WO2010041138A2

WO2010041138A2 - Complex between human insulin and an amphiphilic polymer, and use of said complex for preparing a quick human insulin formulation

Info

Publication number: WO2010041138A2
Application number: PCT/IB2009/007106
Authority: WO
Inventors: Olivier Soula; Rémi SOULA; Gérard Soula
Original assignee: Adocia
Priority date: 2008-10-10
Filing date: 2009-10-12
Publication date: 2010-04-15
Also published as: WO2010041138A3; FR2940802A1

Abstract

The invention relates to a complex between human insulin and an amphiphilic polymer containing carboxyl functional groups, said amphiphilic polymer being selected from among the functionalised polysaccharides mostly made up of glycosidic monomers bonded by (1,6) glycosidic bonds functionalised by at least one tryptophan derivative. The invention also relates to a pharmaceutical composition including at least one complex according to the invention, said formulation capable of being in the form of an injectable solution. The invention relates, more particularly, to the use of a complex according to the invention for the preparation of a formulation of human insulin with a concentration of around 600 µM (100 UI/mL), with the onset of action being in less than 30 minutes, preferably in less than 20 minutes, and more preferably in less than 15 minutes and/or with the glycaemic nadir being in less than 120 minutes, preferably in less than 105 minutes, and more preferably in less than 90 minutes.

Description

Complex between human insulin and an amphiphilic polymer and use of this complex for the preparation of a rapid human insulin formulation

The present invention relates to a fast-acting stable formulation of recombinant human insulin.

Since the production of insulin by genetic engineering, in the early 80s, diabetic patients benefit from human insulin to treat themselves. This product has greatly improved this therapy since the immunological risks associated with the use of non-human insulin, in particular pork, are eliminated.

[0003] Genetic engineering has allowed another improvement in the treatment of diabetes with the development of so-called analogous insulins. These insulins are modified to achieve two objectives that are complementary: on the one hand, have a slow and controlled action within 24 hours: this is the case of Lantus which has a prolonged action much higher than that of human insulin ; - On the other hand, a very fast action after injection: this is the case of insulins Lispro (Lilly), Novolog (Novo) and Apidra (Aventis); which have faster action rates after administration than human insulin. [0004] The control of the speed of action of insulin is a crucial element in the life of patients because they must at each meal avoid situations where they could end up in hyperglycemia or hypoglycemia. It is therefore of the utmost medical importance to match as much as possible the action of insulin administered with the production of glucose brought by food. This is what justifies today the success of ultra rapid analog insulins at the expense of the use of human insulin.

These insulins are modified on one or two amino acids to be more quickly active after a subcutaneous injection. These insulins Lispro (Lilly), Novolog (Novo) and Apidra (Aventis) are stable solutions of insulin with a hypoglycemic response close in terms of kinetics of the physiological response generated by the beginning of a meal. Therefore, patients no longer have to predict their meal time before the injection of a fast insulin. They inject this insulin when they are ready to eat. They can even supplement if necessary this dose at the end of their meal which is very significant for children for whom it is difficult to adjust the dose and control appetite. [0006] Human insulin does not make it possible to obtain a close hypoglycemic response in terms of the kinetics of the physiological response generated by the beginning of a meal, since it assembles in the form of a hexamer while it is active. as monomer and dimer. The dissociation equilibria of dimeric hexamers and dimers to monomers delayed its action by nearly 20 minutes compared with fast-acting insulin, Brange J., et al, Advanced Drug Delivery Review, 35, 1999, 307-335. Human insulin is prepared in the form of hexamers to be stable for 2 years at 4 ° C because in the form of monomers, it has a very high propensity to aggregate and fibrillate which makes it lose its activity, more in this aggregated form, it presents an immunological risk for the patient.

The principle of so-called fast insulin analogues is to form hexamers to ensure the stability of insulin but also to promote the dissociation of the hexamers into monomers to obtain a rapid action.

[0008] ^" The main disadvantage of these insulins is the modification of the primary structure of human insulin This modification leads to variations in the interaction with the insulin receptors present on a very large number of cell lines because it is known that the role of insulin in the body is not limited only to its hypoglycemic activity.While much research has been done in this area, it is not known to determine to date whether these analog insulins have all the physiological properties of human insulin On the other hand, the number of diabetic patients is increasing every day.It is of utmost importance to provide patients with this disease insulin formulations at the lowest price. There is therefore a real and unmet need for a rapid formulation of human insulin that is more effective, safer and cheaper than current formulations on the market. which are either rapid analog insulins or human insulins which have too long a time of action. The company Biodel has proposed a solution to this problem by adding EDTA and citric acid to human insulin Their strategy is to destabilize the hexamer by placing at acidic pH and complexing the ions zinc by EDTA. However, such a formulation has several major disadvantages. The first, due to the acidity of the formulation, is that the human insulin solution must be prepared at the time of injection and this several times a day, whereas today, patients use stable solutions that are ready for use. employment administrable by insulin pens. The second is that to achieve the desired effects, the solution tested by BIODEL is four times more diluted than the international standard of 100 IU / mL for all insulin solutions on the market. The third is a higher frequency of pain at the injection site, pains attributed to the large volume of fluid injected as revealed in phase III clinical studies. The present invention solves the various problems described above since it allows for a stable human insulin formulation at a pH between 5.5 and 7.5 in solution at 100 IU / mL, said formulation to achieve, after administration, a plasma insulin level and / or glucose reduction faster than with human insulin formulations.

The invention consists in forming a complex of human insulin with an amphiphilic polymer having carboxyl functional groups.

The formation of this complex can be carried out by simple mixing of an aqueous solution of insulin and an aqueous solution of amphiphilic polymer.

The invention also relates to the complex between human insulin and an amphiphilic polymer having carboxyl functional groups. It also relates to the use of this complex to prepare human insulin formulations to achieve, after administration, a plasma insulin level and / or glucose reduction faster than human insulin formulations. . The "fast" human insulin formulations on the market at a concentration of 600 μM (100 IU / mL) have an action time of between 30 and 60 minutes and a glycemic nadir of between 2 and 4 hours. The "fast" analog insulin formulations on the market at a concentration of 600 μM (100 IU / mL) have an action time of between 10 and 15 minutes and a glycemic nadir of between 60 and 90 minutes. It relates more particularly to the use of a complex according to the invention for the preparation of a so-called fast insulin formulation.

The invention relates to the use of the complex according to the invention for preparing human insulin formulations at a concentration of 600 μM (100 IU / mL) whose action time is less than 30 minutes, preferably less than 20 minutes, and more preferably less than 15 minutes.

The invention relates to the use of the complex according to the invention for preparing human insulin formulations at a concentration of about 600 μM (100 IU / mL) whose glycemic nadir is less than 120 minutes, preferably less than at 105 minutes, and still preferably less than 90 minutes.

In one embodiment, the amphiphilic polymer comprising carboxyl functional groups is chosen from functionalized polysaccharides consisting mainly of glycoside monomers linked by glycoside bonds of (1,6) type and in one embodiment, the polysaccharide. consisting predominantly of glycosidic monomers linked by glycoside bonds of (1,6) type is a functionalized dextran having carboxyl functional groups. [00022] Said polysaccharides are functionalized by at least one tryptophan derivative, denoted Trp:

said tryptophan derivative being grafted or bound to the polysaccharides by coupling with an acid function, said acid function being an acid function carried by a linker R linked to the polysaccharide by a function F, said function F resulting from the coupling between the R bond and a -OH function of the polysaccharide,

F being either an ester, thionoester, amide, carbonate, carbamate, ether, thioether or amine function, - R being a chain comprising between 1 and 6 carbons, optionally branched and / or unsaturated, comprising one or more heteroatoms, such as O, N and / or S, and having at least one carboxyl function, - Trp being a residue of a derivative of tryptophan, L or D,

^'Product coupling between the amine of tryptophan and at least one acid borne by the group R and / or an acid carried by the polysaccharide having carboxyl functional groups.

According to the invention, the functionalized polysaccharides may correspond to the following general formulas:

polysaccharide

F

[TiP] _n i

Formula I

F resulting from the coupling between the linker R and an OH function of the polysaccharide and being either an ester, thionoester, amide, carbonate, carbamate, ether, thioether or amine function,

R being a chain comprising between 1 and 6 carbons, optionally branched and / or unsaturated, comprising one or more heteroatoms, such as O, N or / and S, and having at least one carboxyl function,

Trp being a residue of a tryptophan derivative, L or D, produces coupling between the amine of the tryptophan derivative and at least one acid carried by the R group and / or an acid carried by the polysaccharide having carboxyl functional groups. , n represents the molar fraction of the Rs substituted with Trp and is between 0.05 and 0.7, preferably 0.1 and 0.5, more preferably 0.3 and 0.4, i represents the mole fraction of the groups FR- [Trp] _n carried per saccharide unit and is between 0 and 2,

when R is not substituted with Trp, then the acid or acids of the R group are cationic carboxylates, alkali preferably as Na ⁺ or K ⁺ , said polysaccharides being amphiphilic at neutral pH.

In one embodiment, the polysaccharide is a dextran. In one embodiment, F is either an ester, a carbonate, a carbamate or an ether. In one embodiment, the polysaccharide according to the invention is characterized in that the group R is chosen from the following groups:

or their alkali metal salts.

In one embodiment, the polysaccharide according to the invention is characterized in that the tryptophan derivative is chosen from the group consisting of tryptophan, tryptophanol, tryptophanamide, 2-indole ethylamine and their cation salts. alkaline. In one embodiment, the polysaccharide according to the invention is characterized in that the tryptophan derivative is chosen from the esters of tryptophan of formula II

Formula II

E being a linear or branched C1-C4 alkyl group.

The polysaccharide may have a degree of polymerization of between 10 and 3000.

In one embodiment, it has a degree of polymerization of between 10 and 400.

In another embodiment, it has a degree of polymerization of between 10 and 200.

In another embodiment, it has a degree of polymerization of between 10 and 50.

In one embodiment, insulin is a recombinant human insulin as described in the European Pharmacopoeia. In one embodiment, the polymer / insulin mass ratios are between 0.1 and 5.

In one embodiment, they are between 0.5 and 2.2.

In one embodiment, they are between 0.7 and 1.3.

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

In one embodiment, the insulin concentration of the solutions is 600 μM or 100 IU / mL.

In one embodiment, the insulin concentration of

600 μM can be reduced by simple dilution, especially for pediatric applications.

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

In the case of local and systemic releases, the modes of administration envisaged are intravenous, subcutaneous, intradermal, transdermal, intramuscular, oral, nasal, vaginal, ocular, oral, pulmonary etc.

The invention also relates to the use of a complex according to the invention for the formulation of a human insulin solution of 100 IU / mL concentration for implantable or transportable insulin pumps.

Example 1: Rapid Analogous Insulin Solution at 100 IU / m L [00043] This solution is a Novo commercial solution sold under the name of Novolog. This product is a fast analog insulin.

Example 2: Human Insulin Solution at 100 IU / mL

This solution is a Novo commercial solution sold under the name of Actrapid. This product is a human insulin.

Example 3 Preparation of a Human Insulin Solution at 200 IU / mL

125.6 mg of insulin (21.6 μmol) comprising 470 μg of Zn ²⁺ are suspended in 8.74 ml of 40 mM acetic acid. The protein is then solubilized by the addition of 1.35 ml of 0.1 N HCl (pH 2.6).

The final concentration is then adjusted to 200 IU / mL (1.2 mM) by adding water.

The final pH of this solution is 2.6 for an acetic acid concentration of 20 mM.

This clear solution is filtered on 0.22 microns.

Example 4 Preparation of Excipients

Preparation of the 200 mM phosphate buffer at pH 7 [00049] A solution of monosodium phosphate is prepared as follows: 1.2 g of NaH ₂ PO ₄ (10 mmol) are solubilized in 50 ml of water in a volumetric flask.

[00050] A solution B of disodium phosphate is prepared as follows

1.42 g of Na ₂ HPO ₄ (10 mmol) are solubilized in 50 ml of water in a volumetric flask. The 200 mM phosphate buffer at pH 7 is obtained by mixing 3 ml of solution A with 7 ml of solution B.

Preparation of a solution of m-cresol 130 mM The m-cresol solution is obtained by solubilizing 0.281 g of m-cresol (2.6 mmol) in 20 ml of water in a volumetric flask.

Preparation of a solution of DTA at 50 mM

The EDTA solution is obtained by solubilizing 0.372 g of EDTA (1 mmol) in 20 ml of water in a volumetric flask.

Preparation of a solution of Tween 20 at 0.8 mM

The solution of Tween 20 is obtained by solubilizing 98 mg of tween 20 (80 micromol) in 100 ml of water in a volumetric flask.

Preparation of a solution of Glycerin at 1.5 M

The Glycerin solution is obtained by solubilizing 13.82 g of glycerin (150 mmol) in 100 ml of water in a volumetric flask.

Preparation of amphiphilic polymer solutions

[00056] Two amphiphilic polymers are employed. Polymer 1 is a sodium dextranmethylcarboxylate modified with the sodium salt of L-tryptophan obtained from a dextran of average molar mass by weight of 10 kg / mol, ie a degree of polymerization of 39 (Pharmacosmos) according to the process described in the patent application FR07.02316. The mole fraction of sodium methylcarboxylate, whether or not modified with tryptophan, i in Formula I, is 1.03. The mole fraction of tryptophan-modified sodium methylcarboxylate, n in formula I, is 0.36. The polymer solution 1 is obtained by solubilizing 4.03 g of polymer 1 (water content = 10%) in 15 ml of water in a 50 ml tube.

[00059] This solution is then adjusted to pH 5.5 with a solution of HCl 0.1 N ^• The polymer solution 1 is transferred to a volumetric flask of 25 ml and the concentration is adjusted to 145 mg / ml, supplementing to the mark with water.

The polymer 2 is a sodium dextranmethylcarboxylate modified with the sodium salt of L-tryptophan obtained from a dextran of average molar mass by weight of 40 kg / mol, ie a degree of polymerization of 154 (Pharmacosmos) according to the process described in the patent application FR07.02316. The mole fraction of sodium methylcarboxylate, whether or not modified with tryptophan, i in Formula I, is 1.03. The molar fraction of tryptophan-modified sodium methylcarboxylate, n in formula I, is 0.37.

The polymer solution 2 is obtained by solubilizing 4.03 g of polymer 2 (water content = 10%) in 15 mL of water in a tube of 50 mL. This solution is then adjusted to pH 5. , With 0.1 N HCl solution.

The polymer solution 2 is transferred to a volumetric flask of 25 ml and the concentration is adjusted to 145 mg / ml, supplementing to the mark with water.

EXAMPLE 5 Preparation of a Human Insulin Solution at 100 IU / mL in the Presence of Polymer 1 For a final volume of 7 mL of formulation with a mass ratio [polymer I] / [insulin] of 1.0 the various reagents are mixed in the amounts specified in the table below and in the following order:

Insulin at 200 IU / mL 3.5 mL

EDTA at 50 mM 28 μL Polymer 1 at 145 mg / mL 174 μL

PH 7 adjustment with NaOH I N 95 μL

200 mM pH 7 350 μL phosphate buffer

Tween 20 0.8 mM 70 μL

Glycerine 1.5 M 793 μL m-cresol 130 mM 1,562 mL

Water (Volume for dilution-volume of soda) 425 μL The final pH is 7 ± 0.3.

This clear solution is filtered on 0.22 microns and is then placed in

+ 4 ° C

Examples 6 to 8 were prepared according to the same procedure by varying the volumes of polymer solution to achieve the polymer / insulin mass ratios indicated in the table, the type of polymer, the presence of EDTA, the type of anti-bacterial agents and / or the presence of Tween and glycerin. The final solutions have a pH of 7, they are isotonic, clear and filtered on 0.22 microns.

Example 9: Kinetics of Aggregation of Insulin

The samples are placed on a rotor at room temperature. In the solution prepared in Example 1, aggregates appear from the hundredth hour. In the solution prepared in Example 5, aggregates only appear from the third hour.

Example 10: Injectability of solutions

[0007Q] All of these solutions are injectable with the usual insulin injection systems. The solutions described in Examples 1, 2 and 5 to 8 are injected just as easily with insulin syringes with 31 gauge needles. The solutions described in Examples 1 to 2 and 5 to 8 are injected equally easily with the Novo insulin pen, marketed under the name Novopen, with 31 gauge needles. Example 11: Protocol for Measuring the Pharmacodynamics of Insulin Solutions

6 domestic pigs of about 50 kg, previously catheterized at the jugular, are fasted 2 to 3 hours before the start of the experiment. In the hour before the injection of insulin, 3 blood samples are taken to determine the basal level of glucose and insulin.

The injection of insulin at a dose of 0.0625 IU / kg is performed subcutaneously in the neck, under the ear of the animal. Blood samples are then made every 10 minutes over 3 hours and every 30 minutes to 5 hours. After each sample, the catheter is rinsed with a dilute solution of heparin.

A drop of blood is taken to determine the blood glucose by means of a glucometer.

The glucose pharmacodynamics curves are then plotted.

Example 12; Pharmacodynamic Results of Insulin Solutions

The results obtained represented in the curves of FIGS. 1 and 2 show that all the complex formulations according to the invention (curves "example 5 to 8") make it possible to obtain an action time (onset of action) of less than 30. minutes and a nadir less than two hours, consistently lower than those of human insulin formulations (curve "Example 2") and substantially comparable to those of rapid insulin formulations (curve "Example 1").

Claims

claims

A complex between human insulin and an amphiphilic polymer comprising carboxyl functional groups, said amphiphilic polymer being chosen from functionalized polysaccharides, mainly consisting of glycoside monomers linked by glycoside bonds of (1,6) type functionalized by at least one of a derivative of tryptophan chosen from the polysaccharides of formula I:

Formula I in which,

F resulting from the coupling between the linker R and a function -OH of the polysaccharide and being either an ester, thionoester, amide, carbonate, carbamate, ether, thioether or amine function,

Trp being a residue of a tryptophan derivative, L or D, produces coupling between the amine of the tryptophan derivative and at least one acid carried by the R group and / or an acid carried by the polysaccharide having functional groups. carboxyl, n represents the molar fraction of Rs substituted with Trp and is between 0.05 and 0.7, preferably 0.1 and 0.5, more preferably 0.3 and 0.4, i represents the molar fraction FR - [Trp] n groups carried per saccharide unit and is between 0 and 2, and when R is not substituted by Trp, then the acid or acids of the R group are cation carboxylates, alkali preferably as Na ⁺ or K ⁺ , said polysaccharides being amphiphilic at neutral pH.

2. Complex according to claim 1, characterized in that the polysaccharide is a dextran.

3. Complex according to any one of the preceding claims, characterized in that F is either an ester, a carbonate, a carbamate or an ether.

4. Complex according to any one of the preceding claims, characterized in that the group R is chosen from the following groups:

or their alkali metal salts.

5. Complex according to any one of the preceding claims, characterized in that the derivative of tryptophan is selected from the group consisting of tryptophan, tryptophanol, tryptophanamide, 2-indole ethylamine and their alkali cation salts.

6. Complex according to any one of the preceding claims, characterized in that the derivative of tryptophan is chosen from the esters of tryptophan of formula II

Formula II

E being a linear or branched C1-C4 alkyl group.

7. Complex according to any one of the preceding claims, characterized in that the insulin is a recombinant human insulin.

8. Complex according to any one of the preceding claims, characterized in that the weight ratios polymer / insulin are between 0.1 and 5, preferably between 0.5 and 2.2 and more preferably they are between 0, 7 and 1.3.

9. Complex according to any one of claims 1 to 8 characterized in that the mass ratios polymer / insulin are between 0.7 and 1.3.

10. Pharmaceutical composition comprising at least one complex according to any one of claims 1 to 9.

11. Composition according to claim 10, characterized in that it is in the form of an injectable solution.

12. Composition according to any one of claims 10 to 11 characterized in that the concentration of the solutions is 600 μM or 100 IU / ml.

13. Use of a complex according to any one of claims 1 to 8 for the preparation of a human insulin formulation at a concentration of about 600 μM (100 IU / mL) whose delay of action is less than 30 minutes, preferably less than 20 minutes, and more preferably less than 15 minutes.

14. Use of a complex according to any one of claims 1 to 8 for the preparation of a human insulin formulation at a concentration of about 600 μM (100 IU / ml) whose glycemic nadir is less than 120 minutes preferably less than 105 minutes, and more preferably less than 90 minutes.

15. Use of a complex according to any one of claims 1 to 8 for the preparation of an insulin formulation at 100 IU / ml for injection pumps.