Quick-acting insulin formulation including a substituted anionic compound

CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 Rapid-acting formulation of insulin comprising a substituted anionic compound [0001] The present invention relates to a rapid-acting formulation of insulin.

[0002] Since the production of insulin by genetic engineering in the early 1980s, diabetic patients have benefited from human insulin to treat themselves.

This product has greatly improved this therapy since the immunological risks linked to the use of non-human insulin, particularly pork insulin, are eliminated.

However, human insulin injected subcutaneously has a hypoglycemic effect only after 60 minutes, which means that diabetic patients treated with human insulin should inject 30 minutes before the meal.

[0003] One of the problems to be solved to improve the health and comfort of diabetic patients is to provide them with insulin formulations which make it possible to provide a hypoglycemic response more quickly than that of human insulin and, if possible, approaching the physiological response of a healthy person.

The secretion of endogenous insulin in healthy individuals is immediately triggered by the increase in blood sugar. The aim is to reduce the time between the insulin injection and the start of the meal as much as possible.

[0004] Today, it is accepted that the provision of such formulations is useful so that the management of the disease is as best possible.

[0005] Genetic engineering has made it possible to provide a response with the development of rapid analog insulins.

These insulins are modified on one or two amino acids to be more quickly absorbed into the blood compartment after a subcutaneous injection.

These insulins lispro (Humalog, Lilly), aspart (Novologe, Novo) and glulisine (ApidraG, Sanofi Aventis) are stable insulin solutions with a faster hypoglycemic response than that of human insulin. Therefore, patients treated with these rapid insulin analogues can inject insulin only 15 minutes before the meal.

[0006] The principle of rapid analog insulins is to form hexamers at a concentration of 100 IU/mL to ensure the stability of the insulin in the commercial product while promoting the very rapid dissociation of these hexamers into monomers after injection under submersion. cutaneous in order to obtain rapid action.

[0007] Human insulin as formulated in its commercial form does not make it possible to obtain a hypoglycemic response close in terms of kinetics to the physiological response generated by the start of a meal (increase in blood sugar), because at the usage concentration (100 IU/mL), in the presence of zinc and other excipients such as phenol or m-cresol, it assembles in the form of a hexamer while it is active in the form of monomer and dimer. Human insulin is prepared in the form of hexamers to be stable for almost 2 years at 4 C because in the form of monomers, it has a very high propensity to aggregate then to fibrillate which causes it to lose its activity.

Furthermore, in this aggregate form, it presents an immunological risk for the patient.

The dissociations of hexamers into dimers and dimers into monomers delay its action by almost 20 minutes compared to a rapid analog insulin (Brangel, et al, Advanced Drug Delivery Review, 35, 1999, 307-335).

[0009] Furthermore, the kinetics of passage of analog insulins into the blood, as well as their kinetics of blood sugar reduction, are not optimal and there is a real need for a formulation having an even shorter action time. in order to approach the kinetics of endogenous insulin secretion in healthy people.

[00010] The company Biodel has proposed a solution to this problem with a formulation of human insulin comprising EDTA and citric acid as described in patent application US200839365. EDTA, through its ability to complex zinc atoms, and citric acid through its interactions with the cationic zones present on the surface of insulin, are described as destabilizing the hexameric form of insulin and thus reducing its absorption time. action.

[00011] However, such a formulation has the particular disadvantage of dissociating the hexameric form of insulin which is the only stable form capable of meeting the stability requirements of pharmaceutical regulations.

[00012] We also know in the name of the applicant, the PCT application W02010/122385 which describes formulations of human insulin or analogue and which makes it possible to resolve the various problems mentioned above by the addition of a substituted polysaccharide comprising carboxyl groups.

[00013] However, the requirements resulting from chronic and intensive use or even pediatric use of such formulations lead those skilled in the art to seek to use excipients whose molar mass and size are as small as possible for facilitate their elimination.

[00014] The polysaccharides described in applications WO 2010/122385A1 and US 2012/094902A1 as excipients are compounds consisting of chains whose lengths are statistically variable and which present a great wealth of possible interaction sites with protein active ingredients.

This richness could lead to a lack of specificity in terms of interaction and a smaller and better defined molecule could make it possible to be more specific on this subject.

[00015] In addition, a molecule with a well-defined skeleton is generally more easily traceable (MS/MS for example) in biological media during pharmacokinetic or ADME experiments (administration, distribution, 2 CA 02889552 2015- 04-24 WO 2014/076423 PCT/FR2013/052736 metabolism, elimination) compared to a polymer which generally gives a very diffuse and noisy signal in mass spectrometry.

[00016] Conversely, it is not excluded that a well-defined and shorter molecule may present a deficit of possible interaction sites with protein active ingredients.

Indeed, due to their reduced size they do not have the same properties as polysaccharide type polymers because there is loss of the polymer effect as demonstrated in the comparative examples of the experimental part, see in particular the tests of solubilization of insulin at the isoelectric point and testing of interaction with a model protein such as albumin.

[00017] Despite these discouraging results, the applicant has succeeded in developing formulations capable of accelerating insulin by using a substituted anionic compound in combination with a polyanionic compound.

[00018] Furthermore, as in the case of the use of polysaccharides, the hexameric nature of insulin is not affected, therefore the stability of the formulations is not affected, as is also confirmed by the examples state of association of human insulin and analogue in circular dichroism in the presence of substituted anionic compound according to the invention.

[00019] The present invention makes it possible to resolve the various problems set out above since it makes it possible in particular to produce a formulation of insulin, human or analog, capable, after administration, of accelerating the passage of human insulin or its analogs into blood and reduce blood sugar more quickly compared to corresponding commercial insulin products.

The invention consists of a composition, in aqueous solution, comprising insulin in hexameric form, at least one substituted anionic compound and a non-polymeric polyanionic compound.

[00021] The term substituted anionic compound means compounds consisting of a saccharide skeleton formed of a discrete number u between 1 and 8 (1 u .15 8) of identical or different saccharide units, linked by identical glycosidic bonds or different, said saccharide units being chosen from the group consisting of hexoses, in cyclic form or in open reduced form, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable.

[00022] In one embodiment, the insulin is in hexameric form.

[00023] In one embodiment, the insulin is human insulin.

3 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [00024] By human insulin is meant an insulin obtained by synthesis or recombination whose peptide sequence is the sequence of human insulin, including allelic variations and counterparts.

[00025] In one embodiment, the insulin is a recombinant human insulin as described in the European Pharmacopoeia and the American Pharmacopoeia.

[00026] In one embodiment, the insulin is an analog insulin.

[00027] By analog insulin is meant a recombinant insulin whose primary sequence contains at least one modification relative to the primary sequence of human insulin.

[00028] In one embodiment, the analog insulin is chosen from the group consisting of insulin lispro (Humaloge), insulin aspart (Novologe, Novorapide) and insulin glulisine (Apidrae).

[00029] In one embodiment, the analog insulin is insulin lispro (Humaloge).

[00030] In one embodiment, the analog insulin is insulin aspart (Novologe, Novorapide).

[00031] In one embodiment, the analog insulin is insulin glulisine (Apidrae).

[00032] In one embodiment, the substituted anionic compound is chosen from substituted anionic compounds, in the isolated state or in a mixture, consisting of a skeleton formed of a discrete number u between 1 and 8 (1 u 8) identical or different saccharide units, linked by identical or different glycosidic bonds, said saccharide units being chosen from the group consisting of hexoses, in cyclic form or in open reduced form, characterized in that they are substituted by:

a) at least one substituent of general formula I:

-[Ri],-[AA]m Formula I = the substituents being identical or different when there are at least two substituents, in which:

= the radical -[AA] designates an amino acid residue, = the radical -R1- being:

4 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 - either a bond and then a = 0, and the amino acid residue -[APt] is directly linked to the skeleton by a G function.

-- either a carbon chain and then a = 1, in C2 to C15 optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function before the reaction with the amino acid, said chain forming with the amino acid residue -[AA] an amide function, and is fixed on the skeleton using a function F resulting from a reaction between a hydroxyl function carried by the skeleton and a function or a substituent carried by the precursor of the radical -R1-, = F is a function chosen from ether, ester or carbamate functions, = G is a carbamate function, = m is equal to 1 or 2, = the degree of substitution of the saccharide units, j, in -[Rda-[AA] , being strictly greater than 0 and less than or equal to 6, 0 <j 5 6 b) and, optionally, one or more substituents -R'1, = the substituent -R'1 being a C2 to C15 carbon chain, optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function in the form of a salt of alkaline cations, said chain being linked to the skeleton by a function F' resulting from a reaction between a hydroxyl function carried by the skeleton and a function or a substituent carried by the precursor of the substituent -R'1, = F' is an ether, ester or carbamate function, = the degree of substitution of the saccharide units, i, in -R'1, being between 0 and 6-j, 0 < i 6- j and, = F and F' are identical or different, = F and G are identical or different, = i+j 6.

= -R'1 identical to or different from -RI-, = The free salifiable acid functions carried by the substituent -R'1 are in the form of salts of alkaline cations, = said identical or different glycosidic bonds being chosen from the group consisting of glycosidic bonds of type (1,1), (1,2), (1,3), (1,4) or (1,6), in an alpha or beta geometry, 5 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [00033} In one embodiment, the substituted anionic compound, in the isolated state or in a mixture, is chosen from substituted anionic compounds consisting of a saccharide skeleton formed of a discrete number u between 1 and 8 (1 u 5.8) of identical or different saccharide units, linked by identical or different glycosidic bonds, said saccharide units being chosen from hexoses, in cyclic form or in open reduced form, characterized:

a) in that they are statistically substituted by:

at least one substituent of general formula I:

Formula I = the substituents being identical or different when there are at least two substituents, in which:

= the radical -[AA]- designates an amino acid residue, said amino acid being chosen from the group consisting of phenylalanine, alpha-methylphenylalanine, 3,4 dihydroxyphenylalanine, tyrosine, alpha-methyltyrosine, 0-methyl-tyrosine, alpha-phenylglycine, 4-hydroxyphenylglycine, 3,5-dihydroxyphenylglycine and their alkaline cation salts, said derivatives being of absolute L or D configuration, -[AA] is fixed on the skeleton of the molecule via a linking arm -R1-- or directly linked to the skeleton by a function G, = -R1- being:

- either a G bond, and then a = 0, - or a carbon chain, and then a = 1, in C2 to C15 optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and carrying at least one acid function before the reaction with the amino acid, said chain forming with the amino acid residue -[AA] an amide bond, and is fixed on the saccharide skeleton using a function F resulting from a reaction between a hydroxyl function carried by the skeleton and a function carried by the precursor of RI, = F is an ether, ester or carbamate function, = G is a carbamate function, = m is equal to 1 or 2, = the degree of substitution, j, in -[(21],--[AA]m being strictly greater than 0 and less than or equal to 6, 0 <j 5 6, 6 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 and, optionally, one or more substituents -W1 = -R'1 being a C2 to C15 carbon chain, optionally substituted and/or comprising at least one heteroatom (such as 0, N and S) and carrying at least one acid function in salt form of alkaline cations said chain being fixed on the saccharide skeleton by a function F' resulting from a reaction between a hydroxyl function carried by the skeleton and a function carried by the precursor of -R'1, = F' is an ether function, ester or carbamate, = the degree of substitution i, in -R'1, being between 0 and 6-j, 0 i < 6-j, and, = -R'1- identical to or different from -R1, = F and F' identical or different, = F' and G identical or different = The free salifiable acid functions are in the form of salts of alkaline cations, b) said identical or different glycosidic bonds being chosen from the group consisting of glycosidic bonds of type (1,1 ), (1,2), (1,3), (1,4) or (1,6), in alpha or beta geometry, c) +j 5. 6 [00034] In one embodiment, m is equal to 1.

[00035] In one embodiment, -R1 and -R'1, identical or different, are a C2 to C8 carbon chain.

[00036] In one embodiment, -R1 and -R'1, identical or different, are a C2 to C4 carbon chain.

[00037] i and j are statistical degrees of substitution and represent the average number of substituents per saccharide unit.

Each saccharide unit having several hydroxyl functions of different reactivity, the distribution of the substituents on the substituted anionic compounds can be different from one saccharide unit to another within the same polyanionic compound.

[00038] In one embodiment 0.3 5 i.

[00039] In one embodiment 0.4 5_ i.

[00040] In one embodiment i 5 3.

[00041] In one embodiment i 2.5.

[00042] In one embodiment 0.3 5 d.

7 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [00043] In one embodiment 0.4 5 j.

[00044] In one embodiment j 5 2.

[00045] In one embodiment j 5. 1.8.

[00046] In one embodiment, we have and j such that 0 < i + j 5 6.

.. [00047] In one embodiment, 0 < i + j 5 5.

[00048] In one embodiment, 0 <i + j 5. 4.

[00049] In one embodiment, 0 <i + j_5 3.

[00050] In one embodiment, 0 <i + j_5 2.5.

[00051] In one embodiment, 0 <i + j 5 2.

I() [00052] In one embodiment, 0.5 _5 i + j 5_ 3.

[00053] In one embodiment, 0.5 5 i + j _5 2.5.

[00054] In one embodiment, 0.5 5 i + j 5 2.

[00055] In one embodiment, 0.6 5 i + j 5. 3.5.

[00056] In one embodiment, 0.8 _5 i + j 5_ 2.5.

[00057] In one embodiment, 0.7 5_ i + j 5 2.5.

[00058] In one embodiment, 0.7 5. i + j _5 2.

[00059] [00060] In one embodiment, 1 < ij 5_ 2.5.

[00061] In one embodiment, 1 <i + j 2.

[00062] In one embodiment, -R1 and -R'l are attached to the skeleton by an ether bond.

[00063] In one embodiment when -R1- is a carbon chain it is directly fixed to the skeleton by an ether bond.

[00064] In one embodiment, when -R1- is a carbon chain it optionally comprises a heteroatom chosen from the group consisting of 0, N and S.

[00065] In one embodiment, -R1- forms an amide bond with the amino acid residue AA, and is directly attached to the skeleton by an ether F function.

.. [00066] In one embodiment, -R1- forms an amide bond with the amino acid residue AA, and is directly attached to the skeleton by a carbamate function F.

[00067] In one embodiment, -R1- forms an amide bond with the amino acid residue AA, and is directly attached to the skeleton by an F ester function.

[00068] In one embodiment, -R1- and -R1 are chosen from the radicals of formulas II and III 8 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 Fts R1 -0* ft'c F14 0 IP R'4 Formula II Formula III, in which;

= o and p Identical or different, greater than or equal to 1 and less than or equal to 12, and = -RI .R'3, and -R'4 identical or different are chosen from the group consisting of a hydrogen atom, a saturated or unsaturated, linear, branched or cyclic C1 to C6 alkyl, a benzyl, a C7 to C10 alkyl-aryl and optionally comprising heteroatoms in the group consisting of 0, N and/or S, or selected functions in the group consisting of carboxylic acid functions, amine, alcohol or thiol.

[00069] In one embodiment, -R1- before attachment to -AA-, is -CH2 COOH.

[00070] In one embodiment, the substituted anionic compounds according to the invention are characterized in that the radical -RI is -C1-12-COOFI.

[00071] In one embodiment, -RI.- before possible attachment to -AA-, comes from citric acid.

[00072] In one embodiment, -R1- before possible attachment to -AA-, is derived from malic acid.

[00073] In one embodiment, -RI comes from citric acid.

[00074] In one embodiment, -RI comes from malic acid.

[00075] In one embodiment, before attachment to -AA, is chosen from the following groups, in which * represents the site of attachment to F 0 9 0H 0 OH OH or their salts of alkaline cations chosen from the group consisting of N / A. or K.

RECTIFIED SHEET (RULE 91) ISA/EP 9 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [00076] In one embodiment, -R'1 is chosen from the following groups, in which * represents the site of attachment to F:

o 4µ 4ç's'i oH oH OH OH OH or their alkaline cation salts chosen from the group consisting of Na + or K.

[00077] In one embodiment, the radical -[AA] is a residue of phenylalanine and its alkaline cation salts of absolute L, D or racemic configuration.

[00078] In one embodiment, the radical -[AA] is a residue of alphamethylphenylalanine and its alkaline cation salts of absolute L, D or racemic configuration.

[00079] In one embodiment, the radical -[AA] is a residue of 3,4 dihydroxyphenylalanine and its alkaline cation salts of absolute L, D or racemic configuration.

[00080] In one embodiment, the radical -[AA] is a residue of tyrosine and its alkaline cation salts of absolute L, D or racemic configuration.

[00081] In one embodiment, the radical -[AA] is a residue of alphamethyl-tyrosine and its alkaline cation salts of absolute L, D or racemic configuration.

[00082] In one embodiment, the radical -[AA] is a residue of 0-methyltyrosine and its alkaline cation salts of absolute L, D or racemic configuration.

[00083] In one embodiment, the radical -[AA] is a residue of alphaphenylglycine and its alkaline cation salts of absolute L, D or racemic configuration.

[00084] In one embodiment, the radical -[AA] is a residue of 4hydroxyphenylglycine and its alkaline cation salts of absolute L, D or racemic configuration.

[00085] In one embodiment, the radical -[AA] is a residue of 3,5dihydroxyphenylglycine and its alkaline cation salts of absolute L, D or racemic configuration.

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [00086] In one embodiment, the radical -[AA] is an amino acid residue in the form of a racemic mixture.

[00087] In one embodiment, the radical -[AA] is an amino acid residue in the form of isolated isomers of absolute D configuration.

[00088] In one embodiment, the radical -[AA] is an amino acid residue in the form of isomers of absolute L configuration, isolated.

[00089] In one embodiment, u is between 1 and 5.

[00090] In one embodiment, u is between 3 and 5.

[00091] In one embodiment, u = 8.

[00092] In one embodiment, u = 7.

[00093] In one embodiment, u = 6.

[00094] In one embodiment, u = 5.

[00095] In one embodiment, u = 4.

[00096] In one embodiment, u = 3.

[00097] In one embodiment, u = 2.

[00098] In one embodiment, u = 1.

.. [00099] In one embodiment, the hexoses are chosen from the group consisting of mannose, glucose, fructose, sorbose, tagatose, psicose, galactose, allose, altrose, talose, idose, gulose, fucose, fuculose, rhamnose, mannitol, sorbitol and galactitol (dulcitol).

[000100] In one embodiment, the glycosidic bonds are of types (1,4) or (1,6).

[000101] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond type (1,1) glycosidic.

[000102] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of different saccharide units chosen from hexoses and linked by a glycosidic bond of type (1,1), said saccharide skeleton being chosen from the group consisting of trehalose and sucrose.

[000103] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond type (1,2) glycosidic.

11 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000104] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed by a discrete number u= 2 of identical or different saccharide units chosen from hexoses linked by a glycosidic bond of type (1,2), said saccharide skeleton being kojibiose.

[000105] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond glycosidic type (1,3).

[000106] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond glycosidic type (1,3), said saccharide skeleton being chosen from the group consisting of nigeriose and laminaribiose.

[000107] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond glycosidic type (1,4).

[000108] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond glycosidic type (1,4), said saccharide skeleton being chosen from the group consisting of maltose, lactose and cellobiose.

[000109] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a glycosidic bond of type (1,6).

[000110] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from hexoses linked by a bond glycosidic type (1,6), said saccharide skeleton being chosen from the group consisting of isomaltose, melibiose and gentiobiose.

[000111] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of identical or different saccharide units chosen from 12 CA 02889552 2015-04 -24 WO 2014/076423 PCT/FR2013/052736 hexoses linked by a glycosidic bond of type (1,6), said saccharide skeleton being isomaltose.

[000112] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of saccharide units, one of which is in cyclic form and the other in open reduced form.

[000113] In one embodiment, the substituted anionic compound is chosen from anionic compounds consisting of a saccharide skeleton formed of a discrete number u=2 of saccharide units, one of which is in cyclic form and the other in open reduced form, said saccharide skeleton being chosen from the group consisting of maltitol and isomaltitol.

[000114] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number 3 5 u < 8 of identical or different saccharide units.

[000115] In one embodiment, the substituted anionic compound according to the invention is characterized in that at least one of the identical or different saccharide units, which make up the saccharide skeleton formed of a discrete number 3 5_ u 5_ 8 of saccharide units, is chosen from the group consisting of hexose units connected by identical or different glycosidic bonds.

[000116] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units, which make up the saccharide skeleton formed of a discrete number 3 5 u 5. 8 of units saccharides, are chosen from hexoses and linked by at least one glycosidic bond of type (1,2).

[000117] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units, which make up the saccharide skeleton formed of a discrete number 3 5 u 5 8 of saccharide units , are chosen from hexoses and linked by at least one glycosidic bond of type (1,3).

[000118] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units, which make up the saccharide skeleton formed of a discrete number 3 5 u 5_ 8 of saccharide units , are chosen from hexoses and linked by at least one glycosidic bond of type (1,4).

[000119] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units, which compose the saccharide skeleton formed of a discrete number 3 5_ u 13 CA 02889552 2015-04 -24 WO 2014/076423 PCT/FR2013/052736 8 of saccharide units, are chosen from hexoses and linked by at least one glycosidic bond of type (1,6).

[000120] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=3 of identical or different saccharide units.

[000121] In one embodiment, the substituted anionic compound according to the invention is characterized in that they comprise at least one saccharide unit chosen from the group consisting of hexoses in cyclic form and at least one .. selected saccharide unit in the group consisting of hexoses in open form.

[000122] In one embodiment, the substituted anionic compound according to the invention is characterized in that the three saccharide units are identical.

[000123] In one embodiment, the substituted anionic compound according to the invention is characterized in that two of the three saccharide units are identical.

[000124] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical saccharide units are chosen from hexoses, two of which are in cyclic form and one in open reduced form and linked by glycosidic bonds of type (1,4).

[000125] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical saccharide units are chosen from hexoses, two of which are in cyclic form and one in open reduced form and linked by glycosidic bonds. of type (1,6).

[000126] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and that the central hexose is linked by a glycosidic bond of type (1, 2) and by a glycosidic bond of type (1,4).

[000127] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and that the central hexose is linked by a glycosidic bond of type (1 ,3) and by a glycosidic bond of type (1,4).

[000128] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and that the central hexose is linked by a glycosidic bond of type (1 ,2) and by a glycosidic bond of type (1,6).

[000129] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses. and that the central hexose is linked by a (1,2) type glycosidic bond and by a (1,3) type glycosidic bond.

[000130] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and that the central hexose is linked by a glycosidic bond of type (1 ,4) and by a glycosidic bond of type (1,6).

[000131] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is erlose.

[000132] In one embodiment, the substituted anionic compound according to the invention is characterized in that the three identical or different saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000133] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltotriose.

[000134] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is isomaltotriose.

[000135] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=4 of identical or different saccharide units.

[000136] In one embodiment, the substituted anionic compound according to the invention is characterized in that the four saccharide units are identical.

[000137] In one embodiment, the substituted anionic compound according to the invention is characterized in that three of the four saccharide units are identical.

[000138] In one embodiment, the substituted anionic compound according to the invention is characterized in that the four saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000139] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltotetraose.

[000140] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and that a terminal hexose is linked by a glycosidic bond of type (1 ,2) and that the others are linked together by a glycosidic bond of type (1,6).

[000141] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses. and linked by a glycosidic bond of type (1,6).

[000142] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=5 of identical or different saccharide units.

[000143] In one embodiment, the substituted anionic compound according to the invention is characterized in that the five saccharide units are identical.

[000144] In one embodiment, the substituted anionic compound according to the invention is characterized in that the five saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000145] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and linked by a glycosidic bond of type (1,4).

[000146] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltopentaose.

[000147] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=6 of identical or different saccharide units.

[000148] In one embodiment, the substituted anionic compound according to the invention is characterized in that the six saccharide units are identical.

[000149] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and linked by a glycosidic bond of type (1,4).

[000150] In one embodiment, the substituted anionic compound according to the invention is characterized in that the six identical or different saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000151] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is nnaltohexaose.

[000152] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=7 of identical or different saccharide units.

16 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000153] In one embodiment, the substituted anionic compound according to the invention is characterized in that the seven saccharide units are identical.

[000154] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and linked by a glycosidic bond of type (1,4).

[000155] In one embodiment, the substituted anionic compound according to the invention is characterized in that the seven saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000156] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltoheptaose.

[000157] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is formed of a discrete number u=8 of identical or different saccharide units.

[000158] In one embodiment, the substituted anionic compound according to the invention is characterized in that the eight saccharide units are identical.

[000159] In one embodiment, the substituted anionic compound according to the invention is characterized in that the identical or different saccharide units are chosen from hexoses and linked by a glycosidic bond of type (1,4).

[000160] In one embodiment, the substituted anionic compound according to the invention is characterized in that the eight saccharide units are hexose units chosen from the group consisting of mannose and glucose.

[000161] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltooctaose.

[000162] In one embodiment, the substituted anionic compound comprising a discrete number of saccharide units is a natural compound.

[000163] In one embodiment, the substituted anionic compound comprising a discrete number of saccharide units is a synthetic compound.

[000164] In one embodiment, the substituted anionic compound according to the invention is characterized in that it is obtained by enzymatic degradation of a polysaccharide followed by purification.

[000165] In one embodiment, the substituted anionic compound according to the invention is characterized in that it is obtained by chemical degradation of a polysaccharide followed by purification.

17 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000166] In one embodiment, the substituted anionic compound according to the invention is characterized in that it is obtained chemically, by covalent coupling lower molecular weight precursors.

[000167] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is sophorose.

[000168] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is sucrose.

[000169] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is lactulose.

[000170] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is maltulose.

[000171] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is leucrose.

[000172] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is rutinose.

[000173] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is isomaltulose.

[000174] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is fucosyllactose.

.. [000175] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is gentianose.

[000176] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is raffinose.

[000177] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is melezitose.

[000178] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is panose.

[000179] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is kestose.

[000180] In one embodiment, the substituted anionic compound according to the invention is characterized in that the saccharide skeleton is stachyose.

[000181] In one embodiment, the polyanionic compound is a non-polymeric polyanionic compound (PNP) whose affinity for zinc is lower than the affinity of insulin for zinc and whose dissociation constant KCica =[PNP compound]r [Ca2+]7[(PNP compound)r-(Ca2+)s] is less than or equal to 10-1'5.

18 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000182] The dissociation constants (Kd) of the different polyanionic compounds with respect to calcium ions are determined by external calibration using an electrode specific for Calcium ions (Mettler Toledo) and a reference electrode.

All measurements are carried out in 150 mM NaCl at pH 7.

Only the concentrations of free calcium ions are determined; calcium ions bound to the polyanionic compound do not induce electrode potential.

[000183] In one embodiment, the polyanionic compound is chosen from the group consisting of polycarboxylic acids and their salts of Na, le, Ca2+ or Mg2+.

[000184] In one embodiment, the polycarboxylic acid is chosen from the group consisting of citric acid, tartaric acid, and their Na, K+, Ca2+ or Mg salts.

[000185] In one embodiment, the polyanionic compound is chosen from the group consisting of phosphoric polyacids and their salts of Na, le, Ca2+ or Mg2+.

[000186] In one embodiment, the polyphosphoric acid is triphosphate and its salts of Na, K+, Ca2+ or Mg2+.

[000187] In one embodiment, the polyanionic compound is citric acid and its Na, K+, Ca2+ or Mg2+ salts.

[000188] In one embodiment, the polyanionic compound is tartaric acid and its salts of Na, K+, Ca2+ or Mg2+.

[000189] In one embodiment, the polyanionic compound is triphosphoric acid and its salts of Na, K+, Ca2+ or Mg2+.

[000190] In one embodiment, the polyanionic compound is a compound consisting of a saccharide skeleton formed of a discrete number of saccharide units obtained from a disaccharide compound chosen from the group consisting of trehalose, maltose , lactose, sucrose, cellobiose, isomaltose, maltitol and isomaltitol.

[000191] In one embodiment, the polyanionic compound consisting of a saccharide skeleton formed of a discrete number of saccharide units is obtained from a compound consisting of a skeleton formed of a discrete number of units saccharides chosen from the group consisting of maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose and isomaltotriose 19 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000192] In one embodiment, the polyanionic compound consisting of a saccharide skeleton formed of a discrete number of units saccharides is chosen from the group consisting of carboxymethylmaltotriose, carboxymethylmaltotetraose, carboxymethylmaltopentaose, carboxymethylmaltohexaose, carboxymethylmaltoheptaose, carboxymethylmaltooactose and carboxymethylisomaltotriose.

[000193] In one embodiment, the ratio (number of moles of acid functions carried by the polyanionic compound/number of moles of anionic compound) is greater than or equal to 3.

[000194] In one embodiment, the ratio (number of moles of acid functions carried by the polyanionic compound/number of moles of anionic compound) is greater than or equal to 4.

[000195] In one embodiment, the ratio (number of moles of acid functions carried by the polyanionic compound consisting of a saccharide skeleton/number of moles of anionic compound) is greater than or equal to 5.

[000196] In one embodiment, the ratio (number of moles of acid functions carried by the polyanionic compound consisting of a saccharide skeleton/number of moles of anionic compound) is greater than or equal to 8.

[000197] In one embodiment, the substituted anionic compound/insulin molar ratios are between 0.6 and 75.

[000198] In one embodiment, the molar ratios are between 0.7 and 50.

[000199] In one embodiment, the molar ratios are between 1.4 and 35.

[000200] In one embodiment, the molar ratios are between 1.9 and 30.

[000201] In one embodiment, the molar ratios are between 2.3 and 30.

[000202] In one embodiment, the molar ratio of substituted anionic compound/insulin is equal to 8.

[000203] one embodiment, the molar ratio of substituted anionic compound/insulin is equal to 12.

[000204] In one embodiment, the molar ratio of substituted anionic compound/insulin is equal to 16.

[000205] In one embodiment, the substituted anionic compound/insulin mass ratios are between 0.5 and 10.

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000206] In one embodiment, the mass ratios are between 0.6 and 7.

[000207] In one embodiment, the mass ratios are between 1.2 and 5.

[000208] In one embodiment, the mass ratios are between 1.6 and 4.

[000209] In one embodiment, the mass ratios are between 2 and 4.

[000210] In one embodiment, the substituted anionic compound/insulin mass ratio is 2.

[000211] In one embodiment, the substituted anionic compound/insulin mass ratio is 3.

[000212] In one embodiment, the substituted anionic compound/insulin mass ratio is 4.

[000213] In one embodiment, the substituted anionic compound/insulin mass ratio is 6 [000214] In one embodiment, the concentration of substituted anionic compound is between 1.8 and 36 mg/mL.

[000215] In one embodiment, the concentration of substituted anionic compound is between 1.8 and 36.5 mg/mL.

[000216] In one embodiment, the concentration of substituted anionic compound is between 2.1 and 25 mg/mL.

[000217] In one embodiment, the concentration of substituted anionic compound is between 4.2 and 18 mg/mL.

[000218] In one embodiment, the concentration of substituted anionic compound is between 5.6 and 15 mg/mL.

[000219] In one embodiment, the concentration of substituted anionic compound is between 7 and 15 mg/mL.

[000220] In one embodiment, the concentration of substituted anionic compound is 7.3 mg/mL.

[000221] In one embodiment, the concentration of substituted anionic compound is 10.5 mg/mL.

[000222] In one embodiment, the concentration of substituted anionic compound is 14.6 mg/mL.

[000223] In one embodiment, the concentration of substituted anionic compound is 21.9 mg/mL.

21 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000224] In one embodiment, the concentration of polyanionic compound is between 2 and 150 mM.

[000225] In one embodiment, the concentration of polyanionic compound is between 2 and 100 mM.

[000226] In one embodiment, the concentration of polyanionic compound is between 2 and 75 mM.

[000227] In one embodiment, the concentration of polyanionic compound is between 2 and 50 mM.

[000228] In one embodiment, the concentration of polyanionic compound is between 2 and 30 mM.

[000229] In one embodiment, the concentration of poiyanionic compound is between 2 and 20 mM.

[0002301 In one embodiment, the concentration of polyanionic compound is between 2 and 10 mM.

[000231] In one embodiment, the concentration of polyanIonic compound is between 5 and 150 mM.

[0002321 In one embodiment, the concentration of polyanionic compound is between 5 and 100 mM.

[000233] In one embodiment, the concentration of polyanionic compound is between 5 and 75 mM.

[000234] In one embodiment, the concentration of polyanionic compound is between 5 and 50 mM.

[000235] In one embodiment, the concentration of polyanionic compound is between 5 and 30 mM.

[000236] In one embodiment, the concentration of polyanionic compound is between 5 and 20 mM.

[000237] In one embodiment, the concentration of polyanionic compound is between 5 and 10 mM.

[000238] In one embodiment, the concentration of polyanionic compound is between 0.5 and 30 mg/mL.

[0002391 In one embodiment, the concentration of polyanionic compound is between 0.5 and 25 mg/mL.

[000240] In one embodiment, the concentration of polyanionic compound is between 0.5 and 10 mg/mL.

[000241] In one embodiment, the concentration of polyanionic compound is between 0.5 and 8 mg/mL.

RECTIFIED SHEET (RULE 91) ISA/EP 22 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000242] In one embodiment, the concentration of polyanlonlque compound is between 1 and 30 mg/mL.

[000243] In one embodiment, the concentration of polyanionic compound is between 1.5 and 2.5 mg/mL.

[000244] In one embodiment, the concentration of polyanionic compound is between 2 and 25 mg/mL.

[000245] In one embodiment, the concentration of polyanionic compound is between 2 and 10 mg/mL.

[000246] In one embodiment, the concentration of polyanionic compound is between 2 and 8 mg/mL.

[000247] In one embodiment, the substituted anionic compound is sodium maltotrlosemethylcarboxylate modified with sodium phenyielaninate, u = 3, i 0.65, j = 1.0.

[000248] In one embodiment, the substituted anionic compound is sodium maltotriosemethylcarboxylate modified with sodium phenylalaninate, u = 3, I = 1.0, j = 0.65.

[000249] In one embodiment, the substituted anionic compound is sodium maltotriosemethylcarboxylate modified with sodium phenialaninate, u = 3, I = 0.46, j -= 1.2.

[000250] In one embodiment, the substituted anlonic compound is sodium maltotrlosemethylcarboxylate modified with sodium phenyialaninate, u .. ¨ 3, i = 0.35, j = 0.65.

[000251] In one embodiment, the polyanionic compound is sodium maltotriosemethylcarboxylate.

[000252] In one embodiment, the polyanionic compound is sodium citrate.

[000253) In one embodiment, the polyanionic compound is triphosphate in acid form or in basic form 50U5 sodium salt or potassium salt form.

In one embodiment, the polyanionic compound is tartrate, in acid form or in basic form in the form of sodium salt or potassium salt [000254] The invention also relates to a pharmaceutical formulation of insulin comprising a composition according to Invention in which insulin is in hexameric form.

RECTIFIED SHEET (RULE 91) ISA/EP 23 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000255] In one embodiment, it relates to a pharmaceutical formulation characterized in that the insulin concentration is included between 240 and 3000 pM (40 to 500 IU/mL).

[000256] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is between 600 and 3000 pM (100 to 500 IU/mL).

[000257] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is between 600 and 2400 pM (100 to 400 IU/mL).

[000258] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is between 600 and 1800 pM (100 to 300 IU/mL).

[000259] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is between 600 and 1200 pM (100 to 200 IU/mL).

[000260] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is 600 pM (100 IU/mL).

[000261] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is 1200 pM (200 IU/mL).

[000262] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is 1800 pM (300 IU/mL).

[000263] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is 2400 pM (400 IU/mL).

[000264] In one embodiment, it concerns a pharmaceutical formulation characterized in that the insulin concentration is 3000 pM (500 IU/mL).

[000265] The invention relates to the use of at least one substituted anionic compound, said compound consisting of a saccharide skeleton formed of a discrete number u between 1 and 8 (1 ...5. u 5. 8) identical or different saccharide units, linked by identical or different glycosidic bonds, said saccharide units being chosen from the group consisting of hexoses, in cyclic form or in open reduced form, said compound comprising partially substituted carboxyl functional groups , the unsubstituted carboxyl functional groups being salifiable to prepare a pharmaceutical formulation of human insulin, in combination with a polyanionic compound, allowing, after administration, to accelerate the passage of insulin into the blood and reduce blood sugar more quickly compared to a formulation free of substituted anionic compound, and possibly anionic compounds.

[000266] In one embodiment, the invention relates to the use of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, to prepare a pharmaceutical formulation of human insulin, in combination with a polyanionic compound, allowing, after administration, to accelerate the passage of human insulin into the blood and to reduce blood sugar more quickly compared to a formulation free of substituted anionic compound, and possibly of anionic compounds.

[000267] In one embodiment, the invention relates to the use of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, to prepare a formulation of analog insulin, in combination with a polyanionic compound, allowing, after administration, to accelerate the passage of analog insulin into the blood and to reduce blood sugar more quickly compared to a formulation free of substituted anionic compound, and possibly of anionic compounds.

[000268] In one embodiment, the insulin is human insulin.

[000269] By human insulin is meant an insulin obtained by synthesis or recombination whose peptide sequence is the sequence of human insulin, including allelic variations and homologs.

[000270] In one embodiment, the insulin is a recombinant human insulin as described in the European Pharmacopoeia and the American Pharmacopoeia.

[000271] In one embodiment, the insulin is an analog insulin.

[000272] By analog insulin is meant a recombinant insulin whose primary sequence contains at least one modification relative to the primary sequence of human insulin.

[000273] In one embodiment the analog insulin is chosen from the group consisting of insulin lispro (Humalogs), insulin aspart (Novolog, Novorapid) and insulin glulisine (Apidre).

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000274] In one embodiment, the analog insulin is insulin Ilspro uma loge).

[000275] In one embodiment, the insulin analogue is insulin aspart (Novologe, Novorapide).

[000276] In one embodiment, the insulin analogue is insulin glulisine (Ap id ray).

[000277] In one embodiment, the use is characterized in that the substituted anionic compound is chosen from substituted anionic compounds in the isolated state or in a mixture, consisting of a saccharide skeleton formed of a discrete number u between 1 and 8 (1 to 8) of identical or different saccharide units, linked by identical or different glycosidic bonds, said saccharide units being chosen from hexoses, in cyclic form or in open reduced form, characterized in that they are substituted by:

a) at least one substituent of general formula I:

Formula I = the substituents being identical or different when there are at least two substituents, in which;

= the radical -[AA] designates an amino acid residue, = the radical -R1- being - either a bond and then a - 0, and the amino acid residue -[AA}- is directly linked to the skeleton by a G function.

- either a carbon chain and then a - 1, in C2 to C15 optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function before the reaction with the amino acid, said chain forming with the amino acid residue -[AAJ- an amide function, and is fixed on the skeleton using a function F resulting from a reaction between a hydroxyl function carried by the skeleton and a function or a substituent carried by the precursor of the radical -Ri-, = F is a function chosen from ether, ester or carbamate functions, = G is a carbamate function, = m is equal to 1 or 2, RECTIFIED SHEET (RULE 91) ISA/EP 26 CA 02889552 2015-04-24 WO 2014 /076423 PCT/FR2013/052736 = the degree of substitution of the saccharide units, j, in -[Ri]a-[AA]on being strictly greater than 0 and Less than or equal to 6.0 js 6 b) and, optionally, one or more substituents = the substituent -R'1 being a C2 to C15 carbon chain, optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function in the form of a salt of alkaline cations, said chain being linked to the skeleton by a function F' resulting from a reaction between a hydroxyl function carried by the skeleton and a function or a substituent carried by the precursor of the substituent -R'1, = F' is an ether, ester, or carbamate function, = the degree of substitution of the saccharide units, I, in -R'1 , being between 0 and 6-j, OeI 6-j and, = F and F' are identical or different, = F and G are identical or different, = i+i 6.

= -R'1 identical to or different from -R1-, = The free salifiable acid functions carried by the -R1 substituent are in the form of salts of alkaline cations, = said identical or different glycosidic bonds being chosen from the group consisting of the bonds glycosidic compounds of type (1,1), (1,2), (1,3), (1,4) or (1,6), in an alpha or beta geometry, (0002781 In one embodiment, the use is characterized in that the substituted anionic compound, in the isolated state or in a mixture, is chosen from substituted anionic compounds consisting of a sacchariclic skeleton formed of a discrete number u between 1 and 8 (1 to 8) of identical or different saccharic units, linked by identical or different glycosidic bonds, said saccharide units being chosen from hexoses, in cyclic form or in open reduced form, characterized:

a) in that they are statistically substituted by;

at least one substituent of general formula I -[Rda-PALõ, Formula I RECTIFIED SHEET (RULE 91) ISA/EP 27 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 = the substituents being identical or different when 'there are at least two substituents, in which:

= the radical -[AM- denotes an amino acid residue, said amino acid being chosen from the group consisting of phenylalanine, alpha-methyl phenylalanine, 3,4 dihydroxyphenylalanine, tyrosine, alpha-methyltyrosine, 0-methyl-tyrosine, alpha-phenylg lycine, 4hydroxyphenylglycine, 3,5-dihydroxyphenylglycine and their alkaline cation salts, said derivatives being of absolute L or D configuration, -[AM- is fixed on the skeleton of the molecule via a linking arm -R1- or directly linked to the skeleton by a function G, = -R1- being - either a bond G, and then has 0 , - either a carbon chain, and then a 1, in C2 to C15 optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and carrying at least one acid function before the reaction with the amino acid, said chain forming with the amino acid residue -CAM- an amide bond, and is fixed on the saccharide skeleton using a function F resulting from a reaction between a hydroxyl function carried by the skeleton and a function carried by the precursor of RI, = F is an ether, ester or carbamate function, = G is a carbamate function, = m is equal to 1 or 2, = the degree of substitution, J, in --(114)3-IAALI being strictly greater than 0 and less than or equal to 6, 0 <i 6, and eventually, one or more substituents -R't = -R't being a C2 to C15 carbon chain, optionally substituted and/or comprising at least one heteroatom (such as 0, N and S) and carrying at least one acid function in the form of alkaline cation salt, said chain being fixed on the saccharide skeleton by a function F' resulting from a reaction between a hydroxyl function carried by the skeleton and a function carried by the precursor of -R'1, = F' is a function ether, ester or carbamate, = the degree of substitution 1, in -R'1, being between 0 and 6-j, 0 ei 6-j, and, = -R't- identical to or different from -R1, RECTIFIED SHEET (RULE 91) ISA /EP 28 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 = F and F' identical or different, = F' and G Identical or different = The free salifiable acid functions are in the form of salts of alkaline cations , b) said identical or different glycosiclic bonds being chosen from the group consisting of glycosIdic bonds of type (1.1), (1.2), (1.3), (1.4) or (1.6), in an alpha or beta geometry, c) i +j 5 6 [000279] In one embodiment, m is equal to 1.

[000280] In one embodiment, -R1 and -R'1, identical or different, are a C1 to C8 carbon chain.

[000281] In one embodiment, -R1 and -R'1, identical or different, are a C1 to C4 carbon chain.

[000282] In one embodiment, -R1 and -RI, identical or different, are a C1 to C2 carbon chain.

[000283] It is known to those skilled in the art that the time of action of insulins is dependent on the insulin concentration.

Only the onset of action values ​​for the 100 IU/mL formulations are documented.

[000284] Regular human insulin formulations on the market at a concentration of 600 el (100,111/mL) have an onset of action of between 50 and 90 minutes and an end of action of approximately 360 to 420 minutes. in humans.

The time to reach peak insulin concentration in the blood is between 90 and 180 minutes in humans.

[000285] Rapid insulin analogue formulations on the market at a concentration of 600 pM (100 IU/mL) have an onset of action of between 30 and 60 minutes and an end of action of approximately 240-300 minutes. in humans.

The time to reach maximum insulin concentration in the blood is between 50 and 90 minutes in humans.

[000286] The invention also relates to a method for preparing a formulation of human insulin having an insulin concentration of between 240 and 3000 uM (40 and 500 IU/mL), whose action time for humans is lower than that of the reference formulation at the same insulin concentration in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step d addition to said formulation of at least one substituted anionic compound, said compound comprising partially substituted RECTIFIED SHEET (RULE 91) ISA/EP 29 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000287] In one embodiment, the insulin is in hexameric form.

.. [000288] [000289] The invention also relates to a method of preparing a formulation of human insulin having an insulin concentration of between 600 and 1200 pM (100 and 200 IU/mL), the time of which action in humans is inferior.. to that of the reference formulation at the same insulin concentration in the absence of substituted anionic compound and of polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one anionic compound substituted, said compound comprising partially substituted carboxyl functional groups, the carboxyl functional groups not.. substituted being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000290] In one embodiment, the insulin is in hexameric form.

[000291] The invention also relates to a method of preparing a... formulation of human insulin having an insulin concentration of 600 pM (100 IU/mL), whose onset of action in humans is less than 60 minutes, characterized in that it comprises (1) an addition step to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation at least one polyanionic compound.

[000292] In one embodiment, the insulin is in hexameric form.

[000293] The invention also relates to a method for preparing a formulation of human insulin having an insulin concentration of 1200 pM (200 IU/mL), the onset of action of which in humans is less than at least less 10% to that of the formulation of human insulin at the same concentration (200 IU/mL) and in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of addition to said formulation of at least one.. substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000294] In one embodiment, the insulin is in hexameric form.

[000295] The invention also relates to a method for preparing a formulation of human insulin having an insulin concentration of 1800 pM (300 IU/mL), whose onset of action in humans is at least 100/0 less than that of the formulation of human insulin at the same concentration (300 IU/mL) and in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000296] In one embodiment, the insulin is in hexameric form.

[000297] The invention also relates to a method for preparing a formulation of human insulin having an insulin concentration of 2400 pM (400 IU/mL), whose onset of action in humans is at least 10% less than that of the formulation of human insulin at the same concentration (400 IU/mL) and in the absence of substituted anionic compound and compound polyanionic characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000298] In one embodiment, the insulin is in hexameric form.

[000299] The invention also relates to a method for preparing a formulation of human insulin having an insulin concentration of 3000 pM (500 IU/mL), whose onset of action in humans is at least 10% less than that of the formulation of human insulin at the same concentration (500 IU/mL) and in the absence of substituted anionic compound and compound polyanionic characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000300] In one embodiment, the insulin is in hexameric form.

31 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 [000301] The invention consists of the preparation of a so-called rapid human insulin formulation characterized in that it comprises (1) a step adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation at least one polyanionic compound.

[000302] In one embodiment, the insulin is in hexameric form.

[000303] The invention also relates to a method for preparing a formulation of human insulin at a concentration of 600 pM (100 IU/mL) whose onset of action in humans is less than 60 minutes, preferably less than 45 minutes, and more preferably less than 30 minutes characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000304] In one embodiment, the insulin is in hexameric form.

[000305] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of between 240 and 3000 pM (40 and 500 IU/mL), whose action time in humans is less than that of the reference formulation at the same insulin concentration in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step d addition to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation at least one polyanionic compound.

[000306] In one embodiment, the insulin is in hexameric form.

[000307] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of between 600 and 1200 pM (100 and 200 IU/mL), including the onset of action in humans. is lower than that of the reference formulation at the same concentration of analogue insulin in the absence of substituted anionic compound substituted anionic compound and 32 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 polyanionic compound , characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000308] In one embodiment, the insulin is in hexameric form.

[000309] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of 600 pmol/L (100 IU/mL), the onset of action of which in humans is less than 30 minutes, characterized in that it comprises (1) a step of adding to said formulation of at least one substituted anionic compound, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000310] In one embodiment, the insulin is in hexameric form.

[000311] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of 1200 pM (200 IU/mL), the onset of action of which in humans is less than at least least 10% to that of the analogous insulin formulation in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one anionic compound substituted, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000312] In one embodiment, the insulin is in hexameric form.

[000313] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of 1800 pM (300 IU/mL), the onset of action of which in humans is less than at least least 10% to that of the analogous insulin formulation in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one anionic compound substituted, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000314] In one embodiment, the insulin is in hexameric form.

[000315] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of 2400 pM (400 IU/mL), the onset of action of which in humans is less than at least least 10% to that of the analogous insulin formulation in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one anionic compound substituted, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000316] In one embodiment, the insulin is in hexameric form.

[000317] The invention also relates to a method for preparing an analog insulin formulation having an insulin concentration of 3000 pM (500 IU/mL), the onset of action of which in humans is less than at least least 10% to that of the analogous insulin formulation in the absence of substituted anionic compound and polyanionic compound characterized in that it comprises (1) a step of adding to said formulation of at least one anionic compound substituted, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable, and (2) a step of adding to said formulation of at least one polyanionic compound.

[000318] In one embodiment, the insulin is in hexameric form.

[000319] The invention consists of the preparation of a so-called very rapid analog insulin formulation, characterized in that it comprises a step of adding to said formulation at least one substituted anionic compound, said compound comprising groups partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable [000320] In one embodiment, the preparation further comprises a step of adding to said formulation of at least one polyanionic compound.

[000321] In one embodiment, the insulin is in hexameric form.

[000322] In one embodiment the analog insulin is chosen from the group consisting of insulin lispro (Humalog), insulin aspart (Novolog, Novorapide) and insulin glulisine (Apidrae).

34 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000323] In one embodiment, the analog insulin is insulin lispro (Humalog).

[000324] In one embodiment, the analog insulin is insulin aspart (Novolog, Novorapid).

.. [000325] In one embodiment, the analog insulin is insulin glulisine (Apidra).

[000326] In one embodiment, the insulin is a recombinant human insulin as described in the European Pharmacopoeia and the American Pharmacopoeia.

[000327] In one embodiment, the insulin is an analog insulin chosen from the group consisting of insulin lispro (Humalog0), aspart (Novolog, NovorapidS) and insulin glulisine (Apidra).

[000328] The composition can also be produced by simply mixing an aqueous solution of human insulin or the like and an aqueous solution of substituted anionic compound mixed with a polyanionic compound.

[000329] In one embodiment, the composition can be produced by simply mixing an aqueous solution of human insulin or the like, an aqueous solution of substituted anionic compound and polyanionic compound in solution or in lyophilisate form.

[000330] In one embodiment, the composition can be produced by simply mixing an aqueous solution of human insulin or the like, a substituted anionic compound in the form of a lyophilisate and a polyanionic compound in solution or in the form of a lyophilisate.

[000331] Preferably this composition is in the form of an injectable solution.

[000332] In one embodiment, the concentration of human insulin or analog is between 240 and 3000 pM (40 to 500 IU/mL).

[000333] In one embodiment, the concentration of human insulin or analogue is between 600 and 3000 pM (100 to 500 IU/mL).

[000334] In one embodiment, the concentration of human insulin or analogue is between 600 and 2400 pM (100 to 400 IU/mL).

[000335] In one embodiment, the concentration of human insulin or analogue is between 600 and 1800 pM (100 to 300 IU/mL).

[000336] In one embodiment, the concentration of human insulin or analogue is between 600 and 1200 pM (100 to 200 IU/mL).

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000337] In one embodiment, the concentration of human insulin or analog is 600 pM (100 IU/mL).

[000338] In one embodiment, the concentration of human insulin or analogue is 1200 pM (200 IU/mL).

[000339] In one embodiment, the concentration of human insulin or analogue of 600 pM (100 IU/mL) can be reduced by simple dilution, in particular for pediatric applications.

[000340] In one embodiment, the concentration of human insulin or analogue is 1800 pM (300 IU/mL).

[000341] In one embodiment, the concentration of human insulin or analogue is 2400 pM (400 IU/mL).

[000342] In one embodiment, the concentration of human insulin or analogue is 3000 pM (500 IU/mL).

[000343] The invention also relates to a pharmaceutical formulation according to the invention, characterized in that it is obtained by drying and/or lyophilization.

[000344] In one embodiment, the compositions according to the invention further comprise the addition of zinc salts at a concentration of between 0 and 500 pM.

[000345] In one embodiment, the compositions according to the invention further comprise the addition of zinc salts at a concentration of between 0 and 300 pM.

[000346] In one embodiment, the compositions according to the invention further comprise the addition of zinc salts at a concentration between 0 and 200 pM.

[000347] In one embodiment, the compositions according to the invention comprise buffers at concentrations of between 0 and 100 mM, preferably between 0 and 50 mM or between 15 and 50 mM.

[000348] In one embodiment the buffer is Tris.

[000349] In one embodiment, the compositions according to the invention further comprise preservatives.

[000350] In one embodiment, the preservatives are chosen from the group consisting of m-cresol and phenol alone or as a mixture.

[000351] In one embodiment, the concentration of the preservatives is between 10 and 50 mM.

[000352] In one embodiment, the concentration of the preservatives is between 10 and 40 mM.

36 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000353] The compositions according to the invention may further comprise additives such as tonic agents such as glycerin, sodium chloride (NaCl), mannitol and glycine.

[000354] The compositions according to the invention may also comprise additives conforming to the pharmacopoeias such as surfactants, for example POLysorbate.

[000355] The compositions according to the invention may further comprise all the excipients conforming to the pharmacopoeias and compatible with the insulins used at the usual concentrations.

[000356] In the case of local and systemic release, the modes of administration envisaged are intravenous, subcutaneous, intradermal or intramuscular.

[000357] Transdermal, oral, nasal, vaginal, ocular, oral and pulmonary administration routes are also envisaged.

[000358] The invention also relates to the use of a composition according to the invention for the formulation of a solution of human insulin or analogue with a concentration of 100 IU/mL intended for implantable or transportable insulin pumps.

[000359] The invention also relates to the use of a composition according to the invention for the formulation of a solution of human insulin or analogue with a concentration of 200 IU/mL intended for implantable or transportable insulin pumps.

[000360] The invention also relates to substituted anionic compounds, in the isolated state or in a mixture, chosen from substituted anionic compounds consisting of a saccharide skeleton formed of a discrete number u between 1 and 8 (1 u 5 8) identical or different saccharide units, linked by identical or different glycosidic bonds, said saccharide units being chosen from hexoses, in cyclic form or in open reduced form, characterized in that they are substituted by a) at least one substituent of general formula Formula = the substituents being identical or different when there are at least two substituents, in which:

= the radical -[AA] designates an amino acid residue, = the radical -R1- being 1 RECTIFIED SHEET (RULE 91) ISA/EP 37 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 - or a bond and then a = 0, and the amino acid residue -(AM- is directly linked to the backbone by a G function.

- either a carbon chain and then a = 1, in C2 to C15 optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function before the reaction with the amino acid, said chain forming with the amino acid residue -[AA]- an amide function, and is fixed on the skeleton using a function F resulting from a reaction between a hydroxyl function carried by the skeleton and a function or a substituent carried by the precursor of the radical -R1-, = F is a function chosen from the ether, ester or carba mate functions, = G is a carbarnate function, = m is equal to 1 or 2, = i degree of substitution of the saccharide units, j, in -E,R11,- [AA],õ, -being strictly greater than O and less than or equal to 6, 0 <j 6 b) and, optionally, one or more substituents -R'1, = the substituent being a C2 to C1 carbon chain. 5, optionally substituted and/or comprising at least one heteroatom chosen from 0, N and S and at least one acid function in the form of a salt of alkaline cations, said chain being linked to the skeleton by a function F' resulting from a reaction between a hydroxyl function carried by the skeleton and a function with a substituent carried by the precursor of the substituent = F is an ether, ester or carbamate function, = the degree of substitution of the saccharide units, i, being between 0 and 6-j, 0 i 61 and, = F and F' are identical or different, = F and G are the same or different, = i+j s. 6.

= Identical or different from -R1-, = The free salifiable acid functions carried by the substituent -R'1 are in the form of salts of alkaline cations, = said identical or different glycosidic bonds being chosen from the group consisting of the glycosidic bonds of type (1,1), (1,2), (1,3), (1,4) or (1,6), in alpha or beta geometry, RECTIFIED SHEET (RULE 91) ISA/EP 38 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 = [000361] In the formula above the different variables have the values ​​cited above.

[000362] The substituted anionic compounds according to the invention can be obtained by random grafting of the substituents onto the saccharyl skeleton.

[000363] In one embodiment, the substituted anionic compounds chosen from anionic compounds substituted by substituents of formulas I or II are characterized in that they can be obtained by grafting the substituents at precise positions on the saccharide units by a process implementing steps for protection/deprotection of alcohol or carboxylic acid groups naturally carried by the skeleton.

This strategy leads to selective grafting, in particular regoselectlf, of the substituents on the backbone.

Protective groups include without limitation those described in the work (Wuts, PGM et al., Greene's Protective Groups in OrganIc Synthesis 2007).

[000364] The saccharide skeleton can be obtained by degradation of a high molecular weight polysaccharide.

Degradation pathways include, but are not limited to, chemical degradation and/or enzymatic degradation.

[000365] The saccharide skeleton can also be obtained by formation of glycosidic bonds between monosaccharide or oligosaccharide molecules using a chemical or enzymatic coupling strategy.

The coupling strategies include those described in the publication (Smoot, Ji- et al., Advances in Carbohydrate Chemistry and Blochemistry 2009, 62, 162-250) and in the book (Lindhorst, TK, Essentials of Carbohydrate Chemistry and Blochemistry 2007 , 157208).

Coupling reactions can be carried out in solution or on a solid support.

The saccharide molecules before coupling can carry substituents of interest and/or be functionalized once coupled together in a statistical or regioselective manner.

[000366] Thus, by way of examples, the compounds according to the invention can be obtained according to one of the following processes:

= the statistical grafting of the substituents onto a saccharide skeleton = one or more glycosylation steps between monosaccharide or oligosaccharide molecules carrying substituents = one or more glycosylation steps between one or more monosaccharide or oligosaccharide molecules carrying substituents and one or more mono molecules -saccharlic or oligosaccharides it one or more steps of introduction of protective groups on alcohols or acids naturally carried by the saccharide skeleton followed by one or more grafting reactions of the substituents and finally a step of elimination of protective groups = one or more glycosylation steps between one or more monosaccharide or oligosaccharide molecules carrying protective groups on alcohols or acids naturally carried by the skeleton saccharide, one or more steps of grafting substituents onto the skeleton obtained then a step of eliminating the protective groups = one or more steps of glycosylation between one or more monosaccharide or oligosaccharide molecules carrying protective groups on alcohols or acids naturally carried by the saccharide skeleton, and one or more mono-saccharide or oligosaccharide molecules, one or more steps of grafting of substituents then a step of eliminating the protective groups.

[000367] The compounds according to the invention, isolated or in a mixture, can be separated and/or purified in different ways, in particular after obtaining them by the processes described above.

[000368] We can in particular cite chromatographic methods, in particular those called preparative or preparative methods such as:

= flash chromatographies or flash chromatography, in particular on silica, and = Chromatographies of the HPLC (high performance liquid chromatography) type (high performance liquid chromatography), in particular RP-HPLC or reverse phase HPLC (high performance liquid chromatography in reverse phase) .

[000369] Selective precipitation methods can also be used.

[000370] The invention is illustrated by the following examples.

Examples 0 t=J o [000371] The structures of the substituted anionic compounds according to the invention are presented in Table 1.

The structures of the ti sa polysaccharide counterexamples are presented in Table 2. ,at 0, and=.) ta AA Substituted anionic compounds R = H, R'1, -[Ri]a-[M]rn Sequence Substitutant Substituent Compound ij ............................................. ........ saccharide -R'1 -1111.211.M1m ______________ P o 0 obe P 0 0) ,0 . j'>==õ :õ.../...., =õ4. = 4, ei m+ 1 0.65 1.0 ..,;:"/S.:'..-e,-;/=.,%.,---( .

. o i",,, 1.9u1 m., ...õ,..

= ,¨.

0 op (A 11P11 io 1 i ro ves....N7ONa eem:

L.,,./:>, L-,t> '.-,-""7, k 2 1,0 0.65 ...-,;5,,,,: e'5,-Ab\ -,;', -4, 0 ...".4,4Q I 1 1 ee" ida ________________________________________________________________________________ ______________ 0 ell r5 i 7,...4%.ye,ONI, tt,s.icil ei .e. 4. Y4 4 Otga , = 4 n: µ 3 0.46 1 1.2 , -- µ.....

6 .., 4, its . , ..) 0, CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 0 , i ¨ 0----- / / ..tZ iZ xz :rz zz 0 K\ _________________________________ 0 / .ç \ , ...., :

8 1 0 .

\ _____________________ VS\ .

______________________ . __________ . __________ .

( , ........... ,... ........ .,. .................. ..... 7 4 1 4 .....µ -:>,\ ) .;......-,...1 t , es :e 1 e.-.):... .r,...

ly Ir \ , = \ , e......, s, e.) .\-.)E, eµ= A ,., $ , te :et . _____________________________________________________________________ .....õ.........________............................_ in in in iti tO 14' Wa iD r.

this you this this.

sort in 1/1 rel eNI 0 CO ce il) ni .4.

E---- _________________________________ - ¨ nt in vo N. co ................ ¨ __________________________________ .. ___________ 42 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013 /052736 1.--g' -6 \ _________________________________ g.

0 - .

= " ... .... e. <!---µ0 'µ.-.....nn ce .,. ,....... , .1 fr 0-4\ ... .0 / ---_, .,,.., :g .

.6 g _... : ) . 0 =...,0 =--..*=0 1S e')''-'1:\'' r^t D it3 iç. .N li :ai 05 õ =f- Nise <it. I¨ ,...,i.à= t ¨1 µ.K? 3:=-==1V.

. .

64,e' / -..

.,.,..c.4 e.---)t..

Y = .

k. .

e _____________ ---+in 1 ,-1 rv `D.. op in aeea cri (Ni .-7 cr.) ce .....

.4.

,..............................................................................

................................. ........._....., 1 . I 1 cr, a 1-1 =e4 rI . . . = 1 43 0 to,J AB Polysaccharides counterexamples o i.., e sa ,à Molar mass 1 0, e to..) Polysaccharides Substituting 1 Substituting cm I j Saccharidic chain average by weight I counterexamples ' - R '1 1 - [Rda-(Mln, I. (kg/mol) i --.. Counterexample polysaccharides AB1, AB2 AB3, AB4 and AB5: R = H, R'1, ERILIAAim AP 0 0 co:N: 0Ma to t 7..-/\..:,..\ µ ( (o 0 ' / i til Ui AB1 0.6 0.46 10 oo I.) RO OR t '-== RO i 1,) op (A ___________________________________________________________ <---- ------------ -------------------------------------- i o- ......... o to:.

ro, AB2 1.01 0.64 0") 5:

o io RO RO OR ,......___ ________________________________________________________________________________ ____________ o ! oNia H "1:1 A63 0.65 0.45 0 RO RO OR') 4to ______________________________ L i. .......................... ......................... 1. ................. CII t,) -4 4) e CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 pp / 0_, ___ x_e.zz __ ----.0 _ ..,.. .¨ gg _____ 0 ..---.0 0 Ln 1-1 , __________________________ IN m tu ci, ...¨... ...¨., cu b I¨ W CC C.).1. 7 0 0 ( _ ....\...- 0 ...... u CC 1 CÉ 0 0 CL CL I e [ te ___ to c;. is .

:. ..

i.

I-1 In I 0 le 1 .4" dt Z In 0 CC CC i i. ..................... 1 CA 02889552 2015-04-24 % VO 2014/076423 PCT/FR2013/052736 AA1.

Compound 1: Sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate [000372] To 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth) dissolved in water at 65 C is added 0.6 g (16 mmol ) of sodium borohydride.

After stirring for 30 min, 28 g (238 mmol) of sodium chloroacetate are added. To this solution are then added dropwise 24 mL of 10 N NaOH (24 mmol) then the mixture is heated at 65 C for 90 minutes. 16.6 g (143 mmol) of sodium chloroacetate are then added to the reaction medium as well as 14 mL of 10N NaOH (14 mmol) dropwise.

After heating for 1 hour, the mixture is diluted with water, neutralized with acetic acid then purified by ultrafiltration on a 1 kDa PES membrane against water.

The molecule concentration of the final solution is determined by dry extract, then an acid/base dosage in a 50/50 (V/V) water/acetone mixture is carried out to determine the degree of methylcarboxylate substitution.

[000373] According to the dry extract: [compound] = 32.9 mg/g [000374] According to the acid/base dosage, the degree of methylcarboxylate substitution is 1.65 per saccharide unit.

[000375] The sodium maltotriosemethylcarboxylate solution is acidified on a Purolite (anionic) resin to obtain maltotriosemethylcarboxylic acid which is then lyophilized for 18 hours.

[000376] 10 g of maltotriosemethylcarboxylic acid (63 mmol of methylcarboxylic acid functions) are solubilized in DMF then cooled to 0 C.

A mixture of ethyl phenylalaninate, hydrochloride salt (8.7 g, 38 mmol) in DMF is prepared.

3.8 g of triethylamine (38 mmol) are added to this mixture.

A solution of NMM (6.3 g, 63 mmol) and EtOCOCI (6.8 g, 63 mmol) is then added to the mixture at 0 C.

The ethyl phenylalaninate solution is then added and the mixture stirred at 10 C.

An aqueous solution of imidazole is added then the mixture heated to 30 C.

The medium is diluted with water then the solution obtained is purified by ultrafiltration on a 1 kDa PES membrane against 0.1 N NaOH, 0.9% NaCl and water.

The molecule concentration of the final solution is determined by dry extract.

A sample of solution is lyophilized and analyzed by 1H NMR in D2O to determine the degree of substitution in methylcarboxylates functionalized with sodium L-phenylalaninate.

[000377] According to the dry extract: [compound 1] = 29.4 mg/g [000378] According to the acid/base dosage, the degree of substitution in sodium methylcarboxylates per saccharide unit is 0.65 .

[000379] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized with sodium L-phenylalaninate per saccharide unit is 1.0.

46 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 AA2.

Compound 2: Sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate [000380] By a process similar to that used for the preparation of compound 1, a sodium maltotriosecarboxylate functionalized with sodium L-phenylalaninate is obtained. According to the acid/base assay, the degree of substitution of sodium methylcarboxylates per saccharide unit is 1.0.

[000381] According to the dry extract: [compound 2] = 20.2 mg/g [000382] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.65.

AA3.

Compound 3: Sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate [000383] By a process similar to that used for the preparation of compound 1, a sodium maltotriosecarboxylate functionalized with sodium L-phenylalaninate is obtained. According to the acid/base assay, the degree of substitution of sodium methylcarboxylates per saccharide unit is 0.46.

[000384] According to the dry extract: [compound 3] = 7.2 mg/g [000385] According to NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is 1.2.

AA4.

Compound 4: Sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate [000386] By a process similar to that used for the preparation of compound 1, a sodium maltotriosecarboxylate functionalized with sodium L-phenylalaninate is obtained. According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 0.35.

[000387] According to the dry extract: [compound 4] = 3.1 mg/g [000388] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.65.

AA5.

Compound 5: Sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate [000389] By a process similar to that used for the preparation of compound 1, a sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate is obtained.

[000390] According to the dry extract: [compound 5] = 10.9 mg/g [000391] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.40.

47 CA 02889552 2015-04-24 W02014/076423 PCT/FR2013/052736 [000392] The degree of substitution of sodium methylcarboxylates per saccharide unit is 1.25.

AA6.

Compound 6: Sodium maltotriosernethylcarboxylate functionalized with sodium L-phenylalaninate [000393] To 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth) dissolved in water at 65 C is added 0.6 g (16 mmol ) of sodium borohydride.

After stirring for 30 min, 28 g (237 mmol) of sodium chloroacetate are added. To this solution are then added dropwise 24 mL of 10 N NaOH (240 mmol).

After heating at 65 C for 90 min, the mixture is diluted with water, neutralized by adding acetic acid then purified by ultrafiltration on a 1 kDa PES membrane against water.

The compound concentration of the final solution is determined by dry extract, then an acid/base dosage in a 50/50 (V/V) water/acetone mixture is carried out to determine the degree of substitution with sodium methylcarboxylate.

[000394] According to the dry extract: [compound] = 14.5 mg/g [000395] According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 1.45.

[000396] The sodium maltotriosemethylcarboxylate solution is acidified on a Purolite (anionic) resin to obtain maltotriosemethylcarboxylic acid which is then lyophilized for 18 hours.

[000397] By a process similar to that used for the preparation of compound 1, a sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate is obtained.

[000398] According to the dry extract: [compound 6] = 10.8 mg/g [000399] According to NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is 0.65.

[000400] The degree of substitution of sodium methylcarboxylates per saccharide unit is 0.8.

AA7.

Compound 7: Sodium maltotriosernethylcarboxylate functionalized with sodium L-phenylalaninate [000401] By a process similar to that described in the preparation of compound 1. 8 g of sodium maltotriosemethylcarboxylate characterized by a degree of substitution in sodium methylcarboxylate of 1. 76 are synthesized and freeze-dried.

[000402] 8 g (58 mmol of hydroxyl functions) of the lyophilisate and 15 g (129 mmol) of sodium chloroacetate are dissolved in water at 65 C. To this solution are added dropwise 13 mL of 10 N NaOH ( 130 mmol) then the mixture is heated at 65 C for 90 minutes. 9 g (78 mmol) of sodium chloroacetate are then added to the reaction medium as well as 8 mL of NaOH lON (80 mmol) dropwise. .

After heating for 1 hour, the mixture is diluted with water, neutralized with acetic acid then purified by ultrafiltration on a 1 kDa PES membrane against water.

The compound concentration of the final solution is determined by dry extract, then an acid/base dosage in a 50/50 (V/V) water/acetone mixture is carried out to determine the degree of substitution in sodium methylcarboxylates.

[000403] According to the dry extract: [compound] = 11.7 mg/g [000404] According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 3.30.

[000405] The sodium maltotriosemethylcarboxylate solution is acidified on a Purolite (anionic) resin to obtain maltotriosemethylcarboxylic acid which is then lyophilized for 18 hours.

[000406] By a process similar to that used for the preparation of Compound 1, a sodium maltotriosemethylcarboxylate functionalized with sodium L-phenylalaninate is obtained.

[000407] According to the dry extract: [compound 7] = 14.9 mg/g [000408] According to H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.65.

[000409] The degree of substitution of sodium methylcarboxylates per saccharide unit is 2.65.

AA8.

Compound 8: Sodium maltopentaosemethylcarboxylate functionalized with sodium L-phenylalaninate [000410] By a process similar to that described in the preparation of compound 1 but carried out with maltopentaose (CarboSynth), 10 g of maltopentaosemethylcarboxylic acid of degree of substitution in methylcarboxylic acid of 1.75 per saccharide unit are obtained then lyophilized.

[000411] By a process similar to that used for the preparation of compound 1, a sodium maltopentaosemethylcarboxylate functionalized with sodium Lphenylalaninate is obtained.

[000412] According to the dry extract: [compound 8] = 7.1 mg/g [000413] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.75.

[000414] The degree of substitution of sodium methylcarboxylates per saccharide unit is 1.0.

49 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 AA9.

Compound 9: Sodium maltooctaosemethylcarboxylate functionalized with sodium L-phenylalaninate [000415] By a process similar to that described in the preparation of compound 1 but carried out with maltooctaose (CarboSynth), 10 g of maltooctaosemethylcarboxylic acid of degree of substitution in methylcarboxylic acid of 1.65 per saccharide unit are obtained then lyophilized.

[000416] By a process similar to that used for the preparation of compound 1, a sodium maltooctaosemethylcarboxylate functionalized with sodium Lphenylalaninate is obtained.

[000417] According to the dry extract: [compound 9] = 26.3 mg/g [000418] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is of 0.65.

[000419] The degree of substitution of sodium methylcarboxylates per saccharide unit is 1.0.

AA10.

Compound 10: Sodium maltotriosemethylcarboxylate functionalized with sodium L-tyrosinate [000420] By a process similar to that described in the preparation of compound 1 but carried out with methyl L-tyrosinate, hydrochloric acid salt (Bachem), a sodium maltotriosemethylcarboxylate, characterized by a degree of sodium methylcarboxylate substitution per saccharide unit of 1.64, is functionalized with sodium tyrosinate.

[000421] According to the dry extract: [compound 10] = 9.1 mg/g [000422] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-tyrosinate per saccharide unit is of 0.81.

The degree of substitution of sodium methylcarboxylates per saccharide unit is 0.83.

AA11.

Compound 11: Sodium maltotriosemethylcarboxylate functionalized with sodium alpha-phenylglycinate [000423] By a process similar to that described in the preparation of compound 1, 10 g of maltotriosemethylcarboxylic acid with degree of substitution into methylcarboxylic acid per saccharide unit of 1 .64 are obtained then freeze-dried.

[000424] 8 g of maltotriosemethylcarboxylic acid (50 mmol of methylcarboxylic acid functions) are solubilized in DMF then cooled to 0 C.

A mixture of sodium alpha-phenylglycinate (Bachem, 5 g; 33 mmol) and triethylamine (33 mmol) is prepared in water.

A solution of NMM (4.9 g; 49 mmol) and EtOCOCI (5.3 g, 49 mmol) is then added to the solution of maltotriosemethylcarboxylic acid 5o CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013 /052736 at 0 C.

The solution of sodium alpha-phenylglycinate and triethylamine is then added and the mixture stirred at 30 C.

An aqueous solution of imidazole (340 g/L) is added after 90 minutes.

The medium is diluted with water then the solution obtained is purified by ultrafiltration on a 1 kDa PES membrane against a NaHCO3/Na2CO3 buffer pH 10.4 150 mM, 0.9% NaCl and water.

The compound concentration of the final solution is determined by dry extract.

A sample of solution is lyophilized and analyzed by 11-1 NMR in D70 to determine the degree of substitution in methylcarboxylates functionalized with sodium alpha-phenylglycinate.

[000425] According to the dry extract: [compound 11] = 9.1 mg/g [000426] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium alpha-phenylglycinate per saccharide unit is 0.52.

[000427] The degree of substitution of sodium methylcarboxylates per saccharide unit is 1.12.

AB Polysaccharides counterexamples ABl.

Polysaccharide 1: Sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate.

[000428] Polysaccharide 1 is a sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate obtained from a dextran with a weight average molar mass of 10 kg/mol (DP = 39, Pharmacosmos) according to the process described in the application patent FR 07/02316 published under number FR2914305.

According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 0.6.

[000429] According to 1H NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is 0.46.

[000430] This polysaccharide corresponds to polysaccharide 1 of application FR0901478.

AB2.

Polysaccharide 2: Sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate.

[000431] Polysaccharide 2 is a sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate obtained from a dextran with a weight average molar mass of 5 kg/mol (DP = 19, Pharmacosmos) according to the process described in the application patent FR 07/02316 published under number FR2914305.

According to the acid/base assay, the degree of substitution of sodium methylcarboxylates per saccharide unit is 1.01.

51 CA 02889552 2015-04-24 W02014/076423 PCT/FR2013/052736 [000432] According to 'I-1 NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is 0.64 .

AB3.

Polysaccharide 3: Sodium dextranmethylcarboxylate functionalized with sodium L-phenylataninate.

[000433] Polysaccharide 3 is a sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate obtained from a dextran with a weight-average molar mass of 5 kg/mol (DP = 19, Pharmacosmos) according to the process described in the application patent FR 07/02316 published under number FR2914305.

According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 0.65.

[000434] According to 111 NMR: the degree of substitution in methylcarboxylates functionalized with sodium L-phenylalaninate per saccharide unit is 0.45.

AB4.

Polysaccharide 4: Sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate.

[000435] Polysaccharide 4 is a sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate obtained from a dextran with a weight-average molar mass of 10 kg/mol (DP = 39, Pharmacosmos) according to the process described in the application patent FR 07/02316 published under number FR2914305.

According to the acid/base assay, the degree of substitution of sodium methylcarboxylates per saccharide unit is 1.01.

[000436] According to "1-1" NMR: the degree of substitution in methylcarboxylates functionalized with sodium L-phenylalaninate per saccharide unit is 0.64.

AB5.

Polysaccharide 5: Sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate.

[000437] Polysaccharide 5 is a sodium dextranmethylcarboxylate functionalized with sodium L-phenylalaninate obtained from a dextran with a weight average molar mass of 5 kg/mol (DP = 19, Pharmacosmos) according to the process described in the application patent FR 07/02316 published under number FR2914305.

According to the acid/base dosage, the degree of substitution of sodium methylcarboxylates per saccharide unit is 0.45.

[000438] According to NMR: the degree of substitution in methylcarboxylates functionalized by sodium L-phenylalaninate per saccharide unit is 0.65.

52 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 AC Polyanionic compound Polyanionic compound 1: Sodium maltotriosemethylcarboxylate [000439] With 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth) dissolved in water at 65 C, 0.6 g (16 mmol) of sodium borohydride is added.

After stirring for 30 min, 28 g (238 mmol) of sodium chloroacetate are added. To this solution are then added dropwise 24 mL of 10 N NaOH (240 mmol) then the mixture is heated at 65 C for 90 minutes. 16.6 g (143 mmol) of sodium chloroacetate are then added to the reaction medium as well as 14 mL of 10 N NaOH (140 mmol) dropwise.

After heating for 1 hour, the mixture is diluted with water, neutralized with acetic acid then purified by ultrafiltration on a 1 kDa PES membrane against water.

The compound concentration of the final solution is determined by dry extract, then an acid/base dosage in a 50/50 (V/V) water/acetone mixture is carried out to determine the degree of substitution with sodium methylcarboxylate.

[000440] According to the dry extract: [polyanionic compound 1] = 32.9 mg/g [000441] According to the acid/base dosage: the degree of substitution in sodium methylcarboxylates per saccharide unit is 1, 65.

B Preparation of Bi solutions.

Novolog rapid insulin analogue solution 100 IU/mL.

[000442] This solution is a commercial insulin aspart solution from Novo Nordisk sold under the name Novologe.

This product is a rapid insulin aspart analogue.

82.

Humalog rapid insulin analogue solution at 100 IU/mL.

[000443] This solution is a commercial insulin lispro solution from Eli Lilly sold under the name Humaloge.

This product is a rapid insulin analogue.

B3.

Actrapid0 regular human insulin solution at 100 IU/mL.

[000444] This solution is a commercial human insulin solution from Novo Nordisk sold under the name Actrapide.

This product is a regular human insulin.

B4.

Humulin R regular human insulin solution at 100 IU/mL.

[000445] This solution is a commercial human insulin solution from Eli Lilly sold under the name Humuline R.

This product is a regular human insulin.

53 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B5.

Preparation of excipient solutions [000446] The non-polymeric polyanionic compounds are selected by measuring their dissociation constant with respect to calcium ions and with respect to their property of not destabilizing the hexameric form of the insulin.

[000447] Concerning the dissociation constant with respect to calcium ions, it is determined as follows.

[000448] Solutions containing 2.5 mM of CaCl2, 150 mM of NaCl and increasing concentrations of polyanionic compound (between 0 and 20 mM) are prepared.

The potential of all these formulations is measured and the concentrations of free calcium ions in the formulations determined.

After linearization by the Scatchard method, the dissociation constants are established.

These data make it possible to compare the affinity of the carboxylates and phosphates of the different polyanionic compounds for Ca.

[000449] Regarding their property of not destabilizing the hexameric form of insulin, this property is measured by circular dichroism in comparison with insulin alone (without anionic compound or polyanionic compound), see the experimental protocols in the experimental part D.

Preparation of a 1.188 M sodium citrate solution.

[000450] A solution of sodium citrate is obtained by dissolving 9.0811 g of sodium citrate (30.9 mmol) in 25 mL of water in a volumetric flask.

The pH is adjusted exactly to 7.4 by adding 1 mL of 1 M HCl.

The solution is filtered through 0.22 μm.

Preparation of a 130 mM m-cresol solution.

[000451] A solution of m-cresol is obtained by dissolving 14.114 g of mcresol (130 mmol) in 986.4 mL of water in a 1 L volumetric flask.

Preparation of a solution of m-cresol and glycerin (96.6 mM m-cresol and 566 mM glycerin).

[000452] 73.3 g of the 130 mM m-cresol solution are added to 5.26 g of glycerin and then diluted by adding 22.25 g of water.

The resulting solution of m-cresol and glycerin is homogenized for 30 minutes then filtered through a 0.22 μm membrane.

Preparation of a 32.7 mM Tween 20 solution.

[000453] A solution of Tween 20 is obtained by dissolving 2.0079 g of Tween 20 (1.636 mmol) in 50 mL of water in a volumetric flask.

The solution is filtered through a 0.22 μm membrane.

54 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B6.

Preparation of a solution of human insulin at 500 IU/mL.

[000454] 15 g of water are added to 563.6 mg of human insulin, then the pH is lowered to acidic pH by adding 5.98 g of 0.1 N HCl.

After complete solubilization of the insulin at an acidic pH, the solution is neutralized to pH 7.2 by adding 8.3 mL of 0.1 N NaOH.

The concentration is then adjusted to 500 IU/mL by adding 0.76 g of water.

The solution is finally filtered through a 0.22 μm membrane.

67.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 1 [000455] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin lispro] of 2.0, the different reagents are added in the quantities specified below and in the following order:

Compound 1 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL [000456] The final pH is adjusted to 7.4 0.4.

[000457] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

68.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 1 and citrate [000458] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 1 lyophilized 730 mg Humaloge commercial solution 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000459] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000460] The final pH is adjusted to 7.4 0.4.

[000461] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B9.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of .. compound 1 and polyanionic compound 1 [000462] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[compound polyanionic 1]/[insulin lispro] of 2.0/2.0/1, the different reagents are added in the quantities specified below and in the following order:

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 Compound 1 lyophilized 730 mg Polyanionic compound 1 lyophilized 730 mg Commercial solution Humaloge100 IU/mL 100 mL [0004631 Polyanionic compound 1 can be used in the acid form or the basic form as sodium salt, potassium salt or other salt compatible with an injectable formulation.

[000464] The final pH is adjusted to 7.4 0.4.

[000465] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B10.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 1 and polyanionic compound 1 [000466] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[polyanionic compound 1 ]/[insulin lispro] of 2.0/5.5/1, the different reagents are added in the quantities specified below and in the following order:

Compound 1 lyophilized 730 mg Polyanionic compound 1 lyophilized 2000 mg Commercial solution Humaloge 100 IU/mL 100 mL [000467] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or of another salt compatible with an injectable formulation.

[000468] The final pH is adjusted to 7.4 0.4.

[000469] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B11.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 2 and citrate [000470] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000471] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[0004721 The final pH is adjusted to 7.4 0.4.

[000473] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

CA 02889552 2015-04-24 %VO 2014/076-123 PCT/FR2013/052736 B12.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 2 and polyanionic compound 1 [000474] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[polyanionic compound 1 ]/[insulin lispro] of 2.0/2.0/1, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Polyanionic compound 1 lyophilized 730 mg Commercial solution Humaloge100 IU/mL 100 mL [000475] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000476] The final pH is adjusted to 7.4 0.4.

[000477] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B13.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 2 and polyanionic compound 1 [000478] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[polyanionic compound 1 ]/[insulin lispro] of 2.0/5.5/1, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Polyanionic compound 1 lyophilized 2000 mg Commercial solution Humaloge 100 IU/mL 100 mL [000479] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or of another salt compatible with an injectable formulation.

[000480] The final pH is adjusted to 7.4 0.4.

[000481] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B14.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 1.

[000482] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin lispro] of 4, the different reagents are added in the specified quantities:

Compound 1 in lyophilized form 1460 mg Humalog commercial solution 100 IU/mL 100 mL [000483] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

57 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B15.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 2.

[000484] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[insulin lispro] of 4, the different reagents are added in the specified quantities:

Compound 2 in lyophilized form 1460 mg Humaloge commercial solution 100 IU/mL 100 mL [000485] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B16.

Preparation of a solution of insulin analogue lispro at 100 IU/mL in the presence of compound 1 and sodium tartrate.

[000486] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin lispro] of 2.0 and a concentration of 80 mM sodium tartrate, the different reagents are added in the specified quantities below :

Compound 1 in lyophilized form 730 mg Humaloge commercial solution 100 IU/mL 100 mL Sodium tartrate 1.552 g [000487] For the tartrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000488] The final pH is adjusted to 7.4 0.4.

[000489] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B17.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 1 and polyanionic compound 1.

[000490] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[polyanionic compound 11/[insulin lispro] of 2/4/1, the different reagents are added in the quantities specified below :

Compound 1 in lyophilized form 730 mg Polyanionic compound 1 in lyophilized form 1460 mg Humaloge commercial solution 100 IU/mL 100 mL [000491] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000492] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

58 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 B18.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 1 and sodium triphosphate.

[000493] For a final volume of 100 mL of formulation, the different reagents are added in the quantities specified below:

Compound 1 in lyophilized form 730 mg Sodium triphosphate 184 mg Commercial solution Humaloge 100 IU/mL 100 mL [000494] For the triphosphate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000495] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B19.

Preparation of an insulin analogue lispro solution at 100 IU/mL in the presence of compound 2 and sodium tartrate.

[000496] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[insulin lispro] of 2.0 and a concentration of 80 mM sodium tartrate, the different reagents are added in the specified quantities below :

Compound 2 in lyophilized form 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium tartrate 1.552 g [000497] For the tartrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000498] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B20.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 2 and polyanionic compound 1.

[000499] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[polyanionic compound 1]/[insulin lispro] of 2/4/1, the different reagents are added in the quantities specified below. below:

Compound 2 in lyophilized form 730 mg Polyanionic compound 1 in lyophilized form 1460 mg Humaloge commercial solution 100 IU/mL 100 mL [000500] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

59 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000501] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B21.

Preparation of an insulin lispro solution at 100 IU/mL in the presence of compound 2 and sodium triphosphate.

[000502] For a final volume of 100 mL of formulation, the different reagents are added in the quantities specified below:

Compound 2 in lyophilized form 730 mg Sodium triphosphate 184 mg Commercial solution Humalog 100 IU/mL 100 mL [000503] For the triphosphate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000504] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B22.

Preparation of an analog insulin solution (insulin lispro) at 200 IU/mL.

[000505] The commercial formulation of insulin lispro (Humaloge) was concentrated using AMICON Ultra-15 centrifuge tubes with a cutoff at 3 kDa.

The amicon tubes were first rinsed with 12 mL of deionized water. 12 mL of the commercial formulation were centrifuged for 35 minutes at 4000 g at 20 C.

The volume of the retentate was measured and the concentration thus estimated. All the retentates were pooled and the overall concentration was estimated (> 200 IU/mL).

.. [000506] The concentration of this concentrated insulin lispro solution was adjusted to 200 IU/mL by adding the commercial formulation of insulin lispro (Humalog8).

The concentrated insulin lispro formulation has the same concentrations of excipients (m-cresol, glycerin, phosphate) as the commercial formulation at 100 IU/mL.

[000507] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B23.

Preparation of an insulin lispro solution at 200 IU/mL in the presence of compound 1 and citrate.

[000508] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin lispro] of 2, the different reagents are mixed in the quantities specified below Insulin lispro at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Sodium citrate solution at 1.188 M 1566 pL CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000509] The final pH is adjusted to 7.4 0.4. The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B24.

Preparation of a solution of insulin lispro at 200 IU/mL in the presence of compound 1 and polyanionic compound 1.

[000510] For a final volume of 100 mL of formulation with a mass ratio [compound 11/[polyanionic compound 1]/[insulin lispro] of 2/2/1, the different reagents are mixed in the quantities specified below.

Insulin lispro at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Lyophilisate of polyanionic compound 1 1460 mg [000511] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, salt of potassium or another salt compatible with an injectable formulation.

[000512] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

825.

Preparation of an insulin lispro solution at 200 IU/mL in the presence of compound 1 and polyanionic compound 1.

[000513] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[polyanionic compound 1]/[insulin lispro] of 2/4/1, the different reagents are mixed in the quantities specified below.

Insulin lispro at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Lyophilisate of polyanionic compound 1 2920 mg [000514] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, salt of potassium or another salt compatible with an injectable formulation.

[000515] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B26.

Preparation of an insulin lispro solution at 200 IU/mL in the presence of compound 2 and polyanionic compound 1.

[000516] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/{polyanionic compound 1}/[insulin lispro] of 2/4/1, the different reagents are mixed in the quantities specified below.

Insulin LisPro at 200 IU/ML 100 ml Lyophilisat of compound 2,1460 mg Lyophilisat of Polyanionic compound 1 2920 mg 61 C 02889552 2015-24-24 WO 2014/076423 PCT/FR2013/052736 [000517] The polyanionic compound 1 can be used under the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000518] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B27.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 1 and tartrate.

[000519] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2 and 80 mM of tartrate, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 1 solution at 36.01 mg/mL 20.27 mL 96.6 mM m-cresol/566 mM glycerin solution 30 mL Water 28.95 mL Sodium tartrate 1.552 g [000520 ] For tartrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000521] The final pH is 7.4 0.4.

This clear solution is filtered through a 0.22 μm membrane and then placed at +4 C.

B28.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 1 and triphosphate.

[000522] For a final volume of 100 mL of formulation, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 1 solution at 36.01 mg/mL 20.27 mL 96.6 mM solution m-creso1/566 mM glycerin 30 mL Water 28.95 mL Sodium triphosphate 184 mg [000523 ] For triphosphate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000524] The final pH is 7.4 0.4.

This clear solution is filtered through a 0.22 μm membrane and then placed at +4 C.

62 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 B29.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 2 and tartrate.

[000525] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2 and 80 mM of tartrate, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 2 solution at 36.01 mg/mL 20.27 mL 96.6 mM solution m-creso1/566 mM glycerin 30 mL Water 28.95 mL Sodium tartrate 1.552 g [000526 ] For tartrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000527] The final pH is 7.4 0.4.

[000528] This clear solution is filtered on a 0.22 μm membrane and then placed at +4 C.

B30.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 2 and triphosphate.

[000529] For a final volume of 100 mL of formulation, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 2 solution at 36.01 mg/mL 20.27 mL 96.6 mM solution m-creso1/566 mM glycerin 30 mL Water 28.95 mL Sodium triphosphate 184 mg [000530 ] For triphosphate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000531] The final pH is 7.4 0.4.

This clear solution is filtered through a 0.22 μm membrane and then placed at +4 C.

B31.

Preparation of a solution of human insulin at 200 IU/mL.

[000532] The commercial formulation of human insulin (Humulin R) was concentrated using AMICON Ultra-15 centrifuge tubes with a cutoff at 3 kDa.

The amicon tubes were first rinsed with 12 mL of deionized water. 12 mL of the commercial formulation were centrifuged for 35 minutes at 4000 g at 20 C.

The volume of the retentate was measured and the concentration thus estimated.

All retentates were pooled and the overall concentration was estimated (>200 IU/mL).

63 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000533] The concentration of this concentrated human insulin solution was adjusted to 200 IU/mL by addition of the commercial formulation of human insulin (Humulin R ).

The concentrated human insulin formulation has the same concentrations of excipients (m-cresol, glycerin) as the commercial formulation at 100 IU/mL.

[000534] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B32.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 1 and citrate.

[000535] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2, the different reagents are mixed in the quantities specified below:

Human insulin at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Sodium citrate solution at 1.188 M 1566 pL [000536] The final p1-1 is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B33.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 1 and polyanionic compound 1.

[000537] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[polyanionic compound 1]/[human insulin] of 2/2/1, the different reagents are mixed in the quantities specified below Insulin human at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Lyophilisate of polyanionic compound 1 1460 mg [000538] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

.. B34.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 1 and polyanionic compound 1.

[000539] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[polyanionic compound 1]/[human insulin] of 2/4/1, the different reagents are mixed in the quantities specified below Human insulin at 200 IU/mL 100 mL Lyophilisate of compound 1 1460 mg Lyophilisate of polyanionic compound 1 2920 mg 64 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000540] The polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000541] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B35.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 2 and citrate.

[000542] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2, the different reagents are mixed in the quantities specified below Human insulin at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg 1.188 M sodium citrate solution 1566 pL .. [000543] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B36.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 2 and polyanionic compound 1.

[000544] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[polyanionic compound 1]/[human insulin] of 2/2/1, the different reagents are mixed in the quantities specified below Insulin human at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg Lyophilisate of polyanionic compound 1 1460 mg [000545] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000546] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B37.

Preparation of a solution of human insulin at 200 IU/mL in the presence of compound 2 and polyanionic compound 1.

[000547] For a final volume of 100 mL of formulation with a mass ratio.. [compound 2]/[polyanionic compound 1]/[human insulin] of 2/4/1, the different reagents are mixed in the quantities specified below Human insulin at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 Lyophilisate of polyanionic compound 1 2920 mg [000548] Polyanionic compound 1 can be used in the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000549] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B38.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 2 and citrate.

[000550] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2 and 9.3 mM citrate, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 2 solution at 36.01 mg/mL 20.27 mL 96.6 mM solution m-creso1/566 mM glycerin 30 mL Water 28.95 mL Sodium citrate solution at 1.188 M 783 pL [000551] For citrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000552] The final pH is 7.4 0.4.

This clear solution is filtered through a 0.22 μm membrane and then placed at +4 C.

B39.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 1 and citrate.

[000553] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2 and 9.3 mM citrate, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 20 mL Compound 1 solution at 36.01 mg/mL 27 mL 96.6 mM solution m-creso1/566 mM glycerin 30 mL Water 28.95 mL Sodium citrate solution at 1.188 M 783 pL [000554] For citrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000555] The final pH is 7.4 0.4.

This clear solution is filtered through a 0.22 μm membrane and then placed at +4 C.

66 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 B40.

Preparation of a solution of insulin aspart at 100 IU/mL in the presence of compound 1 and citrate [000556] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin aspart] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 1 lyophilized 730 mg Novologe commercial solution 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000557] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000558] The final pH is adjusted to 7.4 0.4.

.. [000559] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B41.

Apidra rapid insulin analogue solution at 100 IU/mL.

[000133] This solution is a commercial insulin glulisine solution from SanofiAventis sold under the name Apidrae.

This product is a rapid insulin analogue.

B42.

Preparation of an insulin glulisine solution at 100 IU/mL in the presence of compound 1 and citrate [000560] For a final volume of 100 mL of formulation, with a mass ratio [compound 1]/[insulin glulisine] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 1 lyophilized 730 mg Commercial Apidrae solution 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000561] For citrate, the acid form or the basic form in the form of sodium salt can be used , potassium salt or another salt compatible with an injectable formulation.

[000562] The final pH is adjusted to 7.4 0.4.

[000563] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B43.

Preparation of a solution of insulin aspart at 100 IU/mL in the presence of compound 2 and citrate [000564] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[insulin aspart] of 2 .0 and a concentration of 67 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Commercial solution Novolog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000565] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000566] The final pH is adjusted to 7.4 0.4.

[000567] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B44.

Preparation of an insulin glulisine solution at 100 IU/m1. in the presence of compound 2 and citrate [000568] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[insulin glulisine] of 2.0 and a concentration of 9.3 mM of citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Commercial solution of Apidra 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000569] For the citrate, the acid form or the basic form in the form of sodium salt can be used , potassium salt or another salt compatible with an injectable formulation.

[000570] The final pH is adjusted to 7.4 0.4.

[000571] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B45.

Preparation of an insulin lispro solution at 100 IU/ml. in the presence of compound 5 and citrate [000572] For a final volume of 100 mL of formulation, with a mass ratio [compound 5]/[insulin lispro] of 2.0 and a concentration of 9.3 mM of citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 5 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000573] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000574] The final pH is adjusted to 7.4 0.4.

68 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000575] The clear solution is filtered on a 0.22 pm membrane and stored at 4 C.

846.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 6 and citrate [0005761 For a final volume of 100 mL of formulation, with a mass ratio [compound 6]/[insulin lispro] of 2, 0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 6 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000577] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000578] The final pH is adjusted to 7.4 0.4.

[000579] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

847.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 7 and citrate [000580] For a final volume of 100 mL of formulation, with a mass ratio [compound 7]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 7 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000581] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000582] The final pH is adjusted to 7.4 0.4.

[000583] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B48.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 8 and citrate [000584] For a final volume of 100 mL of formulation, with a mass ratio [compound 8]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 8 lyophilized 730 mg 69 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000585] For citrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000586] The final pH is adjusted to 7.4 0.4.

[000587] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B49.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 9 and citrate [000588] For a final volume of 100 mL of formulation, with a mass ratio [compound 9]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 9 lyophilized 730 mg Commercial solution Humaloge 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 ut_ [000589] For the citrate, the acid form or the basic form in the form of sodium salt, of potassium salt or another salt compatible with an injectable formulation.

[000590] The final pH is adjusted to 7.4 0.4.

B50.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 5 and citrate [000591] For a final volume of 100 mL of formulation, with a mass ratio [compound 5]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 5 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000592] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000593] The final pH is adjusted to 7.4 0.4.

[000594] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B51.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 6 and citrate [0005951 For a final volume of 100 mL of formulation, with a mass ratio [compound 6]/[human insulin] of 2, 0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 6 lyophilized 730 mg Commercial solution Humulin R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000596] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000597] The final pH is adjusted to 7.4 0.4.

[000598] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B-52.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 7 and citrate [000599] For a final volume of 100 mL of formulation, with a mass ratio [compound 7]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 7 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000600] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000601] The final pH is adjusted to 7.4 0.4.

[000602] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B53.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 8 and citrate [000603] For a final volume of 100 mL of formulation, with a mass ratio [compound 8]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 8 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL 71 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000604] For citrate, we may use the acidic form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000605] The final pH is adjusted to 7.4 0.4.

[000606] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B54.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 9 and citrate [000607] For a final volume of 100 mL of formulation, with a mass ratio [compound 9]/[human insulin] of 2 .0 and a concentration of 9.3 rnM of citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 9 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000608] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000609] The final pH is adjusted to 7.4 0.4.

[000610] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B55.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 2 [000611] For a final volume of 100 mL of formulation, with a mass ratio [compound 2]/[human insulin] of 2.0, the different reagents are added in the quantities specified below and in the following order:

Compound 2 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL [000612] The final pH is adjusted to 7.4 0.4.

[000613] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B56.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 7 [000614] For a final volume of 100 mL of formulation, with a mass ratio [compound 7]/[human insulin] of 2.0, the different reagents are added in the quantities specified below and in the following order:

Compound 7 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL [000615] The final pH is adjusted to 7.4 0.4.

72 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000616] The clear solution is filtered on a 0.22 tirn membrane and stored at 4 C.

857.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 10 and citrate [000617] For a final volume of 100 mL of formulation, with a mass ratio [compound 10]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 10 lyophilized 730 mg Fumaloge commercial solution 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000618] For the citrate, the acid form or the basic form in the form of sodium salt, of potassium salt or another salt compatible with an injectable formulation.

[000619] The final pH is adjusted to 7.4 0.4.

[000620] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B58.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 11 and citrate [000621] For a final volume of 100 mL of formulation, with a mass ratio [compound 11]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 11 lyophilized 730 mg Commercial solution Humalog" 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000622] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000623] The final pH is adjusted to 7.4 0.4.

B59.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 10 and citrate [000624] For a final volume of 100 mL of formulation, with a mass ratio [compound 10]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 10 lyophilized 730 mg Commercial solution Humulin R 100 IU/mL 100 mL 73 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 Sodium citrate solution at 1.188 M 783 pL [000625] For the citrate, we may use the acidic form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000626] The final pH is adjusted to 7.4 0.4.

[000627] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

860.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 11 and citrate [000628] For a final volume of 100 mL of formulation, with a mass ratio [compound 11]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order Compound 11 lyophilized 730 mg Commercial solution Humulire R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000629] For citrate, the acid form or the basic form can be used in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000630] The final pH is adjusted to 7.4 0.4.

[000631] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B61.

Preparation of an insulin aspart solution at 200 IU/mL.

[000632] The commercial formulation of insulin aspart (Novologe) was concentrated using AMICON Ultra-15 centrifuge tubes with a cutoff at 3 kDa.

The amicon tubes were first rinsed with 12 mL of deionized water. 12 mL of the commercial formulation were centrifuged for 35 minutes at 4000 g at 20 C.

The volume of the retentate was measured and the concentration thus estimated.

All retentates were pooled and the overall concentration was estimated (>200 IU/mL).

[000633] The concentration of this concentrated insulin aspart solution was adjusted to 200 IU/mL by adding the commercial formulation of insulin aspart (Novolog).

The concentrated formulation of concentrated insulin aspart has the same concentrations of excipients (m-cresol, glycerin) as the commercial formulation at 100 IU/mL.

[000634] By varying the centrifugation time and the final dilution with the commercial formulation, it is possible to prepare formulations of insulin aspart at 300, 400 or 500 IU/mL in the same way.

[000635] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

74 CA 02889552 2015-04-24 %VO 2014/076423 PCT/FR2013/052736 B62.

Preparation of an insulin glulisine solution at 200 IU/mL.

[000636] The commercial formulation of insulin glulisine (Apidra8) was concentrated using AMICON Ultra-15 centrifuge tubes with a cutoff at 3 kDa.

The amicon tubes were first rinsed with 12 mL of deionized water. 12 mL of the commercial formulation were centrifuged for 35 minutes at 4000 g at 20 C.

The volume of the retentate was measured and the concentration thus estimated.

All retentates were pooled and the overall concentration was estimated (>200 IU/mL).

[000637] The concentration of this concentrated insulin glulisine solution was adjusted to 200 IU/mL by adding the commercial formulation of insulin glulisine (Apidra8).

The concentrated insulin glulisine formulation has the same concentrations of excipients (m-cresol, NaCI, TRIS) as the commercial formulation at 100 IU/mL.

[000638] By varying the centrifugation time and the final dilution with the commercial formulation, it is possible to prepare insulin glulisine formulations at 300, 400 or 500 IU/mL in the same way.

[000639] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B63.

Preparation of a solution of insulin aspart at 200 IU/mL in the presence of compound 1 at 14.6 mg/mL and 18.6 mM citrate.

[000640] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin aspart] of 2.0, the different reagents are mixed in the quantities specified below and in the following order:

Lyophilisate of compound 1 1460 mg Insulin aspart at 200 IU/mL 100 mL Sodium citrate solution at 1.188 M 1566 pL [000641] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B64.

Preparation of a solution of human insulin, insulin lispro, insulin aspart or insulin glulisine at 300, 400 and 500 IU/mL.

[000642] Concentrated formulations of human insulin, insulin lispro, insulin aspart or insulin glulisine at 300 IU/mL, 400 IU/mL or 500 IU/mL (as well as at all intermediate concentrations) are prepared on the basis of the protocol of Example B62 relating to the preparation of an insulin glulisine solution at 200 IU/mL.

The commercial insulin formulation is concentrated using AMICON Ultra-15 centrifuge tubes with a cutoff at 3 kDa.

The amicon tubes are first rinsed with 12 mL of deionized water. 12 mL of the commercial formulation are centrifuged at 4000g and CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 20 C.

By varying the centrifugation time it is possible to adjust the final insulin concentration in the formulation.

The volume of the retentate is measured and the concentration thus estimated.

All retentates are pooled and the overall concentration is estimated (> 300, 400 or 500 IU/mL).

[000643] The concentration of this concentrated insulin solution is adjusted to the desired concentration (eg 300 IU/mL, 400 IU/mL or 500 IU/mL) by adding the insulin formulation (Humulin R, Novolog, Humalog or Apidra).

The concentrated formulation of concentrated insulin has the same excipient concentrations as the commercial formulation at 100 IU/mL.

[000644] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B65.

Preparation of an insulin glulisine solution at 200 IU/mL in the presence of compound 1. and citrate.

.. [000645] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin glulisine] of 2, the different reagents are mixed in the quantities specified below and in the following order:

Lyophilisate of compound 1 1460 mg Insulin glulisine at 200 IU/mL 100 mL Sodium citrate solution at 1.188 M 1566 pL [000646] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B66.

Preparation of an insulin aspart solution at 300 IU/ml. in the presence of.. compound 1 and citrate.

[000647] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin aspart] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin aspart at 300 IU/mL 100 mL Lyophilisate of compound 1 2190 mg Sodium citrate 720 mg [000648] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

76 CA 02889552 2015-04-24 %VO 2014/076423 PCT/FR2013/052736 B67.

Preparation of an insulin glulisine solution at 300 IU/mL in the presence of compound 1 and citrate.

[000649] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 300 IU/mL 100 mL Lyophilisate of compound 1 2190 mg Sodium citrate 720 mg [000650] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B68.

Preparation of a solution of insulin aspart at 400 IU/mL in the presence of compound 1 and citrate.

[000651] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin aspart] of 2, the different reagents are mixed in the quantities specified below:

Insulin aspart at 400 IU/mL 100 mL Lyophilisate of compound 1 2920 mg Sodium citrate 960 mg [000652] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B69.

Preparation of an insulin glulisine solution at 400 IU/mL in the presence of compound 1 and citrate.

[000653] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 400 IU/mL 100 mL Lyophilisate of compound 1 2920 mg Sodium citrate 960 mg [000654] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B70.

Preparation of a solution of insulin aspart at 500 IU/mL in the presence of compound 1 and citrate.

[000655] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin aspart] of 2.0, the different reagents are mixed in the quantities specified below:

77 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 Insulin aspart at 500 IU/mL 100 mL Lyophilisate of compound 1 3650 mg Sodium citrate 1200 mg [000656] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B71.

Preparation of an insulin glulisine solution at 500 IU/mL in the presence of compound 1 and citrate.

[000657] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 500 IU/mL 100 mL Lyophilisate of compound 1 3650 mg Sodium citrate 1200 mg [000658] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

872.

Preparation of a solution of human insulin at 300 IU/mL in the presence of compound 1 and citrate.

[000659] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2.0, the different reagents are mixed in the quantities specified below:

Human insulin at 300 IU/mL 100 mL Lyophilisate of compound 1 2190 mg 26 Sodium citrate 720 mg [000660] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

873.

Preparation of an insulin lispro solution at 300 IU/mL in the presence of compound 1 and citrate.

[000661] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin lispro at 300 1.1I/mL 100 mL Lyophilisate of compound 1 2190 mg Sodium citrate 720 mg [000662] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

78 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 B74.

Preparation of a solution of human insulin at 400 IU/mL in the presence of compound 1 and citrate.

[000663] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2.0, the different reagents are mixed in the quantities specified below:

Human insulin at 400 IU/mL 100 mL Lyophilisate of compound 1 2920 mg Sodium citrate 960 mg [000664] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B75.

Preparation of an insulin lispro solution at 400 IU/mL in the presence of compound 1 and citrate.

[000665] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin lispro at 400 IU/mL 100 mL Lyophilisate of compound 1 2920 mg Sodium citrate 960 mg [000666] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

076.

Preparation of a solution of human insulin at 500 IU/mL in the presence of compound 1 and citrate.

[000667] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[human insulin] of 2.0, the different reagents are mixed in the quantities specified below:

Human insulin at 500 11I/mL 100 mL Lyophilisate of compound 1 3650 mg Sodium citrate 1200 mg [000668] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

79 CA 02889552 2015-04-24 %VO 2014/076423 PCT/FR2013/052736 B77.

Preparation of an insulin lispro solution at 500 IU/ml. in the presence of compound 1 and citrate.

[000669] For a final volume of 100 mL of formulation with a mass ratio [compound 1]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin lispro at 500 IU/mL 100 mL Lyophilisate of compound 1 3650 mg Sodium citrate 1200 mg [000670] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B78.

Preparation of an insulin lispro solution at 200 IU/mL in the presence of compound 2 and citrate.

[000671] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin lispro at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg Sodium citrate solution at 1.188 M 1566 pL [000672] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B79.

Preparation of a solution of insulin aspart at 200 IU/mL in the presence of compound 2 and citrate.

[000673] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin aspart] of 2.0 the different reagents are mixed in the quantities specified below:

Insulin aspart at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg Sodium citrate solution at 1.188 M 1566 pL [000674] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B80.

Preparation of a solution of insulin glutisine at 200 tilt mL in the presence of compound 2 and citrate.

[000675] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin glulisine] of 2.0 the different reagents are mixed in the quantities specified below:

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 Insulin glulisine at 200 IU/mL 100 mL Lyophilisate of compound 2 1460 mg Sodium citrate solution at 1.188 M 1566 pL [000676] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B81.

Preparation of a solution of insulin aspart at 300 IU/mL in the presence of compound 2 and citrate.

[000677] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin aspart] of 2.0 the different reagents are mixed in the quantities specified below:

Insulin aspart at 300 IU/mL 100 mL Lyophilisate of compound 2 2190 mg Sodium citrate 720 mg [000678] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

1382.

Preparation of an insulin glulisine solution at 300 IU/mL in the presence of compound 2 and citrate.

[000679] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 300 IU/mL 100 mL Lyophilisate of compound 2 2190 mg Sodium citrate 720 mg [000680] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B83.

Preparation of a solution of insulin aspart at 400 IU/mL in the presence of compound 2 and citrate.

[000681] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin aspart] of 2.0 the different reagents are mixed in the quantities specified below:

Insulin aspart at 400 IU/mL 100 mL Lyophilisate of compound 2 2920 mg Sodium citrate 960 mg [000682] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

81 CA 02889552 2015-04-24 %VO 2014/076-123 PCT/FR2013/052736 884.

Preparation of an insulin glulisine solution at 400 IU/mL in the presence of compound 2 and citrate.

[000683] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 400 IU/mL 100 mL Lyophilisate of compound 2 2920 mg Sodium citrate 960 mg [000684] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

885.

Preparation of a solution of insulin aspart at 500 IU/mL in the presence of compound 2 and citrate.

[000685] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin aspart] of 2.0 the different reagents are mixed in the quantities specified below:

Insulin aspart at 500 IU/mL 100 mL Lyophilisate of compound 2 3650 mg Sodium citrate 1200 mg [000686] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

1386.

Preparation of an insulin glulisine solution at 500 IU/mL in the presence of compound 2 and citrate.

[000687] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin glulisine] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin glulisine at 500 IU/mL 100 mL Lyophilisate of compound 2 3650 mg Sodium citrate 1200 mg [000688] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

82 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B87.

Preparation of a solution of human insulin at 300 IU/mL in the presence of compound 2 and citrate.

[000689] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2, the different reagents are mixed in the quantities specified below:

Human insulin at 300 IU/mL 100 mL Lyophilisate of compound 2 2190 mg Sodium citrate 720 mg [000690] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B88.

Preparation of an insulin lispro solution at 300 IU/mL in the presence of compound 2 and citrate.

[000691] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin lispro] of 2.0 the different reagents are mixed in the quantities specified below:

Insulin lispro at 300 IU/mL 100 mL Lyophilisate of compound 2 2190 mg Sodium citrate 720 mg [000692] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B89.

Preparation of a solution of human insulin at 400 IU/mL in the presence of compound 2 and citrate.

[000693] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2.0, the different reagents are mixed in the quantities specified below:

Human insulin at 400 IU/mL 100 mL Lyophilisate of compound 2 2920 mg Sodium citrate 960 mg [000694] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B90.

Preparation of an insulin lispro solution at 400 IU/mL in the presence of compound 2 and citrate.

[000695] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

83 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 Insulin lispro at 400 IU/mL 100 mL Lyophilisate of compound 2 2920 mg Sodium citrate 960 mg [000696] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B91.

Preparation of a solution of human insulin at 500 IU/mL in the presence of compound 2 and citrate.

[000697] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[human insulin] of 2.0, the different reagents are mixed in the quantities specified below:

Human insulin at 500 IU/mL 100 mL Lyophilisate of compound 2 3650 mg Sodium citrate 1200 mg [000698] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B92.

Preparation of an insulin lispro solution at 500 IU/mL in the presence of compound 2 and citrate.

[000699] For a final volume of 100 mL of formulation with a mass ratio [compound 2]/[insulin lispro] of 2.0, the different reagents are mixed in the quantities specified below:

Insulin lispro at 500 IU/mL 100 mL Lyophilisate of compound 2 3650 mg Sodium citrate 1200 mg [000700] The final pH is adjusted to 7.4 0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at 4 C.

B93.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 3 and citrate [000701] For a final volume of 100 mL of formulation, with a mass ratio [compound 3]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 3 lyophilized 730 mg Humaloge commercial solution 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL 84 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000702] For the citrate, we can use, the acidic form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000703] The final p1-1 is adjusted to 7.4 0.4.

[000704] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

894.

Preparation of a solution of insulin lispro at 100 IU/mL in the presence of compound 4 and citrate [000705] For a final volume of 100 mL of formulation, with a mass ratio [compound 4]/[insulin lispro] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 4 lyophilized 730 mg Commercial solution Humalog 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000706] For the citrate, the acid form or the basic form in the form of sodium salt, potassium salt or another salt compatible with an injectable formulation.

[000707] The final pH is adjusted to 7.4 0.4.

[000708] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

B95.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 3 and citrate [000709] For a final volume of 100 mL of formulation, with a mass ratio [compound 3]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 3 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000710] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000711] The final pH is adjusted to 7.4 0.4.

[000712] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 B96.

Preparation of a solution of human insulin at 100 IU/mL in the presence of compound 4 and citrate [000713] For a final volume of 100 mL of formulation, with a mass ratio [compound 4]/[human insulin] of 2 .0 and a concentration of 9.3 mM citrate, the different reagents are added in the quantities specified below and in the following order:

Compound 4 lyophilized 730 mg Commercial solution Humuline R 100 IU/mL 100 mL Sodium citrate solution at 1.188 M 783 pL [000714] For the citrate, the acid form or the basic form in the form of sodium salt can be used, potassium salt or another salt compatible with an injectable formulation.

[000715] The final pH is adjusted to 7.4 0.4.

[000716] The clear solution is filtered on a 0.22 μm membrane and stored at 4 C.

C Pharmacodynamics and pharmacokinetics Cl: Protocol for measuring the pharmacodynamics of insulin solutions [000717] 12 domestic pigs weighing approximately 50 kg, previously catheterized at the jugular level, are fasted 2.5 hours before the start of the 'experience.

In the hour preceding the insulin injection, 3 blood samples are taken to determine the basal level of glucose and insulin.

[000718] The injection of insulin at a dose of 0.09 IU/kg for insulin lispro and at a dose of 0.125 IU/kg for human insulin and insulin aspart is carried out subcutaneously at neck level, under the ear of the animal using the Novopen insulin pen equipped with a 31 G needle.

[000719] Blood samples are then taken every 4 minutes for 20 minutes then every 10 minutes for up to 3 hours.

After each sample, the catheter is rinsed with a diluted heparin solution.

[000720] A drop of blood is taken to determine the blood sugar level using a glucometer.

[000721] The glucose pharmacodynamic curves are then plotted and the time necessary to reach the minimum blood glucose level for each pig is determined and reported as Tmin glucose.

The average glucose Tmin is then calculated.

[000722] The remaining blood is collected in a dry tube and is centrifuged to isolate the serum.

Insulin levels in serum samples were measured by immuno-enzymatic sandwich ELISA for each pig.

86 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000723] The pharmacokinetic curves are then drawn.

The time required to reach the maximum serum insulin concentration for each pig is determined and reported as insulin Tmax.

The average insulin Tmax is then calculated.

C2: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of Examples B2 and 88 Compound Number of I Example I Insulin Polyanlonic compound pigs B2 Lispro 11 B8 fLispro __ 1 Citrate 9.3 mM __ 10 [000724] The pharmacodynamic results obtained with the formulations described in examples B2 and B8 are presented in Figure 1.

According to the invention, the analysis of these curves show that the formulation of example B8 comprising compound 1 and citrate at 9.3 mM as excipient (curve drawn with the squares corresponding to example B8, Tmin glucose = 30 11 min) makes it possible to obtain a faster action than that of the commercial Humaloge formulation of example 62 (curve drawn with the triangles corresponding to example B2, Tmin glucose = 44 14 min).

[000725] The pharmacokinetic results obtained with the formulations described in Examples 132 and 68 are presented in Figure 2.

According to the invention, the analysis of these curves shows that the formulation of example B8 comprising compound 1 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B8, Tmax insulin = 11 6 min) induces faster absorption of insulin lispro than the commercial Humaloge formulation of example B2 (curve drawn with the triangles corresponding to example 62, Tmax insulin = 18 8 min).

C3: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of examples 62 and B10 Compound Number of __ I Example Insulin Compound B2 Lispro polyanionic pigs $ 11 1 _________________ B10 I Lispro 1 Compound 11 polyanionic 1 [000726] The pharmacodynamic results obtained with the formulations described in examples B2 and 610 are presented in Figure 3.

According to the invention, the analysis of these curves show that the formulation of example 610 comprising compound 1 and polyanionic compound 1 as excipients at 20 mg/mL (curve drawn with the 87 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 squares corresponding to example B10, Tmin glucose = 33 13 min) makes it possible to obtain a faster action than that of the commercial Humalog formulation of example B2 (curve drawn with the corresponding triangles in example B2, Tmin glucose = 44 14 min).

[000727] The pharmacokinetic results obtained with the formulations described in Examples B2 and 810 are presented in Figure 4.

According to the invention, the analysis of these curves shows that the formulation of example B10 comprising compound 1 and polyanionic compound 1 as excipients at 20 mg/mL (curve drawn with the squares corresponding to example 810, Tmax insulin = 15 9 min) induces faster absorption of insulin lispro than the commercial Humalog formulation of example B2 (curve drawn with the triangles corresponding to example B2, Tmax insulin = 18 8 min).

C4: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of Examples B2 and B7 Compound Number of Example Insulin Polyanionic compound pigs B2 I Lispro 12 B7 Lispro 1 1 12 [000728] The pharmacodynamic results obtained with the formulations described in the Examples 82 and B7 are presented in Figure 5.

According to the invention, the analysis of these curves shows that the formulation of example B7 comprising compound 1 as excipient (curve drawn with the squares corresponding to example B7, Tmin glucose = 41 16 min) induces a start of action faster than that of the commercial Humalog formulation of example B2 (curve drawn with the triangles corresponding to example B2, Tmin glucose = 50 14 min).

[000729] The pharmacokinetic results obtained with the formulations described in Examples 132 and 87 are presented in Figure 6. The analysis of these curves shows that the formulation comprising compound 1 as excipient (curve drawn with the squares corresponding to example B2, Tmax insulin = 21 10 min) does not induce faster absorption of insulin lispro than the commercial Humalog formulation of example B2 (curve drawn with the triangles corresponding to example B2 (Tmax insulin = 20 9 min).

Compound 1 alone is therefore not sufficient to induce a significant acceleration in the pharmacokinetics of insulin lispro.

88 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 C5: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of examples B1 and B3 Compound Number of Example 1 Insulin Polyanionic compound pigs B1 Aspart 11 = ... .............. 4 ...........

B3 to Human 11..

[000730] The pharmacodynamic results obtained with the formulations described in Examples B1 and B3 are presented in Figure 7. The analysis of these curves shows that the human insulin formulation of example B3 (curve drawn with the squares corresponding to example B3, Tmin glucose = 61 31 min) does have a slower action than that of the formulation commercial insulin aspart of example B1 (curve drawn with the triangles corresponding to example B1, Tmin glucose = 44 13 min).

[000731] The pharmacokinetic results obtained with the formulations described in Examples B1 and B3 are presented in Figure 8. The analysis of these curves shows that the formulation of human insulin alone in example B3 (curve drawn with the squares corresponding to example B3, Tmax insulin = 36 33 min) indeed induces slower absorption than the commercial formulation. of insulin aspart (Novolog) of example B1 (curve drawn with the triangles corresponding to example Bi, Tmax insulin = 28 13 min).

[000732] These results are consistent with those in the literature with an acceleration in the reduction of blood sugar and insulin absorption for a rapid insulin analog compared to a human insulin.

C6: Pharmacodynamic and pharmacokinetic results of the insulin solutions of Examples B1 and B39.................................. ............... = ..

Compound Number of Example Insulin Polyanionic compound pigs B1 Aspart 14 B39 Human _____ T ..

Citrate 9.3 mM 5 _ [000733] ​​The pharmacodynamic results obtained with the formulations described in Examples B1 and B39 are presented in Figure 9. The analysis of these curves shows that the formulation based on human insulin of example B39 comprising compound 1 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B39, Tmin glucose = 46 9 min) makes it possible to obtain an action similar to that of the commercial formulation of insulin aspart (Novologe) of example 89 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 B1 (curve drawn with the triangles corresponding to example Bi, Tmin glucose = 53 24 min).

[000734] The pharmacokinetic results obtained with the formulations described in Examples B1 and B39 are presented in Figure 10. The analysis of these curves shows that the formulation of example B39 comprising compound 1 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B39, Tmax insulin = 20 7 min) induces an absorption of human insulin similar to that of the commercial formulation of insulin aspart (Novolog) of example B1 (curve drawn with the triangles corresponding to example Bi, Tmax insulin = 22 10 min).

[000735] The time parameters of insulin aspart (Novologe) between examples C5 and C6 being similar, it can be deduced by extrapolation that the formulation of example B39 induces an acceleration of the reduction in blood sugar and blood sugar. absorption of human insulin compared to the commercial formulation of human insulin (example B3).

C7: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of examples B2 and B11 Compound Number of Example Insulin Polyanionic compound pigs I B2 Lispro 26 B11 LisprcT 2 Citrate 9.3 mM 23 [000736] The pharmacodynamic results obtained with the formulations described in Examples 132 and 611 are presented in Figure 13.

According to the invention, the analysis of these curves shows that the formulation of example B11 comprising compound 2 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B11, Tmin glucose = 32 10 min) makes it possible to obtain a faster action than that of the commercial Humalog formulation of example B2 (curve drawn with the triangles corresponding to example B2, Tmin glucose = 41 21 min).

[000737] The pharmacokinetic results obtained with the formulations described in Examples B2 and B1 1 are presented in Figure 14.

According to the invention, the analysis of these curves shows that the formulation of example B11 comprising compound 2 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example 611, Tmax insulin = 13 5 min) induces faster absorption of insulin lispro than the commercial Humalog formulation of example B2 (curve drawn with the triangles corresponding to example 62, Tmax insulin = 22 13 min).

CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 C8: Results of pharmacodynamics and pharmacokinetics of insulin solutions of examples Bi and B38 Compound Number of Example Insulin Polyanionic compound pigs Bi Aspart 37 ..... ...... B38 I Human 2 d Citrate 9.3 mM 31 [000738] The pharmacodynamic results obtained with the formulations described in Examples B1 and B38 are presented in Figure 15. The analysis of these curves shows that the formulation based on human insulin of example B38 comprising compound 2 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B102, Tmin glucose = 47 30 min) makes it possible to obtain an action similar to that of the commercial formulation of insulin aspart (Novologn of example B1 (curve drawn with the triangles corresponding to example Bi, Tmin glucose = 47 15 min).

[000739] The pharmacokinetic results obtained with the formulations described in Examples B1 and B38 are presented in Figure 16. The analysis of these curves shows that the formulation of example B38 comprising compound 2 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B38, Tmax insulin = 22 21 min) induces an absorption of human insulin similar to that of the commercial formulation of insulin aspart (Novolog) of example B1 (curve drawn with the triangles corresponding to example Bi, Tmax insulin = 19 12 min).

[000740] The time parameters of insulin aspart (Novologe) between examples C5 and C8 being close, it can be deduced by extrapolation that the formulation of example B38 induces an acceleration of the reduction in blood sugar and blood sugar. absorption of human insulin compared to the commercial formulation of human insulin (example B3).

C9: Results of pharmacodynamics and pharmacokinetics of the insulin solutions of examples B1 and B53 1 Compound Number of Example Insulin Polyanionic compound pigs Bi Aspart 12 B53 Human Compound 8 Citrate 9.3 mM 8 91 CA 02889552 2015-04-24 WO 2014 /076423 PCT/FR2013/052736 [000741] The pharmacodynamic results obtained with the formulations described in Examples B1 and B53 are presented in Figure 17. The analysis of these curves shows that the formulation based on human insulin of example B53 comprising compound 8 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B53, Tmin glucose = 63 36 min) makes it possible to obtain an action almost as rapid as that of the commercial formulation of insulin aspart (Novologe) of example B1 (curve drawn with the triangles corresponding to example B1, Tmin glucose = 53 19 min).

[000742] The pharmacokinetic results obtained with the formulations described in Examples B1 and B53 are presented in Figure 18. The analysis of these curves shows that the formulation of example B53 comprising compound 8 and citrate at 9.3 mM as excipients (curve drawn with the squares corresponding to example B53, Tmax insulin = 19 12 min) induces an absorption of human insulin similar to that of the commercial formulation of insulin aspart (Novolog) of example B1 (curve drawn with the triangles corresponding to example Bi, Tmax insulin = 19 6 min).

[000743] The time parameters of insulin aspart (Novologs) between examples C5 and C9 being close, we can deduce by extrapolation that the formulation of example B53 induces an acceleration of the drop in blood sugar and blood sugar. absorption of human insulin compared to the commercial formulation of human insulin (example B3).

D Circular dichroism Dl: State of association of insulin lispro evaluated by circular dichroism in the presence of compound 1 [000744] Circular dichroism makes it possible to study the secondary and quaternary structure of insulin.

Insulin monomers are organized into dimers and hexamers. The hexamer is the most physically and chemically stable form of insulin.

There are two hexameric forms, the R6 form and the T6 form.

Insulin lispro presents a strong CD signal at 251 nm characteristic of the R6 hexameric form (the most stable form).

The loss of the CD signal at 251 nm is linked to a destabilization of the hexamer (and therefore the first sign of transformation of the hexamer into a dimer).

[000745] EDTA and the EDTA/citrate mixture completely deconstruct the R6 form of insulin lispro (Figure 11). EDTA therefore has a marked effect on the hexamer.

[000746] On the contrary, citrate alone, compound 1 alone as well as the mixture compound 1/citrate and compound 1/polyanionic compound 1 have almost no impact on the CD signal at 251 nm.

These compounds therefore have almost no impact on the R6 structure of the hexamer and a fortiori on the hexameric structure.

92 CA 02889552 2015-04-24 WO 2014/076-123 PCT/FR2013/052736 D2: State of association of human insulin evaluated by circular dichroism in the presence of compound 1 [000747] Circular dichroism makes it possible to study the structure secondary and quaternary insulin.

Insulin monomers are organized into dimers and hexamers. The hexamer is the most physically and chemically stable form of insulin.

The CD signal at 275 nm is characteristic of the hexameric form of insulin (hexameric signal around -300, dimer signal between 200 and -250 and monomer signal below -200).

The loss of the CD signal at 275 nm is therefore characteristic of a destabilization of the hexamer into dimers or monomers.

[000748] EDTA and the EDTA/citrate combination have a very marked impact on the hexameric structure of human insulin (complete dissociation of the hexamer into dimers, Figure 12).

On the contrary, citrate alone, compound 1 alone, polyanionic compound 1 alone as well as the compound 1/citrate and compound 1/polyanionic compound 1 combinations have no impact on the hexameric structure of human insulin.

Unlike EDTA, human insulin formulations comprising compound 1 and citrate or polyanionic compound 1 do not exhibit dissociation of the human insulin hexamer.

D3: State of association of insulin lispro evaluated by circular dichroism in the presence of compounds 1 to 11 [000749] Circular dichroism makes it possible to study the secondary and quaternary structure of insulin.

Insulin monomers are organized into dimers and hexamers. The hexamer is the most physically and chemically stable form of insulin.

There are two hexameric forms, the R6 form and the T6 form.

Insulin lispro presents a strong CD signal at 251 nm characteristic of the R6 hexameric form (the most stable form).

The loss of the CD signal at 251 nm is linked to a destabilization of the hexamer (and therefore the first sign of transformation of the hexamer into a dimer).

The results obtained are presented in figure 19.

This figure describes on the abscissa:

- A: insulin lispro (100 IU/mL) - B: insulin lispro + 7.3 mg/mL of compound 2 - C: insulin lispro + 7.3 mg/mL of compound 2 + 9.3 mM citrate - D : insulin lispro + 7.3 mg/mL of compound 1 - E: insulin lispro + 7.3 mg/mL of compound 1 + citrate at 9.3 mM - F: insulin lispro + 7.3 mg/mt. of compound 3 - G: insulin lispro + 7.3 mg/mL of compound 3 + citrate at 9.3 mM - H: insulin lispro + 7.3 mg/mL of compound 4 - I: insulin lispro + 7.3 mg /mL of compound 4 + citrate at 9.3 mM 93 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 - J: insulin lispro + 7.3 mg/mL of compound 5 - K: insulin lispro + 7.3 mg/mL of compound 5 + 9.3 mM citrate - L: insulin lispro + 7.3 mg/mL of compound 6 - M: insulin lispro + 7.3 mg/mL of compound 6 + citrate of 9 .3 mM - N: insulin lispro + 7.3 mg/mL of compound 7 - O: insulin lispro + 7.3 mg/mL of compound 7 + citrate at 9.3 mM - P: insulin lispro + 7.3 mg/mL of compound 8 - Q: insulin lispro + 7.3 mg/mL of compound 8 + 9.3 mM citrate - R: insulin lispro + 7.3 mg/mL of compound 9 - S: insulin lispro + 7.3 mg/mL of compound 9 + citrate at 9.3 mM - T: insulin lispro + 7.3 mg/mL of compound 10 - U: insulin lispro + 7.3 mg/mL of compound 10 + citrate at 9.3 mM - V: insulin lispro + 7.3 mg/mL of compound 11 - W: insulin lispro + 7.3 mg/mL of compound 11 + citrate at 9.3 mM and on the ordinate the circular dichroism signal at 251 nm (deg.cm2.dmo1-1).

[000750] Compounds 1 to 11 alone as well as compounds 1 to 11 in combination with citrate have no impact on the CD signal at 251 nm of insulin lispro.

Compounds 1 to 11 therefore have no impact on the R6 structure of the hexamer and a fortiori on the hexameric structure of insulin lispro.

D4: State of association of human insulin evaluated by circular dichroism in the presence of compounds 1 to 11 [000751] Circular dichroism makes it possible to study the secondary and quaternary structure of insulin.

Insulin monomers are organized into dimers and hexamers. The hexamer is the most physically and chemically stable form of insulin.

The CD signal at 275 nm is characteristic of the hexameric form of insulin (hexameric signal around -300, dimer signal between 200 and -250 and monomer signal below -200).

The loss of the CD signal at 275 nm is therefore characteristic of a destabilization of the hexamer into dimers or monomers.

The results obtained are presented in Figure 20.

This figure describes on the abscissa:

[0007521 A: human insulin (100 IU/mL) [000753] B: human insulin + 7.3 mg/mL of compound 2 [000754] C: human insulin + 7.3 mg/mL of compound 2 + citrate 9 .3 mM [000755] D: human insulin + 7.3 mg/mL of compound 1 [000756] E: human insulin + 7.3 mg/mL of compound 1 + 9.3 mM citrate [000757] F: insulin human + 7.3 mg/mL of compound 3 [000758] G: human insulin + 7.3 mg/mL of compound 3 + citrate at 9.3 mM [000759] H: human insulin + 7.3 mg/mL of compound 4 94 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 [000760] I: human insulin + 7.3 mg/mL of compound 4 + citrate 9 .3 mM [000761] J: human insulin + 7.3 mg/mL of compound 5 [000762] K: human insulin + 7.3 mg/mL of compound 5 + citrate at 9.3 mM [000763] L: insulin human + 7.3 mg/mL of compound 6 [000764] M: human insulin + 7.3 mg/mL of compound 6 + citrate at 9.3 mM [000765] N: human insulin + 7.3 mg/mL of compound 7 [000766] 0: human insulin + 7.3 mg/mL of compound 7 + 9.3 mM citrate [000767] P: human insulin + 7.3 mg/mL of compound 8 [000768] Q: human insulin + 7.3 mg/mL of compound 8 + citrate at 9.3 mM [000769] R: human insulin + 7.3 mg/mL of compound 9 [000770] S: human insulin + 7.3 mg/mL of compound 9 + citrate at 9.3 mM [000771] T: human insulin + 7.3 mg/mL of compound 10 [000772] U: human insulin + 7.3 mg/mL of compound 10 + citrate at 9.3 mM [000773] V: human insulin + 7.3 mg/mL of compound 11 [000774] W: human insulin + 7.3 mg/mL of compound 11 + citrate at 9.3 mM and on the ordinate the circular dichroism signal at 275 nm (deg.cm2 .dmo1-1).

[000775] Compounds 1 to 11 alone as well as compounds 1 to 11 in combination with citrate have no impact on the CD signal at 275 nm of human insulin.

Compounds 1 to 11 therefore have no impact on the hexameric structure of human insulin.

E Solubilization of human and analog insulins at the isoelectric point El.

Solubilization of human insulin at its isoelectric point [000776] Human insulin has an isoelectric point of 5.3. At this pH of 5.3 human insulin precipitates.

A test demonstrating the formation of a complex of human insulin with the different compounds is performed at the isoelectric point.

If an interaction exists, it is possible to solubilize insulin at its isoelectric point.

[000777] A solution of human insulin at 200 IU/mL is prepared.

Solutions of compounds at different concentrations (8, 30 or 100 mg/mL) in water are prepared.

An equivolume mixture (50/50) between the human insulin solution and the compound solution is carried out to produce a solution containing 100 IU/mL of human insulin and the desired concentration of compound (4, 15 or 50 mg/ mL).

The pH of the different solutions is adjusted to pH 5.3 by adding 200 mM acetic acid.

[000778] The appearance of the solution is documented.

If the solution is turbid, the compound at the concentration tested does not allow the solubilization of human insulin.

If the solution is translucent, the compound allows the solubilization of human insulin at the concentration tested.

In this way, the compound concentration necessary to solubilize human insulin at its isoelectric point can be determined.

The lower this concentration, the greater the affinity of the compound for human insulin.

[000779] The results obtained are presented in Table 3.

The results show that the compounds and polysaccharides do not have the same properties in terms of solubilization of human insulin.

Solubilization of Solubilization of Solubilization of Compounds human insulin human insulin human insulin (examples) at 100 IU/mL by the at 100 IU/mL by the at 100 IU/mL by the or Polysaccharides compound to 4 compound to 15 compound' to 50 (counter-examples) mg/mL mg/mL mg/mL Counter-examples Polysaccharide 1 Yes Yes Yes Polysaccharide 4 Yes Yes Yes Polysaccharide 3 Yes Yes Yes Polysaccharide 2 Yes Yes Yes Polysaccharide 5 Yes Yes Yes Examples Compound 1 No No Yes Compound 2 No No Yes Compound 3 No No Yes Compound 4 No No Yes Compound 6 No No Yes Compound 8 No No Yes ----Compound 9 No No Yes Compound 10 No No Yes Table 3 E2. Solubilization of insulin lispro at its isoelectric point [000780 ] Insulin lispro has an isoelectric point of 5.3. At this pH insulin lispro precipitates.

A test demonstrating the formation of a complex of insulin lispro with the different compounds is performed at the isoelectric point.

If an interaction exists, it is possible to solubilize insulin lispro at its isoelectric point.

[000781] The commercial formulation of insulin lispro (Humalogn) is dialyzed against 1 mM PO4 buffer (pH 7).

After dialysis, the concentration of insulin lispro is approximately 90 IU/mL.

The compound lyophilisate is weighed and solubilized in the insulin lispro solution to yield formulations containing insulin lispro at 90 IU/mL and 96 CA 02889552 2015-04-24 WO 2014/076423 PCT/FR2013/052736 the compound at the desired concentrations (4, 15 or 50 mg/mL).

The pH of the different solutions is adjusted to pH 5.3 by adding 200 mM acetic acid.

[000782] The appearance of the solution is documented.

If the solution is turbid, the compound at the concentration tested does not allow the solubilization of insulin lispro.

If the solution is translucent, the compound allows the solubilization of insulin lispro at the concentration tested.

In this way, the compound concentration necessary to solubilize insulin lispro at its isoelectric point can be determined.

The lower this concentration, the greater the affinity of the compound for insulin lispro.

[000783] The results obtained are presented in Table 4.

The results show that the compounds and polysaccharides do not have the same properties in terms of solubilization of insulin lispro.

Solubilization of Solubilization of Solubilization of Compounds insulin lispro to insulin lispro to insulin lispro at (examples) 90 IU/mL by the 90 IU/mL by the 90 IU/mL by the or Polysaccharides compound to 4 compound to 15 compound at 50 (counterexamples) mg/mL mg/mL mg/mL Counterexamples Polysaccharide 1 Yes Yes Yes Polysaccharide 3 Yes Yes Yes Polysaccharide 2 Yes Yes Yes Examples Compound 1 No No Yes Yes Compound 2 No No = Compound 3 No No Yes Table 4 F Interaction with albumin: In order to determine the interactions between the different polysaccharides or compounds and a model protein such as albumin, a Centricon assay (50 kDa CutOff membrane) was carried out.

A solution of polysaccharide or compound at 7.3 mg/mL was diluted one third in a solution of BSA (bovine serum albumin) at 20 mg/mL in PBS (concentration in the mixture: 2.43 mg/mL polysaccharide or compound, 13.3 mg/mL albumin and approximately 100 mM salts).

[000784] This mixture was centrifuged on a Centricon to pass approximately half of the volume through the membrane. Albumin is quantitatively retained on the Centricon membrane.

The polysaccharides and compounds analyzed alone pass a large part through the membrane (for the polysaccharides having the largest molar masses, approximately 20% of the polysaccharide is retained ).

[000785] After centrifugation, the polysaccharide or compound is determined by UV in the filtrate.

The percentage of polysaccharide or compound linked to albumin is calculated by the following equation 2 [000786] (1-[polysaccharide or compound in the filtrate in the presence of albumin]/[polysaccharide or compound in the filtrate in the absence of albumlne])*100 [000787] The results obtained are presented in Table 5.

It is very clearly observed that polysaccharides with a molecular mass of 5-15 kDa are strongly retained by albumin in this test.

On the contrary, your compounds of the invention of lower molar mass are significantly less retained by albumin in this test.

ofb Polysaccharide or % Compound Polysaccharide or Compound linked to BsA Counter examples Polysaccharide 4 97 h Polysaccharide 1 95% Polysaccharide 3 77% Polysaccharide 5 86% Polysaccharide Z 82% Examples Compound 2 21% Compound 1 20% Compound 3 27% Compound 4 24 % Compound 5 24% ¨ Compound 6 26% Compound 7 27% Compound 8 27% Compound 9 43% Compound 11 35% Table 5 RECTIFIED SHEET (RULE 91) ISA/EP 98 BRIEF DESCRIPTION OF THE FIGURES [000788] Figure 1: DGIucosis (nnM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B8, glucose Tmin = 30 11 min, curve drawn with triangles corresponding to example B2, glucose Tmin = 44 14 min.

[000789] Figure 2: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B8, insulin Tmax = 11 6 min, curve drawn with triangles corresponding to example B2, insulin Tmax = 18 8 min.

[000790] Figure 3: Dglucose (nnM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B10, glucose Tmin = 33 13 min, curve drawn with triangles corresponding to example B2, glucose Tmin = 44 14 min.

[000791] Figure 4: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B10, insulin Tmax = 15 9 min, curve drawn with triangles corresponding to example B2, insulin Tmax = 18 8 min.

[000792] Figure 5: Dglucose (nnM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B7, glucose Tmin = 41 16 min, curve drawn with triangles corresponding to example B2, glucose Tmin = 50 14 min.

[000793] Figure 6: DIsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B2, insulin Tmax = 21 10 min, curve drawn with triangles corresponding to example B2, insulin Tmax = 20 9 min.

[000794] Figure 7: Dglucose (nnM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B3, glucose Tmin = 61 31 min, curve drawn with the triangles corresponding to example B1, glucose Tmin = 44 13 min.

[000795] Figure 8: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B3, insulin Tmax = 36 33 min, curve drawn with triangles corresponding to example B1, insulin Tmax = 28 13 min.

[000796] Figure 9: Dglucose (nnM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B39, glucose Tmin = 46 9 min, curve drawn with triangles corresponding to example Bi, glucose Tmin = 53 24 min.

[000797] Figure 10: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B39, insulin Tmax = 20 7 min, 99 Date Received/Date Received 2022-07-14 curve drawn with triangles corresponding to example Bi, insulin Tnnax = 22 10 min.

[000798] Figure 11 describes on the x axis, from left to right:

- Humalog - Humalog + 9.3 mM citrate - Humalog + 6 mM EDTA - Humalog + 6 mM EDTA +9.3 mM citrate - Humalog + 7.3 mg/ml Compound 1 - Humalog + 7.3 mg/ml Compound 1 + 9.3 mM citrate - Humalog + 7.3 mg/ml Compound 1 + 20 mg/mi Compound 1 Polyanionic and on the y axis the CD signal at 251 nnn (deg.cm2.dm01-1).

[000799] Figure 12 describes on the x axis, from left to right:

- rhINS - rhINS + 9.3 mM citrate - rhINS + 6 mM EDTA - rhINS + 6 mM EDTA +9.3 mM citrate - rhINS + 7.3 mg/mi Compound 1 - rhINS + 7.3 mg/ml Compound 1 + 9.3 mM citrate - rhINS + 7.3 mg/mi Compound 1 + 20 ring/nnl Compound 1 Polyanionic and on the y axis the CD signal at 275 nnn (deg.cnn2.dm01-1).

[000800] Figure 13: DGIucose (mM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B11, glucose Tmin = 32 10 min, curve drawn with the triangles corresponding to example B2, glucose Tmin = 41 21 min.

[000801] Figure 14: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B11, insulin Tnnax = 13 5 min, curve drawn with the triangles corresponding to example B2, insulin Tnnax = 22 13 min.

[000802] Figure 15: Dglucose (mM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B102, glucose Tmin = 47 30 min, curve drawn with the triangles corresponding to example B1, glucose Tmin = 47 15 min.

[000803] Figure 16: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B38, insulin Tnnax = 22 21 min, curve drawn with the triangles corresponding to example Bi, insulin Tnnax = 19 12 min.

[000804] Figure 17: Dglucose (mM) as a function of time after injection (min.).

Curve drawn with the squares corresponding to example B53, glucose Tmin = 63 36 100 Date Received/Date Received 2022-07-14 min, curve drawn with the triangles corresponding to example B1, glucose Tnnin = 53 19 min.

[000805] Figure 18: DIinsulin (pM) as a function of time after injection (min.).

Curve drawn with squares corresponding to example B53, insulin Tmax = 19 12 min, curve drawn with triangles corresponding to example B1, insulin Tmax = 19 6 min.

[000806] Figure 19 describes on the x axis:

- A: lispro insulin (100 IU/nnL) - B: lispro insulin + 7.3 mg/mL of compound 2 - C: lispro insulin + 7.3 mg/mL of compound 2 + citrate at 9.3 mM - D: lispro insulin + 7.3 mg /mL of compound 1 - E: lispro insulin + 7.3 mg/mL of compound 1 + citrate at 9.3 mM - F: lispro insulin + 7.3 mg/mL of compound 3 - G: lispro insulin + 7.3 mg/mL of compound 3 + citrate at 9.3 mM - H: lispro insulin + 7.3 mg/mL of compound 4 - I: lispro insulin + 7.3 mg/mL of compound 4 + citrate at 9.3 mM - J: lispro insulin + 7.3 mg/mL of compound 5 - K: lispro insulin + 7.3 mg/mL of compound 5 + citrate at 9.3 mM - L: lispro insulin + 7.3 mg/mL of compound 6 - M: lispro insulin + 7.3 mg/ mL of compound 6 + citrate at 9.3 mM - N: lispro insulin + 7.3 nng/rinL of compound 7 - 0: lispro insulin + 7.3 mg/mL of compound 7 + citrate at 9.3 mM - P: lispro insulin + 7.3 mg/mL of compound 8 - Q: lispro insulin + 7.3 ring/rinL of compound 8 + citrate at 9.3 mM - R: lispro insulin + 7.3 ring/nnL of compound 9 - S: lispro insulin + 7.3 mg/mL of compound 9 + citrate at 9.3 mM - T: lispro insulin + 7.3 mg/mL of compound 10 - U: lispro insulin + 7.3 ring/nnL of compound 10 + citrate at 9.3 mM - V: lispro insulin + 7.3 mg/mL of compound 11 - W: lispro insulin + 7.3 mg/mL of compound 11 + citrate at 9.3 mM and on the y axis the circular dichroisnne signal at 251 nnn (deg.crn2.dm01- 1).

[000807] Figure 20 describes on the x axis:

- A: human insulin (100 IU/nriL) - B: human insulin + 7.3 mg/mL of compound 2 - C: human insulin + 7.3 mg/mL of compound 2 + 9.3 mM citrate - D: human insulin + 7.3 mg /mL of compound 1 - E: human insulin + 7.3 nng/nnl_ of compound 1 + citrate at 9.3 mM - F: human insulin + 7.3 mg/mL of compound 3 101 Date Received/Date Received 2022-07-14 - G: human insulin + 7.3 mg/nnL of compound 3 + citrate at 9.3 mM - H: human insulin + 7.3 mg/nnL of compound 4 - I: human insulin + 7.3 mg/nnL of compound 4 + citrate at 9.3 mM - J: human insulin + 7.3 mg/mL of compound 5 - K: human insulin + 7.3 mg/nnL of compound 5 + citrate at 9.3 nriM - L: insulin human + 7.3 nng/nnL of compound 6 - M: human insulin + 7.3 mg/nnL of compound 6 + citrate at 9.3 mM - N: human insulin + 7.3 mginnL of compound 7 - 0: human insulin + 7.3 nng/nnL of compound 7 + citrate at 9.3 mM - P: human insulin + 7.3 mg/nnL of compound 8 - Q: human insulin + 7.3 mg/nnL of compound 8 + citrate at 9.3 mM - R: human insulin + 7.3 mg/nnL of compound 9 - S: human insulin + 7.3 nng/nnL of compound 9 + citrate at 9.3 mM - T: insulin human + 7.3 mg/nnL of compound 10 - U: human insulin + 7.3 mg/nnL of compound 10 + citrate at 9.3 mM - V: human insulin + 7.3 nng/nnL of compound 11 - W: human insulin + 7.3 mginL of compound 11 + citrate at 9.3 mM and on the y axis the circular dichroisnne signal at 275 nnn (deg.cnn2.dmo1-1).

102 Date Received/Date Received 2022-07-14