WO1996023523A1

WO1996023523A1 - Novel hemostatic composition

Info

Publication number: WO1996023523A1
Application number: PCT/KR1995/000189
Authority: WO
Inventors: Seung Ho Kim; Jin Young Lee; Moon Hi Han
Original assignee: Korea Institute Of Science And Technology
Priority date: 1995-02-03
Filing date: 1995-12-30
Publication date: 1996-08-08
Also published as: EP0754053A1; JPH10501554A; US5897860A

Abstract

Disclosed herein is a hemostatic composition comprising thrombin, casein kinase-II, and sphingosine or a sphingosine derivative, which shows an excellent hemostatic or hemopexic activity as well as tissue suture activity.

Description

NOVEL HEMOSTATIC COMPOSITION

BACKGROUND OF THE INVENTION

1 . FIELD OF THE INVENTION The present invention is related to a novel hemostatic composition comprising additives. More specifically, the present invention is related to a hemostatic composition comprising thrombin, plus casein inase II, plus sphingosine or a sphingosine derivative, which permits a rapid clotting and hemostasis.

2. DESCRIPTION OF THE PRIOR ARTS

The hemostasis involves three complex mechanisms, clot formation, rapid constriction of the injured blood vessel and the aggregation of platelets to form a plug on the injured surface of the blood vessel. A clot is formed by a series of transformations involving more than ten different proteins, calcium ion (Ca²⁺) and thromboplastin. When bleeding occurs, fibrinogen, which is highly soluble, is converted into insoluble fibrin monomer by the proteolytic action of thrombin. The fibrin monomers spontaneously associate to form a clot, on which factor IIIa act to aggregate of platelets to form a plug at on the injured surface of the blood vessel. There has been reported that phosphate groups attached to protein of fibrinogen affect the gelation of thrombin(Forsberg, P. 0., Thromb. Res., 53, 1-9, 1989) . And, various protein kinases such as protein kinase A (Engstrim, L., Edlund, B., Rangnarsson, U., Dahlqvist-Edberg, U., and Humble, E., Biochem. Biophys. Res. Ccommun., 96, 1507, 1980), protein kinase C (Papanikolaou, P., Humble, E. And Engstrom, L., FEBS Lett., 143, 199-204, 1982), casein kinase I (Itarte, E., Plana, M., Guasch, M. D., and Martos, C, Biochem. Biophys, Res. Commun., 117, 631-636, 1983), and casein kinase II (Humble, E., Heldin, P., Forsberg, P. O. and Engstrom, L., Arch, Biochem. Biophys., 241, 225-231, 1985) have been reported to be involved in the phosphorylation of fibrinogen. The effect of phosphorylation of fibrinogen on the thrombin-induced gelation of fibrinogen varies depending on the kinds of the kinase used to phosphorylate fibrinogen, and the phosphorylated fibrinogen is hardly cleaved by plasmin regardless the kind of the kinase involved in the phosphorylation thereof (Martin, S. C, et al . , Thromb. Res. 61, 243- 252, 1991) .

Sphingosine, which is one kind of sphingolipid, has important roles in the growth and differentiation of cells. Further, sphingosine also is reported to have an ability to increase the activity of casein kinase II (McDonald, O.Bradley; Hannun, Yusuf A.; Reynolds, E. Hugh and Sahyoun, Naji, Journal of Biological Chem. , Vol. 266, No. 32, pp 21773-21776, 1991) .

A property required to be served as an efficient hemostatic agent includes a fast control of bleeding in order to prevent a contamination of pathogens at the bleeding site or massive hemorrhage during operations. The thrombin preparations has been most widely employed as a hemostatic agent among commercially available different hemostatic compositions.

The inventors conducted extensive studies to develop a more efficient hemostatic composition, which allows a faster hemostasis than conventional hemostatic compositions. As a result thereof, they surprisingly found that, if the thrombin-containing hemostatic compositions contains casein kinase II, a protein phosphorylation enzyme, together with sphingosine or a sphingosine derivative, it can exhibit its hemostatic activity in a shorter period of time.

SUMMARY OF THE INVENTION

Thus, an object of the present invention is to provide a hemostatic composition comprising particular additives, which can promote a formation of fibrin clot .

This object of the present invention can be accomplished by a hemostatic composition comprising thrombin, casein kinase II, and sphingosine or a sphingosine derivative.

BRIEF DESCRIPTION OF DRAWINGS

This invention will be described in more detail with reference with accompanying drawings in which:

FIG. 1 is a graph showing hemostatic activities of hemostatic compositions containing various materials, in which the line -O- indicates a fibrinogen solution; the line -•- indicates a fibrinogen solution containing thrombin; the line -V- indicates a fibrinogen solution containing casein kinase II plus thrombin; and the line -T- indicates a fibrinogen solution containing sphingosine, casein kinase II, and thrombin.

FIG. 2 shows blood coagulation activities of the compositions containing various materials, in which test tube No. 1 contains a buffer "A"; test tube No. 2 contains a mixture containing buffer "A" (0.4 ml), buffer "B" (0.2 ml) and thrombin solution (0.2 ml); test tube No. 3 contains a mixture of buffer "A" (0.2 ml), buffer "B" (0.2 ml), thrombin solution (0.2 ml) and casein kinase II solution (0.2 ml); test tube No. 4 contains a mixture of buffer "A" (0.2 ml), buffer

"B" (0.2 ml), thrombin solution (0.2 ml) and sphingosine solution (0.2 ml); test tube No. 5 contains a mixture of buffer "B" (0.2 ml), thrombin solution (0.2 ml), casein kinase II solution (0.2 ml) and sphingosine solution (0.2 ml); test tube No. 6 contains a mixture of buffer "A" (0.4 ml), buffer "B"

(0.2 ml), sphingosine solution (0.2 ml); and test tube

No. 7 contains a mixture of buffer "A" (0.2 ml), buffer "B" (0.2 ml), casein kinase II solution (0.2 ml) and sphingosine solution (0.2 ml). Each samples was used in an amount of 0.8 ml.

The above and other objects, features and applications of the present invention will be apparent to the ordinary skilled in the art from the following detailed explanation.

DETAILED EXPLANATION OF THE INVENTION

The hemostatic composition according to the present invention comprises thrombin, casein kinase II, and sphingosine or a sphingosine derivative. For the present invention, casein kinase II, a protein phosphorylation enzyme, phosphorylates amino acids of fibrinogen. The phosphorylated fibrinogen is more prone to converted into fibrin by the action of thrombin.

When sphingosine or a sphingosine derivative is incorporated into the composition comprising thrombin and casein kinase II, the former enhances the activity of casein kinase II and thrombin, thereby the composition can stop bleeding in a shorter period of time than the composition without sphingosine or a sphingosine derivative.

The sphingosine derivatives may include, but not limited to, O-phosphorylethanol amine, sphingomyelin, glucosyl sphingosine, galactosyl sphingosine, sphingosine phosphates, erythrosphingenines, threo- sphingenines, stearoyl-sphingomyelin, ceramides, ceramide dihexosides, ceramide tetrahexosides, asia1ogang1ios ides , disialogang1iosides , trisialogangliosides, monosialogangliosides, and the like. These sphingosine derivatives may be used in single or mixtures thereof.

The amount of thrombin, which may be incorporated into the composition according to the present invention, may be more than 0.1 NIHU/ml. The unit of thrombin, "NIHU" means a United States (US) National Institute of Health (NIH, Bethesda, USA) standard unit, and one "NIHU" is equivalent to 1.1 to 1.3 IU of thrombin.

The amount of casein kinase II, which may be incorporated into the composition according to the present invention, may be in the range from 0.01 mU/ml 7 to 1 U/ml, and is preferably about 0.1 mU/ml based on the total amount of the composition. One (1) unit of casein kinase II is defined as the enzyme activity which catalyzes the transfer of 1 μmole phosphate from ATP to a substrate at 37°C in 1 minute.

The amount of sphingosine or its derivative, which may be incorporated into the composition according to the present invention, may be in the range from 0.01 μg/ l to 0.1 μg/ml, and is preferably about 0.05 μg/ml based on the total amount of the composition.

The composition according to the present invention can be formulated into various preparations, for example tablets, capsules, powders, solutions, suspensions and the like, according to the conventional techniques well known to the skilled in the pharmaceutical industries.

The composition according to the present invention can be administered by various routes depending on the purpose, target site or type of its formulations, which can be easily chosen by the skilled in the art.

The composition of the present invention can be used for hemostasis or hemopexis outside the living body as well as inside the living body, for example ulcer. Further, it can be used to treat hemophilia, or as an aid for treating hemophilia. Moreover, it can be used as a hemostatic composition as well as an aid for the conventional hemostatic compositions. It also can be used for suture of injured blood vessel. as well as an inhibitor for growth of bacteria, fungi, or virus.

The degree of formation of fibrin clot by the action of the composition of the present invention is calculated by measuring the changes in the absorbance of the mixtures of fibrinogen solution and the composition. The absorbance is measured using an UV/VIS spectrophotometer. And, the agglutination activity of the composition can be observed with naked eyes by mixing blood plasma of human being and the composition of the invention in accordance with Lee- White method.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will be embodied by way of the following examples. However, these examples are provided for the illustration purpose only and should not be construed as limiting the scope of the invention, which is properly delineated in the accompanying claims. The parts or percents in Examples are based on the weight, unless indicated otherwise.

Example 1

Fibrinogen (CalBiochem, USA) (24 mg) was dissolved into a mixture (100ml) of 100 mM phosphate buffered saline (pH 7.4) and 12 mM MgCl_2, and the resulting solution was evaporated to a final concentration of 1.2 mg fibrinogen/ml. MgCl₂ and ATP were added to 5 ml of the fibrinogen solution to the concentrations of 12 mM and 1 mM, respectively to give a substrate solution.

Thrombin (Sigma, USA) and casein kinase-II

(Boehringer Mannheim, Germany) were diluted with 100 mM phosphate buffered saline (pH 7.4) to the concentrations of 1 NIHU/ml and 1 mU/ml, respectively. Sphingosine (Sigma, USA) was dissolved into a minimum amount of ethanol and diluted with 100 mM phosphate buffered saline to a concentration of 0.1 mg/ml.

In order to measure the fibrin clot formed by thrombin or the composition of the invention, four samples were prepared: Φ fibrinogen solution 0.7 ml; © fibrinogen solution 0.7 ml + thrombin solution 0.1 ml (final concentration 0.1 NIHU/ml); (D fibrinogen solution 0.7 ml + thrombin solution 0.1 ml (final concentration 0.1 NIHU/ml) + casein kinase-II solution 0.1 ml (final concentration 0.1 mU/ml) ; and ® fibrinogen solution 0.7 ml + thrombin solution 0.1 ml (final concentration 0.1 mU/ml) + casein kinase II solution 0.1 ml (final concentration 0.1 mU/ml) + sphingosine (final concentration 10 μg/ml) . 50 mM phosphate buffered saline (pH 7.4) was added to these samples to make the final volume of all the samples to 1 ml. These samples were reacted at 37°C and the absorbencies at 633 nm were measured by using a UV/VIS spectrophotometer (Beckmann Model No. DU-70, USA) at 0, 5, 10, 15, 20, 25 and 30 minutes. The results are shown in FIG. 1. In FIG. 1, the line -O- indicates the fibrinogen solution; the line -•- indicates the fibrinogen solution containing thrombin; the line -V- indicates the fibrinogen solution containing casein kinase II plus thrombin; and the line -T- indicates the fibrinogen solution containing sphingosine, casein kinase II, and thrombin.

As can be seen from FIG. 1, the fibrinogen solution containing sphingosine, casein kinase II and thrombin forms a larger amount of fibrin clot than the fibrinogen solutions containing thrombin in single, or containing thrombin and casein kinase II.

Example 2 In order to estimate the blood coagulation activity of the composition of the invention, same materials as used in Example 1 were employed, except two kinds of buffer solutions were used; the buffer "A" is 50 mM phosphate buffered saline (pH 7.4) and the buffer "B" is 50 mM phosphate buffered saline (pH 7.4) containing ImM ATP and 12 mM MgCl₂. Each of the following samples (2) to (7) was adjusted to have a final concentration of 0.02 mM ATP. A blood coagulation activity of the samples was measured by modified Lee-White method. That is to say, seven test tubes (1) to (7) each containing 1.5 ml of human blood sample were prepared, and 0.1 ml of one of the following samples (1) to (7) was added to each test tube: Sample No. 1, the buffer "A"; sample No. 2, a mixture of buffer "A" (0.4 ml), buffer "B" (0.2 ml) and thrombin solution (0.2 ml); sample No. 3, a mixture of buffer "A" (0.2 ml), buffer "B" (0.2 ml), thrombin solution (0.2 ml) and casein kinase II solution (0.2 ml); sample No. 4, a mixture of buffer "A" (0.2 ml), buffer "B" (0.2 ml), thrombin solution (0.2 ml) and sphingosine solution (0.2 ml); sample No. 5, a mixture of buffer "B" (0.2 ml), thrombin solution (0.2 ml), casein kinase II solution (0.2 ml) and sphingosine solution (0.2 ml); sample No. 6, a mixture of buffer "A" (0.4 ml), buffer "B" (0.2 ml), sphingosine solution (0.2 ml); and sample No. 7, a mixture of buffer "A" (0.2 ml), buffer "B" (0.2 ml), casein kinase II solution (0.2 ml) and sphingosine solution (0.2 ml) .

Test tubes were allowed to stand at room temperature. 10 minutes later, test tubes were declined to observe coagulation of blood. Test tube Nos. 1, 6 and 7 shown no coagulation, while test tube Nos. 2 to 5, which contain thrombin, showed a coagulation. Among them, in particular the test tube No. 5 containing the composition of the invention showed the fast coagulation of blood.

And, when the test tubes were centrifuged at 1,000 rpm for five minutes, test tube Nos. 1, 6 and 7 shown a complete separation of plasma from the precipitated blood cells, while test tube Nos. 2 to 5 shown a slight or no separation. In particular, test tube No. 5 containing the composition of the invention shown no separation at all. These results are shown in FIG. 2.

Example 3

In order to measure the thrombin clotting time of the composition of the invention, 0.4 ml of platelet- rich plasma was placed into seven test tubes (10 X 75 mm) , to which each of seven samples in Example 2 was added in an amount of 0.1 ml. The test tubes were well shaken and the time required to form a fibrin clot was measured. As results, the test tube No. 5 formed a fibrin clot within 2 minutes ± 20 seconds, while the test tube No. 2 formed fibrin clots within 10 minutes ± 30 seconds.

Therefore, the composition of the invention formed fibrin clot as fast as about five times the comparative compositions.

Example 4: Acute toxicity

The composition of the present invention, which was prepared in Example 1, was intraperitoneally administered to ten ICR mice (five males plus five females) in an amount of 10 ml/kg. During seven days after the administration, the number of died animals and behavior of animals were observed. Seven days after the administration, the animals were sacrificed and autopsied to observe the organs with naked eyes. The results are summarized below.

1) There was no died animal and the animals' behaviors were normal during seven days after the administration.

2) Autopsy opinion Skin : Normal Eyes : Normal Liver : Normal Spleen : Normal

Kidney : Normal

Digestive organs : Normal

Genital and urinary organs : Normal

Heart : Normal Lung : Normal

Chest : Normal

Example 5

Two mice were injured with a syringe at coronary artery so that the artery is bleed, and the injured part of each mouse was covered with a cotton soaked with the inventive composition (Inventive) or thrombin solution (Control) of Example 1 for 2 minutes. The bleeding of the wounded part of the mouse of inventive group was stopped as well as the artery was sutured so that blood can be flown therein. By contrary, the wounded part of the control mouse was continued to be bleed.

Example 6

To 10 ml of human blood was added an equal amount of phosphate buffered saline (pH 7.4) containing 154 mM NaCl, and the resulting mixture was subjected to sucrose density gradient (1.077 g/1 ) centrifugation at

1,800 rpm, 20°C for 20 minutes to separate lymphocytes.

The lymphocytes were mixed with 50 ml of the same buffer and subjected to centrifugation (1,600 rpm) at 4°C for 5 minutes to remove impurities. To 100 μl of lymphocyte solution (3 X 10⁵ cells/ml) were added 50 μl of casein kinase II solution (10 mM, 1 mM or 0.1 mM) and 50 μl of ³H-thymidine. The resulting mixture was allowed to stand at room temperature for 3 days, and the radioactivity was measured every day. For comparison, a mixture of concanavalin A ("Con A") (10 μg/ml) and interleukin-2 ("IL-2") (50 IU/ml) was used instead of casein kinase II (CK-II) solution. The results are shown in Table 1. Table 1

(Unit: cpm)

1 day 2 days 3 days

Blank 517 319 485

Con A + IL-2 434 26,854 66,108

10 mM CK-II 478 361 500

1 mM CK-II 406 411 441

O.lmM CK-II 370 293 400

As can be seen from Table 1, casein kinase II does not affect the proliferation of lymphocytes.

As described in the above, the hemostatic composition according to the present invention containing thrombin, casein kinase II and sphingosine forms more fibrin clot in a shorter period of time than the conventional hemostatic composition containing thrombin only. Moreover, the composition according to the present invention shows a hemostatic activity as well as tissue suture activity.

Accordingly, the composition of the present invention can be used for hemostasis or hemopexis outside the living body as well as inside the living body, for example ulcer. Further, it can be used to treat hemophilia, or as an aid for treating hemophilia. Moreover, it- can be used as a hemostatic composition as well as an aid for the conventional hemostatic compositions. It also can be used for suture of wounded blood vessel, as well as inhibitor for growth of bacteria, fungi, or virus.

Claims

1. A hemostatic composition comprising thrombin, casein kinase-II, and sphingosine or a sphingosine derivative.

2. The composition of claim 1, which comprises thrombin, casein kinase-II, and sphingosine.

3. The composition of claim 1, which comprises casein kinase-II in an amount of 0.01 mU/ml to 1 mU/ml, and sphingosine or a sphingosine derivative in amount of 10 ng/ml to 100 ng/ml, based on the total amount of the composition.

4. The composition of claim 2, which comprises casein kinase-II in an amount of 0.01 mU/ml to 1 mU/ml, and sphingosine in amount of 10 ng/ml to 100 ng/ml, based on the total amount of the composition.

5. A pharmaceutical composition for treating ulcer, which comprises thrombin, casein kinase-II, and sphingosine or a sphingosine derivative.

6. A hemostasis aid composition comprising thrombin, casein kinase-II, and sphingosine or a sphingosine derivative. 17

7. A composition for suturing blood vessel, which comprises thrombin, casein kinase-II, and sphingosine or a sphingosine derivative.

8. A composition for treating or aiding treatment of hemophilia, which comprises thrombin, casein kinase- II, and sphingosine or a sphingosine derivative.

9. A composition for inhibiting growth of microorganisms, which comprises thrombin, casein kinase-II, and sphingosine or a sphingosine derivative.