JP2011116752A - Erythropoietin-containing aqueous liquid agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new erythropoietin-containing aqueous liquid agent without containing urea and an amino acid such as histidine, arginine, glycine and the like as a stabilizer. <P>SOLUTION: The erythropoietin-containing aqueous liquid agent contains 10,000-50,000 IU/mL concentration erythropoietin, 0.01-1 mg/mL nonionic surfactant, a buffer and a salt. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an aqueous solution of erythropoietin which is storage-stable in a solution state, and more particularly to an aqueous solution containing erythropoietin which does not contain urea and amino acids such as histidine, arginine and glycine as a stabilizer.

  Erythropoietin is a glycoprotein that promotes the production of red blood cells by acting on erythroid progenitor cells to differentiate into red blood cells. For this reason, erythropoietin is used as a therapeutic agent for human renal anemia and for storing blood by autologous blood in preparation for surgery. The erythropoietin preparations marketed as pharmaceuticals include lyophilized preparations and aqueous liquid preparations, but aqueous liquid preparations are more convenient than lyophilized preparations because they do not require dissolution of the drug at the time of use.

  By supplying erythropoietin as an aqueous liquid, convenience during use is improved. However, since erythropoietin is easily denatured, aggregated, precipitated due to aggregation, and adsorbed on the wall of the container in aqueous solution, the stability as a drug is impaired. In order to distribute aqueous liquids to the market, erythropoietin in aqueous solution It is necessary to improve the stability of Therefore, various attempts have been made to increase the stability of erythropoietin in an aqueous solution. For example, regarding erythropoietin preparations, those containing urea, various amino acids, and nonionic wetting agents have been reported in order to enhance storage stability (see Patent Document 1). According to the data presented in this document, urea is required to obtain good stability of erythropoietin.

  An aqueous solution of erythropoietin containing an amino acid as a stabilizer has been reported (see Patent Document 2). The test results described in this document are L-leucine, L-tryptophan, sodium L-glutamate, L-arginine hydrochloride, L-histidine hydrochloride and L-among amino acids subjected to stability test by accelerated test. While lysine hydrochloride can improve stability, L-phenylalanine and L-cysteine have a significant decrease in stability compared to no addition, and L-serine has a substantial difference from no addition. Indicates that you cannot see.

  In addition, an aqueous solution of erythropoietin containing an amino acid, particularly glycine, as a stabilizer has been reported (see Patent Document 3), and an aqueous solution of erythropoietin containing tryptophan or a tryptophan derivative as a stabilizer (see Patent Document 4), An aqueous solution of erythropoietin containing methionine as a stabilizer (see Patent Document 5) and an aqueous solution of erythropoietin containing a neutral amino acid such as glycine, alanine, leucine or isoleucine as a stabilizer (see Patent Document 6) are reported. ing. Furthermore, an aqueous solution of erythropoietin containing leucine, isoleucine, threonine, glutamic acid, aspartic acid, phenylalanine or glycine as a stabilizer has been reported (see Patent Document 7).

  Commercially available aqueous solutions of erythropoietin contain amino acids such as histidine, arginine and glycine, or urea as a stabilizer for erythropoietin. That is, in order to obtain an aqueous solution of erythropoietin that is stable enough to be distributed on the market, it may be necessary to add an amino acid such as histidine, arginine, or glycine, or urea as a stabilizer of erythropoietin. It was known.

  Japanese Patent Publication No. 7-80782   Japanese Patent Laid-Open No. 10-182481   Special Table 2002-541208   WO2001 / 064241   Special table 2002-527470 gazette   Special table 2007-528842   Special table 2007-536215 gazette

  Under the above background, the object of the present invention is to provide an aqueous solution of erythropoietin that is stable to the extent that it can be distributed to the market without containing amino acids such as histidine, arginine, glycine, and urea as stabilizers. Is to provide.

In the research aimed at the above-mentioned purpose, the present inventors have included erythropoietin in a relatively high concentration in an aqueous liquid preparation, without adding an amino acid such as histidine, arginine, or glycine, or urea as a stabilizer. The present inventors have found that the stability of erythropoietin in an aqueous solution can be enhanced, and the present invention has been completed. That is, the present invention provides the following.
(1) An erythropoietin containing erythropoietin at a concentration of 10,000 to 50,000 IU / mL and a nonionic surfactant of 0.01 to 1 mg / mL, and containing no urea or amino acid as a stabilizer. Aqueous solution.
(2) The aqueous liquid preparation according to (1) above, wherein the concentration of erythropoietin is 12000 to 48000 IU / mL.
(3) The aqueous liquid preparation according to (1) or (2) above, wherein the nonionic surfactant is a polysorbate or a poloxamer.
(4) The aqueous liquid agent according to (1) or (2) above, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.
(5) The aqueous liquid preparation according to (1) to (4) above, further containing a buffer and an isotonic agent and having a pH of 5.5 to 7.2.
(6) The aqueous liquid according to (5) above, wherein the buffer is a phosphate buffer and the tonicity agent is sodium chloride.

  According to the present invention, erythropoietin in an aqueous liquid can be stabilized without containing an amino acid such as histidine, arginine, glycine, or urea as a stabilizer.

  In the present invention, “erythropoietin” refers to human erythropoietin, but is not limited to this, and includes erythropoietin of mammals including domestic animals such as cattle and horses and pets such as dogs and cats. The term “erythropoietin” includes not only wild-type erythropoietin but also analogs of erythropoietin in which one or more amino acid residues constituting erythropoietin are substituted, deleted, or inserted.

  In the present invention, erythropoietin can be produced as recombinant erythropoietin using a gene recombination technique. For example, by incorporating an erythropoietin gene into an expression vector, transforming a cell (eg, a CHO cell derived from Chinese hamster ovary) using this, and culturing the resulting transformed cell, biologically active recombination Methods for producing body erythropoietin are well known to those skilled in the art (WO 2008/068879).

  In the present invention, the concentration of erythropoietin contained in the aqueous liquid is preferably 10,000 to 50,000 IU / mL, more preferably 12,000 to 48,000 IU / mL. Suitably, the concentration of erythropoietin may be 12,000 or 48,000 IU / mL.

  In the present invention, as the nonionic surfactant contained in the aqueous liquid preparation, polysorbate, poloxamer or the like can be used alone or in combination. Polysorbate 20 and polysorbate 80 are particularly suitable as the polysorbate, and poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is particularly suitable as the poloxamer. Further, the concentration of the nonionic surfactant contained in the aqueous liquid agent is preferably 0.01 to 1 mg / mL, more preferably 0.01 to 0.5 mg / mL, and 0.02 More preferably, it is -0.1 mg / mL, and it is especially preferable that it is 0.05 mg / mL.

  In the present invention, the buffer contained in the aqueous solution is not particularly limited as long as it is pharmaceutically acceptable, but a phosphate buffer is preferable, and a sodium phosphate buffer is particularly preferable. The concentration of sodium phosphate is preferably 0.01 to 0.04M. The pH of the aqueous liquid preparation adjusted with a buffer is preferably 5.5 to 7.2, more preferably 5.8 to 7.0, still more preferably 5.8 to 6.2, and particularly preferably 6. .0.

  In the present invention, the isotonic agent contained in the aqueous liquid is not particularly limited as long as it is pharmaceutically acceptable, but sodium chloride and mannitol can be preferably used.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail with reference to an Example, this invention is not intended to be limited to an Example.

[Study on stability of erythropoietin aqueous solution]
An erythropoietin aqueous solution having the composition described in Tables 1 to 3 below was prepared, filled in a glass vial with 0.7 mL each, sealed, and stored in the dark for 7 days at 50 ° C. or 30 days at 40 ° C. . The concentration of erythropoietin in the aqueous solution was set at 1500 IU / mL (Table 1), 12000 IU / mL (Table 2) and 48000 IU / mL (Table 3). About the liquid agent which set the density | concentration of erythropoietin to 1500 IU / mL, pH (6.5), the liquid agent (liquid agent AC) which added 1, 0.5 and 0.25 mg / mL glycine as a stabilizer, A liquid preparation without addition of glycine (Liquid D) was prepared. For the liquid preparations in which the concentration of erythropoietin was set to 12000 IU / mL and 48000 IU / mL, a liquid preparation with 1 mg / mL glycine added as a stabilizer and a liquid preparation without glycine added were prepared. Moreover, pH of the liquid agent was adjusted to 6.0, 6.5, and 7.0. Moreover, recombinant human erythropoietin produced using CHO cells was used as erythropoietin.

[Analysis method]
Water 833mL was added to trifluoroacetic acid (TFA) 0.5mL, and it was set as 0.06% TFA aqueous solution (A liquid). Further, 666 mL of acetonitrile and 167 mL of water were added to 0.5 mL of TFA to obtain a 0.06% trifluoroacetic acid / 80% acetonitrile aqueous solution (solution B). A butylsilylated silica gel column (Vydac 214ATP54, Vydac) (particle size 5 μm, inner diameter 4.6 mm, length 250 mm) as a stationary phase was attached to HPLC system LC-10A (manufactured by Shimadzu Corporation), and erythropoietin material was injected. HPLC was carried out at a flow rate of 1.0 mL / min at 0 ° C. using a mixture of liquid A and liquid B as a mobile phase. The composition of the mobile phase is such that the mixing ratio of liquid A and liquid B is 50/50 for the first 10 minutes, and then the mixing ratio of liquid A and liquid B is linearly shifted to 25/75 over 10 minutes. The ratio was maintained for 20 minutes, then returned to 50/50 and maintained for 10 minutes. Detection was performed with an ultraviolet absorptiometer (measurement wavelength: 215 nm). Under these conditions, it was confirmed that the peak detected at about 20 minutes after the start of HPLC was erythropoietin, and the residual ratio (%) was determined from the ratio of peak areas before and after storage.

〔result of analysis〕
Table 4 shows the residual rate of erythropoietin after storing the erythropoietin-containing aqueous solution at a concentration of 1500 IU / mL at 50 ° C. for 7 days. In the liquid formulation D to which glycine is not added, the residual rate of erythropoietin is remarkably reduced as compared with the liquid formulations (liquid formulations A and B) containing 0.5 mg / mL or more of glycine. It was found that glycine is necessary as a stabilizer in order to keep it stable in This result supports the description in the prior art documents that it is effective to add an amino acid such as glycine as a stabilizer to obtain a stable aqueous solution of erythropoietin.

  Tables 5 and 6 show the residual ratio of erythropoietin after storing the erythropoietin-containing aqueous solution at concentrations of 12000 IU / mL and 48000 IU / mL at 50 ° C. for 7 days. When the concentration of erythropoietin was 12000 IU / mL, the residual rate of erythropoietin was slightly decreased in the liquid preparation containing glycine compared to the liquid preparation containing glycine in the liquid preparation adjusted to pH 7.0. In the liquid preparation adjusted to 6.5, the residual rate of erythropoietin did not differ greatly depending on the presence or absence of glycine (Table 5). Similar results were obtained when the erythropoietin concentration was 48000 IU / mL (Table 6). In particular, in the solution adjusted to pH 6.0, at any concentration, the solution containing no glycine showed a higher residual rate than the solution containing glycine (solutions 1 and 2, solution 7). And 8). These results show that the erythropoietin in the aqueous liquid can be stably maintained without adding a stabilizer such as glycine by setting the concentration of erythropoietin in the aqueous liquid to a relatively high concentration of 12000 IU / mL or more. In particular, at pH 6.0, a solution containing no glycine further increases the stability of erythropoietin. From these results in Table 4 and the description in the prior art literature, it was considered that a stabilizer such as glycine was necessary to stably hold erythropoietin in the aqueous liquid preparation. Is amazing.

  Next, Tables 7 and 8 show the residual ratio of erythropoietin after storing the erythropoietin-containing aqueous solution at 40 ° C. for 30 days. When the concentration of erythropoietin was 12000 IU / mL, the residual rate of erythropoietin did not vary greatly depending on the presence or absence of glycine (Table 7). Similar results were obtained when the erythropoietin concentration was 48000 IU / mL (Table 8). However, at pH 6.5 and 7.0, the erythropoietin residual rate was slightly decreased in the liquid preparation containing no glycine compared to the liquid preparation added with glycine. However, at pH 6.0, the presence or absence of glycine had little effect on the residual rate of erythropoietin.

  According to the present invention, it is possible to provide an erythropoietin-containing aqueous preparation that does not contain amino acids such as histidine, arginine, and glycine, or urea as a stabilizer.

Claims (6)

  An aqueous solution of erythropoietin comprising erythropoietin at a concentration of 10,000 to 50,000 IU / mL and 0.01 to 1 mg / mL of a nonionic surfactant, and containing no urea and amino acids as stabilizers .   The aqueous liquid preparation according to claim 1, wherein the concentration of erythropoietin is 12000-48000 IU / mL.   The aqueous liquid preparation according to claim 1 or 2, wherein the nonionic surfactant is a polysorbate or a poloxamer.   The aqueous liquid preparation according to claim 1 or 2, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.   The aqueous liquid preparation according to claim 1, further comprising a buffer and an isotonic agent and having a pH of 5.5 to 7.2.   The aqueous liquid preparation according to claim 5, wherein the buffer is a phosphate buffer and the tonicity agent is sodium chloride.