LT4947B - Alfa interferon pharmaceutical composition - Google Patents

Abstract

The present invention relates to aqueous solution formulation containing interferon alfa-2b (IFN), which maintains inreased thermal stability and is resistant for autooxidation, and process for making thereof. The formulation comprises (0,01-50) x 106 IU/ml of interferon alfa-2b, 0,08 to 0,1 M of sodium citrates, 0,05 to 0,06 M of glycine, 0,008 to 0,01 % of polysorbate-20, 0,004 to 0,005 % of thimerosal. The pH of solution is maintained as high as 6,0 0,1. This inventon can be used in manufacture of IFN stable, aqueous solution formulation for treatment of some viral and oncological diseases or/and in biotechnology processes for manufacture of genoengineering IFN.

Description

The present invention relates to a pharmaceutical composition comprising a biologically active protein, interferon (IFN) alpha-2, and other components dissolved in water, which provide it with increased thermal stability and resistance to auto-oxidation, and a process for its preparation.

The invention relates to the pharmaceutical field and can be used in the manufacture of IFN dosage forms for injection, drops, lotions and other liquid dosage forms for the therapy, biotechnology of some viral and oncological diseases, and for the production of genetic engineering for IFN.

The production, transport, storage and use of IFN dosage forms present stability issues due to the sensitivity of IFN to environmental physical and chemical factors. They act by aggregation and proteolysis.

oxidation, deamidation. disulfide bond degradation and other processes. As a result, IFN liquid dosage forms (solutions) lose some of their biological activity, resulting in degradation products which, when exceeded, make IFN formulations unusable.

Formulations often containing human serum albumin (HSA) are used to stabilize IFN. BEROFOR (Boehringer Ingelheim, Germany) is the most widely used commercial preparation for interferon alpha in the world market. INTRON A (Sehering, USA). ROFERON A (Hoffmann-La Roche. Switzerland), WELLFERON (Wellcome Foundation, England), which differ in composition or method of production. however, they all have a stabilizing agent, HSA [A. Braun, J. Alsenz (1997) Pharm. Res., Vol. 14th

No. 10, p. 1394-1400]. This is what the next generation of patented interferon solutions have. that they exclude serum-based components, which can be a source of infection with human immunodeficiency virus (HIV). There are only a few patents. Thus, International Application WO 89, 04177 describes a composition of an interferon beta solution. It consists of ammonium acetate buffer pH 5.0 ± 0.1. concentration of isotonic agent NaCl 0.9% and nonionic detergent polysorbate-80 0.03%. Japanese Patent No. 61,276,363 is known which describes a composition of an interferon gamma solution. Its buffer is sodium succinate. pH 5.0. isotonic agent - mannitol 4%.

The closest analogs of the invention are solutions of interferon alpha. This is the European Patent EP 0736303 of Hoffmann-La Roche, which describes solutions for solutions of interferon alpha and its polyethylene glycol derivative. They contain a buffer system of 0.01 - 0.015 M ammonium acetate or sodium lactate, pH 4.5 - 5.5, most preferably pH 5.0 ± 0.1. The nonionic detergent polysorbate-80 is used at a concentration of 0.01-0.5 mg / ml. The preferred isotonizing agent is NaCl or mannitol, and glycerol, arginine, lysine, histidine, methionine or ethanolamine may be used. The antimicrobial agent is benzyl alcohol at a concentration of 8-20 mg / ml. During the preparation of the composition, oxygen is removed from the solution with nitrogen gas. In such a solution after 3 months. remains:

at 5 ° C - 92% - 94%, at 25 ° C - 61% - 88% and at 35 ° C - 44% - 71%, depending on the initial concentration.

European Patent EP 0777495, issued on behalf of the Schering Corporation, describes aqueous solutions of interferons alpha. The biologically active component may be any alpha-type interferon (0.1-100) x ^{10 6} IU / ml. The pH of the buffer system is 4.5 to 7.1, with sodium phosphate buffer pH 6.8 being most suitable. A chelating agent, disodium dihydrogen tetraacetic acid (EDTA) or citric acid, is added, but EDTA 0.1 mg / ml is preferred to keep the solution clear. Non-ionic detergent polysorbate-80 0.1 mg / ml is required to stabilize interferon by preventing potential losses by surface adsorption. The isotonizing agent is NaCl 7.5 mg / ml. The antimicrobial agent is metacresol, phenol, methylparaben, propylparaben, or a mixture of methylpropylparabens. During the preparation of the solution, oxygen must be removed from the solution with a nitrogen gas concentration up to 0.25 ppm and the concentration of oxygen above the solution must not exceed 4%. At 28 ° C, at least 75% of the bioactivity is retained in the solution for 24 months and no turbidity is observed visually.

IFN solutions are used as liquid dosage forms, which should be measured in years, so increasing the stability of IFN in solution is a pressing issue. Patent analysis indicates that the stability of IFN in solutions of known composition is insufficient in terms of long-term storage. Harmful IFN environmental factors heat and oxygen are the engines of degradation processes. Under their influence, the native structure of IFN is altered, methionine residues are oxidized and biological activity is lost.

The object of the present invention is to increase the thermostability and auto-oxidation resistance of an IFN solution.

The object is achieved by the invention of a new composition having the following components:

(a) Interferon alfa-2, active ingredient;

(b) Sodium citrates, an effective concentration buffer system with effective pH. In addition to this function, they serve as chelating, isotonizing, and stabilizing agents;

* 5 (c) glycine, an effective concentration of a chelating, isotonic, and stabilizing agent;

(d) polysorbate-20, a nonionic detergent, an effective concentration stabilizing agent;

(e) thimerosal, an effective concentration stabilizing agent.

In the inventive composition, interferon alfa-2 type may be alpha-2a or alpha-2b, most preferably recombinant human interferon alfa-2b. It has been found that its concentration in the solution should be (0.01 - 50) x10 ⁶ IU / ml, with (0.1 - 30) x 10 ⁶ IU / ml being most suitable.

In the proposed composition, the preferred buffer system is a solution of monosodium and disodium citrates, in contrast to the analog EP 0777495, according to which sodium phosphate solution is preferred. Citrates were found to be most effective in stabilizing IFN at concentrations of 0.08 - 0.1 M compared to phosphates or acetates.

The pH of the IFN solution, which is preferably 6.0 ± 0.1 for the composition of the invention, is essential for the stability of the solution. In contrast, according to EP 0736303 the preferred pH is 5.0 ± 0.1 and according to another EP 0777495 the preferred pH is 6.8.

In addition, citrate anions act as a chelating agent to form complexes with metal cations. The analogue serves EDTA or citric acid for this purpose, but EDTA is most suitable. However, its use is not recommended by the European Agency for the Evaluation of Medicinal Products. In addition to citrates, the composition of the invention has a chelating agent other than glycine. Their overall efficiency has made it possible to forgo EDTA usage.

In the composition of the invention, sodium citrates together with glycine give the IFN solution isotonic at a total concentration of 0.13 to 0.16 M.

Glycine is an IFN stabilizing component with an effective concentration of 0.05-0.06 M. In the proposed composition, it also functions as a chelating and isotonic agent in combination with citrates.

The new pharmaceutical composition uses as an stabilizing component anionic polysorbate-20 at a concentration of 0.008-0.01.

The antimicrobial component is thimerosal, which is preferably present in a concentration of 0.004% to 0.005% in the composition of the invention.

The method of preparing a solution for a new pharmaceutical composition is characterized in that, contrary to the known preparation methods, the buffer system with the addition of a chelating, isotonizing agent, a nonionic detergent and an antimicrobial agent does not remove oxygen from the solution.

The invention is illustrated by the following examples:

example

Preparation of a solution of a new pharmaceutical formulation of interferon

The IFN solution is prepared by dissolving the composition components in water for injection at room temperature. First prepare the buffer from the citric acid solution by neutralizing it with 10 to 20% NaOH solution to pH 6.0 ± 0.1. Thereafter, the necessary amounts of thimerosal and glycine are dissolved by stirring. A solution of polysorbate-20 in a minimal amount of water is then added. Finally, dilute with water and IFN solution to the desired concentration. Under aseptic conditions, the solution is filtered through a membrane filter. The sterile filtrate is dispensed into stoppered vials.

The specific composition of the composition is selected from the data in Table 1.

table

Composition of a new formulation of 1 ml of interferon solution

The component Quantity Interferon alfa-2b (0.01 -50) x10 ⁶ TV Citric acid monohydrate 17-21 mg Sodium hydroxide 10% to pH 5.9-6.1 Glycine 3.8 - 4.5 mg Polysorbate-20 0.08 - 0.1 mg Thimerosal 0.04 - 0.05 mg Water for injections to 1.0 ml

example

Three solutions of IFN alfa-2b (0.1x10 ⁶ IU / ml) were prepared for thermostability studies. One of them (Solution A) according to the lowest values of Example 1 of the present invention, the other two according to analogues: Solution B according to EP 0736303 (see Table 2) and Solution C according to EP 0777495 (see Table 3).

table

Interferon solution B prepared according to EP 0736303

The component Quantity Interferon alfa-2b lxl0 ⁵ TV Ammonium acetate 0.77 mg Sodium chloride 7.21 mg Benzyl alcohol 10.0 mg Polysorbate-80 0.1 mg Acetic acid 0.1 N to pH 5.0 ± 0.1 Sodium hydroxide 0.1 N to pH 5.0 ± 0.1 Water for injections to 1 ml

All three solutions in the stoppered vials were incubated in a water thermostat at 60 ° C. After a time, Hamilton syringe is sampled and IFN biological activity is determined according to the European Pharmacopoeia. The results obtained are shown in Fig.l.

The data obtained show that the stability of the new formulation IFN solution (A) at

60 ° C is significantly higher when compared to its analogs (B, C).

table

Interferon solution C prepared according to EP 0777495

The component Quantity Interferon alfa-2b lxl0 ^{4 5} TV Disodium hydrogen orthophosphate anhydrous 1.8 mg Monosodium dihydrogen orthophosphate monohydrate 1.3 mg Disodium EDTA 0.1 mg Polysorbate-80 0.1 mg m-Cresol 1.5 mg Sodium chloride 7.5 mg Water for injections to 1 ml of solution pH 6.8

Example 3

Three solutions of IFN alpha-2b (1x10 ⁶ IU / ml) were prepared for thermostability studies, which differed from Example 2 in that the concentrations of the components of solution A had the highest values shown in Table 1. Thermostability tests were also performed except that IFN solutions were incubated at 35 ° C. The results obtained are shown in Fig.2.

The results obtained show that at 35 ° C the IFN solution (A) of the new formulation is more stable compared to the IFN analogs (B and C).

example

Three solutions of IFN alpha-2b (18x10 ⁶ IU / ml) were prepared for thermostability studies, which differ from Example 2 in that the concentrations of the components of Solution A have the highest values shown in Table 1. Thermostability tests were also performed except that IFN solutions were incubated at 25 ° C. The results obtained are shown in Figs. 3. The results obtained show that at 25 ° C the new formulation of IFN solution (A) is more stable compared to the analogue IFN solutions (B and C).

example

For the determination of oxidized products according to Example 2. solutions of IFN alfa-2b at concentrations of 10 and 18 million IU / ml were prepared (A, B, C). Oxygen was not removed from all solutions during preparation. The solutions were incubated at 5 ° C and 25 ° C for 18 months. Then

- 5 is the value of oxidised IFN in solutions by high performance chromatography (HPLC) according to the European Pharmacopoeia. The results obtained are shown in Table 4.

table

Amount of oxidized IFN formed in solutions of various formulations incubated at 4 ° C and 25 ° C for 18 months.

IFN solution name IFN concentration, IU / ml Oxidized IFN quantity,%, 4 ° C Oxidized IFN quantity,% 25 ° C A - The invention 10x10 ⁶ 1.9 2.6 composition 18x10 ⁶ 2.0 2.4 B - By analogy 10x10 ⁶ 10.6 11.9 EP 0736303 18x10 ⁶ 8.7 11.2 C by analog 10x10 ⁶ 8.9 23.7 EP 0777495 18x10 ⁶ 7.1 26.6

Note: 1.9% oxidized form was found in the IFN stock solution.

The results obtained show that the known interferon solutions in terms of resistance to auto-oxidation cannot match those of the invented formulation IFN.

Claims (2)

An interferon alpha pharmaceutical composition comprising a buffering system and / or stabilizing agents and capable of containing a chelating agent, a tonic to an agent, a nonionic detergent and an antimicrobial agent, characterized in that the buffer system comprises sodium citrates and the compositions contain effective glycine, polysorbate -20 and thimerosal concentrations. 2. A pharmaceutical composition according to claim 1, which is an aqueous solution comprising: recombinant human interferon alfa-2b sodium citrate glycine polysorbate-20 thimerosal (0.01-50) x 10 IU / ml, 0.08-0, 1M, 0.05-0.06 M, 0.008-0.01%, 0.004-0.005%. Pharmaceutical composition according to claim 1 or 2, characterized in that the pH of the solution is 6.0 ± 0.1. The preparation of a solution of an interferon alpha pharmaceutical composition according to claims 1-3 Method 2-5, comprising preparing a buffer, dissolving the stabilizing agents and / or a non-ionic detergent and an antimicrobial agent, deprotecting the oxygen with an inert gas, and adding the active ingredient solution, wherein the addition of the interferon alpha solution does not remove oxygen.