JPS5911564B2 - Method for producing sustained release complex - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a sustained release complex in which a physiologically active substance is included in a protein derived from human serum.

A sustained release complex (hereinafter sometimes referred to as a "sustained release agent") in which a physiologically active substance is encapsulated in a protein such as collagen or gelatin.

), for example, JP-A-49-102827
, No. 50-42025, various methods have been proposed in the past.

However, the sustained release performance of sustained release agents manufactured by conventional methods of this type, that is, the duration of efficacy of physiologically active substances, is limited to a few hours to more than 10 days, and in this point, proteins as biocompatible materials cannot be used sufficiently. This was because it could not be used effectively.

Furthermore, although the proteins employed in the conventional methods can be digested in vivo, they are antigenic and cannot be said to be fully satisfactory as materials for intra-tissue administration.

The present inventors conducted research to produce a sustained-release agent that does not cause side effects to tissues even when administered intra-tissue and has a long release duration of physiologically active substances. It has been discovered that human serum-derived proteins that are digested by humans are suitable materials for the purpose of the present invention.

Furthermore, the present inventors have discovered that sustained release properties can be further expanded by denaturing human serum-derived proteins in the process of producing a sustained-release agent by bringing a physiologically active substance into contact with human serum-derived proteins.

Therefore, the present invention has been made based on the above findings.

According to the present invention, one or more physiologically active substances and one
A novel sustained-release agent is produced by bringing a species or two or more types of human serum protein aqueous solutions into contact and then applying physical manipulation to denature the human serum protein and clathrate the physiologically active substance into the protein. Ru.

Alternatively, the sustained-release complex produced as described above according to the present invention is brought into contact with an aqueous solution comprising one or more physiologically active substances and one or more human serum proteins, and then heat-treated. to denature the human serum proteins, form a sustained-release complex around the sustained-release complex as a core, and if desired, repeat this operation sequentially to produce a physiologically active substance with a multilayer structure. It is possible to produce a sustained-release composite containing clathrates.

The appropriate quantitative ratio of the components used in carrying out the present invention is 0.1 to 500 parts by weight of an aqueous human serum protein solution of 0.1 to 50% by weight per 1 to 100 parts by weight of the physiologically active substance.

Possible methods for denaturing proteins include chemical methods such as addition of acids, alkalis, urea, organic solvents, surfactants, heavy metal salts, etc., and physical methods such as heat, pressure, ionizing radiation, and freezing. However, in the present invention, physical means were adopted in consideration of the influence on living organisms, particularly human tissues.

The most preferred means of modification in the practice of this invention are heat and a combination of heat and ionizing radiation.

In particular, heat treatment is the most desirable method because human serum proteins can be denatured in a short time and no impurities are mixed into the sustained release agent formed.

In this case, the processing temperature ranges from room temperature to 100°C, preferably from 30°C to 90°C, but this processing temperature is naturally correlated with the processing time, and changes depending on the temperature or time. can be done.

That is, the denaturation of human blood proteins can be changed to a desired degree by appropriately combining treatment temperature and treatment time.

In carrying out the present invention, the contact between the physiologically active substance and the human serum protein aqueous solution can be carried out by various methods.

For example, it is effective to mix the two or to immerse the physiologically active substance in the aqueous layer, but by increasing the viscosity of the aqueous solution, brushing, spraying, etc. may be selected as desired.

Human serum proteins used in carrying out the present invention are mainly exemplified by human serum albumin, human serum globulin, and the like.

In the present invention, for example, film-like, sheet-like,
The sustained-release agent can be formed into various shapes such as particulate, powder, and rod-like, and can also be formed into a multilayer structure in which the physiologically active substance has a concentration gradient.

By the way, as one of the conventional techniques, albumin is heated at 100° in advance.
A method has been reported in which albumin is coagulated into small globules by heat treatment at about C, and then the albumin in the form of spherules is brought into contact with a physiologically active substance to impregnate the albumin with the physiologically active substance to produce a sustained release agent. There is.

However, when albumin is coagulated in advance as in this method, it is difficult to impregnate the coagulant albumin with a sufficient amount of physiologically active substances, and a relatively high temperature is required for coagulation of albumin. The problem of negative effects on albumin remains.

The present invention represents a significant improvement in this respect as well.

That is, in the present invention, the entire system consisting of an aqueous solution of a physiologically active substance and human serum protein is heat-treated at a relatively low temperature, and a sufficient amount of the physiologically active substance is dispersed and fixed in a coagulated body of human serum protein, and then packaged. .

The physiologically active substances used in the present invention include pleomycin hydrochloride, mitomycin C1 adriamymine, carbazylquinone, lomustine, ifosfamide, thioinosine, cytarabine, fluorouracil, 1-(2-tetrahydrofuryl)=5-fluorouracil, cytotin, Chlorambutyl, cifromomannitol, thioteba,
Cyclophosphamide, acetylniline, noradrenaline, serotonin, kallikrene, gastrin, secretin, adrenaline, insulin, glucagon, β-methasone, indomethacin A CT H.

Growth hormone, gonadotropic hormone, oxytocin, vanpressin, thyroxine, Hanamaru hormone, follicular hormone,
Progesterone, adrenocortical hormone, prostaglandin, antihistamine, antihypertensive agent, vasodilator, vascular reinforcing agent, stomachic digestive agent, intestinal regulation agent, contraceptive, disinfectant for the skin, agent for parasitic skin diseases, anti-inflammatory agent Examples include vitamin preparations, various enzyme preparations, vaccines, antiprotozoal agents, interferon inducers, anthelmintics, fish disease drugs, agricultural chemicals, auxin gibberellin, cytokinin, abscisic acid, and insect pheromones.

The present invention will be specifically explained below using comparative examples and examples.

In addition, the elution test of physiologically active substances from sustained release agents is 1010.
U, S-P,
It was carried out according to the following.

Example 1 Cyclophosphamide 500 da, human serum albumin 50
10 rILl of water was added to a glass ampoule containing 0 Da and mixed.

The mixture was left in a 50° C. hot bath for 3 minutes to undergo heat shrinkage and heat denaturation, and then taken out from the ampoule and air-dried.

The resulting mixture of heat-denatured Alhumin containing cyclophosphamide was rod-shaped, and the elution of cyclophosphamide from this mixture in vitro is shown in Figure 1 (solid line a).
.

Example 2 The mixture of heat-denatured albumin containing cyclophosphamide prepared in step 11 was placed in a glass ampoule, the inside of the ampoule was purged with nitrogen gas, the tube was sealed, and Co-60 was heated at room temperature.
The specimen was irradiated with gamma rays from a 300° C. for 1 hour at a dose rate of 5×10'R/hr.

The resulting mixture of irradiated heat-denatured albumin containing cyclophosphamide was rod-shaped, and the elution of cyclophosphamide from this mixture in vitro was shown in Figure 1 (
Solid line b).

Example 3 A glass ampoule containing Adriamycin (ADM) 50r119 and human serum albumin 200m9 water
ml was added and mixed.

Heat shrinkage and heat denaturation were performed by leaving the ampoule containing the mixture in a 60° C. hot bath for 1 minute, and the mixture was taken out from the ampoule and air-dried.

The resulting mixture of heat-denatured albumin containing ADM was in the form of a hard rod, and the elution of ADM from this mixture in vitro is shown in FIG.

Example 4 2 ml of water was added to a glass ampoule containing ADM 50~ and human serum albumin 50~ and mixed well.

Heat shrinkage and heat denaturation were performed by placing the mixture in a 50°C hot bath for 30 seconds.

Furthermore, this mixture was added to human serum albumin 200~, A
5 ml of water was added to the glass ampoule containing DMIO~ and mixed.

The ampoule was inserted into the center of the mixture, left in a 50°C hot bath for 1 minute to perform heat shrinkage and heat denaturation, and the mixture was taken out from the ampoule and air-dried.

The resulting mixture of heat-denatured albumin containing ADM is in the form of a hard rod and ADM from this mixture in vitro.
The elution is shown in Figure 3.

Example 5 Heat-denatured albumin containing ADM prepared in Example 3 was mixed with mitomycin (MMC) 50~, human serum albumin 3
5 ml of water was added to a glass ampoule containing 0.00 m9 and mixed.

The ampoule was inserted into the center of the mixture, left in a 50° C. hot bath for 1 minute to perform heat shrinkage and heat denaturation, and the mixture was taken out from the ampoule and air-dried.

The resulting heat-denatured albumin containing M'MC and ADM was in the form of a hard rod, and MMC from this mixture in vitro.
The elution of 1 and ADM is shown in FIG.

(Solid line C is MMC, and d is ADM.) Example 6 The sample prepared in Example 3 was inserted into the peritoneal cavity of an ICR female mouse in which Ehrlich ascites carcinoma cells were implanted into the peritoneal cavity. , we investigated its lifespan.

As a result, 100% of the mice died around 15 days when the mouse was not inserted, and 70% of the mice remained alive even after 7 months had passed when the mouse was inserted, demonstrating a remarkable effect in prolonging life. .

Seven months later, when the inserted sample was removed from the mouse, its size was 1% of the original sample, and no rejection phenomenon was observed around the inserted tissue.

Example 6 The sample prepared in Example 4 was inserted subcutaneously into an ICR female mouse in which skin cancer had been formed on the back.

As a result, 100% of the mice died within 1 month when the mice were not injected, but when they were injected, 60% of the mice were still alive even after - years had passed, indicating a remarkable effect in prolonging survival. .

% of the sample taken from the mouse after 1 year
Furthermore, no rejection phenomenon was observed around the inserted tissue.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 to 4 are graphs showing the results of elution tests of physiologically active substances from sustained-release preparations prepared according to the present invention.

Claims (1)

[Claims] After contacting an aqueous solution consisting of 11 or more physiologically active substances and one or more human serum proteins, heat treatment is performed at a temperature of 30°C to 90°C to obtain the human serum proteins. A method for producing a sustained-release complex containing a physiologically active substance, which comprises dispersing, immobilizing, and encapsulating a physiologically active substance in a denatured human serum protein matrix. After contacting an aqueous solution consisting of 21 or more physiologically active substances and one or more human serum proteins, heat treatment is performed at a temperature of 30°C to 90°C to denature the human serum proteins to produce denatured human serum. A sustained release complex in which a physiologically active substance is dispersed and immobilized and included in a protein matrix is brought into contact with an aqueous solution consisting of one or more physiologically active substances and one or more human serum proteins. Heat treatment is performed at a temperature of 30° C. to 90° C. to denature the human serum protein, form a sustained release complex around the sustained release complex as a core, and repeat this operation sequentially if desired. A method for producing a sustained-release composite containing a physiologically active substance, which has a multilayer structure consisting of: After contacting an aqueous solution consisting of 31 or 2 or more physiologically active substances and 1 or 2 or more human serum proteins, heat treatment is performed at a temperature of 30°C to 90°C, and ionizing radiation is irradiated to remove the human serum protein. A method for producing a sustained-release complex containing a physiologically active substance, which comprises denaturing serum proteins, dispersing and immobilizing the physiologically active substance in a denatured human serum protein matrix, and packaging it. After contacting an aqueous solution consisting of 41 or more physiologically active substances and one or more human serum proteins, heat treatment is performed at a temperature of 30°C to 90°C to transform the human serum proteins into a denatured human serum protein matrix. A sustained release complex in which a physiologically active substance is dispersed and immobilized and encapsulated is brought into contact with an aqueous solution consisting of one or more physiologically active substances and one or more human serum proteins. ℃
After heat treatment at a temperature of ~90°C, the human serum proteins are denatured by irradiation with ionizing radiation to form a sustained release complex around the core of the sustained release complex, and if desired. A method for producing a sustained-release composite containing a physiologically active substance, which has a multilayer structure and consists of sequentially repeating this operation.