JP2008024722A - Agents for preventing and treating thrombocytopenia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide agents for preventing and treating thrombocytopenia, thrombocytopenic purpura and various diseases that tend to cause bleeding due, presumably, to thrombocytopenia. <P>SOLUTION: Agents for preventing and treating thrombocytopenia, containing a parathyroid hormone (PTH) or at least one PTH derivative selected from peptides which have the same activities as those of PTH and are the partial peptides of PTH, wherein one or more other amino acids are substituted or added to one part of the constituting amino acids, as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing parathyroid hormone (PTH) or a PTH derivative as an active ingredient.

  Platelets are derived from hematopoietic stem cells in the same manner as other mature blood cells such as erythrocytes and neutrophils, and are produced by the differentiation and proliferation of these cells. In the early stage of the hematopoietic process, hematopoietic stem cells differentiate and proliferate into megakaryocytes via megakaryocyte precursor cells. Megakaryocytes are thought to form proplatelets after maturation, which eventually become platelets and are released into the peripheral blood. It has been clarified that various hematopoietic factors and cytokines are involved in these series of platelet production processes. For example, it has been experimentally shown that interleukin 3 is involved in the stage from stem cells to megakaryocyte progenitor cells, interleukin 6 is involved in maturation of megakaryocytes, and thrombopoietin is involved in the maturation of stem cells to megakaryocytes. It is speculated that some factor is also involved in the process of platelet production and release from mature megakaryocytes, but this factor has not been clarified yet.

  Thrombocytopenia occurs due to inhibition of any of the above processes for platelet formation. The cause can be roughly divided into two. That is, hematopoietic cell abnormality and hematopoietic factor abnormality. In the former, the differentiation or proliferation of hematopoietic cells is inhibited by innate or acquired causes, such as aplastic anemia or myelodysplastic syndrome due to innate causes, bone marrow transplantation as due to acquired causes Or by administration of chemotherapeutic agents. On the other hand, periodic thrombocytopenia is known as a result of abnormal hematopoietic factors.

  Currently, platelet transfusion is the most effective method for treating thrombocytopenia. However, it does not always supply a sufficient amount of platelets, and there is a risk of infection with viruses, etc., and effective prevention. Development of agents and therapeutic agents is desired.

  On the other hand, parathyroid hormone (PTH) is known as one of the important hormones in bone metabolism. Conventionally, many effects of PTH on bones have been reported, but there are few reports on the effects on the hematopoietic system. As an effect on the hematopoietic system, there is a report of Meytes et al. (J. Clin. Invest. Vol. 67, 1263-1269; 1981). It shows that it inhibits colony formation in vitro by CFU-GM which is a cell. Therefore, the effect of PTH on platelet hematopoiesis has not been clarified so far.

  An object of this invention is to provide the preventive agent and therapeutic agent of thrombocytopenia. A further object of the present invention is to provide a drug effective for the treatment or prevention of diseases associated with thrombocytopenia.

  As a result of intensive studies, the present inventors have found that parathyroid hormone (PTH) or a PTH derivative is effective in the treatment of thrombocytopenia and completed the present invention.

  That is, the present invention relates to a prophylactic agent and a therapeutic agent for thrombocytopenia containing parathyroid hormone (PTH) or a PTH derivative as an active ingredient. The present invention further relates to a prophylactic and therapeutic agent for thrombocytopenia containing human PTH (1-84) or one or more of its derivatives as an active ingredient. Furthermore, the present invention relates to a preventive and therapeutic agent for thrombocytopenia containing parathyroid hormone (PTH) or human PTH (1-34) as an active ingredient. Furthermore, the present invention relates to a prophylactic agent and a therapeutic agent for thrombocytopenia containing parathyroid hormone (PTH) as an active ingredient. Furthermore, the present invention relates to a preventive agent and a therapeutic agent for thrombocytopenia containing human PTH (1-84) as an active ingredient. Furthermore, the present invention relates to a preventive agent and a therapeutic agent for thrombocytopenia containing a parathyroid hormone (PTH) derivative as an active ingredient. Furthermore, the present invention relates to a preventive agent and a therapeutic agent for thrombocytopenia containing human PTH (1-34) as an active ingredient. In addition to thrombocytopenia, the prophylactic and therapeutic agents for thrombocytopenia of the present invention include various types caused by thrombocytopenia, such as thrombocytopenic purpura and various diseases that exhibit bleeding tendency thought to be caused by thrombocytopenia. Targeting the prevention and treatment of various diseases.

  The parathyroid hormone (PTH) in the present invention includes natural PTH, PTH produced by genetic engineering techniques, and chemically synthesized PTH, preferably human PTH (human) consisting of 84 amino acid residues. PTH (1-84)). In addition, the PTH derivative is a partial peptide of the above PTH, a PTH itself or a partial amino acid of the partial peptide substituted with another amino acid, or a PTH itself or a part of the partial peptide of the partial peptide is deleted. Or a peptide obtained by adding one or more amino acids to PTH itself or a partial peptide thereof, and the like and having the same activity. Examples of the partial peptide of PTH include human PTH (1-34), human PTH (1-64), human PTH (35-84), bovine PTH (1-34) and the like. PTH (1-34) is a partial peptide of PTH consisting of 34 amino acids from the N-terminus of PTH to the 34th amino acid.

  Preferred examples of amino acid substitution include substitution of the constituent amino acid at position 8 with leucine or norleucine, substitution of the constituent amino acid at position 18 with leucine or norleucine, substitution of the constituent amino acid at position 34 with tyrosine, and the like. .

Preferable examples of parathyroid hormone (PTH) or PTH derivatives used as preventive and therapeutic agents for thrombocytopenia in the present invention include human PTH (1-84), human PTH (1-34), human PTH (1 -38), human PTH (1-37), such as human PTH (1-34) -NH ₂ and the like, it is more preferably a human PTH (1-84), human PTH (1-34), most preferably An example is human PTH (1-84).

  The preventive and therapeutic agent for thrombocytopenia means a therapeutic or prophylactic agent, preferably a therapeutic agent for thrombocytopenia, thrombocytopenic purpura, and various diseases that have a tendency to bleed due to thrombocytopenia. . Here, thrombocytopenia includes thrombocytopenia due to radiation therapy, thrombocytopenia associated with bone marrow transplantation, and the like. Furthermore, thrombocytopenia, aplastic anemia, autoimmune thrombocytopenic purpura due to selective suppression of megakaryocytes caused by drugs (phenylbutazone, gold preparations, tolbutamide, chemotherapeutic agents) and viral infections Thrombocytopenia due to systemic bone failure such as myelodysplastic syndrome, leukemia, multiple myeloma, megakaryoblastic anemia, drug-induced immune thrombocytopenia, posttransfusion purpura, secondary immune platelets It includes thrombocytopenia due to decrease.

  As the dosage form of the drug of the present invention, in addition to injections (eg, liquid preparations, freeze-dried preparations, etc.) produced by the usual method for preparing peptides, for example, they are included in microcapsules or contained in gel sheets. The dosage form is expected to be effective and slow-acting. In formulating, pharmaceutically acceptable auxiliary ingredients can be added. A preparation modified with polyethylene glycol for the purpose of increasing the blood half-life is also possible.

  These auxiliary ingredients include bases, stabilizers, preservatives, preservatives, emulsifiers, suspending agents, solubilizers, solubilizers, lubricants, flavoring agents, coloring agents, fragrances, soothing agents, soothing agents. Specific examples include, for example, calcium carbonate, lactose, sucrose, sorbit, mannitol, starch, amylopectin, cellulose derivatives, gelatin, cocoa butter, and distilled for injection. Examples include water, aqueous sodium chloride solution, Ringer's solution, glucose solution, and human serum albumin.

  When adjusting the drugs of the present invention using these auxiliary ingredients, for example, in the Pharmaceutical Additives List (issued by the Tokyo Pharmaceutical Manufacturers Association Medical Regulations Committee and the Osaka Pharmaceutical Manufacturers Association Medical Regulations Research Committee) As it is, the auxiliary component may be appropriately selected and used. Moreover, the usage-amount of an auxiliary | assistant component should just be suitably selected according to dosage form etc. in the range accept | permitted pharmacologically.

  The method of administering the drug of the present invention can be performed systemically or locally, but preferred examples include systemic administration by subcutaneous administration, intravenous administration, intranasal administration, transpulmonary administration, and the like. In principle, the administration period is a period that is clinically determined to be thrombocytopenia, and it is possible to continue the administration after recovery according to the judgment of the clinician according to the etiology. Furthermore, when thrombocytopenia is predicted, for example, when a chemotherapeutic agent is administered, it can be administered prophylactically even if thrombocytopenia does not occur. The administration frequency can be from once a month to every day, preferably about once every 2 weeks to about 5 times per week or every day.

  The dose of PTH of the present invention varies depending on the indication disease, symptoms, etc., but in systemic administration, it is about 1 μg to 1000 μg per kg body weight, preferably 5 μg to 200 μg per kg body weight.

  Hereinafter, the present invention will be described in more detail with reference to examples. Human PTH (1-84) used in the examples is disclosed in JP-T-4-505259 and J.P. Biol. Chem. , 265, 15854 (1990). In addition, human PTH (1-34) is available from Peptide Institute Inc. We purchased more.

Example 1 Mouse Single-Dose Test [Experimental Animal] In the experiment, 9-week-old male C57B1 / 6N was purchased from Nippon Charles River and used.

[Preparation of Drug Solution] Human PTH (1-84) contains 0.05% citrate buffer solution (pH = 5, twin 80, so that the final concentration is 1,0.2, 0.04 μg / ml). ) Was prepared and used as a drug solution.

[Administration] The above-mentioned drug solution and citrate buffer solution as a control were each administered to 15 mice at a rate of 10 ml / kg from the tail vein. Therefore, the dosage is 0, 0.4, 2, 10 μg / kg. Administration started at 10 am.

[Blood Collection / Measurement of Peripheral Blood Cells] Five mice were selected from each group at 3, 6, and 9 hours after administration, and blood was collected from the orbital vein. Separately, blood was collected from untreated mice (4 mice) at 10:00 am on the same day, and values before administration were obtained. The peripheral blood cell count was measured using an automatic blood cell counter F-800 (Toa Medical Electronics).

[Results] The results are shown in FIG. In the PTH 10 μg / kg administration group, a significant increase in the number of platelets was observed relative to the control at 6 and 9 hours after administration. Thus, an increase in the number of platelets after PTH administration is seen in a short time after administration.

Example 2 Mice Daily Administration Test [Experimental Animal] For the experiment, male C57B1 / 6N of 9 weeks old was purchased from Nippon Charles River and used.

[Preparation of Administration Drug Solution] Preparation of administration drug solution was basically carried out in the same manner as in Example 1, and the drug solution concentrations were 5, 20, 80 μg / ml.

[Administration] The mice were divided into 4 groups, and each of them was subcutaneously abdomen once a day with the above-mentioned drug solution and a citrate buffer solution as a control at a rate of 10 ml / kg. Therefore, the daily dose is 0, 50, 200, 800 μg / kg. Administration started at 10 am.

[Blood Collection / Measurement of Peripheral Blood Cell Count] On the 5th, 7th and 9th days after the start of administration, 5 to 6 mice were selected from each group, and blood was collected from the orbital vein. Blood collection was performed 24 hours after the final administration. Separately, blood was also collected from untreated mice (5 mice) to obtain values for normal mice. The peripheral blood cell count was measured using an automatic blood cell counter F-800 (Toa Medical Electronics).

[Results] The results are shown in FIG. On the fifth day, the platelet count only tended to be higher in the 200 and 800 μg / kg administration groups, but on the seventh day, the platelet count was significantly higher in all administration groups compared to the control. Therefore, it has been clarified that PTH has an effect of increasing platelets even when administered daily.

Example 3 Effect of PTH Derivative [Experimental Animal] In the experiment, 10-week-old male C57B1 / 6N was purchased from Charles River Japan and used.

[Preparation of administered drug solution] Preparation of administered drug solution of human PTH (1-84) and human PTH (1-34) was basically carried out in the same manner as in Example 1, and the drug solution concentration was 20 μg / ml.

[Administration] Eighteen mice were divided into three groups, and each of the above-mentioned drug solution and citrate buffer solution as a control was subcutaneously administered to the abdomen once a day for 6 days at a rate of 10 ml / kg. Therefore, the daily dose is 0.200 μg / kg. Administration started at 10 am.

[Blood Collection / Measurement of Peripheral Blood Count] Blood was collected from the orbital vein of each mouse 24 hours after the final administration. The peripheral blood cell count was measured using an automatic blood cell counter F-800 (Toa Medical Electronics).

[Results] The results are shown in FIG. It was revealed that PTH (1-34) has a platelet-increasing action equivalent to PTH (1-84). From this result, it can be expected that a shorter PTH partial peptide or a PTH partial peptide having an intermediate size between them, such as PTH (35-84) and PTH (1-64), have the same effect.

Example 4 Rabbit Daily Administration Test [Experimental Animal] Japanese white rabbit JW / CSK 12-15 weeks old 6 males were used. These animals are aluminum rabbit bracket cages under conditions of temperature 24 ± 2 ° C, humidity 50 ± 10%, lighting 5:00 lighting 19:00 lighting, ventilation 15 times / hour in the animal house under SPF environment. A single head was raised on Rb-1 (W350 × D500 × H350 mm). As a feed, a rabbit radiation-sterilized feed RM (Funabashi Farm) was fed at 120 g / day. Moreover, tap water was freely ingested using an automatic water dispenser.

[Preparation of Administration Drug Solution] Human PTH (1-84) was donated at a concentration of 200 μg / ml at 2 ml / vial. The solvent was similarly donated at 2 ml / vial. These drugs were stored at −135 ° C. until use.

[Administration method] PTH 200 μg / kg was subcutaneously administered to the back of the rabbit neck for 13 days. The solvent was similarly administered in the same volume as the dose of rh-PTH solution converted from body weight.

[Blood Collection / Measurement of Peripheral Blood Count] Before administration and before administration of PTH on 2, 6, 9, and 13 days after administration, 0.5 ml of blood was collected from the rabbit marginal vein. Blood is immediately dispensed into blood collection container SB-41S (Toa Medical Electronics Co., Ltd.) for blood test, and after anticoagulation treatment, platelet count is measured using automatic blood cell counter F-800 (Toa Medical Electronics). did.

[Results] The solvent was administered to 3 rabbits and PTH 200 μg / kg was administered daily to 3 rabbits, and the platelet count was measured 0, 2, 6, 9 and 13 days after administration. The result is shown in FIG. The platelets increased from 2 days after administration, and on the 6th day after administration, the increase in platelets was about 2 to 4 times the pre-dose value. In addition, on the 13th day after administration, the value almost returned to the value before administration.

  The preventive and therapeutic agent for thrombocytopenia containing the parathyroid hormone (PTH) or PTH derivative of the present invention as an active ingredient exhibits a tendency to bleed due to thrombocytopenia, thrombocytopenic purpura, and thrombocytopenia. It is useful as a therapeutic or prophylactic agent for various diseases.

It is a figure which shows the platelet increase effect (mouse single administration) of PTH. It is a figure which shows the platelet increase effect (mouse daily administration) of PTH. It is a figure which shows the platelet increase effect (mouse daily administration) of a PTH derivative. It is a figure which shows the platelet increase effect (rabbit daily administration) of PTH.

Claims (7)

  Parathyroid hormone (PTH) or a PTH derivative selected from peptides having partial activity of the same amino acid as PTH, which is a partial peptide of PTH, with one or more of the constituent amino acids substituted or added A prophylactic and therapeutic agent for thrombocytopenia containing one or more of these as an active ingredient.   The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, wherein the parathyroid hormone (PTH) is selected from natural PTH, PTH produced by genetic engineering techniques, or chemically synthesized PTH. Parathyroid hormone (PTH) or a PTH derivative is human PTH (1-84), human PTH (1-34), human PTH (1-38), human PTH (1-37), human PTH (1-34). preventive and therapeutic agents for thrombocytopenia of claim 1 selected from the group consisting of -NH _2.   The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, wherein the parathyroid hormone (PTH) is human PTH (1-84).   The preventive and therapeutic agent for thrombocytopenia according to claim 1, wherein the thyroid hormone (PTH) derivative is human PTH (1-34).   Parathyroid hormone (PTH) or partial peptide of PTH is replaced with leucine or norleucine of the constituent amino acid at position 8 of the partial peptide of PTH or PTH, or leucine or norleucine of the constituent amino acid at position 18 of the partial peptide of PTH or PTH The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, wherein the agent has at least one of PTH and substitution of tyrosine of a constituent amino acid at position 34 of PTH or a partial peptide of PTH.   The partial peptide of PTH is selected from the group consisting of human PTH (1-34), human PTH (1-64), human PTH (35-84), and bovine PTH (1-34). Preventive and therapeutic agents for thrombocytopenia.

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