JPH10101574A - Treating and improving medicine for proliferative organ disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine capable of displaying excellent effect on proliferative organ diseases such as carcinoma, sarcoma and myoma by making the medicine contain an erythropoietin antagonist as an active ingredient. SOLUTION: This medicine contains an erythropoietin antagonist, preferably an antierythropoietin antibody or an erythropoietin receptor protein (preferably in particular a soluble erythropoietin receptor protein) as an active ingredient. It is preferable that this medicine is topically and directly administrated to cancered or tumoral tissues or organs in forms of liquids and solutions, e.g., injection and the like. If tumor focuses exist in topical parts, it is preferable to administrate to the cancer having a size of little finger head (about 1g) in an amount of about 30-50μg per one administration. This process is useful for treatment and reformation of every cancerous ulters, e.g. uterine cancer, uterine myoma and skin carcinoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an agent for treating and improving proliferative organ diseases. According to the present invention, a therapeutic and / or ameliorating agent having an excellent effect on proliferative organ diseases such as carcinoma, sarcoma, and fibroid is provided.

[0002]

2. Description of the Related Art Conventionally, surgical resection, irradiation, administration of anticancer drugs, or a combination thereof has been used as a treatment for proliferative organ diseases such as cancer and proliferative lesions in tissues or organs. However, basic and detailed research on the biological characteristics of cancer itself has lagged behind the vast advances in cancer diagnostic technology. Therefore, drastic treatment means have not been established yet.

[0003] Erythropoietin is involved in blood cell proliferation and differentiation. Unlike other cytokines, erythropoietin is not produced in blood cells, but is produced in the kidney or liver and released into the blood. Erythropoietin acts on erythroid progenitor cells, erythroid burst-forming cells (BFU-E) and erythroblast colony-forming cells (CFU-E), promoting their differentiation and proliferation, and inducing erythrocyte production. (Krantz SB, Blood, Vol. 77,
pp419-434 (1991)). When erythropoietin binds to the erythropoietin receptor present on the cell membrane of progenitor cells,
A signal is transmitted into the cell nucleus, which is considered to cause erythrocyte differentiation, that is, intracellular accumulation of globin mRNA, production of hemoglobin, and erythrocyte differentiation (D'Andrea AD et a
l., Cell, Vol.57, pp277-285 (1989)). However, the details of the mechanism have not been elucidated yet, and there are many points to be resolved in the future.

As a site where erythropoietin expresses its gene in a tissue other than a site related to erythroblasts, an embryo body immediately after implantation (Yasuda Y. et al., Develop. Growth Diff.
er., Vol. 35, pp711-722 (1993)), human, monkey and mouse brain (Marti HH et al., Eur. J. Neu. Sci., Vol.
8, pp666-676 (1996)). The present inventors have also found that the erythropoietin receptor gene is expressed in mouse decidua in addition to erythroblasts (Yasuda).
Y. et al., Develop. Growth Differ., Vol. 35, pp711-
722 (1993)). At present, the functions of erythropoietin or erythropoietin receptor genes at sites other than these blood cell systems have not been elucidated.

[0005] When the embryo is implanted in the endometrium of the decidua, the intima at the implantation site undergoes decidual change, and surrounds the embryo. Since the erythropoietin receptor gene is expressed in the decidua and the erythropoietin is not expressed, it is considered that the erythropoietin receptor is produced in the decidua and binds to erythropoietin in the bloodstream to transmit an erythropoietin signal. A search for normal human endometrium revealed no expression of the erythropoietin gene. However, expression of erythropoietin and erythropoietin receptor at the protein level was observed. Therefore, in human normal endometrium, erythropoietin is taken in from the blood, as in decidua, and is considered to be involved in the normal physiology of the uterus. On the other hand, cervical cancer, body cancer,
Erythropoietin mRNA for fibroids, ovarian cancer, and ovarian cysts
Was confirmed by RT-PCR and Southern blotting. These tissue searches revealed that erythropoietin, erythropoietin receptor protein, and proliferative nuclear antigen were present in the cancer cells. Therefore, it was speculated that erythropoietin is involved in cancer cell proliferation.

[0006] Erythropoietin is produced by genetic recombination and has been effectively used for the treatment of anemia, particularly for the treatment of anemia in renal dialysis patients and for preparation of autologous blood transfusion at the time of surgery. Erythropoietin receptor is expected to be used as an agonist for anemia or an antagonist such as erythrocytosis (WO90 / 08822), and it is described that it can be used for hypererythropoietinemia and hypererythropoietinemia. However, there is no known effect of treating cancer in tissues and organs.

[0007]

According to the above facts, the inventors of the present invention have proposed that if cancer cells can block the binding between erythropoietin and erythropoietin receptor produced from cancer tissue in a cancer tissue extirpated from a diseased lesion, cancer cells will As a result of intensive studies based on the presumption that it will disappear, soluble erythropoietin receptor (extracellular domain) having erythropoietin antagonism was injected into cancer tissue and the reaction was observed.
The disappearance of proliferating nuclear antigen in cancer tissue and death due to apoptosis of cancer cells were recognized, and the present invention was completed. That is, an object of the present invention is to provide a therapeutic / ameliorating agent for a proliferative organ disease containing an erythropoietin antagonist as an active ingredient. Specifically, containing an anti-erythropoietin antibody or erythropoietin receptor protein as an active ingredient,
An object of the present invention is to provide an agent for treating and improving proliferative organ diseases. More specifically, a treatment for proliferative organ disease, comprising a soluble erythropoietin receptor protein as an active ingredient.
It is an object to provide an improving agent.

[0008]

SUMMARY OF THE INVENTION The present invention relates to an agent for treating and improving proliferative organ diseases, which comprises an erythropoietin antagonist as an active ingredient. More specifically, the present invention relates to a therapeutic or ameliorating agent for proliferative organ disease, comprising an anti-erythropoietin antibody or an erythropoietin receptor protein as an active ingredient. More specifically, the present invention relates to a therapeutic or ameliorating agent for proliferative organ disease, comprising a soluble erythropoietin receptor protein as an active ingredient. The present invention provides a therapeutic and / or ameliorating agent having an excellent effect on proliferative organ diseases such as carcinoma, sarcoma, and fibroid.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Erythropoietin antagonists are used as an active ingredient of the agent for treating and improving proliferative organ diseases according to the present invention as described above. Such erythropoietin antagonists include anti-erythropoietin antibodies and erythropoietin receptor proteins. Erythropoietin receptor protein can be prepared by known methods (WO90 / 08822, JP-A-6-38787, Chemistry and Biology, 31 (4), pp. 270-274 (1993), US Pat. No. 5,292,545). Can be manufactured. Among the erythropoietin receptor proteins, a soluble erythropoietin receptor protein is particularly preferably used. This erythropoietin receptor protein is disclosed in
No. 8787. As the anti-erythropoietin antibody, a monoclonal or polyclonal antibody obtained by a conventional method is used. Both the erythropoietin receptor protein and the anti-erythropoietin antibody bind to erythropoietin in an organ or tissue, have an action of blocking the binding between erythropoietin and the erythropoietin receptor, and are considered to be functionally equivalent substances.

[0010] As described above, the erythropoietin receptor protein is a known protein, and its DNA sequence and amino acid sequence are described in the above-mentioned patent publications and other documents. The erythropoietin receptor protein in the present invention may be any protein that has a binding property to human erythropoietin and suppresses the production of autocrine (autocrine) of erythropoietin, that is, an erythropoietin antagonist. Therefore, a human erythropoietin receptor protein or an erythropoietin affinity fragment, a mouse erythropoietin receptor protein having high homology to the human erythropoietin receptor protein, a fragment thereof, or an analog thereof may be used. As the anti-erythropoietin antibody, an anti-erythropoietin poly or monoclonal antibody obtained by a conventional method is used.

[0011] The agent for treating or improving proliferative organ diseases of the present invention comprises:
It is used for treating or improving proliferative organ diseases such as carcinomas, sarcomas, and fibroids. Specifically, uterine cancer, uterine fibroids,
Skin cancer, etc., and other cancerous tumors such as adenocarcinoma such as rectum and lung front, stomach cancer, osteoblastoma, leiomyosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, and cancerous mesothelial tumor Can be used for treatment and improvement. It is preferred that the therapeutic ameliorating agent for proliferative organ disease be directly administered locally to a cancerous or tumorous tissue or organ in the form of a liquid such as an injection. The dose varies depending on the degree of invasion of the lesion, but when a cancer lesion is localized, about 30 to 50 μg should be administered at a time to a small finger size (about 1 g) cancer. In cases where the invasion is widespread or for metastatic cancer, 20 to 50 μg is intravenously or intraperitoneally 3 to 5 times a day.
The administration may be repeated once and the administration may be continued for several days. In this manner, reduction or disappearance of the cancer lesion is recognized.

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the present invention.

[0013]

Example 1 Production of Soluble Erythropoietin Receptor Protein The method of JP-A-6-38787 was followed. That is, according to the method of Example 2 of JP-A-6-38787, an sEPO-R expression vector pcmEPR sol · dhfr was constructed (hereinafter, the soluble erythropoietin receptor is sometimes referred to as sEPO-R), and this gene was transformed into Escherichia coli MC1061. / P3 was transfected. This strain was deposited at Micro Engineering Laboratory (currently the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology) as Micro Engineering Laboratories No. 12814. Similarly, an sEPO-R expression vector pcmEPR Msol.dhfr was constructed according to the method of Example 3, and this gene was transfected into E. coli MC1061 / P3. This strain was deposited at the Microtechnical Laboratory as Microbiological Research Bacterial Deposit No. 13813. In the present invention, the plasmid was removed from these Escherichia coli and transfected into CHO.dhfr ^- cells. That is, sEPO-R expression vector pcmEPRsol-dhfr, pcmEPRMsol
· The dhfr CHO by the calcium phosphate method (dhfr ^-) was introduced into a cell, the soluble erythropoietin receptor protein (SEPO-
R) Producer cells were selected. The sEPO-R gene was amplified by methotrexate (MTX), and the sEPO-R high-producing cell line N1
I got 4.2. The obtained cell line is grown in a culture medium containing 10% dialyzed fetal calf serum and 100 nM MTX in a nucleic acid-free α-MEN synthetic medium, and then contains 0.5% dialyzed fetal bovine serum.
The medium was replaced with OPTI-MEMI medium (manufactured by Gibco) to produce sEPO-R. 6 ml of EPO-immobilized CH-
After adding Sepharose 4B (15 mg EPO / ml gel) and gently contacting overnight at 4 ° C, the immobilized carrier was washed with 10 times the amount of PBS, and sEPO-R adsorbed on the carrier was treated with 1.5 M MgCl ₂ . PBS including
Eluted. The eluate was concentrated by ultrafiltration and the solvent was replaced with PBS, and then TSKgel G3000SW (Tosoh) was used.
Perform molecular weight fractionation by HPLC, partially purified sEPO-R (0.6 mg)
I got The obtained sEPO-R was subjected to molecular weight measurement using SDS-PAGE and Sephadex G-75 gel filtration method.
33 kDa.

[0014]

Example 2 Preparation of Injection The erythropoietin receptor protein obtained in Example 1 was dissolved in physiological saline to prepare a 200 μg / ml solution. This was filtered, dispensed into sterile microtubes in 200 μl aliquots, and stored frozen. This was used as an erythropoietin receptor protein injection. When used, this is dissolved in physiological saline before use.

[0015]

Example 3 Inhibition of Cancer Cell Growth by Soluble Erythropoietin Receptor Protein
Effect 1. Preparation of Administration Solution and Cancer Tissue Piece The erythropoietin receptor protein injection prepared in Example 2 was melted, mixed with physiological saline containing 0.5% Evans blue (Chroma) at a ratio of 2.5%, and colored. This was used as a liquid for administration. In addition, as a control, one containing no erythropoietin receptor protein was similarly colored and used as a control solution. As the cancer tissue piece, a human cervical squamous cell carcinoma tissue piece removed by surgery was washed with phosphate buffered saline and stored at 4 to 8 ° C.

[0016] 2. Injection of administration solution into cancer tissue pieces The cancer tissue pieces prepared in Example 3-1 were washed with an α-MEM medium (Gibco) supplemented with 100 IU / ml penicillin and 80 μg / ml streptomycin. The weight of the tissue piece was measured and divided into a size of 10 to 100 mg to prepare 12 test cancer tissue pieces. Four of these tissue pieces were observed as untreated pieces and fixed with Zamboni solution. 4 pieces of remaining 8 pieces
The administration solution and the control solution prepared in Example 3-1 were infused at a dose of 0.45 to 0.8 μl / mg tissue, respectively, into a administration group and a control group. Inject the micrometer syringe (8070
1. Hamilton Co., Ltd.) and a 32G injection needle (90131, Hamilton Co., Ltd.) were used to perform several 5 μl injections at several locations on tissue sections. The state of the injection was confirmed by blue coloring of the tissue piece and outflow of the injection liquid. After injecting administration solution and control solution, 37 ℃, 5% C
After culturing under O ₂ for 90 minutes, the same injection solution as the first injection was injected as the second injection. After further culturing for 60 minutes under the same conditions, the same injection solution as in the first injection was injected as the third injection. After the third injection, the cells were cultured for 60 minutes under the same conditions. After observing two of the four tissue pieces in each group, a Zamboni solution (Zamboni La
nd De Martino C., J. Cell Biol., Vol. 35, p148A (196
7)) fixed (administration group: 3SEPOR-210, control group: 3Sal-21
0). These tissues were used as tissue specimens according to the method described in Example 3-3 below. Fig. 1 shows the state of the tissue in the administration group.
FIG. 2 shows the state of the tissue of the control group.

The remaining two pieces of each group were subjected to a fourth injection (the same amount of the same injection as the first injection), and a culture solution of α-MEM medium containing 10% calf serum (Flow) was added. Mesh on microplate (organ culture grid # 3014,
Falcon, which was obtained by bending a triangle and processing it like a hexagonal table) was placed, and a filter (pore size: 0.45 μm) (JH, Millipore) was placed on top of it. At this time, the amount of the culture solution was such that each tissue piece was immersed in almost half, and after culturing for 8.5 hours under the same conditions, observation was performed.
fixed with oni solution (administration group: 4SEPOR-12H, control group: 3Sal
-12H). These tissues were used as tissue specimens according to the method described in Example 3-3 below. FIG. 3 shows the state of the tissue in the administration group, and FIG. 4 shows the state of the tissue in the control group.

[0018] 3. Preparation of Tissue Section and Microscope The samples obtained in Example 3-2 were each frozen to prepare a tissue section. Tissue sections are cryostat (Leica)
7 μm sections at a time, and 5 consecutive sections at 175 μm apart
One set of the sections was placed on two slide glasses and subjected to immunostaining. That is, in order to observe the growth of cancer cells in a tissue section, an anti-PCNA mouse monoclonal antibody PC-10 (DAK
O-PCNA, Proliferating Cell Nuclear Antigen; PC-10)
And stained with a Delafield-Hematoxylin solution (Merck) to check the state of nuclei. From the stained tissue sections prepared in this manner, a fixed area (56 × 10 ⁻⁴ mm ² ) was arbitrarily selected from 120 sections per specimen using only the cancer infiltration foci as a control, and the number of PC-10-positive cells, nuclear degeneration (mainly Apoptosis-like degeneration), and the number of degenerated degeneration cells. Table 1 shows the results.

[0019]

[Table 1]

In the administration group (3SEPOR-210) in which three injections were performed,
As compared with the control group and the untreated group, a decrease was observed in the number of PC-10 positive cells, and an increase was observed in the number of degenerated nuclei and degenerated degenerated cells. In contrast, the control group (3Sal
-210), no significant difference was observed in any of the number of PC-10 positive cells, the number of degenerated nuclei, and the degenerated degenerative cells as compared with the untreated group. In the administration group (4SEPOR-12H) and the control group (4Sal-12H) that were injected four times, as a result of immunostaining, disappearance of carcinoma vesicle sites was remarkably observed in the former (light staining), whereas in the latter, Hardly ever seen. From these results, it was confirmed that the erythropoietin soluble protein has an effect of suppressing the growth of cancer cells in a short time and eliminating or killing the cancer cells.

[0021]

Example 4 Soluble erythropoietin receptor or erythropoietin
In vivo cancer cell growth inhibitory effect of anti-cancer antibody The tumor cell inhibitory effect of soluble erythropoietin receptor, which was observed in vitro, was demonstrated to be tumorigenic after transplantation by implanting tissue explants extracted from patients into nude mice. soluble erythropoietin receptor or erythropoietin antibodies against the tumor was injected (hereinafter, sometimes referred to R _2), reduction in tumorigenicity was confirmed test loss effect.

That is, a 5-week-old nude mouse (male, BALC / C
jcl-nu) were bred under SPF conditions, and fed with sterile water and sterile food ad libitum, and used for transplantation at 6 weeks of age.
As transplants, two cases of cervical cancer (squamous cell carcinoma: one case of non-keratinized type, one case of adenocarcinoma) and two cases of endometrial carcinoma (adenocarcinoma) were used. After the operation, the cancer tissue pieces aseptically collected were washed with a phosphate buffer and stored in a cold phosphate buffer (4 to 8 ° C.) until the start of transplantation. 100 U / ml penicillin and 80 μg / ml at the start of transplantation
The cells were washed in a MEM culture solution (Gibco) supplemented with streptomycin. Then, 5 ×
The pieces cut into 5 × 5 mm ³ were used for transplantation.

An incision was made in the prepared graft at the back of the mouse (between both scapulas) when transplanting one mouse, and at the back and lower occipital region when two grafts were transplanted per mouse. The graft was inserted subcutaneously, and the incision was adhered with Novactane spray (Astra). After transplantation, the growth of the graft (major axis and minor axis) was measured over time (3 times / week). Graft on day 7 after grown to three times the size of the time of implantation were injected sEPO-R or R _2. Also, as a control group,
The same volume of saline was injected. Table 2 shows these injection amounts and injection methods. The result is shown in FIG. 5 as a tumor growth curve. As shown in this figure, it was confirmed that all the groups suppressed tumor growth. In the table,
The sEPO-R infusion is shown as S, and the saline infusion is shown as saline. The needle indicates a needle-only insertion procedure.

[0024]

[Table 2]

R ₂ treatment and three groups of subjects (1 and 2, 3
And 4, 5, and 6) were examined for the dose effect of suppressing tumor growth. The suppression degree was calculated based on the following equation. Degree of inhibition = (mean value of tumor reduction by R ₂ injection / (mean value of tumor reduction by saline injection))

FIG. 6 shows the dose effect of growth inhibition by the continuous treatment of R ₂ three times. In this figure, especially the experimental groups 5 and 6
, A remarkable degree of inhibition (5.84) was confirmed. In addition, when a significant difference test of the degree of growth inhibition was performed for these,
In all experimental groups, a significant difference was observed between the treatment group and the control group (Table 3).

[0027]

[Table 3]

Next, external observation of the tumors removed from these experimental groups was performed. FIG. 7 shows the results. As a result, all the resected tumors were wrapped in the capsule (FIGS. 7a and b), and sEPO-R and R
_{In the two} treatment groups, a large gap or space was observed between the tumor tissue and the capsule (FIG. 7a), and in the control group, a narrow gap or space was identified between the tumor tissue and the capsule (FIG. 7b). or,
Tumor tissue necrosis was observed in all cases in the treatment group, and non-necrotic areas were not present in a smaller area than in the necrotic areas, whereas necrotic areas were observed in 80% of the control group, but non-necrotic areas The part occupied a wider area of the tumor tissue. In addition, angiogenesis in the tumor tissue was mainly observed in the capsular portion in 87.5% of the treated groups, whereas the presence in the tumor tissue (FIG. 7b) was observed in the control group in 80% of the cases. Was.

Further, in all of these examples, immunostaining of the excised specimen was performed using various antibodies, and nuclear staining was further performed using a hematoxylin solution. These sections were used to assay changes in tissue organization, changes in erythropoietin-expressing cells, and apoptosis. 8 and 9 show the results.
Shown in As a result, by sEPO-R and R _2, erythropoietin-positive cells are the deaths in extensive site of the tumor tissue, are you convert unstructured tissue was observed.
That is, it was revealed that this site was caused by apoptotic death due to fragmentation of the nucleus of the tumor cell. In addition, with the death of apoptosis, the structure of the tissue was destroyed and a defective site was formed, and many large and small holes were formed. In addition, tumor tissue at non-necrotic sites also showed apoptotic death. On the other hand, in the control group, a necrotic image and apoptotic death of the tumor tissue were locally observed, but the non-necrotic tumor tissue was actively growing.
Moreover, the tumor tissue was hardly changed by the needle insertion treatment. Regardless of the concentration of R _2, the tumor tissue showed a necrotic image including glandular structures by the R ₂ treatment, and as a result, showed a tissue defect due to multiple focal necrosis. Extensive anatomical necrotic sites at the center of the tumor were interspersed with nuclear fragments and nuclei indicating nuclear enrichment. At this site, apoptotic bodies were accumulated innumerably. At the non-necrotic site at the tumor margin, the glandular cells were surrounded by apoptotic bodies and neutrophils, showing a progressive necrosis image. On the other hand, in the control group and the needle insertion treatment, no change was observed in most tumor tissues.

The present invention provides a therapeutic and / or ameliorating agent having an excellent effect on proliferative organ diseases such as carcinoma, sarcoma, and fibroid.

[Brief description of the drawings]

FIG. 1A is an immunostained image of a tissue of an administration group fixed in 1 hour after treatment with an erythropoietin receptor protein administration solution three times in Example 3. FIG.

[Explanation of Symbols] *: Site of dead degenerated cell population b. FIG.

[Explanation of symbols]

 ←: apoptotic dead cells c figure: a shows a high magnification image of the specimen.

[Explanation of symbols]

 Yajiri: Nuclear fragments

FIG. 2a: In Example 3, after treatment with a control solution three times,
The immunostaining image of the tissue of the control group fixed for 1 hour is shown.

[Explanation of symbols]

 *: Cancer infiltration site b diagram: a sample shows a middle enlarged image of the site indicated by the arrow.

[Explanation of symbols]

 ←: Presence of nucleus enrichment c figure: a shows a strongly magnified image of the cancer infiltration foci of the specimen a.

[Explanation of symbols]

 ← (Large): Swelling of the cytoplasm ← (Small): Light staining of the nucleus

FIG. 3a shows an immunostained image of a tissue fixed 8.5 hours after treatment with erythropoietin receptor protein four times in Example 3.

[Explanation of symbols]

*: Site where PC-10 positive cells disappeared. FIG. B: High magnification image of PC-10 cell disappearance site (*) in a sample.

FIG. 4a shows an immunostained image of a tissue fixed 8.5 hours after treatment with erythropoietin receptor protein four times in Example 3.

[Explanation of symbols]

 *: Cancer infiltration site b figure: a shows a strongly magnified image of cancer infiltration foci (*) of specimen a.

[Explanation of symbols]

←: PC-10 positive cells and degenerative degenerated cells In the figure, the sample a has a magnification of 58.4 and the sample b has a magnification of 292.
And the C sample shows a magnification of 584, respectively.

FIG. 5 shows a tumor growth curve of Example 4.

[Explanation of symbols]

 ↓: At the time of transdermal injection

FIG. 6 shows the dose effect of growth inhibition by 3 consecutive treatments of R _{2 of} Example 4.

FIG. 7a shows an example of a macro-tissue image (× 14) of experimental group 7 (sEPO-R treated group) in Example 4.

[Explanation of symbols]

← (Large): Separated part ← (Small): Necrotic part Yajiri: Non-necrotic part *: Coating b figure: Macroscopic image (× 12) of Example 8 (saline injection group) in Example 4 An example is shown.

[Description of Signs] ←: Necrotic site Yajiri: Blood vessel *: Capsule c figure: A tissue section of FIG. 7a stained with an anti-erythropoietin antibody (× 32).

[Explanation of symbols]

←: Tumor tissue defect d view of necrosis: tissue sections Figure 7b dyed with a R ₂ (× 80)
Is shown. .

[Explanation of symbols]

 ←: Tumor of glandular structure with necrotic cells Yajiri: Erythropoietin-positive cells

FIG. 8a: Experimental group 5 (R ₂ 16 mg) in Example 4.
An example (× 32) of a histological image of the (treatment group) is shown.

[Explanation of symbols]

←: Tissue destruction due to necrosis Yajiri: Nuclear accumulation image of dead cells in the necrotic area b figure: An enlarged image (× 320) of the non-necrotic area surrounded by a square in FIG. 8A.

[Explanation of symbols]

←: Nuclear fragment Yajiri: Fusion of dead nucleus c figure: apoptosis positive image of non-necrotic part in FIG. 8a (× 320)
Is shown.

[Explanation of symbols]

←: Apoptotic bodies aggregated in the nucleus accumulation area where necrosis is progressing. D figure: An example (× 32) of the tissue image of Example 6 (saline injection group) in Example 4 by anti-CD34 staining. .

[Explanation of symbols]

 ←: Dissected tumor Yajiri: Tumor tissue in non-necrotic area e-diagram: An enlarged image (× 320) of the tumor tissue in FIG. 8D is shown.

[Explanation of symbols]

 ←: Dividing cells

FIG. 9 a: Experimental group 3 (R ₂ 8 mg) in Example 4.
An example (× 20) of the histological image of (treatment) is shown.

[Explanation of symbols]

←: Tissue defect due to necrosis of tumor tissue Yajiri: Accumulation of necrotic cells b diagram: An example (× 20) of the histological image of experimental group 3 (R ₂ 16 mg treatment) in Example 4.

[Explanation of symbols]

←: Tissue defect due to tumor tissue necrosis Yajiri: Necrosis of glandular epithelium *: Unstructured site c figure: An enlarged image (× 200) of staining with the anti-erythropoietin antibody in FIG. 9b is shown.

[Explanation of symbols]

*: Unstructured site (reduction of erythropoietin-positive cells) ←: Nuclear fragment d diagram: Positive apoptosis reaction (× 200) at the site in FIG. 9b.

[Explanation of symbols]

←: Apoptotic body Yajiri: Nucleus dense image e figure: An example of a tissue image of the experimental group 4 (saline injection group) in Example 4 by staining with an anti-erythropoietin antibody (× 20)
Is shown.

[Explanation of symbols]

←: Proliferating part of epithelial-like cells f: An example (× 160) of a tissue image of the proliferating part in FIG.

[Explanation of symbols]

←: Neutrophil ingress Yajiri: Angiogenesis image g diagram: An example (× 20) of a tissue image of the experimental group 4 (needle group) in Example 4 by staining with an anti-erythropoietin antibody is shown.

[Explanation of symbols]

 ←: Adenocarcinoma h diagram: An enlarged image (× 160) of FIG. 9g is shown. .

[Explanation of symbols]

 Yajiri: Angiogenesis image

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 16/22 C07K 16/22

Claims (5)

[Claims] 1. A therapeutic or ameliorating agent for a proliferative organ disease, comprising an erythropoietin antagonist as an active ingredient. 2. The therapeutic or ameliorating agent for proliferative organ disease according to claim 1, wherein the erythropoietin antagonist is an anti-erythropoietin antibody. 3. The therapeutic or ameliorating agent for proliferative organ disease according to claim 1, wherein the erythropoietin antagonist is an erythropoietin receptor protein. 4. The agent for treating or improving a proliferative organ disease according to claim 3, wherein the erythropoietin receptor protein is a soluble erythropoietin receptor protein. 5. The agent according to claim 1, wherein the proliferative organ disease is a cancer or a malignant tumor.