JPS6259221A - Composition containing tissue plasminogen activating factor - Google Patents

Abstract

PURPOSE:The titled composition consisting essentially of a partial hydrolyzate of gelatin linked to a diisocyanate in the form of an aqueous solution or useful therefor. CONSTITUTION:A composition, containing a tissue plasminogen activating factor (tPA), i.e. a substitute blood plasma component without antigenicity obtained by partially hydrolyzing gelatin to give as a low-molecular polypeptide and linking the resultant polypeptide to a diisocyanate and having 35,000 average molecular weight as an essential component in the form of an aqueous solution or useful therefor. If the concentration of the tPA is high, the viscosity of the aqueous solution is increased to make the handling difficult. The tPA is incorporated to give preferably <=10%, more preferably <=5% concentration when used as an aqueous solution. A saccharide or amino acid may be freely incorporated to give a freeze-dried pharmaceutical.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to a tissue plasminogen activator-containing composition. That is, the present invention is an invention that can be used in the medical field, and more specifically, the present invention is an invention that can be used in the medical field, and more specifically, it is an invention that can be used in the medical field to utilize tissue plasminogen activator, which is used for the purpose of treating various diseases of the circulatory system caused by the formation of blood clots. Relates to a composition invented to make it more accessible in the field.

(2) Prior Art and Problems Tissue plasminogen activator, ie, tissue plasminogen activator, is generally abbreviated as tPA, and this abbreviation will be used in the following description.

It is well known that for the purpose of eliminating disorders of the circulatory system caused by the formation of blood clots, a group of substances called plasminogen activators are used to dissolve blood clots by the fibrinolytic system.

That is, active plasmin is generated by plasminogen activator, and the fibators of blood clots are streptokinase and urokinase. However, these drugs are not specific for blood clots and destroy not only fibrin but also blood proteins such as fibrinogen, prothrombin, factor 5, and factor 8, so they may cause the side effect of internal bleeding. For this reason, streptokinase and urokinase are administered by directly injecting them into the thrombus through a catheter, which is a complicated operation and is gradually becoming obsolete. However, on the other hand, there are plasminogen activators extracted from normal and cancerous tissues, which, depending on the extraction source, are urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) according to the present invention. )are categorized.

The tPA group has a much higher affinity for fibrin than the uPA group (and is therefore known to be useful for therapeutic purposes. Therefore, the preparations necessary to maximize the above therapeutic effects of tPA) The provision of technology has come to be desired.

However, since tPA is a protein that is extremely poorly soluble in water, it is extremely difficult to dissolve tPA in water and prepare it for administration, such as an injection. This is the biggest problem in putting tPA into medical practice. Since tPA is a drug that must be administered directly to the circulatory system, it cannot be put into practical use unless sufficient water solubility is achieved. However, the reality is that no satisfactory solution technology has yet been provided regarding the water solubilization of tPA. As a conventional technology regarding tPA preparations, Japanese Patent Application Laid-Open No. 59-196824
Although the invention described in the publication has been disclosed, a technology that solves the problem of water solubilization of tPA has not yet been achieved.

(3) Means for Solving the Problems In view of the above-mentioned problems, the present inventor conducted various studies on techniques for improving the solubility of tPA in water. As a result, by adding gelatin partial hydrolyzate combined with diisocyanate, tPA
The present invention was completed based on the knowledge that the solubility of the compound in water increases dramatically. That is, the purpose of the present invention is to
The objective is to improve the solubility of A in water, and to achieve this objective, the present invention discloses a technique of adding a partially hydrolyzed gelatin combined with a diisocyanate.

The present invention will be explained in detail below.

In the present invention, tPA may be obtained by extraction from natural products, or may be obtained from cells or cells artificially produced by genetic recombination technology. That is, it is commonly used as a human tissue plasminogen activator.

The present invention is not limited by the source of the product as long as it is available.

More specifically, the diisocyanate-bonded gelatin partial hydrolyzate according to the present invention is a non-antigenic plasma substitute component made by partially hydrolyzing gelatin to produce a low-molecular-weight polypeptide and bonding this polypeptide with diisocyanate.
Usually, those having an average molecular weight of 35,000 are used. Those commercially available from Hoechst Co. under the registered trademarks of Haθmaccθl and Hemacel may be used. For details of this substance, reference is made to the description in German Patent No. 1 118 792 and German Patent No. 1 155 134. As shown by the experimental examples described below, the solubility of tPA increases with the concentration of the substance, so there is no particular limitation on the concentrations of the substance and tPA in the composition of the present invention. However, when this substance becomes highly concentrated, the viscosity of the aqueous solution increases, making it difficult to handle. % or less in the composition of the present invention.

Other ingredients in the composition of the invention 9 such as fillers, stabilizers,
Buffers 9. Components such as tonicity agents may be incorporated freely as long as they do not significantly impair the purpose of the present invention, and the present invention is not limited by these. For example, in order to make the composition of the present invention into a lyophilized preparation, saccharides and amino acids may be freely added.

The main form of the composition of the present invention is an aqueous liquid.

Prepared in advance to form an aqueous solution. That is, forms for aqueous liquids are also included.

The following 1) to IV) are shown with specific examples of composition forms.
can be mentioned.

1) Gelatin partial hydrolyzate combined with tPA and diisocyanate in the same aqueous solution (hereinafter referred to as "partial hydrolyzate").

11) tPA and the additive according to the present invention are prepared as separate aqueous liquids, but 2) both are given in combination so that they are combined at the time of use. tPA powder (e.g. lyophilized powder); 9 The additive according to the present invention may be prepared as an aqueous liquid; 1) both may be combined and mixed together at the time of use; tPA powder (e.g. lyophilized powder); ) and a powder (eg lyophilized powder) of the additive according to the invention are prepared as one unit. A suitable aqueous solution is prepared for dissolving this at the time of use, and it is free to provide further combinations.

The preparation of the composition according to the invention depends on the desired composition form.

It may be carried out according to the usual manufacturing method for each. -An example is as follows. A tPA aqueous solution is dispensed into vials and lyophilized to prepare a vial of tPA-containing powder. Separately, an aqueous solution of the additive according to the present invention is dispensed into a vial and prepared.

The composition of the present invention is made by combining both.

(4) Effect The effect of the present invention is to increase the solubility of tPA in water, thereby providing a highly concentrated aqueous solution of tPA. Therefore, as a specific example, a high-potency tPA-containing injection is provided. According to the experimental example described later.

The solubility of tPA in water, expressed in titer units (u), was 2144 u/ml when gelatin partial hydrolyzate combined with diisocyanate was not added, but when this material was added, it was 1% of that. 34042 u 1m
l, 2.5% at 59845 u 1ml, 5%
106923u/ml at 10%, 138038t at 10%
It was 1/ml. In other words, a dramatic increase in solubility is observed.

(5) Examples The present invention will be explained in more detail by the examples described below.

Example 1 pH 7.5 (7) 0.03 M phosphate buffer 100 m
An aqueous solution containing 10 g of gelatin partial hydrolyzate bound with diisocyanate, 0 & 10,000 units of Mayutolumi, and 10 million units of tPAI in 1. Dispense ml into vials, freeze-dry, and seal.

Separately, prepare an ampoule containing 2 ml of distilled water for injection and use it as an ampoule for dissolution.

Example 2 10 million units of aseptically prepared tPA powder and 10 g of sterile mannitol are uniformly mixed, filled and sealed so that 100,000 units of tPA per vial.

Separately, 0.01M phosphate buffer (pH 7,5) 20
A solution containing 10 g of partially hydrolyzed gelatin bound with diisocyanate in 0 ml is prepared aseptically and dispensed into ampoules in 2 ml portions to prepare ampoules for dissolution.

Example 3 100 ml 0.03M phosphate buffer Partial hydrolyzate of gelatin combined with 19-diisocyanate 10. !
i': An aqueous solution containing 4 g of glycine is prepared aseptically, freeze-dried, and then powdered. Add 10,000,000 units of tPA powder to this powder, mix uniformly, fill and seal so that 100,000 units of tPA per vial.

Separately, prepare an ampoule containing 2 mg of distilled water for injection and use it as an ampoule for dissolution.

Effect of the invention The present invention will be explained in detail by the following experimental examples.

Experimental Example 1 Take 1000 u4 of Ri'tPA in a small test tube and add 55 μl of water to it.
0 μl or 5% aqueous solution 5 of the following additives (1) to (6)
Add 0 μl, mix well, centrifuge, take a certain amount of supernatant, and add 0.1 μl. M) Lis-HCl buffer (pI (8, BSA
The activity of tPA was measured using a fibrin plate.

(1) Hydroxyethyl starch (abbreviated as HES)
(2) Low-substituted hydroxyethyl starch (abbreviated as Hespander) (3) Ethylene oxide propylene oxide copolymer (abbreviated as Pluronic) (4) Pullulan (5) Dextran (6) Partially hydrolyzed gelatin combined with diisocyanate (Hemacel) ) The results are shown in Figure 1. FIG. 1 is a bar graph showing the solubility of tPA in water or in a 5% aqueous solution of each additive in u/1 frl.

It is clear from FIG. 1 that the partially hydrolyzed gelatin combined with diisocyanate has the effect of significantly increasing the solubility of tPA in water.

Experimental Example 2 In Experimental Example 1, 1% Hemacel aqueous solution, 2.5% Hemacel aqueous solution, 596 Hemacel aqueous solution, and 10% Hemacel aqueous solution were used to replace the 5% aqueous solution of additives (1) to (6). The procedure was carried out in the same manner as described in Experimental Example 1 except that the relationship between the concentration of the hemacel aqueous solution and the solubility of tPA in water was determined.

Results The results are shown in Table 1. When Table 1 is illustrated, it becomes as shown in Figure 2.

FIG. 2 is a graph showing the solubility of tPA in u/ml in Hemacell aqueous solutions at various concentrations.

It is clear from FIG. 2 that the solubility of tPA in water increases as the concentration of gelatin partial hydrolyzate combined with diisocyanate increases.

Table 1

[Brief explanation of the drawing]

Figure 1 shows t in water or in a 5% aqueous solution of each additive.
It is a bar graph showing the solubility of PA. FIG. 2 is a graph showing the relationship between the concentration of Hemacel aqueous solution and the solubility of tPA in Hemacel aqueous solution.

Claims (2)

[Claims] (1) An aqueous liquid or a tissue plasminogen activator-containing composition for an aqueous liquid, which contains a partially hydrolyzed gelatin combined with a diisocyanate as an essential component. (2) The tissue plasminogen activator-containing composition according to claim 1, wherein the gelatin partial hydrolyzate combined with diisocyanate has an average molecular weight of 35,000.