JPS62123130A - Tpa-containing drug composition having increased water solubility - Google Patents

Abstract

PURPOSE:The titled composition obtained by blending a tissue Plasminogen Activator (tPA for shot) with a specific gelatin partial hydrolyzate and a basic amino acid, etc. CONSTITUTION:A tPA (one obtained by extraction from animal tissue, a microorganism artificially prepared by gene recombination technology or culture mixture of animal cell) is blended with <=10wt% gelatin hydrolyzate crosslinked with a diisocyanate and 1-5wt% one or more basic amino acids or their salts (e.g., arginine, ornithine, lysine, histidine, etc.,) to give a tPA-containing drug composition. Since tPA is extremely slightly soluble protein, addition of a solubilizer such as surface active agent, etc., is required for production of injection, etc., from tPA, however, the composition has water solubility increased in such a degree as to satisfy medical use and keeps safety.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a tissue plasminogen activator (tissue plasminogen activator).
ue Plasminogen Activator
(hereinafter referred to as tPA)]. More specifically, 9 the present invention comprises gelatin partially hydrolyzed U, tPA crossed with diisocyanate, and koji (malt).
It is characterized by containing εJ (shown as gelatin decomposition product) and iff or more basic amino acids or salts thereof. The present invention relates to a pharmaceutical composition containing tPA with 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 blood clots. Therefore, tPA is effective against diseases of the circulatory system caused by the formation of blood clots. It is known to be a therapeutically useful substance.

However, since tPA is a protein that is extremely poorly soluble in water, preparations in which tPA is dissolved in water and administered.

For example, when preparing an injection, it is necessary to add an appropriate solubilizer to make it soluble in water. Conventionally, surfactants and the like have been used as means for solubilizing such poorly soluble drugs, but the use of these agents in multiple bases poses a safety problem.

Therefore, it is an object of the present invention to maintain safety.

and water-soluble to a degree that is satisfactory for medical use.
The objective is to provide a tPA-containing pharmaceutical composition.

Means for Solving the Problems The inventors of the present invention have found that gelatin decomposition products increase the solubility of tPA in water, and have previously filed a patent application (Japanese Patent Application No. 60-198, 629). . After further investigation, the present inventors found that by adding L or more basic amino acids or their salts to tPA in addition to the gelatin decomposition product, the solubility of tPA in water was mutually increased. The present invention has been completed based on the discovery that there is a further increase in the

In the present invention, tPA may be obtained by extraction from animal tissue fibers, or may be obtained from cultures of microorganisms or animal cells artificially created by genetic recombination technology. Well, the invention is not limited by its origins.

The gelatin decomposition product used in the present invention is:

It can be manufactured according to the method described in US Pat. No. 3,057,782. Average molecular ffi 35 cross-linked with hexamethylene diisocyanate
It is preferable to use a gelatin decomposition product of about 1,000 ml.

Examples of the basic amino acids used in the present invention include arginine, ornithine, lysine, and histidine, and salts of these basic amino acids include:
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 amounts of the gelatin decomposition product and the basic amino acid or their salts can be appropriately selected depending on the desired amount of tPA dissolved in water. Since the gelatin decomposition product increases its concentration in water and increases the viscosity of the aqueous solution, it is generally desirable to blend it in an amount such that the concentration in the aqueous solution is 10% or less, preferably 5% or more. On the other hand, basic amino acids and their salts affect the osmotic pressure of aqueous solutions and become hypertonic as their concentration increases, so from the standpoint of administration to living organisms, a concentration of 1 to 5% is generated in aqueous solutions. Although it is generally preferred to include such amounts, higher or lower amounts can be used if desired.

The composition of the present invention may contain, if necessary, auxiliary ingredients commonly used in formulation, such as fillers, stabilizers, buffers, tonicity agents, and the like.

The pharmaceutical compositions of the present invention include not only solid or aqueous compositions containing tPA, a gelatin decomposition product, and one or more basic amino acids or salts thereof, but also, for example, ready-to-use injections. T like in
The tPA and the gelatin decomposition product and/or the basic amino acid or salt thereof were packaged separately, such as a lyophilized vial containing PA and a dissolution ampoule containing an aqueous solution of the gelatin decomposition product and the basic amino acid or salt thereof. It also includes compositions in the form.

The effect of the combination of tPA and basic amino acids or their salts on increasing the water solubility of tPA was confirmed in a first experiment.

Add 1.000 U of tPA to a small test tube, add 50 μL of an aqueous solution containing gelatin decomposition product (cross-linked with hexamethylene diisocyanate; average molecular Jt 35,000) and/or basic amino acid hydrochloride, or water (control). ) was added and mixed well, centrifuged, and one aliquot of the supernatant was collected, containing 0.1 M BSA! - Dilute with Lis-HCl buffer (pH 8) and use fibrin plate method [T. Astrup:
Archives Fist of φ Biochemistry and Biophysics (Arch, Biochem, B
iophys, ), 40.346 (1952))
tPA activity was measured according to the following. The results obtained are shown in the following table.

9. From the above table, the use of a combination of gelatin decomposition products and basic amino acids or their salts according to the present invention is effective against additive-free control (water) or gelatin decomposition products and basic amino acids. It is clear that this method has a significant effect on improving tPA solubility compared to the use of amino acids or their salts alone.

The present invention will be explained in more detail with reference to the following examples.

The gelatin decomposition product used in the examples is a partially hydrolyzed gelatin crosslinked with hexamethylene diisocyanate (average molecular ffi 35.000).

Example 1 In 100 mL of 0.03 M phosphate buffer at pH 7.5, 2 g of L-arginine hydrochloride, 5 g of gelatin hydrolyzate
g, aseptically preparing an aqueous solution of tPA1 tens of millions of gold units;
Dispense 1 mL into vials and lyophilize.

and use it as an ampoule for dissolution.

By adding the contents of the ampoule to the vial and mixing them before use, this injection becomes a homogeneous aqueous solution containing 100,000 units of tPA. The gelatin decomposition product concentration of this aqueous solution was 2.5% (W/V), and the arginine hydrochloride concentration was 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 tpAI was prepared aseptically in 100 ml of 0.03 M phosphate buffer at pH 7.5, and 1 mL of the solution was prepared aseptically. Dispense into vials and lyophilize.

Seal.

Separately, prepare an ampoule containing distilled water for injection '2at,
Use as an ampoule for dissolution.

Example 3 In 100 ml of 0.03M phosphate buffer at pH 7.5, 2 g of L-histidine hydrochloride, 5 g of gelatin hydrolyzate
y, aseptically preparing an aqueous solution containing 10 million monomers of tpA,
Dispense into vials and lyophilize.

Seal.

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

Example 4 In 100 ml of 0.03 M phosphate buffer at pH 7.5, 1.6 g of L-lysine hydrochloride and 5 g of gelatin hydrolyzate.

An aqueous solution containing tPA 1 unit was prepared aseptically.

Dispense 1 rrtl into vials, lyophilize, and seal.

Separately, an ampoule containing 2 aL of distilled water for injection is prepared and used as an ampoule for dissolution.

Example 5 In 100 ml of 0.03 M phosphate buffer at pH 7.5,
3 g of L-arginine aspartate, 5 g of gelatin hydrolyzate, and an aqueous solution of 10 million gold units of tPA1 were prepared aseptically, and 1 rttL each was dispensed into vials.

Lyophilize and seal.

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

Example 6 In 100 ml of 0.03 M phosphate buffer at pH 7.5, 2.6 g of L-lysine aspartate, 5 g of gelatin hydrolyzate, and an aqueous solution of tPA1 were prepared aseptically. , 1+LL into vials, lyophilize, and seal.

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

Example 7 100 rnL of 0.03 M phosphate buffer at pH 7.5
Inside, 0.8 g of L-arginine hydrochloride, 1.2 g of L-histidine hydrochloride, 5 g of gelatin hydrolyzate, tPAI
An aqueous solution of tens of millions of gold units is prepared aseptically, dispensed in imL portions into vials, freeze-dried, and sealed.

Separately, an ampoule containing 2rfLL of distilled water for injection is prepared and used as an ampoule for dissolution.

Example 8 1.8 g of L-arginine and 5 g of gelatin hydrolyzate were dissolved in 70 ml of distilled water for injection, and the pH was adjusted to 7 with hydrochloric acid.
．． Adjust from 0 to 8.0, tPAl in tens of millions of gold.

After dissolving, add distilled water for injection to bring the total volume to 100 ml. This liquid was sterilized and filtered, and dispensed into vials in imL portions.
Lyophilize and seal.

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

Mix evenly with 脣Manitou 7, and add 1 tPA per vial.
Fill to 0,000 units and seal.

Another 0.01M phosphate buffer (pH 7.5) 20
4 g of L-arginine hydrochloride and 10.0 g of gelatin hydrolyzate in 0 at. Aseptically prepare a solution containing 2rttL.
Dispense each into ampoules and use them as ampoules for dissolution.

Claims (1)

[Claims] A tissue plasminogen activator comprising a tissue plasminogen activator (tPA) combined with a gelatin partial hydrolyzate cross-linked with a diisocyanate and one or more basic amino acids or salts thereof. Pharmaceutical composition containing.