JPH0741432A - Novel sustained release medicine composition - Google Patents

Abstract

PURPOSE:To provide a novel sustained release medicine composition using safe fibrinogen or fibrin enabling the continuous release of a medicine for a long period as a medicine-dispersing medium. CONSTITUTION:The sustained release medicine composition is obtained in that a medicine-sealed liposome or lipid microshpere is homogeneously dispersed in fibrinogen or fibrin. The diameter of each lipsome or lipid microsphere particle is preferably larger than approximately 200nm diameter of each mesh of the net structure of the fibrin, especially 200-1000nm, when the lipsome or lipid microsphere comes into contact with a body fluid. The lipsome or lipid microsphere is preferably a lipsome or lipid microsphere containing a lipid, e.g. an acidic phospholipid, especially stearylamine-containing acidic phospholipid, which is negatively charged when coming into contact with the body fluid. The composition homogeneously dispersed in the fibrinogen is produced as a flowable liquid composition or solid fine particulate composition and subsequently administered into a living body. The composition homogeneously dispersed in the fibrin is produced as a semi-solid composition or semi-solid fine particulate composition and subsequently administered in a living body.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel sustained-release pharmaceutical composition. More specifically, the present invention relates to a sustained-release pharmaceutical composition in which liposomes or lipid microspheres encapsulating a drug are uniformly dispersed in fibrinogen or fibrin.

[0002]

BACKGROUND OF THE INVENTION Fibrinogen is a fiber protein having a molecular weight of about 340,000.
When this fibrinogen reacts with thrombin, Factor XIIIa, Ca ^2+, etc., which accounts for about 7% of plasma proteins, it becomes a gel-like mass (hereinafter referred to as fibrin) due to polymerization / crosslinking reaction. This fibrin has good biocompatibility, biodegradability, no toxicity, and excellent bioadhesion. Therefore, fibrin has already been used as an adhesive agent for living tissue mainly in the surgical field for the purpose of adhering tissue, closing the tissue and promoting wound healing.

In addition to these properties, (1) fibrinogen, even if administered alone in the body, thrombin, F. XIIIa, Ca ^2+, etc. can be converted into fibrin in the body, or (2) Fibrinogen and thrombin, etc. can be contacted in the body and rapidly converted into fibrin in situ, and further (3) Reaction conditions Since it has characteristics such that fibrin can be molded into a fine particle form by the method described above, it is attracting attention as a controlled release material for sustained-release preparations (Senderoff, R.
I. et al., J. Parent, Sci. Tech., 45 (1), 2, 1991.).

[0004] In fact, Monden et al. (Cancer, 69 (3), 636, 1) reported that the drug was directly encapsulated in fibrin.
992.) and Yamamoto et al. (Medical Postgraduates,
23 (7), 411, 1985.), intratumor administration of an anticancer drug-encapsulated fibrin shows a higher therapeutic effect than administration of the drug alone. In addition, according to a report by Sugitachi et al. (Cancer and Chemotherapy, 18 (11), 1817, 1991), intraperitoneal administration of antitumor agent-encapsulated fibrin to tumor-bearing mice resulted in a survival time difference as compared with single administration of drug. Extension is allowed.

Regarding the releasability of the drug from these conventional drug-encapsulated fibrin, the above-mentioned Senderoff et al. Have described the releasability of dexamethasone-containing fibrin microparticles to a tris buffer (pH = 7.5, 37 ° C.) in vitro. 80% of dexamethasone contained in about 5 hours
It is reported that the above is released (J. Paren
t. Sci. Tech., 45 (1), 2, 1991).

Monden et al., Yamamoto et al. And Sugitach, supra.
Although the report of i et al. describes the pharmacological effect of the drug, it does not mention at all how long the drug was retained in fibrin and continued to be released.

On the other hand, Miyazaki et al. Examined the release property of prednisolone from the fibrin membrane and found that nearly 100% was released in 3 hours (Chem. Pharm. B.
ull., 28 (7), 2261, 1980). Furthermore, Miyazaki et al. Investigated the miosis effect of pilocarpine by inserting a fibrin membrane containing pilocarpine into the lower eyelid of rabbits.
It disappears after 5 hours, whereas it lasts for 8 hours when a fibrin membrane is applied (Chem. Pharm. B).
ull., 30 (9), 3405, 1982). That is, it is considered that the drug release from fibrin is relatively fast in any of these conventional reports.

By the way, the inventors of the present invention observed that after fibrin was formed subcutaneously in rats, the weight of fibrin remaining at the formation site was measured over time, and that fibrin almost disappeared after about 28 days. ing. Therefore, the maximum possible duration of release of the drug contained within fibrin was expected to be approximately 28 days, consistent with the disappearance time of the fibrin body.

However, in reality, various drugs having different molecular weights are included in fibrin, and then in vitro.
Tris buffer (pH 7.
4) When the present inventors examined the releasability of the drug into, the time required for 100% release was 246.
It was about 4 hours at FUdR of 2 and about 24 hours at CYTOCHROME c having a molecular weight of 12,500. That is, it was confirmed that the release rate of the drug from fibrin was extremely high in the range of the molecular weight of the drug that is usually used, and the drug was rapidly diffused and released in a relatively short time. That is, before the elapse of about 28 days, which is the maximum sustained release period of fibrin as a sustained-release preparation that can be exerted in vivo,
It was revealed that most of the drugs contained in fibrin were released from fibrin by diffusion and the potential of fibrin as a release controlling material was not fully utilized.

Therefore, it is desired to maximize the potential of fibrin as a sustained-release material by controlling the diffusion and release of the drug from fibrin and matching the drug release property with the biodegradability of fibrin. It is rare.

On the other hand, Japanese Patent Application Laid-Open No. 61-56134 discloses a fluid composition in which a cytotoxic drug is dispersed in collagen and / or fibrinogen, and when the composition is locally administered, the encapsulated drug is gradually released. It is described to be released. In addition, it is described that a cytotoxic drug is made into liposome to be present in collagen or fibrinogen, and that the release of the drug is delayed by this liposome formation. However, there is no specific exemplification of liposome formation, and no mention is made of the type of liposome, its particle size, let alone its specific effect.

Further, Weiner, AL et al. Suggest that a drug-containing liposome is administered together with collagen to release the drug slowly (J. Pharm. Sci. 74 (9), 922, 1
985), there is no specific description or suggestion of the combination of drug-containing liposome with fibrin and / or fibrinogen, the type of liposome in such a case, its particle size, let alone its effect.

Nakai et al. Have also proposed a sustained-release preparation comprising a combination of hyaluronic acid gel and drug-containing lipid microspheres (small fat particles) (J. Controlled).
Release, 1992. (in press)), hyaluronic acid gel is not a natural product and its safety is unknown.

On the other hand, as a result of electron microscopic analysis of the mesh diameter of the mesh structure of fibrin, the present inventors found that this mesh diameter was about 2
Be less than or equal to 00 nm (see FIG. 2 below),
Therefore, it is thought that if the size of the drug is made larger than this diameter, it will be difficult for the drug to diffuse from fibrin, and as a result of examining various methods, encapsulating the drug in liposomes or lipid microspheres having a size larger than the mesh size of fibrin. It has been found that the present invention makes it possible to delay the release of a drug from fibrin so that the disappearance in the living body can be matched with the disappearance of fibrin itself, and thus the present invention has been reached.

An object of the present invention, therefore, is to provide a safe, sustained-release pharmaceutical composition capable of sustained drug release for a long period of time.

A further object of the present invention is to provide a sustained-release pharmaceutical composition containing fibrinogen or fibrin as a drug dispersion medium, which enables safe and long-term drug release.

[0017]

Means for Solving the Problems That is, the present invention is a sustained-release pharmaceutical composition in which liposomes or lipid microspheres encapsulating a drug are uniformly dispersed in fibrinogen. Further, the present invention is a sustained-release pharmaceutical composition in which liposomes or lipid microspheres encapsulating a drug are uniformly dispersed in fibrin.

The fibrinogen used in the present invention is
It is a soluble fibrous glycoprotein having a molecular weight of about 340,000 and is produced from blood of mammals such as human or bovine, which occupies about 7% of plasma protein, by a known method such as ethanol or ammonium sulfate fractionation precipitation method. Say something. Alternatively, it may be prepared by another technique, for example, recombinant DNA technique.

The fibrin used in the present invention is a fibrinogen in which thrombin acts on the fibrinogen. XIIIa, C
It is a substance that physically and chemically produces a solid and stable semi-solid fibrin produced by the action of a ^{2+. It} is usually formed by adding 30 to 500 units of thrombin to 80 mg of fibrinogen. To be done.

Generally, a liposome (liposome) is an endoplasmic reticulum composed of a bilayer of lipids and having an aqueous layer inside, and is formed when a naturally-occurring lipid and a corresponding synthetic lipid are dispersed in water. And the number of lipid bilayers, multilamellar liposomes (MLV, particle size = 400-35)
00 nm), small unilamellar vesicles (SUV, particle size = 20-50 nm), large unilamellar vesicles (LU)
V, particle size = 200 to 1000 nm) and the like.

As the liposome of the present invention, among the above-mentioned liposomes, those having a particle diameter (diameter) of 200 nm or more, for example, multilamellar liposomes and large unilamellar vesicles can be mentioned as preferable ones. 20 in diameter
When it is less than 0 nm, it is smaller than the mesh size of fibrin, and the drug tends to be released and released from fibrin. The average particle diameter of the liposome of the present invention is preferably 200 nm or more, and more preferably 200 nm to 1000 nm. Since the sustained-release pharmaceutical composition of the present invention is administered in vivo and comes into contact with body fluid in the administration part, the particle diameter of such liposomes is about 200 nm when the fibrin network structure is in contact with such body fluid. It means that the diameter is 200 nm or more.

As the lipid constituting the liposome of the present invention, generally known lipids can be used, and examples thereof include phospholipids, glycolipids, cholesterol and derivatives thereof. Specific examples of phospholipids include natural phospholipids such as soybean lecithin and egg yolk lecithin; synthetic phospholipids such as dipalnitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleylphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, and phosphatidylinositol. Examples include lipids, hydrogenated lecithin and other natural phospholipids that have been hydrogenated. Examples of glycolipids include palmityl glucoside, stearyl glucoside, myristyl glucoside, cholesteryl maltoside, and cholesteryl glucoside.

The charge on the surface of the liposome is adjusted by the composition of the lipids constituting the liposome. Usually, liposomes are made of the above-mentioned lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and the like and lipids containing cholesterol as a main component, and these lipids are electrically neutral when they come into contact with body fluids. When negatively charged when contacting with a body fluid, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylinositol, diacetylphosphoric acid and the like are usually added to the above lipids. Stearylamine is usually added when positively charged.

According to the knowledge of the present inventors, when the sustained-release pharmaceutical composition of the present invention is administered as a fluid liquid composition, the negatively charged liposomes interact with fibrinogen to delay the release. There is an advantage to bring.
Therefore, it is desirable to add an acidic phospholipid such as phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, and phosphatidylserine diachitylphosphate to the liposome of the present invention in addition to the lipid constituting the ordinary liposome, in order to make it negatively charged. Among these, phosphatidylserine is preferable. These charged substances are usually 0.1 to 0.
About 2 mol is added.

That is, as the liposome of the present invention, a liposome that is not charged when contacted with a body fluid, a positively charged liposome, or a negatively charged liposome can be used. Among them, a negatively charged liposome, for example, an acidic phospholipid is used. Preferred examples include liposomes containing lipids that are negatively charged when contacted with body fluids such as.

The method for producing such liposomes is usually as follows. That is, a predetermined amount of lipid is weighed and dissolved in an organic solvent (chloroform, ether, etc.), and then the solvent is distilled off under reduced pressure using a rotary evaporator in an eggplant-shaped flask to form a lipid thin film on the bottom of the flask. . It is then formed by swelling the lipids and then sonicating to obtain the final dispersion in water.

To encapsulate the drug in the liposome, the same operation as described above is performed to form a lipid thin film at the bottom of the flask, and then the desired drug aqueous solution and buffer are added and shaken vigorously. When the drug is highly lipophilic, the drug is entrapped in the hydrophobic portion of the lipid bilayer of the liposome, and when the drug is highly water soluble, the drug is entrapped in the lipid bilayer of the liposome. It is said to be distributed in.

The diameter of the liposome can be adjusted as follows. The liposomes are prepared by a production method such as a French press method, an ultrasonic treatment, a freeze-thaw method, a cholic acid removal method, etc., which can obtain a uniform size, and further gel filtration (Sepharose 4B, 2B, etc.), ultrafiltration, etc. By using, the size can be made uniform.

The lipid microsphere used in the present invention is an oil-in-water type (O / O) in which soybean oil is dispersed in an aqueous phase using phospholipid as an emulsifier.
W) In the emulsion, the dispersed phase oil droplets usually have a diameter of about 200n.
Stable fine particles of m are formed. The lipid microspheres are obtained by emulsifying an oil phase such as soybean oil with a suitable emulsifier such as phospholipid such as lecithin and purified water. Usually a lipophilic drug is encapsulated within the phase of lipid microspheres. To encapsulate the drug, the drug may be previously dissolved in the oil phase and emulsified in the above production method.

The particle size of the lipid microspheres is adjusted by selecting the emulsification method. It is possible to adjust the particle diameter up to about 1 μm with a normal emulsifying machine (such as a homomixer), and up to about 200 nm with a precision emulsifying machine (such as a Manton-Gaulin emulsifying machine or a microfluidizer).

As the lipid microspheres of the present invention, similar to the liposomes described above, lipid microspheres having no charge when contacted with body fluid, positively charged lipid microspheres, or negatively charged lipid microspheres are used. Preferred examples thereof include lipid microspheres negatively charged like the above-mentioned liposome. Thus, the liposomes or rebid microspheres encapsulating the drug constituting the granule-retarding pharmaceutical composition of the present invention can be obtained as a liquid dispersion.

The sustained-release pharmaceutical composition obtained by uniformly dispersing the drug-encapsulated liposomes or ribid microspheres in fibrinogen is, for example, a uniform fluid liquid composition or solid fine particle composition. Or administered to a living body, or a sustained-release pharmaceutical composition obtained by uniformly dispersing liposomes or rebid microspheres encapsulating the drug of the present invention in fibrin is, for example, a semisolid composition. Alternatively, it is produced as a semisolid fine particle composition and administered to a living body.

First, a uniform fluid liquid composition is prepared, for example, by dissolving fibrinogen in a liquid dispersion of liposomes or lipid microspheres containing the drug prepared by the above-mentioned method. In this case, fibrinogen may be added as a solid to the liquid dispersion of the liposome or lipid microspheres and dissolved by stirring, but fibrinogen is previously dissolved in a solvent such as water, and the obtained fibrinogen solution is added to the liposome or It may be added to the liquid dispersion of lipid microspheres. Further, a liquid dispersion of liposomes or lipid microspheres encapsulating a drug is lyophilized to a solid by a known method, and water or the like is added to the solid mixture of the solid and fibrinogen at the time of use to uniformly flow the mixture. Liquid composition may be used.

Among the sustained-release pharmaceutical compositions in which the liposomes or lipid microspheres encapsulating the drug of the present invention are uniformly dispersed in fibrin, the semi-solid composition is
For example, it is manufactured by the following method. First, when a liquid composition of fibrinogen as described above is administered to the body, it reacts with thrombin or the like derived from the body in situ to be converted into fibrin, and liposomes or lipid microspheres encapsulating the drug are contained in the fibrin. The sustained-release pharmaceutical composition of the present invention is uniformly dispersed. Alternatively, a liquid dispersion or a solid drug-encapsulated liposome or rebid microspheres, solid or solution fibrinogen is added to prepare a fibrinogen-containing liquid composition, and this is mixed with a thrombin-containing liquid composition in advance. A sustained-release pharmaceutical composition can also be obtained by forming liposomes or lipid microspheres in which the drug of the present invention is encapsulated by forming fibrin.

Further, to a liposome or lipid microsphere in which a liquid dispersion or solid drug is encapsulated, solid or solution fibrinogen is added, and this is mixed with a thrombin-containing liquid composition at the time of administration to obtain fibrin in the body. A sustained-release pharmaceutical composition may be formed by rapidly forming liposomes or lipid microspheres encapsulating the drug of the present invention in fibrin.
In this case, a two-liquid mixed injection needle is usually used to prevent administration difficulties due to fibrin production, and the two liquids mixed in the container are rapidly delivered to the body to form fibrin.

On the other hand, a sustained-release pharmaceutical composition in which liposomes or lipid microspheres encapsulating a drug are uniformly dispersed in fibrinogen, and a solid fine particle composition is the above-mentioned liquid composition of fibrinogen. Known methods, for example Chem. Pharm. Bull., 34 (6), 263.
2, 1986. For example, fibrinogen containing lipid microspheres is homogenized in cottonseed oil containing a surfactant and then at about 90 ° C.
The solid fine particle composition can be formed by heating the mixture to a solid state and uniformly dispersing the fine particle molded product in water with a known dispersant after isolation and washing.

Among the sustained-release pharmaceutical compositions in which liposomes or lipid microspheres encapsulating the drug of the present invention are uniformly dispersed in fugen, semisolid fine particle compositions are described in, for example, J. Porent. Sci. Tech., 45 (1),
2, 1991, fibrin microparticles are prepared by adding fibrinogen in solid or solution form to liposomes or rebid microspheres encapsulating a drug, and mixing this with a thrombin-containing liquid dispersion, and then A semi-solid fine particle composition can be produced by dispersing these fine particles in water using a known dispersant.

In the present invention, as a drug encapsulated in liposomes or lipid microspheres and uniformly dispersed in fibrinogen or fibrin, since the composition of the present invention is biodegradable, it can be directly subcutaneously or intramuscularly or dermally. In addition, a drug that is administered on the mucous membrane and acts systemically in the vicinity thereof or locally in the bloodstream is desirable.

As such drugs, the drugs of the present invention include drugs other than those having cytotoxicity, such as anti-inflammatory drugs such as aspirin, indomethacin, ibuprofen, tiaramid, ketoprofen, diclofenac sodium, piroxicam, fenbufen, Steroidal anti-inflammatory drugs such as dexamethasone mefenamic acid; as antibiotics, lincomycin, amikacin, amoxicillin, ampicillin, cefaclor, cefadroxil,
Cephalexin, fosfomycin, amphotericin B,
Rifampicin, etc .; hormones, insulin,
Calcitonin, testosterone, estradiol, progesterone, etc .; as cytokines, G-CSF,
Interleukins, erythropoietin and the like; autacoids prostaglandins of; bone, cyclopentenone as rheumatoid drugs, activated vitamin D ₃ and its derivatives such as 1,24- (OH) ₂ - Vitamin D _3, 12-OH
- vitamin _{D 3, 1,25- (OH) 2} - vitamin D ₃
Etc. Among these, hormonal agents such as insulin, steroidal anti-inflammatory drugs such as dexamethasone, active vitamin D ₃ and its derivatives are preferred.

The sustained-release pharmaceutical composition in which the liposome or lipid microspheres encapsulating the drug of the present invention is uniformly dispersed in fibrinogen or fibrin is mainly administered subcutaneously or intramuscularly, or on the skin or mucous membrane. However, it is mixed with a commonly used additive other than the above composition to form a sustained-release pharmaceutical composition, which is supplied to the clinical site. Such commonly used additives include citrates, acetates, phosphates, etc. as buffering agents to keep the pH of the solution within a certain range; isotonic agents for making the osmotic pressure of the solution equal to that of body fluids. Salts, glucose, etc. as agents; L-ascorbic acid, sodium pyrosulfite, EDTA, etc. as stabilizers for suppressing chemical decomposition and physical changes of the preparation; prevent contamination and decomposition of preparations by microorganisms Preservatives therefor include benzoic acid, benzoic acid esters, benzalkonium chloride, benzethonium chloride and the like.

INDUSTRIAL APPLICABILITY The present invention provides a novel and safe sustained-release pharmaceutical composition capable of long-term drug release, which has great clinical value.

[0042]

EXAMPLES The present invention will be described in detail below with reference to examples and reference examples, but the present invention is not limited thereto.

[0043]

Reference Example 1 Releasability of various drugs from fibrin Fibrinogen was prepared by adding 250 μl of Tris buffer (pH 7.4) in which 11 kinds of drugs shown in FIG. 1 were dissolved in appropriate amounts in 20 mg of fibrinogen (including F.XIIIa). Was dissolved. 250 μ of thrombin solution in this solution
1 (62.5 IU) was added to the drug-containing fibrin (5
00 μl) was obtained. 10 ml of this obtained fibrin
In Tris buffer (pH 7.4) at 37 ℃
After shaking, the drug concentration in the Tris buffer was measured over time, and the diffusion constant (K value) was determined by the following formula.

[0044]

[Equation 1]

The relationship between the diffusion constant (K value) of various drugs and the molecular weight is shown in FIG. From FIG. 1, for example, the molecular weight of 246.
The K value of FUdR, which is 2, is about 100 (h ^-1/2 ), the K value of cytochrome c is about 30 (h ^-1/2 ), and the former shows about the release of these drugs from fibrin. It can be seen that in 4 hours, the latter is completely released from each fibrin in about 24 hours.

[Example 1, Comparative Example 1] (1) Tris was added to 500 μg of insulin (Sigma).
250 μl of s buffer (pH 7.4) was added and dissolved. Fibrinogen (derived from human plasma, Fact
or XIIIa; Chemotherapy and Serum Therapy Research Institute (Bolheal) 20 mg was added and dissolved while heating to 37 ° C. to obtain an insulin solution. Separately 40 in 62.5 units of thrombin (Human plasma origin, Volheal manufactured by Institute of Chemotherapy and Serum Therapy)
A thrombin solution was prepared by adding 250 μl of Tris buffer (pH 7.4) containing mM CaCl ₂ (Wako Pure Chemical Industries, Ltd.). Fibrin containing insulin (500 μl) by mixing this thrombin solution and insulin solution
(Semi-solid composition) was obtained (A: Comparative Example 1).

(2) A liposome (Lip) containing the same amount of insulin as described above was prepared by Pick, U. Report (Arc
h. Biochem. Biophys., 212, 196, 1981.). As a lipid when adjusting the lipolyme, phosphatidylcholine (PC) (NOF Corporation) is used for Lip that does not have an electric charge, and lipid (SA) (Wako Pure Chemical Industries) mixed with PC and stearylamine (SA) for Lip that has a positive charge (SA) Is 1/6 in molar ratio with respect to PC), and P for negatively charged Lip is P
A mixed lipid of C and phosphatidylserine (PS) was used. (PS / PC = 1/6: molar ratio). The Lip (particle size = 200 to 1000 nm) thus obtained is T
Suspended in ris buffer. Then, the Lip-containing fibrin was prepared in the same manner as in the above (1) except that a fluid liquid composition obtained by dissolving fibrinogen in a Tris buffer suspension of Lip containing insulin thus obtained was used as an insulin solution. (Semi-solid composition) was prepared (Example 1, BD). The fibrin containing Lip (without charge) consisting of PC is B, the fibrin containing Lip (positive charge) consisting of (PC + SA) is C, (PC + P)
The Lip (negative charge) -containing fibrin composed of S) was designated as D.

(3) The above A, B, C and D were placed in Tris buffer (pH 7.4) and shaken at 37 ° C.
The release rate (%) was determined by quantifying insulin in the s buffer with HPLC over time. The results are shown in Table 1.

[0049]

[Table 1]

Example 2 and Comparative Example 2 (1) 25 μg of dexamethasone (Sigma) was used.
The suspension was suspended in 0 μl of Tris buffer (pH 7.4), 300 mg of phosphatidylcholine (PC) was further added, and the mixture was emulsified with Polytron (manufactured by KINEMATICA). Fibrin was prepared using this solution in the same manner as in Example 1 (A; Comparative Example 2).

(2) To 31.25 μg of dexamethasone, 250 mg of purified soybean oil was added and dissolved, 30 mg of PC was added, and homogenized (Polytron, 10 ⁴ rpm × 60 min) while heating (80 ° C.), and Tris buffer was added. The total volume was 1.25 ml. This solution was emulsified using a microfluidizer and then filtered to obtain a lipid microsphere (LM) Tris buffer suspension having a diameter of about 1 μm.

LM using (PC + SA) and LM using (PC + PS) were obtained in the same manner as in Example 1, respectively. 250 μl of each LM was collected and treated in the same manner as in Example 1 to obtain LM-containing fibrin (semisolid composition) (Examples 2, BD). B for each LM-containing fibrin using PC, C for (PC + SA), (PC +
What used PS) was set to D.

(3) The release rate (%) of dexamethasone from A, B, C, and D fibrin was 0.02% PC /
It was determined by measuring the amount of dexamethasone in Tris buffer (pH 7.4) (37 ° C) with HPLC over time. The results are shown in Table 2.

[0054]

[Table 2]

[Example 3, Comparative Example 3] (1) In the same manner as in Example 1, an insulin solution and a thrombin solution were obtained. The same amount of the fibrin and thrombin solution as in Example 1 was subcutaneously injected into the mouse using a two-liquid mixed syringe to form fibrin in the mouse subcutaneously (A; Comparative Example 3).

(2) PC, (PC + S
A), a Lip-containing fibrinogen solution prepared by using (PS + PC) (fluid liquid composition) was subcutaneously injected into a mouse using the same amount of thrombin solution and a two-liquid mixed syringe (Example 3, BD). The fibrin formed in this way was obtained using B and (P
The one using (C + SA) was designated as C, and the one using (PC + PS) was designated as D.

(3) With respect to the above four fibrins (semi-solid compositions), the fibrin residual rate (%) was measured with time by removing the fibrin from the mouse subcutaneously. The residual insulin ratio (%) in fibrin was measured with time in the same manner as in Example 1. Table 3 shows each result
It was shown to.

[0058]

[Table 3]

[Examples 4 and Comparative Example 4] Four kinds of fibrin (half) obtained in the same manner as in Example 2 (A (Comparative Example 4); B, C, D (Examples 4, BD)) The solid composition) was incised into the skin of a mouse and inserted subcutaneously. It was extracted with time, and the residual rate (%) of fibrin was measured. The dexamethasone residual rate (%) in fibrin was measured in the same manner as in Example 2. The results of each are shown in Table 4.

[0060]

[Table 4]

[Example 5, Comparative Example 5] Four types of fibrinogen solutions (A (Comparative Example 5); B, C, D (Examples 5, BD)) obtained in the same manner as in Example 1 ( A fluid liquid composition) was subcutaneously injected into each mouse to form fibrin (semi-solid composition). Peel off the skin at the injection site,
The attached fibrin was taken out. The residual rate (%) of the taken out fibrin was measured with time. The residual insulin ratio (%) in fibrin was measured over time by operating in the same manner as in Example 1. The results of each are shown in Table 5.

[0062]

[Table 5]

[Example 6 and Comparative Example 6] Four types (A, B, C, D) of fibrinogen solutions (fluid liquid compositions) obtained by the same operation as in Example 2 were subcutaneously injected into mice. Injected to form fibrin (semi-solid composition). Subcutaneous fibrin extraction was carried out in the same manner as in Example 5, and the residual rate (%) of fibrin and the residual rate (%) of dexamethasone in fibrin were determined in the same manner as in Example 2 over time. The results of each are shown in Table 6.

[0064]

[Table 6]

Example 7 The same operation as in Example 1 was carried out to obtain Lip containing insulin-containing PC, (PC + SA) and PS. Lip-containing fibrin fine particles (semi-solid fine particle-shaped composition) are fine particles of ethyl acetate / 0.05.
Sendernoff except washed with% PC then lyophilized
Report (J. Parent. Sci. Tech., 45 (1), 2, 1991)
Got according to. PC, (PC + SA), (PC + PS)
The fibrin fine particles containing Lip are composed of A, B, and
The fibrin microparticles obtained by performing the same operation as above using C as C and using a solution containing the same amount of insulin as Lip instead of Lip. A, B, C and D were suspended in 500 μl physiological saline (Otsuka Pharmaceutical Co., Ltd.) and subcutaneously administered to mice. Ogawa
Bulletin (J. Chem. Pharm. Bull., 36 (7), 2576, 198
By taking out the fibrin microparticles at the administration site over time according to 8) and quantifying the remaining amount by HPLC,
The residual insulin rate (%) in the fibrin microparticles was determined. The results are shown in Table 7.

[0066]

[Table 7]

[Example 8] The same operation as in Example 2 was carried out to prepare PC containing dexamethasone, (PC + SA), (PC
Tris of LM (diameter about 1 μm) using + PS) as an emulsifier
An s buffer suspension was obtained. The LM-containing fibrin fine particles (semi-solid fine particle composition) were obtained by the same operation as in Example 7 except that dexamethasone and LM were used. P
L with C, (PC + SA) and (PC + PS) as emulsifiers
The M-containing fibrin fine particles were designated as A, B and C, respectively. LM
The fibrin microparticles prepared by using a solution containing the same amount of dexamethasone instead of the above was designated as D. The residual amount of dexamethasone in the fine fibrin particles over time was quantified by the same operation as in Example 7, and the residual rate (%) was determined. The results are shown in Table 8.

[0068]

[Table 8]

[Embodiment 9] The same operation as in Embodiment 1 is performed, and P
Lip consisting of C (particle diameter = 200 to 1000 n
m) containing fibrin was obtained (A). Using PC as lipid
Report by Sunamoto et al. (Biochem. Biophys. Res. Commun.,
94 , 1367, 1980.) Lip (particle diameter = 20
~ 35 nm) was obtained. This Lip-containing fibrin (semi-solid composition) was obtained in the same manner as in Example 1 (B). The insulin release rate (%) from each fibrin was determined in the same manner as in Example 1. The results are shown in Table 9
It was shown to.

[0070]

[Table 9]

[Reference Example 2] The same operation as in Example 1 was carried out, and P
Lip consisting of C (particle diameter = 200 to 1000 n
m) -containing fibrin (A) and insulin-containing fibrin (B) (semisolid composition) were obtained. Lip containing the same amount of insulin as in Example 1 (particle size = 200 to 1000 n)
m) containing collagen gel (C) and insulin containing collagen gel (D) are reported by Weiner AL et al. (J. Pharm.
Sci., 74 (9), 922, 1985). Insulin-containing collagen gel is a solution containing Tris buffer (pH 7.
4) was used. Above A, B, C, D
The insulin release rate (%) was calculated by operating in the same manner as in Example 1. The results are shown in Table 10.

[0072]

[Table 10]

[Brief description of drawings]

FIG. 1 shows the relationship between the molecular weight of a drug and the diffusion constant (K) in Reference Experiment 1.

FIG. 2 shows an electron micrograph of the network structure of fibrin. From Fig. 2, the mesh size of the fibrin mesh is about 2
It is estimated that the thickness is 00 nm or less.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshiki Suzuki 4-3-2 Asahigaoka, Hino City, Tokyo Teijin Limited Tokyo Research Center

Claims (8)

[Claims] 1. A sustained-release pharmaceutical composition comprising a drug-encapsulated liposome or lipid microspheres uniformly dispersed in fibrinogen. 2. A sustained-release pharmaceutical composition comprising a drug-encapsulated liposome or lipid microspheres uniformly dispersed in fibrin. 3. The sustained-release pharmaceutical composition according to claim 1, wherein the composition is a fluid liquid composition or a solid particulate composition. 4. The sustained-release pharmaceutical composition according to claim 2, wherein the composition is a semi-solid composition or a semi-solid particulate composition. 5. The sustained-release pharmaceutical composition according to claim 3, wherein the fine particles have a diameter in the range of 10 to 100 μm. 6. The sustained-release pharmaceutical composition according to claim 1 or 2, wherein the drug is a drug other than a drug having cytotoxicity. 7. The sustained-release pharmaceutical composition according to claim 1 or 2, wherein the liposome or the lipid microsphere has a diameter of 200 nm or more when brought into contact with a body fluid. 8. The sustained-release pharmaceutical composition according to claim 1, wherein the liposome or lipid microsphere contains a lipid that is negatively charged when contacted with a body fluid.