KR20060108040A - Hg-csf aqueous formulation - Google Patents

Abstract

The present invention provides a solution formulation containing phosphorus granulocyte colony stimulating factor (hG-CSF) as an active ingredient and a nonionic surfactant, tonicity agent and buffer as a stabilizer and having an acidity maintained within a range of pH 4.2 to 4.8. As such, the hG-CSF solution formulation can prevent monomer loss due to precipitation during long-term storage and can prevent pain and necrosis of tissues when administered to a human body.

Description

Phosphorus Granulocyte Colony Stimulator Solution Formulation {hG-CSF Aqueous Formulation}

The present invention relates to a stable solution formulation of phosphorus granulocyte colony stimulating factor, and specifically includes hG-CSF as an active ingredient and a nonionic surfactant, tonicity agent, and buffer as a stabilizer, and an acidity of pH 4.2 to 4.8. It provides a hG-CSF solution formulation which can prevent the loss of the monomer and the necrosis of the tissue during administration and the loss of the monomer, such as by precipitation during long-term storage.

Granulocyte colony stimulating factor is a hematopoietic factor that acts on granulocyte progenitor cells in the bone marrow to promote differentiation and proliferation into neutrophils (neutrophils leukocytes). Neutropenia occurs when the number of neutrophils falls below approximately 1.5 × 10 ⁹ / L, which is associated with a variety of diseases, including congenital defects, drug treatment (anti-cancer treatment), and bone marrow suppression after infection. And can sometimes cause infections caused by bacteria or fungi that require hospitalization. Granulocyte colony stimulating factors have been shown to reduce and prevent the duration of neutropenia, and in 1991, the US Food and Drug Administration (NEF) reported that neutrophils were associated with neutropenia during cytotoxic chemotherapy or after chemotherapy. ^TM ; Filgrastim, r-met-huG-CSF). Therefore, the use of nufogen in bone marrow transplantation has been approved worldwide and used in the US for the treatment of chronic congenital neutropenia, and clinical trials for the treatment of ADIS patients or other infectious diseases using the nufogen. This is in progress.

Granulocyte colony stimulating factors naturally come in two forms: glycated proteins with 174 amino acids and glycated proteins with 177 amino acids. Both types of proteins have five cysteine residues, four of which are linked by two intramolecular disulfide bonds, and one is in a free cysteine group. Due to these structural features, there are many problems in its stability, such as promoting precipitation reactions through aggregation and multiplex formation between protein molecules at high temperatures.

In the case of glycated hG-CSF obtained through animal cell culture, as known from EP 1060746, oligosaccharides in the molecule play a role in reducing the hydrophobicity of hG-CSF to stabilize proteins from multimer formation or aggregation, The surfactants used in general protein solution formulations can solve these problems. However, the production cost of glycated protein through animal cell culture has a problem that is several times that of the production of non-glycosylated protein through recombinant E. coli fermentation, which is a very important factor in the development and commercialization of stable formulation of unglycosylated hG-CSF molecules. The solution has been studied by many researchers.

USP 5919443 discloses lyophilized hG-CSF formulations, but lyophilized formulations are being replaced by solution formulations due to their drawbacks. Lyophilized products are inconvenient to be re-dissolved in water for injection, and the lyophilization process is added to the production process compared to the solution formulation, which requires more capital investment. For this reason, lyophilized formulations have been required to develop hG-CSF solution formulations due to their low market competitiveness compared to solution formulations.

USP 5104651 proposes an acidic solution condition of pH 2.7 to 4 without the use of buffers as a method of stabilizing hG-CSF, and specifies that the pH of the hG-CSF protein has the maximum stability of 3.5 to 3.7. However, in spite of the results in the above patents, Nuphogen (trademark; Amgen, USA), a product marketed by the patentee, does not use the optimal pH range specified in the above patents, which is caused by injection. It is indirectly indicated that the acidic pH of the solution formulation can cause pain in the patient and necrosis of the tissue.

Therefore, there is a high need for the development of a solution formulation that is stable and close to neutral acidity during the storage period of aglycosylated hG-CSF, which is strongly hydrophobic and sensitive to free cysteine aggregation and multimer formation reactions.

Therefore, an object of the present invention is to solve the problems of the prior art and the technical problems that have been requested from the past.

That is, an object of the present invention is to prevent aggregation and multimer formation during long-term preservation, and hG- to prevent pain and tissue necrosis of a patient during administration due to having a pH (acidity) close to neutrality. To provide a CSF solution formulation.

The hG-CSF solution formulation according to the present invention for achieving this purpose, contains a phosphorus granulocyte colony stimulating factor (hG-CSF) as an active ingredient and a nonionic surfactant, isotonic agent and buffer solution as a stabilizer and acidity It is maintained in the range of pH 4.2 to 4.8.

After various experiments and in-depth studies, the inventors have used a stabilizer of such composition as a hG-CSF solution formulation to maintain a certain range of acidity, as is known in the art. It has been found that aggregation or formation of multiplexes can be prevented in the patient, and that pain and tissue necrosis of the patient can be prevented upon administration of the solution formulation.

Solution formulations according to the invention can be preferably used, in particular for injection.

Since the hG-CSF in the solution formulation of the present invention can be expressed and purified through animal cell culture or E. coli fermentation by natural and genetic recombination methods, the hG-CSF that can be used in the present invention can be used for all types of hG-CSF. Contains the CSF.

The solution formulation of the present invention uses a nonionic surfactant as one of the stabilizers to prevent aggregation and multimer formation of hG-CSF. Preferred examples of such nonionic surfactants include polysorbate-based, poloxamer-based, Triton-based surfactants, and the like, which can be used in one or two or more combinations. Especially, a poloxamer type nonionic surfactant is preferable. Examples of poloxamer-based nonionic surfactants include Pluronic F-68 and Pluronic F-127. Among them, Pluronic F-68 is particularly preferable.

The isotonic agent, another component of the stabilizer in the solution formulation of the present invention serves to properly maintain the osmotic pressure when the hG-CSF is administered into the body, and also has the side effect of further stabilizing the hG-CSF in solution. Examples of the tonicity agent include water-soluble inorganic salt tonicity agents such as sodium chloride and calcium chloride, and sugar alcohol tonicity agents such as Mannitol, Sorbitol, Lactose, Mannose, Maltose, Trehalose, Glucose, Glycerol, Raffinose, and Sucrose. Preferred isotonic agents for use in the present invention are sugar alcohols isotonic agents, and among them, Sorbitol is particularly preferred.

In the solution formulation of the present invention, the buffer, the remaining component of the stabilizer, contributes to enhancing the stability of the protein by maintaining the pH of the formulation in the target pH range so that the pH of the hG-CSF solution formulation does not change rapidly. The side effect of further stabilizing hG-CSF is also shown depending on the type of. As the target pH range is pH 4.2 to 4.8, buffer solutions having a buffering effect in the pH range include, for example, Acetate, Citrate, Succinate, Tartarate, and Benzoate, preferably Acetate, Citrate, Succinate, and Tartarate buffers. Solutions may be used, with Citrate buffer being particularly preferred.

The components of the solution formulations according to the invention are more effective when used in specific compositions.

The active ingredient hG-CSF is included in the pharmacologically effective amount in the solution formulation, preferably from 150 μg / ml to 600 μg / ml.

Preferred concentrations of the nonionic surfactant are 0.1% (w / v) or less, particularly preferably 0.001 to 0.05% (w / v).

In general, the concentration of the buffer and the surfactant is low, so the concentration of the tonicity agent is preferably 5% to match the osmotic pressure.

Since hG-CSF is a hydrophobic protein and is generally unstable at high salt concentrations, the preferred salt concentration of the buffer solution is 50 mM or less, and more preferably, the concentration is 2.5 to 10 mM.

The composition of the present invention can be administered via several routes.

By “administration” in the present invention is meant introducing any substance into the patient in any suitable way, the route of administration of the composition of the present invention being administered via any general route as long as the drug can reach the desired tissue. Can be. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, pulmonary administration, rectal administration, and the like may be performed, but is not limited thereto. However, since oral administration of protein drugs is digestible, oral compositions have disadvantages such as coating the active agent or formulating them to be protected from degradation in the stomach, and therefore, may be preferably administered in the form of injections. Preferably subcutaneously. In addition, the compositions of the present invention may be administered by any device in which the active agent may migrate to target cells.

The actual dosage of the solution formulation according to the invention can be determined according to several relevant factors such as the disease to be treated, the route of administration, the age, sex and severity of the disease of the patient.

In the following Examples, the present invention will be described in more detail, but since these are only examples, the scope of the present invention is not limited thereto.

Example 1 Influence of Surfactant as Stabilizer

hG-CSF 600 μg / ml and Sobitol 5% (w / v) were added to 10 mM acetate buffer solution at pH 4.5, and the formulation was prepared to include a surfactant with the composition shown in Table 1, and each 0.8 ml vial. After filling in, it was stored in a 25 ℃ incubator. To determine the effect of type and concentration of surfactant on the stability of hG-CSF, it was opened after 2 months, 100 μl of each formulation was taken, and ultrafiltration high performance liquid chromatography (SE-HPLC) The monomer was quantified using. The ratio of the residual monomer amount after 2 months to the initial monomer amount was calculated and shown in Table 1.

From the results of Table 1, the type and concentration of nonionic surfactant affects the stabilization of hG-CSF, but overall precipitation does not occur due to such composition, in particular, Pluronic F-68 0.01% (w / In the case of v), it can be seen that there is little change in the amount of monomer during the storage period of 25 ℃ 2 months.

Example 2 Results of Stability Test at 25 ° C. for 6 Months with Different Kinds of Isotonic Agents and Pluronic F-68

Formulation was prepared to include hG-CSF 600 μg / ml in 2.5 mM Citrate buffer solution at pH 4.5, and to include stabilizers with the composition shown in Table 2 below. 0.8 ml of these solution formulations were filled in vials, stored in a 25 ° C. incubator, and opened after 6 months. 100 μl of each formulation was compared to compare the stability of the formulations by ultrafiltration, high performance liquid chromatography. Monomers were quantified. The ratio of the residual monomer amount after 6 months after the initial monomer amount was calculated and shown in Table 2 below.

From the results of Table 2, when Maltose and Sorbitol are used as isotonic agents, and the concentration of Pluronic F-68 serving as a protein stabilizer is shown in Table 2, the recovery rate of residual monomers of 95% or more in a 25 ° C 6-month stability test It can be seen that. Therefore, it can be seen that hG-CSF is more stabilized when Maltose or Sorbitol is used as an isotonic agent and Pluronic F-68 is used as a surfactant when stored at room temperature for 6 months.

The hG-CSF solution formulation according to the present invention comprising a phosphorus granulocyte colony stimulating factor and a nonionic surfactant, isotonic agent and buffer as a stabilizer, hG-CSF protein is stored for a long time in solution. A solution of hG-CSF which is closer to the neutral acidity which can prevent patient pain and necrosis of tissue during administration is improved while improving the problem of monomer loss due to precipitation, which is a characteristic denaturation, etc.

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

Claims (12)

A hG-CSF solution formulation containing phosphorus granulocyte colony stimulating factor (hG-CSF) as an active ingredient and a nonionic surfactant, isotonic agent and buffer as a stabilizer and having an acidity maintained within a range of pH 4.2 to 4.8. The hG-CSF solution formulation of claim 1, wherein the solution formulation is an injectable formulation. The hG-CSF solution formulation of claim 1, wherein the hG-CSF is natural or recombinant hG-CSF. The method of claim 1, The nonionic surfactant is one or two or more selected from the group consisting of polysorbate-based, poloxamer-based, and triton-based; The isotonic agent is one or more selected from the group consisting of Mannitol, Sorbitol, Lactose, Mannose, Maltose, Trehalose, Glucose, Glycerol, Raffinose and Sucrose; The buffer solution is one or more selected from the group consisting of Acetate, Succinate, Citrate, Tartarate and Benzoate; HG-CSF solution formulation, characterized in that. 5. The hG-CSF solution formulation of claim 4, wherein the nonionic surfactant is a poloxamer based nonionic surfactant. 6. The hG-CSF solution formulation of claim 5, wherein said poloxamer based nonionic surfactant is Pluronic F-68. The hG-CSF solution formulation of claim 4 wherein the tonicity agent is Sorbitol. 5. The hG-CSF solution formulation according to claim 4, wherein the buffer is Citrate. The hG-CSF solution formulation according to claim 1, wherein the concentration of hG-CSF is 150 μg / ml to 600 μg / ml. The hG-CSF solution formulation of claim 1, wherein the concentration of nonionic surfactant is 0.001 to 0.05% (w / v). The hG-CSF solution formulation of claim 1 wherein the concentration of the tonicity agent is 5%. The hG-CSF solution formulation according to claim 1, wherein the salt concentration of the buffer solution is 2.5 to 10 mM.