HK1197734A

HK1197734A - Injectable solution of at least one type of basal insulin

Info

Publication number: HK1197734A
Application number: HK14111422.9A
Authority: HK
Inventors: 奥利维耶．索拉; 格拉尔德．索拉; 杰夫．托纳尔; 亚历山大．盖斯勒
Original assignee: 阿道恰公司
Priority date: 2011-08-10
Filing date: 2012-08-09
Publication date: 2015-02-13

Description

Injectable solution of at least one basal insulin

The present invention relates to the treatment of diabetes by injection of insulin.

In recent years, insulin therapy or treatment of diabetes by injection of insulin has made significant progress, particularly by the development of new insulin that provides correction of the patient's blood glucose, which can better mimic the physiological activity of the pancreas.

To meet the daily insulin demand, diabetics currently have two planned complementary acting insulins available: prandial (or fast acting) insulin and basal (or slow acting) insulin.

Prandial insulin allows rapid control (metabolism and/or storage) of glucose provided by meals and snacks. The patient must inject prandial insulin prior to each food intake, i.e., about 2 to 3 injections per day. The most commonly used prandial insulins are: recombinant human insulin,(insulin aspart from Novonordst (Novonordst)),(insulin lispro from lilly) and(insulin glulisine from Senoft-Anvants (SANOFI-AVENTIS)).

Basal insulin maintains the patient's blood glucose homeostasis outside the food intake period. It basically functions by blocking the endogenous production of glucose (hepatic glucose). The daily dose of basal insulin is typically 40-50% of the total daily insulin requirement. The dose is dispensed by 1 or 2 injections evenly distributed throughout the day, depending on the basal insulin used. The most commonly used basal insulin is(insulin detemir) from Novonide) and(insulin glargine from Xenophenanthrene-Anvant).

It should be noted that, for completeness, NPH (NPH insulin, Neutral ProtamineHagedorn; Humuline) Is the oldest basal insulin. This formulation is the result of precipitation of human insulin (anionic at neutral pH) using the cationic protein protamine. These microcrystals are dispersed in an aqueous suspension and slowly dissolve after subcutaneous injection. This slow dissolution ensures a sustained release of insulin. However, this release does not ensure a constant concentration of insulin over time. The release profile is bell-shaped and lasts only 12 to 16 hours. Thus, injections were given twice daily. This NPH basal insulin is more potent than modern basal insulinAndthe efficacy is much worse. NPH is a basal insulin with moderate action.

With the advent of fast insulin analogues, the principle of NPH was altered to produce a "pre-mix" product, which provided both a fast and a moderate effect. NovoLog(Novonide) and Humalilog(Paris) is a fast insulin analogue comprising a moiety complexed with protamineAndthe formulation of (1). These preparations therefore contain microcrystals of insulin which are believed to act intermediately and a proportion of insulin which remains soluble and acts rapidly. These formulations do indeed provide the advantage of rapid insulin but they also have the disadvantage of NPH, i.e. the duration of action is limited to 12 to 16 hours and a bell-shaped insulin release. However, these products enable a single injection of moderate acting basal insulin and fast acting prandial insulin to a patient. In fact, many patients wish to reduce their number of injections.

Basal insulin, which is currently sold and currently under clinical development, can be classified according to the technical scheme for obtaining sustained action, and two approaches are currently used.

The first, insulin detemir, binds albumin in vivo. It is an analogue soluble at pH7 that contains a fatty acid (tetradecanoyl) side chain attached at position B29, which enables this insulin to be combined with albumin in vivo. The sustained action is mainly due to this affinity for albumin after subcutaneous injection.

However, its pharmacokinetic profile does not allow it to cover the entire day, which means that twice daily injections are usually required.

Other basal insulins soluble at pH7 (e.g.) Are currently under development.Also included is a fatty acid side chain (hexadecanediacyl- γ -L-Glu) attached to insulin.

The second, insulin glargine, mode is precipitation at physiological pH. This is a human insulin analogue obtained by extending the carboxy-terminal part of the B-chain of human insulin with two arginine residues and replacing the asparagine residue a21 with a glycine residue (US 5656722). It is contemplated that the addition of two arginine residues will adjust the pI (isoelectric point) of insulin glargine at physiological pH, thereby rendering the insulin analog insoluble in physiological media.

Substitution of a21 is contemplated to make insulin glargine stable at acidic pH, enabling its formulation into the form of injectable solutions at acidic pH. During subcutaneous injection, the process of insulin glargine from an acidic pH (pH4-4.5) to physiological pH (neutral pH) causes it to precipitate under the skin. The slow re-dissolution of the insulin glargine microparticles ensures a slow and sustained action.

The hypoglycemic effect of insulin glargine is essentially constant over a24 hour period, which allows most patients to limit themselves to once daily injections.

Insulin glargine is currently considered to be the best basal insulin on the market.

However, the acidic pH of the basic insulin (its isoelectric point is between 5.8 and 8.5) formulation of the insulin glargine type prevents any pharmaceutical combination with other proteins, in particular prandial insulin, since the latter is unstable at acidic pH.

However, to date no one has managed to dissolve the basal insulin of these insulin glargine types (which has an isoelectric point between 5.8 and 8.5) at neutral pH, while maintaining a pH-independent solubility differential between the in vitro medium (containing these insulins) and the in vivo medium (under the skin).

In particular, the working principle of the basic insulin of the insulin glargine type outlined above (i.e. that it is soluble at acidic pH, precipitates at physiological pH) discourages the skilled person from moving away from any solution of pH6-8 that would dissolve insulin of the insulin glargine type while maintaining the essential properties of insulin glargine type precipitating in subcutaneous media.

Furthermore, prandial insulin cannot be formulated at acidic pH because under these conditions prandial insulin undergoes deamidation side reactions at position a21, making it unable to meet the united states pharmacopoeia (USPharmacopeia) requirements, i.e. less than 5% by-products after 4 weeks at 30 ℃.

Furthermore, this acidic pH of the base insulin formulation of the insulin glargine type (which has an isoelectric point between 5.8 and 8.5) hinders even any temporary combination with prandial insulin at neutral pH.

This limitation of insulin glargine use has been demonstrated by recent clinical studies proposed at 69 th american diabetes association science conference held in 5-9 th of 6 th of us, 5 th to 9 th of louisiana, 2009. A dose of insulin glargine and a dose of prandial insulin (in this case insulin lispro) were mixed together just before injection (e.cengiz et al, 2010, Diabetes Care, 33(5), 1009-12). This experiment can confirm that the pharmacokinetic and pharmacodynamic profiles of prandial insulin are significantly delayed, which can cause postprandial hyperglycemia and nocturnal hypoglycemia. This study does confirm the incompatibility of insulin glargine with the fast acting insulin currently on the market.

Additionally, commercial insulin glargine-based products from the company sunofil-amphetaThe instructions of (a) clearly indicate to the user that the insulin should not be mixed with the solution of prandial insulin, possibly due to a serious risk of altering the pharmacokinetics and pharmacodynamics of the insulin glargine and/or mixed prandial insulin.

However, from a therapeutic point of view, clinical studies (abstract 2163-PO and abstract number 0001-LB, especially those performed by Senoffie-Anvate) disclosed during the 70 th annual American Diabetes Association (ADA) scientific conference in 2010 indicated a combinationTreatment ratios of insulin glargine and prandial insulin were based on NovologOr HumalogThe type "pre-mixed" products are much more effective in treatment.

For the combination of insulin glargine and fast insulin, the Biodel company in patent application US7718609 describes in particular a composition comprising a basal insulin and a prandial insulin at pH3.0 to 4.2 in the presence of a chelating agent and a polybasic acid. This patent teaches how to make prandial insulin compatible at acidic pH in the presence of insulin of the insulin glargine type. It does not teach how to prepare a combination of insulin glargine type insulin and prandial insulin at neutral pH.

From an analysis of the compositions described in the literature and in the patents, it can be seen that the insolubility at pH7 of the basal insulin of the insulin glargine type is a prerequisite for its slow action. Therefore, all proposed solutions for combining it with other products (e.g. prandial insulin) are based on tests of the dissolution or stability of prandial insulin at acidic pH, see e.g. WO 2007/121256 and WO 2009/021955.

Surprisingly, the composition according to the invention enables the dissolution of basal insulin with an isoelectric point between 5.8 and 8.5 at pH7.

Surprisingly, the composition according to the invention allows to maintain the duration of the hypoglycemic activity of basal insulin having an isoelectric point between 5.8 and 8.5, despite its dissolution at pH7 prior to injection. This noteworthy property is due to the fact that insulin glargine, dissolved at pH7 in the composition of the present invention, precipitates in the subcutaneous medium by virtue of the change in the composition of the medium. The factor causing precipitation of insulin glargine is no longer a change in pH but a change in the composition of the environment during the passage of the pharmaceutical composition into the physiological medium.

By solving this problem of solubility at pH7, the present invention enables:

-proposing an injectable composition in the treatment of diabetes comprising: basal insulin with isoelectric point between 5.8 and 8.5 in the form of homogeneous solution of pH7 while maintaining its biological activity and action profile;

-proposing a composition in the form of a formulation comprising a combination of a basal insulin having an isoelectric point between 5.8 and 8.5 and a prandial insulin and not altering the profile of action of the prandial insulin soluble at pH6-8 and unstable at acidic pH, while maintaining the basal profile of action inherent to the basal insulin;

-an injectable composition is proposed for the treatment of diabetes, further comprising a combination of basal insulin with an isoelectric point between 5.8 and 8.5 and a derivative or analogue of gastrointestinal hormone (such as GLP-1 or "glucagon-like peptide-1");

-for the patient, reducing their number of injections;

-for said composition, satisfying the requirements of the united states pharmacopeia and the european pharmacopeia.

Surprisingly, in the insulin combination of insulin glargine and prandial insulin which is the subject of the present invention, the rapid action of prandial insulin is maintained despite the precipitation of insulin glargine in the subcutaneous medium.

The invention relates to a composition in the form of an injectable aqueous solution, having a pH of between 6.0 and 8.0, comprising at least:

a) a basal insulin having an isoelectric point pI of 5.8 to 8.5;

b) dextran of formula I or formula II substituted with a group bearing a carboxylic acid load (charge) and a hydrophobic group:

wherein:

● R is-OH or a group selected from:

○-(f-[A]-COOH)_n；

○-(g-[B]-k-[D])_md comprises at least one alkyl chain containing at least 8 carbon atoms;

● n represents the degree of substitution of glucoside units by-f- [ A ] -COOH and 0.1. ltoreq. n.ltoreq.2;

● m represents the degree of substitution of-g- [ B ] -k- [ D ] for glucoside units and 0< m.ltoreq.0.5;

● q denotes the degree of polymerisation of the glucoside units, in other words the average number of glucoside units per polysaccharide chain, and 3. ltoreq. q.ltoreq.50;

●-(f-[A]-COOH)_n：

O-A-is a straight or branched chain group containing 1 to 4 carbon atoms; the-A-group:

bonded to the glucoside unit via a functional group f selected from ether, ester and carbamate functional groups;

●-(g-[B]-k-[D])_m：

O-B-is a linear or branched, at least divalent group comprising from 1 to 4 carbon atoms; the-B-group:

bonded to the glucoside unit via a functional group g selected from ether, ester and carbamate functional groups;

o is bonded to the-D group via the functional group k; k is selected from ester, amide and carbamate functional groups; the-D group:

● is-X (-l-Y)_pA group, X is an at least divalent group comprising 1 to 12 atoms selected from C, N and O atoms, optionally bearing a carboxyl or amine function and l or is derived from an amino acid, a diol, a diamine or a mono or polyethylene glycol mono or diamine; y is a linear or cyclic alkyl, alkylaryl or arylalkyl radical having from 8 to 30 carbon atoms, optionally substituted by one or more C₁To C₃Alkyl substitution; p is ≧ 1, and l is a functional group selected from ester, amide, and carbamate functional groups;

● f, g and k are the same or different;

● the free acid functional group is Na⁺And K⁺In the form of a salt of an alkali metal cation of (a);

● and, when p is 1, if Y is C₈To C₁₄Alkyl, then q m.gtoreq.2, if Y is C₁₅Alkyl, then q m is more than or equal to 2; and if Y is C₁₆To C₂₀Alkyl, then q m is more than or equal to 1;

● and, when p.gtoreq.2, if Y is C₈To C₉Alkyl, then q m.gtoreq.2, if Y is C₁₀To C₁₆Alkyl, then q m is more than or equal to 0.2;

wherein:

● R is OH or- (f- [ A)]-COOH)_nGroup (b):

O-A-is a straight or branched chain group containing 1 to 4 carbon atoms; the group-A-:

o n represents a degree of substitution of the glucoside unit with-f- [ A ] -COOH, and 0.1. ltoreq. n.ltoreq.2;

● R' is selected from the following groups:

○-C(O)NH-[E]-(0-[F])_t；

○-CH₂N(L)_z-[E]-(0-[F])_t；

wherein:

z is a positive integer equal to 1 or 2,

l is selected from:

■ -H, and z is equal to 1, and/or

■ -COOH and z is equal to 1 or 2, if f is an ether function,

■ -CO- [ A ] -COOH and z is equal to 1 if f is an ester function, and

■ -CO-NH- [ A ] -COOH and z is equal to 1 if f is a carbamate functional group;

○-[E]-(0-[F])_t：

■ -E-is a straight or branched chain at least divalent radical comprising 1 to 8 carbon atoms and optionally comprising heteroatoms such as O, N or S;

■ -F-is a straight-chain alkyl or cycloalkyl, alkylaryl or arylalkyl radical of 12 to 30 carbon atoms, optionally substituted by one or more C₁To C₃Alkyl substitution;

■ 0 is a functional group selected from ether, ester, amide or carbamate functional groups;

■ t is a positive integer equal to 1 or 2;

● when z is 2, the nitrogen atom is in the form of a quaternary ammonium.

In one embodiment, when p ═ 1, if Y is C₂₁To C₃₀And then q m.gtoreq.1. In one embodiment, when p ═ 1, if Y is C₂₁To C₃₀And then q m is 0.1. In one embodiment, - (f- [ A)]-COOH)_nThe radicals are such that:

● -A-is a group containing 1 carbon atom, the-A-group being bonded to the glucoside unit via an ether function f.

In one embodiment, - (g- [ B)]-k-[D])_mThe radicals are such that:

● -B-is a group containing 1 carbon atom, which-B-group is bonded to the glucoside unit via an ether function g, and

● X is a group derived from an amino acid.

In one embodiment, - (f- [ A)]-COOH)_nThe radicals are such that:

● -A-is a group containing 1 carbon atom, which-A-group is bonded to the glucoside unit via an ether function f, and

●-(g-[B]-k-[D])_mthe radicals are such that:

● X is a group derived from an amino acid,

● k is an amide functional group.

In one embodiment, the dextran substituted with a group bearing a carboxylic acid load and a hydrophobic group is a dextran of formula III:

wherein:

● R is-OH or a group selected from:

○-(f-[A]-COOH)_n；

●-(f-[A]-COOH)_n：

●-(g-[B]-k-[D])_m：

bonded to the group-D via a functional group k selected from ester, amide and carbamate functional groups; the-D group:

● is-X (-l-Y)_pA group, X is an at least divalent group comprising 1 to 12 atoms selected from C, N and O atoms, optionally bearing a carboxyl or amine function and l or is derived from an amino acid, a diol, a diamine or a mono or polyethylene glycol mono or diamine; y is a straight-chain alkyl or cycloalkyl, alkylaryl or arylalkyl radical having from 8 to 20 carbon atoms, optionally substituted by one or more C₁To C₃Alkyl substitution; p is ≧ 1, and l is a functional group selected from ester, amide, and carbamate functional groups;

● f, g and k are the same or different;

● and when p.gtoreq.2, if Y is C₈To C₁₁Alkyl, then q m.gtoreq.2, if Y is C₁₂To C₁₆Alkyl, then q m.gtoreq.0.3.

In one embodiment, the dextran substituted with a group bearing a carboxylic acid load and a hydrophobic group is a dextran of formula IV:

wherein:

● R is-OH or a group selected from:

○-(f-[A]-COOH)_n；

● n represents the degree of substitution of the hydroxyl-OH function by-f- [ A ] -COOH per glucoside unit and 0.1. ltoreq. n.ltoreq.2;

● m represents the degree of substitution of the hydroxyl-OH function by-g- [ B ] -k- [ D ] per glucoside unit and 0< m.ltoreq.0.5;

● q denotes the degree of polymerisation of the glucoside units, in other words the average of the glucoside units in each polysaccharide chain

The average number is more than or equal to 3 and less than or equal to 50;

●-(f-[A]-COOH)_n：

●-(g-[B]-k-[D])_m：

● is an-X (-l-Y) p group, X is an at least divalent group comprising 1 to 12 atoms selected from C, N and O atoms, optionally bearing a carboxyl or amine functional group and l or is derived from an amino acid, a diol, a diamine or a mono or polyethylene glycol mono or diamine; y is 8 to 30 carbonsLinear or cyclic alkyl, alkylaryl or arylalkyl radicals of atoms, optionally substituted by one or more C₁To C₃Alkyl substitution; p is ≧ 1, and l is a functional group selected from ester, amide, and carbamate functional groups;

● f, g and k are the same or different;

● and, when p is 1, if Y is C₈To C₁₄Alkyl, then q m.gtoreq.2, if Y is C₁₅Alkyl, then q m is more than or equal to 2; and if Y is C₁₆To C₃₀Alkyl, then q m is more than or equal to 1; ● and when p.gtoreq.2, if Y is C₈To C₉Alkyl, then q m.gtoreq.2, if Y is C₁₀To C₁₆Alkyl, then q m.gtoreq.0.2.

The structure diagram corresponds to the diagram usually used to indicate the glucan (a polysaccharide consisting mainly of (1, 6) linkages between the glucoside units), which is the diagram used. Dextran also typically contains about 5% of (1, 3) linkages, which are intentionally not shown, but which are of course also included within the scope of the present invention.

The term "basal insulin" having an isoelectric point between 5.8 and 8.5 is understood to mean insulin which is insoluble at pH7 and has a duration of action of 8 to 24 hours or more in a standard model of diabetes.

These basal insulins having an isoelectric point of 5.8 to 8.5 are recombinant insulins, the primary structure of which is modified mainly by introducing basic amino acids such as arginine or lysine. Which are described, for example, in the following patents, patent applications or publications: WO 2003/053339, WO 2004/096854, US5656722 and US 6100376.

In one embodiment, the basal insulin having an isoelectric point between 5.8 and 8.5 is insulin glargine.

In one embodiment, the composition according to the invention comprises 100 IU/ml (i.e. about 3.6 mg/ml) basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises 40 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises 200 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises 300 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises 400 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises 500 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 0.2 and 5.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 0.2 and 4.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 0.2 and 3.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 0.5 and 3.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 0.8 and 3.

In one embodiment, the weight ratio of basal insulin to substituted dextran having an isoelectric point between 5.8 and 8.5 (i.e., substituted dextran/basal insulin) is between 1 and 3.

In one embodiment, the concentration of substituted dextran is 1 to 100 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 80 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 60 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 50 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 30 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 20 mg/ml.

In one embodiment, the concentration of substituted dextran is 1 to 10 mg/ml.

In one embodiment, the concentration of polysaccharide is 5 to 20 mg/ml.

In one embodiment, the concentration of the polysaccharide is from 5 to 10 mg/ml.

In one embodiment, the composition according to the invention further comprises prandial insulin. Prandial insulin is soluble at pH7.

The term "prandial insulin" is understood to mean "fast" insulin or "normal" insulin.

"fast" prandial insulin is insulin that must meet the demand caused by ingestion of protein and sugar at the time of a meal, which must function in less than 30 minutes.

In one embodiment, the "normal" prandial insulin is selected from(human insulin) and(human insulin).

"fast acting" prandial insulin is insulin obtained recombinantly and which is modified to reduce its duration of action.

In one embodiment, the "fast acting" prandial insulin is selected from insulin lisproInsulin glulisineAnd insulin aspart

In one embodiment, the composition according to the invention comprises a total of 100 IU/ml (i.e. about 3.6 mg/ml) of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 40 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 200 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 300 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 400 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 500 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 600 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 700 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the composition according to the invention comprises a total of 800 IU/ml of insulin combined from prandial insulin and basal insulin having an isoelectric point between 5.8 and 8.5.

For the above formulations of 40 to 800 IU/ml, the ratio of basal insulin to prandial insulin having an isoelectric point of 5.8 to 8.5 (expressed as a percentage relative to the total amount of insulin) is, for example, 25/75, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20 and 90/10. However, any other ratio may be produced.

For a formulation comprising 100 IU/ml total insulin, the ratio of basal insulin to prandial insulin with an isoelectric point between 5.8 and 8.5 is, for example, 25/75, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20 or 90/10 in IU/ml. However, any other ratio may be produced.

In an embodiment, the composition according to the invention further comprises GLP-1, a GLP-1 analog or a GLP-1 derivative.

In one embodiment, the GLP-1 analog or derivative is selected from the group consisting of: exenatide (exenatide) developed by Gift and Amelin Pharmaceuticals orLiraglutide (liraglutide) developed by Novonide orOr lixisenatide developed by sunofilTheir analogs or derivatives and their pharmaceutically acceptable salts.

In one embodiment, the GLP-1 analog or derivative is exenatide orAnalogs or derivatives thereof and pharmaceutically acceptable salts thereof.

In one embodiment, the GLP-1 analog or derivative is liraglutide or liraglutideAnalogs or derivatives thereof and pharmaceutically acceptable salts thereof.

In one embodiment, the GLP-1 analog or derivative is lixisenatide orAnalogs or derivatives thereof and pharmaceutically acceptable salts thereof.

The term "analogue", when used in reference to a peptide or protein, is understood to refer to a peptide or protein in which one or more constituent amino acid residues have been replaced by other amino acid residues and/or one or more constituent amino acid residues in the peptide or protein have been deleted and/or one or more constituent amino acid residues have been added to the peptide or protein. For this definition of analogs, the percent identity accepted is 50%.

The term "derivative" when used in reference to a peptide or protein is understood to mean a peptide or protein or analogue that is chemically modified by a substituent that is not present in the peptide or protein or analogue in reference, that is, a peptide or protein that is modified by creating a covalent bond to introduce a substituent.

In one embodiment, the concentration of GLP-1, GLP-1 analog, or GLP-1 derivative is from 0.01 to 10 mg/ml.

In one embodiment, the exenatide, an analogue or derivative thereof as well as a pharmaceutically acceptable salt thereof is in a concentration of 0.05 to 0.5 mg/ml.

In one embodiment, the concentration of liraglutide, analogs or derivatives thereof, and pharmaceutically acceptable salts thereof is from 1 to 10 mg/ml.

In one embodiment, the concentration of lixisenatide, analogs or derivatives thereof, and pharmaceutically acceptable salts thereof, is from 0.01 to 1 mg/ml.

In one embodiment, the composition according to the invention is produced by mixing a commercial solution of basal insulin having an isoelectric point between 5.8 and 8.5 and a commercial solution of GLP-1, a GLP-1 analogue or a GLP-1 derivative in a volume ratio between 10/90 and 90/10.

In one embodiment, the composition according to the invention comprises a daily dose of basal insulin and a daily dose of GLP-1, a GLP-1 analog or a GLP-1 derivative.

In one embodiment, the composition according to the invention comprises 500 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml exenatide.

In one embodiment, the composition according to the invention comprises 500 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 1 to 10 mg/ml liraglutide.

In one embodiment, the composition according to the invention comprises 500 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml lixisenatide.

In one embodiment, the composition according to the invention comprises 100 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml exenatide.

In one embodiment, the composition according to the invention comprises 100 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 1 to 10 mg/ml liraglutide.

In one embodiment, the composition according to the invention comprises 100 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml lixisenatide.

In one embodiment, the composition according to the invention comprises 40 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml exenatide.

In one embodiment, the composition according to the invention comprises 40 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 1 to 10 mg/ml liraglutide.

In one embodiment, the composition according to the invention comprises 40 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml lixisenatide.

In one embodiment, the composition according to the invention comprises 200 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml exenatide.

In one embodiment, the composition according to the invention comprises 200 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 1 to 10 mg/ml liraglutide.

In one embodiment, the composition according to the invention comprises 200 IU/ml basal insulin having an isoelectric point between 5.8 and 8.5 and 0.05 to 0.5 mg/ml lixisenatide.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 0 to 5000 μ M.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 50 to 4000 μ M.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 200 to 3000 μ M.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 0 to 1000 μ M.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 20 to 600 μ M.

In one embodiment, the composition according to the invention further comprises a zinc salt in a concentration of 50 to 500 μ M.

In one embodiment, the composition according to the invention comprises a buffer selected from Tris, citrate and phosphate in a concentration of 0 to 100mM, preferably 0 to 50mM or 15 to 50 mM.

In one embodiment, the composition according to the invention further comprises a preservative.

In one embodiment, the preservative is selected from m-cresol and phenol, alone or as a mixture.

In one embodiment, the concentration of the preservative is 10 to 50 mM.

In one embodiment, the concentration of the preservative is 10 to 40 mM.

The compositions according to the invention may also comprise additives, for example tonicity agents (agents de)) Such as glycerol, NaCl, mannitol and glycine.

The composition according to the invention may also comprise additives according to pharmacopoeia, for example surfactants, for example polysorbates.

The composition according to the invention may also comprise all excipients according to pharmacopoeia compatible with the insulin used at the concentration used.

In one embodiment, 0.3. ltoreq. n.ltoreq.1.7.

In one embodiment, 0.7. ltoreq. n.ltoreq.1.5.

In one embodiment, 0.9. ltoreq. n.ltoreq.1.2.

In one embodiment, 0.01. ltoreq. m.ltoreq.0.5.

In one embodiment, 0.02. ltoreq. m.ltoreq.0.4.

In one embodiment, 0.03. ltoreq. m.ltoreq.0.3.

In one embodiment, 0.05. ltoreq. m.ltoreq.0.2.

In one embodiment, 3 ≦ q ≦ 50.

In one embodiment, 3 ≦ q ≦ 40.

In one embodiment, 3 ≦ q ≦ 30.

In one embodiment, 3 ≦ q ≦ 20.

In one embodiment, 3 ≦ q ≦ 10.

In one embodiment, - (f- [ A)]-COOH)_nThe radicals are selected from the following sequences (enchainement), f having the meaning given above:

in one embodiment, - (g- [ B)]-k-[D])_mThe radicals are selected from the following sequences, g, k and D having the meanings given above:

in one embodiment, D is wherein the X group is at least a divalent group from an amino acid.

In one embodiment, D is wherein the X group is an at least divalent group derived from an amino acid selected from the group consisting of glycine, leucine, phenylalanine, lysine, isoleucine, alanine, valine, aspartic acid and glutamic acid.

The group derived from an amino acid may be levorotatory or dextrorotatory.

In one embodiment, D is wherein the X group is an at least divalent group from a mono or polyethylene glycol.

In one embodiment, D is wherein the X group is an at least divalent group derived from ethylene glycol.

In one embodiment, D is where the X group is an at least divalent group derived from a polyethylene glycol selected from the group consisting of diethylene glycol (dienethine) and triethylene glycol (thiethylene).

In one embodiment, D is wherein the X group is an at least divalent group from a mono or polyethylene glycol amine.

In one embodiment, D is where the X group is at least a divalent group from a mono or poly (ethylene glycol) amine selected from the group consisting of ethanolamine, diglycolamine (di (thienylneglycolamine), and triglycolamine).

In one embodiment, D is wherein the X group is at least a divalent group from a mono or polyethylene glycol diamine.

In one embodiment, D is wherein the X group is at least a divalent group derived from ethylenediamine.

In one embodiment, D is where the X group is at least a divalent group derived from a mono or poly (ethylene glycol) diamine selected from the group consisting of diethylene glycol diamine (di (thiethylene) diamine) and triethylene glycol diamine (thiethylene diamine).

In one embodiment, D is where the Y group is an alkyl group from a hydrophobic alcohol.

In one embodiment, D is where the Y group is an alkyl group from a hydrophobic alcohol selected from octanol (octyl alcohol), 3, 7-dimethyloctan-1-ol, decanol (decyl alcohol), dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), and hexadecanol (cetyl alcohol).

In one embodiment, D is where the Y group is an alkyl group from a hydrophobic acid.

In one embodiment, D is where the Y group is an alkyl group from a hydrophobic acid selected from the group consisting of capric acid, lauric acid, myristic acid and palmitic acid.

In one embodiment, D is wherein the Y group is a group derived from a sterol.

In one embodiment, D is wherein the Y group is a group derived from a sterol selected from the group consisting of cholesterol and derivatives thereof.

In one embodiment, D is wherein the Y group is a group derived from tocopherol.

In one embodiment, D is wherein the Y group is a group from a tocopherol derivative selected from the racemate, the L isomer, or the D isomer of alpha-tocopherol.

In one embodiment, D is such wherein the X group is derived from glycine, p ═ 1, the Y group is derived from octanol, and the functional group l is an ester functional group.

In one embodiment, D is where the X group is from glycine, the p ═ 1, the Y group is from dodecanol, and the functional group l is an ester functional group.

In one embodiment, D is such wherein the X group is derived from glycine, p ═ 1, the Y group is derived from cetyl alcohol, and the functional group l is an ester functional group.

In one embodiment, D is such where the X group is derived from phenylalanine, p ═ 1, the Y group is derived from octanol, and the functional group l is an ester functional group.

In one embodiment, D is such wherein the X group is derived from phenylalanine, p ═ 1, the Y group is derived from 3, 7-dimethyloctan-1-ol, and the functional group l is an ester functional group.

In one embodiment, D is where the X group is from aspartic acid, p ═ 2, the Y group is from octanol, and the functional group l is an ester functional group.

In one embodiment, D is where the X group is from aspartic acid, p ═ 2, the Y group is from decanol, and the functional group l is an ester functional group.

In one embodiment, D is where the X group is from aspartic acid, p ═ 2, the Y group is from dodecanol, and the functional group l is an ester functional group.

In one embodiment, D is such that the X group is derived from ethylenediamine, the Y group is derived from dodecanoic acid, and the functional group l is an amide functional group.

In one embodiment, D is such where the X group is derived from diglycolamine, the p ═ 1, the Y group is derived from dodecanoic acid, and the functional group l is an ester functional group.

In one embodiment, D is where the X group is derived from triethylene glycol diamine, the p ═ 1, the Y group is derived from dodecanoic acid, and the functional group l is an amide functional group.

In one embodiment, D is where the X group is derived from triethylene glycol diamine, the p ═ 1, the Y group is derived from hexadecanoic acid, and the functional group l is an amide functional group.

In one embodiment, D is such wherein the X group is derived from leucine, p ═ 1, the Y group is derived from cholesterol, and the functional group l is an ester functional group.

In one embodiment, D is such where the X group is derived from ethylenediamine, the p ═ 1, the Y group is derived from cholesterol, and the functional group l is a carbamate functional group.

In one embodiment, the E group is an at least divalent group derived from an amino acid selected from the group consisting of glycine, leucine, phenylalanine, lysine, isoleucine, alanine, valine, serine, threonine, aspartic acid, and glutamic acid.

The group derived from an amino acid may be levorotatory or dextrorotatory.

In one embodiment, the E group is an at least divalent group derived from a mono or polyethylene glycol amine.

In one embodiment, the E group is at least a divalent group derived from a mono or polyethylene glycol amine selected from the group consisting of ethanolamine, diglycolamine, and triglycolamine.

In one embodiment, the E group is at least a divalent group derived from a mono or polyethylene glycol diamine.

In one embodiment, the E group is at least a divalent group derived from ethylenediamine.

In one embodiment, the E group is at least a divalent group derived from a mono or polyethylene glycol diamine selected from diethylene glycol diamine and triethylene glycol diamine.

In one embodiment, the F group is an alkyl group derived from a hydrophobic alcohol.

In one embodiment, the F group is a group derived from a hydrophobic alcohol selected from the group consisting of dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), and hexadecanol (cetyl alcohol).

In one embodiment, the F group is a group derived from a hydrophobic acid.

In one embodiment, the F group is a group derived from a hydrophobic acid selected from dodecanoic acid, tetradecanoic acid, and hexadecanoic acid.

In one embodiment, the F group is a group derived from a sterol.

In one embodiment, the F group is a group derived from a sterol selected from the group consisting of cholesterol and derivatives thereof.

In one embodiment, the F group is a group derived from tocopherol.

In one embodiment, the F group is a group derived from a tocopherol derivative selected from the group consisting of the racemate, the L isomer, or the D isomer of alpha-tocopherol.

In one embodiment, the E group is derived from ethylenediamine, t ═ 1, o is a carbamate functional group, and the F group is derived from cholesterol.

In one embodiment:

○-(f-[A]-COOH)_nis such that A is-CH₂-a group, and f is an ether functional group;

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k is an amide function, and D is such that X is derived from glycine, l is an ester function, and Y is derived from octanol;

q is 38, n is 0.9, and m is 0.2.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group k is an amide function, and D is such that X is derived from glycine, p ═ 1, l is an ester function, and Y is derived from cetyl alcohol;

q is 19, n is 1.0, and m is 0.1.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group k is an amide function and D is such that X is derived from phenylalanine, p ═ 1, l is an ester function and Y is derived from octanol;

q is 38, n is 1.0, and m is 0.1.

In one embodiment:

q is 19, n is 1.0, and m is 0.2.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group, k is an amide function, and D is such that X is derived from phenylalanine, p ═ 1, l is an ester function, and Y is derived from 3, 7-dimethyloct-1-ol;

q is 38, n is 1.0, and m is 0.1.

In one embodiment:

○-(f-[A]-COOH)_nis such that A is-CH₂-a group of,and f is an ether functional group;

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group k is an amide function and D is such that X is derived from aspartic acid, p ═ 2, l is an ester function and Y is derived from octanol;

q is 38, n is 1.05, and m is 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group, k is an amide function, and D is such that X is derived from aspartic acid, p ═ 2, l is an ester function, and Y is derived from decanol;

q is 38, n is 1.05, and m is 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group k is an amide function, and D is such that X is derived from aspartic acid, p ═ 2, l is an ester function, and Y is derived from dodecanol;

q ═ 19, n ═ 1.05, and m ═ 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k being an amide functionAnd D is where X is from ethylenediamine, p ═ 1, l is an amide function, and Y is from dodecanoic acid;

q is 38, n is 1.0, and m is 0.1.

In one embodiment:

○-(f-[A]-COOH)_nis such that A is-CH₂-CH₂-a group, and f is an ester function;

○-(g-[B]-k-[D])_mis such that g is an ester functionality and B is-CH₂-CH₂-the group k is an amide function, and D is such that X is derived from glycine, p ═ 1, l is an ester function, and Y is derived from dodecanol;

q is 38, n is 1.3, and m is 0.1.

In one embodiment:

○-(f-[A]-COOH)_nis such that A is-CH₂-a group, and f is a carbamate functional group;

○-(g-[B]-k-[D])_mis such that g is a carbamate functional group and B is-CH₂-the group k is an amide function and D is such that X is derived from aspartic acid, p ═ 2, l is an ester function and Y is derived from octanol;

q is 38, n is 1.3, and m is 0.1.

In one embodiment:

q is 4, n is 0.96, and m is 0.07.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k is an amide function, and D is such that X is derived from diglycolamine, p ═ 1, l is an ester function, and Y is derived from dodecanoic acid;

q is 38, n is 1.0, and m is 0.1.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group, k is an amide function, and D is such that X is derived from triethylene glycol diamine, p ═ 1, l is an amide function, and Y is derived from dodecanoic acid;

q is 38, n is 1.0, and m is 0.1.

In one embodiment:

○o-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-the group, k is an amide function, and D is such that X is derived from triethylene glycol diamine, p ═ 1, l is an amide function, and Y is derived from hexadecanoic acid;

q is 38, n is 1.05, and m is 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and is-CH₂-the group k is an amide function, and D is such that X is derived from glycine, p ═ 1, l is an ester function, and Y is derived from cetyl alcohol;

q ═ 19, n ═ 1.05, and m ═ 0.05.

In one embodiment:

q is 38, n is 0.37, and m is 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k is an amide function, and D is such that X is derived from leucine, p ═ 1, l is an ester function, and Y is derived from cholesterol;

q is 19, n is 1.61, and m is 0.04.

In one embodiment:

q is 19, n is 1.06, and m is 0.04.

In one embodiment:

q is 19, n is 0.66, and m is 0.04.

In one embodiment:

q is 19, n is 0.46, and m is 0.04.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k is an amide function, and D is such that X is derived from leucine, p ═ 1, l is an ester function, and Y is derived from biliary cirrhosisAn alcohol;

q is 4, n is 1.61, and m is 0.05.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is an ether function and B is-CH₂-a group, k is an amide function, and D is such that X is derived from ethylenediamine, p ═ 1, l is a carbamate function, and Y is derived from cholesterol;

q is 19, n is 1.61, and m is 0.04.

In one embodiment:

○-(g-[B]-k-[D])_mis such that g is a carbamate functional group and B is-CH₂-a group, k is an amide function, and D is such that X is derived from leucine, p ═ 1, l is an ester function, and Y is derived from cholesterol;

q is 19, n is 1.96, and m is 0.04.

In one embodiment:

○-[E]-(o-[F])_tis such wherein E is derived from ethylenediamine, o is a carbamate functional group, and F is derived from cholesterol;

q ═ 19, and n ═ 1.65.

In one embodiment:

○-(f-[A]-COOH)_nis thatWherein A is-CH₂-a group, and f is an ether functional group;

q is 38, n is 0.99, and m is 0.05.

In one embodiment:

q is 4, n is 1.41, and m is 0.16.

In one embodiment:

q is 4, n is 1.50, and m is 0.07.

In one embodiment:

q is 4, n is 1.05, and m is 0.05.

In one embodiment, the composition according to the invention comprises a glucan selected from the following glucans of formula I, III or IV:

sodium dextran methylcarboxylate (dextran sulfate sodium) modified with octyl glycinate,

sodium dextran-methylcarboxylate modified with cetyl glycinate,

sodium dextran-methylcarboxylate modified with octyl phenylalanine ester,

sodium dextran-methylcarboxylate modified with 3, 7-dimethyl-1-octyl phenylalanine,

sodium dextran-methylcarboxylate modified with dioctyl aspartate,

sodium dextran-methylcarboxylate modified with didecyl aspartate,

sodium dextran-methylcarboxylate modified with dilauryl aspartate,

sodium dextran-methylcarboxylate modified with N- (2-aminoethyl) dodecanamide,

dextran sodium succinate (dextran sulfate sodium) modified with lauryl glycine,

-N- (sodium methylcarboxylate) dextran carbamate modified with dioctyl aspartate,

sodium dextran-methylcarboxylate modified with dilauryl aspartate,

sodium dextran-methylcarboxylate modified with 2- (2-aminoethoxy) ethyl dodecanoate,

sodium dextran-methylcarboxylate modified with 2- (2- {2- [ dodecanoylamino ] ethoxy } ethoxy) ethylamine,

sodium dextran-methylcarboxylate modified with 2- (2- {2- [ hexadecylamido ] ethoxy } ethoxy) ethylamine,

sodium dextran methylcarboxylate modified with leucine cholesteryl ester,

sodium dextran-methylcarboxylate modified with cholesteryl 1-ethylenediamine-carboxylate,

n- (sodium methylcarboxylate) dextran carbamate modified with leucine cholesteryl ester.

In one embodiment, the composition according to the invention comprises a glucan selected from the group consisting of glucans of formula II below:

sodium dextran-methylcarboxylate modified with cholesteryl 1-ethylenediamine carboxylate grafted by reductive amination on the reducing chain ends.

The invention also relates to a single dose formulation comprising a basal insulin having an isoelectric point between 5.8 and 8.5 and a prandial insulin having a pH between 6.6 and 7.8.

The invention also relates to a single dose formulation comprising a basal insulin having an isoelectric point between 5.8 and 8.5 and a prandial insulin having a pH between 7 and 7.8.

In one embodiment, the prandial insulin is selected from the group consisting of:(human insulin) and(human insulin).

In one embodiment, the prandial insulin is selected from the group consisting of: insulin lisproInsulin glulisineAnd insulin aspart

By means of the change in the appearance of the solution, the dissolution of basal insulins with an isoelectric point of 5.8 to 8.5 at pH6.6 to 7.8 can be observed and controlled in a simple manner with the naked eye for the polysaccharides of formula I, II, III or IV.

By means of the change in the appearance of the solution, the dissolution of basal insulin with an isoelectric point of 5.8 to 8.5 at a pH of 7 to 7.8 can be observed and controlled in a simple manner with the naked eye with the aid of the polysaccharides of formula I, II, III or IV.

Furthermore, and equally important, the applicant company is able to demonstrate that basal insulins with isoelectric points of 5.8 to 8.5, which dissolve in the presence of polysaccharides of the formulae I, II, III or IV, do not lose their slow insulin action.

The preparation of the composition according to the invention appears to have the advantage of being able to be carried out by simply mixing an aqueous solution of a basal insulin having an isoelectric point between 5.8 and 8.5, a solution of a prandial insulin and an aqueous solution or a polysaccharide of formula I, II, III or IV in lyophilized form. The prepared pH was adjusted to pH7 if necessary.

The preparation of the composition according to the invention appears to have the ability to be carried out by simply mixing the polysaccharide of formula I, II, III or IV in aqueous solution, aqueous solution or lyophilized form and the prandial insulin in aqueous solution or lyophilized form of a basal insulin having an isoelectric point between 5.8 and 8.5.

In one embodiment, the mixture of basal insulin and polysaccharide is concentrated by ultrafiltration before mixing with an aqueous solution or lyophilized form of prandial insulin.

The composition of the mixture is adjusted, if necessary, among these excipients by adding to the mixture a concentrated solution of these excipients, such as glycerol, m-cresol, zinc chloride and tween. The pH of the preparation is adjusted to 7 if necessary.

Drawings

Figures 1 to 6 show the results obtained in the form of a pharmacokinetic profile of glucose. The axis of the coordinate axis represents the D-glucose (in mM) as a function of time (in minutes) after injection.

FIG. 1: with the composition according to the invention polysaccharides(75/25) (■) in comparison, sequential administrationAnd(□) mean + standard deviation of mean.

FIG. 2:independent curves (tested in 6 pigs).

FIG. 3: polysaccharidesIndependent curves (tested in 6 pigs).

FIG. 4: with the composition according to the invention polysaccharides(■) sequential administration in comparisonAnd(□) mean + standard deviation of mean.

FIG. 5:independent curves (tested in 6 pigs).

FIG. 6: polysaccharidesIndependent curves (tested in 5 pigs).

Figures 7 to 12 show the results obtained in the form of a pharmacokinetic profile for glucose. The axis of the coordinate axis represents the D-glucose (in mM) as a function of time (in hours) after injection.

FIG. 7: with the composition according to the invention described in example B28 (0.53 IU/kg)In contrast, sequential application(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

FIG. 8: with the composition according to the invention described in example B27 (0.47 IU/kg)In contrast, sequential application(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

FIG. 9: with the composition according to the invention described in example B29 (0.53 IU/kg)In contrast, sequential application(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

FIG. 10: with the composition according to the invention described in example B31 (0.48 IU/kg)In contrast, sequential application(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

FIG. 11: with the composition according to the invention described in example B30 (0.64 IU/kg)In contrast, sequential application(100 IU/ml, 0.24U/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

FIG. 12: with the composition according to the invention described in example B32 (0.53 IU/kg)In contrast, sequential application(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4IU／kg)average + standard deviation of the average.

Examples

Part A: polysaccharides

Table 1 below gives, in a non-limiting manner, examples of polysaccharides that can be used in the compositions according to the invention.

TABLE 1

Example a 1: preparation of polysaccharide 1

16g (i.e. 296mmol of hydroxyl) dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasosmos) was dissolved in water at 420 g/l. To the solution was added 30ml of 10N NaOH (296 mmol). The mixture was warmed to 35 ℃ and 46g (396mmol) of sodium chloroacetate were added. The temperature of the reaction mixture was raised to 60 ℃ at 0.5 ℃/min and then held at 60 ℃ for 100 minutes. The reaction medium is diluted with 200ml of water, neutralized with acetic acid and purified by ultrafiltration through a 5kda pes membrane using 6 volumes of water. The final solution was measured by dry extraction (exit sec) to determine the concentration of the polysaccharide, then by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of methyl carboxylate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 31.5 mg/g.

According to acid/base titration: the average number of methyl carboxylate units per glucoside unit was 1.1.

The sodium dextran methylcarboxylate solution was passed through Purolite resin (anionic) to give dextran methylcarboxylate, which was then lyophilized for 18 hours.

Octyl glycinate p-toluenesulfonate was obtained according to the method described in patent US 4826818.

10g of dextran-methyl carboxylic acid (44.86mmol of methyl carboxylic acid) were dissolved in DMF at 60g/l and then cooled to 0 ℃. 3.23g octyl glycinate p-toluenesulfonate (8.97mmol) were suspended in DMF at 100 g/l. Subsequently, 0.91g triethylamine (8.97mmol) was added to the suspension. Once the polysaccharide solution was at 0 deg.C, then NMM (5.24g, 51.8mmol) in DMF (530g/l) and 5.62g (51.8mmol) EtOCOCOCl were added. After 10 minutes of reaction, octyl glycinate suspension was added. The medium was then kept at 10 ℃ for 45 minutes. The medium was subsequently heated to 30 ℃. To the reaction medium were added an imidazole solution (10.38g in 17ml water) and 52ml water. The polysaccharide solution was ultrafiltered through a10 kDa PES membrane using 15 volumes of 0.9% NaCl solution and 5 volumes of water. The concentration of the polysaccharide solution was determined by dry extraction. A portion of the solution is lyophilized and concentrated in D₂In O through¹H NMR analysis was performed to determine the degree of substitution of octyl glycinate to methyl carboxylate in each glucoside unit.

According to the dry extraction: [ polysaccharide 1] ═ 36.4mg/g

According to acid/base titration: n is 0.9

According to¹H NMR：m＝0.2。

Example a 2: preparation of polysaccharide 2

Cetyl glycinate p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with cetyl glycinate, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 5 kg/mol (q 19, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 2] ═ 15.1mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example a 3: preparation of polysaccharide 3

The octyl phenylalanine p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with octyl phenylalanine ester, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasosmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 3] ═ 27.4mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.1。

Example a 4: preparation of polysaccharide 4

Sodium dextran methylcarboxylate modified with octyl phenylalanine ester, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 5 kg/mol (q ═ 19, pharmasosmos), was obtained by a method similar to that described in example A3.

According to the dry extraction: [ polysaccharide 4] ═ 21.8mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.2。

Example a 5: preparation of polysaccharide 5

Phenylalanine 3, 7-dimethyl-1-octyl p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with phenylalanine 3, 7-dimethyl-1-octyl ester, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasosmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 5] ═ 24.3mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.1。

Example a 6: preparation of polysaccharide 6

The dioctyl aspartate p-toluenesulfonate salt was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with dioctyl aspartate synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, PHARMACOSMOS) was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 6] ═ 22.2mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example a 7: preparation of polysaccharide 7

The didecyl aspartate p-toluenesulfonate salt was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with didecyl aspartate, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 7] ═ 19.8mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example A8: preparation of polysaccharide 8

Dilauryl aspartate p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with dilauryl aspartate, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 5 kg/mol (q ═ 19, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 8] ═ 22.8mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example a 9: preparation of polysaccharide 9

N- (2-aminoethyl) dodecanoamide was obtained from methyl dodecanoate (SIGMA) and ethylenediamine (ROTH) according to the process described in patent US 2387201.

Sodium dextran-methylcarboxylate modified with N- (2-aminoethyl) dodecanoamide, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 9] ═ 23.8mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.1。

Example a 10: preparation of polysaccharide 10

Weight average molecules were determined from the weight average molecules according to the method described in Sanchez-Chaves et al 1998 article (Manual et al, Polymer,1998, 39(13), 2751-Dextran sodium succinate was obtained in an amount of about 10 kg/mol (q 38, pharmasosmos). According to D₂In O/NaOD¹H NMR, average number of succinate groups per glucoside unit 1.4.

Lauryl glycinate p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran succinate modified with lauryl glycine was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 10] ═ 16.1mg/g

According to acid/base titration: n is 1.3

According to¹H NMR：m＝0.1。

Example a 11: preparation of polysaccharide 11

12g (i.e. 0.22mol hydroxyl groups) dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasosmos) was dissolved in a DMF/DMSO mixture. The mixture was warmed to 80 ℃ with stirring. 3.32g (0.03mol) of 1, 4-diazabicyclo [2.2.2] octane and then 14.35g (0.11mol) of ethyl isocyanatoacetate were introduced gradually. After 5 hours of reaction, the medium was diluted in water and purified by ultrafiltration through a 5kDa PES membrane using 0.1N NaOH, 0.9% NaCl and water. The final solution is assayed by dry extraction to determine the concentration of polysaccharide; this was then determined by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of N-methyl carboxylate carbamate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 30.5mg/g

According to acid/base titration: the average number of N-methyl carboxylate carbamate units per glucoside unit was 1.4.

N- (sodium methylcarboxylate) dextran carbamate modified with dioctyl aspartate was obtained by a method similar to that described in example A1.

According to the dry extraction: [ polysaccharide 11] ═ 17.8 mg/g

According to acid/base titration: n is 1.3

According to¹H NMR：m＝0.1。

Example a 12: preparation of polysaccharide 12

Sodium dextran methylcarboxylate modified with dilauryl aspartate, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 1 kg/mol (q ═ 4, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 12] ═ 12.3mg/g

According to acid/base titration: n is 0.96

According to¹H NMR：m＝0.07。

Example a 13: preparation of polysaccharide 13

Dodecanoic acid 2- (2-aminoethoxy) ethyl ester p-methylbenzenesulfonate was obtained according to the process described in patent US 4826818.

Sodium dextran methylcarboxylate modified with 2- (2-aminoethoxy) ethyl dodecanoate was obtained by a method similar to that described in example a1, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, PHARMACOSMOS).

According to the dry extraction: [ polysaccharide 13] ═ 25.6mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.1。

Example a 14: preparation of polysaccharide 14

2- (2- {2- [ dodecanoylamino ] ethoxy } ethoxy) ethylamine was obtained from methyl dodecanoate (SIGMA) and triethylene glycol diamine (HUNSTMAN) according to the method described in patent US 2387201.

Sodium dextran methylcarboxylate modified with 2- (2- {2- [ dodecanoylamino ] ethoxy } ethoxy) ethylamine, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q ═ 38, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 14] ═ 24.9mg/g

According to acid/base titration: n is 1.0

According to¹H NMR：m＝0.1。

Example a 15: preparation of polysaccharide 15

2- (2- {2- [ hexadecylamido ] ethoxy } ethoxy) ethylamine was obtained from methyl palmitate (SIGMA) and triethylene glycol diamine (HUNSTMAN) according to the method described in patent US 2387201.

Sodium dextran methylcarboxylate modified with 2- (2- {2- [ hexadecylamido ] ethoxy } ethoxy) ethylamine was obtained by a method similar to that described in example a1, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q ═ 38, pharmasmos).

According to the dry extraction: [ polysaccharide 15] ═ 22.2mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example a 16: preparation of polysaccharide 16

According to the dry extraction: [ polysaccharide 16] ═ 23mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Example a 17: preparation of polysaccharide 17

10g (i.e. 185mmol of hydroxyl groups) of dextran with a weight average molecular weight of about 10 kg/mol (q 38, PHARMACOSMOS) was dissolved in water at 420 g/l. To the solution was added 19ml of 10N NaOH (185 mmol). The mixture was warmed to 35 ℃ and then 8.6g (74mmol) of sodium chloroacetate were added. The temperature of the reaction mixture was raised to 60 ℃ at 0.5 ℃/min and then held at 60 ℃ for 100 minutes. The reaction medium is diluted with 200ml of water, neutralized with acetic acid and purified by ultrafiltration through a 5klDa PES membrane using 6 volumes of water. The final solution was assayed by dry extraction to determine the concentration of polysaccharide and then by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of methyl carboxylate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 35.1mg/g

According to acid/base titration: the average number of methyl carboxylate units per glucoside unit was 0.42.

Sodium dextran-methylcarboxylate modified with cetyl glycinate was obtained by a method similar to that described in example A1

According to the dry extraction: [ polysaccharide 17] ═ 18 mg/g

According to acid/base titration: n is 0.37

According to¹H NMR：m＝0.05。

Example a 18: preparation of polysaccharide 18

From dextran with a weight average molecular weight of 5 kg/mol (q 19, pharmasosmos) 10g of sodium dextran-methylcarboxylate characterized by a degree of substitution of the methylcarboxylic acid per glucoside unit of 1.10 was synthesized according to a method similar to that described for polysaccharide 1, and then lyophilized.

8g (i.e., 64mmol of hydroxyl groups) of sodium dextran methylcarboxylate characterized by a degree of substitution of the methylcarboxylate per glucoside unit of 1.05 was dissolved in water at 1000 g/l. 6ml of 10N NaOH (64mmol) were added. The mixture was heated to 35 ℃ and 7.6g of sodium chloroacetate (65mmol) were added. The mixture was gradually warmed to 60 ℃ and held at this temperature for an additional 100 minutes. The mixture was diluted with water, neutralized with acetic acid, and purified by ultrafiltration through a 5kDa PES membrane using water. The final solution was assayed by dry extraction to determine the concentration of polysaccharide and then by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of methyl carboxylate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 45.8mg/g

According to acid/base titration: the average number of methyl carboxylate units per glucoside unit was 1.65.

Leucine cholesteryl ester p-methylbenzenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with leucine cholesteryl ester was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 18] ═ 21mg/g

According to acid/base titration: n is 1.61

According to¹H NMR：m＝0.04。

Example a 19: preparation of polysaccharide 19

Sodium dextran methylcarboxylate modified with leucine cholesteryl ester, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 5 kg/mol (q ═ 19, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 19] ═ 19.4mg/g

According to acid/base titration: n is 1.06

According to¹H NMR：m＝0.04。

Example a 20: preparation of polysaccharide 20

16g (i.e. 296mmol of hydroxyl) dextran with a weight average molecular weight of about 5 kg/mol (q 19, pharmasosmos) was dissolved in water at 420 g/l. To this solution was added 30ml of 10N NaOH (296 mmol). The mixture was warmed to 35 ℃ and 26g (222mmol) of sodium chloroacetate were added. The temperature of the reaction mixture was gradually raised to 60 ℃ and maintained at 60 ℃ for 100 minutes. The reaction medium is diluted with water, neutralized with acetic acid and purified by ultrafiltration through a 5kDa PES membrane using water. The final solution was assayed by dry extraction to determine the concentration of polysaccharide and then by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of methyl carboxylate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 33.1mg/g

According to acid/base titration: the average number of methyl carboxylate units per glucoside unit was 0.70.

According to the dry extraction: [ polysaccharide 20] ═ 18.9mg/g

According to acid/base titration: n is 0.66

According to¹H NMR：m＝0.04。

Example a 21: preparation of polysaccharide 21

16g (i.e. 296mmol of hydroxyl) dextran with a weight average molecular weight of about 5 kg/mol (q 19, pharmasosmos) was dissolved in water at 420 g/l. To this solution was added 30ml of 10N NaOH (296 mmol). The mixture was warmed to 35 ℃ and 18g (158mmol) of sodium chloroacetate were added. The temperature of the reaction medium is gradually raised to 60 ℃ and then maintained at 60 ℃ for 100 minutes. The reaction medium is diluted with water, neutralized with acetic acid and purified by ultrafiltration through a 1kDa PES membrane using water. The final solution was assayed by dry extraction to determine the concentration of polysaccharide and then by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of methyl carboxylate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 52.6mg/g

According to acid/base titration: the average number of methyl carboxylate units per glucoside unit was 0.50.

According to the dry extraction: [ polysaccharide 21] ═ 18.9mg/g

According to acid/base titration: n is 0.46

According to¹H NMR：m＝0.04。

Example a 22: preparation of polysaccharide 22

Sodium dextran methylcarboxylate modified with leucine cholesteryl ester, synthesized according to the method described in example a18 using dextran with a weight average molecular weight of about 1 kg/mol (q ═ 4, pharmasmos), was obtained by a method similar to that described in example a 18.

According to the dry extraction: [ polysaccharide 22] ═ 20.2mg/g

According to acid/base titration: n is 1.61

According to 1H NMR: and m is 0.04.

Example a 23: preparation of polysaccharide 23

1-Ethylenediaminecarboxylic acid cholesterol ester hydrochloride was obtained according to the method described in the patent (Akiyoshi, K et al, WO 2010/053140).

Sodium 1-ethylenediaminecarboxycholesteryl ester-modified dextran-methylcarboxylate synthesized according to the method described in example a18 using dextran with a weight average molecular weight of about 5 kg/mol (q ═ 19, pharmasmos) was obtained by a method similar to that described in example a 18.

According to the dry extraction: [ polysaccharide 23] ═ 20.1mg/g

According to acid/base titration: n is 1.61

According to¹H NMR：m＝0.04。

Example a 24: preparation of polysaccharide 24

12g (i.e. 0.22mol hydroxyl groups) dextran with a weight average molecular weight of about 5 kg/mol (q 19, pharmasosmos) was dissolved in a DMF/DMSO mixture. The mixture was warmed to 80 ℃ with stirring. 3.32g (0.03mol) of 1, 4-diazabicyclo [2.2.2] octane and then 26.8g (0.21mol) of ethyl isocyanatoacetate were introduced gradually. After 5 hours of reaction, the medium was diluted in water and purified by ultrafiltration through a 5kDa PES membrane using 0.1N NaOH, 0.9% NaCl and water. The final solution is assayed by dry extraction to determine the concentration of polysaccharide; this was then determined by acid/base titration in 50/50 (V/V) water/acetone to determine the average number of N-methyl carboxylate carbamate units per glucoside unit.

According to the dry extraction: [ polysaccharide ] ═ 30.1mg/g

According to acid/base titration: the average number of N-methyl carboxylate carbamate units per glucoside unit was 2.0.

Leucine cholesterol ester modified N- (sodium methyl carboxylate) dextran carbamate was obtained by a similar method as described in example a 1.

According to the dry extraction: [ polysaccharide 24] ═ 17.9mg/g

According to acid/base titration: n is 1.96

According to¹H NMR：m＝0.04。

Example a 25: preparation of polysaccharide 25

10g of dextran with a weight average molecular weight of about 5 kg/mol (q 19, PHARMACOSMOS, 3.2mmol of chain ends) were dissolved in DMSO at 80 ℃. To the reaction medium were added 4.8g 1-Ethylenediaminecarboxylic acid cholesterol ester hydrochloride (9.5mmol), 0.96g triethylamine (9.5mmol) and 2.0g sodium cyanoborohydride (32mmol), and the reaction medium was stirred at 80 ℃ for 24 hours. After cooling, the mixture was precipitated from dichloromethane and then acetone and dried under vacuum. According to¹H NMR was carried out to obtain dextran modified at the chain end with 1-ethylenediaminecholesteryl carboxylate. Sodium methyl carboxylate of dextran characterized by having a degree of substitution of methyl carboxylate per glucoside unit of 1.65 and being modified at the chain end by cholesterol ester of 1-ethylenediamine carboxylic acid was synthesized by a method similar to that described in example a18 using dextran modified at the chain end by cholesterol ester of 1-ethylenediamine carboxylic acid.

According to the dry extraction: [ polysaccharide 25] ═ 13.7mg/g

According to acid/base titration: n is 1.65

According to¹H NMR: each polypeptide chain carries a 1-ethylenediamine-carboxylic acid cholesterol ester group grafted to the reducing chain end.

Example a 26: preparation of polysaccharide 26

Leucine cholesterol p-toluenesulfonate was obtained according to the method described in patent US 4826818.

Sodium dextran methylcarboxylate modified with leucine cholesteryl ester, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 10 kg/mol (q 38, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 26] ═ 26.6mg/g

According to acid/base titration: n is 0.99

According to¹H NMR：m＝0.05。

Example a 27: preparation of polysaccharide 27

Sodium dilauryl aspartate-modified dextran-methyl-carboxylate, synthesized according to the method described in example a18 using dextran with a weight average molecular weight of about 1 kg/mol (q ═ 4, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 27] ═ 16.7mg/g

According to acid/base titration: n is 1.41

According to¹H NMR：m＝0.16。

Example a 28: preparation of polysaccharide 28

According to the dry extraction: [ polysaccharide 28] ═ 25mg/g

According to acid/base titration: n is 1.50

According to¹H NMR：m＝0.07。

Example a 29: preparation of polysaccharide 29

Sodium dextran methylcarboxylate modified with didecyl aspartate, synthesized according to the method described in example a1 using dextran with a weight average molecular weight of about 1 kg/mol (q ═ 4, pharmasmos), was obtained by a method similar to that described in example a 1.

According to the dry extraction: [ polysaccharide 29] ═ 15mg/g

According to acid/base titration: n is 1.05

According to¹H NMR：m＝0.05。

Examples

And part B: demonstrating the Properties of the compositions according to the invention

Example B1: fast acting insulin analogues at 100 IU/mlSolution of (2)

The solution is a commercial solution of insulin aspart, known by the company Novonid in the United states by the nameSold under the name in EuropeAnd (5) selling. The product is a fast acting insulin analogue.

Example B2: fast acting insulin analogues at 100 IU/mlSolution of (2)

The solution is a commercial solution of insulin lispro, known by the name of the salsaAnd (5) selling. The product is a fast acting insulin analogue.

Example B3: fast acting insulin analogues at 100 IU/mlSolution of (2)

The solution is a commercial solution of insulin glulisine, known by the company sunofil-ampaite under the nameAnd (5) selling. The product is quickActing on insulin analogues.

Example B4: slow acting insulin analogues of 100 IU/mlSolution of (2)

The solution is a commercial solution of insulin glargine, known by the name Xenoffy-AnthrateAnd (5) selling. The product is a slow acting insulin analogue.

Example B5: 100 IU/ml human insulinSolution of (2)

The solution is named by NovonideCommercial solutions are sold. The product is human insulin.

Example B6: solubilisation with substituted dextran at pH7 and 100 IU/ml

Exactly 20mg of polysaccharide 4 described in example A4 were weighed out. Placing the lyophilisate in 2ml of commercial preparationIn (1). A brief precipitation occurred, but after about 30 minutes the solution became clear. The pH of the solution was 6.3. The pH was adjusted to 7 with 0.1N sodium hydroxide solution. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃.

Example B7: substituted dextran of pH775/25 preparation of the composition

Mixing 0.25ml(for its commercial formulation) was added to 0.75ml of the polysaccharide prepared in example B6To the solution to form 1ml of a composition of pH7. The composition was clear, demonstratingAndgood solubility under these formulation conditions. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃.

Example B8: substituted dextran of pH775/25 preparation of the composition

Mixing 0.25ml(for its commercial formulation) was added to 0.75ml of the polysaccharide prepared in example B6To the solution to form 1ml of a composition of pH7. The composition was clear, demonstratingAndgood solubility under these formulation conditions. The clear solution was filtered through a 0.22 μm filter and then placed at +4 deg.CThe following steps.

Example B9: substituted dextran of pH775/25 preparation of the composition

Mixing 0.25ml(for its commercial formulation) was added to 0.75ml of the polysaccharide prepared in example B6In solution to form 1ml of a composition having a pH of 7. The composition was clear, demonstratingAndgood solubility under these formulation conditions. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃.

Example B10: substituted dextran of pH775/25 preparation of the composition

Example B11: substituted dextran of pH760/40 preparation of the composition

Mixing 0.4ml(for its commercial formulation) was added to 0.6ml of the polysaccharide prepared in example B6To the solution to form 1ml of a composition of pH7. The composition was clear, demonstratingAndgood solubility under these formulation conditions. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃.

Example B12: substituted dextran of pH740/60 preparation of the composition

Mixing 0.6ml(for its commercial formulation) was added to 0.4ml of the polysaccharide prepared in example B6To the solution to form 1ml of a composition of pH7. The composition was clear, demonstratingAndgood solubility under these formulation conditions. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃.

Example B13:precipitation of

Mixing 1mlAdded to a 2ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurs, which well corresponds toThe mechanism of action (precipitation due to increase in pH after injection).

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measured. As a result, 86% of the total amount was foundIn the form of a precipitate.

Example B14: substituted glucansPreparation of the composition

1ml of the polysaccharide prepared in example B6The solution was added to a 2ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measured. As a result, 85% ofIn the form of a precipitate.This percentage of precipitation was the same as the results obtained in the control described in example B13.

In each 100 IU/mlThe same dissolution and precipitation tests as described in examples B6 and B14 were performed on other substituted dextrans at the same concentration of 10 mg/ml polysaccharide. 20mg of polysaccharide in lyophilized form are weighed out accurately. Placing the lyophilisate in 2ml of commercial preparationIn (1). A brief precipitation occurred, but after about 30 minutes to several hours (depending on the nature of the polysaccharide) the solution became clear. The pH of the solution was 6.3. The pH was adjusted to 7 with 0.1N sodium hydroxide solution. The clear solution was filtered through a 0.22 μm filter and then placed at +4 ℃. The results are collated in Table 2.

TABLE 2

Practice ofExample B15: substituted dextran of pH775/25 precipitation of the composition

1ml of the substituted dextran prepared in example B775/25 composition (containing 7.5 mg/ml polysaccharide, 75 IU/ml)And 25 IU/ml) Added to a 2ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

The pellet was separated from the supernatant by centrifugation at 4000 rpm. Subsequently, the content of the supernatant is measuredSimilar to the results obtained in the control described in example B13.

Example B16: precipitation of various compositions of substituted dextrans with different properties

Other tests were performed in the presence of other substituted dextrans under the same conditions as in example B15.

The results are combined in Table 3 below and observed to be maintainedDissolution and precipitation of (3).

TABLE 3

Example B17: precipitation of multiple compositions differing in properties of prandial insulin

By mixing 0.75ml of the polysaccharide prepared in example B6The solution was mixed with 0.25ml of prandial insulin to prepare a composition to form 1ml of substituted dextranPrandial insulin composition (containing 7.5 mg/ml polysaccharide, 75 IU/ml)And 25 IU/ml prandial insulin).

The composition was added to a 2ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

The pellet was separated from the supernatant by centrifugation at 4000 rpm. Subsequently, the content of the supernatant is measured. In the presence of the four prandial insulins tested,the precipitation was about 90%.This percentage of precipitation was similar to the results obtained in the control described in example B13; the results are incorporated in table 4.

TABLE 4

Example B18: preparation of concentrated solutions of slow acting insulin analogs (insulin glargine).

Will be referred to by the Senoffy-Anthrat companyCommercial solutions of insulin glargine sold are passed through a 3kDa membrane prepared from regenerated cellulose (sold by Millipore corporation)Ultra-15) ultrafiltration for concentration. At the end of this ultrafiltration phase, the concentration of insulin glargine in the retentate was determined by reverse phase high performance liquid chromatography (RP-HPLC). The final concentration of insulin glargine was then adjusted by adding a commercial 100 IU/ml insulin glargine solution to achieve the desired final concentration. The process enables concentrated solutions of insulin glargine of different concentrations greater than 100 IU/ml to be obtained, using C_{Insulin glargine}Represents, for example, C_{Insulin glargine}200, 250, 300 and 333 IU/ml. The concentrated solution was filtered through a 0.22 μm filter and then stored at +4 ℃.

Example B19: dialysis of commercial solutions of fast acting insulin analogs (insulin lispro)

Will be named by the gift companyCommercial solutions of insulin lispro sold by regenerated cellulose (sold by Millipop corporation)Ultra-l5) was subjected to dialysis by ultrafiltration of the 3kDa membrane prepared. Dialysis was performed in l mM phosphate buffer pH7. At the end of the ultrafiltration phase, the concentration C of insulin lispro in the retentate was determined by reverse phase high performance liquid chromatography (RP-HPLC)_{Dialyzed Humalo}g. The dialysis solution was stored in a refrigerator at-80 ℃.

Example B20: lyophilization of commercial forms of a solution of a fast-acting insulin analog (insulin lispro)

The volume is V_HumalogA commercial form of a solution of fast acting insulin lispro at a concentration of 100 IU/ml is placed in a pre-autoclave sterilizedIn (1). Will be provided withAfter leaving at-80 ℃ for about 1 hour in a refrigerator, it was lyophilized overnight at a temperature of 20 ℃ and a pressure of 0.31 mbar (mbar).

The sterile lyophilisate thus obtained is stored at ambient temperature.

Example B21: lyophilization of dialyzed commercial solutions of fast-acting insulin analog (insulin lispro)

The volume is V_{Humalilog by dialysis}At a concentration of C_{Humalilog by dialysis}The solution of fast-acting insulin obtained according to example B19 was placed in a sterilized autoclaveIn (1). Will be provided withAfter a storage time of about 1 hour at-80 ℃ in a refrigerator, the mixture was lyophilized overnight at a temperature of 20 ℃ and a pressure of 0.31 mbar.

The sterile lyophilisate thus obtained is stored at ambient temperature.

Example B22: the substituted dextran/insulin glargine composition of pH7 was prepared using substituted dextran according to the method of using insulin glargine in liquid form (in solution) and polysaccharide in solid form (lyophilized)

Accurately weighing W_{Polysaccharides}The polysaccharide 18 of (1). Placing the lyophilized product in a volume V_{Insulin glargine}To the concentrated insulin glargine solution prepared according to example B18, a concentration C exhibiting polysaccharides was obtained_{Multiple purpose} _Candy(mg／ml)＝W_{Polysaccharides}／V_{Insulin glargine}And the concentration of insulin glargine is C_{Insulin glargine}(IU/ml). The solution was milky white. The pH of the solution was about 6.3. The pH was adjusted to 7 by adding concentrated NaOH and the solution was then placed in an oven at 37 ℃ for about 1 hour under static conditions. Volume V_{Polysaccharide/insulin glargine}This solution of clear appearance was placed at +4 ℃.

Example B23: the substituted dextran/insulin glargine composition of pH7 was prepared using substituted dextran according to the method of using liquid form (solution) insulin glargine and liquid form (solution) polysaccharide

A concentrated solution of m-cresol, glycerol and Tween 20 was added to the solution exhibiting the concentration C_{Polysaccharide mother liquor}To a mother liquor of polysaccharide 20 at a pH of 7, to obtain a solution in a content equal to that in a10 ml bottleIn the presence of these excipients in the amounts described in commercial solutions, with a concentration C_{Polysaccharide mother liquor/excipient}(mg/ml) polysaccharide solution.

Volume V_LantusConcentration 100 IU/ml by nameCommercial solution addition of slow acting insulin glargine soldVolume V into sterile flask_{Polysaccharide mother liquor/excipient}Concentration C_{Polysaccharide mother liquor/excipient}(mg/ml) in polysaccharide solution. Turbidity appeared. The pH was adjusted to pH7 by addition of 1M NaOH, and the solution was then placed in an oven at 37 ℃ for about 1 hour under static conditions. This solution, which is clear in appearance, is placed at +4 ℃.

Example B24: concentrated polysaccharide/insulin glargine compositions having a pH of 7 were prepared using substituted dextrans according to the method for concentrating dilute compositions

Through a 3kDa membrane prepared from regenerated cellulose (sold by Millipore corporation)Ultra-15) ultrafiltration, dilute polysaccharide 20/glargine insulin composition of pH7 described in example B23 was concentrated. At the end of this ultrafiltration phase, the retentate was cleaned and the concentration of insulin glargine in the composition was determined by reverse phase chromatography (RP-HPLC). If necessary, the concentration of insulin glargine is subsequently adjusted to the desired value by dilution in a solution of the m-cresol/glycerol/tween 20 excipient showing a concentration of each entity equal to that inConcentrations described in commercial solutions (10ml bottles). The appearance is clear and the insulin glargine concentration C is shown_{Insulin glargine}(IU/ml) and polysaccharide C_{Polysaccharides}(mg/ml) A concentrated solution of pH7 was placed at +4 ℃.

Example B25: preparation of a substituted dextran/insulin glargine/insulin lispro composition at pH7 from a commercial form of fast acting insulin lispro

Volume V_{Polysaccharide/insulin glargine}Prepared according to example B22, exhibiting insulin glargine concentration C_{Sweet taste} _{Insulin glargine}(IU/ml) and polysaccharide 18 concentration C_{Polysaccharides}(mg/ml) polysaccharide/insulin glargine solution pH7 added by volume V_{Insulin lispro}The lyophilized insulin lispro lyophilizate obtained (the preparation of which is described in example B19) is subjected to a procedure such that the ratio V is obtained_{Polysaccharide/insulin glargine}／V_{Insulin lispro}＝100／C_{Insulin lispro}In which C is_{Insulin lispro}Is the target insulin lispro concentration (IU/ml) in the composition. The solution was clear. The zinc content of the formulation is adjusted to the desired concentration C by adding a concentrated zinc chloride solution_Zinc(μ M). The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The solution was clear, demonstrating good solubility of insulin glargine and insulin lispro under these formulation conditions. The solution was filtered through a 0.22 μm filter and placed at +4 ℃.

Example B26: starting from fast acting insulin lispro obtained by dialysis of commercial solutions, a substituted dextran/insulin glargine/insulin lispro composition of pH7 was prepared

Will V_{Polysaccharide/pancreas Glycine}Insulin glargine concentration C apparent prepared according to example B24 in island volume_{Sweet taste} _{Insulin glargine}(IU/ml) and polysaccharide 20 concentration C_{Polysaccharides}(mg/ml) polysaccharide/insulin glargine solution pH7 added by volume V_{Humalilog by dialysis}The lyophilized insulin lispro lyophilizate obtained (the preparation of which is described in example B21) is thus such that the ratio V is_{Polysaccharides}/_{Insulin glargine}／V_{Dialyzed Humalo}＝C_{Dialyzed Humalo}／C_{Is prepared from} _{Insulin glauber}In which C is_{Dialyzed Humalo}Is the insulin concentration (IU/ml) of insulin lispro obtained at the end of the commercial solution dialysis (this stage is described in example B19), C_{Insulin lispro}Is the target concentration of insulin lispro (IU/ml) in the composition. The solution was clear. The zinc content of the formulation is adjusted to the desired concentration C by adding a concentrated zinc chloride solution_Zinc(μ M). The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The formulation was clear, demonstrating good solubility of insulin glargine and insulin lispro under these formulation conditions. The solution was filtered through a 0.22 μm filter and placed at +4 ℃.

Example B27: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting a insulin glargine concentration of 200 IU/ml and an insulin lispro concentration of 33 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 85/15)

A concentrated 200 IU/ml insulin glargine solution was prepared according to example B18. A pH7 composition of polysaccharide 18(13 mg/ml)/insulin glargine 300 IU/ml was prepared from polysaccharide 18 according to the preparation described in example B22. The polysaccharide 18/insulin glargine 200 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation method described in example B25. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) ═ 750 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B28: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting a concentration of insulin glargine of 200 IU/ml and a concentration of insulin lispro of 66 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 75/25)

A concentrated 200 IU/ml insulin glargine solution was prepared according to example B18. A pH7 composition of polysaccharide 18(13 mg/ml)/insulin glargine 300 IU/ml was prepared from polysaccharide 18 according to the preparation described in example B22. The polysaccharide 18/insulin glargine 200 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation method described in example B25. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) 1500 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B29: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting a concentration of insulin glargine of 300 IU/ml and a concentration of insulin lispro of 100 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 75/25)

A concentrated 300 IU/ml insulin glargine solution was prepared according to example B18. A pH7 composition of polysaccharide 18(23 mg/ml)/insulin glargine 300 IU/ml was prepared from polysaccharide 18 according to the preparation described in example B22. The polysaccharide 18/insulin glargine 300 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation method described in example B25. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) ═ 2000 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B30: preparation of a substituted dextran/insulin glargine/insulin lispro composition having a pH7 exhibiting a concentration of 250 IU/ml insulin glargine and a concentration of 150 IU/ml insulin lispro (ratio as a percentage of insulin: insulin glargine/insulin lispro is 63/37)

A concentrated 300 IU/ml insulin glargine solution was prepared according to example B18. Preparation of polysaccharide 18 pH7 (19 mg/m 1)/insulin glargine 300I from polysaccharide 18 according to the preparation described in example B22U/ml of composition. The polysaccharide 18/insulin glargine 250 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation method described in example B25. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) 1500 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B31: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting an insulin glargine concentration of 333 IU/ml and an insulin lispro concentration of 67 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 83/17)

A concentrated 333 IU/ml insulin glargine solution was prepared according to example B18. A pH7 composition of polysaccharide 18(20 mg/ml)/insulin glargine 300 IU/ml was prepared from polysaccharide 18 according to the preparation described in example B22. The polysaccharide 18/insulin glargine 333 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation method described in example B25. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) ═ 2000 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B32: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting a concentration of insulin glargine of 300 IU/ml and a concentration of insulin lispro of 100 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 75/25)

A concentrated 300 IU/ml insulin glargine solution was prepared according to example B18. A pH7 composition of polysaccharide 18(23 mg/ml)/insulin glargine 300 IU/ml was prepared from polysaccharide 19 according to the preparation described in example B22. The polysaccharide 19/insulin glargine 300 IU/ml composition was added to insulin lispro lyophilizate lyophilized from a solution of the commercial form of the fast acting analog according to the preparation described in example B26. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) 3000 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Example B33: preparation of a substituted dextran/insulin glargine/insulin lispro composition of pH7 exhibiting a concentration of insulin glargine of 300 IU/ml and a concentration of insulin lispro of 100 IU/ml (ratio as percentage of insulin: insulin glargine/insulin lispro is 75/25)

A pH7 polysaccharide 20(23 mg/ml)/insulin glargine 300 IU/ml composition was prepared from polysaccharide 20 according to the preparation described in example B23. The polysaccharide 20/insulin glargine 300 IU/ml composition was added to a solution lyophilized insulin lispro lyophilizate of a fast acting analog obtained from commercial solution dialysis according to the preparation method described in example B26. The solution was clear. The zinc content of the preparation is adjusted to a concentration C by adding a concentrated zinc chloride solution_Zinc(μ M) 1500 μ M. The final pH was adjusted to 7 by addition of concentrated NaOH or HCl.

The composition is described in table 5.

Table 5: substituted dextran/insulin glargine/insulin lispro composition at pH7

Example B34: preparation of multiple substituted dextran/insulin glargine/insulin lispro compositions exhibiting different insulin glargine concentrations and insulin lispro concentrations and pH7 of different relative proportions of the two insulins

1ml of the substituted dextran prepared in examples B27 to B33The composition was added to a 2ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measuredThe percentage of precipitation was similar to the control described in example B13. The results are summarized in table 6.

Example B35: chemical stability of the composition

Will be in the examplesSubstituted glucans described in B7, B27, B28 and B29Prandial insulin compositions and corresponding controls were left at 30 ℃ for 4 weeks. Regulations require 95% native (undegraded) insulin after 4 weeks at 30 ℃.

After 4 weeks, the formulations studied meet the specifications defined by the legislation. The results are incorporated in table 7.

TABLE 7

Thus, whatever the formulation studied, a percentage of native insulin greater than 95% is obtained, which complies with the requirements of the legislation.

Example B36: injectability of solutions

The entire composition prepared can be injected using a standard system for injecting insulin. Solutions described in examples B7 to B12 and compositions described in examples B27 to B33 utilized insulin syringes equipped with 31 gauge needles and named by noh and knodBoth insulin pens are sold for injection without difficulty.

Example B37: protocol for measuring pharmacokinetics of insulin solutions

A clinical study was performed on pigs to evaluate two compositions according to the invention:

example B7 formulated with polysaccharide 4(6 mg/ml)(75/25), and

formulated with polysaccharide 4(6 mg/ml) as described in example B8(75／25)。

Relative to injection at the same time but will(pH4) and then prandial insulinOrThe hypoglycemic effects of these compositions were compared in the case of separate injections.

6 domestic pigs weighing about 50kg previously cannulated in the jugular vein were fasted for 2 to 3 hours before the experiment was started. During one hour prior to insulin injection, three blood samples were taken to determine basal insulin levels.

Using a needle equipped with a 31-gauge needleThe Yita insulin pen injects insulin into the neck part by subcutaneous injection at the ear of the animal with the dose of 0.4 IU/kg.

Blood samples were then collected after 4, 8, 12, 16, 20, 30, 40, 50, 60, 90, 120, 240, 360, 480, 600, 660 and 720 minutes. After each sample was collected, the cannula was rinsed with dilute heparin solution.

A drop of blood was taken to determine blood glucose using a blood glucose meter. The pharmacokinetics of glucose were then plotted.

The results obtained are shown in the form of pharmacokinetic curves for glucose represented in figures 1 to 6.

Formulated with polysaccharide 4(6 mg/ml)(75／25)。

FIG. 1: with the composition according to the invention polysaccharides(75/25) sequential administration in comparisonAndaverage + standard deviation of the average.

FIG. 2:independent curves.

FIG. 3: polysaccharidesIndependent curves.

Figure 1 shows the mean curve and standard deviation of the mean of the porcine blood glucose reductions tested for each formulation. The reduction in blood glucose was similar for the first 30 minutes for both formulations, indicating that the presence of polysaccharide did not interfereFast acting nature of (2).

On the other hand, between 90 minutes and 10 hours (600 minutes), the sequential applicationAndresulting in unequal reductions in blood glucose with the same plateau response in three pigs and different responses in the other three pigs (figure 2).In contrast, polysaccharides are utilizedThe 6 pigs tested for the formulation had the same response (figure 3). This is reflected in the analysis of the Coefficient of Variation (CV) between 60 minutes and 10 hours forThe control averaged 54% (21% to 113%) for the polysaccharide12% (5% to 25%).

Formulated with polysaccharide 4(6 mg/ml)(75／25)。

FIG. 4: and applying the composition according to the invention polysaccharidesIn contrast, sequential applicationAndaverage + standard deviation of the average.

FIG. 5:independent curves.

FIG. 6: polysaccharidesIndependent curves.

Figure 4 shows the mean curve and standard deviation of the mean of the porcine blood glucose reductions tested for each formulation. Two kinds of systemThe reduction in blood glucose was similar for the first 30 minutes of the agent, indicating that the presence of the polysaccharide did not interfereFast acting nature of (2). On the other hand, between 60 minutes and 8 hours (480 minutes), the application is carried out sequentiallyAndresulting in unequal reduction of blood glucose with the same plateau response in four pigs and a different response in the other two pigs (figure 5). In contrast, polysaccharide 4 is usedThe 5 pigs tested for the formulation had the same response (figure 6). This is reflected in the analysis of the Coefficient of Variation (CV) of the blood glucose lowering data between 60 minutes and 8 hours forThe control averages 54% (31% to 72%) based on polysaccharide 4%15% (6% to 28%). Thus, the presence of polysaccharide 4 is greatly reducedFor variability in blood glucose lowering.

Example B38: protocol for measuring the pharmacokinetics of insulin solutions. Clinical practice in dogs

The previous study was conducted to evaluate six compositions according to the invention:

for simultaneous but separate injection of 100 IU/ml(pH4) andand then 100 IU/ml of prandial insulin is injectedTo compare the hypoglycemic effects of these compositions.

10 domestic dogs (Beagle) weighing about 12kg were fasted for 18 hours before the experiment was started. One hour prior to insulin injection, three blood samples were taken to determine basal glucose levels.

Using a needle equipped with a 31-gauge needleAn Yita island insulin pen subcutaneously injects a dose of 0.53 IU/kg (unless otherwise mentioned in the examples below) of insulin into the neck of an animal by subcutaneous injection.

Blood samples were then collected after 10, 20, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900 and 960 minutes. The first sample was collected using a cannula (up to 180 minutes) and then directly from the jugular vein. After each sample was collected, the cannula was rinsed with dilute heparin solution.

A drop of blood was taken to determine the blood glucose with the aid of a blood glucose meter. The pharmacokinetics of glucose were then plotted.

The results obtained are in the form of pharmacokinetic profiles of glucose shown in figures 7 to 12.

Solution of example B28

FIG. 7: in comparison with the composition according to the invention described in example B28 (0.53 IU/kg), sequential administration(100 IU/ml, 0.13 IU/kg) andaverage of (100 IU/ml, 0.4 IU/kg) + Standard deviation of averageThe poor curve.

Figure 7 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. Both curves were similar up to 12 hours, resulting in a rapid decrease in blood glucose, indicating that the polysaccharide did not affectThe rapid effect of (2), the significant rise back between peaks due toThe plateau phase was due to insulin glargine, which was then followed up to 12 hours, indicating that the basal effect of insulin glargine was well preserved.

Solution of example B27

FIG. 8: in comparison with the composition according to the invention described in example B27 (0.47 IU/kg), sequential administration(100 IU/ml, 0.13 IU/kg) andaverage of (100 IU/ml, 0.4 IU/kg) plus standard deviation of the average.

Figure 8 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. In this comparison, basal insulin of the compositionThe dosage of (A) is the same as that of the control, andthe dose of (c) was half of that of the control. The decrease in glucose was greater in the case of formulation B27 compared to a control comprising2 times of the total weight of the powder. On the other hand, the duration of the plateau phase is shorter in the case of the combination relative to the control. This indicates that in this composition, a portion after injectionNot precipitated and reacted withAct together.

Solution of example B29

FIG. 9: in comparison with the composition according to the invention described in example B29 (0.53 IU/kg), sequential administration(100 IU/ml, 0.13 IU/kg) andaverage of (100 IU/ml, 0.4 IU/kg) plus standard deviation of the average.

Figure 9 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. The two curves are similar, resulting in a rapid decrease in blood glucose, indicating that the polysaccharide does not affectThe rapid effect of (2), the significant rise back between peaks due toThe plateau period is due toThen theUntil 13 hours, indicating that the basal effect of insulin glargine is well preserved.

Solution of example B31

FIG. 10: in comparison with the composition according to the invention described in example B31 (0.48 IU/kg), sequential administration(100 IU/ml, 0.13 IU/kg) andaverage of (100 IU/ml, 0.4 IU/kg) plus standard deviation of the average.

Figure 10 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. In this comparison, basal insulin of the compositionThe dosage of (A) is the same as that of the control, andthe dose of (c) was half of that of the control. The decrease in glucose was greater in the case of the control compared to the combination corresponding to example B31. In view of the combinationThe concentration of (c) was half of the control, and this response was not expected. In addition, the first and second substrates are,the duration of the plateau phase was the same as the control in the case of the combination. This indicates that in this composition and by comparison with the composition described in example B29 (FIG. 9), it is possible to adjust the combinationWithout changing the amount ofThe basic role of (1).

Solution of example B30

FIG. 11: in comparison with the composition according to the invention described in example B30 (0.64 IU/kg), sequential administration(100 IU/ml, 0.24 IU/kg) andaverage of (100 IU/ml, 0.4 IU/kg) plus standard deviation of the average.

Figure 11 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. The two curves are similar, resulting in a rapid decrease in blood glucose, indicating that the polysaccharide does not affectThe rapid effect of (2), the significant rise back between peaks due toThe plateau period is due toThen theUntil 10 hours, indicating that the basal effect of insulin glargine is well preserved.

Solution of example B32

FIG. 12: in comparison with the composition according to the invention described in example B32 (0.53 IU/kg), sequential administration(100 IU/ml, 0.13 IU/kg) and(100IU／ml，0.4 IU/kg) of the total weight of the pellets.

Figure 12 shows the mean curve and standard deviation of the mean for the blood glucose reduction of the dogs tested for each formulation. The two curves were similar up to 10 hours, resulting in a rapid decrease in blood glucose, indicating that the polysaccharide did not affectThe rapid effect of (2), the significant rise back between peaks due toThe plateau period is due toThis is followed by a insulin glargine plateau indicating that the basal action of insulin glargine is maintained for up to 10 hours.

In summary, figures 7 to 12 show that by adjusting the composition of the polysaccharide and the concentrations of insulin lispro and insulin glargine, the same curve can be achieved for fast acting and basal insulin at different ratios for the double injection. The duration of basal insulin can also be adjusted without affecting the fast acting insulin, or the amount of fast acting insulin can be adjusted without affecting the action of basal insulin.

Examples

Part C: demonstrating the Properties of a composition comprising a GLP-1 analogue or derivative according to the invention

Example C1: 0.25 mg/ml GLP-1 analog exenatideSolutions of

The solution is named by the salsaExenatide solution for sale

Example C2: 6 mg/ml GLP-1 derivative liraglutideSolutions of

The solution is named by NovonidLiraglutide solutions on the market

Example C3: 100 IU/ml and pH7 using substituted dextran at a concentration of 10 mg/mlSolution of

Accurately 20mg of substituted dextran selected from those described in table 1 was weighed out. This lyophilizate was placed in 2ml of the insulin glargine solution of example B4 to obtain a solution with a polysaccharide concentration equal to 10 mg/ml. After mechanical stirring on a roller at ambient temperature, the solution became clear. The pH of the solution was 6.3. The pH was adjusted to 7 using 0.1N sodium hydroxide solution. The clear solution was filtered through a membrane (0.22 μm) filter and then placed at +4 ℃.

Summarizing: a clear solution of 100 IU/ml insulin glargine at a concentration of 20 and 40mg/ml pH7 of substituted dextran was also obtained according to the same protocol as described in example C3. Therefore, the amounts of those lyophilized polysaccharides described in table 1 were exactly weighed out. This lyophilizate was placed in the insulin glargine solution of example B4 to obtain a solution with a concentration of substituted dextran equal to 20 or 40mg/ml as described in table 8. After mechanical stirring on a roller at ambient temperature, the solution became clear. The pH of the solution was below 7. The pH was then adjusted to 7 using 0.1N sodium hydroxide solution. The clear final solution was filtered through a membrane (0.22 μm) filter and then placed at +4 ℃.

Table 8: a100 IU @, pH7 was prepared using a concentration of 10, 20, or 40mg/ml of the substituted dextranmlSolution of (2)

Example C4: pH7.570/30 preparation of the composition

0.09ml of the exenatide solution of example C1 is added to 0.21ml of the insulin glargine solution of example B4, which after mixing gives 0.3ml of a composition with a pH of 4.5. Comprises 70 IU/mlAnd 0.075 mg/mlThe composition of (A) is clear, demonstrating that under these formulation conditions (pH4.5)Andgood solubility. The pH was then adjusted to 7.5 using 0.1N sodium hydroxide solution. The composition subsequently became cloudy, demonstrating that at pH7.5Poor solubility of the composition.

pHs of 4.5, 5.5, 6.5, 8.5 and 9.5 were also prepared by a similar protocol to that described in example C4A composition is provided. For each of these compositions, 0.09ml was carried outThe exenatide solution of example C1 is added to 0.21ml of the insulin glargine solution of example B4 to obtain after mixing 0.3ml of a composition with a pH of 4.5. The composition was clear, demonstrating that under these formulation conditions (pH4.5)Andgood solubility. The pH was adjusted to 5.5 or 6.5 or 8.5 or 9.5 using 0.1N sodium hydroxide solution. After adjusting the pH, the composition at 5.5 was slightly cloudy, the compositions at 6.5-7.5 and 8.5 were very cloudy, and the composition at pH9.5 was clear. These compositions were left at +4 ℃ for 48 hours. After 48 hours at +4 ℃, only the composition at ph4.5 remained clear. 70/30 of different pH valuesThe visual appearance of the composition after 48 hours is summarized in table 9.

Table 9: 70/30 of different pH valuesVisual appearance of the composition after 48 hours

Example C5: 70/30 at pH7.5Preparation of the composition

0.09ml of liraglutide solution of example C2 was added to 0.21ml of insulin glargine solution of example B4 to obtain 0.3ml of a composition having a pH of 7 after mixing. Compositions containing 70 IU/ml insulin glargine and 1.8 mg/ml exenatide were turbid, demonstrating under these formulation conditions (pH4.5)Andpoor solubility. The pH was adjusted to 7.5 using 0.1N sodium hydroxide solution. After adjusting the pH, the composition remained cloudy. The composition was left at +4 ℃ for 48 hours.

70/30, pH4.5-5.5-6.5-8.5 and 9.5, was also prepared by a protocol similar to that described in example C5A composition is provided. For each of these compositions, 0.09ml of the liraglutide solution of example C1 was added to 0.21ml of the insulin glargine solution of example B4 to give 0.3ml of the composition having a pH of 7. The composition was cloudy, demonstrating that under these formulation conditions (pH7)Andpoor solubility. The pH is adjusted to 4.5 or 5.5 or 6.5 with 0.1N hydrochloric acid solution or to pH9.5 with 0.1N sodium hydroxide solution. After pH adjustment, the compositions at pH4.5-5.5 and 6.5 were cloudy, demonstrating that under these formulation conditionsAndpoor solubility. These compositions were left at +4 ℃ for 48 hours. After 48 hours at +4 ℃, only the composition at ph9.5 cleared. 70/30 of different pH valuesThe visual appearance of the composition after 48 hours is summarized in table 10.

Table 10: 70/30 of different pH valuesVisual appearance of the composition after 48 hours

Example C6: substituted dextran-70/30 at pH7Preparation of the composition

0.09ml of the exenatide solution of example C1 is added to 0.21ml of the substituted glucan-To the solution, 0.3ml of a composition of pH5.3 was obtained. The pH was adjusted to 7 using 0.1N sodium hydroxide solution. Contains 7 mg/ml polysaccharide, 70 IU/mlAnd 0.075 mg/mlThe composition of (A) is clear, demonstrating the presence of a substituted dextran at pH7Andgood solubility. The clear solution was placed at +4 ℃.

Summarizing: also by the same protocol as described in example C6 at V of 90/10, 50/50, 30/70 and 10/90_Lantus／V_ByettaPreparing a substituted dextran of pH7A composition is provided. Thus, the volume V_ByettaExenatide solution of example C1 added to volume V_LantusThe substituted glucan prepared in example C3 @In solution to obtain a composition adjusted to pH7 with 0.1N sodium hydroxide solution. The resulting composition (see Table 11) was clear, demonstrating that in the presence of a substituted dextran at pH7Andgood solubility. The clear solution was placed at +4 ℃.

Example C7: substituted dextran-100/50 at pH7Preparation of the composition

0.150ml of the exenatide solution according to example C1 is lyophilized, and then 0.3ml of the substituted glucan prepared in example C3 is added to the lyophilizateSolution to obtain a composition adjusted to pH7 with 0.1N sodium hydroxide solution. Contains 10 mg/ml polysaccharide and 100 IU/mlAnd 0.125 mg/mlThe composition of (A) is clear, demonstrating at pH7 in the presence of a substituted dextranAndgood solubility. The clear solution was placed at +4 ℃.

Table 11: of the compositions obtained in examples C6 and C7Substituted glucans andto a final concentration of

Example C8: substituted dextran-70/30 at pH7Preparation of the composition

0.09ml of the liraglutide solution of example C2 was added to 0.21ml of the substituted glucan ≥ er prepared in example C3To the solution, 0.3ml of a composition of pH7.6 was obtained. The pH was adjusted to 7 using 0.1N sodium hydroxide solution. Contains 7 mg/ml polysaccharide, 70 IU/mlAnd 1.8 mg/mlThe composition of (A) is clear, demonstrating the presence of a substituted dextran at pH7Andgood solubility. The clear solution was placed at +4 ℃.

Summarizing: also by the same protocol as described in example C6 at V of 90/10, 50/50, 30/70 and 90/10_Lantus／V_VictozaPreparing a substituted dextran of pH7A composition is provided. Thus, the volume V_VictozaThe liraglutide solution of example C2 was added to volume V_LantusThe substituted glucan prepared in example C3 @In solution to obtain a composition adjusted to pH7 with 0.1N hydrochloric acid solution.

The resulting composition (see Table 12) was clear, demonstrating that in the presence of a substituted dextran at pH7Andgood solubility. The clear solution was placed at +4 ℃.

Example C9: substituted dextran-100/50 at pH7Preparation of the composition

0.150ml of the liraglutide solution of example C2 was lyophilized, and then 0.3ml of the substituted glucan prepared in example C3 was added to the lyophilizateComposition of solution, process for producing the sameThe pH was adjusted to 7 using 0.1N sodium hydroxide solution. Contains 10 mg/ml polysaccharide and 100 IU/mlAnd 3 mg/mlThe composition of (A) is clear, demonstrating the presence of a substituted dextran at pH7Andgood solubility. The clear solution was placed at +4 ℃.

Table 12: in the compositions obtained in examples C8 and C9Substituted glucans andto a final concentration of

Example C10: substituted dextran-60/20/20 at pH7Preparation of the composition

Exactly 20mg of the lyophilized polysaccharide 4 described in example A3 were weighed out. The lyophilizate is placed in 2ml of the insulin glargine solution of example B4. After mechanical stirring on a roller at ambient temperature, the solution became clear. The pH of the solution was 6.3. The pH was adjusted to 7 using 0.1N sodium hydroxide solution. 0.2ml of the exenatide solution of example C1 and 0.2ml of the insulin glulisine solution of example B3 were added to the solution0.6ml of a substituted glucan prepared above-In the solution, 1ml of a composition having a pH of 7 was obtained. This contained 6 mg/ml polysaccharide, 60 IU/ml20IU／mlAnd 0.05 mg/mlThe composition of (A) is clear, demonstrating the presence of a substituted dextran at pH7Andgood solubility. The clear solution was filtered through a membrane (0.22 μm) and then placed at +4 ℃.

Example C11:precipitation of

Mixing 0.250mlTo a solution of 0.5ml PBS (phosphate buffered saline) containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium.

A precipitate appears, which well conforms toThe mechanism of action (precipitation due to increase in pH after injection).

Centrifugation was carried out at 4000 rpm to allow the precipitation to proceed toAnd separating clear liquid. Subsequently, the content of the supernatant is measured. As a result, 90% ofIn the form of a precipitate.

Example C12: substituted glucan-Precipitation of the composition

0.250ml of the substituted glucan prepared in example C3 is-The solution was added to 0.5ml PBS solution containing 20mg/ml BSA. The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measured. As a result, 90% ofIn the form of a precipitate.This percentage of precipitation of (a) is the same as the percentage of precipitation obtained for the control described in example C11.

Example C13: substituted glucansPrecipitation of the composition

0.250ml of the substituted dextran prepared in example C6The composition was added to a 0.5ml PBS solution containing 20mg/ml BSA. The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measuredAndthe percentage of precipitation was similar to the control described in example C11.

Example C14: substituted dextran-70/30Precipitation of the composition

0.250ml of the substituted dextran prepared in example C8The composition was added to a 0.5ml PBS solution containing 20mg/ml BSA (bovine serum albumin). The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measuredAndall-grass ofThe ratio was similar to the control described in example C11.

Example C15: precipitation of different compositions that alter the properties of substituted dextrans

Further tests were carried out in the presence of further dextran under the same conditions as examples C13 and C14.

Using up to 20mg/ml of substituted dextran and 70/30The results of the compositions are combined in table 13 below. Observe and maintainDissolution and precipitation of (3).

Table 13: using up to 20mg/ml of substituted dextran and 70/30Results of dissolution and precipitation tests obtained for the compositions

Using up to 20mg/ml of substituted dextran and differentThe results of the compositions are combined in table 14 below. Observe and maintainDissolution and precipitation of (3).

Table 14: using up to 20mg/ml of substituted dextran and differentResults of dissolution and precipitation tests obtained for the compositions

Using up to 40mg/ml of substituted dextran and 70/30The results of the compositions are incorporated in table 15 below. Observe and maintainDissolution and precipitation of (3).

Table 15: using up to 40mg/ml of substituted dextran and 70/30Results of dissolution and precipitation tests obtained for the compositions

Using up to 20mg/ml of substituted dextran and differentThe results of the compositions are combined in table 16 below. Observe and maintainDissolution and precipitation of (3).

Table 16: using up to 20mg/ml of substituted dextran and differentResults of dissolution and precipitation tests obtained for the compositions

Example C16: substituted dextran-60/20/20 at pH7Precipitation of the composition

0.250ml of the substituted dextran prepared in example C10The composition was added to a 0.5ml PBS solution containing 20mg/ml BSA. The PBS/BSA mixture mimics the composition of the subcutaneous medium. Precipitation occurred.

Centrifugation was performed at 4000 rpm to separate the pellet from the supernatant. Subsequently, the content of the supernatant is measuredThe percentage of precipitation was similar to the control described in example C11.

Claims

1. Composition in the form of an injectable aqueous solution having a pH of from 6.0 to 8.0, comprising at least:

a) basal insulin with isoelectric point pI of 5.8 to 8.5;

b) dextran substituted with a group bearing a carboxylic acid load and a hydrophobic group of formula I or formula II:

wherein:

● R is-OH or a group selected from:

○-(f-[A]-COOH)_n；

● q represents the degree of polymerisation of the glucoside units, in other words the average number of glucoside units per polysaccharide chain, and 3. ltoreq. q.ltoreq.50;

●-(f-[A]-COOH)_n：

●-(g-[B]-k-[D])_m：

● f, g and k are the same or different;

● and, when p is 1, if Y is C₈To C₁₄Alkyl, then q m.gtoreq.2, if Y is C₁₅Alkyl, then q m is more than or equal to 2; and if Y is C₁₆To C₂₀Alkyl, then q m is more than or equal to 1; ● and, when p.gtoreq.2, if Y is C₈To C₉Alkyl, then q m.gtoreq.2, and if Y is C₁₀To C₁₆Alkyl, then q m is more than or equal to 0.2;

wherein:

● R is-OH or- (f- [ A)]-COOH)_nGroup (b):

bonded to the glucoside unit via a functional group f selected from ether, ester or carbamate functional groups;

● R' is selected from the following groups:

○-C(O)NH-[E]-(o-[F])_t；

○-CH₂N(L)_z-[E]-(o-[F])_t；

wherein:

z is a positive integer equal to 1 or 2,

l is selected from:

■ -H, and z is equal to 1, and/or

■ - [ A ] -COOH and z is equal to 1 or 2, if f is an ether function,

■ -CO- [ A ] -COOH and z is equal to 1 if f is an ester function, and

○-[E]-(o-[F])t：

■ o is a functional group selected from ether, ester, amide or carbamate functional groups;

■ t is a positive integer equal to 1 or 2;

● when z is 2, the nitrogen atom is in the form of a quaternary ammonium.

2. The composition according to claim 1, characterized in that the dextran substituted with groups bearing carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula I.

3. The composition according to claim 1, characterized in that the dextran substituted with groups bearing carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula II.

4. Composition according to either of claims 1 and 2, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula I, wherein- (f- [ a [)]-COOH)_nThe radicals are selected from the following sequences, f having the meaning given above:

5. the composition according to any one of claims 1, 2 and 4, characterized in that said charged carboxylic acid is chargedAnd the dextran substituted with a hydrophobic group is selected from the group consisting of dextrans of formula I, wherein, -) g- [ B]-k-[D])_mThe radicals are selected from the following sequences, g, k and D having the meanings given above:

6. composition according to any one of claims 1, 2 and 4, 5, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula I, wherein- (g- [ B]-k-[D])_mThe radicals are such that:

● -B-is a group containing 1 carbon atom, the-B-group being bonded to the glucoside unit via an ether function g, and X is a group derived from an amino acid.

7. Composition according to any one of claims 1, 2 and 4 to 6, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula I wherein the X group is an at least divalent group derived from an amino acid selected from glycine, phenylalanine, lysine, isoleucine, alanine, valine, aspartic acid and glutamic acid.

8. Composition according to any one of claims 1, 2 and 4 to 7, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group consisting of dextrans of formula I wherein the Y group is selected from hydrophobic alcohols, hydrophobic acids, sterols or tocopherols.

9. Composition according to any one of claims 1, 2 and 4 to 8, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group of dextrans of formula I, wherein the Y group is a sterol selected from cholesterol derivatives.

10. The composition according to any one of claims 1 and 3, characterized in that the dextran substituted with carboxylic acid bearing groups and hydrophobic groups is selected from the group of dextrans of formula II, wherein the R' group is such wherein the-E-group is derived from a diamine.

11. Composition according to any one of claims 1, 3 and 10, characterized in that the dextran substituted with groups bearing a carboxylic acid load and hydrophobic groups is selected from the group of dextrans of formula II, wherein the R' group is such wherein the-F-group is derived from a cholesterol derivative.

12. The composition according to any one of claims 1, 2 and 4 to 9, characterized in that the glucan substituted with the carboxylic acid-loaded groups and the hydrophobic groups is selected from the group consisting of glucans of formula I as follows:

-sodium dextran-methylcarboxylate modified with octyl glycinate,

sodium dextran-methylcarboxylate modified with cetyl glycinate,

sodium dextran-methylcarboxylate modified with octyl phenylalanine ester,

sodium dextran-methylcarboxylate modified with dioctyl aspartate,

sodium dextran-methylcarboxylate modified with didecyl aspartate,

sodium dextran-methylcarboxylate modified with dilauryl aspartate,

sodium dextran-methylcarboxylate modified with N- (2-aminoethyl) dodecanamide,

-dextran sodium succinate modified with lauryl glycine,

sodium dextran-methylcarboxylate modified with dilauryl aspartate,

sodium dextran methylcarboxylate modified with leucine cholesteryl ester,

13. The composition according to any one of claims 1, 3, 10 and 11, characterized in that the dextran substituted with groups bearing carboxylic acid load and hydrophobic groups is selected from the group consisting of dextran of formula II and is:

14. Composition according to any one of the preceding claims, characterized in that the basal insulin with isoelectric point between 5.8 and 8.5 is insulin glargine.

15. Composition according to any one of the preceding claims, characterized in that it comprises between 40 and 500 IU/ml of basal insulin having an isoelectric point between 5.8 and 8.5.

16. Composition according to any one of the preceding claims, characterized in that it further comprises prandial insulin.

17. Composition according to any one of the preceding claims, characterized in that it comprises from 40 IU/ml to 800 IU/ml of total insulin.

18. The composition according to any one of claims 16 and 17, characterized in that it comprises 25/75, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20 and 90/10 in a ratio, expressed as a percentage, of a basal insulin having an isoelectric point between 5.8 and 8.5 and a prandial insulin.

19. The composition according to any one of claims 1 to 15, characterized in that the composition further comprises GLP-1, a GLP-1 analogue or a GLP-1 derivative.

20. Composition according to any one of the preceding claims, characterized in that it further comprises a zinc salt in a concentration ranging from 0 to 5000 μ Μ.

21. Composition according to any one of the preceding claims, characterized in that it further comprises a buffer chosen from Tris, citrate and phosphate in a concentration ranging from 0 to 100mM, preferably from 0 to 50 mM.

22. Composition according to any one of claims 16 to 18, characterized in that the prandial insulin is selected from the group formed by human insulin, insulin glulisine, insulin lispro and insulin aspart.

23. A single dose formulation comprising the composition of any one of claims 1 to 18 and 20 to 22 at a pH of 6.6 to 7.8 and prandial insulin.

24. A single dose formulation comprising a composition according to any one of claims 1 to 15 and 19 to 21 and GLP-1, a GLP-1 derivative or a GLP-1 analogue at a pH of 6.6 to 7.8.

25. Single dose formulation according to claim 23, characterized in that the prandial insulin is selected from the group comprising human insulin.

26. The single dose formulation according to claim 23, characterized in that said prandial insulin is selected from the group comprising insulin lispro, insulin glulisine and insulin aspart.