JP4663837B2 - Pharmaceutical composition for preventing / treating bleeding disorders associated with thrombocytopenia, comprising factor VIII as the main component - Google Patents

Description

【図面の簡単な説明】
【図１】血小板減少動物モデルを用いた本願医薬組成物の効果を示す図である。[0001]
[Industrial application fields]
The present invention relates to a pharmaceutical composition comprising a blood-derived component as a main active ingredient. Specifically, the present invention relates to a pharmaceutical composition for bleeding disorders caused by platelet disorders such as thrombocytopenia or abnormal platelet function. More specifically, the present invention relates to a pharmaceutical composition for preventing / treating bleeding symptoms associated with platelet disorders containing blood coagulation factor VIII (hereinafter sometimes referred to as factor VIII or FVIII) as an active ingredient.
[0002]
[Prior art and problems to be solved by the invention]
Hemostasis is only achieved when blood vessels, platelets and blood clotting factors function normally. Thus, these functional declines cause a tendency to bleed, and significant declines cause excessive bleeding and are life-threatening.
Platelets are present in blood in the range of 1 to 360,000 / μL, and quickly gather at the damaged vascular injury site and play an important role in controlling hemostasis. However, if there is an abnormality in the hemostatic function of this platelet or the number of circulating platelets decreases to 50,000 / μL or less due to various causes, a bleeding tendency is induced. Severe thrombocytopenia of 10,000 / μL or less leads to spontaneous bleeding, which may increase the amount of bleeding and affect life. In particular, thrombocytopenia that develops with anticancer chemotherapy, which has been increasing in recent years, is a serious side effect, and its hemostasis management has become an important issue in cancer treatment (Junji Nishimura. History of Medicine 184: 448 -452, 1998).
[0003]
When thrombocytopenia is classified by mechanism, it can be divided into four main categories. 1) It is caused by a decrease in platelet production in the bone marrow, and there are cases where normal hematopoietic tissue is replaced by a tumor due to aplastic anemia, bone marrow hematopoiesis suppression after administration of an anticancer agent, acute leukemia, and the like. 2) Due to increased destruction of platelets, a typical disease is idiopathic thrombocytopenic purpura (ITP). In addition, there are thrombocytopenia due to drug allergy and neonatal thrombocytopenia due to platelet blood type incompatible pregnancy. 3) Some are due to increased consumption of platelets. In cases of disseminated intravascular coagulation (DIC) or thrombotic thrombocytopenic purpura (TTP), a large amount of platelets is consumed at the site of peripheral thrombus formation, resulting in decreased platelet count. Come on. 4) Something is due to abnormal platelet distribution. When platelets regenerated in the bone marrow are released to the periphery in a normal person, about 1/3 is captured in the spleen without circulating in the periphery. On the other hand, when there is cirrhosis or giant splenomegaly, most of the newly formed platelets are trapped in the spleen. As a result, even if platelets are produced in the bone marrow, platelets cannot be supplied enough to the periphery, resulting in thrombocytopenia (Kurata Yoshiyuki. General Clinical 47: 2742-2748, 1998). In addition, as platelet dysfunction, platelet asthenia caused by IIb / IIIa deficiency of platelet membrane protein, Bernard-Soulier disease of Ib deficiency, and the like are known.
[0004]
Hemostatic disturbances associated with platelet disorders result in increased bleeding tendency as shown in mucosal bleeding from the nasal / oral area or gastrointestinal route and wounds, ulcers and leaching from the injection site. Bleeding due to thrombocytopenia is widespread, especially during surgery, and thrombocytopenia becomes a serious problem during and after surgery. If the platelet count drops below 50,000 / μL, even small surgery such as extraction can cause severe bleeding. At significantly lower platelet counts of 10,000 / μL or less, spontaneous bleeding occurs without special events such as surgery or trauma, for example, even if the bleeding occurs in the skull, it is even fatal.
[0005]
By the way, at present, transfusion of platelet concentrate is the only effective treatment for bleeding symptoms caused by thrombocytopenia or abnormal platelet function. As an actual treatment mode, it is basically administered at 50,000 / μL or more at the time of surgery or when active bleeding is observed, and at 20,000 / μL or more in other cases. (Ministry of Health and Welfare, Pharmaceutical Affairs Bureau, Japanese Red Cross Society: About proper use of platelet products). However, when such concentrates are administered in large quantities and frequently, platelets collected from multiple blood donors are used. As a result, many patients who repeatedly receive platelet transfusions develop HLA antigens of mixed white blood cells and alloantibodies against platelet blood types, resulting in a platelet transfusion refractory state where the effect of platelet transfusion is weakened. Furthermore, in the platelet concentrate, lymphocytes that cause GVHD after blood transfusion and viruses that cause hepatitis and AIDS cannot be completely ruled out. Therefore, at present, platelet transfusion is the only effective treatment for bleeding symptoms caused by thrombocytopenia or abnormal platelet function, but there is an urgent need for a drug that can safely control hemostasis instead of platelet transfusion. .
[0006]
[Structure of the invention]
[Means for Solving the Problems]
In view of the above problems, the present inventors have conducted extensive research to develop a prophylactic / therapeutic agent for bleeding symptoms associated with platelet disorders. As a result, surprisingly, the purified factor VIII, which has not been attempted before, The present invention has been found to improve the hemostatic ability reduced by in vivo administration to almost non-existing animals, and has found that it can be sufficiently applied as a pharmaceutical composition for the prevention and treatment of hemostatic disorders associated with thrombocytopenia or functional abnormalities. It came to complete.
[0007]
Factor VIII, which is the main component of the pharmaceutical composition of the present invention, is synthesized mainly in the liver, and its concentration in plasma is 0.1 μg / mL, which is the lowest among coagulation factors. In the bloodstream, it forms a complex with vWF and circulates, and is protected from degradation by proteases such as activated protein C by forming the complex (Fay, PJ et al. J Biol Chem 266: 2172). -2176,1991). Since congenital deficiency of factor VIII is known as hemophilia A and presents severe bleeding symptoms due to coagulation disorders, blood-derived and recombinant factor VIII concentrates are used for hemostasis management in hemophilia A patients. Factor VIII functions not as a protease but as a coagulation cofactor, and has the effect of remarkably increasing the catalytic efficiency of the limited decomposition (activation) reaction of blood coagulation factor X by activated blood coagulation factor IX. The domain structure of factor VIII is homologous to A1 (amino acid residues 1-328), A2 (380-711), A3 (1694-2019) and discoidin lectin having homology with blood coagulation factor V and ceruloplasmin. It consists of C1 (amino acid residues 2020-2172), C2 (2173-3232) and B domain. Furthermore, factor VIII has two acidic sequences, which are called a1 (331-372) and a2 (1649-1689), respectively, and a2 functions as a binding site for vWF. The cofactor activity of factor VIII is exerted by the dissociation of factor VIII from vWF by limited degradation by thrombin, and is enhanced by limited degradation of Arg372-Ser373 binding in the factor VIII molecule (Eaton, D. et al. Biochemistry 25: 505-512, 1986).
[0008]
The method for producing Factor VIII, which is the main component of the pharmaceutical composition of the present invention, is not particularly limited, and can be produced by, for example, a method of separating from human blood or a method of producing by gene recombination technology.
[0009]
The following method is mentioned as a manufacturing method of blood-derived factor VIII. For example, there is a method of purifying factor VIII from a cryoprecipitate obtained by cold thawing fresh frozen human plasma by affinity chromatography using an anti-vWF monoclonal antibody or an anti-factor VIII monoclonal antibody. (Rotblat, F., et al., Biochemistry 24: 4294-4300, 1985).
[0010]
In order to maximize the activity of the factor VIII prepared by the method described above, it can be lyophilized and stored with suitable stabilizers, or the factor VIII solution can be frozen and stored. Is possible. In the present invention, factor VIII as an active ingredient can be combined with a known appropriate excipient, and the preparation for prevention / treatment of hemostatic disorders associated with thrombocytopenia of the present invention can be obtained by a known method.
[0011]
The administration target of the factor VIII-containing preparation of the present invention is not particularly limited as long as it is a patient with hemostasis disorder associated with thrombocytopenia or functional decline. The effective dose of factor VIII varies depending on, for example, the age, symptoms and severity of the subject of administration, but a hemostatic effect can be expected in the range of 50 units / kg to 100 units / kg in factor VIII activity. As the administration method, single administration (Bolus) or intravenous infusion is optimal.
When factor VIII is used as a prophylactic / therapeutic method for hemostasis disorder associated with thrombocytopenia, factor VIII can be administered alone, or in combination with vWF or with vWF / FVIII complex to increase the effect It can be expected as an effective means.
[0012]
The blood-derived FVIII used in this example is FVIII from which vWF has been separated and removed, or vWF / FVIII complex containing vWF, and its multimer pattern is similar to that of plasma. The vWF / FVIII complex is administered to patients with hemophilia or von Willebrand disease in clinical settings, and its safety has been confirmed.
[0013]
EXAMPLES Hereinafter, although this invention is demonstrated further in detail along an Example, these Examples do not limit the scope of the present invention.
[0014]
EXAMPLES Hereinafter, although this invention is demonstrated further in detail along an Example, these Examples do not limit the scope of the present invention.
[0015]
Preparation example After cryoprecipitate obtained by cold thawing of fresh frozen human plasma was purified as a vWF / FVIII complex by cation exchange chromatography and precipitation fractionation, 350 mM calcium chloride was present. VWF and FVIII were dissociated by gel filtration using Sephacryl S-500 below.
[0016]
Example 1
(Effect of the present pharmaceutical composition using a thrombocytopenic animal model)
The hemostatic effect on bleeding tendency caused by platelet reduction by FVIII administration could be confirmed by the following examples.
This experiment was mainly conducted according to a method established as a model that reflects thrombocytopenia associated with anticancer drug administration (Kuter, DJ et al. Blood 85: .2720-2730, 1995). It is considered to be an evaluation system suitable as a typical thrombocytopenic bleeding model. Rats (Wistar strain, male, body weight 180-230 g) were subcutaneously administered on the back with “Busulfan” (trade name) as an anticancer agent in an amount of 12.5 mg / kg to induce a thrombocytopenic state. On the 14th day when the decrease in the platelet count was most remarkable, it was fixed to the dorsal position under anesthesia with pentobarbital, and a 3 mm diameter tail was cut with a cutter blade and bled. The bleeding blood was sucked through a filter, and the amount of bleeding was determined by hemoglobin determination and evaluated. The blood loss of normal rats having a platelet count of about 600,000 / μL is 112.2 ± 61.0 μL, while the amount of blood loss increases in the test animals as the platelet count decreases and the platelet count is 0.1 ±. At 0.1000 / μL, it increased significantly to 1202.8 ± 188.3 μL.
Rats were thrombocytopenic as in control animals and then administered human FVIII. For human FVIII, recombinant FVIII (manufactured by BAYER Yakuhin, “KOGENATE” (trade name)) is used, and the administration dose is 10, 20 U / kg, 10 minutes before the start of evaluation from the rat external jugular vein. did. Similarly, human FVIIa was administered at 500 U / kg for evaluation. The results are summarized in Table 1 and FIG. The blood loss of thrombocytopenic rats was significantly reduced depending on the dose of FVIII administered compared to the control animal group. In one of the animals administered FVIII at 20 U / kg, hemostasis was completely stopped within the observation time of 30 minutes, and the amount of bleeding was as low as that of normal rats. Although a reduction in the amount of bleeding was also observed in the group administered with a high dose of 500 U / kg FVIIa, whose hemostatic effect was shown in Japanese Patent Publication No. 7-80783, administration of FVIII exceeded that effect.
[0017]
[Table 1]

[Brief description of the drawings]
FIG. 1 is a graph showing the effect of the pharmaceutical composition of the present application using a thrombocytopenic animal model.

Claims (4)

 A composition having an effective amount of blood coagulation factor VIII (hereinafter sometimes referred to as factor VIII or FVIII) for hemostasis as a medicinal ingredient, and not mainly caused by a blood coagulation factor defect or a von Willebrand factor defect A pharmaceutical composition for preventing or treating bleeding disorders caused by a decrease in circulating platelet count or abnormal platelet function.  Diseases related to decreased circulating platelet count or abnormal platelet function include aplastic anemia, suppression of bone marrow hematopoiesis after administration of anticancer agents, acute leukemia, idiopathic thrombocytopenic purpura, thrombocytopenia due to drug allergy, neonatal thrombocytopenia The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the following: disease, disseminated intravascular coagulation syndrome, thrombotic thrombocytopenic purpura, abnormal platelet distribution, asthenia gravis and Bernard-Soulier disease.  The pharmaceutical composition according to claim 1 or 2, which comprises factor VIII at least at a concentration of 5 units / mL with clotting activity.  The pharmaceutical composition according to any one of claims 1 to 3, comprising Factor VIII corresponding to 5 to 400 units / kg body weight in terms of clotting activity as a dose necessary for hemostasis.

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