JP2001342146A - Liquid fibrinogen preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fibrinogen-based preparation preservable stably in the form of a solution at room or normal temperature. SOLUTION: This liquid preparation is a fibrinogen-based one, containing a calcium ion source, and preferably optionally, a guanidino group-containing substance, and additionally, a blood coagulation factor XIII and a fibrinolysis inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a fibrinogen-containing preparation which is a component of a tissue adhesive existing as a two-part preparation. More specifically, the present invention relates to a liquid fibrinogen preparation that can be stored at room temperature or at room temperature in a solution state.

[0002]

BACKGROUND OF THE INVENTION Fibrinogen is a very important coagulation factor that is present at the end of the so-called coagulation cascade. Fibrinogen is converted by thrombin from its soluble form to insoluble fibrin, which makes a significant contribution to hemostasis and wound healing, for example, in the activation of the coagulation system after injury.

[0003] Fibrinogen is of importance for hemostasis and wound healing and is used clinically as an intravenous formulation in replacement therapy in congenital and acquired fibrinogen deficiency, for example, by increasing the concentration of fibrinogen in the blood. Prevent severe bleeding. Furthermore, in recent years,
By mixing fibrinogen with thrombin,
It is used in surgical procedures as an adhesive for suturing soft organs such as the liver or spleen, or as a suture aid, and at the same time has a wide range of clinical applications.

As disclosed in the claims and detailed description of the invention in Japanese Patent Publication No. 63-40546, fibrinogen and blood coagulation X
Methods for producing tissue adhesives containing factor III (factor XIII) are known. It is known that lyophilized fibrinogen or a lyophilizate of a plasma protein mixture containing a high proportion of fibrinogen gradually redissolves only at elevated temperatures during reconstitution with a lysate.

[0005] In order to obtain a sufficient adhesive action as a fibrin adhesive, it is necessary to dissolve fibrinogen in a high concentration, and the higher the concentration of fibrinogen that can be coagulated, the more advantageous. However, it takes time to prepare such a high-concentration fibrinogen solution from a lyophilized fibrinogen solution, and it cannot be used for emergency use. Furthermore, if the time required for the operation of dissolving the freeze-dried fibrinogen is prolonged, it is feared that the patient may be adversely affected.

Japanese Patent Laid-Open Publication No. Sho 63-40547 discloses a technique for freezing a fibrinogen solution having the above-mentioned composition at a low temperature to enhance the stability. In order to provide a preparation containing a higher concentration of fibrinogen, attention was paid to a solvent that increases the solubility of fibrinogen, and 6-10% (w
A technique of preparing a fibrinogen solution of / v), storing it in a frozen state, and thawing it before use has been developed (Japanese Patent Publication No. 57-149229). Although the above-mentioned frozen formulation has improved convenience in use compared to the freeze-dried formulation, it still has problems such as the fact that it requires time for thawing before use, and that it requires equipment for storage in a frozen state. Was included.

Therefore, Japanese Patent Application Laid-Open No. Hei 7-730
No. 73, as a component of tissue adhesive, and further as a fibrinogen concentrate for intravenous administration, to be able to respond to emergency use, stably in solution under cold storage conditions avoiding freezing. Techniques for preparing a fibrinogen-based preparation that can be stored are disclosed. However, although the liquid preservative formulation has improved convenience in use, there is still room for improvement in that it requires time to return to room temperature when used, and that it requires facilities for storage at low temperatures. Was.

[0008] Under such circumstances, an object of the present invention is to prepare a preparation containing fibrinogen as a main component which can be stably stored in a solution state under room temperature or room temperature storage conditions. In addition, room temperature and normal temperature are defined in the Japanese Pharmacopoeia general rules as 1-30 ° C. for room temperature and 15-25 ° C. for normal temperature.

The problem with the above problem is that fibrinogen is decomposed over time by storage in a solution state, and the degree of decomposition depends on the storage temperature. Table 1 and FIG. 1 show the results of evaluation of the degree of fibrinogen degradation under each storage temperature condition, based on the change in the purity of the coagulable protein, which is an index of fibrinogen degradation. as a result,
When stored at a low temperature of 15 ° C or less, the purity of the coagulable protein, which indicates the degree of fibrinogen degradation, hardly changes, but is kept at 25 ° C.
In the above storage, the purity of the coagulating protein decreases with time,
The degree is more remarkable as the storage temperature is higher. From this, the fibrinogen solution is allowed to reach the temperature range of room temperature (1 to
30 ℃), especially high temperature range above normal temperature (15 ℃ or more)
In the case of storage for a long time, it is important to suppress the decomposition of fibrinogen.

[0010]

[Table 1]

[0011]

The inventors of the present invention have conducted intensive studies in view of the above-mentioned problems. As a result, when a small amount of a calcium ion source is allowed to coexist in a fibrinogen solution, the fibrinogen solution can be used in a high temperature region at room temperature (15 ° C. or higher). In storage, the knowledge that the decomposition of fibrinogen can be suppressed without causing gelation due to the presence of calcium ions was obtained, and by pre-sterilizing and storing a fibrinogen solution in a coating device in advance and storing, the room temperature and high temperature range above normal temperature (15-30 ° C.) It has made it possible to prepare fibrinogen which can be stored stably in a solution state during storage and with simplicity. Further, since the fibrinogen solution tends to gel in a low-temperature environment, a substance having a guanidino group having a gelation-suppressing action is added to the above-mentioned calcium ion source-added fibrinogen solution. It has been found that gelation can be suppressed without significantly reducing the stabilizing effect, and it has been found that the fibrinogen solution can be stably stored even at room temperature and low temperature range (1 to 15 ° C) lower than room temperature. From the above, liquid fibrinogen preparation that can be stably stored at room temperature by adding an optimal concentration of calcium ion source to the fibrinogen solution, more preferably coexist with a substance having an effect of inhibiting gelation at an optimal concentration. As a result, the present invention has been completed which provides a liquid fibrinogen preparation which can be stably stored under a wide temperature range of room temperature including low temperature conditions. Hereinafter, the present invention will be described in more detail.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The fibrinogen preparation of the present invention which is stably stored in a solution state may be a suitable buffer, for example, a citrate buffer (for example, a p buffer containing 0.3 M sodium chloride).
H7.0 (0.04M citrate buffer) in 5% (w /
v) A major feature is that a fibrinogen solution having the above concentration contains a calcium ion source. Calcium ion sources include calcium chloride, calcium hydroxide,
Examples thereof include calcium dihydrogen phosphate and calcium sulfate, and calcium chloride is particularly preferred. The amount of the calcium ion source to be added is selected in a concentration range of 0.05 to 2 mM, and particularly preferably 0.2 to 1 mM. The mechanism of action of the suppression of fibrinogen degradation by the calcium ion source is presumed to be that calcium ions cause slight cross-linking or structural change of the γ chain, and the fibrinogen molecule becomes structurally resistant to protease attack. You. It is known that gelation occurs when calcium ions are present in a fibrinogen solution at a certain concentration or higher. However, the above-mentioned fibrinogen solution containing a low-concentration calcium ion source is not less than room temperature (15-30 ° C.), which is a high temperature region at room temperature. Decomposition accompanying high-temperature storage is suppressed without increasing the gelation or viscosity in the storage in step (1).

Although the fibrinogen solution gels in a low-temperature environment, the effect of a substance having a guanidino group as a gelling inhibitor during low-temperature storage has been disclosed (Japanese Patent Application Publication No. Hei 7-1995). No. 173073), which can be added for the purpose of avoiding gelation accompanying storage in a low temperature range (1 to 15 ° C.) at room temperature. The substance having a guanidino group here is not particularly limited, but arginine, guanidine and the like are particularly preferable embodiments,
These can be added alone or in combination. The addition amount is preferably a concentration of 0.1 to 2.0 M, and is preferably 0.1 to 2.0 M.
Addition at a concentration of 1-1.0 M is the optimal embodiment.

[0014] Fibrinogen, which is the main component of the preparation of the present invention, is prepared by a known method. As such, for example, a method in which the solubility reduction effect of fibrinogen by glycine is combined with a cold ethanol precipitation method (Blomback, B. and Blomback, M., Arkiv Kemi, 10 , p.415)
443, (1956)) and a glycine precipitation method using glycine alone (Kazal, LA, et al., Proc. Soc. Exp. Med., 11
3 , p. 989-994, (1963)).

The fibrinogen preparation of the present invention may further contain blood coagulation factor XIII and a fibrinolysis inhibitor for the purpose of forming a crosslink in the fibrin gel-matrix and inhibiting fibrinolysis. Fibrinolysis inhibitor is not particularly limited, aprotinin, alpha ₂ plasmin inhibitor tranexamic acid, epsilon, etc. aminocaproic acid. There are no particular restrictions on the order of the steps of adding the additives according to the present invention and the additional additives, as long as the above-mentioned additives are contained at a predetermined concentration in the final preparation. After freeze-drying and heat treatment for virus inactivation, it is preferably added at the time of final preparation after reconstitution.

In the above solution composition, the fibrinogen solution can be stably stored in a solution state under room temperature storage conditions. Room temperature is defined as 1 to 30 ° C., and varies within this range depending on the season and region, but is generally room temperature (15 to 25 ° C.).
° C).

Further, the liquid fibrinogen preparation can be used by being previously dispensed into a coating device such as a syringe. After sterilizing filtration of the fibrinogen solution of the present invention, the solution is dispensed into a sterilized syringe and the tip thereof is capped, so that the preparation can be directly used without being transferred to another container during clinical use. In this case, a syringe made of a synthetic resin such as polypropylene can be suitably used from the viewpoint of convenience.

The above-mentioned liquid fibrinogen preparation of the present invention is used not only alone, but also in combination with a component containing thrombin as a main component to constitute a biological adhesive for human and animal tissues. Applied in clinical settings.

Hereinafter, the present invention will be described specifically with reference to Test Examples and Examples, but the present invention is not limited to these Examples.

[0020]

EXAMPLES Test Example 1 0.04M at pH 7.0 containing 0.3M sodium chloride
Various concentrations of calcium chloride were added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a citrate buffer, and the stability was tested over time at a temperature of 30 ° C. Stability was assessed by visual assessment of the condition, turbidity, coagulable protein purity, and kinematic viscosity (measured at 20 ° C.) for the three storage periods up to six months later, with values before storage. Was. The coagulable protein purity was measured according to the coagulable protein content and purity test, which is a test of dried human fibrinogen fraction products described in the Ministry of Health and Welfare's “Biological Standards”.

The results are shown in Table 2 (fibrinogen solution 30
Table 3 and FIG. 2 (measured values of the purity of the coagulable protein over time when the fibrinogen solution was stored at 30 ° C.), and Table 4 and FIG. 3 (the fibrinogen solution was stored at 30 ° C.). (Measured value of kinematic viscosity over time). The storage at 30 ° C. caused the purity of the coagulable protein to decrease over time, but the addition of calcium chloride showed an effect of suppressing this change over time in a concentration-dependent manner, which proved to be effective for the storage stability of fibrinogen. did. Regarding the visual characteristics, gelation of the samples with 1 mM and 2 mM calcium chloride added progressed with time, but no change was observed with time in any of the other samples and no difference due to the concentration of calcium chloride added. However, the measured kinematic viscosity was 0.5 m
It increases with time in the sample containing M calcium chloride. In this sample, the kinematic viscosity measurement condition (20 ° C.) is changed from the storage temperature (30 ° C.).
The viscosity increased when the temperature was lowered, suggesting that the upper limit of calcium chloride addition that does not lead to gelation depends on the storage temperature, and that samples with lower concentration of calcium chloride also gel at lower temperature storage. Was done.

[0022]

[Table 2]

[0023]

[Table 3]

[0024]

[Table 4]

Test Example 2 0.04 M at pH 7.0 containing 0.3 M sodium chloride
Various concentrations of calcium chloride were added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a citrate buffer, and the stability was tested over time at a temperature of 10 ° C. Stability was evaluated by visual state determination of the stored specimen.

The results are shown in Table 5 (Fibrinogen solution 1
Over time when stored at 0 ° C.). The fibrinogen solution gelled by storage at 10 ° C., and the addition of calcium chloride promoted the gelation in a concentration-dependent manner. In addition, the time required for heating the gelled sample at a temperature of about 37 ° C. to return to a solution state was extended in a manner dependent on the concentration of calcium chloride added, so that calcium chloride enhances gelation. confirmed.

[0027]

[Table 5]

Test Example 3 In Test Examples 1 and 2, it was suggested that the gelation was enhanced by the addition of calcium chloride, and the effect of adding arginine, which is an embodiment of a substance having a gelation inhibitory effect, was examined. In order to do so, the following test was conducted.
0.04M at pH 7.0 with 0.3M sodium chloride
Various concentrations of calcium chloride and arginine were added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a citrate buffer, and stability was tested over time at temperatures of 10 ° C and 30 ° C. Was. Stability was determined by visual observation, storage of the coagulable protein, and kinematic viscosity (20 points at 4 points for up to 4 weeks for specimens stored at 10 ° C. and 3 points at up to 6 months for specimens stored at 30 ° C.).
(Measured in ° C) was compared to the value before storage.

The results are shown in Table 6 (Fibrinogen solution 1
Table 7 (measured values of the purity of the coagulating protein when the fibrinogen solution was stored at 10 ° C. over time), and Table 8 (the kinematic viscosity when the fibrinogen solution was stored at 10 ° C.) Table 9 (Changes in properties over time when fibrinogen solution is stored at 30 ° C.),
Table 10 (measured values of the purity of the coagulable protein over time when the fibrinogen solution was stored at 30 ° C.) and Table 11 (measured values over time of the kinematic viscosity when the fibrinogen solution was stored at 30 ° C.) are shown.

When stored at 10 ° C., it was confirmed that the samples having any calcium chloride and arginine concentrations did not decrease the coagulable protein purity and could be stored stably. It has been found that gelation and increase in kinematic viscosity during storage can be suppressed by adding 0.3 M or more arginine in the presence of 1 mM or less calcium chloride. 30 ° C
In storage, by adding 0.5 mM or more of calcium chloride, a decrease in the coagulation protein purity due to storage can be effectively prevented, and even if arginine at a concentration of 0.3 M or less coexists in these samples, this stabilization can be achieved. It was confirmed that the effect was not affected. The increase in the kinematic viscosity of each sample was dependent on the concentration of calcium chloride added, but it was suggested that the addition of about 0.3 M arginine could prevent the addition of calcium chloride of 1 mM or less.

[0031]

[Table 6]

[0032]

[Table 7]

[0033]

[Table 8]

[0034]

[Table 9]

[0035]

[Table 10]

[0036]

[Table 11]

Test Example 4 In Test Example 3, it was suggested that an appropriate combination of calcium chloride and arginine was effective in stabilizing liquid fibrinogen under a wide temperature range. Regarding the combination of arginine, the stability at 1 ° C., which is the lowest temperature at room temperature, was examined. PH 7.0 containing 0.3 M sodium chloride.
To a fibrinogen solution dissolved at a concentration of 8% (w / v) in a 04 M citrate buffer, 0.5 mM of calcium chloride and 0.3 M of arginine were added, and the stability was maintained over time at a temperature of 1 ° C. Was tested. Stability was evaluated by comparing the condition of the sample stored for up to one month by visual observation, the purity of the coagulating protein, and the kinematic viscosity (measured at 20 ° C.) with the values before storage.

The results are shown in Table 12 (time-dependent change when the fibrinogen solution was stored at 1 ° C.). Properties,
There was no significant change in the coagulation protein purity and the kinematic viscosity before and after storage, confirming that the protein can be stored stably.

[0039]

[Table 12]

Example 1 A cryoprecipitate obtained from citrate plasma was solubilized,
The fibrinogen precipitate precipitated using a 1.5 M glycine solution was dissolved in a 0.025 M citrate buffer at pH 8.0 containing 0.15 M sodium chloride at 37 ° C. After cooling the fibrinogen solution to -2 ° C, 8% (v
/ V) Ethanol was added and the precipitate was collected. The resulting precipitate was dissolved at 37 ° C. in a 0.04 M citrate buffer at pH 7.0 containing 0.3 M sodium chloride, 0.5 mM calcium chloride, 0.3 M arginine and 1000 KIE / ml aprotinin. Undissolved parts were removed by centrifugation. A fibrinogen concentration of about 8% (w /
After adjusting to v), the mixture was aseptically filtered and dispensed into containers.

Example 2 Solubilized cryoprecipitate obtained from citrated plasma
The fibrinogen precipitate precipitated using a 1.5 M glycine solution was dissolved in a 0.025 M citrate buffer at pH 8.0 containing 0.15 M sodium chloride at 37 ° C. After cooling the fibrinogen solution to -2 ° C, 8% (v
/ V) Ethanol was added and the precipitate was collected. The resulting precipitate was washed with a pH 7.0 solution containing 0.075 M sodium chloride.
It was dissolved at 37 ° C. in 0 mM citrate buffer. After adjusting the fibrinogen concentration to about 2% (w / v), 25 units of factor XIII were added per ml of fibrinogen solution. The solution was sterile filtered, lyophilized and subsequently subjected to a dry heat treatment, and the resulting lyophilized powder was treated with a pH 7.0 solution containing 0.5 mM calcium chloride, 0.3 M arginine and 1000 KIE / ml aprotinin. It was solubilized to a fibrinogen concentration of about 8% (w / v). This solution was aseptically filtered, 3 ml was dispensed into a sterilized 5 ml syringe made of polypropylene, and the tip was capped to obtain a liquid fibrinogen preparation in a syringe.

[0042]

The advantage of the present invention is that it enables the storage of a liquid fibrinogen preparation at room temperature without the necessity of dissolving fibrinogen or transferring it to a syringe or the like, and in addition to improving the convenience in emergency use, This eliminates the need for special equipment for storing the formulation.

[Brief description of the drawings]

FIG. 1. Each temperature of the fibrinogen solution (4 ° C., 15 ° C.)
FIG. 7 is a graph showing the change over time in the purity of a coagulating protein with storage at 25 ° C., 25 ° C., 30 ° C., and 37 ° C.).

FIG. 2 is a graph showing the change over time in the purity of a coagulable protein when a calcium chloride-added fibrinogen solution at each concentration is stored at 30 ° C.

FIG. 3 Kinematic viscosity (measurement temperature: 20 ° C.) when calcium chloride-added fibrinogen solution of each concentration is stored at 30 ° C.
FIG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/22 A61P 41/00 A61P 7/04 A61K 37/465 41/00 37/547 F-term (Reference) 4C076 AA12 BB21 CC09 DD23 DD26 DD30 DD51 DD60 FF15 FF36 GG45 4C084 AA03 BA44 DC03 DC11 DC20 MA01 MA02 MA05 MA16 MA65 NA03 ZA532 ZA892 ZB212 ZC512

Claims (15)

[Claims] 1. A method for stabilizing fibrinogen, comprising adding a calcium ion source to a fibrinogen solution at a final concentration of 0.05 to 2 mM. 2. The method according to claim 1, wherein the calcium ion source is 0.2 to 1 m.
2. The method for stabilizing fibrinogen according to claim 1, wherein the stabilizing agent is contained at an M concentration. 3. A liquid fibrinogen preparation which can be stored in a solution under normal temperature storage conditions, comprising fibrinogen as a main component and a calcium ion source. 4. The method according to claim 1, wherein the calcium ion source is 0.05 to 2
The liquid fibrinogen preparation according to claim 3, which is contained at a concentration of mM. 5. The method according to claim 1, wherein the calcium ion source is 0.2 to 1 m.
The liquid fibrinogen preparation according to claim 3, which is contained at a concentration of M. 6. A liquid fibrinogen preparation which can be stored in a solution under room temperature and room temperature storage conditions, comprising fibrinogen as a main component and a substance having a calcium ion source and a guanidino group. 7. The method according to claim 1, wherein the calcium ion source is 0.05 to 2
The liquid fibrinogen preparation according to claim 6, which is contained at a concentration of mM. 8. The method according to claim 1, wherein the calcium ion source is 0.2 to 1 m.
The liquid fibrinogen preparation according to claim 6, which is contained at a concentration of M. 9. The liquid fibrinogen preparation according to claim 6, wherein the substance having a guanidino group is selected from arginine and guanidine. 10. The substance having a guanidino group in 0.1 g.
The liquid fibrinogen preparation according to claim 6, which is contained at a concentration of 1 to 2.0 M. 11. The substance having a guanidino group in 0.1 g
The liquid fibrinogen preparation according to claim 6, which is contained at a concentration of 1 to 1.0M. 12. The liquid fibrinogen preparation according to claim 3, further comprising blood coagulation factor XIII. 13. The liquid fibrinogen preparation according to claim 3, which further comprises a fibrinolytic inhibitor. 14. A biological adhesive for human and animal tissues by combining with a component containing thrombin as a main component.
4. The liquid fibrinogen preparation according to any one of 3. 15. The liquid fibrinogen preparation according to claim 3, which is previously dispensed into a coating device.