mass (Mn) of 2300 g/mol

[1452] Co-polyamino acid B24-1: poly-L-benzylglutamate obtained from the polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by molecule 4 and modified at the extremities thereof by molecule A1.

[1453] To a suspension of molecule 4 (9.92 mmol) in anhydrous DMF (80 mL) cooled to 0 C. is rapidly added a solution of -benzyl-L-glutamate N-carboxyanhydride (26.11 g, 99.2 mmol) in anhydrous DMF (20 mL) at 0 C. After 24 h of stirring at 0 C., a freshly prepared solution of molecule A1 (16.1 g, 49.6 mmol), HATU (18.9 g, 49.6 mmol) and DIPEA (8.64 mL, 49.6 mmol) in DMF (80 mL) is added to the medium and the mixture is stirred from 0 C. to 25 C. for 3.5 h. The resin is filtered, washed successively with DMF (3100 mL), isopropanol (1100 mL) and DCM (3100 mL). The resin obtained is then treated with an 80:20 DCM/HFIP mixture (120 mL). After 30 min of stirring at ambient temperature, the resin is filtered and washed successively with DCM (3100 mL). The solvents are evaporated at reduced pressure to produce co-polyamino acid B24-1

Co-Polyamino Acid B24

[1454] By means of a similar method to that used for the hydrogenation step of co-polyamino acid B16 applied to co-polyamino acid B24-1 (27.4 g), with a saponification step at pH 12 for 50 min but without the carbofiltration step, co-polyamino acid B24 is obtained.

[1455] Dry extract: 14.1 mg/g

[1456] DP (estimated as per .sup.1H NMR): 14

[1457] As per .sup.1H NMR: i=0.143

[1458] The calculated average molar mass of co-polyamino acid B24 is 2899 g/mol.

[1459] HPLC-Organic SEC (PEG calibrator): Mn=2300 g/mol.

EXAMPLE B25

Co-Polyaminoacid B25Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A22 and having a Number Average Molar Mass (Mn) of 3050 g/mol

[1460] By means of a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B18-1 (30.0 g) and molecule A22 (8.56 g) using a four-fold greater quantity of 300 g/L sodium carbonate solution to precipitate the polymer after the hydrogenolysis step, co-polyamino acid B25 is obtained.

[1461] Dry extract: 23.7 mg/g

[1462] DP (estimated as per .sup.1H NMR): 24

[1463] As per .sup.1H NMR: i=0.074

[1464] The calculated average molar mass of co-polyamino acid B25 is 4743 g/mol.

[1465] HPLC-Organic SEC (PEG calibrator): Mn=3050 g/mol

EXAMPLE B26

Co-Polyaminoacid B26Sodium Poly-L-Glutamate Modified at the Two Extremities thereof by Molecule A23 and having a Number Average Molar Mass (Mn) of 3400 g/mol

[1466] By means of a similar method to that used for the preparation of co-polyamino acid B25 applied to co-polyamino acid B17-1 (25.78 g) and to molecule A23 (8.27 g), co-polyamino acid B21 is obtained.

[1467] Dry extract: 11.8 mg/g

[1468] DP (estimated as per .sup.1H NMR): 24

[1469] As per .sup.1H NMR: i=0.073

[1470] The calculated average molar mass of co-polyamino acid B21 is 4902 g/mol.

[1471] HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol

EXAMPLE B27

Co-Polyamino Acid B27Butyltetracarboxylic Acid Substituted with Molecule A24 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2500 g/mol

[1472] ##STR00117##

[1473] Molecule 53: Product obtained by polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine then capped with molecule 2.

[1474] A solution of BocEDA (12.00 g, 74.9 mmol) in DMF (12 mL) is prepared. In a reaction vessel, -benzyl-L-glutamate N-carboxyanhydride (78.87 g, 300.0 mmol) is solubilized in DMF (165 mL) at 25 C. The mixture is then stirred until complete dissolution, cooled to 10 C., then the BocEDA solution is introduced rapidly. The reaction medium is stirred at 0 C. for 3 h then are introduced successively DMF (100 mL), molecule 2 (26.73 g, 89.88 mmol), HOPO (9.99 g, 89.88 mmol) and EDC (17.23 g, 89.88 mmol). The reaction mixture is stirred at 0 C. for 1 h, from 0 C. to 20 C. for 2 h then at 20 C. for 16 h. It is then poured onto a 1:1 2-propanol/H.sub.2O solution (10 V) under stirring. After 3 h, the white precipitate is filtered on a sintered filter, washed with a 1:1 2-propanol/H.sub.2O mixture (2360 mL) and dried at 30 C. at reduced pressure.

[1475] Yield: 70 g (71%)

[1476] .sup.1H NMR (TFA-d, ppm): 0.99 (3H); 1.34-1.59 (16H); 1.68-2.85 (36H); 3.52-3.62 (2H); 3.79-3.99 (4H); 4.70-4.92 (5.75H); 5.20-5.38 (9.5H); 7.36-7.52 (23.75H).

[1477] DP (estimated as per .sup.1H NMR): 4.75

[1478] The calculated average molar mass of molecule 53 is 1481.0 g/mol.

Molecule A24

[1479] By means of a similar method to that used for the preparation of molecule A13 applied to molecule 53 (34.00 g, 22.96 mmol), a white solid of molecule A24 in hydrochloride salt form is obtained.

[1480] Yield: 29.40 g (90%)

[1481] .sup.1H NMR (TFA-d, ppm): 1.00 (3H); 1.35-1.61 (16H); 1.79-1.93 (2H); 2.05-2.90 (25H); 3.53-3.65 (2H); 3.79-4.02 (4H); 4.74-4.94 (5.75H); 5.20-5.43 (9.5H); 7.32-7.58 (23.75H).

[1482] DP (estimated as per .sup.1H NMR): 4.75

[1483] The calculated average molar mass of molecule A13 in hydrochloride salt form is 1417.2 g/mol.

Co-Polyamino Acid B27-1:

[1484] By means of a similar method to that used for the preparation of co-polyamino acid B15-1 applied to molecule A24 (11.9 g, 8.40 mmol) and to BTCA (0.41 g, 1.75 mmol) in solution in DMF, a white solid is obtained after drying at 30 C. at reduced pressure.

Co-Plyamino Acid B27

[1485] By means of a similar method to that used for the preparation of co-polyamino acid B15 applied to co-polyamino acid B27-1 (9.31 g, 1.64 mmol), under hydrogen pressure (6 bar) and with a saponification step at pH 12 for 1 h prior to the ultrafiltration step, co-polyamino acid B27 is obtained.

[1486] Dry extract: 19.9 mg/g

[1487] DP (estimated as per .sup.1H NMR): 4.75

[1488] As per .sup.1H NMR: i=3.7

[1489] The calculated average molar mass of co-polyamino acid B27 is 4085.8 g/mol.

[1490] HPLC-Organic SEC (PEG calibrator): Mn=2500 g/mol.

EXAMPLE B28

Co-Polyamino Acid B28Tricarballylic Acid Substituted with Molecule A25 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2200 g/mol

[1491] ##STR00118##

[1492] Molecule 54: Product obtained by polymerization of -benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine then capped with molecule A1.

[1493] By means of a similar method to that used for the preparation of molecule 53 applied to BocEDA (6.00 g, 37.45 mmol), to -benzyl-L-glutamate N-carboxyanhydride (39.44 g, 150.00 mmol) and to molecule A1 (14.63 g, 44.94 mmol), a white solid of molecule 54 is obtained.

[1494] Yield: 23.71 g (40%)

[1495] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.12-2.76 (57.6H); 3.06-4.50 (12.15H); 4.90-5.25 (10.3H); 5.91-8.49 (32.9H).

[1496] DP (estimated as per .sup.1H NMR): 5.15

[1497] The calculated average molar mass of molecule 54 is 1596.8 g/mol.

Molecule A25

[1498] By means of a similar method to that used for the preparation of molecule A13 applied to molecule 54 (23.29 g, 14.59 mmol), a translucent solid of molecule A25 in hydrochloride salt form is obtained.

[1499] Yield: 19.08 g (85%)

[1500] .sup.1H NMR CDCl.sub.3, ppm): 0.87 (3H); 1.17-1.32 (20H); 1.48-1.63 (2H); 1.69-2.78 (29.6H); 3.15-4.40 (12.15H); 4.89-5.18 (10.3H); 7.06-9.13 (31.9H).

[1501] DP (estimated as per .sup.1H NMR): 5.15

[1502] The calculated average molar mass of molecule A25 in hydrochloride salt form is 1533.1 g/mol.

Co-Polyamino Acid B28-1:

[1503] By means of a similar method to that used for the preparation of co-polyamino acid B15-1 applied to molecule A25 (3.93 g, 2.56 mmol) and to tricarballylic acid (TCA, 125.2 mg, 0.71 mmol) in solution in DMF, a white solid is obtained after drying at 30 C. at reduced pressure.

Co-Polyamino Acid B28

[1504] By means of a similar method to that used for the preparation of co-polyamino acid B15 applied to co-polyamino acid B28-1 (2.98 g, 0.65 mmol) and with a saponification step at pH 12 for 1 h prior to the ultrafiltration step, co-polyamino acid B28 is obtained.

[1505] Dry extract: 25.8 mg/g

[1506] DP (estimated as per .sup.1H NMR): 5.15

[1507] As per .sup.1H NMR: i=3.0

[1508] The calculated average molar mass of co-polyamino acid B28 is 3559.2 g/mol.

[1509] HPLC-Organic SEC (PEG calibrator): Mn=2200 g/mol.

EXAMPLE B29

Co-Polyamino Acid B294,7,10-trioxa-1,13-tridecanediamine (TOTA) substituted with molecule A12 wherein the esters are deprotected and having a number average molar mass (Mn) of 2000 g/mol

Co-Polyamino Acid B29-1:

[1510] To a solution of molecule A12 (3.70 g, 1.98 mmol) in chloroform (31 mL) at ambient temperature are added successively HOBt (304 mg, 1.98 mmol) and 4,7,10-trioxa-1,13-tridecanediamine (TOTA, 208 mg, 0.94 mmol). The mixture is cooled to 0 C. then EDC (380 mg, 1.98 mmol) is added. The reaction medium is stirred for 15 min at 0 C. followed by 18 h at ambient temperature. The organic phase is washed with a 0.1 N NaOH aqueous solution (228 mL), and the organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated at reduced pressure. The solid obtained is solubilized in CHCl.sub.3 (40 mL) and the solution is added dropwise to IPE (400 mL) under stirring. The suspension is placed in an ice bath without stirring for 17 h. The suspension is centrifuged at 3200 rpm for 10 min at 25 C. The colorless supernatant is removed and the solid obtained is concentrated at reduced pressure.

[1511] Yield: 4.59 g (quant.)

[1512] .sup.1H NMR CDCl.sub.3, ppm): 0.88 (12H); 1.12-1.58 (192H); 1.58-2.17 (48H); 2.17-2.62 (44H); 3.08 (2H); 3.13-3.38 (6H); 3.48 (4H); 3.53-3.66 (12H); 3.74-3.83 (4H); 3.92 (2H); 4.00-4.12 (4H); 4.12-4.33 (10H); 4.37 (2H); 6.72-6.84 (4H); 7.06 (2H); 7.31 (2H); 7.52 (2H); 7.82 (2H); 7.94 (2H); 8.57-8.69 (4H).

Co-Polyamino Acid B29

[1513] Molecule B29-1 (3.67 g, 0.93 mmol) is solubilized in TFA (11.5 mL) and the solution is stirred at ambient temperature for 6 h. The solution is poured dropwise onto IPE (18 mL) at 5 C. then water (18 mL) is added. The suspension is placed in an ice bath under stirring for 15 h. The suspension is filtered and triturated with IPE (10 mL) and water (210 mL). The residue is dried at reduced pressure then solubilized in a 1 N NaOH solution (56 mL) with regular addition of 1 N NaOH to maintain the pH at 7. The solution is diluted to 20 g/L theoretical with water then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1514] Dry extract: 8.0 mg/g

[1515] The calculated average molar mass of co-polyamino acid B29 is 3520 g/mol.

[1516] HPLC-Organic SEC (PEG calibrator): Mn=2000 g/mol.

EXAMPLE B30

Co-Polyamino Acid B30Tricarballylic Acid Substituted with Molecule A26 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2100 g/mol

[1517] Co-polyamino acid B30-1: By means of a similar method to that used for the preparation of co-polyamino acid B15-1 applied to molecule A26 (10.87 g, 11.33 mmol) and to tricarballylic acid (TCA, 0.605 g, 3.43 mmol) in solution in DMF, a white solid is obtained after 2 consecutive precipitations of the product in solution in DMF in a 50:50 H.sub.2O/MeCN mixture (10V), filtration, trituration with a 50:50 H.sub.2O/MeCN mixture followed by drying at reduced pressure at 30 C.

Co-Polyamino Acid B30

[1518] Co-polyamino acid B30-1 (8.53 g, 2.95 mmol) is solubilized in TFA (30 mL), and the solution is stirred for 3 h at ambient temperature then is poured dropwise onto water under stirring (300 mL). After 1 h, the white precipitate is retrieved by filtration, triturated with water and dried at reduced pressure. The solid obtained is then solubilized in water (350 mL) by adjusting the pH to 7 adding an aqueous solution of 1 N sodium hydroxide. The solution is filtered on a 0.2 m filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 S/cm. The solution obtained is filtered on a 0.2 m filter and stored at 2-8 C.

[1519] Dry extract: 28.8 mg/g

[1520] The molar mass of co-polyamino acid B30 is 2585 g/mol.

[1521] HPLC-Organic SEC (PEG calibrator): Mn=2100 g/mol.

EXAMPLE B31

Co-Polyamino Acid B31Sodium Poly-L-Glutamate Modified at the Extremities thereof by Molecule A27 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3800 g/mol

[1522] By means of a similar method to that used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B18-1 (28.6 g) and to molecule A27 (6.799 g), co-polyamino acid B31 is obtained.

[1523] Dry extract: 20.5 mg/g

[1524] DP (estimated as per .sup.1H NMR): 24

[1525] As per .sup.1H NMR: i=0.075

[1526] The calculated average molar mass of co-polyamino acid B31 is 4591 g/mol.

[1527] HPLC-Organic SEC (PEG calibrator): Mn=3800 g/mol

Part C: Compositions

EXAMPLE C1

Preparation of a 0.6 mg/mL Pramlintide Solution Containing m-cresol (29 mM) and Glycerin (174 mM) at pH 6.6

[1528] A 5 mg/mL concentrated pramlintide solution is prepared by dissolving pramlintide in powder form purchased from Ambiopharm. This solution is added to a concentrated solution of excipients (m-cresol, glycerin) so as to obtain the aimed final concentration. The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

EXAMPLE C2

Preparation of a 0.6 mg/mL Pramlintide Solution Containing co-polyamino Acid B6, m-cresol (29 mM) and Glycerin (174 mM) at pH 6.6

[1529] A concentrated solution of co-polyamino acid B6 and excipients is prepared by adding concentrated solutions of excipients (m-cresol, glycerin) to a concentrated solution of co-polyamino acid B6.

[1530] A 5 mg/mL concentrated pramlintide solution at pH 4 is added to this concentrated solution of co-polyamino acid B6 and excipients so as to obtain the final compositions 2-1 to 2-5 (Table 1). The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

TABLE-US-00004 TABLE 1 Compositions and visual appearance of pramlintide solutions at pH 6.6 at different co-polyamino acid B6 concentrations. Co-polyamino acid B6 B6/pramlintide ratio concentration Visual appearance of Solution mol/mol mg/mL mM solution C1 Clear C2-1 1.5 1.2 0.23 Clear C2-2 2 1.6 0.30 Clear C2-3 2.5 2.0 0.38 Clear C2-4 3 2.3 0.46 Clear C2-5 4 3.1 0.61 Clear

EXAMPLE C3

Preparation of a 0.6 mg/mL Pramlintide Solution Containing Different co-polyamino Acids According to the Invention, m-cresol (29 mM) and Glycerin (174 mM) at pH 6.6

[1531] By means of a similar protocol to that described in example C2, 0.6 mg/mL pramlintide solutions containing different co-polyamino acids according to the invention, m-cresol (29 mM) and glycerin (174 mM) at pH 6.6, C3-1 to C3-26 are obtained.

TABLE-US-00005 TABLE 2a Compositions and visual appearance of 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of different co-polyamino acids. Ratio of Co-polyamino co-polyamino Visual acid acid/ appearance Co-polyamino concentration pramlintide of Solution acid mg/mL mM mol/mol solution C3-1 B2 3.9 0.75 5 clear 7.9 1.5 10 clear 11.8 2.28 15 clear C3-2 B3 7.5 1.5 10 clear 11.3 2.3 15 clear C3-3 B5 1.5 0.3 2 clear 2.3 0.45 3 clear 3 0.6 4 clear C3-4 B4 5.3 1.53 10 clear C3-6 B9 1.8 0.3 2 clear 2.6 0.46 3 clear C3-7 B16 6.2 1.3 8 clear C3-8 B17 5.6 1.2 8 clear C3-9 B19 6.4 1.3 8 clear C3-10 B20 11.2 2.3 15 clear C3-11 B21 3.9 0.76 5 clear C3-13 B23 4.0 0.76 5 clear C3-14 B24 4.4 1.5 10 clear C3-15 B25 3.8 0.8 5 clear C3-16 B26 2.6 0.52 3 clear C3-17 B1 15.3 2.28 15 clear C3-18 B14 2.1 0.30 2 clear C3-19 B14 2.9 0.42 3 clear C3-20 B19 6 1.21 8 clear C3-21 B23 12 2.31 15 clear C3-22 B27 3.1 0.76 5 clear C3-23 B28 1.1 0.3 2 clear C3-24 B29 1.1 0.3 2 clear C3-25 B30 0.8 0.3 2 clear C3-26 B31 10.5 2.3 15 clear

EXAMPLE C4

Preparation of a 0.6 mg/mL Pramlintide and 100 IU/mL Human Insulin Solution Containing m-cresol (29 mM), Glycerin (174 mM) and Zinc Chloride (229 M) at pH 6.6

[1532] The 5 mg/mL concentrated pramlintide solution C1 is added to a concentrated solution of excipients (m-cresol, glycerin, zinc chloride). A 500 IU/mL human insulin solution is prepared by dissolving human insulin in powder form purchased from Amphastar. This solution is added to the concentrated solution of Pramlintide and excipients so as to obtain the sought final concentration. The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

EXAMPLE C5

Preparation of a 0.6 mg/mL Pramlintide and 100 IU/mL Human Insulin Solution Containing Co-Polyamino Acid B6, m-cresol (29 mM), Glycerin (174 mM) and Zinc Chloride (229 M) at pH 6.6

[1533] A concentrated solution of co-polyamino acid B6 and excipients is prepared by adding concentrated solutions of excipients (m-cresol, glycerin, zinc chloride) to a concentrated solution of co-polyamino acid B6.

[1534] A 5 mg/mL concentrated pramlintide solution at pH 4 followed by a 500 IU/mL human insulin solution are added to the concentrated solution of co-polyamino acid B6 and excipients so as to obtain the sought final composition (Table 3). The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

[1535] Solutions C5-1 to C5-5 are prepared according to the above protocol.

TABLE-US-00006 TABLE 3 Compositions and visual appearance of 0.6 mg/mL pramlintide and human insulin solutions at pH 6.6 at different co-polyamino acid B6 concentrations. B6/pramlintide Co-polyamino acid ratio B6 concentration Visual appearance of Solution mol/mol mg/mL mM solution C4 Turbid C5-1 1.5 1.2 0.23 Clear C5-2 2 1.6 0.30 Clear C5-3 2.5 2.0 0.38 Clear C5-4 3 2.3 0.46 Clear C5-5 4 3.1 0.61 Clear

EXAMPLE C6

Preparation of a 0.6 mg/mL pramlintide and 100 IU/mL human insulin solution containing different co-polyamino acids according to the invention, m-cresol (29 mM), glycerin (174 mM) and zinc chloride (229 M) at pH 6.6

[1536] By means of a similar method to example C5, a 0.6 mg/mL pramlintide and 100 IU/mL human insulin solution containing a co-polyamino acid according to the invention, m-cresol (29 mM), glycerin (174 mM) and zinc chloride (229 M) at pH 6.6 is obtained.

[1537] Solutions C6-1 to 3 and C6-6 to C6-9 are prepared according to the above protocol.

TABLE-US-00007 TABLE 4a Compositions and visual appearance of 0.6 mg/mL pramlintide and 100 IU/mL human insulin solutions at pH 6.6 in the presence of different co-polyamino acids. Ratio of Co-polyamino co-polyamino Visual acid acid/ appearance Co-polyamino concentration pramlintide of Solution acid mg/mL mM mol/mol solution C6-1 B2 3.9 0.75 5 clear 7.9 1.5 10 clear C6-2 B3 7.5 1.5 10 clear 11.3 2.30 15 clear C6-3 B5 1.5 0.3 2 clear 2.3 0.45 3 clear 3 0.6 4 clear C6-6 B9 1.8 0.3 2 clear 2.6 0.46 3 clear C6-7 B1 15.3 2.28 15 clear C6-8 B14 2.9 0.42 3 clear C6-9 B23 9.6 1.8 12 clear C6-10 B27 3.1 0.76 5 clear C6-11 B28 1.6 0.46 3 clear C6-12 B29 1.6 0.46 3 clear C6-13 B30 1.2 0.46 3 clear C6-14 B31 13.8 3 20 clear

EXAMPLE C7

Preparation of a 0.6 mg/mL Pramlintide Solution Containing Co-Polyamino Acid B2, M-Cresol (29 mM), Glycerin (174 mM), Sodium Chloride and Optionally Zinc Chloride at pH 6.6

[1538] A concentrated solution of co-polyamino acid B2 and excipients is prepared by adding concentrated solutions of excipients (m-cresol, glycerin, NaCl, zinc chloride) to a concentrated solution of co-polyamino acid B2.

[1539] A 5 mg/mL concentrated pramlintide solution at pH 4 is added to this concentrated solution of co-polyamino acid B2 and excipients so as to obtain the final compositions C7-1 to C7-10 (Table 5). The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

TABLE-US-00008 TABLE 5 Compositions and visual appearance of 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of co-polyamino acid B2, and optionally sodium chloride and zinc chloride. Co-polyamino acid Ratio of co- Visual Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] appearance Solution acid mg/mL mM acid/pramlintide (mM) (mM) of solution C7-1 B2 2.4 0.46 3 50 clear C7-2 B2 2.4 0.46 3 100 clear C7-3 B2 2.4 0.46 3 50 0.5 clear C7-4 B2 2.4 0.46 3 50 1 clear C7-5 B2 2.4 0.46 3 1 clear

TABLE-US-00009 TABLE 5a Compositions and visual appearance of 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of co-polyamino acid B2, sodium chloride and optionally zinc chloride. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] Visual Solution acid mg/mL mM acid/pramlintide (mM) (mM) appearance C7-6 B2 6.3 1.2 8 25 clear C7-7 B2 4.7 0.9 6 25 1 clear C7-8 B2 3.9 0.76 5 50 clear C7-10 B2 2.4 0.46 3 100 clear

EXAMPLE C8

Preparation of a 0.6 mg/mL Pramlintide Solution Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), Glycerin (174 mM), NaCl and Zinc Chloride at pH 6.6

[1540] By means of a similar protocol to that described in example C7, 0.6 mg/mL pramlintide solutions containing different co-polyamino acids according to the invention, m-cresol (29 mM) and glycerin (174 mM), optionally sodium chloride and zinc chloride at pH 6.6, C8-1 to C8-4 and C8-9a, C8-10a and C8-11 are obtained.

TABLE-US-00010 TABLE 6a Compositions and visual appearance of 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of different co-polyamino acids, sodium chloride and optionally zinc chloride. Co-polyamino acid Ratio of co- Visual Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] appearance Solution acid mg/mL mM acid/pramlintide (mM) (mM) of solution C8-1 B1 15.3 2.28 15 100 clear C8-2 B1 15.3 2.28 15 100 1 clear C8-3 B1 15.3 2.28 15 100 2 clear C8-4 B3 11.3 2.3 15 100 clear C8-9a B20 11.2 2.3 15 25 1 clear C8-10a B21 3.9 0.76 5 25 clear C8-11 B21 3.9 0.76 5 25 1 clear

EXAMPLE C8a

Preparation of a 0.6 mg/mL Pramlintide Solution Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), Glycerin (174 mM) and NaCl (50 mM) at pH 6.6

[1541] By means of a similar protocol to that described in example C7, 0.6 mg/mL pramlintide solutions containing different co-polyamino acids according to the invention, m-cresol (29 mM) and glycerin (174 mM), sodium chloride at pH 6.6, C8a-1 to C8a-10 are obtained.

TABLE-US-00011 TABLE 6c Compositions and visual appearance of 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of different co-polyamino acids, sodium chloride. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] Visual Solution acid mg/mL mM acid/pramlintide (mM) appearance C8a-1 B16 6.2 1.3 8 50 clear C8a-2 B17 2.8 0.61 4 50 clear C8a-3 B19 6.4 1.3 8 50 clear C8a-4 B20 6.0 1.2 8 25 clear C8a-7 B23 4.0 0.76 5 25 clear C8a-8 B24 2.2 0.76 5 50 clear C8a-9 B14 2.1 0.30 2 25 clear C8a-10 B19 6 1.21 8 50 clear

EXAMPLE C9

Preparation of a 0.6 mg/mL Pramlintide and 100 IU/mL Human Insulin Solution Containing Co-Polyamino Acid B2, M-Cresol (29 mM), Glycerin (174 mM), Sodium Chloride (50 mM) and Zinc Chloride (1 mM)

[1542] A concentrated solution of co-polyamino acid B2 and excipients is prepared by adding concentrated solutions of excipients (m-cresol, glycerin, sodium chloride, zinc chloride) to a concentrated solution of co-polyamino acid B2.

[1543] A 5 mg/mL concentrated pramlintide solution at pH 4 followed by a 500 IU/mL human insulin solution are added to this concentrated solution of co-polyamino acid B2 and excipients so as to obtain the sought final composition. The final pH is adjusted to pH 6.6 by adding NaOH/HCl.

[1544] Solution C9 is prepared according to the above protocol.

EXAMPLE C10

Preparation of a 0.6 mg/mL Pramlintide and 100 IU/mL Human Insulin Solution Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), glycerin (174 mM), and Different Sodium Chloride and Zinc Chloride Concentrations

[1545] By means of a similar method to example C9, 0.6 mg/mL pramlintide and 100 IU/mL human insulin solutions containing different co-polyamino acids according to the invention, m-cresol (29 mM), glycerin (174 mM), sodium chloride and zinc chloride at pH 6.6 are obtained.

[1546] Solutions C9 and C10-1 to C10-7 are prepared according to the above protocol.

TABLE-US-00012 TABLE 7a Compositions and visual appearance of 0.6 mg/mL pramlintide and 100 IU/mL insulin solutions at pH 6.6 in the presence of different co-polyamino acids, sodium chloride and zinc chloride. Co-polyamino acid Ratio of co- Visual Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] appearance Solution acid mg/mL mM acid/pramlintide (mM) (mM) of solution C9 B2 2.4 0.46 3 50 1* clear C10-1 B1 15.3 2.28 15 100 * clear C10-2 B1 15.3 2.28 15 100 1* clear C10-3 B1 15.3 2.28 15 100 2* clear C10-4 B3 11.3 2.3 15 100 * clear C10-5 B3 11.3 2.3 15 100 1* clear C10-6 B19 6 1.21 8 50 * clear C10-7 B23 6 1.15 8 25 * clear *composition comprising 0.23 mM of ZnCl.sub.2 from the 100 IU/mL human insulin solution

EXAMPLE C11

Results of Visual Observations on Mixture and Fibrillation Measurements by ThT

Principle

[1547] The poor stability of a peptide may give rise to the formation of amyloid fibrils, defined as organized macromolecular structures. These may potentially result in gel formation in the sample.

[1548] The thioflavin T (ThT) fluorescence monitoring test is used to analyze the physical stability of the solutions. Thioflavin is a small molecular probe having a characteristic fluorescence signature when it binds to amyloid type fibrils (Naiki et al. (1989) Anal. BioChem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284).

[1549] This method makes it possible to monitor fibril formation for low ThT concentrations in undiluted solutions. This monitoring is carried out under accelerated stability conditions: under stirring and at 37 C.

Experimental Conditions

[1550] The samples were prepared just before starting measurement. The preparation of each composition is described in the associated example. Thioflavin T was added in the composition using a concentrated stock solution so as to induce a negligible dilution of the composition. The Thioflavin T concentration in the composition is 2 M.

[1551] A 150 L volume of the composition was introduced into a well of a 96-well plate. Each composition was analyzed in three tests (triplicate) in the same plate. The plate was sealed with transparent film so to prevent the evaporation of the composition.

[1552] This plate was then placed in the enclosure of a plate reader (EnVision 2104 Multilabel, Perkin Elmer). The temperature is set to 37 C., and lateral stirring of 960 rpm with 1 mm amplitude is applied.

[1553] A fluorescence intensity reading in each well is carried out with an excitation wavelength of 442 nm, and an emission wavelength of 482 nm over time.

[1554] The fibrillation process is conveyed by a significant increase in fluorescence after an interval known as the latent period.

[1555] For each well, this interval was determined graphically as the intersection between the baseline of the fluorescence signal and the slope of the fluorescence curve as a function of time determined during the initial significant increase in fluorescence. The value of the latent period reported corresponds to the mean of the latent period measurements made on three wells.

[1556] An example of a graphic determination is shown in FIG. 1.

[1557] In this figure, the determination of the latent period (LT) by monitoring the fluorescence of Thioflavin T is represented graphically, on a curve having on the y-axis the fluorescence value (in a.u arbitrary units) and on the x-axis the time in minutes.

EXAMPLE CA1

Stability of 0.6 mg/mL Pramlintide Solutions at pH 6.6 in the Presence of Co-Polyamino Acid B6 at Different Concentrations

[1558]

TABLE-US-00013 TABLE 8 Lag time measurement by ThT of solutions C1 and C2-1 to C2-5. Co-polyamino acid B6 B6/pramlintide ratio concentration Lag time Solution mol/mol mg/mL mM (h) C1 1 C2-1 1.5 1.2 0.23 >20 C2-2 2 1.6 0.30 >60 C2-3 2.5 2.0 0.38 >60 C2-4 3 2.3 0.46 >60 C2-5 4 3.1 0.61 >60

[1559] The pramlintide solution at pH 6.6 (C1) devoid of co-polyamino acid has a short lag time; the lag times of the solutions containing co-polyamino acid B6 are greater.

EXAMPLE CA2

Stability of 0.6 mg/ml Pramlintide Solutions at pH 6.6 in the Presence of Different Co-Polyamino Acids

[1560]

TABLE-US-00014 TABLE 9a Lag time measurement by ThT of compositions C3-1 to C3-4 and C3-6, C3-15 and 16. Co-polyamino Ratio of acid co-polyamino Co-polyamino concentration acid/pramlintide Lag time Solution acid mg/mL mM mol/mol (h) C3-1 B2 3.9 0.75 5 5 > t > 3 7.9 1.5 10 10 > t > 7 11.8 2.28 15 >30 C3-2 B3 7.5 1.5 10 >2 11.3 2.3 15 5 > t > 4 C3-3 B5 1.5 0.3 2 >10 2.3 0.45 3 >30 3 0.6 4 >50 C3-4 B4 5.3 1.53 10 >40 C3-6 B9 1.8 0.3 2 >20 2.6 0.46 3 >50 C3-15 B25 3.8 0.8 5 >30 C3-16 B26 2.6 0.52 3 >30 C3-22 B27 3.1 0.76 5 >30 C3-23 B28 1.1 0.3 2 >30 C3-24 B29 1.1 0.3 2 >45 C3-25 B30 0.8 0.3 2 >40

[1561] The pramlintide solution at pH 6.6 (C1) devoid of co-polyamino acid has a short lag time. The co-polyamino acids according to the invention make it possible to obtain lag times greater than 2 h under the test conditions.

EXAMPLE CA3

Stability of 0.6 mg/ml Pramlintide and 100 IU/mL Human Insulin Solutions at pH 6.6 in the Presence of Co-Polyamino Acid B6 at Different Concentrations

[1562]

TABLE-US-00015 TABLE 10 lag time measurement by ThT of compositions C5-1 to C5-5. Co-polyamino acid B6 B6/pramlintide ratio concentration Lag time Solution mol/mol mg/mL mM (h) C4 * C5-1 1.5 1.2 0.23 >1 C5-2 2 1.6 0.30 >5 C5-3 2.5 2.0 0.38 >10 C5-4 3 2.3 0.46 >10 C5-5 4 3.1 0.61 >10 * lag time not measured due to turbid solution

[1563] One 0.6 mg/ml pramlintide and 100 IU/mL human insulin solution at pH 6.6 devoid of co-polyamino acid is turbid (C4). The clear 0.6 mg/ml pramlintide and 100 IU/ml human insulin solutions at pH 6.6 in the presence of co-polyamino acid B6 have lag times greater than 1 h at a molar ratio of B6/pramlintide of 1.5, potentially greater than 10 h for molar ratios of B6/pramlintide greater than 2.5.

EXAMPLE CA4

Stability of 0.6 mg/ml Pramlintide and 100 IU/mL Human Insulin Solutions at pH 6.6 in the Presence of Different Co-Polyamino Acids

[1564]

TABLE-US-00016 TABLE 11a Lag time measurement by ThT of compositions C6-1 to C6-3 and C6-6. Ratio of Co-polyamino co-polyamino acid acid/ Co-polyamino concentration pramlintide Lag time Solution acid mg/mL mM mol/mol (h) C6-1 B2 3.9 0.75 5 5 > t > 3 7.9 1.5 10 >5 C6-2 B3 7.51.4 1.50.15 10 >3 C6-3 B5 1.5 0.3 2 >3 2.3 0.45 3 >5 3 0.6 4 >5 C6-6 B9 1.8 0.3 2 >2 2.6 0.46 3 >5

[1565] The pramlintide and human insulin solution at pH 6.6 (C4) is turbid. The co-polyamino acids make it possible to obtain lag times greater than 2 h under the test conditions.

EXAMPLE CA5

Stability of 0.6 mg/mL Pramlintide Solutions Containing Co-Polyamino Acid B2, M-Cresol (29 mM), Glycerin (174 mM), Optionally Sodium Chloride and Zinc Chloride at pH 6.6

[1566]

TABLE-US-00017 TABLE 12 Lag time measurement by ThT of compositions C3-1 and C7-1 to C7-5 Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] Lag time Solution acid mg/mL mM acid/pramlintide (mM) (mM) (h) C3-1 B2 3.9 0.75 5 5 > t > 3 C7-1 B2 2.4 0.46 3 50 >15 C7-2 B2 2.4 0.46 3 100 35 > t > 25 C7-3 B2 2.4 0.46 3 50 0.5 >25 C7-4 B2 2.4 0.46 3 50 1 >40 C7-5 B2 2.4 0.46 3 1 >5

[1567] The pramlintide solution at pH 6.6 (C1) devoid of co-polyamino acid has a short lag time; adding a salt alone or in combination with zinc chloride to the solutions containing co-polyamino acid B2 makes it possible to obtain markedly greater lag times than solution C3-1 containing B2/pramlintide devoid of salt and zinc.

EXAMPLE CA5a

Stability of 0.6 mg/mL Pramlintide Solutions Containing Co-Polyamino Acid B2, M-Cresol (29 mM), Glycerin (174 mM), Optionally Sodium Chloride and Zinc Chloride at pH 6.6

[1568]

TABLE-US-00018 TABLE 12a Lag time measurement by ThT of compositions C3-1 and C7-2, C7-4 and C7-6 to C7-8. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] Lag time Solution acid mg/mL mM acid/pramlintide (mM) (mM) (h) C3-1 B2 7.9 1.5 10 10 > t > 7 C7-6 B2 6.3 1.2 8 25 >30 C7-7 B2 4.7 0.9 6 25 1 >30 C7-8 B2 3.9 0.76 5 50 >30 C7-4 B2 2.4 0.46 3 50 1 >40 C7-2 B2 2.4 0.46 3 100 >30

[1569] Adding salt alone or in combination with zinc chloride in the solutions containing B2 makes it possible to lower the ratio of co-polyamino acid B2/pramlintide and obtain lag times greater than 30 h.

EXAMPLE CA6

Stability of 0.6 mg/mL Pramlintide Solutions Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), Glycerin (174 mM), Optionally NaCl and Zinc Chloride at pH 6.6

[1570]

TABLE-US-00019 TABLE 13a Lag time measurement by ThT of compositions C8-1 to C8-4, C8-9a, C8-10a, C8-11 and C3-2, C3-10, C3-11 and C3-17. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] Lag time Solution acid mg/mL mM acid/pramlintide (mM) (mM) (h) C3-17 B1 15.3 2.28 15 <1 C8-1 B1 15.3 2.28 15 100 >3 C8-2 B1 15.3 2.28 15 100 1 >10 C8-3 B1 15.3 2.28 15 100 2 >14 C3-2 B3 11.3 2.3 15 <5 C8-4 B3 11.3 2.3 15 100 >20 C3-10 B20 11.2 2.3 15 <5 C8-9a B20 11.2 2.3 15 25 1 >30 C3-11 B21 3.9 0.76 5 <6 C8-10a B21 3.9 0.76 5 25 >20 C8-11 B21 3.9 0.76 5 25 1 >30

[1571] Adding salt alone or in combination with zinc chloride in the solutions containing a co-polyamino acid makes it possible to increase the lag times significantly compared to those obtained with solutions free from salt or zinc.

EXAMPLE CA6a

Stability of 0.6 mg/mL Pramlintide Solutions Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), Glycerin (174 mM) and Optionally NaCl (25 or 50 mM) at pH 6.6

[1572]

TABLE-US-00020 TABLE 13c Lag time measurement by ThT of compositions C3-7 to C3-9, C3-13 to C3-14, C3-18, C8a-1 to C8a-3 and C8a-7 to C8a-9. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] Lag time Solution acid mg/mL mM acid/pramlintide (mM) (h) C3-7 B16 6.2 1.3 8 <10 C8a-1 B16 6.2 1.3 8 50 >30 C3-8 B17 5.6 1.2 8 <15 C8a-2 B17 2.8 0.61 4 50 >30 C3-9 B19 6.4 1.3 8 <6 C8a-3 B19 6.4 1.3 8 50 >30 C3-13 B23 4.0 0.76 5 <15 C8a-7 B23 4.0 0.76 5 25 >30 C3-14 B24 4.4 1.5 10 <10 C8a-8 B24 2.2 0.76 5 50 >30 C3-18 B14 2.1 0.3 2 <35 C8a-9 B14 2.1 0.3 2 25 >60

[1573] Adding salt (25 or 50 mM) in the solutions containing co-polyamino acid/pramlintide makes it possible to lower the ratio of co-polyamino acid/pramlintide and obtain lag times greater than 30 h.

EXAMPLE CA7

Physical Stability of 0.6 mg/mL Pramlintide and 100 IU/mL Human Insulin Solutions Containing Different Co-Polyamino Acids According to the Invention, M-Cresol (29 mM), Glycerin (174 mM), and Different Sodium Chloride and Zinc Chloride Concentrations

[1574]

TABLE-US-00021 TABLE 14a Lag time measurement by ThT of compositions C6-1, C6-2, C6-7, C9 and C10-1 to C10-5. Co-polyamino acid Ratio of co- Co-polyamino concentration polyamino [NaCl] [ZnCl.sub.2] Lag time Solution acid mg/mL mM acid/ (mM) (mM) (h) C6-1 B2 3.9 0.75 5 * <5 C9 B2 2.4 0.46 3 50 1* >10 C6-7 B1 15.3 2.28 15 * <1 C10-1 B1 15.3 2.28 15 100 0.23 >4 C10-2 B1 15.3 2.28 15 100 1* >9 C10-3 B1 15.3 2.28 15 100 2* >10 C6-2 B3 2.8 0.3 15 * <4 C10-4 B3 11.3 2.3 15 100 0.23 >10 C10-5 B3 11.3 2.3 15 100 1.2* >15 *composition comprising 0.23 mM of ZnCl.sub.2 from the 100 IU/mL human insulin solution indicates data missing or illegible when filed

[1575] Adding and zinc chloride in the solutions containing a co-polyamino acid makes it possible to increase the lag times significantly compared to those obtained with solutions free from salt or zinc.

EXAMPLE CB1

Physical Stability in Vial and Cartridge at 30 C. of 0.6 mg/mL Pramlintide Solutions in the Presence of Co-Polyamino Acid, M-Cresol (29 mM), Glycerin (174 mM) and Different NaCl Concentrations at pH 6.6.

[1576] Solutions C3-19, C8a-10 and C3-20 are filtered (0.22 m). 1 mL of solution is introduced into 3 mL self-injection pen glass cartridges and into 3 mL glass vials. The cartridges and the vials are placed in an oven at 30 C. under static conditions then are observed every 2 weeks.

TABLE-US-00022 TABLE 15 Physical stability results in vial and in cartridge at 30 C. of compositions with 0.6 mg/mL pramlintide in the presence of co-polyamino acid B14, B19 and B23 and different NaCl concentrations. Co-polyamino Physical stability Physical stability Co-polyamino acid concentration NaCl 30 C. in vial 30 C. in cartridge Solu tion acid mg/mL ratio (mM) (weeks) (weeks) C1 <7 C3-19 B14 2.9 3 >12 C8a-10 B19 6 6 50 >9 >12 C3-20 B23 12 15 >12 >12

[1577] The 0.6 mg/mL pramlintide solution at pH 6.6 exhibits a physical stability in vials of less than 7 weeks at 30 C. The 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of co-polyamino acids B19 and B23 (optionally with NaCl) exhibit a physical stability at 30 C. of greater than 9 weeks in vials.

[1578] The 0.6 mg/mL pramlintide solutions at pH 6.6 in the presence of co-polyamino acid and optionally salt exhibit a physical stability at 30 C. of greater than 12 weeks in cartridges.

EXAMPLE CB2

Physical Stability in Vial and Cartridge at 30 C. of 0.6 mg/mL Pramlintide and 100U/mL Human Insulin Solutions in the Presence of Co-Polyamino Acid, M-Cresol (29 mM), Glycerin (174 mM), Zinc Chloride (229 M) and different NaCl concentrations at pH 6.6.

[1579] Solutions C6-8, C3-20, C10-6 and C6-9 are filtered (0.22 m). 1 mL of solution is introduced into 3 mL self-injection pen glass cartridges and into 3 mL glass vials. The cartridges and the vials are placed in an oven at 30 C. under static conditions then are observed every 2 weeks.

TABLE-US-00023 TABLE 16 Physical stability results in vial and in cartridge at 30 C. of compositions with 0.6 mg/mL pramlintide and 100 U/mL human insulin in the presence of co-polyamino acid B14, B9 and B23 and different NaCl concentrations. Physical Co-polyamino stability acid 30 C. in Co-polyamino concentration NaCl cartridge Solution acid mg/mL ratio (mM) (weeks) C6-8 B14 2.9 3 >12 C3-20 B19 6 6 >2 C10-6 B19 6 6 50 >12 C6-9 B23 9.6 12 >12 C10-7 B23 6 8 25 >12

[1580] The 0.6 mg/mL pramlintide and 100 IU/mL insulin solution at pH 6.6 is turbid.

[1581] The 0.6 mg/mL pramlintide and 100 IU/mL human insulin solution at pH 6.6 in the presence of co-polyamino acid and optionally salt exhibit a physical stability at 30 C. of greater than 4 weeks in vials and greater than 2 weeks in cartridges. Adding salt in solution C10-6 containing co-polyamino acid B19 makes it possible to increase the stability in cartridges at 30 C. significantly compared to that obtained with solution C3-20 free from salt.

EXAMPLE CB3

Physical Stability in Cartridge at 37 C. of 0.6 mg/mL Pramlintide Solutions in the Presence of Co-Polyamino Acid, M-Cresol (29 mM), Glycerin (174 mM) and Different NaCl Concentrations at pH 6.6.

[1582] Solutions C3-19, C8a-10 and C3-21 are filtered (0.22 m). 1 mL of solution is introduced into 3 mL self-injection pen glass cartridges. The cartridges are placed in an oven at 37 C. under static conditions then are observed every 2 weeks.

TABLE-US-00024 TABLE 17 Physical stability results in cartridge at 37 C. of compositions with 0.6 mg/mL pramlintide in the presence of co-polyamino acid and different NaCl concentrations. Physical Co-polyamino stability acid 37 C. in Co-polyamino concentration NaCl cartridge Solution acid mg/mL ratio (mM) (weeks) C3-19 B14 2.9 3 >12 C8a-10 B19 6 6 50 >9 C3-21 B23 12 15 >9

[1583] The 0.6 mg/mL pramlintide solution at pH 6.6 in the presence of co-polyamino acid and optionally salt exhibits a physical stability at 30 C. of greater than 7 weeks in cartridges.

EXAMPLE CB4

Physical Stability in Cartridge at 37 C. of 0.6 mg/mL Pramlintide and 100U/mL Human Insulin Solutions in the Presence of Co-Polyamino Acid, M-Cresol (29 mM), Glycerin (174 mM), Zinc Chloride (229 M) and Different NaCl Concentrations at pH 6.6.

[1584] Solutions C6-8, C10-6, C6-9 and C10-7 are filtered (0.22 m). 1 mL of solution is introduced into 3 mL self-injection pen glass cartridges. The cartridges are placed in an oven at 37 C. under static conditions then are observed every 2 weeks.

TABLE-US-00025 TABLE 18 Physical stability results in cartridge at 37 C. of compositions with 0.6 mg/mL pramlintide and 100 U/mL human insulin in the presence of co-polyamino acid and different NaCl concentrations. Physical stability Co-polyamino acid 37 C. in Co-polyamino concentration NaCl cartridge Solution acid mg/mL ratio (mM) (weeks) C6-8 B14 2.9 3 >8 C10-6 B19 6 6 50 >6 C6-9 B23 9.6 12 >9 C10-7 B23 6 8 25 >9

[1585] The 0.6 mg/mL pramlintide and 100 IU/mL insulin solution at pH 6.6 is turbid.

[1586] The 0.6 mg/mL pramlintide and 100 IU/mL human insulin solution at pH 6.6 in the presence of co-polyamino acid and salt exhibit a physical stability at 37 C. of greater than 6 weeks in cartridges.