JP2001081040A - Tissue plasminogen activator-containing composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition capable of stabilizing a naturai tissue plasminogen activator (t-PA), etc., and prolonging the half-life period of the natural t-PA in blood, and useful for a thrombolytic agent by including the natural t-PA, etc., and a specific saccharides. SOLUTION: This composition comprises (A) the natural t-PA or (B) a modified t-PA and (C) mannose-based saccharides. The content of the ingredient A or B is preferably 0.005-1 wt.%, especially preferably 0. 1-0.3 wt.%. The content of the ingredient C is preferably 0.05-50 wt.%, especially preferably 1-20 wt.%. The ingredient A may be not only a human natural t-PA but also various kinds of t-PAs such as glycosylated t-PA, nonglycosylated t-PA, etc., obtained by a genetic engineering technique. The ingredient B is the t-PA obtained by improving the natural t-PA through a point mutation method, etc., or modified for enhancing the biological activity of the natural t-PA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a natural tissue plasminogen activator (hereinafter referred to as natural t-PA) having excellent stability or a modified form thereof (hereinafter referred to as modified t-P).
A)).

[0002]

2. Description of the Related Art Native t-PA has a high affinity for fibrin, activates plasminogen bound to fibrin, and dissolves thrombus very efficiently. Further, in thrombolytic therapy for thrombosis causing myocardial infarction or cerebral infarction, a modified t-PA formulation has been developed as a more thrombolytic thrombolytic agent.

It is known that natural t-PA has a sugar chain recognized in the blood by a mannose receptor, mainly a mannose receptor in the liver, and is rapidly removed. The half-life of natural t-PA in blood is known. There is a demand for the development of a formulation that improves the drug.

[0004] As one means for compensating for the drawbacks of natural t-PA, a modified t-PA that has been modified using genetic engineering techniques and has an extended half-life in blood has been developed. However, natural t-PA is a protein that is extremely poorly soluble in water, and in particular, modified t-PA is more sparingly soluble than natural t-PA, and is administered at the time of preparation or dissolved in water. This makes it extremely difficult to formulate preparations such as injections.

[0005] Glycoproteins are generally unstable to heat, and natural t-PA and modified t-PA are no exception. Stabilization technology is indispensable for developing such substances as pharmaceuticals.

[0006] As a method for solubilizing natural or modified t-PA, arginine hydrochloride or a salt thereof is added to dissolve and stabilize t-PA in neutral to weak alkaline conditions, and the solution is adjusted to pH 3 or higher. There is known a method of dissolving by adjusting to ~ 5. However, in the method of dissolving in neutral to weakly alkaline conditions, the protein structure becomes unstable, and in the method of dissolving the solution by adjusting the liquid property to pH 3 to 5, the protein is rapidly decomposed in the lyophilized state and the solution state. ,
There is a problem of deactivation.

On the other hand, as a method for stabilizing natural or modified t-PA, a method of adding albumin, a method of adding a decomposed product of albumin, and a method of adding acid-treated gelatin are known. However, in these methods, t-
There is a problem in the solubility of PA and the effectiveness when the concentration of t-PA is high.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to improve the short blood half-life of native t-PA and to improve the solubility of native t-PA and modified t-PA.
-It is an object of the present invention to provide a PA composition which can be made into a solution having a concentration that can be used as a medicament, and a stabilized composition of these substances.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above-mentioned object, and as a result, the mannose-based saccharides have remarkably improved the solubility and stability of natural t-PA and modified t-PA. At the same time, it was found that the biological activity was increased in native t-PA, and the present invention was completed. That is, the gist of the present invention is a t-PA-containing composition comprising natural t-PA or modified t-PA and a mannose-based saccharide.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The natural t-PA used in the present invention includes not only human-derived natural t-PA but also various t-PAs such as glycosylated and non-glycosylated t-PA obtained by genetic engineering techniques. Can be used. The modified t-PA used in the present invention is obtained by improving the natural t-PA by a point mutation method or the like, or
It is t-PA that has been modified to enhance the biological activity or the like of PA, and any modified t-PA may be used. For example, the following modified t-PA can be mentioned. (1) The k1 region of t-PA is missing, and Arg at position 275 is G
(2) a modified t-PA in which the F region and the G region of t-PA are deleted, Gly at position 183, and Ser at position 186 are substituted with Ser and Thr, respectively.
(3) The F- and G-regions of t-PA are partially deleted and
Modified t-PA in which Asn at position 17 has been substituted with Glu, (4)
Modified t-PA in which Val at position 245 of t-PA is substituted with Met
PA and (5) a modified t-PA in which Cys at position 84 of t-PA is substituted with Ser.

[0011] The mannose-based saccharide used in the present invention is D-mannose, wherein 2 to 10 D-mannose are α-mannose.
Alternatively, β-linked oligosaccharides and the like can be mentioned, and α-bonds include α-1,1, α-1,2, α-1,3, α-1,
4, α-1,6 and β bonds are β-1,1, β-1,
2, β-1,3, β-1,4, β-1,6.

The composition of the present invention comprises natural t-PA or modified t-PA and a mannose-based saccharide, if necessary, and an additive, and this mixture is dissolved in water (purified water). Liquid preparations or preparations for dissolving at the time of use, which are further dried by a drying means such as freeze-drying, may be mentioned.

The amount of natural t-PA or modified t-PA in the composition of the present invention is 0.005 to 1% by weight, preferably 0.01 to 0.5% by weight, more preferably ,
The content may be 0.1 to 0.3% by weight.

The amount of the mannose saccharide is as follows:
The appropriate amount is 0.05 to 50% by weight, preferably 0.5 to 30% by weight, and more preferably 1 to 20% by weight in practical use.

[0015] Additives added as necessary include amines such as monoethanolamine, diethanolamine, triethanolamine and ethylenediamine and salts thereof, amino acids such as arginine, and lower alkyl esters of amino acids such as glycine ethyl ester. Agent, sucrose, trehalose, sorbit, xylit,
Non-reducing sugars such as mannitol, stabilizers such as gelatin,
Tris-HCl buffer, phosphate, carbonate, borate, citrate,
Buffering agents such as barbituric acid and amino acids, tonicity agents composed of electrolytes of inorganic salts such as sodium chloride, potassium chloride, sodium monohydrogen phosphate, sodium dihydrogen phosphate, polyoxygen; Polyoxyethylene sorbitan fatty acid esters such as ethylene sorbitan monostearate and polyoxyethylene sorbitan monopalmitate; sorbitan fatty acid esters such as sorbitan monostearate and sorbitan sesquiolate; polyoxyethylene hydrogenated castor oil; polyoxyethylene polyoxypropylene condensation Surfactants such as materials,
Examples include excipients such as cellulose, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxymethylcellulose.

These additives are appropriately selected, and two or more of the same additives may be contained. When the composition of the present invention is used as a ready-to-use formulation, a solubilizing agent or the like can be added to the solution, or the solution can be used as a buffer. When the composition of the present invention is used as an intravenous injection, it is necessary to adjust the osmotic pressure ratio to a range where the composition can be used as an intravenous injection.

[0017]

Next, the present invention will be described in detail with reference to examples. In Examples, natural t-PA derived from recombinant CHO cells ("Glutopa" manufactured by Tanabe Seiyaku) was used. Also,
The modified t-PA was obtained by deleting the F region, the G region, and the K1 region and replacing the Arg at position 275 with Glu.
ogy Progress, 10 (5): 472-479 (1994).

Example 1 0.9 containing modified t-PA at a concentration of 0.5 mg / ml
Wt% sodium chloride and 0.01 wt% Tween 80
A 0.1 M phosphate buffer solution (pH 7.2) containing
10% by weight of D-mannose (manufactured by Ishizu Pharmaceutical Co.) in the solution
And then add 4 to a glass vial.
The solution was dispensed in ml and a freeze-dried product was prepared using a freeze-dryer. In addition, Tween 80 is a product name of polyoxyethylene higher fatty acid ester manufactured by Kao Atlas Co., Ltd.

Example 2 According to the method of Example 1, β-1,4 was prepared according to the formulation shown in Table 1.
A freeze-dried modified t-PA of a ready-to-use type containing mannobiose (manufactured by Ishizu Pharmaceutical Co., Ltd.) was produced.

[0020]

[Table 1]

Example 3 According to the method of Example 1, a natural t-type of a ready-to-use type containing D-mannose (manufactured by Ishizu Pharmaceutical Co., Ltd.) in the formulation shown in Table 2
-A freeze-dried product of PA was produced.

[0022]

[Table 2]

Example 4 According to the method of Example 1, β-1,4 was prepared according to the formulation shown in Table 3.
A freeze-dried product of natural t-PA that was dissolved in use and contained mannobiose (manufactured by Ishizu Pharmaceutical Co., Ltd.) was prepared.

[0024]

[Table 3]

Comparative Examples 1-4 According to the method of Example 1, freeze-dried t-PA of the ready-to-use type was prepared according to the formulations shown in Tables 4-7.

[0026]

[Table 4]

[0027]

[Table 5]

[0028]

[Table 6]

[0029]

[Table 7]

Reference Example 1 The freeze-dried products produced in Example 1, Example 2, and Comparative Examples 1 to 3 were left under various temperature conditions, and the appearance and the dissolution were observed. In addition, the residual titer was determined by Clot L
ysis) method to determine stability. Tables 8 to 1 show the results.
It is shown in FIG. Note that Initial in the table indicates the initial condition.

[0031]

[Table 8]

[0032]

[Table 9]

[0033]

[Table 10]

[0034]

[Table 11]

[0035]

[Table 12]

As is apparent from Tables 8 to 12, the modified t
-By adding D-mannose or β-1,4 mannobiose to PA and freeze-drying, it is possible to produce a preparation having higher stability than when glycine, maltose or lactose is used as a stabilizer. did it.

Reference Example 2 A freeze-dried product of natural t-PA was weighed, and was weighed at 0.01% by weight.
A 50 mM phosphate buffer at pH 7.0 containing Tween 80 was added and mixed well. This was centrifuged, and the supernatant was collected to measure the protein concentration. As a result, the concentration of native t-PA was 0.066 mg / ml. Further, while maintaining the pH at 7.0, the concentration of phosphoric acid was 500 m
M, the concentration of natural t-PA is 0.267.
The solubility was as low as mg / ml. Next, D-mannose (manufactured by Ishizu Pharmaceutical Co., Ltd.), β-1,4 mannobiose (manufactured by Ishizu Pharmaceutical Co., Ltd.), and β-1,4 mannotriose (manufactured by Ishizu Pharmaceutical Co., Ltd.) were used, each containing 0.01 wt% Tween 80 Using 50 mM phosphate buffer at pH 7.0 containing natural t-
A similar experiment was performed for PA.

FIG. 1 shows the results. FIG. 1 is a graph showing the relationship between the concentration of mannose-based saccharides and the solubility of natural t-PA. From FIG. 1, it can be seen that natural t-P is added to the mannose saccharide.
It can be seen that there is an effect of increasing the solubility of A.

When the same experiment was carried out for the modified t-PA, the solubility in 50 mM phosphate buffer at pH 7.0 was 0.234 mg / ml, but it contained 10% by weight of mannose. 6.0 mg / ml in 50 mM phosphate buffer, 7.0 mg / m in 50 mM phosphate buffer containing 10% by weight β-1,4 mannobiose.
5, containing 10% by weight of β-1,4 mannotriose
It is 7.0 mg / ml or more in 0 mM phosphate buffer, indicating that the mannose-based saccharide also increases the solubility of the modified t-PA.

Reference Example 3 The lyophilized preparation of natural t-PA produced in Examples 3 and 4 and Comparative Example 4 was dissolved in distilled water for injection and added to rats.
The administration was performed by rapid intravenous injection so as to be μg / kg. After administration, the concentration of t-PA in rat plasma was measured over time.

FIG. 2 shows the results. FIG. 2 is a diagram showing the time-dependent change in the t-PA concentration in plasma. 2. From FIG. 2, it can be seen that the t-PA preparation containing mannose or β-1,4 mannobiose has a longer half-life in blood than the t-PA preparation containing lactose, and exhibits excellent drug efficacy at the same dose. It became clear to do.

[0042]

EFFECT OF THE INVENTION In the composition of the present invention, natural t-P
A or modified t-PA is significantly stabilized,
It is possible to prolong the blood half-life of native t-PA. Therefore, the present invention is useful as providing a clinically usable preparation of natural t-PA or modified t-PA.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of the concentration of a mannose-based saccharide on the solubility of modified t-PA.

FIG. 2 is a graph showing a time-dependent change in the concentration of native t-PA in plasma in rats to which the t-PA preparation of the present invention was administered.

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Claims (1)

[Claims] 1. A tissue plasminogen activator-containing composition comprising a natural tissue plasminogen activator or a modified tissue plasminogen activator and a mannose-based saccharide.