CN1183962C - 具有动员活性的制剂 - Google Patents
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Abstract
本发明公开了一种增加哺乳动物外周血液循环中干细胞和祖细胞数量的方法。其特征在于，施用去纤维蛋白多核苷酸，并联合或依次施用至少一种能够动员造血祖细胞的造血因子，优选为粒细胞集落刺激因子(G-CSF)。
Description
本发明涉及一种新的能够增加哺乳动物外周血液循环中干细胞和祖细胞数量的制剂；其特征在于，该制剂含有去纤维蛋白多核苷酸(defibrotide)和至少一种能够动员(mobilize)造血祖细胞的造血因子，优选为G-CSF。
发明领域
近年来，大量研究用来探讨增加哺乳动物(尤其是人类)外周血循环中干细胞和造血祖细胞的含量的可能性。实际上，干细胞和/或造血祖细胞的获得在一些领域是非常重要的，这些领域诸如有循环造血祖细胞的自体移植、循环造血祖细胞的异体移植以及在循环造血细胞的基因治疗的过程中。
尽管出生后，干细胞和祖细胞几乎只存在于骨髓中，但它们仍然具有迁移性质；也就是说，在生理条件下它们可以通过骨髓腔进入血液循环。该过程即通常所说的“动员”，通过各种处理例如使用细胞因子特别是粒细胞集落生长因子(G-CSF)，可以在哺乳动物中扩增。该过程的反过程(称作“归巢”)发生在例如造血细胞移植后接受放射治疗的病人中；然而，动员和归巢的机制仍然不明确(C.F.Craddock等人，“在灵长类和小鼠中VLA4整联蛋白抗体动员长效种群恢复细胞和增大细胞因子诱导的迁移(Antibodies to VLA4 IntegrinMobilize Long-Term Repopulating Cells and Augment Cytokine-InducedMobilization in Primates and Mice)”，Blood，Vol.90，n.12，1997，pp.4779-4788；F.Prosper等，“外周血祖细胞的动员和归巢与α4β1整联蛋白的表达和功能的可逆减量调节有关(Mobilization and Homing of PeripheralBlood Progenitors is Related to Reversible Downregulation of α4β1Integrin Expression and Function)”，J.Clin.Invest.，Vol.101，n.11，1998，pp.2456-2467；M.Vermeulen等，“粘着分子在鼠造血干细胞和祖细胞的归巢和动员中的作用(Role of Adhesion Molecules in the Homing andMobilization of Murine Hermatopoietic Stem and Progenitor Cells)”，Blood，Vol.92，n.3，1998，pp.894-900)。
G-CSF(CAS登记号为143011-2-7/Merck Index，1996，page 4558)是一种造血生长因子，其在粒系祖细胞的增殖和分化中是不可缺少的；该因子是一种分子量为18-22KDa、通常响应各种细胞的特定刺激而产生的糖蛋白，其中这些细胞包括单核细胞、成纤维细胞和内皮细胞。术语“去纤维蛋白多核苷酸”(CAS登记号83712-60-1)通常是指从动物和/或植物组织中提取的一种多脱氧核糖核苷酸(US3,770,720和US3,899,481)；该多脱氧核糖核苷酸一般是以碱金属盐(通常为钠盐)的形式存在。去纤维蛋白多核苷酸主要用作抗凝剂(US3,829,567)，但它还可用于其它用途，如用于治疗急性肾功能不全(US4,694,134)和急性心肌缺血(US4,693,995)。美国专利US4,985,552和US5,223,609中描述了一种制备去纤维蛋白多核苷酸的工艺方法，通过此工艺制成的产品具有稳定和确切的物理-化学特性，同时也无任何不良副作用。
根据本发明的目的，术语“去纤维蛋白多核苷酸”应被理解为从动物和/或植物组织中特别是从哺乳动物的器官中提取出来的任一种寡核苷酸和/或多核苷酸。优选地，去纤维蛋白多核苷酸是根据上述专利中所描述的方法生产出来的，因此这些方法本身也应作为本说明书的一部分；更为优选的是，去纤维蛋白多核苷酸是用美国专利US4,985,552和US5,223,609中所描述的方法生产的
发明详述
人们业已惊奇地发现，通过施用去纤维蛋白多核苷酸，并同时或依次施用一种具有动员造血祖细胞的能力的造血因子，可以增加干细胞和造血祖细胞的迁移。
从实施例中可以看出，通过施用去纤维蛋白多核苷酸，并同时或依次施用一种具有动员造血祖细胞能力的造血因子，所获得的动员能力大大高于单独服用造血因子所获得的动员能力。
因此，本发明的主题为一种制剂，其包括作为“活性成分”的去纤维蛋白多核苷酸和至少一种具有动员造血祖细胞能力的造血因子，优选为G-CSF。在优选实施方案中，该制剂由一种可注射的水溶液组成。另外，该制剂也可以由两种不同的溶液构成，其中一种含有去纤维蛋白多核苷酸，另一种含有具有动员造血祖细胞能力的造血因子。因此本发明的制剂可以是一种可以同时、单独或依次使用上文所述活性成分的复合制剂，以增加哺乳动物外周血循环中干细胞和/或造血祖细胞的数量。
本发明的第二个主题是，去纤维蛋白多核苷酸联合至少一种具有动员造血祖细胞能力的造血因子在制备制剂中的应用。该制剂能够增加哺乳动物、优选为人类外周血循环中干细胞和/或造血祖细胞的数量。
本发明的另一个主题是，一种增加哺乳动物外周血循环中干细胞和/或造血祖细胞数量的方法，其特征在于，给哺乳动物施用去纤维蛋白多核苷酸，并同时或依次服用至少一种具有动员造血祖细胞能力的造血因子。本发明实验中所用的造血因子为G-CSF；然而，本发明并不排除通过使用G-CSF之外的、具有动员造血祖细胞能力的其它造血因子也可以得到类似的结果，这些造血因子诸如有粒细胞巨噬细胞集落生长因子(GM-CSF)、Flt3配体(FL)、干细胞生长因子(SCF)、血小板生成素(TP0)、白介素8(IL-8)以及其它的本领域技术人员所知晓的造血因子。
在实验的第一阶段与G-CSF联合使用的去纤维蛋白多核苷酸是Crinos Spa提供的ProciclideTM，该产品按照美国专利US4,985,552和US5,223,609中所描述的方法生产。
至于施用上述两种活性成分的方法并不受到本发明目的的限制。也就是说，可以按照本领域公知的方法和剂量给予哺乳动物(特别是人类)去纤维蛋白多核苷酸和具有动员造血祖细胞能力的造血因子；通常可以通过口服、肌肉注射、腹腔注射、皮下注射或静脉内注射给药。其中静脉注射为优选的给药途径。
上述两种活性成分也可以同时或依次施用。也就是说，第一种情况是，通过使用单一制剂给予上述两种活性成分，该单一抑制剂包括上述两种活性成分，并可以任选地在其中加入本领域公知的赋型剂和/或辅助剂；另一种情况是，上述两种活性成分可以依次给予，也就是说，使用两个不同的制剂，其中一个含有具有动员造血祖细胞能力的造血因子、优选为G-CSF，而另一个含有去纤维蛋白多核苷酸。
通常，皮下注射G-CSF，其剂量为5-24ug/kg，而DEF可以通过连续的静脉输注给予，剂量为5-15mg/kg/hr，连续输注2-7天。
从本申请的实施例中可以看出(但本发明并不局限于这些实施例)，给鼠(最常见的实验用哺乳动物模型)和猴联合施用G-CSF和去纤维蛋白多核苷酸，所获得的动员能力比单独施用G-CSF所获得的动员能力高得多。这一点对于那些希望获得高水平动员能力的治疗领域来说是非常有利的。
实施例1
本试验用于评价施用G-CSF和/或去纤维蛋白多核苷酸(DEF)对鼠科动物血液中存在的白细胞(WBC)数量的影响。对年龄在6-8周、体重为20-25克的BALB/c鼠腹膜内注射给予G-CSF(5ug/鼠/天)，DEF(1mg/鼠/天)，或联合使用G-CSF(5ug/鼠/天)和增加剂量的DEF(1，10，15mg/鼠/天)。对未给予G-CSF和/或DEF的对照组的小鼠腹膜内注射稀释至0.1％的鼠血清白蛋白的盐溶液(PBS/MSA)。对小鼠进行上述治疗5天，在经上述治疗的3天或5天后、或停止上述治疗的3天后将小鼠处死。本试验的结果见图1。下列标记代表各组小鼠：G-CSF(■)(n＝24)，DEF1(○)(n＝3)，G-CSF+DEF1(◆)(n＝13)，G-CSF+DEF10(▲)(n＝6)，G-CSF+DEF15(●)(n＝23)。给予PBS/MSA的对照组小鼠的平均白细胞计数为2.87±0.2×106/毫升血。用平均数±平均数的标准差(SEM)表示各组数据。
实施例2
研究实施例1中的小鼠每毫升血中形成总的集落细胞(CFC)的动员动力学。给予PBS/MSA的对照组小鼠的平均CFC计数为39±12/毫升血。数据见图2，并用平均数±SEM表示，其是由每个时间点上对每个动物的样品进行双份培养而得出的。
实施例3
实施例1中小鼠的每105个外周血的棕黄层细胞(buffy-coat cell)中总CFC(CFU-GM+BFU-E+CFU-Mix+HPP-CFC)的频数改变。对照组小鼠中的平均CFC计数为3.5±1；数据见图3，并用平均数±SEM表示，其是由每个时间点上对每个动物的样品进行双份培养而得出的。
实施例4
实施例1中经过5天治疗的小鼠每毫升血中总CFC(CFU-GM+BFU-E+CFU-Mix+HPP-CFC)数量。数据见图4，并用平均数±SEM表示，其是对每个动物的样品进行双份培养而得出的。
实施例5
实施例1中小鼠的每105个外周血的棕黄层细胞中总CFC(CFU-GM+BFU-E+CFU-Mix+HPP-CFC)的频数。数据见图5，并用平均数±SEM表示，其是对每个动物的样品进行双份培养而得出的。
实施例6
该实验用于评价施用G-CSF和/或DEF对猴外周血循环中造血祖细胞/干细胞(高增殖潜能集落刺激细胞，或HPP-CFC)数量的影响。实验中使用4-6岁的、健康的猕猴(Macaca Mulatta)，其血液学和临床化学的检测指标均正常。皮下注射G-CSF，剂量为100μg/kg，连续使用2个周期，每周期5天；在第二个周期中静脉输注DEF，剂量为15mg/kg/hr，连续5天。进行采血、G-CSF给药、换药时对受试动物实施麻醉。本实验的结果见图6。从该图中可以看出，联合使用G-CSF和去纤维蛋白多核苷酸的猴子中的HPP-CFC的动员能力是单独使用G-CSF的猴子的8倍。
结论
从图1可以很容易地看出，联合使用G-CSF和去纤维蛋白多核苷酸使小鼠血液循环中白细胞的含量显著增加。需要特别指出的是，单独使用去纤维蛋白多核苷酸对血液循环中白细胞的含量并无积极作用。因此，联合使用G-CSF和去纤维蛋白多核苷酸能够使白细胞数量惊人地增加，并存在着剂量-效果关系，其并不仅仅是两个相互独立效应的加和。
图2至图5清楚地表明在小鼠中联合使用G-CSF和去纤维蛋白多核苷酸而达到的动员水平是单独使用G-CSF的动员水平的10倍至100倍。最后，图6证实，联合使用G-CSF和去纤维蛋白多核苷酸使猴子中干细胞(HPP-CFC)的动员能力是单独使用G-CSF的8倍。
两个活性成分的联合效应存在着剂量-效果关系，因为动员能力的增加与施用的去纤维蛋白多核苷酸的量呈现一定的比例；所有情况下动员能力的最高峰均在施用药物的大约第5天左右出现。
Claims (9)
1.一种制剂，其含有作为活性成分的去纤维蛋白多核苷酸，以及至少一种具有动员造血祖细胞能力的造血因子。
2.如权利要求1所述的制剂，其特征在于，具有动员造血祖细胞能力的造血因子是G-CSF。
3.如权利要求1所述的制剂，其特征在于，所述制剂为水溶液。
4.如权利要求1所述的制剂，其特征在于由两种不同的、可单独服用的制剂构成，其中一种含有具有动员造血祖细胞能力的造血因子，另一种含有去纤维蛋白多核苷酸。
5.一种作为复合制剂使用的制剂，含有去纤维蛋白多核苷酸和至少一种具有动员造血祖细胞能力的造血因子，用于同时、单独或依次施用以增加哺乳动物外周血液循环中的干细胞和/或造血祖细胞的数量。
6.权利要求5所述的制剂，其中具有动员造血祖细胞能力的造血因子为G-CSF。
7.如权利要求1-6任一项所述的制剂，进一步含有常用的赋形剂和/或辅助剂。
8.与至少一种具有动员造血祖细胞能力的造血因子联合使用的去纤维蛋白多核苷酸在制备能够增加哺乳动物外周血液循环中的干细胞和/或造血祖细胞数量的制剂中的应用。
9.如权利要求8所述的应用，其特征在于，具有动员造血祖细胞能力的造血因子是G-CSF。
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| EP00830293A EP1147777A1 (en) | 2000-04-18 | 2000-04-18 | Combination of defibrotide and G-CSF and its use to activate haematopoietic progenitors |
| EP00830293.7 | 2000-04-18 |
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| US8771663B2 (en) | 2000-04-18 | 2014-07-08 | Gentium Spa | Formulation having mobilising activity |
| EP1656156A2 (en) * | 2003-08-13 | 2006-05-17 | Children's Hospital Medical Center | Mobilization of hematopoietic cells |
| EP1656451A2 (en) | 2003-08-13 | 2006-05-17 | Children's Hospital Medical Center | Chimeric peptides for the regulation of gtpases |
| EP2103689A1 (en) * | 2008-03-19 | 2009-09-23 | Gentium S.p.A. | Synthetic phosphodiester oligonucleotides and therapeutical uses thereof |
| RU2376985C1 (ru) * | 2008-07-17 | 2009-12-27 | Владислав Николаевич Ласкавый | Средство для активации стволовых клеток |
| AU2010363814B2 (en) | 2010-11-12 | 2016-05-19 | Gentium S.R.L. | Defibrotide for use in prophylaxis and/or treatment of Graft versus Host Disease (GVHD). |
| AU2012383169B2 (en) | 2012-06-22 | 2017-12-21 | Gentium S.R.L. | Euglobulin-based method for determining the biological activity of defibrotide |
| EP3026122A1 (en) | 2014-11-27 | 2016-06-01 | Gentium S.p.A. | Cellular-based method for determining the potency of defibrotide |
| CN106567694A (zh) * | 2016-11-14 | 2017-04-19 | 北京国泰通源技术有限公司 | 智能控水短节 |
| CN110352246A (zh) * | 2018-01-12 | 2019-10-18 | 赛尔美格公司 | 产生细胞囊和细胞囊管的方法 |
| KR20220071274A (ko) | 2019-11-13 | 2022-05-31 | 후지필름 가부시키가이샤 | 가식 필름, 성형물, 및 전자 디바이스 |
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| IT1190313B (it) | 1986-04-17 | 1988-02-16 | Crinos Industria Farmaco | Procedimento per l'ottenimento di polidesossiribonucleotidi chimicamente definiti e riproducibili e prodotto farmacologicamente attivo risultante |
| IT1223322B (it) * | 1987-10-23 | 1990-09-19 | Crinos Industria Farmaco | Metodo per prevenire la formazione di coaguli sanguigni nel circuito extracorporeo di apparecchi di dialisi a composizione utile per esso |
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| IT1231509B (it) * | 1989-09-07 | 1991-12-07 | Crinos Industria Farmaco | Composizione farmceutica ad uso topico per la terapia della fragilita' capillare. |
| US5199942A (en) * | 1991-06-07 | 1993-04-06 | Immunex Corporation | Method for improving autologous transplantation |
| US5977083A (en) * | 1991-08-21 | 1999-11-02 | Burcoglu; Arsinur | Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states |
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| US5856444A (en) * | 1994-11-30 | 1999-01-05 | Chugai Seiyaku Kabushiki Kaisha | Thrombocytopoiesis stimulating factor |
| CA2260014A1 (en) * | 1996-07-10 | 1998-01-15 | Meiji Milk Products Co., Ltd. | Novel use of mk family as hematopoietic factor |
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| US6573372B2 (en) * | 1999-01-07 | 2003-06-03 | Heska Corporation | Feline immunoglobulin E molecules and compositions there of |
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