JPH0669960B2 - Pharmaceutical composition containing tPA with increased water solubility - Google Patents

Description

TECHNICAL FIELD The present invention relates to tissue plasminogen activator [tissue P
lasminogen Activator (hereinafter referred to as tPA)]-containing pharmaceutical composition. More specifically, the present invention is characterized by incorporating a partial gelatin hydrolyzate (hereinafter referred to as a gelatin hydrolyzate) cross-linked with tPA to tPA and one or more basic amino acids or salts thereof. And a pharmaceutical composition containing tPA having increased water solubility.

Conventional technology and its problems tPA acts on plasminogen in vivo to generate plasmin, and this plasmin destroys and dissolves the fibrin network of the thrombus. It is known to be a useful substance for treatment.

However, since tPA is a protein that is extremely sparingly soluble in water,
When tPA is dissolved in water and administered, for example, as an injection, it is necessary to add an appropriate solubilizer to make it soluble in water. Conventionally, as a means for solubilizing such a poorly soluble drug,
Although surfactants and the like have been used, using a large amount of these is problematic in terms of safety.

Therefore, it is an object of the present invention to maintain safety and to improve the solubility of water in a tPA that is satisfactory for use in medicine.
It is to provide a pharmaceutical composition containing the same.

Means for Solving Problems The present inventors have found that a gelatin decomposition product enhances the solubility of tPA in water, and have applied for a patent in advance (Japanese Patent Application No. 60-198,629). As a result of further studies, the present inventors have found that the solubility of tPA in water is compensated by adding tPA to one or more basic amino acids or salts thereof in addition to a gelatin degradation product. To 1
The inventors have found that the number is increasing and completed the present invention.

In the present invention, tPA may be obtained by extracting from animal tissue, or may be obtained from a culture of microorganisms or animal cells artificially created by gene recombination technology. The present invention is not limited by its source.

The gelatin decomposition product used in the present invention is described in US Pat.
It can be manufactured according to the method described in the specification of 3,057,782. It is preferable to use a gelatin decomposition product having an average molecular weight of about 35,000 cross-linked with hexamethylene diisocyanate.

Examples of the basic amino acid used in the present invention include arginine, ornithine, lysine, and histidine, and the salts of these basic amino acids are
It may be a salt with an inorganic acid such as a hydrochloride, or a salt with an organic acid such as an acetate, an aspartate or a glutamate.

In the present invention, the blending amount of the gelatin decomposition product and the basic amino acid or a salt thereof depends on the target dissolution amount of tPA in water and can be appropriately selected. Degradation products of gelatin increase the viscosity in aqueous solution as the concentration in water increases, so the concentration in aqueous solution is generally 10% or less, preferably 5% or less.
It is desirable to add an amount such that the amount is not more than%. On the other hand,
Basic amino acids and their salts affect the osmotic pressure of aqueous solutions and become hypertonic as their concentration increases. Therefore, from the viewpoint of administration to living organisms, the amount that produces a concentration of 1 to 5% in aqueous solutions should be selected. Incorporation is generally preferred, but higher or lower amounts can be used, if desired.

The composition of the present invention may optionally contain auxiliary components usually used for formulation, such as a bulking agent, a stabilizer, a buffering agent, an isotonicity agent and the like.

The pharmaceutical composition of the present invention is not only a solid or aqueous composition containing tPA, a gelatin degradation product, and one or more basic amino acids or salts thereof, but also, for example, an injection prepared immediately before use. A lyophilized vial containing tPA and a gelatin decomposition product and a basic amino acid or a salt thereof such as an ampoule for dissolution containing tPA and a gelatin decomposition product and / or a basic amino acid or a salt thereof are separately packaged. Also included are compositions in the form provided.

The combination of tPA with basic amino acids or their salts is tPA.
The effect of increasing the water solubility of was confirmed by the following experiments.

Take 1,000 U each of tPA in a small test tube and add 50 μl of an aqueous solution containing gelatin degradation product (cross-linked with hexamethylene diisocyanate; average molecular weight 35,000) and / or basic amino acid hydrochloride, or water (control). After mixing well, centrifuge and collect a fixed amount of
Dilute with 0.1M Tris-HCl buffer (pH 8) containing A, and use the fibrin plate method [T.Astrup:
Archives of Biochemistry-and-bio-physics (Arch. Biochem. Biophys.) , 40, 346
(1952)], the tPA activity was measured. The results obtained are shown in the following table.

From the above table, the combined use of the gelatin decomposition product and the basic amino acid or the salt thereof according to the present invention is remarkable as compared with the additive-free control (water) or the gelatin decomposition product and the basic amino acid or the salt thereof alone. It is clear that it has a tPA solubility improving effect.

The present invention will be described more specifically by the following examples.
The gelatin hydrolyzate used in the examples is a gelatin partial hydrolyzate (average molecular weight 35,000) cross-linked with hexamethylene diisocyanate.

Example 1 An aqueous solution containing 2 g of L-arginine hydrochloride, 5 g of gelatin hydrolyzate and 10 million units of tPA was aseptically prepared in 100 ml of 0.03 M phosphate buffer having a pH of 7.5, and dispensed in 1 ml aliquots. Then freeze-dry and seal.

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

This injection is made into a homogeneous aqueous solution containing 100,000 units of tPA by adding the content of the ampoule for dissolution to a vial and mixing them. The concentration of gelatin decomposed product in this aqueous solution is 2.5
% (W / V) and the concentration of arginine hydrochloride is 1% (W / V).

Example 2 An aqueous solution containing 1.6 g of L-ornithine hydrochloride, 5 g of gelatin hydrolyzate and 10 million units of tPA was aseptically prepared in 100 ml of 0.03 M phosphate buffer having a pH of 7.5, and divided into 1 ml vials. Pour, freeze-dry and seal.

Separately, make an ampoule containing 2 ml of distilled water for injection and use it as a dissolving ampoule.

Example 3 An aqueous solution containing 2 g of L-histidine hydrochloride, 5 g of gelatin hydrolyzate and 10 million units of tPA was aseptically prepared in 100 ml of 0.03 M phosphate buffer having a pH of 7.5, and dispensed into vials in 1 ml portions. Then freeze-dry and seal.

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

Example 4 L-Lysine hydrochloride in 100 ml of 0.03M phosphate buffer pH 7.5
Aseptically prepare an aqueous solution containing 1.6 g, 5 g of gelatin hydrolyzate, and 10 million units of tPA, dispense 1 ml aliquots, freeze-dry, and seal.

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

Example 5 In 100 ml of 0.03M phosphate buffer of pH 7.5, L-arginine
Prepare an aqueous solution containing 3 g of aspartate, 5 g of gelatin hydrolyzate, and 10 million units of tPA aseptically, dispense 1 ml aliquots, freeze-dry and seal.

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

Example 6 An aqueous solution containing 2.6 g of L-lysine / aspartate, 5 g of gelatin hydrolyzate, and 10 million units of tPA was aseptically prepared in 100 ml of 0.03 M phosphate buffer having a pH of 7.5, and 1 ml vials were used. Dispense, freeze-dry and seal.

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

Example 7 Aseptically an aqueous solution containing 0.8 g of L-arginine hydrochloride, 1.2 g of L-histidine hydrochloride, 5 g of gelatin hydrolyzate and 10 million units of tPA in 100 ml of 0.03 M phosphate buffer having a pH of 7.5. Prepared, 1 ml
Dispense each into vials, freeze-dry and seal.

Separately, make an ampoule containing 2 ml of distilled water for injection and use it as a dissolving ampoule.

Example 8 L-Arginine (1.8 g) and gelatin hydrolyzate (5 g) were dissolved in 70 ml of distilled water for injection, the pH was adjusted to 7.0 to 8.0 with hydrochloric acid, and tPA was added.
Add 10 million units, add distilled water for injection after dissolution, bring the total amount to 1
Set to 00 ml. This solution is sterilized, dispensed in 1 ml aliquots, lyophilized and sealed.

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

Example 9 10 million units of aseptically prepared tPA powder and 4 g of sterile mannitol are uniformly mixed, filled so that tPA is 100,000 units per vial, and sealed.

Separately, prepare a solution containing 4 g of L-arginine hydrochloride and 10 g of gelatin hydrolyzate in 200 ml of 0.01M phosphate buffer (pH 7.5) aseptically, and dispense 2 ml aliquots into ampoules for dissolution. To do.

 ─────────────────────────────────────────────────── --Continued from the front page (56) Reference JP-A-58-224687 (JP, A) JP-A-60-184026 (JP, A) JP-A-60-181028 (JP, A) JP-A 61- 236730 (JP, A) JP 62-59221 (JP, A) JP 62-81326 (JP, A)

Claims (1)

[Claims] 1. A tissue plasminogen activator (tPA)
A pharmaceutical composition containing tissue plasminogen activator, which comprises a partially hydrolyzed gelatin partially cross-linked with diisocyanate and one or more basic amino acids or salts thereof.