JPS61215331A - Thrombolytic agent - Google Patents

Abstract

PURPOSE:To provide an inexpensive and high-safety thrombolytic agent containing semi-alkali protease or its polyalkylene glycolmodified derivative as an active component. CONSTITUTION:The objective agent contains semi-alkali protease or its polyalkylene glycol-modified derivative (e.g. semi-alkali protease modified with polyethylene glycol) as an active component. It may further contain one or more stabilizing carrier for drug preparation such as a polymer (e.g. amino acid). Enzymes such as urokinase have hitherto been used as thrombolytic agents, however, the enzymatic thrombolytic agent is expensive because the active component is extracted from living body and is administered only in the form of injection from the viewpoint of absorbability and stability. Semi- alkali protease can be produced industrially in a mass by the cultivation of Aspergillus melleus and the absorbability and stability of the agent in the oral administration can be improved by modifying with polyalkylene glycol.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a pharmaceutical, particularly a thrombolytic agent containing an enzyme as an active ingredient.

(Prior Art) Thrombolytic agents are often required in the treatment of adult diseases or traumatic diseases associated with circulatory system disorders, and various thrombolytic agents have been developed and used for this purpose. Enzymes are sometimes used as the active ingredient of thrombolytic agents, such as urokinase, which has been recognized to be effective, but the active ingredients of conventional enzyme-based thrombolytic agents of this type are extracts from living organisms. However, it has disadvantages in that it is expensive and its dosage form is limited to injections from the viewpoints of absorption and stability.

(Problems to be Solved by the Invention or Problems to be Solved by the Invention) The basic object of the present invention is to provide an enzyme preparation that is highly safe but can be easily and inexpensively produced, and therefore is an inexpensive thrombolytic agent. Our goal is to provide the following.

Another object of the present invention is to provide a thrombolytic agent that can be easily formulated into oral or parenteral formulations using conventional formulation techniques and is therefore not limited by dosage form.

(Means and effects for solving the problems) According to the present invention, the above-mentioned problems and problems can be solved by using a blood clot containing a semi-alkaline protease or a modified polyalkylene glycol thereof as an active ingredient. Solved by lysing agents. The thrombolytic agent according to the invention may also contain at least one pharmaceutical stabilizing carrier.

That is, the present invention is based on the discovery that semi-alkaline protease, which has been known as a proteolytic enzyme with anti-inflammatory and anti-tumor effects, also has thrombolytic activity.

Semi-alkaline protease is a white crystalline substance with a molecular weight of about 30,000 and an isoelectric point of 6°5.17 [314 amino acids].

It can be industrially produced in large quantities by culturing Aspergillus m5lleus, and therefore can be obtained at low cost. This semi-alkaline protease can be modified with polyalkylene glycols, such as polyethylene glycol, to reduce its antigenicity and improve its absorption upon oral administration, as well as its stability in the blood. It becomes possible to increase it significantly. If a polymeric substance such as an amino acid, albumin, hydroxypropyl methyl cellulose, etc. is used as a carrier during formulation, the storage stability of semi-alkaline protease or its modified product can be improved, and stable and sustained action can be provided. .

The thrombolytic agent according to the present invention can be administered orally in the form of tablets, capsules, powders, granules, etc. using conventional formulation techniques, or parenterally in the form of injections, suppositories, ointments, etc. It can be administered directly. Furthermore, the thrombolytic agent according to the present invention can be used in combination with other drugs, such as anticancer drugs, to enhance their effects.

The dosage depends on the severity of the disease, administration method, dosage form, etc.
In the case of oral administration, the dose for adults is preferably within the range of 1-1000 mg/day, particularly about 100 mg/day.

(Examples, etc.) Next is an example of manufacturing modified semi-alkaline protease.

The present invention will be explained in more detail with reference to formulation examples, pharmacological test examples, and the like.

J (Production of modified semi-alkaline protease) 2-o-methoxypolyethylene glycol-4゜6-dichloro-8
- 200 mg of triazine and 25 mg of semi-alkaline protease were mixed in 0.1 M borate buffer under cold conditions (4
℃) for 2 hours, followed by ultrafiltration and freeze-drying to obtain polyethylene glycol-modified semi-alkaline protease.

Tablets Various ingredients were blended according to the following formulation, mixed, and compressed into tablets. An enteric coating of methacrylic acid polymer was then applied in a conventional manner to obtain oral tablets.

Semi-alkaline protease 20 (mg) Avicel
20 Bafila-101,20 Magnesium stearate 2 Lactose 58 Total 120mg/tablet 1 2 (External cream) Semi-alkaline protease is added and dissolved in 4% bovine serum albumin solution to give a 2.0% concentration of semi-alkaline protease. A solution was prepared and then lyophilized. This composition was blended with excipients and the like according to the following formulation, and a cream for external use was prepared by a conventional method.

The above composition 0.5 (g) Diethyl sepatate 8.0 Sperm wax 5.0 Sodium polyoxyethylene oil ether phosphate 6.0 Vaseline -1-Total 100 g 1-year-old (suppositories) The following The ingredients were blended according to the prescription and suppositories were prepared by a conventional method.

Semi-alkaline protea-zepolyethylene glycol modified product 30 (IIlg) Oil-based base (cocoa butter) -1 to 1 - per unit 1700
(IIlg) Theory 1 (Fibrin degrading activity, in vitro test) a)
Test drug 1) Semi-alkaline protease (Seaprose 5J manufactured by Amano Pharmaceutical Co., Ltd.) 2) Trypsin (originated from bovine pancreas) (manufactured by Miles, Laboratories, Ltd.) 3) α-Chymotrypsin (originated from bovine pancreas) (Miles, Laboratories) , Ltd.) b) Measurement method 0.01 M phosphate buffer containing 0.15 M NaCl (
Test drug was dissolved at various concentrations in pH 7.5), Part 1
Plasminogen-free fibrin plate 5 (
Kitasato Laboratory - Put it in the hole of Daiichi Chemical Co., Ltd.'s "Kitaken" lot number 8304). Next, this plate was placed in a humid box and subjected to reaction treatment at 37°C for 4 hours. The area of the resulting dissolution window was measured (however, the hole size (3 x 3) was subtracted), and the fibrin decomposition effect of the test drug was compared and examined from this measured value.

C) Results and Discussion The results are as shown in Figure 1. All of the test drugs exhibited fibrin-degrading activity, and the strength of the activity was in the order of trypsin > semi-alkaline protease > α-chymotrypsin.

Although trypsin generally has a fibrin-degrading action, it was found that this action of semi-alkaline protease is comparable to that of trypsin.

Theory 2 (Thrombolytic effect, in vivo test) a) Test method A male mongrel dog weighing 10-15 kg was used as the experimental animal.
After fasting for 4 hours, the mice were immobilized by administering a muscle relaxant under anesthesia. Thereafter, the size of the thrombus created in the femoral artery under artificial respiration was measured before and after oral administration (10 units/kg) of semi-alkaline protease ("Seaprose SJ" manufactured by Amano Pharmaceutical Co., Ltd.) and modified semi-alkaline protease.
, 6.9 and 12 hours later to calculate the thrombolysis rate.

In addition, to prevent blood clotting, heparin should be administered at
It was continuously infused intravenously at 0 units/kg and thereafter at 100 units/kg/hr.

b) Results and discussion The results are as shown in Figure 2. When compared with the control group (no drug administration group), an increase in the thrombolytic rate was observed in the test group from 3 hours after drug administration. Furthermore, when comparing modified and unmodified drugs, the modified drug was found to be superior.

These facts clearly demonstrate that semi-alkaline protease has a thrombolytic effect.

[Brief explanation of drawings]

Figure 1 shows semi-alkaline protease, which is the active ingredient of the thrombolytic agent according to the present invention, and trypsin and α, which are the contrast substances.
- A graph showing a comparison of the fibrinolytic activity with that of chymotrypsin. Fig. 2 is a graph showing the thrombolysis rate of semi-alkaline protease and its modified product over time.

Claims (5)

[Claims] (1) A thrombolytic agent characterized by containing semi-alkaline protease or a modified polyalkylene glycol derivative thereof as an active ingredient. (2) The thrombolytic agent according to claim 1, wherein the polyalkylene glycol modified product is a polyethylene glycol modified semi-alkaline protease. (3) characterized by containing semi-alkaline protease or its modified polyalkylene glycol as an active ingredient and at least one type of stabilizing carrier for formulation;
Thrombolytic agents. (4) The thrombolytic agent according to claim 3, wherein the stabilizing carrier for the formulation is a polymeric substance. (5) The thrombolytic agent according to claim 4, wherein the polymeric substance is an amino acid, an albumin, or a cellulose.