METHOD FOR PREPARING NANOPRECIPITATES OF LOW MOLECULAR WEIGHT PEPTIDE OR PROTEIN

Abstract

The present invention relates to a method for the non-denaturing preparation of peptide or protein nanoprecipitates, or of peptide or protein and metal ion nanocoprecipitales, in which said protein or said peptide has a molecular weight no higher than 20 kDa, preferably no higher than 15 kDa, advantageously no higher than 10 kDa, and more advantageously no higher than 8 kDa. Said method includes a step of preparing a mixture of an aqueous solution of peptides or proteins, a nonsolvent of the peptide or protein, and optionally a water-soluble metal salt. The present invention also relates to a nanoprecipitate that can be obtained by the method according to the invention, as well as to a pharmaceutical composition comprising same, for use in the treatment or prevention of diabetes.

Claims

1. Method of non-denaturing preparation of peptide or protein nanoprecipitates or of peptide or protein and metal ion nanocoprecipitates, having a mean diameter of less than 1 μm, comprising the following steps: a) preparation of a mixture of an aqueous solution of peptides or proteins, a nonsolvent of the peptides or proteins, and optionally a water-soluble metal salt; b) gentle stirring of the mixture obtained in step a); c) solid-liquid separation of the mixture obtained in step b); and d) optionally, collection of the peptide or protein nanoprecipitates or the peptide or protein and metal ion nanocoprecipitates, wherein said peptides or proteins have a molecular weight of 20 kDa or less, preferably of kDa or less, advantageously of 10 kDa or less, more advantageously of 8 kDa or less, and said nonsolvent is selected from polyethylene glycols or polyethylene glycol derivatives having a molecular weight of less than 2,000 Da, advantageously between 200 and 2,000 Da, more advantageously of 550 Da, and organic diols selected from the group of hexylene glycol, butane-1,4-diol, pentane-1,5-diol, ethohexadiol, 2-methylpentane-2,4-diol(hexylene glycol), 3-cyclopentene-1,2-diol, cis-4-cyclopentene-1,3-diol, trans-1,4-dioxane-2,3-diol, 1,3-dioxane-5,5-dimethanol, (3S,4S)-pyrrolidine-3,4-diol, (3R,4R)-(−)-1-benzyl-3,4-pyrrolidinediol, (3S,4S)-(+)-1-benzyl-3,4-pyrrolidinediol, 3-cyclopentene-1,2-diol, 2-methyl-butane-1,3-diol.

2. Method according to claim 1, characterized in that the mean diameter of the nanoprecipitates or nanocoprecipitates is between 5 nm and 500 nm, particularly between 5 and 200 nm, more particularly between 5 and 170 nm.

3. Method according to claim 1, characterized in that said metal on is selected from Zn, Mg, Ca, Mn, Fe, U or Cu ions, advantageously said metal ion is Zn or Mn.

4. Method according to claim 1, characterized in that said nonsolvent is selected from PEG 550, glycofurol or hexylene glycol, particularly from PEG 550 and hexylene glycol, even more particularly said nonsolvent is PEG 550.

5. Method according to claim 1, characterized in that said peptides or proteins are therapeutic.

6. Method according to claim 1, characterized in that said peptides or proteins are selected from human insulin, growth hormone, glucagon, peptide hormones or a therapeutically effective derivative or fragment thereof.

7. Method according to claim 1, characterized in that said water-soluble metal salt is selected from ZnCl2, MgCl2, CaCl2, MnCl2, FeCl2, LiCl and CuSO4, advantageously ZnCl2 or MnCl2.

8. Method according to claim 1, characterized in that said solid/liquid separation of step c) is a tangential filtration or a centrifugation.

9. Peptide or protein nanoprecipitates or peptide or protein and metal ion nanocoprecipitates, obtainable by the preparation method according to claim 1.

10. Nanoprecipitates or nanocoprecipitates according to claim 9, characterized in that the peptide or the protein is human insulin or a derivative or a therapeutically effective fragment.

11. Nanoprecipitates or nanocoprecipitates according to claim 9, for use as a medicinal product.

12. Nanoprecipitates or nanocoprecipitates according to claim 9, for use as a medicinal product for single parenteral administration such that the duration during which the plasma concentration of said peptides or proteins is within the therapeutic window is greater than the duration during which the plasma concentration after a single parenteral administration of the same peptides or proteins not prepared according to the method of preparation as claimed in claims 1 to 8 is within the therapeutic window.

13. Sustained-release pharmaceutical composition comprising nanoprecipitates or nanocoprecipitates according to claim 9.

14. Sustained-release pharmaceutical composition according to claim 13, for use as a medicinal product.

15. Pharmaceutical composition according to claim 13, characterized in that the peptide or the protein is selected from human insulin, growth hormone, glucagon, peptide hormones or a therapeutically effective derivative or fragment thereof.

Description

DESCRIPTION OF THE FIGURES

[0093] FIG. 1. Insulin nanoprecipitation yield as a function of insulin mass used.

[0094] FIG. 2. Insulin nanocoprecipitation yield as a function of insulin mass, in the presence of manganese chloride, MnCl.sub.2 (condition (1) 0.12 mg of MnCl.sub.2; condition (2) 0.25 mg of MnCl.sub.2).

[0095] FIG. 3. Insulin nanocoprecipitation yield as a function of insulin mass, in the presence of calcium chloride, CaCl.sub.2 (condition (1) 0.12 mg of CaCl.sub.2; condition (2) 0.25 mg of CaCl.sub.2).

[0096] FIG. 4. Insulin nanocoprecipitation yield as a function of insulin mass, in the presence of zinc chloride, ZnCl.sub.2 (condition (1) 0.12 mg of ZnCl.sub.2; condition (2) 0.25 mg of ZnCl.sub.2).

[0097] FIG. 5. Influence of zinc chloride concentration on insulin nanoprecipitation yield.

[0098] FIG. 6. Influence of temperature on insulin nanoprecipitation or nanocoprecipitation yield with 0.12 mg of ZnCl.sub.2.

[0099] FIG. 7. Optical microscopy photographs of polymer microspheres loaded with insulin nanoprecipitates.

[0100] FIG. 8. Profile of insulin release from insulin nanoprecipitates with glycofurol (.diamond-solid.) or PEG 550 (.square-solid.) according to the invention, from a 1% hyaluronic acid gel.

[0101] FIG. 9. Profile of insulin release from insulin nanoprecipitates with glycofurol (.diamond-solid.) or PEG 550 (.square-solid.) according to the invention, from a 2% hyaluronic acid gel.

[0102] FIG. 10. Profile of insulin release from an insulin and metal ion nanocoprecipitate according to the invention prepared with glycofurol and ZnCl.sub.2(), glycofurol and MnCl.sub.2 (.diamond-solid.), PEG 550 and ZnCl.sub.2 (∘) or PEG 550 and MnCl.sub.2 (⋄), within a hyaluronic acid gel in comparison with the release profile of insulin in native form from a hyaluronic acid gel (.square-solid.).

[0103] FIG. 11. Insulin release profile as a function of the presence or absence of a step of re-suspension of the nanoprecipitates. This suspension step is carried out before dispersion of the insulin nanoprecipitates within the hyaluronic acid matrix. The condition without prior dilution (.diamond-solid.) corresponds exclusively to insulin in nanoprecipitated form. The condition with prior dilution (.square-solid.) corresponds to a mixture of nanoprecipitated insulin and non-precipitated insulin in solution.

[0104] FIG. 12. Size distribution of insulin nanoprecipitates obtained by dynamic light scattering.

[0105] FIG. 13. Evolution of glycemia in rat following subcutaneous administration of liquid insulin (Humuline®, Eli Lilly) and of Humuline® nanoprecipitates at a dose of 5 IU/kg/d. In this case, the Humuline® nanoprecipitates are administered in a 1% hyaluronic acid gel.

[0106] FIG. 14a. Evolution of glycemia in rat following subcutaneous administration of liquid insulin (Humuline®, Eli Lilly) and of Humuline® nanoprecipitates at a dose of 5 IU/kg/d. In this case, the Humuline® nanoprecipitates are administered in a polymer solution (175 mg/ml PLGA-PEG-PLGA polymer, 20% Capmul®, triacetin).

[0107] FIG. 14b. Evolution of insulinemia in rat following subcutaneous administration of liquid insulin (Humuline®, Eli Lilly) and of Humuline® nanoprecipitates at a dose of 5 IU/kg/d. In this case, the Humuline® nanoprecipitates are administered in a polymer solution (175 mg/ml PLGA-PEG-PLGA polymer, 20% Capmul®, triacetin). FIGS. 14a and 14b are derived from data collected from the same animals within a single experiment.

[0108] FIG. 15. Evolution of glycemia in rat following subcutaneous administration of liquid insulin (Humuline®, Eli Lilly) and of Humuline® nanoprecipitates at a dose of 5 IU/kg/d. In this case, the Humuline® nanoprecipitates are administered in a 1% hyaluronic acid, 10% Pluronic F127 gel.

[0109] FIG. 16. Scanning electron microscopy image of lyophilized insulin nanoprecipitates.

[0110] FIG. 17. Profiles of interleukin-2 (molecular mass of 15 kDa) release from three matrices containing interleukin-2 nanoprecipitates prepared according to the invention. (rhombuses: 3% propylene glycol alginate, squares: 10% hyaluronic acid, triangles: 4% Carbopol).

[0111] The following examples aim at illustrating the present invention.

EXAMPLES

Example 1: Preparation of Insulin Nanoprecipitates

[0112] In a flask, a solution of 0.468 mg of human insulin (Umuline Rapide® from Lilly at 100 IU/ml) is contacted with a PEG 550 solution (an organic nonsolvent of insulin), The preparation obtained is mixed by gentle stirring then the flask is placed in an ice bath for 30 minutes at 4° C. The mixture is then centrifuged using a range of centrifugal force between 10,000 and 50,000 g. Finally, the nanoprecipitates are collected and the quantity of insulin is determined by HPLC.

[0113] The nanoprecipitation yield is expressed as a percentage in relation to the insulin mass introduced at the start.

[0114] To measure the influence of insulin quantity on nanoprecipitation yield, the method above is repeated with an insulin mass of 0.936 mg, 1.404 mg, 1.874 mg and 2.340 mg, and the yield is calculated for each nanoprecipitation.

[0115] The results are shown in FIG. 1. It can thus be noted that the smaller the quantity of insulin to precipitate, the higher the nanoprecipitation yield.

[0116] The mean diameter of the nanoprecipitates obtained is measured by dynamic light scattering and is illustrated in FIG. 12. It can be noted that this mean diameter is less than 200 nm.

[0117] FIG. 16 is a scanning optical microscopy image of lyophilized insulin nanoprecipitates. The lyophilizate consists of all the insulin nanoprecipitates prepared according to the method described above, agglomerated to each other. It can be observed that each nanoprecipitate has a spherical shape and has a diameter between 50 and 100 nm. These sizes are close to those determined by the light scattering techniques.

Example 2: Influence of the Presence of a Water-Soluble Salt During Nanoprecipitation Nanocoprecipitation of Insulin and MnCl.SUB.2., CaCl.SUB.2 .or ZnCl.SUB.2

[0118] To measure the influence of the presence of a water-soluble salt on the yield of the insulin nanoprecipitation according to the invention, as a function of the quantity of salt and the quantity of insulin, three salts were studied: manganese chloride (MnCl.sub.2), calcium chloride (CaCl.sub.2) and zinc chloride (ZnCl.sub.2). For each salt, four solutions to nanoprecipitate were prepared according to the quantities given in Table 1 below:

TABLE-US-00001 TABLE 1 Composition of solutions A, B C and D to nanoprecipitate Solution A B C D Human insulin (mg) 0.135 0.540 PEG 550 (mg) 510 Salt (mg) 0.12 0.25 0.12 0.25

[0119] For each solution, human insulin (Umuline Rapide® from Eli Lilly at 100 IU/ml) is contacted with PEG 550 and the salt, in the quantities presented in Table 1. The preparation obtained is mixed by gentle stirring then the flask is placed in an ice bath for 30 minutes at 4° C. The mixture is then centrifuged using a range of centrifugal force between 10 000 and 50 000 g. Finally, the nanocoprecipitates are collected and the quantity of insulin is determined by HPLC.

[0120] The nanocoprecipitation yield is expressed as a percentage in relation to the quantity of insulin introduced at the start.

[0121] The results are shown in FIGS. 2, 3 and 4: [0122] from FIGS. 2 and 3, it can be seen that the use of MnCl.sub.2 or CaCl.sub.2 makes it possible to obtain insulin nanoprecipitates with a yield close to 60-70% irrespective of the quantity of MnCl.sub.2, CaCl.sub.2 or insulin used; [0123] from FIG. 4, it can be seen that the use of ZnCl.sub.2 makes it possible to obtain insulin nanoprecipitates with a yield close to 80-90% irrespective of the quantity of MnCl.sub.2 or insulin used.

[0124] The use of MnCl.sub.2, CaCl.sub.2 or ZnCl.sub.2 thus has a dual advantage: [0125] maximization of the nanoprecipitation yield, [0126] formation of nanocoprecipitates having properties different from the nanoprecipitates prepared without salts.

[0127] In a general way, FIGS. 2, 3 and 4 illustrate the flexibility of the method according to the invention with respect to the nature of the salt used. Moreover, the same method (identical insulin mass, identical nonsolvent volume and nature) makes it possible to obtain different products in terms of physicochemical nature and biochemical properties by changing only the salt.

[0128] Nanocoprecipitation of Insulin and ZnCl.sub.2

[0129] To supplement the results obtained in Example 2, new solutions are prepared according to the method of Example 2, in the quantities given in Table 2 below:

TABLE-US-00002 TABLE 2 Composition of solutions E, F, G, H, I, J, K, L, M, N, O, P to nanoprecipitate Solution E F G H I J K L M N O P Insulin (mg) 0.936 1.104 1.872 PEG 550 (mg) 510 ZnCl.sub.2 (mg) 0 0.12 0.18 0.25 0 0.12 0.18 0.25 0 0.12 0.18 0.25

[0130] The nanocoprecipitation yield is expressed as a percentage in relation to the insulin mass introduced at the start.

[0131] The results are shown in FIG. 5. From these results, it is to be noted that the highest yields are obtained for the largest masses of insulin. The effect of zinc chloride concentration is thus negligible.

[0132] We can thus conclude that: [0133] zinc chloride makes it possible to optimize the nanoprecipitation yield, the yields obtained being close to 100%; [0134] zinc chloride makes it possible to form nanocoprecipitates having specific properties.

Example 3: Influence of Temperature on Nanoprecipitation or Nanocoprecipitation Yield

[0135] To measure the influence of temperature on the yield of the insulin nanoprecipitation according to the invention in the presence or absence of a water-soluble salt, the nanoprecipitation yields of two different solutions were studied for two different temperature conditions: at 4° C. and at room temperature. The first solution is prepared according to the method described in Example 1 and comprises 1.404 mg of human insulin (Umuline Rapide® from Lilly at 100 IU/ml). The second solution is prepared according to the method described in Example 2 and comprises 1.874 mg of human insulin (Umuline rapide@ from Lilly at 100 IU/ml) and 0.12 mg of zinc chloride.

[0136] The nanoprecipitation yield is expressed as a percentage in relation to the insulin mass introduced at the start.

[0137] The results are shown in FIG. 6. They illustrate the fact that the nanoprecipitation can be carried out at room temperature, which introduces two important concepts: [0138] flexibility of the method according to the invention and thus ease of use and of scaling; [0139] notable difference with the methods described in the literature where temperature is a critical factor.

Example 4: Profile of Insulin Release from Insulin Nanoprecipitates or Nanocoprecipitates According to the Invention from a Hyaluronic Acid Gel

[0140] To study the profile of insulin release from insulin nanoprecipitates according to the invention formulated within a polymer matrix, as well as the influence of the nature of the insulin nonsolvent, the insulin nanoprecipitates obtained according to the method described in Example 1, using PEG 550 or glycofurol as insulin nonsolvent, is formulated within a 1% hyaluronic acid gel.

[0141] FIG. 7 shows the microspheres comprising the insulin nanoprecipitates (optical microscopy).

[0142] FIG. 8 shows the release of insulin from said insulin nanoprecipitates formulated within the 1% hyaluronic acid gel matrix. These results show that [0143] the release occurs over more than 20 days, and [0144] the nature of the nonsolvent influences the rate of release; the use of PEG 550 as insulin nonsolvent makes it possible to obtain a plasma insulin concentration close to 70% over more than 20 days, thus higher than when glycofurol is used (plasma concentration close to 30-40% over more than 20 days).

[0145] FIG. 9 shows the release of insulin from said insulin nanoprecipitates formulated within the 2% hyaluronic acid gel matrix.

[0146] Likewise, to study the profile of insulin release from a nanocoprecipitate of insulin and water-soluble salt according to the invention formulated within a polymer matrix, as well as the influence of the nature of the insulin nonsolvent and of the water-soluble salt, the insulin nanocoprecipitates obtained according to the method described in Example 2, using PEG 550 or glycofurol as insulin nonsolvent, and ZnCl.sub.2 or MnCl.sub.2 as water-soluble salt, are formulated within a 1% hyaluronic acid gel.

[0147] The profiles of insulin release from said nanocoprecipitates of insulin and water-soluble salt formulated within the 1% hyaluronic acid gel matrix are observed in FIG. 10 in comparison with the release profile of insulin in native form. These results show that [0148] the release of insulin from the nanocoprecipitates occurs over more than 20 days whereas the release of insulin in native form is less than 1 day; [0149] the nature of the nonsolvent influences the rate of release. The use of PEG 550 makes it possible to obtain a higher plasma insulin concentration than when glycofurol is used; and [0150] the nature of the water-soluble salt also influences the rate of release. The use of ZnCl.sub.2 makes it possible to obtain a higher plasma insulin concentration than when MnCl.sub.2 is used.

Example 5: Insulin Release Profile as a Function of the Presence or Absence of a Step of Re-Suspension of the Nanocoprecipitates According to the Invention

[0151] This study is carried out to determine the advantage of the reversibility of the nanoprecipitation or nanocoprecipitation method according to the invention. Indeed, when the nanoprecipitates or nanocoprecipitates according to the invention are suspended in solution, they regain their native, i.e. non-precipitated, form.

[0152] For this study, the profile of insulin release from nanocoprecipitates of insulin and MnCl.sub.2 according to the invention, formulated in a hyaluronic acid gel matrix (condition without dilution), is compared with the release profile of these same nanocoprecipitates suspended in solution before being formulated in a hyaluronic acid gel matrix (condition with dilution). The results are shown in FIG. 11.

[0153] The reversibility of the nanoprecipitation or nanocoprecipitation according to the invention is a crucial process for several key reasons: [0154] the biological activity of the protein or peptide is retained; [0155] the ability of the nanoprecipitates to be an in vivo source of biotherapeutics from depot forms; [0156] the ability to modulate the rate of release of the protein or peptide by playing with this reversibility (nanoprecipitates, partially suspended nanoprecipitates, peptide or protein free in solution).

Example 6: Study of the Evolution of Glycemia and of Insulinemia in Rat Following the Administration of Insulin Nanoprecipitates

[0157] Three groups of six rats catheterized in the femoral vein were injected subcutaneously with the formulations tested at a dose of 5 IU/kg/d: [0158] insulin (Humuline®) nanoprecipitates as prepared in Example 1 in a 1% hyaluronic acid gel (FIG. 13); [0159] insulin (Humuline®) nanoprecipitates as prepared in Example 1 in a polymer solution (175 mg/ml PLGA-PEG-PLGA polymer, 20% Capmul®, triacetin) (FIGS. 14a and 14b); and [0160] insulin (Humuline®) nanoprecipitates as prepared in Example 1 in a 1% hyaluronic acid, 10% Pluronic F127 gel (FIG. 15).

[0161] A control group of six rats catheterized in the femoral vein was injected subcutaneously with commercial Umuline® (Eli Lilly) solution at 5 IU/kg.

[0162] Blood samples were taken at precise times before and after injection of the test and control systems according to following schedule: [0163] Three days before injection of the systems tested, basal glycemia was determined for all the rats of the study. [0164] Six minutes before injection, blood samples were taken and designated as being the samples at T=0 minute. [0165] After injection of the systems tested, blood samples were taken at the following times: 5 minutes, 30 minutes, 1 hour, 3 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours.

[0166] The glycemias were determined using the Accu-Chek Active® system from Roche. The insulinemias were determined using an ELISA kit marketed by Mercodia.

[0167] FIGS. 13 to 15 show the evolution of the glycemia or the insulinemia in rat following subcutaneous administration of liquid insulin (Humuline®, Eli Lilly) and of nanoprecipitates as prepared above.

[0168] The experiments carried out make it possible to compare the biological action of the insulin contained in the various formulations. The commercial liquid insulin formulation has a rapid action on glycemia between 30 and 60 minutes and presents a risk of causing hypoglycemias. Indeed, the pharmacokinetic profile has a peak activity at 30 minutes. Most of the insulin dose is delivered between 5 minutes and 1 hour after injection, which greatly increases the risk of hypoglycemia. The formulations based on insulin nanoprecipitates have a slower action on glycemia and make it possible, in certain cases, to limit the risks of the occurrence of hypoglycemias. Such is the case, for example, of the formulation based on insulin nanoprecipitates presented in FIG. 14a: the action of insulin is slowed down and the glycemia is controlled better because most of the insulin dose is delivered between 5 minutes and 12 hours.

[0169] The quantitative parameters for the formulation of Umuline® nanoprecipitates within a polymer carrier in comparison with the commercial Umuline® formulation (FIGS. 14a and 14b) are as follows:

TABLE-US-00003 Duration of Minimum Time corresponding action in glycemia in to the minimum Formulation hours mg/dL glycemia in hours Liquid insulin 8-12 58 0.5 (Umuline ®) Humuline ® 24 75 3 nanoprecipitates in a polymer carrier

[0170] Concerning the evolution of the insulinemia (FIG. 14b), the use of a formulation based on insulin nanoprecipitates makes it possible to smooth the pharmacokinetic profile and to increase the plasma circulation time of the insulin by a factor of 3.

Example 7: Preparation of Interleukin-2 Nanoprecipitates

[0171] Interleukin-2 (PROLEUKIN®—Novartis) is precipitated by addition of glycofurol (CAS 3169-2-85-0). For each test, a fixed volume of 1 ml of glycofurol is used; only the volume of the interleukin-2 solution varies. The solutions are left on ice for 30 minutes then centrifuged for 30 minutes at 21,382 G at 4° C. Nanoprecipitation tests are carried out for quantities of interleukin-2 ranging from 49 μg to 297 μg.

[0172] The supernatants are removed by aspiration then the interleukin-2 nanoprecipitates are taken up in 100 μl of Milli-Q water, and a 100 μl volume is assayed by addition of 5 ml of Bradford reagent. The samples are left in the dark for 5 minutes then absorbance is measured at 595 nm. The standard range was prepared over an interleukin-2 concentration range of 5 to 20 μg/ml, R.sup.2=0.99). The results of the assays are presented in Table 3 below:

TABLE-US-00004 TABLE 3 Nanoprecipitation yield Volume of interleukin-2 Volume of glycofurol Nanoprecipitation solution (1.1 mg/ml) in μl in ml yield in % 45 1 77 90 1 72 135 1 93 180 1 76 Mean yield in % 80

[0173] The nanoprecipitation yield is expressed as a percentage in relation to the mass of interleukin-2 introduced at the start.

[0174] The mean diameter of the nanoprecipitates obtained is measured using a Nanosizer Z from Malvern. This mean diameter is 14 nm.

Example 8: Study of Interleukin-2 Release from Gel Matrices Containing Interleukin-2 Nanoprecipitates

[0175] The release experiments are carried out at 37° C. 198 μg of interleukin-2 nanoprecipitates, prepared according to the method described in Example 7, is mixed with 200 μl of gel matrix (10% hyaluronic acid, 3% propylene glycol alginate or 4% Carbopol) then placed in 1.5 ml polypropylene tubes. 1 ml of sterile DPBS (Dulbecco's Phosphate Buffered Saline, pH 7.4) supplemented with mannitol (1 g/l) and SDS (sodium dodecyl sulfate, 150 mg/1) is then added. Samples (1 ml) are taken at regular time intervals over a period of 30 days; after each sample is taken fresh medium is added to maintain a constant total volume. The assays are carried out using a commercially available ELISA kit (Thermo Scientific® Human IL-2 ELISA Kit).

[0176] FIG. 17 shows the release of interleukin-2 (molecular mass of 15 kDa) from three matrices containing interleukin-2 nanoprecipitates prepared according to the invention. These results show that the release occurs over more than 30 days.

[0177] This figure demonstrates that the formulations based on interleukin-2 nanoprecipitates make it possible to obtain sustained-releases of interleukin-2 in vitro. As a function of the type of polymer employed, the rates of release can be more or less rapid. In this example, the formulations based on hyaluronic acid and Carbopol make it possible to obtain continuous releases of interleukin-2 with no “burst” effect for durations equal to 30 days.