JP2005035921A - Preparation containing tissue plasminogen activation factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a high-quality preparation containing a tissue plasminogen activation factor that is preservable for a longer period of time. <P>SOLUTION: The preparation containing a tissue plasminogen activation factor comprises a tissue plasminogen activation factor and both of (1) lysine or its salt and (2) an acidic amino acid or its salt or (3) lysine glutamic acid and/or lysine aspartic acid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a preparation containing tissue plasminogen activator.

  Conventionally, urokinase isolated and purified from urine or cultured kidney cells, and streptokinase isolated from the culture solution of β-hemolytic streptococci have been used as thrombolytic agents for the treatment of thrombosis. However, these are inferior in affinity for fibrin, so they are often administered in large amounts to obtain the necessary effects in treatment, and side effects such as bleeding due to destruction of clotting factors by plasmin generated in the bloodstream appear. It is known.

In recent years, tissue plasminogen activator (hereinafter referred to as TPA) found in human or animal tissues and cell cultures derived from those tissues has a high affinity for fibrin, a small amount, and thrombolysis. It is expected as a thrombolytic agent with high activity and few side effects. Recently, TPA has been produced and used by genetic engineering means. As a method for obtaining these tissue plasminogen activators, in the case of natural type, Japanese Patent Publication No. 1-343549, Japanese Patent Publication No. 3-57749, Japanese Patent Laid-Open No. 5-292963, Yoshida, Furumoto et al. Production of t-PA by mass culture ”, BIO INDUSTRY, 1999, Vol16 No.1, P.34-42, etc. are known. -230084, Japanese Patent Laid-Open No. 63-192320, Japanese Patent Laid-Open No. 2-167076, and the like are known.

  Tissue plasminogen activator is a protein that is difficult to make into a high-concentration aqueous solution by itself, and methods for improving solubility include Japanese Patent Publication No. 5-69507, Japanese Patent Publication No. 6-69960, Kosho 62-36332 is known.

  Furthermore, as methods for improving the stability of tissue plasminogen activator, Japanese Patent Publication No. 4-82132, Japanese Patent Publication No. 6-72105, Japanese Patent Application Laid-Open No. 2001-81040, Japanese Patent Publication No. 9-511495, etc. are known. ing.

Japanese Patent Publication No. 1-43549 Japanese Patent Publication No. 3-57749 JP-A-5-292963 Japanese Unexamined Patent Publication No. 63-230083 JP 63-230084 JP-A 63-192320 Japanese Patent Laid-Open No. 2-167076 Japanese Patent Publication No. 5-69507 Japanese Patent Publication No. 6-69960 JP 62-36332 A Japanese Patent Publication No. 4-82132 Japanese Patent Publication No. 6-72105 Japanese Patent Laid-Open No. 2001-81040 JP 9-511495 gazette Yoshida, Furumoto et al., "Production of t-PA by mass culture of normal human lung cells", BIO INDUSTRY, 1999, Vol16 No.1, P.34-42

  Although the solubility of TPA can be increased by adding lysine, as a result of intensive studies by the present inventors, unexpectedly, when this solution is freeze-dried and formulated, it is stored for a longer period of time. It was found that the activity decreased. An object of the present invention is to provide a higher-quality preparation containing tissue plasminogen activator that can be stored for a longer period of time.

  As a result of intensive studies to solve the above problems, the present inventor has found that a preparation containing a tissue plasminogen activator and containing lysine or a salt thereof, and further an acidic amino acid or a salt thereof can be stored for a long period of time. It has been found that the activity is retained, and the present invention has been made based on this finding.

That is, the present invention contains both (1) lysine or a salt thereof and (2) (1) (2) in an acidic amino acid or a salt thereof together with a tissue plasminogen activator, or (3) It is a tissue plasminogen activator-containing preparation characterized by containing lysingtamic acid and / or lysine aspartic acid.
The present invention is also a tissue plasminogen activator-containing preparation characterized in that the tissue plasminogen activator is derived from a tissue culture solution of human normal lung cells.

The present invention also provides a tissue plasminogen activator-containing preparation characterized in that the acidic amino acid or a salt thereof is glutamic acid or a salt thereof.
The present invention also provides a tissue plasminogen activator-containing preparation comprising a tissue plasminogen activator, comprising (1) lysine or a salt thereof, and (3) lysingtamic acid.
The present invention is also a tissue plasminogen activator-containing preparation characterized by being a lyophilized preparation.
Furthermore, the present invention is a tissue plasminogen activator-containing preparation characterized by containing a saccharide.

The present invention also includes (1) lysine or a salt thereof and (2) both (1) and (2) in an acidic amino acid or a salt thereof together with a tissue plasminogen activator, or (3) lysine It is a method of stabilizing a tissue plasminogen activator characterized by containing glutamic acid and / or lysine aspartic acid.

  The tissue plasminogen activator-containing preparation of the present invention can retain its activity even during long-term storage, and can supply a preparation with higher quality.

Hereinafter, the present invention will be specifically described.
The TPA used in the present invention may be not only a natural type derived from human or animal tissue but also TPA obtained by genetic engineering techniques. That is, in the present invention, TPA can be understood as a substance having a pharmacological action that activates and converts plasminogen into plasmin, and that plasmin causes dissolution of fibrin, which is a main constituent of thrombus.

Specifically, for example, TPA extracted from human or animal-derived tissue or tissue culture solution derived from these tissues can be used. For example, cells having the ability to produce tissue plasminogen activator, such as normal diploid cells from human fetus kidney, intestine, lung, heart, ureter, skin, foreskin and whole fetus, human Extract from tissue culture fluid using placenta-derived cells or human kidney, intestine, lung, thyroid, heart, ureter, skin-derived normal diploid cells, human melanoma cells or tumor cells with similar properties be able to. Particularly preferred is extraction from a tissue culture solution using normal diploid cells derived from human fetal kidney, lung and foreskin.

For example, according to Yoshida, Furumoto et al., “Production of t-PA by mass culture of normal human lung cells”, BIO INDUSTRY, 1999, Vol16 No.1, P.34-42, a finger structure from the N-terminus of the molecule, The structure of human natural t-PA consisting of 527 amino acid residues with an epiderma growth factor structure, two first and second kringle structures and a serine protease structure is shown.

  In addition, TPA can be used by genetic engineering techniques. As a method for obtaining TPA, deletion or substitution of one or many amino acids of natural TPA is performed using DNA recombination to change the amino acid sequence. Improved TPA can be obtained. For example, JP-A-63-230083 describes a method for obtaining a mutant TPA by substituting Ser at the amino acid position number 183 and Ser at position 186 of natural TPA with Ser and Thr, respectively. Obtainable. In addition, the methods described in JP-A-63-230084, JP-A-63-192320, and JP-A-2-67076 can be mentioned.

The TPA described above is preferably purified to an extent suitable for use as a medicine. Examples of the purification method include methods usually used in protein chemistry, such as adsorption methods using carriers, ion exchange methods, and fractionation. Precipitation methods, gel filtration methods, electrophoresis methods, various affinity chromatograms, particularly methods using specific antibodies, etc. are exemplified as preferred examples, and these may be purified alone or in combination.

More specifically, fibrin sepharose column chromatography using sepharose bound to fibrin, CM sepharose column chromatography using sepharose bound to carboxymethyl group, lysine sepharose column chromatography using sepharose bound to lysine. , Ligand exchange chromatography using zinc chelate sepharose, lectin column chromatography using sepharose bound to concanavalin A, antibody affinity bound to antibody specifically binding to plasminogen activator used in the present invention Examples include chromatography, DEAE sepharose column chromatography, gel filtration using cross-linked dextran particles.

  An example of a specific method for separating and purifying plasminogen activator used in the present invention is represented by Reference Example 1 of this application in tissue culture solution, tissue extract, or recombinant cell culture solution. An example of performing a purification method is given, and in some cases, a CM sepharose column or an anti-TPA monoclonal antibody column may be further repeated, or purification may be performed on a DEAE sepharose column.

It is also preferable to use Sephacryl S-200. Further, the precipitate formed by adding ammonium sulfate to the above raw material is separated, dissolved in a rhodammonium solution containing sodium chloride, passed through an anti-urokinase Ig-G Sepharose column, further adsorbed on a lysine Sepharose column, and ε-aminocapron An example is a method in which an acid is eluted with an elution solvent and then passed through an anti-urokinase Ig-G Sepharose column.

  The lysine (also referred to as lysine) of the present invention may be either L-form, D-form, or a racemate that is a mixture thereof, but is preferably L-form. The salt of lysine is preferably a water-soluble salt. For example, an acid addition salt thereof is a preferred example. In the case of an acid addition salt, the acid that can be added is usually exemplified by hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, oxalic acid, citric acid, tartaric acid, etc., preferably hydrochloric acid, acetic acid, phosphoric acid, Preferably hydrochloric acid is used. These lysine and its salt can utilize an appropriate hydrate other than an anhydride. Specifically, lysine hydrate can be mentioned as a preferred example.

  Examples of the acidic amino acid of the present invention include glutamic acid and aspartic acid, which may be either L-form or D-form, or a racemate that is a mixture thereof. preferable. The salt of glutamic acid and aspartic acid is preferably a water-soluble salt. For example, acid addition salts thereof are preferred examples. In the case of an acid addition salt, examples of acid that can be added are usually hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, oxalic acid, citric acid, tartaric acid, etc., preferably hydrochloric acid, acetic acid, phosphoric acid, Preferably hydrochloric acid is used.

Moreover, as another salt, an ammonium salt is mentioned as a preferable example. Further, for example, a salt with an alkali metal or an alkaline earth metal is also a preferred example. Examples of the alkali metal include sodium, potassium, lithium and the like, preferably sodium and potassium, and more preferably sodium. A salt with an alkaline earth metal is also a preferred example. Examples of the alkaline earth metal include magnesium, calcium, beryllium and the like, preferably magnesium and calcium, and more preferably calcium. As these acidic amino acids and salts thereof, appropriate hydrates can be used in addition to anhydrides. Specific examples include sodium glutamate monohydrate and sodium aspartate monohydrate.

  In addition, when implementing a combination of lysine or a salt thereof, and an acidic amino acid or a salt thereof, it is also preferable to use ricintamate anhydride, ricintamate dihydrate, or the like as a whole. In addition, the use of lysine aspartic acid or its hydrate is also a preferred example.

  In the TPA-containing preparation of the present invention, saccharides can be added in addition to the above additives. Examples thereof include glucose, mannitol, sorbitol, trehalose, sucrose, maltose and the like used as excipients or stabilizers, tonicity agents and the like. Preferred are mannitol, trehalose and sucrose, and particularly preferred are trehalose and sucrose. Other excipients, excipients, surfactants, tonicity agents, buffering agents, thickeners, preservatives, preservatives, soothing agents, pH adjusters, adsorption inhibitors, acceptable as pharmaceutical additives Etc. may be added as appropriate. Although the kind of said additive is not necessarily clear, as a surfactant, polysorbate 80, polysorbate 20, a poloxamer etc. are mentioned as a preferable example. Further, albumin, gelatin and the like are preferable as the adsorption inhibitor.

In producing the TPA-containing preparation of the present invention, an appropriate method can be adopted. For example, an appropriate amount of TPA and the above-mentioned additive or a solution thereof are mixed simultaneously or sequentially to obtain a mixed solution of all the compositions. It can be prepared by preparing and freeze-drying by a known method.

  For example, the above-mentioned TPA and additives and other component additives may be added directly to the TPA-containing solution, or the additive and other component additives may be added in advance to water, water for injection, or an appropriate solution. Examples of the method include a method of adding to a TPA-containing solution after dissolution in a buffer solution. The timing of addition may be any time, but preferably includes a purification process of TPA or exemplified during the formulation process. It is also preferable to add lysine in the purification process of TPA and then add an acidic amino acid at the end of purification or in the formulation step. In the purification process, lysine and acidic amino acid may optionally be added to lysingtamic acid or It is also preferable to add aspartic acid.

When the TPA-containing preparation of the present invention is expressed in international units (hereinafter also referred to as IU) as the amount of TPA, the preparation usually contains 500,000 IU or more, preferably 1 million IU or more, preferably 1.3 million IU. The above is more preferable, 1.6 million IU or more is particularly preferable, and 2 million IU or more, 3 million IU or more, and 3.6 million IU or more are also preferable examples.

  In preparing a preparation containing 1.6 million IU of TPA, when the amount of lysine or a salt thereof present in the preparation is expressed by the amount of free lysine, the lower limit thereof is preferably, for example, 1 mg or more, more preferably Is 5 mg or more, more preferably 10 mg or more, and in some cases, 20 mg or more, or 30 mg or more. The upper limit is usually 1000 mg or less, preferably 500 mg or less, more preferably 100 mg or less, and in some cases 50 mg or less. Furthermore, when preparing preparations having different TPA contents, it is also preferable to obtain the ratio by calculation with reference to the example of the amount added.

  Further, for example, when preparing a preparation containing 1.6 million IU of TPA, when the amount of acidic amino acid or salt thereof present in the preparation is expressed by the amount of free amino acid, the lower limit is, for example, 1 mg or more. Examples are preferable, more preferably 5 mg or more, still more preferably 10 mg or more, and in some cases 20 mg or more, or 30 mg or more. The upper limit is usually 1000 mg or less, preferably 500 mg or less, more preferably 100 mg or less, and in some cases 50 mg or less. Furthermore, when preparing preparations having different TPA contents, it is also preferable to obtain the ratio by calculation with reference to the example of the amount added.

  Furthermore, the addition ratio of lysine or a salt thereof and an acidic amino acid or a salt thereof is not particularly limited, but the lower limit of the free amino acid is 1 part by weight of the free lysine and 0.01 parts by weight or more. Preferably, it is 0.1 parts by weight or more, more preferably 1 part by weight or more, or 2 parts by weight or more, and particularly preferably 5 parts by weight or more. The upper limit is 100 parts by weight or less, preferably 50 parts by weight or less, and more preferably 10 parts by weight or less.

  The TPA-containing preparation of the present invention preferably includes lysingtamic acid together with TPA. The lower limit of the amount added is, for example, 1 mg or more, preferably 5 mg or more, more preferably 10 mg or more, particularly preferably 20 mg or more or 30 mg or more when preparing a preparation containing TPA 1.6 million IU. Examples of the upper limit include 1000 mg or less, preferably 500 mg or less, more preferably 100 mg or less, and particularly preferably 50 mg or less. Furthermore, when preparing preparations having different TPA contents, it is also preferable to obtain the ratio by calculation with reference to the example of the amount added.

  In addition, in the TPA-containing preparation of the present invention, when a saccharide is added together with TPA, the addition amount is, for example, 1 mg or more, preferably 5 mg or more, as a lower limit when preparing a preparation containing TPA 1.6 million IU. Preferably 10 mg or more, particularly preferably 20 mg or more or 30 mg or more. Examples of the upper limit include 1000 mg or less, preferably 500 mg or less, more preferably 100 mg or less, and particularly preferably 50 mg or less.

  In formulating the TPA-containing preparation of the present invention, the pH of the TPA-containing solution in the process is particularly preferably about neutral. Furthermore, the lower limit of the pH is typically pH 4 or higher, preferably pH 5 or higher, more preferably pH 6 or higher. Further, the upper limit of pH is usually 9 or less, preferably 8 or less, more preferably 7 or less. In order to achieve the above-mentioned pH, an appropriate pH adjuster or the like may be added as necessary. Examples of the pH adjuster include hydrochloric acid, acetic acid, citric acid, phosphoric acid, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, potassium hydroxide and the like, preferably hydrochloric acid, sodium hydroxide and the like. . The adjustment time is not particularly limited, but is preferably just before the sterile filtration step. Moreover, the purification process of TPA may be sufficient.

A solution containing TPA and additives prepared in the formulation step as described above and further containing other appropriate additives as required is preferably aseptically filtered through, for example, a 0.22 μm filter. The solution containing TPA thus obtained may be filled in a container and sealed as it is to prepare a preparation, but lyophilization is more preferable. The freeze-drying method is not particularly limited, and can be freeze-dried by a usual method.

The container is not particularly limited as long as it is a material and shape suitable for aseptic filling that can be used as a container for pharmaceuticals. For example, glass vials (and stoppers), glass syringes (and rubber caps and stoppers) ), Glass ampules and the like, and plastic containers can also be used. Examples of the plastic material include polyvinyl chloride, polyethylene, polypropylene, and cyclic polyolefin. Preferably, cyclic polyolefin is used. In the present invention, it is preferable to provide a freeze-dried preparation enclosed in a glass vial.

Further, for example, a kit preparation in which an aqueous solution for dissolution is combined with or attached to a freeze-dried preparation is preferable. The freeze-dried preparation and the aqueous solution for dissolution are filled in separate containers, for example, freeze-dried. A preferable example is a kit preparation composed of a combination of a glass vial or ampoule filled with a sterile preparation and a prefilled syringe filled with an aqueous solution for dissolution aseptically. In addition, a two-chamber syringe called a glass or plastic double chamber or dual chamber (one syringe has two chambers, for example, one chamber is lyophilized and the other chamber is filled with an aqueous solution for dissolution. Are also preferred as kit formulations. In addition, as a different type of two-chamber type syringe, a type in which two chambers are separated at the time of production and distribution and is dissolved by combining the two chambers at the time of use is preferable.

  When the lyophilized preparation is dissolved upon use, it may be dissolved in distilled water for injection, physiological saline, 5% glucose injection, sugar infusion, electrolyte infusion, amino acid infusion, or the like. Particularly preferable examples include physiological saline and 5% glucose injection. The amount of dissolution is not particularly limited, but is typically 1 mL or more, preferably 5 mL or more, more preferably 10 mL or more, or 20 mL or more per 1.6 million IU of TPA. The upper limit is typically 50 mL or less, preferably 30 mL or less, more preferably 20 mL or less, or 15 mL or less. Furthermore, it is particularly preferred that the amount is usually about 20 mL. A particularly preferred example is usually about 25 mL per 3.6 million IU of TPA.

  In use, the lyophilized preparation may be dissolved and then diluted into an infusion solution, but it is preferably used without dilution. Examples of the infusion include physiological saline, 5% glucose injection, sugar infusion, electrolyte infusion, and amino acid infusion. The amount of infusion is not particularly limited, and examples thereof include 10 mL to 500 mL per 1.6 million IU of TPA.

  Examples of the administration route of TPA include arterial injection, intravenous injection, drip injection and the like. In the case of coronary thrombus dissolution in acute myocardial infarction, it is preferable to inject directly into the coronary artery using a catheter or the like, and the injection time at this time is 20 mL of physiological saline or 5% glucose injection solution per 1.6 million IU It is preferable to inject in about 10 minutes without dilution when dissolved.

  Next, the present invention will be described with reference examples, examples, comparative examples, and experimental examples.

The present invention will be described based on examples.

[Reference Example 1]
The tissue plasminogen activator used in Examples 1 to 8 and Comparative Examples 1 to 5 was obtained by the following method. Human fetal lung cells were implanted in a 12L spinner flask at a density of 10 ⁵ cells / mL with Cytodex I (bead carrier for cell culture, registered by Amersham Biosciences) at 37 ° C, 5% carbon dioxide 8L of medium MEM containing 10% calf serum is added as a growth medium in air containing, and suspension culture is performed with stirring at a rotation speed of 40 rpm. After culturing for 8 days and allowing the cells to grow sufficiently, the bead carrier to which the cells have adhered is washed with physiological saline, and the medium is replaced with medium 198 L containing 0.75% animal peptone without serum and cultured. Every 3rd day, the culture medium containing the plasminogen activator used in the present invention is recovered while changing the medium.

  The culture solution thus obtained is adsorbed on a CM Sepharose column equilibrated with 20 mM acetate buffer (pH 4.0) containing 0.2 M NaCl. After washing, elution is performed by increasing the salt concentration, and a solution containing the plasminogen activator used in the present invention is collected. This solution is adjusted to pH and adsorbed to an anti-TPA monoclonal antibody affinity column equilibrated with 20 mM Tris hydrochloride buffer (pH 7.4) containing 0.5 M NaCl. After washing, the plasminogen activator is eluted by lowering the pH. Then, a solution containing tissue plasminogen activator was obtained.

[Example 1]
The solution obtained in Reference Example 1 was subjected to gel filtration on a column of Sephacryl S-200 equilibrated with 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl and 10 mM lysine hydrochloride, and tissue plasminol A solution (about 470000 IU / mL) containing a gene activator was obtained. Weigh 35.7mL of this solution in a 100mL graduated cylinder, weigh 315mg of glutamic acid Na (manufactured by Ajinomoto Co., Inc.), dissolve in 20mL of water for injection, add water for injection to 63mL, add a stir bar, and do not disturb Was stirred as such. This solution was filtered into a 100 mL beaker using Millipore Sterivex-GV (0.22 μm). Each vial was filled with 6 mL, and a rubber stopper was half stoppered. After freeze-drying using a lyophilizer Triomaster A04, the inside of the vial was filled with nitrogen gas to -50 kPa and sealed to obtain a preparation containing about 1.600000 IU of tissue plasminogen activator.

[Example 2]
A tissue plasminogen activator-containing preparation was obtained in the same manner except that 315 mg of sodium aspartate (manufactured by Ajinomoto Co., Inc.) was used instead of Na glutamate in Example 1.

[Example 3]
A tissue plasminogen activator-containing preparation was obtained in the same manner as in Example 1 except that 315 mg of rimonotamic acid (Ajinomoto Co., Inc.) was used instead of Na glutamate.

[Example 4]
A tissue plasminogen activator-containing preparation was obtained in the same manner as in Example 1, except that 315 mg of rimonotamic acid and 315 mg of D-mannitol (manufactured by Towa Kasei Kogyo Co., Ltd.) were used instead of Na glutamate.

[Example 5]
A tissue plasminogen activator-containing preparation was obtained in the same manner as in Example 1 except that 315 mg of rimonotamic acid and 315 mg of trehalose (Hayashibara Co., Ltd.) were used instead of Na glutamate.

[Example 6]
A tissue plasminogen activator-containing preparation was obtained in the same manner as in Example 1 except that 315 mg of rimonotamic acid and 10.5 mg of polysorbate 80 (manufactured by Wako Pure Chemical Industries, Ltd.) were weighed in place of Na glutamate.

[Example 7]
The solution obtained in Reference Example 1 was subjected to gel filtration on a column of Sephacryl S-200 equilibrated with 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl and 30 mM rimonotaminic acid to obtain tissue plasminogen. An activator-containing solution (about 470000 IU / mL) was obtained. 35.7 mL of this solution was weighed into a 100 mL graduated cylinder, and water for injection was added to make 63 mL. This solution was filtered into a 100 mL beaker using Millipore Sterivex-GV (0.22 μm). Each vial was filled with 6 mL, and a rubber stopper was half stoppered. After freeze-drying using a lyophilizer Triomaster A04, the inside of the vial was filled with nitrogen gas to -50 kPa and sealed to obtain a preparation containing about 1.600000 IU of tissue plasminogen activator.

[Example 8]
The solution obtained in Reference Example 1 was subjected to gel filtration on a column of Sephacryl S-200 equilibrated with 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl and 10 mM lysine hydrochloride, and tissue plasminol A solution (about 470000 IU / mL) containing a gene activator was obtained. In a 200 mL graduated cylinder, 76.6 mL of this solution was weighed, 225 mg of rimonotamic acid was weighed, 30 mL of water for injection was added and dissolved, water for injection was added to 135 mL, a stir bar was added, and the mixture was stirred so as not to be irritated. This solution was filtered into a 200 mL beaker using Millipore Sterivex-GV (0.22 μm). Each vial bottle was filled with 13.5 mL, and a rubber stopper was half stoppered. After freeze-drying using a lyophilizer Triomaster A04, the inside of the vial was filled with nitrogen gas up to -50 kPa and sealed to obtain a preparation containing tissue plasminogen activator of about 3.600000 IU.

[Example 9]
As a genetically modified TPA, “Glutopa Note 6 million” manufactured by Mitsubishi Pharma Corporation was used. Dissolve in the attached water for injection and perform gel filtration on a column of Sephacryl S-200 equilibrated with 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl and 10 mM lysine hydrochloride. A solution containing recombinant tissue plasminogen activator was obtained. In a 100 mL graduated cylinder, 35.7 mL of this solution was weighed, 315 mg of glutamic acid Na was weighed, 20 mL of water for injection was added and dissolved, water for injection was added to 63 mL, a stir bar was added, and the mixture was stirred so as not to be irritated. This solution was filtered into a 100 mL beaker using Millipore Sterivex-GV (0.22 μm). Each vial was filled with 6 mL, and a rubber stopper was half stoppered. After freeze-drying using a lyophilizer Triomaster A04, the inside of the vial was filled with nitrogen gas to -50 kPa and sealed to obtain a preparation containing a recombinant tissue plasminogen activator.

[Comparative Example 1]
A tissue plasminogen activator-containing composition was obtained in the same manner as in Example 1 except that Na glutamate was not used.
[Comparative Example 2]
A tissue plasminogen activator-containing composition was obtained in the same manner except that 315 mg of glycine (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of Na glutamate of Example 1.
[Comparative Example 3]
A tissue plasminogen activator-containing composition was obtained in the same manner except that 315 mg of alanine (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of Na glutamate of Example 1.
[Comparative Example 4]
A tissue plasminogen activator-containing composition was obtained in the same manner except that 315 mg of lysine hydrochloride was used instead of Na glutamate in Example 1.
[Comparative Example 5]
A tissue plasminogen activator-containing composition was obtained in the same manner except that 315 mg of arginine hydrochloride (Wako Pure Chemical Industries, Ltd.) was used instead of Na glutamate in Example 1.

[Experiment 1]
The preparation containing 1.6 million IU of TPA obtained in Examples 1 to 7, the preparation containing 3.6 million IU of TPA obtained in Example 8, and the composition obtained in Comparative Examples 1 to 5 were stored at 60 ° C. for 4 weeks. The residual titer rate (%) from the start was determined, and the storage stability was examined. In addition, the titer measurement was performed according to the method “USP26, P.71-72” described in Assay for biological potency of Alteplas. The results are shown in Table 1. In addition, the quantity of the additive of Example 8 was described as a conversion quantity with respect to 1.6 million IU of TPA.

[Experiment 2]
In the same manner as in Experimental Example 1, the residual titer (%) from the start after storage at 40 ° C. for 3 months was determined. The results are shown in Table 2.

  From the results of Experimental Examples 1 and 2, either (1) lysine or a salt thereof and (2) an acidic amino acid or a salt thereof (1) (2) together with a tissue plasminogen activator, or (3) It has been found that the tissue plasminogen activator-containing preparation characterized by containing lysingtamic acid and / or lysine aspartic acid retains its activity under severe storage at 60 ° C or 40 ° C. In addition, the formulation obtained in Example 9 was also subjected to the titer residual rate according to Experimental Examples 1 and 2, and the same results as above were obtained. Therefore, it is estimated that the plasminogen activator-containing preparation of the present invention retains the activity for a period of 2 to 3 years or more under room temperature storage conditions around 25 ° C. Further, from the results of the TPA compositions obtained in the comparative examples, it is clear that the addition of neutral amino acids or basic amino acids has no or very little effect on storage stabilization.

The tissue plasminogen activator-containing preparation of the present invention is useful as a thrombolytic agent,
It is possible to provide a preparation that can be stored for a long period of time.

Claims (7)

(1) lysine or a salt thereof, and (2) both (1) and (2) in an acidic amino acid or a salt thereof together with tissue plasminogen activator, or (3) lysingtamic acid and / or lysine A tissue plasminogen activator-containing preparation characterized by containing aspartic acid. The tissue plasminogen activator-containing preparation according to claim 1, wherein the tissue plasminogen activator is derived from a tissue culture solution of human normal lung cells. The tissue plasminogen activator-containing preparation according to claim 1 or 2, wherein the acidic amino acid or a salt thereof is glutamic acid or a salt thereof. A tissue plasminogen activator-containing preparation comprising a tissue plasminogen activator, comprising (1) lysine or a salt thereof, and (3) lysingtamic acid. The tissue plasminogen activator-containing preparation according to any one of claims 1 to 4, which is a freeze-dried preparation. The tissue plasminogen activator-containing preparation according to any one of claims 1 to 5, further comprising a saccharide. (1) lysine or a salt thereof, and (2) both (1) and (2) in an acidic amino acid or a salt thereof together with tissue plasminogen activator, or (3) lysingtamic acid and / or lysine A method of stabilizing a tissue plasminogen activator characterized by containing aspartic acid.