JP4839021B2 - Leukocyte and / or hematopoietic stem / progenitor cell mobilization agent - Google Patents

Description

  The present invention relates to a novel use of N-acetylneuraminic acid sulfate. Specifically, the present invention relates to a mobilizing agent for leukocytes and / or hematopoietic stem / progenitor cells, containing the substance as an active ingredient, in other words, a leukocyte increasing agent and / or a mobilizing agent for hematopoietic stem / progenitor cells from bone marrow to peripheral blood.

  A granulocyte colony-stimulating factor (G-CSF) preparation is known as a leukocyte increasing agent and an agent for mobilizing hematopoietic stem / progenitor cells to the peripheral blood. It is used for the treatment of infectious diseases of immunocompromised patients and leukopenia and aplastic anemia when administered with anticancer agents. In clinical practice, it has been reported that G-CSF is used as a leukocyte increasing agent (Patent Document 1) or a hematopoietic stem cell mobilizing agent (Non-Patent Document 1). Moreover, it has been reported that AMD3100 which is a CXCR4 antagonist enhances the hematopoietic stem cell mobilization effect by G-CSF (non-patent document 2). At the research level using mice, it has been reported that one of the sulfated polysaccharides, fucoidan, exhibits a rapid hematopoietic stem cell mobilization effect (Non-Patent Documents 3 and 4).

  As genetically modified G-CSF preparations, Gran (Soba Wine Co., Ltd.), Neutrogin (Chugai Pharmaceutical Co., Ltd.), Neuup (Kyowa Hakko Kogyo Co., Ltd.) and the like are already on the market. Moreover, leucoprole (Kyowa Hakko Kogyo Co., Ltd.), inotine (Towa Pharmaceutical Co., Ltd.), leucon (Sankyo Co., Ltd.), etc. are already on the market as leukopenia therapeutic agents.

  G-CSF has a molecular weight of about 18,000 to 22,000. It consists of 174 amino acids (rarely 178) for humans and 178 amino acids for mice. It is a glycoprotein to induce. G-CSF has an action of prolonging the survival and function of mature neutrophils, but also enhances the formation of erythroblasts by erythropoietin and blast colonies by interleukin-3. Examples of such cells that produce G-CSF include macrophages, stromal cells, monocytes, T lymphocytes, fibroblasts, and vascular endothelial cells.

  Since G-CSF preparations are protein preparations, they are economically burdensome. In addition, when G-CSF is used as a mobilizer for hematopoietic stem / progenitor cells from bone marrow to peripheral blood, it must be administered for at least 4 days every day in hospital, and side effects such as bone pain, fever, and headache have been reported. Has been. In addition, it is said that about 10% of healthy donors have a low mobilization effect of hematopoietic stem / progenitor cells, and it is difficult to collect hematopoietic stem / progenitor cells sufficient for transplantation. Furthermore, the use as a leukocyte increasing agent requires frequent administration, and patients and doctors have been burdened and burdened.

  On the other hand, many reports have been made on N-acetylneuraminic acid sulfate and the like, and colominic acid, which is a mixture of natural N-acetylneuraminic acid homopolymers, can be produced as a raw material (Patent Documents 2 and 3). ). Colominic acid is a substance discovered by Barry et al. In 1957, and is a high molecular weight polymer having a molecular weight of about 30,000 and having sialic acid (acetylneuraminic acid) as a structural unit and linked by α2 → 8. It is very important in serotyping such as E. coli and Neisseria meningitidis. It is also important as a raw material for pharmaceuticals and cosmetics. Examples of pharmaceuticals include anti-HIV agents (patent documents 2 and 3), anti-rotavirus agents (patent document 4), anti-inflammatory agents (patent document 5), antithrombotic agents (patent document 6), fibroblast growth factor ( Patent Document 7) and the like are disclosed.

  However, with regard to N-acetylneuraminic acid sulfate, etc., there are reports on the recruitment of leukocytes and / or hematopoietic stem / progenitor cells, specifically the leukocyte increase agent and the mobilization agent of hematopoietic stem / progenitor cells from bone marrow to peripheral blood. Absent.

Japanese Patent No. 2718426 JP-A-6-279503 Japanese Patent No. 3062906 Japanese Patent Laid-Open No. 9-278660 Japanese Patent Laid-Open No. 9-25576 Japanese Patent Laid-Open No. 10-158174 Japanese Patent Laid-Open No. 10-310602 Blood, 89, 7, p.2233-2258, 1997 Transfusion, 45, p.295-300, 2005 Blood, 96, 7, P.2460-2468, 2000 PNAS, 97, 12, p.6544-6549, 2000 Leuk Lymphoma, 31, p.61-69, 1998 Cytokine, 8, p.702-709, 1996

  The present invention relates to a leukocyte and / or hematopoietic stem / progenitor cell mobilizing agent that is economical and has reduced side effects compared to administration of a G-CSF preparation. It is an object to provide a mobilization agent for peripheral blood from blood vessels. In particular, it is an object of the present invention to provide a preparation with reduced side effects such as headache, fever and bone pain when a G-CSF preparation is used as a mobilizing agent for hematopoietic stem / progenitor cells.

  As a result of intensive studies to solve the above problems, the present inventors have found that N-acetylneuraminic acid sulfate, N-acetylneuraminic acid homopolymeric acid sulfate, etc. It has been found that it has a mobilizing action on peripheral blood and a leukocyte increasing action, and has completed the present invention.

[式中、Ｒは、同一又は異なって水素原子又はＳＯ_３Ｈを示し、ｎは１〜１０００の整数を示す。但し、Ｎ−アセチルノイラミン酸残基１分子あたりのＳＯ_３Ｈ基の数は０．１〜３である。]
で表されるＮ−アセチルノイラミン酸ホモポリマー硫酸エステル、又はその薬学的に許容される塩である、前項１〜３のいずれか一に記載の動員剤。 That is, this invention consists of the following.
1. A leukocyte and / or hematopoietic stem / progenitor cell mobilization agent comprising N-acetylneuraminic acid sulfate as an active ingredient.
2. 2. The mobilizing agent according to 1 above, wherein the leukocyte mobilizing agent is a leukocyte increasing agent.
3. 2. The mobilization agent according to 1 above, wherein the mobilization agent for hematopoietic stem / progenitor cells is a mobilization agent from the bone marrow of the hematopoietic stem / progenitor cells to the peripheral blood.
4). The N-acetylneuraminic acid sulfate is represented by the following general formula (I):

In the formula, R may be the same or different and represent a hydrogen atom or SO ₃ H, n is an integer of 1 to 1,000. However, the number of SO ₃ H groups per molecule of N-acetylneuraminic acid residue is 0.1-3. ]
The mobilizing agent according to any one of the preceding items 1 to 3, which is an N-acetylneuraminic acid homopolymer sulfate represented by formula (I) or a pharmaceutically acceptable salt thereof.

  The preparation containing the N-acetylneuraminic acid sulfate ester of the present invention as an active ingredient is recognized to have increased colony forming cells (CFU-C) and leukocytes in peripheral blood even when administered as a single agent. The effects of mobilizing hematopoietic stem / progenitor cells from the bone marrow to peripheral blood and increasing leukocytes were confirmed. In addition, when the mobilizing agent of the present invention was administered in combination after administration of the G-CSF preparation, an increase in colony forming cells was significantly observed as compared to the case of using the G-CSF preparation alone. Thereby, the mobilization agent of this invention is excellent as a substitute or combined preparation of a G-CSF formulation.

The “N-acetylneuraminic acid sulfate” of the present invention includes N-acetylneuraminic acid sulfate and N-acetylneuramin homopolymer sulfate, and also includes these pharmaceutically acceptable salts. used.
In the “recruitment agent for leukocytes and / or hematopoietic stem / progenitor cells” of the present invention, the term “mobilization” is used in the present specification in the sense that cells accumulate not only from the cell type but also from other sites. . For example, leukocytes and / or hematopoietic stem / progenitor cells are used to mobilize when leukocytes accumulate in peripheral blood or hematopoietic stem / progenitor cells accumulate from bone marrow into peripheral blood. The leukocyte and / or hematopoietic stem / progenitor cell mobilizing agent in the present invention is, in other words, a leukocyte increasing agent and / or hematopoietic stem / progenitor cell mobilizing agent from the bone marrow to the peripheral blood. The mobilizing agent for leukocytes and / or hematopoietic stem / progenitor cells of the present invention (hereinafter sometimes simply referred to as “mobilizing agent of the present invention”) contains N-acetylneuraminic acid sulfate as an active ingredient. N-acetylneuraminic acid homopolymer sulfate or a pharmaceutically acceptable salt thereof is included as an active ingredient.

  In the present invention, as N-acetylneuraminic acid sulfate, for example, those described in Patent Documents 2 to 7 can be used. The raw material is not particularly limited, but a homopolymer having a certain degree of polymerization (n) may be used. For example, a mixture of natural N-acetylneuraminic acid homopolymer such as colominic acid is used. May be. Further, the origin of acquisition is not particularly limited, and it may be extracted and purified from nature, or may be synthesized by a chemical method.

In the N-acetylneuraminic acid sulfate of the present invention, the number of SO ₃ H groups per molecule of N-acetylneuraminic acid residue is preferably 0.1 to 3. Specifically, such a compound can be represented by the following general formula (I). Here, R in the formula is the same or different and represents a hydrogen atom or SO ₃ H, and n represents an integer of 1 to 1000, preferably an integer of ₃ to 200, more preferably an integer of 6 to 100. The N-acetylneuraminic acid sulfate of the present invention may be a single compound in which n is only one kind, and a plurality of N-acetylneuramins having different values of n within the range of 5 to 1000 It may be a mixture of acid homopolymer sulfates. The molecular weight is preferably 5,000 to 50,000, more preferably 8,000 to 30,000. The molecular weight can be measured by HPLC using pullulan, sialic acid oligomer or the like as a standard substance.

  The N-acetylneuraminic acid sulfate in the present invention can be produced according to the following method. With respect to 1 part by weight of N-acetylneuraminic acid or N-acetylneuraminic acid homopolymer, 0.5 to 200 parts by weight of catalyst and 0.2 to 30 parts by weight of sulfating agent are present in the presence or absence of a solvent. React. The reaction time is about 0.1 to 48 hours, and the reaction temperature is about -40 to 90 ° C.

Examples of the catalyst include pyridine, dimethylaminopyridine, triethylamine and the like. Examples of the sulfating agent include chlorosulfonic acid, piperidine sulfate, sulfatrioxide · trimethylamine complex, and the like. When an excessive amount of pyridine or the like is used as a catalyst, it is not necessary to use a solvent. The solvent is not necessarily used, but when used, dimethylformamide, dimethylsulfoxide, formamide and the like can be used.
After completion of the sulfation reaction, known methods such as various chromatographies such as concentration, gel filtration, ion exchange, reprecipitation, dialysis and the like can be performed.

  In the present invention, pharmaceutically acceptable salts include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as magnesium and calcium. These salts can also be produced by neutralizing a sulfation reaction solution of N-acetylneuraminic acid or a homopolymer thereof with a salt such as sodium hydroxide or potassium carbonate, and once releasing sulfated sugar. After obtaining as an acid, it may be used as a salt form. Furthermore, you may make into the form of a salt using an ion exchange resin, an ion exchange gel, and an ion exchange cellulose column.

  The N-acetylneuraminic acid sulfate of the present invention can be used as a mobilizing agent for leukocytes and / or hematopoietic stem / progenitor cells together with various pharmaceutically acceptable additives. Although the mobilizing agent of the present invention is not particularly limited, for example, it can be used as pharmaceutical preparations in various forms such as injections, oral preparations such as tablets, capsules, granules, fine granules, and emulsions. Can do. Moreover, the mobilization agent of the present invention can be used as a health food or a health drink having the ability to mobilize hematopoietic stem / progenitor cells from bone marrow to peripheral blood or increase white blood cells in addition to the pharmaceutical preparation.

  When the mobilizing agent of the present invention is prepared as an injection, a pH adjuster, a buffer, a stabilizer, and an isotonic agent can be appropriately added as additives. The injection is administered, for example, intravenously, intramuscularly or subcutaneously. Moreover, when preparing as an oral agent, an excipient | filler, a disintegrating agent, a lubricant, a binder, a flavoring agent, a flavoring agent, etc. can be mix | blended suitably.

  The mobilizing agent of the present invention can be used alone or in combination with a preparation already on the market. When the mobilizing agent of the present invention is used as a single agent, the dosage varies depending on the age, sex, weight, symptoms, etc. of the patient to be administered, but N-acetylneuraminic acid sulfate contained as an active ingredient is In general, it is appropriately selected from about 0.1 to 7000 mg, preferably 20 to 1000 mg for oral preparations, and about 0.1 to 7000 mg, preferably about 20 to 2000 mg for injections per dosage unit for human adults. can do. Moreover, when using together with the formulation already marketed, it can determine suitably in relation to a symptom, the kind of marketed agent used together, and dosage. The timing of administration of the mobilization agent of the present invention can be appropriately selected according to the symptoms, and can be appropriately determined in relation to the concomitant drug. For example, when used in combination with a commercially available G-CSF preparation, the mobilization agent of the present invention can be administered after the administration of the G-CSF preparation.

The pharmacological evaluation of the hematopoietic stem / progenitor cell mobilizing agent and leukocyte increasing agent of the present invention can be performed as follows.
The hematopoietic stem / progenitor cell mobilization agent can be evaluated by a colony formation test. For example, the effect of mobilization of hematopoietic stem / progenitor cells from bone marrow to peripheral blood is performed by separating the peripheral blood from the collected blood by the specific gravity centrifugation method and confirming the increase of colony forming cells in the peripheral blood Can do.
As a leukocyte increasing agent, the number of leukocytes in peripheral blood can be counted and evaluated with a hemocytometer. Further, evaluation may be performed by a known leukocyte count measurement method. By the above method, neutropenia caused by cancer chemotherapy, neutropenia associated with myelogenesis syndrome, congenital / idiopathic neutropenia, and human immunodeficiency virus (HIV) infection are hindered. Application to various neutropenia such as neutropenia accompanying neutropenia and immunosuppressive therapy (for example, kidney transplantation) can be evaluated.

EXAMPLES The present invention will be described below with reference to examples, but it is obvious that the present invention is not limited to these examples.
Example 1
1) Preparation of N-acetylneuraminic acid homopolymer sulfate ester To 20 mL of dimethylformamide, 90 mg of colominic acid having an average molecular weight of about 17,000, 477 mg of sulfatrioxide-pyridine complex and 37 mg of dimethylaminopyridine were added and stirred at 30 ° C. for 24 hours. did. After the reaction, the reaction mixture was ice-cooled, and the reaction solution was added dropwise to 50 mL of 1M sodium hydrogen carbonate to neutralize. After neutralization, the solid matter was separated by filtration, and dialyzed using a dialysis tube (VISKASE SALES, fractional molecular weight 12,000-14,000).
After dialysis, it was passed through a sodium ion type ion exchange resin (Amberlite IR-120B, manufactured by Organo Corporation) column. The column effluent was concentrated under reduced pressure and freeze-dried to obtain 126 mg of the title compound having an average molecular weight of about 22,000 as a powder.

2) Confirmation of mobilization effect of hematopoietic stem / progenitor cells on peripheral blood and increase effect of leukocytes The N-acetylneuraminic acid homopolymer sulfate prepared above was dissolved in phosphate buffer (PBS) at a concentration of 10 mg / mL. did. N-acetylneuraminic acid homopolymer sulfate was administered to C57BL / 6 mice at a dose of 100 mg / kg from the tail vein, and the mobilization effect of hematopoietic stem / progenitor cells on peripheral blood and the leukocyte increase effect were examined. In the following examples, N-acetylneuraminic acid homopolymer sulfate was similarly administered.

The effect of mobilization of hematopoietic stem / progenitor cells on peripheral blood is collected after a certain period of time after the tail vein administration, peripheral blood mononuclear cells are separated by specific gravity centrifugation, and colony assay with methylcellulose is used. This was confirmed by calculating colony-forming unit in culture (CFU-C).
The colony assay with methylcellulose was performed by the following method. The assay was performed according to the method of use of the product, using a measurement kit of METHOD GF M3534 of STEMCELL TECHNOLOGIES INC. (Vancouver, Canada). In this product, growth factors necessary for dividing and proliferating hematopoietic progenitor cells to form colonies are added in advance to a methylcellulose semi-solid medium. Peripheral blood mononuclear cells that have been collected and separated by specific gravity centrifugation are added to a culture dish and cultured for 10 days at 37 ° C. under 5% CO ₂ , and cell clusters formed by 40 or more cells are identified as colonies. Defined and counted under an inverted microscope, this was designated as CFU-C.
The effect of increasing leukocytes was confirmed by diluting peripheral blood 10 times with PBS, lysing erythrocytes by equal dilution with 6% acetic acid, and counting the number of leukocytes using a hemocytometer.

  The results are shown in FIG. When N-acetylneuraminic acid homopolymer sulfate was administered via the tail vein, it was confirmed that hematopoietic progenitor cells were mobilized to peripheral blood with a peak at 30 minutes. The white blood cell count peaked at 1 hour after administration, and returned to a steady level after 5 hours.

(Example 2)
It has been reported that the effect of mobilization of hematopoietic stem / progenitor cells by G-CSF is greatly influenced by genetic background, C57BL / 6 mice are less likely to be recruited, and DBA / 2 mice are more likely to be recruited. N-acetylneuraminic acid homopolymer sulfate (100 mg / kg) was administered to C57BL / 6 mice and DBA / 2 mice via the tail vein. As a control, PBS was similarly administered. Blood was collected 30 minutes after administration, and CFU-C was calculated in the same manner as in Example 1.

  The results are shown in FIG. When N-acetylneuraminic acid homopolymer sulfate was administered, the mobilization effect of CFU-C was higher in DBA / 2 mice than in C57BL / 6 mice, as in the G-CSF results. However, in any of the mice, a higher mobilization effect was observed when N-acetylneuraminic acid homopolymer sulfate was administered compared to the control.

(Example 3)
Regarding the mobilizing agent of the present invention, the necessity of a sulfate group was examined. DBA / 2 mice were administered N-acetylneuraminic acid homopolymer sulfate (100 mg / kg). As a comparative example, colominic acid (100 mg / kg) containing no sulfate group as described in Example 1 was administered in the same manner as a control, and PBS. Blood was collected 30 minutes after administration, and CFU-C was calculated in the same manner as in Example 1.

  The results are shown in FIG. In the system administered with colominic acid containing no sulfate group, mobilization of hematopoietic progenitor cells to peripheral blood was not observed. Thus, it was confirmed that the addition of sulfate groups is necessary for the mobilization of hematopoietic stem / progenitor cells.

(Example 4) Combined effect with G-CSF (filgrastim) G-CSF was dissolved in PBS + 0.1% bovine serum albumin (BSA) at a concentration of 15 μg / mL, and a single dose of 125 μg / mL was obtained. A dose of kg was administered to C57BL / 6 mice twice a day every 12 hours. Two hours and thirty minutes after the final administration, PBS or N-acetylneuraminic acid homopolymer sulfate (100 mg / kg) was administered, blood was further collected 30 minutes later, and peripheral blood mononuclear cells were collected by specific gravity centrifugation. CFU-C was calculated by colony assay using methylcellulose.

  The results are shown in FIG. In the case of administration of G-CSF alone, mobilization of CFU-C was hardly observed after 2 days, but about 2,000 / mL of CFU-C was observed after administration for 4 days. In the system in which N-acetylneuraminic acid homopolymer sulfate was used in combination after administration of G-CSF, the same mobilization effect as that on Day 4 of G-CSF single administration was observed on the second day. In addition, when G-CSF was administered for 4 days, N-acetylneuraminic acid homopolymer sulfate was used in combination, and a CFU-C mobilization effect of 4 times or more was observed compared to single administration.

  As described above, the preparation containing the N-acetylneuraminic acid sulfate ester of the present invention as an active ingredient can increase leukocytes in peripheral blood and colony-forming cells (CFU-C) even when administered as a single agent. Increased. This confirmed the effect of increasing leukocyte activity and mobilizing hematopoietic stem / progenitor cells from bone marrow to peripheral blood. In addition, when the mobilizing agent of the present invention was used in combination after administration of the G-CSF preparation, an increase in the number of cells forming excellent colonies was observed as compared with the case of using the G-CSF preparation alone. Furthermore, the appearance of colony cells was confirmed earlier compared to the case of using the G-CSF preparation alone. That is, the mobilization agent of the present invention is also effective as an alternative to the G-CSF formulation. When used in combination with the G-CSF formulation, the administration period of the G-CSF formulation can be shortened and the amount used can be reduced. It was suggested that As a result, the use of the mobilizing agent of the present invention reduces the burden on the patient compared to the single use of the G-CSF preparation, is economically superior, and can be used industrially.

It is a figure which shows the colony formation ability and leukocyte increase effect of N-acetylneuraminic acid homopolymer sulfate. (Example 1) It is a figure which shows the difference in the number of colony forming cells by the kind (genetic difference) of a mouse | mouth. (Example 2) It is a figure which shows the difference in the number of colony forming cells by the presence or absence of a sulfate group about N-acetylneuraminic acid homopolymer. Example 3 It is a figure which shows the difference in the number of colony forming cells in the case of G-CSF independent, and combined use of N-acetylneuraminic acid homopolymer sulfate. (Example 4)

Explanation of symbols

PBS control (phosphate buffer)
SCA N-acetylneuraminic acid homopolymer sulfate CA colominic acid

Claims (4)

A leukocyte and / or hematopoietic stem / progenitor cell mobilization agent comprising N-acetylneuraminic acid sulfate as an active ingredient. The mobilizing agent according to claim 1, wherein the leukocyte mobilizing agent is a leukocyte increasing agent. The mobilization agent according to claim 1, wherein the mobilization agent for hematopoietic stem / progenitor cells is a mobilization agent from the bone marrow to peripheral blood of hematopoietic stem / progenitor cells. The N-acetylneuraminic acid sulfate is represented by the following general formula (I):

In the formula, R may be the same or different and represent a hydrogen atom or SO ₃ H, n is an integer of 1 to 1,000. However, the number of SO ₃ H groups per molecule of N-acetylneuraminic acid residue is 0.1-3. ]
The mobilization agent according to any one of claims 1 to 3, which is an N-acetylneuraminic acid homopolymer sulfate represented by formula (I) or a pharmaceutically acceptable salt thereof.

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