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

Leukocyte and / or hematopoietic stem / progenitor cell mobilization agent Download PDF

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JP4839021B2
JP4839021B2 JP2005171039A JP2005171039A JP4839021B2 JP 4839021 B2 JP4839021 B2 JP 4839021B2 JP 2005171039 A JP2005171039 A JP 2005171039A JP 2005171039 A JP2005171039 A JP 2005171039A JP 4839021 B2 JP4839021 B2 JP 4839021B2
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義雄 片山
四郎 久保西
和眞 池田
光音 谷本
信也 山口
泰弘 太田
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Description

本発明はＮ−アセチルノイラミン酸硫酸エステルの新規用途に関する。具体的には、前記物質を有効成分として含む、白血球及び／又は造血幹・前駆細胞の動員剤、いいかえると白血球増加剤及び／又は造血幹・前駆細胞の骨髄から末梢血への動員剤に関する。   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.

白血球増加剤及び造血幹・前駆細胞の末梢血への動員剤として、顆粒球コロニー刺激因子 （ｇｒａｎｕｌｏｃｙｔｅ ｃｏｌｏｎｙ−ｓｔｉｍｕｌａｔｉｎｇ ｆａｃｔｏｒ：Ｇ−ＣＳＦ）製剤が知られており、同種及び自家末梢血幹細胞の採取や、免疫不全患者の感染症や抗癌剤投与時の白血球減少症及び再生不良性貧血の治療に使用される。臨床においては、Ｇ−ＣＳＦが、白血球増加剤（特許文献１）や造血幹細胞動員剤（非特許文献１）として使用されることが報告されている。また、ＣＸＣＲ４アンタゴニストであるＡＭＤ３１００が、Ｇ−ＣＳＦによる造血幹細胞動員効果を増強させることが報告されている（非特許文献２）。マウスを使用した研究レベルにおいては、硫酸化多糖の一種、フコイダンが急速な造血幹細胞動員効果を示すことが報告されている（非特許文献３、４）。   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).

遺伝子組換Ｇ−ＣＳＦ製剤として、グラン（麒麟麦酒株式会社）、ノイトロジン（中外製薬株式会社）、ノイアップ（協和発酵工業株式会社）等がすでに市販されている。また、白血球減少症治療剤として、ロイコプロール（協和発酵工業株式会社）、イノチン（東和薬品株式会社）、ロイコン（三共株式会社）等が既に販売されている。   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は、分子量は約18,000〜22,000で、ヒトの場合１７４個（まれに１７８個）、マウスで１７８個のアミノ酸で構成され、血液成分の白血球の一種である好中球の分化増殖を誘導する糖タンパク質である。Ｇ−ＣＳＦは、成熟好中球の生存の延長や機能の亢進作用を有するが、エリスロポエチンによる赤芽球、インターロイキン３による芽球コロニーの形成も増強する。このようなＧ−ＣＳＦを産生する細胞としては、マクロファージ、ストローマ細胞、単球、Ｔリンパ球、繊維芽細胞、血管内皮細胞などが挙げられる。   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.

Ｇ−ＣＳＦ製剤はタンパク質製剤であるため、経済的に負担が大きい。また、造血幹・前駆細胞の骨髄から末梢血への動員剤としてＧ−ＣＳＦ製剤を使用する場合、入院で連日最低４日間の投与が必要であり、骨痛、発熱、頭痛等の副作用が報告されている。また、健常人ドナーの約１０％で造血幹・前駆細胞の動員効果が低いといわれており、移植に十分な造血幹・前駆細胞を採取するのは困難である。さらに、白血球増加剤としての使用は、頻回投与が必要であり、患者及び医師は苦痛と負担を強いられてきた。   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.

一方、Ｎ−アセチルノイラミン酸硫酸エステル等については、多く報告されており、天然のＮ−アセチルノイラミン酸ホモポリマーの混合物であるコロミン酸を原料として生産することができる（特許文献２，３）。コロミン酸は、１９５７年にバリー等により発見された物質で、シアル酸（アセチルノイラミン酸）を構成単位とし、α２→８で結合した分子量３万程度の高分子ポリマーである。大腸菌、髄膜炎双球菌などの血清型分類において非常に重要である。また、医薬品や化粧品の原料としても重要である。医薬品としては、例えば抗ＨＩＶ剤（特許文献２，３）、抗ロタウイルス剤（特許文献４）、抗炎症剤（特許文献５）、抗血栓剤（特許文献６）、線維芽細胞増殖因子（特許文献７）等が開示されている。   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.

しかしながら、Ｎ−アセチルノイラミン酸硫酸エステル等について、白血球及び／又は造血幹・前駆細胞の動員剤、詳しくは白血球増加剤や造血幹・前駆細胞の骨髄から末梢血への動員剤については報告はない。   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.

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

本発明は、Ｇ−ＣＳＦ製剤の投与に比べて経済的であり副作用の軽減化された白血球及び／又は造血幹・前駆細胞動員剤、詳しくは白血球増加剤及び／又は造血幹・前駆細胞の骨髄から末梢血への動員剤を提供することを課題とする。とりわけ、Ｇ−ＣＳＦ製剤を造血幹・前駆細胞の動員剤として使用する場合の頭痛、発熱、骨痛等の副作用が軽減化された製剤を提供することを課題とする。   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.

本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、Ｎ−アセチルノイラミン酸の硫酸エステル及びＮ−アセチルノイラミン酸ホモポリマー酸硫酸エステル等が、造血幹・前駆細胞の末梢血への動員作用及び白血球増加作用を有することを見いだし、本発明を完成した。   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.

すなわち本発明は以下よりなる。
１．Ｎ−アセチルノイラミン酸硫酸エステルを有効成分として含有することを特徴とする白血球及び／又は造血幹・前駆細胞の動員剤。
２．白血球の動員剤が、白血球増加剤である前項１に記載の動員剤。
３．造血幹・前駆細胞の動員剤が、造血幹・前駆細胞の骨髄から末梢血への動員剤である前項１に記載の動員剤。
４．前記Ｎ−アセチルノイラミン酸硫酸エステルは、下記一般式（Ｉ）：

Figure 0004839021
[式中、Ｒは、同一又は異なって水素原子又はＳＯ３Ｈ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ＳＯ３Ｈ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):
Figure 0004839021
 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.

本発明のＮ−アセチルノイラミン酸硫酸エステルを有効成分として含む製剤は、単剤で投与しても、末梢血でのコロニーの形成細胞（ＣＦＵ−Ｃ）の増加及び白血球の増加が認められ、造血幹・前駆細胞の骨髄から末梢血への動員作用及び白血球増加作用を有するという効果が確認された。また、Ｇ−ＣＳＦ製剤投与後に、上記本発明の動員剤を併用して投与すると、Ｇ−ＣＳＦ製剤単独使用の場合に比べて、コロニーの形成細胞の増加が有意に認められた。これにより、本発明の動員剤は、Ｇ−ＣＳＦ製剤の代替品又は併用製剤として優れている。   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.

本発明の「Ｎ−アセチルノイラミン酸硫酸エステル」は、Ｎ−アセチルノイラミン酸硫酸エステル及びＮ−アセチルノイラミンホモポリマー硫酸エステルを含み、さらにこれらの薬学的に許容できる塩をも含む概念で使用される。
本発明の「白血球及び／又は造血幹・前駆細胞の動員剤」において、「動員」なる用語は、本明細書において、細胞の種類に限らず他の部位から細胞が集積するという意味で使用する。例えば白血球が末梢血に集積したり、造血幹・前駆細胞が骨髄から末梢血に集積することを白血球及び／又は造血幹・前駆細胞が動員するという意味で使用される。本発明における白血球及び／又は造血幹・前駆細胞の動員剤は、いいかえれば白血球増加剤及び／又は造血幹・前駆細胞の骨髄から末梢血への動員剤である。本発明の白血球及び／又は造血幹・前駆細胞の動員剤（以下、単に「本発明の動員剤」という場合もある。）は、Ｎ−アセチルノイラミン酸硫酸エステルを有効成分として含み、いいかえれば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できる塩を有効成分として含む。 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.

本発明において、Ｎ−アセチルノイラミン酸硫酸エステルは、例えば特許文献２〜７に記載のものを使用することができる。原料は特に限定されるものではないが、重合度（ｎ）が一定の数であるホモポリマーを用いても良く、例えばコロミン酸のような天然のＮ−アセチルノイラミン酸ホモポリマーの混合物を用いても良い。また取得の由来も特に限定されず、天然から抽出、精製したものでも良く、化学的手法により合成したものであっても良い。   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.

本発明のＮ−アセチルノイラミン酸硫酸エステルは、Ｎ−アセチルノイラミン酸残基１分子あたりのＳＯ３Ｈ基の数が０．１〜３であることが好ましい。このような化合物として、具体的には以下に示す一般式（Ｉ）で表すことができる。ここにおいて、式中Ｒは、同一又は異なって水素原子、又はＳＯ３Ｈを示し、ｎは１〜１０００の整数、好ましくは３〜２００の整数、より好ましくは６〜１００の整数を示す。本発明のＮ−アセチルノイラミン酸硫酸エステルは、ｎが１種のみである単一化合物であっても良く、また、５〜１０００の範囲内でｎの値が異なる複数のＮ−アセチルノイラミン酸ホモポリマーの硫酸エステルの混合物であってもよい。好ましい分子量としては、平均分子量が5,000〜50,000であり、より好ましくは8,000〜30,000である。分子量は、プルラン、シアル酸オリゴマーなどを標準物質とし、ＨＰＬＣにより測定することができる。 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.

Figure 0004839021
Figure 0004839021

本発明におけるＮ−アセチルノイラミン酸硫酸エステルは、以下の方法に従い製造することができる。Ｎ−アセチルノイラミン酸若しくはＮ−アセチルノイラミン酸ホモポリマー１重量部に対し、溶媒の存在下又は不存在下に触媒０．５〜２００重量部、硫酸化剤０．２〜３０重量部を反応させる。反応時間は０．１〜４８時間程度であり、反応温度は−４０〜９０℃程度である。   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.

触媒としては、ピリジン、ジメチルアミノピリジン、トリエチルアミンなどが挙げられる。硫酸化剤としては、クロロスルホン酸、ピペリジン硫酸、サルファトリオキシド・トリメチルアミン錯体などが挙げられる。触媒としてピリジンなどを過剰量使用する場合には、溶媒を使用する必要はない。溶媒は、必ずしも使用する必要はないが、使用する場合には、ジメチルホルムアミド、ジメチルスルホキシド、ホルムアミドなどを使用することができる。
硫酸化反応終了後は、公知の方法、例えば濃縮、ゲルろ過、イオン交換などの各種クロマトグラフィー、再沈殿、透析などの処理を行うことができる。 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.

本発明において、薬学的に許容される塩とは、ナトリウム、カリウム、リチウムなどのアルカリ金属塩、マグネシウム、カルシウムなどのアルカリ土類金属塩などが挙げられる。これらの塩は、Ｎ−アセチルノイラミン酸若しくはそのホモポリマーの硫酸化反応液を水酸化ナトリウム、炭酸カリウムなどの塩で中和することで製造することもでき、また、いったん硫酸化糖の遊離の酸として得た後、それを用いて塩の形態としてもよい。更に、イオン交換樹脂、イオン交換ゲル、イオン交換セルロースカラムを用いて塩の形態にしても良い。   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.

本発明のＮ−アセチルノイラミン酸硫酸エステルは、薬学的に許容される種々の添加剤とともに、白血球及び／又は造血幹・前駆細胞の動員剤として用いることができる。本発明の動員剤は、特に限定されるものではないが、例えば、注射剤や、錠剤、カプセル剤、顆粒剤、細粒剤、乳剤などの経口剤などの種々の形態の医薬製剤として用いることができる。また、本発明の動員剤は、前記医薬製剤のほか、造血幹・前駆細胞の骨髄から末梢血への動員能又は白血球増加能を有する健康食品、健康飲料としても用いることができる。   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.

本発明の動員剤を注射剤として調製する場合、添加剤としては、ｐＨ調整剤、緩衝剤、安定化剤、等張化剤を適宜配合することができる。該注射剤は、例えば静脈内、筋肉内又は皮下に投与される。また、経口剤として調製する場合は、賦形剤、崩壊剤、滑沢剤、結合剤、矯臭剤、矯味剤などを適宜配合することができる。   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.

また、本発明の動員剤は、単剤で、又は既に市販されている製剤と併用して使用することができる。本発明の動員剤を単剤として使用する場合の投与量は、投与すべき患者の年齢、性別、体重、症状などにより変動するが、有効成分として含有されるＮ−アセチルノイラミン酸硫酸エステルは、一般的には、ヒト成人に対する１投与単位当たり、経口剤では０．１〜７０００ｍｇ程度、好ましくは２０〜１０００ｍｇ、注射剤では０．１〜７０００ｍｇ、好ましくは２０〜２０００ｍｇ程度の間から適宜選択することができる。また、既に市販されている製剤と併用して使用する場合は、症状ならびに併用される市販剤の種類、投与量との関係において、適宜決定することができる。本発明の動員剤の投与時期は、症状に応じて適宜選択することができ、また併用剤との関係において適宜決定することができる。例えば、市販のＧ−ＣＳＦ製剤と併用する場合には、Ｇ−ＣＳＦ製剤の投与後に本発明の動員剤を投与することができる。   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.

本発明の造血幹・前駆細胞動員剤及び白血球増加剤としての薬理学的評価は、次のようにして行うことができる。
造血幹・前駆細胞動員剤としては、コロニー形成試験により評価することができる。例えば、造血幹細胞・前駆細胞の骨髄から末梢血への動員効果は、採血した血液を比重遠沈法にて末梢血を分離し、末梢血中のコロニー形成細胞の増加を確認することにより行うことができる。
白血球増加剤としては、末梢血中の白血球数を血球計算板によりカウントし、評価することができる。さらに公知の白血球数測定方法により評価してもよい。上記方法により、癌化学療法による好中球減少症、骨髄形成症候群に伴う好中球減少症、先天性・特発性好中球減少症、ヒト免疫不全ウイルス（ＨＩＶ）感染症の治療に支障をきたす好中球減少症、免疫抑制療法（例えば腎移植）に伴う好中球減少症等の各種好中球減少症への適用を評価することができる。 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.

以下実施例により本発明を説明するが、本発明はこれらに限定されるものではないことは明らかである。
（実施例１）
１）Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルの調製
ジメチルホルムアミド２０ｍＬに平均分子量約17,000のコロミン酸９０ｍｇ、サルファトリオキシド・ピリジン複合体４７７ｍｇ及びジメチルアミノピリジン３７ｍｇを加え、３０℃にて２４時間撹拌した。反応後氷冷し、反応液を１Ｍ炭酸水素ナトリウム５０ｍＬに滴下し、中和した。中和後、濾過により固形物を分離し、透析チューブ（VISKASE SALES社製、分画分子量12,000〜14,000）を用いて、透析を行った。
透析後、ナトリウムイオン型のイオン交換樹脂（アンバーライトＩＲ−１２０Ｂ、オルガノ（株）社製）カラムに通した。カラム流出液を減圧下で濃縮し、凍結乾燥により、平均分子量約22,000の標記化合物１２６ｍｇを粉体として得た。 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）造血幹・前駆細胞の末梢血への動員効果及び白血球の増加効果の確認
上記調製したＮ−アセチルノイラミン酸ホモポリマー硫酸エステルをリン酸緩衝液（ＰＢＳ）に１０ｍｇ／ｍＬの濃度で溶解した。C57BL/6マウスにＮ−アセチルノイラミン酸ホモポリマー硫酸エステルを１００ｍｇ／ｋｇの用量で、尾静脈より投与し、造血幹・前駆細胞の末梢血への動員効果及び白血球の増加効果を調べた。以下の実施例でも同様に、Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルの投与を行った。 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.

造血幹・前駆細胞の末梢血への動員効果は、上記尾静脈投与後の一定時間経過後に採血し、比重遠沈法にて末梢血単核球を分離し、メチルセルロースによるコロニーアッセイ法にて、コロニー形成細胞（colony-forming unit in culture: ＣＦＵ−Ｃ）を算定することにより確認した。
メチルセルロースによるコロニーアッセイは以下の手法により行った。STEMCELL TECHNOLOGIES INC.（バンクーバー、カナダ）のMETHOCULT GF M3534の測定キットを用いて、製品の使用方法に従いアッセイを行った。本製品には、造血前駆細胞を分裂・増殖させてコロニーを形成するのに必要な増殖因子が予めメチルセルロース半固形培地に添加されている。採血し、比重遠沈法にて分離した末梢血単核球を培養皿に加え、３７℃５％ＣＯ２の条件で１０日間培養し、４０個以上の細胞で形成された細胞塊をコロニーと定義して、倒立顕微鏡下でカウントし、これをＣＦＵ−Ｃとした。
白血球の増加効果は、末梢血をＰＢＳにて１０倍希釈し、６％酢酸との等量希釈で赤血球を溶血させ、血球計算板にて白血球数を算定することにより確認した。 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.

その結果を図１に示した。Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルを尾静脈投与すると、３０分をピークとして、造血前駆細胞が末梢血に動員されることが確認された。また、白血球数は投与後１時間でピークとなり、５時間後には定常レベルに戻った。   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.

（実施例２）
Ｇ−ＣＳＦによる造血幹・前駆細胞動員効果は、遺伝的背景に大きく左右され、C57BL/6マウスは動員されにくく、DBA/2マウスは比較的動員されやすいことが報告されている。C57BL/6マウス及びDBA/2マウスに、Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステル（１００ｍｇ／ｋｇ）を尾静脈投与した。対照として、同様にＰＢＳを投与した。投与後３０分で採血し、実施例１と同手法にてＣＦＵ−Ｃを算出した。 (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.

その結果を図２に示した。Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルを投与すると、Ｇ−ＣＳＦの結果と同様に、C57BL/6マウスに比べ、DBA/2マウスのほうがＣＦＵ−Ｃの動員効果が高かった。しかしながら、いずれのマウスにおいても、Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルを投与したほうが、対照と比較して高い動員効果が認められた。   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.

（実施例３）
本発明の動員剤について、硫酸基の必要性について検討した。DBA/2マウスに、Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステル（１００ｍｇ／ｋｇ）を投与した。比較例として、実施例１に記載の硫酸基を含まないコロミン酸（１００ｍｇ／ｋｇ）を、対照として、ＰＢＳを同様に投与した。投与後３０分で採血し、実施例１と同手法にてＣＦＵ−Ｃを算出した。 (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.

その結果を図３に示した。硫酸基を含まないコロミン酸を投与した系では、造血前駆細胞の末梢血への動員作用は認められなかった。これにより、造血幹・前駆細胞の動員には、硫酸基の付加が必要であることが確認された。   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.

（実施例４）Ｇ−ＣＳＦ（フィルグラスチム：filgrastim）との併用効果
Ｇ−ＣＳＦを、ＰＢＳ＋０．１％ウシ血清アルブミン（ＢＳＡ）で１５μｇ／ｍＬの濃度で溶解し、一回投与量１２５μｇ／ｋｇの用量で、C57BL/6マウスに１日２回、１２時間おきに投与した。最終回投与後２時間３０分に、ＰＢＳ又はＮ−アセチルノイラミン酸ホモポリマー硫酸エステル（１００ｍｇ／ｋｇ）を投与し、さらに３０分後に採血し、比重遠沈法にて末梢血単核球を分離し、メチルセルロースによるコロニーアッセイ法にて、ＣＦＵ−Ｃを算定した。 (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.

その結果を図４に示した。Ｇ−ＣＳＦ単独投与の場合は、２日間投与ではＣＦＵ−Ｃの動員は殆ど認められなかったが、４日間投与により、約２，０００／ｍＬのＣＦＵ−Ｃの動員が認められた。Ｇ−ＣＳＦの投与後にＮ−アセチルノイラミン酸ホモポリマー硫酸エステルを併用した系では、２日目で、Ｇ−ＣＳＦ単独投与の４日目と同様の動員効果を観察した。また、Ｇ−ＣＳＦを４日間投与後にＮ−アセチルノイラミン酸ホモポリマー硫酸エステルを併用することにより、単独投与に比べて４倍以上のＣＦＵ−Ｃ動員効果が認められた。   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.

以上説明したように、本発明のＮ−アセチルノイラミン酸硫酸エステルを有効成分として含む製剤は、単剤で投与しても、末梢血中の白血球の増加効果及びコロニー形成細胞（ＣＦＵ−Ｃ）の増加が認められた。これにより白血球増加作用及び造血幹・前駆細胞の骨髄から末梢血へ動員作用を有するという効果が確認された。また、Ｇ−ＣＳＦ製剤投与後に本発明の動員剤を併用して投与すると、Ｇ−ＣＳＦ製剤単独使用の場合に比べて、すぐれたコロニーの形成細胞の増加が認められた。さらに、Ｇ−ＣＳＦ製剤単独使用の場合に比べて早期にコロニー細胞の出現が確認された。つまり、本発明の動員剤は、Ｇ−ＣＳＦ製剤の代替品としても有効であり、Ｇ−ＣＳＦ製剤と併用使用とした場合は、Ｇ−ＣＳＦ製剤の投与期間の短縮、使用量の軽減が図られることが示唆された。これにより本発明の動員剤の使用は、Ｇ−ＣＳＦ製剤の単独使用に比べて患者負担が軽減化され、経済的にも優れており、産業上利用可能である。   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.

Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステルのコロニー形成能及び白血球増加効果を示す図である。（実施例１）It is a figure which shows the colony formation ability and leukocyte increase effect of N-acetylneuraminic acid homopolymer sulfate. (Example 1) マウスの種類（遺伝的な違い）によるコロニー形成細胞数の違いを示す図である。（実施例２）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) Ｎ−アセチルノイラミン酸ホモポリマーについて、硫酸基の有無によるコロニー形成細胞数の違いを示す図である。（実施例３）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 Ｇ−ＣＳＦ単独の場合とＮ−アセチルノイラミン酸ホモポリマー硫酸エステルの併用とのコロニー形成細胞数の違いを示す図である。（実施例４）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)

符号の説明Explanation of symbols

ＰＢＳ 対照（リン酸緩衝液）
ＳＣＡ Ｎ−アセチルノイラミン酸ホモポリマー硫酸エステル
ＣＡ コロミン酸 PBS control (phosphate buffer)
SCA N-acetylneuraminic acid homopolymer sulfate CA colominic acid

Claims (4)

Ｎ−アセチルノイラミン酸硫酸エステルを有効成分として含有することを特徴とする白血球及び／又は造血幹・前駆細胞の動員剤。 A leukocyte and / or hematopoietic stem / progenitor cell mobilization agent comprising N-acetylneuraminic acid sulfate as an active ingredient. 白血球の動員剤が、白血球増加剤である請求項１に記載の動員剤。 The mobilizing agent according to claim 1, wherein the leukocyte mobilizing agent is a leukocyte increasing agent. 造血幹・前駆細胞の動員剤が、造血幹・前駆細胞の骨髄から末梢血への動員剤である請求項１に記載の動員剤。 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. 前記Ｎ−アセチルノイラミン酸硫酸エステルは、下記一般式（Ｉ）：
Figure 0004839021
 [式中、Ｒは、同一又は異なって水素原子又はＳＯ３Ｈ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ＳＯ３Ｈ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、請求項１〜３のいずれか一に記載の動員剤。 The N-acetylneuraminic acid sulfate is represented by the following general formula (I):
Figure 0004839021
 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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