JPH09301887A - Preventive and therapeutic agent for thrombocytopenia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a preventive and therapeutic agent for thrombocytopenia by containing parathyroid hormone(PTH) or a PTH derivative. SOLUTION: This preventive and therapeutic agent for thrombocytopenia contains humane PTH (1-84) or its derivative, or PTH or humane PTH (1-34) as an effective ingredient. It is effective for preventing and curing patients suffering from thrombocytopenia, thrombocytopenic peliosis and various diseases expressing bleeding tendency which is seemed to be caused by decrease of platelets. A formulation of the agent can be prepared by a usual formulating method of a peptide in forms of an injection, an encapsulation in microcapsules, an inclusion in a get-like sheet, etc., as the formulation for localization and slow acting. An administration frequency is possible for once a month to an every day administration, preferably once/two weeks to five times/a week, or the everyday administration. The dose is 1-1000μg/kg body weight.

Description

Detailed Description of the Invention

[0001]

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

[0002]

BACKGROUND OF THE INVENTION Like other mature blood cells, erythrocytes and neutrophils, platelets are derived from hematopoietic stem cells.
Produced by proliferation. In the initial stage of this hematopoietic process, hematopoietic stem cells differentiate and proliferate into megakaryocytes via megakaryocyte precursor cells. After maturation, megakaryocytes are thought to form proplatelets, which eventually become platelets and are released into the peripheral blood. It has been clarified that various hematopoietic factors and cytokines are involved in the 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 megakaryocyte maturation, and thrombopoietin is involved in stem cell to megakaryocyte maturation. It is speculated that some factor is involved in the process of platelet production and release from mature megakaryocytes, but this factor has not been clarified yet.

Thrombocytopenia is caused by the inhibition of any of the above processes of platelet hematopoiesis. The cause is roughly divided into two. That is, an abnormality in hematopoietic cells and an abnormality in hematopoietic factors. In the former, the differentiation / proliferation of hematopoietic cells is inhibited by congenital or acquired causes, aplastic anemia and myelodysplastic syndrome as congenital causes, and bone marrow transplantation as acquired causes. It is known to administer chemotherapeutic agents. on the other hand,
Periodic thrombocytopenia is known as a cause of abnormal hematopoietic factors.

As a therapeutic method for thrombocytopenia, platelet transfusion is the most effective means at present, but it is not always possible to supply a sufficient amount of platelets, and there is a risk of infection with viruses and the like. Development of effective prophylactic and therapeutic agents is desired.

On the other hand, parathyroid hormone (PTH) is known as one of the important hormones in bone metabolism.
Although many effects of PTH on bone have been reported so far, there are almost no reports on effects on the hematopoietic system. The action on the hematopoietic system is reported by Meytes et al. (J. Clin. Invest. 67: 1263).
-1269; 1981) in which PTH is an erythroid progenitor cell BFU-E and a granulocyte / macrophage progenitor cell CFU-GM in vitro.
It was shown to inhibit colony formation in tro. Therefore, the effect of PTH on platelet hematopoiesis has not been clarified so far.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a preventive agent and a therapeutic agent for thrombocytopenia. Another object of the present invention is to provide a drug effective for treating or preventing diseases associated with thrombocytopenia.

[0007]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have found that parathyroid hormone (PTH) or PTH
The inventors have found that the derivative is effective in treating thrombocytopenia and completed the present invention.

That is, the present invention relates to parathyroid hormone (P
The present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing TH) or a PTH derivative as an active ingredient. Furthermore, the present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing one or more human PTH (1-84) or its derivative as an active ingredient. The invention further provides parathyroid hormone (PTH) or human PTH (1-34).
The present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing as an active ingredient. Furthermore, the present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing parathyroid hormone (PTH) as an active ingredient. Furthermore, the present invention relates to human PT
The present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing H (1-84) as an active ingredient. Furthermore, the present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing a parathyroid hormone (PTH) derivative as an active ingredient. Furthermore, the present invention relates to a prophylactic and therapeutic agent for thrombocytopenia containing human PTH (1-34) as an active ingredient. The prophylactic and therapeutic agents for thrombocytopenia of the present invention include, in addition to thrombocytopenia, thrombocytopenic purpura, various diseases caused by thrombocytopenia such as various diseases showing bleeding tendency that is thought to be caused by thrombocytopenia. For the prevention and treatment of the above diseases.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Parathyroid hormone (PTH) in the present invention includes natural PTH, PTH produced by a genetic engineering method, and chemically synthesized PTH, preferably 84 amino acid residues Human PTH consisting of a group
(Human PTH (1-84)) is shown. The PTH derivative is a partial peptide of the above PTH, PTH itself or a partial amino acid of the partial peptide substituted with another amino acid, or PTH itself or a partial amino acid of the partial peptide is deleted. And a peptide obtained by adding one or more kinds of amino acids to PTH itself or a partial peptide thereof, and the like, mean a peptide having similar activity. Examples of the partial peptide of PTH include human PTH (1-34) and human PTH (1
-64), human PTH (35-84), bovine PTH (1
-34) and the like. What is PTH (1-34)?
The partial peptide of PTH which consists of 34 amino acids from the N terminal of TH to the 34th amino acid is shown.

Preferred examples of amino acid substitution include substitution of constituent amino acids at position 8 with leucine and norleucine, substitution of constituent amino acids at position 18 with leucine and norleucine, substitution of constituent amino acid at position 34 with tyrosine, etc. Can be given.

Preferred examples of parathyroid hormone (PTH) or PTH derivatives used as prophylactic and therapeutic agents for thrombocytopenia in the present invention include human PTH (1-
84), human PTH (1-34), human PTH (1-3
8), human PTH (1-37), human PTH (1-3
4) -NH _{2 and the} like, and more preferably human P
TH (1-84) and human PTH (1-34), with human PTH (1-84) being most preferred.

The preventive and therapeutic agents for thrombocytopenia are
It means a therapeutic or prophylactic drug, preferably a therapeutic drug, for thrombocytopenia, thrombocytopenic purpura, and various diseases showing a bleeding tendency which is considered to be caused by thrombocytopenia. Here, thrombocytopenia includes thrombocytopenia due to radiation therapy, thrombocytopenia associated with bone marrow transplantation, and the like. Furthermore, drugs (phenylbutazone, gold preparations, tolbutamide, chemotherapeutic agents)
Thrombocytopenia due to selective suppression of megakaryocytes due to infection with virus or virus, aplastic anemia, autoimmune thrombocytopenic purpura, myelodysplastic syndrome, leukemia, multiple myeloma, megakaryoblastic anemia It also includes thrombocytopenia due to systemic osteocongestion, drug-induced immune thrombocytopenia, post-transfusion purpura, thrombocytopenia due to secondary immune thrombocytopenia and the like.

The dosage form of the drug of the present invention is an injection (for example, a liquid preparation, etc.) produced by a conventional peptide preparation method.
In addition to a lyophilized formulation), a dosage form that is expected to have localized and delayed action, such as encapsulation in microcapsules or inclusion in a gel-like sheet, is also possible. When formulating,
Pharmaceutically acceptable auxiliary components can be added. Further, a preparation modified with polyethylene glycol for the purpose of increasing the half-life in blood is also possible.

The auxiliary components include bases, stabilizers, preservatives, preservatives, emulsifiers, suspending agents, solubilizers, solubilizers, lubricants, corrigents, coloring agents, fragrances, and soothing agents. Examples include agents, excipients, binders, thickeners, buffers, and the like. Specific examples include calcium carbonate, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, cocoa butter, Examples include distilled water for injection, sodium chloride aqueous solution, Ringer's solution, glucose solution, human serum albumin and the like.

When the drug of the present invention is prepared by using these auxiliary ingredients, for example, a list of pharmaceutical additives (Tokyo Pharmaceutical Industry Association Medical Law Committee and Osaka Pharmaceutical Industry Association Medical Law Research Committee) Issue), the auxiliary component may be appropriately selected and used. The amount of the auxiliary component to be used may be appropriately selected depending on the dosage form and the like within a pharmaceutically acceptable range.

The administration method of the drug of the present invention may be systemic administration or local administration. Preferred examples include systemic administration such as subcutaneous administration, intravenous administration, intranasal administration and transpulmonary administration. As a general rule, the administration period is a period clinically judged to be thrombocytopenia, and it is also possible to continue administration after recovery depending on the etiology of the disease, according to the judgment of the clinician.
Furthermore, when thrombocytopenia is predicted, for example, when a chemotherapeutic agent is administered, prophylactic administration can be performed even if thrombocytopenia has not occurred. The frequency of administration can be from once a month to daily administration, preferably once every 2 weeks to 5 times.
Administration is once / weekly or daily.

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

[0018]

The present invention will be described in more detail with reference to the following examples. Human PTH (1-84) used in the examples is
Japanese Patent Publication No. 4-505259 and J. Biol. C
hem. , 265, 15854 (1990). Also,
Human PTH (1-34) is a Peptide Inst
itute Inc. I bought more.

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

[Preparation of Administration Solution] Human PTH (1-8
For 4), a citrate buffer solution (pH = 5, containing 0.05% of Twin 80) having a final concentration of 1,0.2,0.04 µg / ml was prepared and used as a drug solution for administration. .

[Administration] 10 ml of each of the above drug solution and citrate buffer solution as a control was administered to 15 mice.
/ Kg was administered through the tail vein. Therefore, the dose is 0, 0.4, 2, 10 μg / kg. Administration is 1 am
It started at midnight.

[Blood collection and measurement of peripheral blood cell count] 3, after administration
Five mice were selected from each group at 6th and 9th hours, and blood was collected from the orbital vein. Separately, blood was collected from untreated mice (4 mice) at 10 am on the same day, and the value before administration was obtained. Peripheral blood cell count is automatic blood cell counter F-80
It measured using 0 (Toa Medical Electronics).

[Results] The results are shown in FIG. PTH 10μ
In the g / kg administration group, a significant increase in the number of platelets was observed at 6 and 9 hours after administration compared with the control. Thus, an increase in the number of platelets after administration of PTH can be seen within a short time after administration.

Example 2 Daily Mouse Administration Test [Experimental Animal] For the experiment, 9-week-old male C57B1 / 6N was purchased from Charles River Japan and used.

[Preparation of administration liquid] The administration liquid is prepared basically in the same manner as in Example 1, and the concentration of the administration liquid is 5, 20,
It was set to 80 μg / ml.

[Administration] The mice were divided into four groups,
The above drug solution and a citrate buffer solution as a control were administered subcutaneously to the abdomen once a day at a rate of 10 ml / kg. Therefore, the daily dose is 0, 50, 20
It becomes 0,800 μg / kg. The administration started at 10 am.

[Blood collection and measurement of peripheral blood cell count] On the 5th, 7th and 9th days after the start of administration, 5 to 6 mice were taken from each group.
Each animal was selected and blood was collected from the orbital vein. Blood was collected 24 hours after the final administration. Separately from this, 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. Platelet count is
On the 5th day, there was only a tendency to increase in the 200 and 800 μg / kg administration groups, but on the 7th day, it was significantly higher than that of the control in all the administration groups.
Therefore, it was clarified that PTH has a thrombocytosis effect even when administered daily.

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

[Preparation of Solution for Administration] Human PTH (1-8
4), the preparation of the drug solution for administration of human PTH (1-34) is basically carried out in the same manner as in Example 1, and the drug solution concentration is 20 μg /
ml.

[Administration] Eighteen mice were divided into three groups, and the above drug solution and a citrate buffer solution as a control were 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. The administration started at 10 am.

[Blood collection and measurement of peripheral blood cell count] 2 after the final administration
Blood was collected from the orbital vein of each mouse at 4 hours.
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. PTH (1-
34) was found to have a thrombocytosis effect equivalent to that of PTH (1-84). From this result, it can be expected that a shorter PTH partial peptide or a PTH partial peptide having a size intermediate between the two, such as PTH (35-84) and PTH (1-64), will also have the same action.

Example 4 Daily Rabbit Administration Test [Experimental Animal] Japanese White Rabbit JW / CSK 12-1
Six 5-week-old males were used. These animals had a temperature of 24 ± 2 ° C and a humidity of 50 ± 10 in an animal house under an SPF environment.
%, Lighting was turned on at 5:00, turned off at 19:00, and ventilation was carried out 15 times / hour under the conditions of an aluminum rabbit bracket cage Rb-1 (W350 × D500 × H350 mm). As a feed, a radiation sterilized feed RM for rabbits (Funabashi Farm) was fed at 120 g / day per animal.
As for water supply, tap water was freely ingested using an automatic water supply device.

[Preparation of Administration Solution] Human PTH (1-8
4) was donated at 2 ml / vial at a concentration of 200 μg / ml. The solvent was also provided 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 neck of the rabbit for 13 days. The solvent was similarly administered in the same volume as the dose of the rh-PTH solution converted from body weight.

[Blood collection and measurement of peripheral blood cell count] Before administration and before administration of PTH 2, 6, 9, and 13 days after administration, 0.5 ml of blood was collected from the marginal vein of the rabbit ears. Blood was immediately dispensed into a blood test blood collection container SB-41S (Toa Medical Electronics Co., Ltd.), and after performing anticoagulation treatment, an automatic blood cell counter F-80.
The number of platelets was measured using 0 (Toa Medical Electronics).

[Results] Solvent was added to 3 rabbits and PTH was added to 3 rabbits.
200 μg / kg was administered every day, and 0, 2, 6,
The platelet count was measured on the 9th and 13th days. The result is shown in Figure 4.
Shown in Platelets increased from 2 days after the administration, and 6 days after the administration, the platelets increased about 2 to 4 times the pre-administration value. On the 13th day after administration, the value almost returned to the value before administration.

[0039]

The preventive and therapeutic agents for thrombocytopenia containing the parathyroid hormone (PTH) or PTH derivative of the present invention as an active ingredient are considered to be caused by thrombocytopenia, thrombocytopenic purpura and thrombocytopenia. It is useful as a therapeutic or prophylactic drug for various diseases showing bleeding tendency.

[Brief description of drawings]

FIG. 1 is a graph showing the platelet increasing effect of PTH (single administration of mouse).

FIG. 2 is a graph showing the platelet increasing action of PTH (daily daily administration to mice).

FIG. 3 is a graph showing the platelet increasing action of PTH derivatives (daily daily administration to mice).

FIG. 4 is a graph showing the platelet increasing effect of PTH (daily rabbit daily administration).

Claims (7)

[Claims] 1. Parathyroid hormone (PTH) or PT
A prophylactic and therapeutic agent for thrombocytopenia containing one or more H derivatives as active ingredients. 2. Parathyroid hormone (PTH) is human PT
The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, which is H (1-84). 3. The PTH derivative is human PTH (1-34).
The preventive and therapeutic agent for thrombocytopenia according to claim 1, wherein 4. The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, which contains parathyroid hormone (PTH) as an active ingredient. 5. Parathyroid hormone (PTH) is human PT
The prophylactic and therapeutic agent for thrombocytopenia according to claim 4, which is H (1-84). 6. The prophylactic and therapeutic agent for thrombocytopenia according to claim 1, which contains a parathyroid hormone (PTH) derivative as an active ingredient. 7. The parathyroid hormone (PTH) derivative is human PTH (1-34).
The preventive and therapeutic agents for thrombocytopenia described.