JPH04360840A - Therapeutic agent for thrombocytopenia - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for thrombocytopenia useful as a therapeutic agent substitutive for the existing blood transfusion therapy due to its action on bone marrow to promote the maturation of blood platelet-producing cells (megarokaryocytic cells) and simultaneous increase in precursor cells of the megarokaryocytes by its use with a hematopoietic factor such as IL-6 in combination. CONSTITUTION:A therapeutic agent for thrombocytopenia containing (A) one or more substances selected from a polypeptide BUF-3 and further BUF-4 and BUF-5 which are its analogs produced by culturing a human malignant monocytic cell in the coexistence of a specific differentiation inducer such as actinomycin A as an active ingredient and preferably further (B) one or more substances selected from interleukin (IL)-6, IL-3, IL-1 and granulocytic colony stimulating factors as an adjuvant. The aforementioned agent is safe and effective without causing rejection or viral infection in principle. Furthermore, since the above-mentioned active ingredient is a protein derived from humans, antigenicity is low and allergy hardly occurs. Thereby, use for a long period can be carried out.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a therapeutic agent for thrombocytopenia.

0002

BACKGROUND OF THE INVENTION Conventionally, platelet transfusions have been used to treat thrombocytopenia and bleeding disorders. In platelet transfusion therapy, platelets, which are the raw materials, are provided by blood donations, but the supply of materials is not always satisfactory due to factors such as a lack of donated blood. In addition, frequent blood transfusions are painful for patients, and major histocompatibility antigen mismatches,
It is known that incompatible blood types such as contaminated red blood cells often cause rejection reactions and shock symptoms. Furthermore, patients may become infected with viruses derived from blood transfusion materials and develop viral diseases such as hepatitis, interstitial pneumonia, and AIDS, which poses a problem when performing blood transfusions.

On the other hand, recently, polypeptide interleukin 6 (hereinafter abbreviated as IL-6, IL-6 is BCDF,
Although it is also called BSF-2, in the present invention, it will be unified as IL-6. ) was shown to have a platelet-producing effect, and could be used as a therapeutic agent in place of platelet transfusion in the treatment of thrombocytopenia and bleeding disorders (Japanese Patent Application Laid-Open No. 3-101624). In addition, the same applies to interleukin 3 (hereinafter referred to as IL-3), interleukin 1 (hereinafter referred to as IL-1), and granulocyte colony stimulating factor (hereinafter referred to as G-CSF) other than IL-6. Effectiveness has been reported. Therefore, hematopoietic factors such as IL-6 are useful as therapeutic agents for thrombocytopenia, but if a new factor that is effective for platelet production is discovered in combination with factors such as IL-6 or alone, even better therapeutic effects will be achieved. You can expect it.

0004

[Problems to be Solved by the Invention] A more effective therapeutic agent for thrombocytopenia that can replace platelet transfusion and has fewer side effects is desired. The object of the present invention is to identify factors effective for platelet production from among various proteins produced by human cells, either alone or in combination with factors such as IL-6, and to provide a new drug useful for the treatment of thrombocytopenia. It's about doing.

[0005]

[Means for Solving the Problems] In order to solve the above problems, the present inventors searched for activities effective for platelet production in the products of various human cells. It was discovered that the polypeptide BUF-3 or its analogs BUF-4 and BUF-5, which are produced by culturing in the presence of a substance, are effective for platelet production in mice, and the present invention has been achieved. It was completed. That is, the present invention provides polypeptide BUF-3
, BUF-4, and BUF-5 as an active ingredient. The physicochemical properties of the polypeptides BUF-3, BUF-4 and BUF-5 are as follows.

(1) Physical and chemical properties of polypeptide BUF-3 (hereinafter referred to as BUF-3) (a) Structure; homodimer of monomer A (see SEQ ID NO: 1 in the sequence listing) (b) Molecular weight; monomer 16 ± 1 kd as a homodimer (SDS electrophoresis in the presence of 1% mercaptoethanol) 25 ± 1 kd as a homodimer (SDS electrophoresis in the absence of mercaptoethanol) (c) Thermal stability; heating at 65°C for 60 minutes Stable (d) Pronase resistance; Inactivated by pronase treatment (e) Amino acid sequence; The amino acid sequence of monomer A is shown in SEQ ID NO: 1 in the sequence listing (2) Polypeptide BUF-4 (hereinafter referred to as BUF-4) Physical and chemical properties (a) Structure: Heterodimer of monomer A and monomer B (see SEQ ID NO: 2 in the sequence listing) (b) Molecular weight: 16 ± 1 kd as a monomer (in the presence of 1% mercaptoethanol, (SDS electrophoresis method) 25 ± 1 kd as a heterodimer (SDS electrophoresis method in the absence of mercaptoethanol) (c) Thermostability; stable by heating at 65°C for 60 minutes (d) Pronase resistance; inactivated by pronase treatment (e) Amino acid sequence; The amino acid sequence of monomer A is shown in SEQ ID NO: 1 of the sequence listing, and the amino acid sequence of monomer B is shown in SEQ ID NO: 2 of the sequence listing. (3) Polypeptide BUF-5 (hereinafter referred to as BUF-5) (b) Molecular weight: 16 ± 1 kd as a monomer (SDS electrophoresis method in the presence of 1% mercaptoethanol) 25 ± 1 kd as a homodimer (SDS electrophoresis method in the absence of mercaptoethanol) (c) Thermostability; stable by heating at 65°C for 60 minutes (d) Pronase resistance; inactivated by pronase treatment (e) Amino acid sequence; The amino acid sequence of monomer B is shown in SEQ ID NO: 2 in the sequence listing.

[0007] BUF-3 and BUF-4 according to the present invention
, BUF-5 means that even if the substance does not have exactly the same amino acid sequence as shown in SEQ ID NOs: 1 and 2 in the sequence listing, if it has a thrombocytopenia therapeutic effect, the substance can be considered as the BUF of the present invention.
-3, BUF-4 and BUF-5. That is, polypeptides with a structure in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing are replaced with other amino acids, and one or more amino acids in the sequence are N-terminal. Also included are polypeptides added to the C-terminus, and also polypeptides consisting of a continuous amino acid sequence in which one or more amino acids are missing from the N-terminus or C-terminus of the sequence.

BUF-3 is a polypeptide purified using as an indicator its differentiation-inducing effect on mouse friend virus-induced leukemia cells F5-5. In addition to the activity of causing mouse leukemia cells to differentiate and mature into normal cells (Japanese Patent Application Laid-open No. 62-240700, 62-234097), BUF-3 also has
Anemia prevention effect (JP-A-62-240700) and follicle-stimulating hormone secretion effect (Nature, 321,776)
-779, (1986)).

[0009] BUF-3 is EDF (Erythro
id Differentiation Fact
or) and FRP (FSH Rereasing
However, in the present invention, the conventional name BUF-3 will be used. On the other hand, it has already been reported that BUF-4 has a follicle-stimulating hormone secretion effect (Nature, 321, 7
76-779, (1986)). Although BUF-4 is also called activin, the name BUF-4 will be used in the present invention. Furthermore, BUF-5 is a substance disclosed in JP-A-63-119679. BUF-3 as mentioned above
, BUF-4, and BUF-5 are already known to have effects such as follicle-stimulating hormone secretion, but there have been no reports on platelet production as in the present invention.

[0010] BUF-3, BUF-4 and BUF-5 of the present invention all have an excellent platelet production promoting effect when administered alone or in combination with hematopoietic factors such as IL-6 in animal experiments. Furthermore, it is considered to be safe and effective for the treatment of thrombocytopenia, as it does not show toxicity to cultured mouse and human cells.

The therapeutic agent for thrombocytopenia of the present invention is BUF-3.
, BUF-4, and BUF-5 as an active ingredient, it may contain the above active ingredients alone, or it may contain two or more substances in combination. It may be something you do. Furthermore, one or more substances among IL-6, IL-3, IL-1 and G-CSF may be included as an auxiliary agent. Especially IL-6
It exhibits excellent effects when used in combination with. The therapeutic agent for thrombocytopenia of the present invention is most effective when used before, after, or simultaneously with the administration of a factor such as IL-6, but it can also be expected to be effective when used alone. This therapeutic agent is mainly administered parenterally (intravenously, subcutaneously, intramuscularly, transdermally, transmucosally). The dosage of the active ingredient is BUF
-3, When using only one substance, BUF-4 or BUF-5, it is usually about 0.01 mg to 100 mg per day for adults, regardless of which substance is used.
This can be administered once or in several doses. In addition, when administering two or more types in combination (i.e.,
(a) BUF-3 and BUF-4, (b) BUF-3 and B
UF-5, (c) BUF-4 and BUF-5, (d) BU
F-3, BUF-4, and BUF-5) are also generally administered at a dose of about 0.01 per day for adults, since the medicinal efficacy of each substance is almost the same.
mg to 100 mg may be administered once or divided into several doses. Of course, the dosage will vary depending on the patient's platelet count, medical condition, patient's weight, and other factors recognized by those skilled in the art, so it is not necessary to strictly adhere to the above dosages and may be determined on a case-by-case basis. Furthermore, when a hematopoietic factor such as IL-6 is used as an auxiliary agent, there is no particular restriction on its dosage, but it is usually sufficient to administer about 0.01 mg to 100 mg per day for adults.

[0012] The active ingredient such as BUF-3 used in the present invention is formulated by a conventional method, and is mainly used as an injection, but it can also be formulated into dosage forms such as capsules and tablets. good. When preparing injections, the main drug BUF-
3 and/or BUF-4 and/or BUF-5, if necessary, add auxiliary agents such as IL-6, pH adjusters, buffers, stabilizers, preservatives, etc., and administer intravenously or subcutaneously by a conventional method. It may be prepared as an intramuscular injection. In addition, when preparing oral preparations, excipients are added to the main drug BUF-3 and/or BUF-4 and/or BUF-5, and if necessary, I
It may be made into tablets, capsules, etc. by adding auxiliary agents such as L-6, binders, disintegrants, coloring agents, etc. in a conventional manner. BUF-3 and/or BUF in formulations such as injections, tablets, capsules, etc.
The content of -4 and/or BUF-5 is usually 0.01-100 parts by weight, preferably 0.1 parts by weight when the formulation is 100.
-10 parts by weight is sufficient.

Next, BUF-3 and BU used in the present invention
The manufacturing method of F-4 and BUF-5 will be explained below. First, regarding BUF-3, the human malignant monocytic cells that produce BUF-3 include artificially malignant human leukemia cells or human bone marrow cells, and more specifically, the following. There is. Human chronic myeloid leukemia cells include U-937 cells (ATCC CRL 1593
, Int. J. Cancer17:565 (197
6)), K562 cells (Blood45:321(19
75)), acute monocytic leukemia cells include THP-1 cells (Int. J. Cancer 26:171-176 (
1980)). Of course, B.U.
Human leukemia cells other than those mentioned above may be used as long as they produce F-3. Now, the specific differentiation-inducing substance is a substance that has the effect of inducing the differentiation of malignant monocytic cells into macrophages and granulocytic cells and producing BUF-3 when brought into contact with these cells, and specifically, actinomycin D, mitomycin C, concanavalin A
and specific differentiation inducers such as phorbol ester (TPA).

The method for producing BUF-3 of the present invention is carried out by culturing malignant monocytic cells in the coexistence of at least one or more of the above-mentioned specific differentiation-inducing substances, and BUF-3 is produced in a culture medium. Produced inside (extracellularly). The medium for culturing the cells is a normal medium for culturing animal cells, usually at a density of 1 to 5 x 106 cells/ml, and 35 to 50 cells/ml.
It is carried out at 38° C. in a stream of 4 to 6% carbon dioxide gas with gentle stirring. A specific differentiation-inducing substance may be added to the medium from the beginning of normal culture, or may be added from the middle of culture. The amount added varies depending on the type of differentiation-inducing substance, but in the case of actinomycin D, mitomycin C, etc., it is 0.1 to 10 μg/ml, and in the case of TPA, it is 1 to 500 μg/ml.
It is μg/ml. When cultured in this manner for 1 to 5 days, BUF-3 is accumulated in the culture solution.

[0015] BUF-3 may also be produced using a recombinant DNA method. Recombinant DNA method is B
A eukaryotic animal cell (specifically, IFO-50146, etc.) transformed with a plasmid containing the gene encoding UF-3, i.e., monomer A, is cultured in a culture medium,
The BUF-3 is produced in a culture solution (Japanese Patent Application Laid-Open No. 64-2580, Biochem.B).
iophys. Res. Commun. 151,230
-235 (1988)).

Production of BUF-4 and BUF-5 is carried out in accordance with the production of BUF-3 by the recombinant DNA method. B
The method for producing UF-4 is to culture a eukaryote transformed with a plasmid containing the gene encoding BUF-4, that is, monomer A and monomer B, in a culture solution, and BUF-4 can be manufactured by
3-119679). In addition, the method for producing BUF-5 is to culture a eukaryotic organism transformed with a plasmid containing the gene encoding BUF-5, that is, monomer B, in a culture solution, and to produce BUF-5 in the culture solution. It may be manufactured (Japanese Unexamined Patent Publication No. 63-119679).

Now, BUF-3, BUF-4, or BUF-5 produced in this manner is purified according to a conventional polypeptide purification method. For example, a crude polypeptide sample can be obtained by concentrating the culture solution by ultrafiltration, salting out the polypeptide from this concentrated solution, and performing ion exchange chromatography using an anion exchanger after dialysis. Most of the contaminant proteins are removed from this crude sample by hydrophobic chromatography or chromatofocusing. Furthermore, by combining the two, the purification ratio can be further improved. The specimen purified in this way was subjected to reverse phase high performance liquid chromatography (
HPLC) or Superrose or MonoQ
FPLC equipped with HR5/5 column (Fast Protein Peptide Pol manufactured by Pharmacia)
ynucleotide Liquid Chro
It can be purified by high performance gel filtration using a chromatography system or ion exchange chromatography. In addition to the general purification method for polypeptides as described above, a purification method developed by the present inventors that makes full use of an organic solvent containing an organic acid at a predetermined concentration (Japanese Patent Application No. 131268/1982) can be used for purification. I don't mind if you do.

BUF-3 is a Friend virus-induced leukemia cell F5-5 (Bibl. Haemat., 43, 3).
7 (1976)), it has a differentiation-inducing effect on
Qualitative and quantitative analysis of BUF-3 can be performed using this effect, and analysis using F5-5 can be performed using Proc. Natl. A
cad. Sci. , 71, 98, (1975). Also, the activity display is F5-5
The reciprocal of the dilution rate of the sample stock solution at which cell differentiation is clearly confirmed is defined as the activity per 1.0 ml of the stock solution. When BUF-3 is produced by the method of this invention, the culture solution exhibits an activity of 4 to 1000 units/ml. In this way, the desired BUF
-3 will be produced. The details of this method can be found in Japanese Unexamined Patent Publication No. 62-2.
It is described in Japanese Patent Application Laid-open No. 34097 and Japanese Patent Application Laid-Open No. 62-24070.

The present invention will be explained in detail below with reference to Examples.

(Example 1) C57/BL6 mice (male,
9 weeks old, using Nippon Charles River Co., Ltd., 4 in 1 group.
was used as a test animal. Experimental groups are control group, BUF
-3 single administration group, IL-6 single administration group, and BUF-
There were 4 groups: a group administered in combination with IL-6 and IL-6. BUF-
The third administration was performed in the following manner. That is, BUF-3 obtained by the above method was dissolved in distilled water for injection containing 10 mM acetic acid to prepare a 50 μg/ml administration solution, and a mini osmotic pump (model 2001 manufactured by ALZA, USA) was prepared.
) was filled. On the first day of the experiment, the mice in the BUF-3 alone administration group and the BUF-3 and IL-6 combination administration group underwent laparotomy under ether anesthesia, and the mini-osmotic pump was transferred into the abdominal cavity, and the abdomen was immediately opened. was sutured. Control group and I
A mini osmotic pump filled with distilled water for injection containing only 10 mM acetic acid was intraperitoneally transferred to mice in the L-6 single administration group using the same method. A mini-osmotic pump is a device that continuously releases a filled dosage solution at a constant rate over a period of 7 days. Model 2001 used in this experiment has a release rate of 1
μl/hour, BUF-3 is continuously administered intraperitoneally at a rate of 50 ng/hour until the 8th day of the experiment.

[0021] IL-6 administration was carried out in the following manner. J.
Biochem. , 104, 30-34, (1989)
IL-6 obtained by the method described in C57/B
It was dissolved in physiological saline for injection containing 0.2% mouse serum obtained from L6 mice to prepare a 5 μg/ml administration solution. From day 7 of the experiment, IL-6 monoadministration group and B
Mice in the UF-3 and IL-6 combination administration group were subcutaneously injected with the above solution in an amount of 0.1 ml (0.5 μg of IL-6) twice a day for 5 days. Control group and BUF-
Injectable physiological saline containing only 0.2% mouse serum was administered to the 3 single administration group in the same manner.

[0022] After the mice in each experimental group were sacrificed on the 12th day of the experiment, the femurs were immediately removed and the CFU-Meg in the bone marrow was removed.
The number of cells (precursor cells of megakaryocytic cells that produce platelets) is calculated by Exp. Hematol. , 7, 345-351, (1
979). The measurement results are shown in Table 1. CF of BUF-3 and IL-6 combination administration group
The average U-Meg number was 18.3 per 40,000 bone marrow cells, which was not only significantly higher than the average of 10.0 in the control group, but also significantly higher than the average of 12.0 in the IL-6 alone administration group. However, it was significantly more common.

[0023]

[Table 1]

Next, the maturity level of megakaryotic cells in the bone marrow of mice of each experimental group was compared using the following method. In other words, since the degree of maturation of megakaryocyte cells is reflected in the cell size, a micrograph was prepared at a magnification of 400 times from a section of the excised femur, and the diameter of each megakaryocyte cell on the photograph was measured using a caliper. It was measured. Table 2 shows the average diameters of 69 to 72 megakaryocyte cells for each experimental group. In both the BUF-3 single administration group and the BUF-3 and IL-6 combination administration group, the average diameter was 7.0 mm, which was significantly larger than the control group's 6.6 mm. Maturation was accelerated.

[0028]

[Table 2]

(Example 2) The administration effect of BUF-5 was investigated under the same conditions as in Example 1 above. 9 week old male C57/
BL6 mice were used as test animals, with 4 mice per group. The experimental groups are a control group, a BUF-5 single administration group, and an IL-6 administration group.
There were 4 groups: a single administration group and a combined administration group of BUF-5 and IL-6. BUF-5 administration was carried out in the same manner as BUF-3 in Example 1, using an administration solution in which BUF-5 obtained by the method described above was dissolved in distilled water for injection containing 10 mM acetic acid to give a concentration of 50 μg/ml. Ta. Furthermore, IL-6 administration was performed under the same conditions as in Example 1.

[0030] The number of CFU-Meg cells in the bone marrow is BUF-
5 and IL-6 in the control group and the IL-6 combination administration group.
-6 was significantly more than the group administered alone. In addition, the average diameter of megakaryocyte cells in the BUF-5 alone administration group and in the BUF-
The value was significantly higher in the group administered with 5 and IL-6 in combination than in the control group and the group administered with IL-6 alone.

[0031]

[Effects of the present invention] BUF-3 and BUF-4 according to the present invention
A therapeutic agent for thrombocytopenia containing one or more of BUF-5 and BUF-5 as active ingredients not only acts on the bone marrow to promote the maturation of megakaryocytes that produce platelets, but also promotes the maturation of megakaryocytes that produce platelets.
When used in combination with hematopoietic factors such as, it increases megakaryocyte precursor cells and is therefore effective in treating thrombocytopenia. In addition, there are problems with platelet transfusion, which is the conventional treatment method.
In principle, rejection reactions and viral infections do not occur. Therefore, the therapeutic agent for thrombocytopenia of the present invention can be used as an excellent therapeutic agent in place of the conventionally used platelet transfusion. B
Since UF-3, BUF-4, and BUF-5 are human-derived proteins, they have low antigenicity and are less likely to cause allergies, so they can be used for a long period of time. Although the therapeutic agent for thrombocytopenia according to the present invention exhibits its full effect when used in combination with IL-6,
Effects can be expected when used alone or in combination with other hematopoietic factors.

[Sequence list]

Claims (2)

[Claims] [Claim 1] Polypeptide BUF-3, BUF-4
and BUF-5, a therapeutic agent for thrombocytopenia containing one or more substances as an active ingredient. [Claim 2] Further interleukin 6 as an auxiliary agent,
The therapeutic agent for thrombocytopenia according to claim 1, which contains one or more substances selected from among interleukin-3, interleukin-1, and granulocyte colony-stimulating factor.