JPH1045616A - Sustained release preparation for injection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject preparation capable of not coagulating at a room temperature, forming a fibrin clot after subcutaneous administration and gradually releasing a medicine by adjusting the blending ratio of a fibrinogen and a thrombin to a proper value. SOLUTION: This preparation comprises (A) a medicinal active ingredient, (B) a fibrinogen, (C) a blood coagulation 13 factor and (D) a thrombin in the blending ratio of the component D to the component B equivalent to 1-30 units, preferably 1.2-6 units of the component D to 80mg of the component B. The preparation further contains (E) calcium ion and the component A is a protein medicine, especially interleukin 2 or interferon-γ. The preparation further contains (F) hyaluronic acid. Preferably, a kit for making the objective preparation when necessary is composed of a basic constitution of a lyophilized product containing the component B and the component C and a lyophilized product containing the component D (the component A is contained in either of the lyophilized products).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sustained-release preparation for injection which is useful for maintaining the efficacy of various pharmaceutically active ingredients and a kit for preparing the same when used. More specifically, a pharmaceutically active ingredient,
It is a sustained-release preparation for injection containing fibrinogen, blood coagulation factor 13, and thrombin, and is particularly suitable for subcutaneously or intramuscularly administering a proteinaceous drug.

[0002]

2. Description of the Related Art As preparations for maintaining the efficacy of a pharmaceutically active ingredient, there have already been reported some preparations utilizing a mechanism of forming a fibrin clot (hereinafter, referred to as fibrin clot or simply clot) by coagulation of fibrinogen. References (BIOTHERAPY, 5 (6), 1086-1090, 1991, Takahashi et al.)
Include the sustained release effect of the IL-2 · Fibrin preparation and the mouse Colon26
Antitumor effects have been reported for subcutaneously transplanted tumors. Specifically, as an in vitro test, IL-2 prepared by mixing equivalent amounts of solution A (aprotinin solution containing IL-2, fibrinogen, and blood coagulation factor 13) and solution B (calcium chloride solution containing thrombin).・ Fibrin preparation is solidified to study sustained release, but the mixing ratio of fibrinogen and thrombin is 250 units of thrombin with respect to 80 mg of fibrinogen. Furthermore, subcutaneous administration to mice is performed as an in vivo test, and the solution A and the solution B are separately injected with two syringes.

[0003] JP-A-6-336444 discloses a sustained-release pharmaceutical composition containing fibrinogen and / or fibrin, but applicable drugs are limited to fat-soluble drugs. Indispensable surfactant. Furthermore, the blending ratio of fibrinogen to thrombin when preparing fibrin is described as 30 to 500 units of thrombin with respect to 80 mg of fibrinogen. JP-A-7-41432 discloses a sustained-release pharmaceutical composition in which a drug-encapsulated liposome or lipid microsphere is uniformly dispersed in fibrinogen or fibrin. The compounding ratio of fibrinogen to thrombin is described as 30 to 500 units of thrombin for 20 mg of fibrinogen.

[0004] Commercially available biological tissue adhesives also apply the process of fibrin formation by fibrinogen and thrombin. For example, the package insert of Tissile (Immuno) contains the formulation ratio of fibrinogen and thrombin as formulation components. However, it is disclosed that thrombin is 500 units or 4 units per 90 mg of fibrinogen. However, such a biological tissue adhesive is capable of adhering and closing a living tissue, and is used when suturing or joining is difficult at the time of an operation or when blood, body fluid, etc. leaks. It is not intended for sustained release of drugs.

[0005]

In a conventional sustained-release preparation utilizing a fibrin clot formation mechanism, the compounding ratio of thrombin to fibrinogen is relatively high. Tested at a rate of about 250 units.
The lower limit of the amount of added thrombin is usually at least 30 units or more of thrombin with respect to 80 mg of fibrinogen. As a result, fibrin mixed with thrombin may form a fibrin clot outside the body in a relatively short period of time and coagulate, making it impossible to administer it. Therefore, when administered as an injection, drugs,
After preparing a mixture containing fibrinogen and thrombin, it is necessary to quickly set the mixture in a syringe and administer it immediately, or to separately inject a solution containing fibrinogen and a solution containing thrombin.
Even if the two liquids are set in a two-liquid mixing type syringe, they must be administered promptly after mixing. Such a situation is not satisfactory as a clinical administration operation and leads to mental suppression not only for doctors but also for patients.

[0006] Accordingly, there has been a need for the development of sustained-release preparations which can be easily injected without requiring special techniques.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in view of the above-mentioned problems, and as a result, if the mixing ratio of fibrinogen and thrombin is appropriately adjusted, the injection release property that does not require any special technique at the time of use is considered. The present inventors have found that a preparation can be obtained and completed the present invention. That is, the present invention is a sustained-release preparation for injection comprising a pharmaceutically active ingredient, fibrinogen, blood coagulation factor 13, and thrombin, wherein the mixing ratio of fibrinogen and thrombin is 80 mg of fibrinogen.
A ratio of thrombin to more than 1 unit and less than 30 units (hereinafter referred to as the preparation of the present invention). After the preparation of the present invention is mixed and prepared as an injection solution, it does not immediately coagulate at room temperature and, after subcutaneous administration, forms a fibrin clot before the drug is released and takes the drug into the clot. Injectable composition capable of gradually releasing a drug.

Preferred forms of the preparation of the present invention include: a. The mixing ratio of fibrinogen and thrombin is such that thrombin is 1.2 mg with respect to 80 mg of fibrinogen.
A formulation having a ratio corresponding to ~ 6 units; b. A formulation further containing calcium ions; c. A formulation wherein the pharmaceutically active ingredient is a proteinaceous drug; d. A formulation wherein the pharmaceutically active ingredient is interleukin-2 or interferon-γ; e. A formulation further comprising a thrombin inhibitor; f. A formulation wherein the thrombin inhibitor is gabexate mesylate; g. A formulation further comprising hyaluronic acid; h. Preparations for subcutaneous injection; and the like.

Further, the present invention also provides a kit for preparing the preparation of the present invention before use (hereinafter, referred to as the kit of the present invention). The basic configuration of the kit consists of the following A and B. A: Lyophilized product containing fibrinogen and blood coagulation factor 13. B: Lyophilized product containing thrombin. The lyophilized product of A or B contains a pharmaceutically active ingredient, and the compounding ratio of fibrinogen to thrombin is a ratio corresponding to an amount of more than 1 unit of thrombin and less than 30 units per 80 mg of fibrinogen. is there. Preferred forms of the kit of the present invention include: a. A kit containing a preparation solution containing calcium ions separately from the lyophilized products of A and B; b. A kit or the like further containing a thrombin inhibitor in the freeze-dried product of either A or B is exemplified.

Hereinafter, the preparation and the kit of the present invention will be described in more detail. As the pharmaceutically active ingredient applicable to the present invention, it is administered subcutaneously or intramuscularly by injection, and locally acts in the vicinity thereof or moves into the blood and acts systemically. The desired drug can be widely applied. Specifically, calcitonin, elcitonin, bone morphogenetic protein (BMP), parathyroid hormone, etc. as bone-related peptides, atrial natriuretic peptide (ANP), ANP-degrading enzyme inhibitor, B-type natriuretic peptide, C-type natriuretic peptide Endothelin-related substances, such as endothelin-receptor antagonists, endothelin-converting enzymes, thrombopoietin (TPO), etc., platelet growth factors, tumor necrosis factor (TNF) as lymphokines, and various interleukins (IL-1,2) , 3,4,5,6,7,8,9,
10, 11, 12, 13, 15, etc.), various interferons (IFN-α, β, γ), colony stimulating factors (CS
F), various growth factors such as macrophage activating factor (MAF), such as nerve growth / trophic factor, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor I (IGFI), blood vessels Formation factor, platelet-derived growth factor (PDGF), blood stem cell growth factor (SC
F), hepatocyte growth factor (HGF), transforming growth factor, etc .; gastrointestinal hormones such as gastrin, glucagon, secretin, bombesin, motilin, cholecystokinin, etc .; and cancer-related substances such as methionase, neocarzinostatin, etc. Examples include insulin and its derivatives, erythropoietin (EPO), human growth hormone, antisense drugs, vaccines, immunosuppressants and the like. Among them, preferred are protein drugs such as interleukins and interferons. The compounding amount of the pharmaceutically active ingredient may be appropriately set in consideration of the type, target disease, patient condition, number of administrations, and the like.

The mixing ratio of thrombin to fibrinogen is a ratio corresponding to an amount of more than 1 unit and less than 30 units of thrombin for 80 mg of fibrinogen. More preferably, the ratio is more than 1 unit and 6 units or less, more preferably 1.2 to 6 units, most preferably 2 to 4 units of thrombin for 80 mg of fibrinogen. When the preparation of the present invention is administered in vivo, activated thrombin acts on fibrinogen to produce fibrin monomer and then fibrin polymer, which are cross-linked by blood coagulation factor 13 in the presence of calcium ions. and finally a fibrin clot is formed in a form incorporating part or all of the pharmaceutically active ingredient. Thereafter, sustained drug efficacy is achieved by the sustained release of the drug from within the clot.

However, when the mixing ratio of thrombin in the preparation of the present invention is 30 units or more with respect to 80 mg of fibrinogen, after preparing an aqueous solution, the mixture is brought to room temperature (about 20 ° C.).
) In a very short time (for example, about 18 seconds). Therefore, sufficient time is not ensured for setting the aqueous solution in the syringe and administering the solution to the living body, and thus not only careful drug administration cannot be performed, but also, in some cases, coagulation in the syringe prior to administration results in improper administration. Fall into a possible situation.
On the other hand, the mixing ratio of thrombin is fibrinogen 80m.
If it is 1 unit or less with respect to g, for example, the fibrin clot formation time at 37 ° C. takes about 13 minutes or more,
Coagulation after being administered in vivo is significantly delayed. As a result, most of the encapsulated drug is rapidly diffused and released from the administration site before the clot is formed, so that it is difficult to achieve sustained efficacy. As described above, even if the mixing ratio of thrombin exceeds any of the limited ranges, the practical use of the sustained-release preparation for injection is remarkably reduced or unusable.

The mixing ratio of blood coagulation factor 13 is usually
Blood coagulation factor 13 is about 0.05 to 100 units, preferably about 0.1 to 8 per 80 mg of fibrinogen.
0 units, more preferably about 0.25-60 units.
Although it is considered that there is no particular problem with the proportion of the blood coagulation factor 13 being large, if the proportion is too small, the fibrin clot becomes soft and adversely affects the sustained release of the drug. The preparation of the present invention may optionally contain calcium ions. In order for fibrin clots to be formed after the preparation of the present invention is administered in vivo, the presence of calcium ions is essential. If not, a fibrin clot can be formed. However, from the viewpoint of formulation design that controls the sustained-release pattern at a higher level according to the type of drug, it is preferable to contain calcium ions as a formulation component in advance. In this case, the mixing ratio of calcium ions is usually about 5 to 200 mmol, preferably about 10 to 100 mmol per 80 mg of fibrinogen.
mmol, more preferably about 30-70 mmol. The calcium ion can be formulated as a calcium salt such as calcium chloride.

The preparation of the present invention further comprises a stabilizer for the preparation, pH
Additives such as a regulator, an isotonic agent, a preservative, and a regulator for sustained release of a drug can be optionally contained. As a stabilizer, for example,
Examples include human serum albumin, aprotinin, L-ascorbic acid, sodium pyrosulfite, α-tocopherol and the like. Examples of the pH adjuster include hydrochloric acid, citrate, acetate, phosphate, and various amino acid salts. Examples of the tonicity agent include salt, glucose, mannitol and the like. Examples of the preservative include benzyl alcohol, benzoic acid or esters thereof, and benzalkonium chloride.

Examples of the regulator for sustained release include guanidine benzoic acid derivatives (eg, gabexate mesylate, camostat mesylate, nafamostat mesylate, patamostat mesylate, etc.), argatroban, hirudin, etc.
Examples thereof include substances capable of suppressing the action of thrombin (hereinafter, referred to as thrombin inhibitors), microparticles of hyaluronic acid, phospholipids, and lactic / glycolic acid-based polymers (microspheres). The types and amounts of the above additives are
What is necessary is just to set suitably considering the purpose of addition, the kind and amount of a pharmaceutically active ingredient, the kind of other additive used together, etc.

Next, a method for preparing the preparation of the present invention will be described. The preparation of the present invention may be in the form of a powder mixture or an aqueous solution that is injectable and viscous so long as it contains the above-mentioned components and optional additives.
The method and order of compounding various components and additives are not particularly limited as long as the compounded mixture can be injected in the presence of water, but a more preferable preparation method is a method using the kit of the present invention. Hereinafter, the kit of the present invention and a method for using the kit will be described.

As described above, the kit of the present invention contains A: a freeze-dried product containing fibrinogen and blood coagulation factor 13, and B: a freeze-dried product containing thrombin, as described above. The dried product further contains a pharmaceutically active ingredient.
The mixing ratio of fibrinogen and thrombin is a ratio corresponding to an amount of more than 1 unit and less than 30 units of thrombin for 80 mg of fibrinogen. More preferably, the ratio is more than 1 unit and 6 units or less of thrombin with respect to 80 mg of fibrinogen, further preferably 1.2 to 6 units, and most preferably 2 to 6 units.
4 units.

The mixing ratio of blood coagulation factor 13 is usually
Blood coagulation factor 13 is about 0.05 to 100 units, preferably about 0.1 to 8 per 80 mg of fibrinogen.
0 units, more preferably about 0.25-60 units.
The kit of the present invention may optionally contain the above-mentioned additives and one or more injection solutions. Further, the preparation may optionally contain a calcium salt such as calcium chloride. In that case,
The mixing ratio of the calcium salt is usually fibrinogen 8
About 5 to 200 mmol, preferably about 1 to 0 mg.
0-100 mmol, more preferably about 30-70 mm
ol.

The freeze-drying method of each of A and B is not particularly limited as long as it does not adversely affect the quality of the ingredients, and well-known freeze-drying methods can be applied. The preparation of the present invention is prepared by mixing the freeze-dried products of A and B in the presence or absence of water. The mixing may be performed before the lyophilized products of A and B are set in the syringe, or after both are set in the syringe in a non-contact state. As a syringe in the latter case, for example,
Kit-type syringes described in WO94 / 12227, WO95 / 17916 and the like can be used. The formulation of the present invention
Since it does not immediately coagulate when present in an aqueous solution at room temperature, it has a relatively long time before administration as compared with conventional sustained-release preparations for injection. However, the time from preparation of the formulation of the present invention in an aqueous solution inside or outside a syringe until administration into a living body is preferably within about 1.5 minutes,
More preferably, less than about 1 minute is recommended. It is also desirable to keep the aqueous solution below about 20 ° C, preferably below about 4 ° C, until administration.

[0020]

EXAMPLES Examples of the present invention will be described below, but they do not limit the present invention in any way. In addition, immunonas (Shionogi) was used for rIL-2 and Immunomacs (Shionogi) was used for IFN-γ. Example 1 : Relationship between the amount of thrombin added and clot formation time In the preparation of the present invention comprising fibrinogen, blood coagulation factor 13, thrombin and calcium ions as a basic composition,
The clot formation time when the amount of added thrombin was changed was measured. (Method of measuring clot formation time) (1) 80 mg of fibrinogen and 60 units of blood coagulation factor 13 were dissolved in 1 ml of a buffer solution (pH 7.5) and cooled on ice. (2) Thrombin was dissolved in a calcium chloride solution (5.88 mg / ml) and cooled with ice. (3) 0.1 ml of each of the above (1) and (2) was added at room temperature (about 20 ml).
C) or 37 ° C., and after mixing gently, the time when the fluidity disappeared when the container was turned over was defined as the clot formation time. The clot formation time at 37 ° C. was measured while keeping the container warm in a water bath adjusted to 37 ° C.

(Results) The results are shown in Table 1.

[Table 1] Amount of thrombin added (unit) 0.5 1 1.2 2 3 4 5 6 30 Room temperature of 300 clots 23.5 16.7 10.0 4.4 2.0 1.8 1.3 1.5 0.3 0.3 Forming time (min) 37 ° C 15.7 13.0 5.5 4.8 2.0 1.8 1.7 1.0 0.2 0.2 Clot formation time is affected by the amount of thrombin added,
The larger the amount of addition, the shorter the formation time at both room temperature and 37 ° C. However, by setting the thrombin amount to less than 30 units, the clot formation time at room temperature could be delayed, and in particular, by setting it to 6 units or less, it was possible to control it to 1 minute or more. Also, by setting the amount of thrombin to be more than 1 unit, the clot formation time at 37 ° C. assuming body temperature could be controlled to less than 13 minutes, and by setting it to 1.2 units or more, it could be controlled within 5.5 minutes. .

Example 2 : Relationship between the amount of added thrombin and the release of encapsulated drug [0022] A fluorescently labeled dextran (FITC-DEX) having a molecular weight of about 10,000 was encapsulated in a clot, and the effect of thrombin on the release of FITC-DEX was examined. . (Test method) (1) 80 mg of fibrinogen, 60 units of blood coagulation factor 13 and FITC-DEX (4 mg dissolved in 1 ml of phosphate buffered saline)
Was dissolved in 1 ml of a buffer solution (pH 7.5). (2) 1 ml of thrombin in calcium chloride solution (5.88 mg / ml)
And dissolved. (3) Take 0.1 ml of each of the above liquids (1) and (2) in a polypropylene centrifuge tube (10 ml), and after 5 minutes at 37 ° C,
8 ml of a phosphate-buffered physiological saline kept at room temperature was added. After storage at 37 ° C., the concentration of FITC-DEX in the solution after 5 minutes was quantified using a fluorometer (excitation wavelength: 494 nm, emission wavelength: 517 nm).

(Results) The results are shown in Table 2 below.

Table 2 Amount of thrombin (unit) 0.4 1.7 6.8 27.3 Release rate after 5 minutes (%) 100 10 10 10 When the amount of thrombin added is 0.4 units, the drug release rate after 5 minutes is It reached 100% and did not solidify at all.
On the other hand, at 1.7 units or more, the release rate was only 10%, indicating that clots were formed quickly.

Example 3 Release of rIL-2 from Clot in Vivo The preparation of the present invention containing rIL-2 (Immuneth, Shionogi) was administered subcutaneously to mice, and rIL- remaining in the formed clot was administered. The time course of the two levels and plasma concentration was determined. (Test method) (1) Fibrinogen 24 mg, blood coagulation factor 13 18 units,
Thrombin 0.9 units, calcium chloride 1.76 mg, and rI
After preparing about 0.6 ml of an aqueous solution containing 10166 JRU of L-2,
It was set in a syringe and administered subcutaneously on the back of C3H / HeN mice. (2) After a certain period of time, blood was collected from the vena cava under ether anesthesia, and the clot was recovered. (3) The clot was sufficiently crushed with a homogenizer and the supernatant after centrifugation was used as a sample. The blood was separated from plasma, and the rIL-2 concentration contained in each was quantified by ELISA (Amersham quantification kit).

(Results) FIG. 1 shows the time-dependent change in the residual amount of rIL-2 in the clot after administration, and FIG. 2 shows the rIL-2 concentration in plasma. RIL-2 remains in the clot for a long time, and even after one week
0.2% remained. Compared with the control aqueous solution, the plasma concentration of the preparation of the present invention was maintained without a sudden rise in the plasma concentration immediately after administration, and the bioavailability (A
UC: area under the plasma concentration-time curve) increased, and the absorption rate improved.

Example 4 : When hyaluronic acid was added
In vivo release of rIL-2 For two preparations of the present invention having different contents of fibrinogen, blood coagulation factor 13, thrombin, calcium chloride and rIL-2 (different amounts of gabexate mesylate), the rIL of sodium hyaluronate was The effect of -2 on release was investigated in mice. (Test method) (1) Fibrinogen 24 mg, blood coagulation factor 13 18 units,
Thrombin 0.9 units, calcium chloride 1.76 mg, and rI
L-2 was encapsulated at 28000 JRU, and a preparation I of the present invention containing no gabexate mesylate and a preparation II of the present invention containing 1.2 mg of gabexate mesilate were prepared. (2) Each of the above preparations or a preparation containing about 2 mg of sodium hyaluronate (about 0.6 ml) was subcutaneously administered to the back of a C3H / HeN mouse. (3) Blood was collected from the vena cava 2 and 4 hours after administration, and r
IL-2 concentration was quantified by ELISA (Amersham quantification kit).

(Results) The results are shown in Table 3.

[Table 3] By adding hyaluronic acid, any formulation
Plasma concentrations decreased at 2 hours and increased at 4 hours. This means that the release of rIL-2 from the clot was delayed by the addition of hyaluronic acid.

Example 5 : Release of IFN-γ from clot in vivo Recombinant interferon-gamma (IFN-γ)
Of the present invention containing 100,000 JRU was administered subcutaneously to the back of the mouse, and the residual amount in the clot and the change in plasma concentration over time were measured up to 24 hours after the administration. (Test Method) (1) IFN-γ was encapsulated in the preparation of the present invention having the same composition as that of Example 3, and 0.6 ml was administered subcutaneously to the back of C3H / HeN mice. IFN-
The dose of γ was adjusted to 100,000 JRU. (2) After a certain period of time, blood was collected from the vena cava under ether anesthesia, and the clot was recovered. (3) The clot was broken with plasmin after shredding. The IFN-γ concentration in the supernatant and the plasma after the centrifugation was quantified by ELISA (quantification kit manufactured by BioSource).

(Results) FIG. 3 shows the time-dependent change in the amount of IFN-γ remaining in the clot after administration, and FIG. 4 shows the IFN-γ concentration in plasma. About 30% of IFN-γ remained in the clot even after 24 hours. The plasma concentration remained almost plateau after 4 hours.

Example 6 : Antitumor activity of rIL-2-containing preparation The rIL-2-containing preparation of the present invention was subcutaneously administered to the back of a mouse transplanted with myeloma cells, and the tumor weight was measured one week later. The antitumor effect was evaluated. (Test Method) (1) Mouse myeloma cells X5563 were subcultured intraperitoneally in C3H / HeN mice, and 2 × 10 ⁵ cells were subcutaneously transplanted subcutaneously to the back of syngeneic mice. (2) fibrinogen 24 mg, blood coagulation factor 13 18 units,
The preparation of the present invention (about 0.6 ml) containing 0.9 units of thrombin, 1.76 mg of calcium chloride, and various concentrations of rIL-2 was administered subcutaneously to the back of the above mice (one week after transplantation). (3) One week after administration of the preparation, blood was collected from the vena cava under ether anesthesia, and a tumor mass was recovered. (4) Heparin was added to the blood and centrifuged to separate plasma, and rIL
-2 concentration was quantified by ELISA (Amersham quantification kit). (5) The weight of the tumor mass was measured. The tumor activity was expressed as a ratio to the tumor weight of the control to which physiological saline was administered, and expressed as a T / C value with the administration day being 1.

(Results) The results are shown in FIG. In a single dose of the control aqueous solution, the T / C value was
The value was 0.7, however, in the case of the preparation of the present invention, it was reduced to 0.5 or less, and enhancement of the tumor effect was observed.

Example 7 : Antitumor effect of rIL-2 inclusion preparation on human solid tumors The present invention contains rIL-2 in a system in which human hepatocellular carcinoma cells (HuH-7) are implanted subcutaneously in the back of nude mice. The antitumor effect of the formulation was evaluated. (Test method) (1) Human hepatocellular carcinoma cells (HuH-7) were subcultured subcutaneously on the back of nude mice, and 100 mg thereof was transplanted subcutaneously on the back 7 days after transplantation.
The preparation of the present invention having the same composition as in Example 3 (the content of rIL-2 was appropriately changed) was continuously administered around the tumor for 10 days (from the 7th day to the 16th day after transplantation). As a control, an aqueous solution of rIL-2 was used. In each case, five mice were used per group. (2) The size of the tumor was measured over time.

(Results) T /
FIG. 6 shows the C change, and FIG. 7 shows the T / C change when an aqueous solution of rIL-2 was administered as a control. In the preparation of the present invention, rIL-
It exhibited superior antitumor activity at lower doses than the aqueous solution of 2.

Example 8 A kit of the present invention containing rIL-2 composed of the following combinations is prepared. A: 80 mg of fibrinogen, blood coagulation factor 13
A lyophilized vial containing 0 units, 19 mg human serum albumin, and about 10,000-360,000 JRU of rIL-2. B: Lyophilized vial containing 3 units of thrombin. C: 1 ml of an aqueous calcium chloride solution (containing 5.9 mg as calcium chloride) Example 9 A kit of the present invention containing IFN-γ composed of the following combinations is prepared. A: 80 mg of fibrinogen, blood coagulation factor 13
0 units, human serum albumin 19 mg, and IFN-γ
Lyophilized vial containing about 1 to 3 million JRU. B: Lyophilized vial containing 3 units of thrombin. C: 1 ml of an aqueous solution of calcium chloride (containing 5.9 mg as calcium chloride)

[0035]

The preparation of the present invention hardly coagulates until administration, and can be administered as an aqueous solution, but when it enters the body after injection, it immediately becomes a gel or a solid state to form a fibrin clot. Pharmaceuticals contained in fibrin clots are gradually released in the body along with their decomposition. Sustained release enhances the pharmacologic effect of the drug because it can reduce frequent dosing and can also maintain drug levels in tissues at a constant level. In addition, by suppressing a rapid increase in tissue concentration, side effects can be reduced.

[0036]

[Brief description of the drawings]

FIG. 1 is a graph showing the time course of the residual amount of rIL-2 in a clot when a formulation of the present invention containing rIL-2 is subcutaneously administered to mice. The vertical axis is the residual rate (%) in the clot,
The horizontal axis represents time (hr).

FIG. 2 is a graph showing the time course of plasma rIL-2 concentration when a formulation of the present invention (clot formulation) containing rIL-2 and a control formulation (aqueous solution) are subcutaneously administered to mice. The vertical axis represents plasma concentration (JRU / ml), and the horizontal axis represents time (hr).

FIG. 3 is a graph showing the time-dependent change in the amount of residual IFN-γ in a clot when the present preparation containing IFN-γ is subcutaneously administered to mice. The vertical axis represents the residual rate (%) in the clot, and the horizontal axis represents time (hr).

[0037]

FIG. 4 is a graph showing the time-dependent changes in the plasma IFN-γ concentration when the preparation of the present invention containing IFN-γ is subcutaneously administered to mice. The vertical axis represents the plasma concentration (JRU / m
l), the horizontal axis represents time (hr).

FIG. 5 shows the tumor weight after one week when the formulation of the present invention (clot formulation) containing rIL-2 and the control formulation (aqueous solution) were subcutaneously administered to mice transplanted with myeloma cells.
It is a graph which shows the result investigated by dosage. The vertical axis indicates the ratio (T / C) to the tumor weight of the control to which physiological saline was administered, and the horizontal axis indicates the dose of rIL-2 (× 10 ⁴ JRU / mouse).

FIG. 6. Formulations of the invention containing rIL-2 and rIL-2 as control
Clot preparation containing no hepatocellular carcinoma cells (HuH
FIG. 11 is a graph showing the results of examining the relative tumor weight ratio when -7) was administered continuously for 10 days (from day 7 to day 16 of implantation) in the vicinity of the tumor of a nude mouse implanted subcutaneously on the back. The vertical axis indicates the ratio (T / C) to the tumor weight of the control to which physiological saline was administered, and the horizontal axis indicates the number of days (days) after cell transplantation.

[0038]

FIG. 7 shows an aqueous solution containing rIL-2 as a control preparation of the present invention in the vicinity of a tumor of a nude mouse in which human hepatocellular carcinoma cells (HuH-7) were subcutaneously transplanted subcutaneously for 10 consecutive days (day 7 after transplantation). 15 is a graph showing the result of examining the relative tumor weight ratio when the compound was administered from the first day to the 16th day). The vertical axis represents the ratio (T / T) to the tumor weight of the control to which physiological saline was administered.
C), the horizontal axis shows the number of days (days) after cell transplantation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location A61K 47/42 A61K 37/66 H

Claims (9)

[Claims] 1. A sustained-release preparation for injection containing a pharmaceutically active ingredient, fibrinogen, blood coagulation factor 13, and thrombin, wherein the mixing ratio of fibrinogen and thrombin is 1 unit of thrombin with respect to 80 mg of fibrinogen. A formulation characterized by a ratio corresponding to an amount of more than 30 units. 2. The preparation according to claim 1, wherein the mixing ratio of fibrinogen to thrombin is a ratio corresponding to 1.2 to 6 units of thrombin with respect to 80 mg of fibrinogen. 3. The preparation according to claim 1, further comprising calcium ions. 4. The preparation according to claim 1, wherein the pharmaceutically active ingredient is a protein drug. 5. The preparation according to claim 4, wherein the pharmaceutically active ingredient is interleukin-2 or interferon-γ. 6. The method according to claim 1, further comprising hyaluronic acid.
A formulation as described. 7. The preparation according to claim 1, which is for subcutaneous injection. 8. The method according to claim 1, wherein the basic configuration includes:
A kit for preparing the above-described preparation at the time of use: A: a lyophilized product containing fibrinogen and blood coagulation factor 13; and B: a lyophilized product containing thrombin (pharmaceutical agent in any of the above lyophilized products A or B) The active ingredient is contained, and the compounding ratio of fibrinogen and thrombin is a ratio corresponding to an amount of more than 1 unit and less than 30 units of thrombin for 80 mg of fibrinogen.) 9. The kit according to claim 8, further comprising a preparation solution containing calcium ions, separately from the freeze-dried product of A or B.