CN116478268A - Bmp2蛋白保护液、bmp2蛋白制剂及其制备方法 - Google Patents
Bmp2蛋白保护液、bmp2蛋白制剂及其制备方法 Download PDFInfo
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A—HUMAN NECESSITIES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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Abstract
本发明首先提供了一种在常温下长期运输,保存,使用骨形态发生蛋白BMP2蛋白保护液,以及BMP2蛋白制剂及其制备方法。本发明提供的BMP2蛋白保护液和BMP2蛋白制剂通过提供pH值为4.0‑6.8的酸性环境,其包含的精氨酸保护并提升BMP2在常温25℃及37℃下的成骨诱导活性。
Description
技术领域
本发明涉及医药技术领域,特别是指一种骨形态发生蛋白BMP2蛋白保护液、BMP2蛋白制剂及其制备方法。
背景技术
骨形态发生蛋白BMP2是骨形成蛋白家族成员之一,具有多种生物学功能,在体内不仅可以诱导骨的形成,而且还是胚胎发育阶段必需的细胞调节因子。
BMP主要分布在动物的骨组织中。现在已经从牛、兔、猪、羊、狗、猴和人的脱钙骨基质中分离出BMP,并在人和兔的牙及肾组织中也提取到了BMP。在自然界中。现已经发现的BMP亚型就有20个以上,除BMP1外,均属于TGFβ超家族成员,彼此之间无论在基因、蛋白质序列或其高级结构,还是在生物学功能都非常相似。各种动物的BMP基因之间有很高的保守性,提示这是一个进化过程中非常保守和重要的调控分子家族。现在已克隆出来的BMP亚型中,一级结构中都有和TGFβ一样的非常保守的七个半胱氨酸残基,这些残基组成了三对链内二硫键和一对链间二硫键。这些二硫键对于维持分子的天然活性构象具有决定作用,使用还原剂将二硫键打开后,其骨诱导活性将完全丧失。天然BMP2是糖蛋白,但是BMP分子的糖基化修饰与它的诱骨活性无关,去糖基化后并不影响BMP2的活性。
人BMP2的cDNA全长1191bp;编码396个氨基酸,由信号肽序列、前肽区和成熟肽三部分组成。其中,成熟肽单体114个氨基酸,等电点约:8.6~8.8。BMP2不但在硬组织的发育、修复发挥至关重要的作用,还广泛参与几乎所有器官、组织的发生及发育成熟。因其强大的骨诱导活性,常将BMP2与一些载体材料如生物陶瓷(羟基磷灰石、磷酸三钙、生物活性玻璃)、天然组织材料(胶原、脱钙骨基质)、机高分子材料(聚乳酸、羟基丙酸多聚体等)复合,进行大段骨缺损修复、脊柱融合手术、治疗胫骨中段骨折或陈旧性骨折等难治性骨折。无论在动物实验还是临床实验,都取得了显著优于对照组的结果。随着基础实验和临床研究的进一步深入,可以预期BMP2会有广阔的应用前景
BMP2在机体内存在半衰期短、易向周围组织扩散或随血液扩散流失等缺点,导致其无法长期稳定的作用于机体。为了增强BMP2在作用部位的持续性和稳定性,目前常用的方法是利用基因重组技术制备BMP2,使其能够与细胞外基质材料、天然高分子材料、合成高分子材料和金属材料表面特异性的结合。
BMP2在生理条件下很难溶解,酸性溶液可以提高BMP2的溶解度,但现有的BMP2保护试剂其pH为极端酸性环境。不利于BMP2对细胞或组织的促成骨作用。已有报道口服左旋精氨酸(L-精氨酸,L-arginine)促进矮小儿童身高增长的作用【1】。精氨酸能够提供一氧化氮(NO),而NO是一种可高度弥散的自由基,作为第2信使和神经递质,是细胞和细胞间信息传递的重要调节因子。NO可以调节成骨细胞的增殖及其功能活性,从而在维持骨形态和骨重建中起着重要作用【2】。并且左旋精氨酸能促进骨质疏松骨折愈合,同时改善骨质疏松【3】。精氨酸与壳聚糖、DNA纳米粒控释PELA微球的结合应用于成骨的研究也有报道【4】。精氨酸也被广泛用于蛋白保护剂,比如精氨酸可以提升血红蛋白色素稳定性【5】,精氨酸的添加还可以提升肌原纤维蛋白氧化稳定性及凝胶性能,以及提高肉蛋白的氧化稳定性,改善肉制品的嫩度和持水性【6】,Kim NA等【7】介绍了精氨酸可以在蛋白液体制剂中作为保护剂【7】。但精氨酸溶液偏碱性,相较于乙酸等溶液会极大的降低BMP2的溶解度,不利于BMP2的应用。因此,需要开发出一种即含有精氨酸又能不影响BMP2溶解度还能提供一种pH温和的长期运输,保存,使用骨形态发生蛋白BMP2溶液或冻干粉的组合物。
引用索引:
【1】蒋明玉.口服精氨酸促进长骨线性生长的作用及机制的研究[D].复旦大学.
【2】镐英杰,王珍,李秀群,等.左旋精氨酸对大鼠成骨细胞的影响[J].中华实验外科杂志,2007,24(9):1.
【3】张世旭,路考生,石昌浩,等.左旋精氨酸对骨质疏松骨折骨愈合及血生化的影响[J].中国组织工程研究,2013,17(28):6.
【4】徐晓龙.精氨酸—壳聚糖/DNA纳米粒控释PELA微球的制备及其成骨诱导效能的实验研究[D].南方医科大学,2016.
【5】宋璇.精氨酸—血红蛋白色素稳定性及应用研究[D].西北农林科技大学.
【6】马文慧、李保玲、曹云刚、黄峻榕.不同浓度精氨酸对肌原纤维蛋白氧化稳定性及凝胶性能的影响[C]//中国食品科学技术学会第十七届年会.
【7】Kim NA.[Int J Pharm,2016,513(1-2):26]
发明内容
本发明提供了一种用于在常温下长期运输、保存时保证骨形态发生蛋白BMP2蛋白成骨诱导活性的溶液或冻干粉。
本发明首先提供了一种用于保护BMP2蛋白稳定性的组合物的制备方法,其包括:
1)将适量精氨酸溶于水中,使精氨酸浓度为0.001-0.5M;
2)加入适量酸,调整pH值4.0-6.8,即得BMP2蛋白保护液。
优选地,所述精氨酸浓度为0.001-0.5M;更优选地,精氨酸浓度为0.01M,或0.05M。
进一步优先地,步骤2)中,所述酸为乙酸。
本发明还提供了根据上述制备方法制备得到的BMP2蛋白保护液。
本发明的另一方面提供了一种BMP2蛋白制剂的制备方法,其包括:将BMP2蛋白组合物溶解于上述的BMP2蛋白保护液中,即得BMP2蛋白溶液。
优选地,所述BMP2蛋白组合物中,BMP2蛋白含量为0mg/ml-20mg/ml。
更优选地,以mg:mL计,BMP2蛋白与BMP2蛋白保护液的比例为0mg/ml-15mg/ml
在根据本发明的另一实施方案中,还包括:
将所述BMP2蛋白溶液放置于冷冻干燥机中冻干,得到BMP2蛋白冻干粉,复溶时在BMP2冻干粉中加入蛋白保护液,以mg:mL计BMP2蛋白冻干粉与蛋白保护液的比例为0mg/ml-15mg/ml。
本发明进一步提供了根据上述的制备方法制备得到的BMP2蛋白制剂。
本发明的上述技术方案的有益效果如下:
上述方案中,本发明提供的BMP2蛋白保护液通过提供pH值为4.0-6.8的酸性环境,其包含的精氨酸保护BMP2在常温25℃及37℃下存储的蛋白活性,提供良好的促成骨环境。
附图说明
图1为在根据本发明实施例1和2制备的蛋白保护液在25℃保存BMP2蛋白1月、2月、4月和8月后,碱性磷酸酶ALP活性检测试剂盒检测其对iMEF细胞成骨诱导活性,检测405nm波长下吸光值,吸光值越大代表碱性磷酸酶ALP活性越高即反映BMP2活性越高,吸光值越小代表碱性磷酸酶ALP活性越低即反映BMP2活性越低,蛋白保护液1和2使BMP2在25℃中储存1/2/4/8月后其促成骨活性明显高于常规的乙酸和盐酸。
图2为在根据本发明实施例1和2制备的蛋白保护液在37℃保存BMP2蛋白1月、2月、4月和8月后,碱性磷酸酶ALP活性检测试剂盒检测其对iMEF细胞成骨诱导活性,检测405nm波长下吸光值,吸光值越大代表碱性磷酸酶ALP活性越高即反映BMP2活性越高,吸光值越小代表碱性磷酸酶ALP活性越低即反映BMP2活性越低,蛋白保护液1和2使BMP2在37℃中储存1/2/4/8月后其促成骨活性明显高于常规的乙酸和盐酸。
图3为用茜素红对不同条件培养的iMEF细胞进行染色,以显示矿化结节,茜素红染色颜色越深代表BMP2成骨诱导活性越强,茜素红染色颜色越浅代表BMP2成骨诱导活性越弱,如图蛋白保护液1和2使BMP2具有更强的促iMEF细胞成骨分化活性。
图4为大鼠肌袋异位诱导成骨实验,4周时X射线照片检测,结果显示有明显显影,蛋白保护液1和2使含BMP2的丝素蛋白成骨材料具有更强的异位成骨性能。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
试剂与材料:
BMP2干粉购自金斯瑞生物科技有限公司
ALP(碱性磷酸酶)活性检测试剂盒购自碧云天生物技术有限公司
茜素红染色试剂盒购自Solarbio公司
BCA蛋白检测试剂盒购自碧云天生物技术有限公司
SD大鼠购自重庆医科大学动物实验中心
实施例1BMP2蛋白保护液制备方法
配置BMP2蛋白保护液1:
1)配制步骤:溶解或者稀释精氨酸以及乙酸,使BMP2蛋白保护液组合物最终含有0.01M/L精氨酸以及乙酸,通过乙酸调节蛋白保护液的pH值为4.5。
2)示例:
称取0.1742g精氨酸,溶解在10mL双蒸水中,以乙酸调节pH值到4.5。
实施例2BMP2蛋白保护液制备方法
1)配置BMP2蛋白保护液2:
溶解或者稀释精氨酸以及乙酸,使BMP2蛋白保护液组合物最终含有0.05M/L精氨酸以及乙酸,利用乙酸调节蛋白保护液的pH值为6.0。
2)示例:称取0.871g精氨酸溶解在10mL双蒸水中,以乙酸调节pH值到6.0。
实施例3蛋白保护液性能检测
将0.05mg BMP2干粉分别溶解在0.1ml的BMP2蛋白保护液1、0.1ml的BMP2蛋白保护液2中,以及0.1ml的50mM乙酸和0.1ml的1mM盐酸中,充分溶解后分别将0.1ml溶液各自分装到4支1.5ml的EP管中,每支25ul,然后放入-80°冰箱预冻过夜然后放入真空冷冻干燥机中冻干。冻干后放在25℃恒温培养箱中,分别储存1月、2月、4月和8月后各取出一支,然后处理iMEF细胞3天。采用ALP(碱性磷酸酶)活性检测试剂盒检测细胞中碱性磷酸酶的表达,来显示不同蛋白保护液下保存不同时间后BMP2的活性。结果如图1所示,蛋白保护液1和2可使BMP2在25℃中储存1/2/4/8月后其促成骨活性明显高于常规的乙酸和盐酸。
实施例4蛋白保护液性能检测
将0.05mg BMP2干粉分别溶解在0.1ml的BMP2蛋白保护液1、0.1ml的BMP2蛋白保护液2中,以及0.1ml的50mM乙酸和0.1ml的1mM盐酸中,充分溶解后分别将0.1ml溶液各自分装到4支1.5ml的EP管中,每支25ul,然后放入-80°冰箱预冻过夜然后放入真空冷冻干燥机中冻干。冻干后放在37℃恒温培养箱中,分别储存1月、2月、4月和8月后各取出一支,然后处理iMEF细胞3天。采用ALP(碱性磷酸酶)活性检测试剂盒检测细胞中碱性磷酸酶的表达,来显示不同蛋白保护液下保存不同时间后BMP2的活性。结果如图2所示,蛋白保护液1和2可使BMP2在37℃中储存1/2/4/8月后其促成骨活性明显高于常规的乙酸和盐酸。
实施例5茜素红染色实验检测蛋白保护液性能
用茜素红对不同条件培养的iMEF细胞进行染色,以显示矿化结节。茜素红染色研究中的细胞培养条件与iMEF细胞ALP活性检测实验相似。接种后约24小时,将培养基转换为成骨诱导培养基以诱导骨形成,根据实施例1、2、4配制蛋白保护液1、蛋白保护液2、ph3.5的50mM乙酸和ph3.5的1mM盐酸,分别溶解37℃条件下储存8个月的BMP2,然后分别加入铺有iMEF细胞的6孔板中。细胞培养13天后,用茜素红染色试剂盒(Solarbio,中国)对细胞进行染色。当茜素红染料与钙离子反应时,可以形成橙红色的络合物。结果如图3所示蛋白保护液1和2使BMP2具有更强的促iMEF细胞成骨分化活性。
实施例6大鼠肌袋异位诱导成骨实验检测蛋白保护液性能
选取300g左右的雌性SD大鼠(购自重庆医科大学动物实验中心),根据实施例1、2、4配制100ul蛋白保护液1、100ul蛋白保护液2和100ul ph3.5的50mM乙酸,分别溶解37℃条件下储存8个月的BMP2,以不含BMP2的100ul双蒸水为空白对照,分别滴加到体积为200ul的丝素蛋白材料上,环氧乙烷灭菌后,植入大鼠股四头肌肌袋,4周后X射线照片检测成骨情况。
具体实验步骤如下:1.对SD大鼠行腹腔内注射麻醉,将手术区域备皮、消毒;2.选取大鼠左右大腿后方中上侧,作一切口长约1cm,分离筋膜后暴露大鼠股四头肌,钝性分离股四头肌肌肉,顺肌纤维方向做一长、深均0.5cm的肌袋(勿穿破肌肉),3.植入含有BMP-2的丝素蛋白材料,同时植入含有相同体积双蒸水的丝素蛋白材料作为阴性对照,缝合肌袋及皮肤,标记。4周时X射线照片检测,成骨材料都会促进异位骨形成,X线检测有明显显影,但蛋白保护液1和2使含BMP2的丝素蛋白成骨材料具有更强的异位成骨性能,参见图4。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种BMP2蛋白保护液的制备方法,其特征在于,包括:
1)将适量精氨酸溶于水中,使精氨酸浓度为0.001-0.5M;
2)加入适量酸,调节pH值4.0-6.8,即得BMP2蛋白保护液。
2.如权利要求1所述的制备方法,其特征在于,所述精氨酸浓度为0.001-0.5M;优选地,精氨酸浓度为0.01M或者0.05M。
3.如权利要求1或2所述的制备方法,其特征在于,步骤2)中,所述酸为乙酸。
4.根据权利要求1-3中任一项所述的制备方法得到的BMP2蛋白保护液。
5.一种BMP2蛋白制剂的制备方法,其特征在于,包括:将BMP2蛋白组合物溶解于如权利要求4所述的BMP2蛋白保护液中,即得BMP2蛋白溶液。
6.如权利要求5所述BMP2蛋白制剂的制备方法,其特征在于,所述BMP2蛋白组合物中,BMP2蛋白含量为0mg/ml-20mg/ml。
7.如权利要求5所述BMP2蛋白制剂的制备方法,其特征在于,以mg:mL计,BMP2蛋白与BMP2蛋白保护液的比例为0mg/ml-20mg/ml。
8.如权利要求5所述的BMP2蛋白制剂的制备方法,其特征在于,还包括:
将所述BMP2蛋白溶液冻干,得到BMP2蛋白冻干粉。
9.根据权利要求5-8中任一项所述的制备方法制备得到的BMP2蛋白制剂。
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