CN107135653A - 大肠杆菌素变体 - Google Patents
大肠杆菌素变体 Download PDFInfo
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- CN107135653A CN107135653A CN201580054115.1A CN201580054115A CN107135653A CN 107135653 A CN107135653 A CN 107135653A CN 201580054115 A CN201580054115 A CN 201580054115A CN 107135653 A CN107135653 A CN 107135653A
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- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
描述了大肠杆菌素变体及其在治疗病毒性出血热中的用途。本文描述了用于通过施用大肠杆菌素多肽治疗全身炎症反应综合征或病毒性出血热的方法。本文描述了包含SEQ ID NO:2‑9和11‑18中任一个的氨基酸序列的多肽。还描述了：一种多肽，所述多肽包含前面是甲硫氨酸的SEQ ID NO:11‑18中任一个的氨基酸序列；一种多肽，所述多肽包含SEQ ID NO:11‑18中任一个的具有至多5个单氨基酸改变或缺失的氨基酸序列，前提是该多肽不包含SEQ ID NO:10的氨基酸序列。
Description
背景
病毒性出血热(VHF)指与由属于以下四种不同的有包膜、反义、单链RNA病毒科之一的病毒引起的发热和出血素质(bleeding diathesis)相关的临床疾患：丝状病毒科，布尼亚病毒科，黄病毒科和沙粒病毒科。这四个科中的很多病毒在A类别生物威胁列表上，因为它们可导致高发病率和死亡率，并且是气溶胶传播高度传染性的[1]。这些病毒引起类似的疾病谱，具有相似的潜在病理生理学[2、3]。在4-10天的潜伏期后，患有VHF的患者突然发展为发热，伴有突出的持续性症状，诸如虚脱、脱水、肌痛和全身不适。随着疾病的进展，患者发展出临床出血征象，诸如瘀点状出血、斑状丘疹，伴有凝结障碍。在疾病的终末阶段期间，致死病例发展为弥漫性血管内凝血(DIC)、严重出血、低血压、多器官衰竭和休克。
患有严重VHF的患者通常死于终末临床病程，所述终末临床病程通常与也称为脓毒症的全身炎症反应综合征(SIRS)无法区分，所述全身炎症反应综合征是严重细菌和病毒感染的常见后遗症。一些VHF病毒特别容易引起SIRS；它们包括丝状病毒科中的埃博拉病毒(EBOV)和马尔堡病毒(MARV)、布尼亚病毒科中的裂谷热病毒(RVFV)和汉坦病毒、以及黄病毒科中的登革病毒[4、5]。
概述
本文描述了用于通过施用大肠杆菌素多肽治疗全身炎症反应综合征或病毒性出血热的方法。
本文描述了一种多肽，所述多肽包含SEQ ID NO：2-9和11-18中任一个的氨基酸序列。还描述了以下：一种多肽，所述多肽包含前面是甲硫氨酸的SEQ ID NO：11-18中任一个的氨基酸序列；一种多肽，所述多肽包含SEQ ID NO:11-18中任一个的具有至多5个单氨基酸改变或缺失的氨基酸序列，条件是该多肽不包含SEQ ID NO:10的氨基酸序列；一种多肽，所述多肽具有至多4个单氨基酸改变，条件是该多肽不包含SEQ ID No：10的氨基酸序列；一种多肽，所述多肽具有至多3个单氨基酸改变，条件是该多肽不包含SEQ ID No：10的氨基酸序列；一种多肽，所述多肽具有至多2个单氨基酸改变，条件是该多肽不包含SEQ ID No：10的氨基酸序列；一种多肽，所述多肽具有不超过1个氨基酸改变，条件是该多肽不包含SEQID No：10的氨基酸序列；一种多肽，所述多肽包含SEQ ID NO：2-9和11-18中任一个的具有至多5个单氨基酸改变或缺失的氨基酸序列，条件是该多肽不包含SEQ ID No：1或10的氨基酸序列；一种多肽，所述多肽具有至多4个单氨基酸改变，条件是该多肽不包含SEQ ID No：1或10的氨基酸序列；一种多肽，所述多肽具有至多3个单氨基酸改变，条件是该多肽不包含SEQ ID No：1或10的氨基酸序列；一种多肽，所述多肽具有至多2个单氨基酸改变，条件是该多肽不包含SEQ ID No：1或10的氨基酸序列；一种多肽，所述多肽具有不超过1个氨基酸改变，条件是该多肽不包含SEQ ID No：1或10的氨基酸序列；以及前面是甲硫氨酸的任何此类多肽。
还描述了包含本文描述的多肽和药学上可接受的载体或赋形剂的药物组合物。还公开了用于治疗被导致病毒性出血热的微生物感染的患者的方法，该方法包括施用本文描述的药物组合物或多肽。在多种实施方案中，患者被来自选自由以下组成的组的科的病毒感染：丝状病毒科、布尼亚病毒科、黄病毒科和沙粒病毒科；并且患者被选自埃博拉病毒(EBOV)、马尔堡病毒(MARV)、裂谷热病毒(RVFV)、汉坦病毒和登革热病毒的病毒感染。
还描述了包含编码本文描述的多肽的序列的核酸分子以及还包含可操作地连接至编码多肽的序列的表达调控序列的此类核酸分子。还描述了包含本文描述的核酸分子的重组细胞和产生多肽的方法，所述方法包括在适于表达编码的多肽的条件下培养本文描述的重组细胞，和从重组细胞分离所编码的多肽。
在以下的附图和说明中阐述了本发明的一种或更多种实施方案的细节。本发明的其他特征、目标和优势将由说明书和附图，以及由权利要求而明显。
附图
图1A-B.NB109和NB101对人血液体外凝结的影响。将递增浓度的NB109和NB101在37℃与人血浆样品一起预孵育持续15min。(A)PT测定和(B)APTT测定利用ACL100自动凝血计使用标准试剂进行。凝固时间的延长计算为(候选处理样品中的值)/(对照样品中的值)，并对药物浓度作图。PT和APTT的分别为对照的1.2倍和1.5倍的凝固时间用虚线表示。误差线表示SEM，n＝来自3个不同供体的3种血浆。
图2.NB101和NB109在小鼠内毒素血症模型中的作用。使BALB/c雌性小鼠(N＝5)经历两次注射脂多糖(LPS)作为DIC的模型。在t＝0小时，将5μg/小鼠先导剂量的LPS注射到足垫中，然后24小时后是400μg/小鼠的腹膜内诱发剂量。在诱发前1小时施用NB101、NB109或PBS。按小时监测小鼠的存活，持续直至诱发后70小时。利用Graphpad Prism 4.2通过Kaplan-Meier Log Rank测试来评价存活曲线中的统计学差异。星号表示NB101和PBS之间以及NB142和PBS之间的显著差异*p<0.05，***p<0.0001。
图3.在CLP模型中，NB109对动物存活的影响。对小鼠进行CLP外科手术。在CLP前18小时皮下给予NB109处理(预荷载)，并且接着每日两次。第2组接受60mg/kg NB109用于预荷载，并且接着40mg/kg。第3组接受30mg/kg NB109用于预荷载，并且接着20mg/kg。通过皮下注射进行液体复苏，每日1ml持续5天。每12小时观察存活。
图4A-B.NB101和NB109在豚鼠中的EBOV感染中的作用。在第0天，通过皮下注射1000pfu的EBOV来感染品系13豚鼠(N＝2)。通过i.p.注射施用化合物先导(compoundleads)，从感染后24小时开始，每天一次，持续7天。每天监测存活和体重。利用GraphpadPrism 4.2通过Kaplan-Meier Log Rank测试评价生存曲线中的统计学差异(*p<0.05)。
图5.用NB109处理的小鼠中的凝结参数。给予单i.p.剂量的NB109的BALB/c小鼠。在给药后4、8和24小时，分析PT和aPTT。在每个时间点展示了来自每组3-4只小鼠的平均值和标准偏差。*：单数据点。**:>180秒。
图6.在经由不同递送途径的小鼠中的NB-109血浆浓度。通过静脉内注射(i.v.)、腹膜内注射(i.p.)和皮下注射(s.c.),以72mg/kg向小鼠(n＝3)施用NB109。在不同的时间点获取血液样品。数据以平均值±SD显示。
图7.单剂量施用后，豚鼠中的NB109的血浆浓度和凝固参数。平均值±SD，n＝3
图8.单剂量施用后，豚鼠中的NB109的血浆浓度和凝固参数在每次给药后24小时和最后一次给药后96小时收集的血液样品。平均值±SD，n＝3。
图9.在小鼠LPS模型中的作用。在t＝0小时，BALB/c小鼠(N＝10)接受注射到足垫中的40μg先导剂量的LPS，然后24小时后是400μg的腹膜内(i.p.)诱发剂量的LPS。在诱发剂量之前1小时，用经i.p递送的45mg/kg NB101、NB109或NB142处理小鼠。在诱发后2、4和6小时，从动物取血以获得血浆细胞因子水平。利用Graphpad Prism 4.2通过Kaplan-MeierLog Rank测试来评价存活曲线中的统计学差异。星号表示NB101和PBS之间以及NB142和PBS之间的显著差异*p<0.05，***p<0.0001。
图10A-C.在聚(I:C)攻击的小鼠中，NB109和NB142对动物存活的影响。将10周龄的雌性BALB/c小鼠随机分成媒介物组和药物处理组(n＝6)。从第0天到第3天，i.p.施用200μg的聚(I:C)(聚肌苷酸:聚胞苷酸),每天两次。如图中所示，在第0天、第1天或第2天开始,以45mg/kg/天i.p.给予NB109或NB142处理，每天一次。
图11.在聚(I:C)攻击的小鼠中，NB101、NB109和NB142对细胞因子和D-二聚体的影响。在聚(I:C)攻击之前1小时，对BALB/c i.p.注射45mg/kg NB101、NB109、NB142或媒介物。在0时刻(t＝0hr)，每只小鼠注射200μg聚(I:C)或PBS。
图12.NB142和NB109在豚鼠中的EBOV感染中的作用。在第0天，通过皮下注射1000pfu的EBOV感染品系13豚鼠(N＝3)。通过i.p.注射施用先导化合物，从感染后24小时开始每天一次持续7天。每天监测存活和体重。利用Graphpad Prism 4.2通过Kaplan-MeierLog Rank测试评价生存曲线中的统计学差异(*p<0.05)。
图13.候选物的药效动力学。
使BALB/c雌性小鼠(N＝15)经历两次LPS注射。在t＝0小时，将5μg/小鼠先导剂量的LPS注射到足垫中，然后24小时后是400μg/小鼠的腹膜内诱发剂量。在诱发前1小时，施用单剂量的45mg/kg的NB101、NB109或NB142或者PBS。在所示出的处理后时间点，进行aPTT&PT测量。
图14.NB109生产工艺流程图。
详细说明
下文描述了对野生型大肠杆菌素(NB101；SEQ ID NO：1)和大肠杆菌素变体(NB109；SEQ ID No：2)的研究。NB109在所谓的反应性中心环(“RCL”；氨基酸82-88；突变的氨基酸数涉及缺少前20个氨基酸(MKTILPAVLFAAFATTSAWA；SEQ ID NO:19)的成熟大肠杆菌素序列即SEQ ID NO:1，如SEQ ID NO：10所示)的P1位置处的一个氨基酸残基上与大肠杆菌素不同，M84R。
NB101是靶向丝氨酸弹性蛋白酶(也被称为中性粒细胞弹性蛋白酶(NE)或粒细胞弹性蛋白酶(GE))凝血因子(Xa、XIIa、VIIa)和激肽释放酶(表1)的广谱蛋白酶抑制剂。除了NB101的经由NE抑制的潜在抗炎功能之外，其还直接靶向“SIRS三角(SIRS triangle)”中的两个组分：凝血因子和激肽释放酶。但是，NB101不抑制纤维蛋白溶解。因此，对RCL的P1位置处的所有可能的点突变进行筛选，得到NB109。不同于NB101，NB109抑制纤维蛋白溶酶和凝血酶。结果，它直接靶向“SIRS三角”中的所有三个组分。
表1.NB101和NB109的抑制常数Ki(nM)*
NB109与大肠杆菌素共有化学和物理性质。NB109具有相当数目的带负电荷的残基(Asp+Glu)和带正电荷的残基(Arg+Lys)，并且计算出的pI为6.85[61]。一个单位的化合物活性被定义为在标准测定条件下抑制50％胰蛋白酶所需化合物的量。根据该定义，NB109具有1x105单位/mg的比活度，这与NB101相等。
在人血浆中的抗凝结活性
测试NB101和NB109以确定其抑制血液凝结的能力，特别是其经由抑制炎症和激肽释放酶-激肽系统抑制内源性血液凝结途径的能力。通过进行PT测定(凝血酶原时间；外源性凝结途径)和aPTT测定(部分激活促凝血酶原激酶时间；内源性凝结途径)，针对人血液体外凝结测试这些剂。两种分子均表现出强有力的剂量依赖性抗凝结作用，并且可能是由于NB109抵抗凝血酶的活性，其效力比NB101强约2倍(图1)。另外，NB109和NB101两者均展示出对内源性凝结途径(如通过aPTT测量的)比对外源性途径(通过PT测量的)更强的抑制(大约两倍)(图1)。
重要的是要注意，PT和aPTT升高是候选物的预期药理作用。PT或APTT升高自身并不意味着自发性出血是不利影响。自发性出血倾向与未受抑制的纤维蛋白溶解和增加的血管通透性有关[62]。NB101和NB109可具有降低的自发性出血风险，因为它们抑制血管高渗透性或血管高渗性和纤维蛋白溶解两者。
抵抗SIRS的体内效果
在小鼠内毒素血症模型中测试NB101和NB109，所述模型是由相距24小时施用的两次连续全身性内毒素(LPS)暴露引起的致死性休克模型。病理生理学上，该模型的特征在于炎症、出血、组织坏死和DIC[63]。
所有的媒介物处理的小鼠都在LPS攻击的一天内快速死亡，但用NB101和NB109处理具有显著的存活益处(图2)。在该研究中，NB101和NB109都以类似的方式增加了动物的存活，并且它们均很有利地与目前的标准抗-DIC处理，低分子量肝素(LMWH)进行了比较。在LPS诱发前两次给予LMWH只将被处理的小鼠的30小时存活提高25％(在处理组中50％存活以及在对照组中25％存活)[64]。
盲肠结扎和穿刺(CLP)是SIRS的另一种常用动物模型。在CLP模型中，SIRS由肠损伤并被通常存在于肠中的多种细菌感染之后的腹膜炎产生。它被认为更好地模拟了脓毒症的自然成因[65]。在初步研究中，NB109在CLP模型中获得了显著(p<0.005)的存活优势(图3)。
抵抗VHF的体内效果
在用EBOV的扎伊尔(Zaire)毒株感染的豚鼠中评价NB101和NB109。媒介物处理的动物总是到第9天时死亡。NB101和NB109处理在感染后24小时开始，并通过腹膜内注射给予,一天一次，持续7天。虽然NB101不影响动物存活或体重减轻，但NB109达到了50％存活，并拯救存活动物免于致死性体重减轻(图4)。该结果提供了概念验证。总而言之，体外和体内结果表明NB109和NB101是作为抗SIRS和抗VHF化合物和药物制剂的潜在的有效候选物。
安全&PK研究-对原代细胞的影响
将高达250μM的NB109与包括原代人肾近端小管细胞、肾皮质上皮细胞、肺血管内皮细胞或肝细胞在内的人原代细胞以及细胞系A549和BEAS-2B的集合一起孵育。在4天的孵育内，利用MTS测定评价细胞毒性。
Nb109不引起细胞毒性，并且对细胞的成活力无影响。
对溶血的影响
检查NB109的经由补体的活化的间接溶血或直接溶血。作为补体介导的溶血的阳性对照，使用针对红细胞(RBC)的物种特异性抗体以活化经典补体途径并引发信号级联，导致RBC的裂解。为了评价NB109的直接溶血活性，洗涤RBC以去除任何补体蛋白，并且然后用含有NB109的热灭活的血浆或血清重悬。在人血液中，高达1mg/ml的浓度的NB109不诱发溶血反应，不诱发直接的溶血反应也不诱发补体介导的溶血反应。
在小鼠中NB109处理的系统安全性
在5组16只BALB/c小鼠中评价在小鼠中NB109系统性处理的安全性和耐受性。四组中的每组分别接受5mg/kg、10mg/kg、15mg/kg和90mg/kg的NB109的一次腹腔内注射；第五组接受PBS。在给药后4小时和24小时处死小鼠，并进行尸检、凝结分析和临床化学。
尸检后，所有动物似乎都是正常的而没有出血迹象。如预期的，凝结参数以剂量依赖性方式受影响；该效应在治疗后4小时达到峰值，并在处理后24小时前恢复到基线(图5)，这表明NB109在24小时内从血液清除。与在人血液中观察到的一致，aPTT对NB109更敏感，并且在5mg/kg观察到效果，而PT直到15mg/kg不受影响。PT在aPTT恢复到基线水平之前恢复到基线水平。应该注意的是，PT和aPTT的升高是药理作用，并且不被认为是不利影响。
在豚鼠中的重复剂量毒性研究
通过腹膜内施用以0.1mg/kg/天、0.5mg/kg/天、1.5mg/kg/天和5mg/kg/天的剂量对Hartley豚鼠给予NB109，持续7天。安全性参数包括临床迹象、血清化学、凝结时间和尸检。
所有动物经NB109处理而存活，并且贯穿该研究进程，NB109处理的动物的所有临床观察都是正常的。在组间体重变化方面不存在显著差异，并且所有组都显示出显著的体重增加(到研究结束时19-23％)。所有NB109处理的动物的尸检都不显著。
在≥1.5mg/kg时，在第2天，存在肌酸磷酸激酶(CPK)的轻微且瞬时的升高趋势，但该值到第7天时恢复到正常范围。在≥1.5mg/kg时，在第14天，观察到AST的轻微升高，但其他肝酶和胆红素是正常的。所有其他临床实验室参数都在正常范围内。在1.5mg/kg和低于1.5mg/kg的剂量，未观察到凝结参数的改变(注意豚鼠具有降低的FVII水平，因此具有比其他物种长的PT)。在5.0mg/kg时，从第一次给药后8小时开始观察到升高的PT和aPTT值，并继续到在第7天的最后一次给药后8小时。所有PT和aPTT值到第14天时恢复正常。
初步药代动力学分析
在其中通过不同途径施用NB109的小鼠中进行初步药代动力学(PK)研究。数据在图6中示出。相对于IP或SC注射，用IV施用的初始血浆浓度要高得多。腹膜内注射导致比通过SC注射注射相同剂量显著更高的浓度，意味着通过SC途径的NB109的生物利用度将不太理想。鉴于IV施用的血浆浓度数据的变化，提供PK暴露的任何估量是不可能的。然而，IP途径的血浆浓度适用于药代动力学建模(WinNonlin软件，Cary，NC)。通过IP途径的NB109的消除半衰期(t1/2)为7.6小时。
在豚鼠中进行NB109的研究，以评价单剂量施用和重复剂量施用后血浆药物浓度和血液凝结参数之间的关系。将NB109以5mg/kg的剂量IV施用至Hartley豚鼠(n＝3)。在单剂量IV后，NB109的血浆水平和延长的aPTT之间存在极佳的相关性(图7)。虽然aPTT密切反映了到注射后8小时时几乎恢复到背景水平的血浆药物水平，但PT在该时间点维持延长。
再次以每天5mg/kg的剂量进行重复给药，持续5天。血浆药物水平在第3次给药后似乎略有增加，但数据的变化使得难以确定对于药物累积的任何结论(图8)。血液凝结参数与血浆NB109之间具有很好的相关性。到第7天(给药后96小时)时，所有参数都恢复到基线。
其他大肠杆菌素变体
开发并测试了表2中所示的构建体并在下文进一步的描述。在表3中，构建体的氨基酸序列显示为SEQ ID No.1-9。
表2.肽的抑制常数Ki(nM)*
表3.具有前导序列的初级和优化的先导候选物的氨基酸序列
表4.无前导序列的初级和优化的先导候选物的氨基酸序列
在内毒素血症模型中的效果
由于鼠内毒素血症模型的简单性，其被用作一线筛选模型。在该模型中，所有潜在优化的先导候选物都保护动物；NB142、NB137、NB147和NB178似乎是最有效的先导候选物。有趣的是，在该模型中，NB142显著地优于NB101或NB109(图9)。除了具有最高的动物存活率之外，NB142在抑制炎性细胞因子IL-6和TNF-α方面也是最有效的(图9)。
在VHF模型中的初步效果
表3中显示的若干种肽，在用模拟外来RNA分子的肌苷聚合物聚(I:C)注射攻击的小鼠中。由于VHF病毒都是RNA病毒，因此该模型被设计以再现宿主对病毒RNA分子的反应。与VHF病毒相似，聚(I:C)注射引发SIRS的征象，包括炎性细胞因子的释放、升高的D-二聚体(指示DIC的纤维蛋白溶解产物)以及在肝、肺和肾中丰富的微血栓。
未处理的动物总是在第一次聚(I：C)注射后五天内死亡。当在聚(I：C)注射之前开始处理时，NB109、NB142、NB137和NB147都显著地防止了动物死亡。当NB109处理在聚(I：C)注射之后开始时，当它在攻击后的第一天首次给予时，它是有效的(图1)。即使当在聚(I：C)攻击后48小时开始仅两次处理，NB104也延长动物存活(图10)。该结果表明，NB142和NB109两者均能够在诱发时防止SIRS，但NB142可更有效地治疗已确立的与VHF相关的SIRS。
在同一模型中，当在聚(I：C)攻击之前给予时，NB101、NB109和NB142都显著地抑制由聚(I：C)触发的炎性细胞因子和D-二聚体。然而，在三个候选物中，NB142在抑制炎性细胞因子IL-6和TNF-α(错误！未找到引用源。 )方面是最有效的。
接下来，在感染EBOV的Zaire毒株的豚鼠的研究中比较了NB109和NB142。虽然媒介物处理的动物总是在第9天之前死亡，但1mg/kg/天的NB142和5mg/kg/天的NB109实现了显著的67％的存活。再次，NB142以在较低剂量具有较好的存活以及显著的体重增加显示出较好的效果(图12)。该研究结果的力度还在于这一事实，NB109和NB142处理是以未经优化的处理剂量并且在感染后24小时开始施用方案。
NB142、NB101和NB109的初步药效动力学
NB142与NB101和NB109在体内具有不同的药效动力学(PD)。尽管NB101和NB109均导致PT升高，但NB142不影响PT(图13)。所有三个候选物都以不同的效力导致aPTT的升高。PD结果表明NB101和NB109抑制外源性凝结途径和内源性凝血途径两者，而NB142则似乎特异性地影响内源性凝结途径。
感染EBOV的恒河猴的血液学监测显示，EBOV HF中的消耗性凝血病是由于内源性凝结途径的激活，而不是外源性凝结途径的激活[66]。内源性凝血途径由炎性细胞因子和激肽释放酶直接激活，并通过纤维蛋白溶酶激活增强。Nb142具有抗炎作用。它还有效地抑制激肽释放酶和纤维蛋白溶酶，同时饶过(sparing)凝血酶。因此，它可以抑制触发内源性凝血的上游事件而不恶化消耗性凝血病。因此，NB142可具有用于VHF治疗的优选PD特征。
药物材料
肽可以使用高密度、补料分批的大肠杆菌(E.coli)发酵工艺，然后周质提取、离子交换层析和过滤步骤以去除内毒素来制备。
发酵工艺
可以评价两种微生物表达系统的先导化合物产生：大肠杆菌和酵母。利用使用葡萄糖作为碳源的时间依赖性补料分批大肠杆菌发酵工艺生产NB109，所述发酵工艺每升发酵产生～0.2gm纯化的NB109。先导化合物还可以利用使用甘油作为碳源的溶解氧依赖性补料控制系统来产生。该发酵工艺已经导致>9克/升不同蛋白药物候选物的表达。该后一工艺可被容易地扩大。它使用包括USP级试剂的经认证无动物成分的半确定成分培养基。
作为细菌表达系统的替代，酵母菌株诸如毕赤酵母(P.pastoris)和多形汉逊酵母(H.polymorpha)也可以作为用于产生先导化合物的系统来评价。当与微生物系统相比时，这些系统具有高等真核生物表达系统的优点，诸如更好的蛋白加工、折叠和分泌，并且仍然具有快速生长和严格调控的启动子。肽可以通过分泌到酵母培养基中来表达，以大大简化纯化工艺。作为本发明的一部分，已经产生了毕赤酵母菌株以将先导化合物分泌到酵母培养基中。这些菌株是甲醇可诱导的，并且适于发酵。
通过调查多种分泌前导序列诸如α-交配因子、α-淀粉酶、葡糖淀粉酶、菊粉酶和转化酶酵母信号序列，并转化多种野生型和蛋白酶缺陷型酵母菌株，进一步优化毕赤酵母系统是可能的。可以从以96孔和24孔形式生长的小规模培养物的上清液进行菌落的筛选。在转移到发酵之前，选择的克隆可以在摇瓶培养基中生长。可以使用体积为4升至20升的BioFlo 3000和BioFlo Iv发酵罐建立发酵工艺。用于诱导表达的甲醇进料可以通过可得的YSI 2700Select Biochemistry Analyzer与甲醇探针来定量。发酵优化可以改变培养基和进料组成、pH、温度、进料时间过程和诱导时间以达到期望的蛋白表达水平。
纯化工艺
从大肠杆菌发酵的纯化工艺包括周质提取，然后是用于纯化的离子交换层析步骤和用于减少内毒素的离子交换过滤步骤。该纯化已经对本文描述的肽起作用。在图14中展示了该工艺的细节。
另外的下游步骤可以包括但不限于亲和层析、疏水相互作用层析、尺寸排阻层析和另外的离子交换步骤。初步筛选可以在96孔过滤器板中进行，以在不使用机器人技术下获得高通量。待评价的结合条件可包括层析树脂、盐、离子强度和pH。微量洗脱物可以使用可得的96孔UV分光光度计通过UV吸光度来分析总浓度，并通过48-样品SDS-PAGE(Invitrogen，Carlsbad，CA)与考马斯亮蓝染色来分析纯度。选择条件可以扩大到在可得的FPLC上使用标准1-10ml柱的层析术。工艺中间体的产率和纯度可以使用以下描述的释放测试的子集来监测，包括SDS-PAGE、HPLC和活性。
开发还可以聚焦于使纯化工艺适应于酵母表达系统，并添加另外的纯化步骤以提高纯度。另外的步骤可以包括但不限于疏水相互作用层析、反相层析和另外的离子交换步骤。
预制剂和制剂开发
先导化合物可以被开发成无菌、无防腐剂(non-preserved)的单位剂量肠胃外产品。目前的数据表明，先导化合物可以在宽的pH和温度范围内是非常稳健和稳定的。
预计剂量
基于NB142在豚鼠EBOV模型中的1mg/kg/天的有效剂量，人的治疗剂量可以是约0.2mg/kg/天。对于7天的最长疗程，预计总药物消耗将为84毫克(对于60kg个体)至280毫克(对于200kg个体)。
已描述了本发明的很多实施方案。尽管如此，将理解的是，可以做出多种改变而不偏离本发明的精神和范围。
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Claims (25)
1.一种多肽，所述多肽包含SEQ ID NO：2-9和11-18中任一个的氨基酸序列。
2.一种多肽，所述多肽包含前面是甲硫氨酸的SEQ ID NO：11-18中任一个的氨基酸序列。
3.一种药物组合物，所述药物组合物包含根据权利要求1或权利要求2所述的多肽和药学上可接受的载体或赋形剂。
4.一种用于治疗被导致病毒性出血热的微生物感染的患者的方法，所述方法包括施用根据权利要求3所述的药物组合物。
5.根据权利要求4所述的方法，其中所述患者被来自选自由以下组成的组的科的病毒感染：丝状病毒科，布尼亚病毒科，黄病毒科和沙粒病毒科。
6.根据权利要求5所述的方法，其中所述患者被选自埃博拉病毒(EBOV)、马尔堡病毒(MARV)、裂谷热病毒(RVFV)、汉坦病毒和登革热病毒的病毒感染。
7.一种多肽，所述多肽包含SEQ ID NO：2-9和11-18中任一个的具有至多5个单氨基酸改变或缺失的氨基酸序列，条件是所述多肽不包含SEQ ID No：1或10的氨基酸序列。
8.根据权利要求7所述的多肽，所述多肽具有至多3个单氨基酸改变，条件是所述多肽不包含SEQ ID No：1或10的氨基酸序列。
9.根据权利要求7所述的多肽，所述多肽具有至多3个单氨基酸改变，条件是所述多肽不包含SEQ ID No：1或10的氨基酸序列。
10.根据权利要求7所述的多肽，所述多肽具有至多2个单氨基酸改变，条件是所述多肽不包含SEQ ID No：1或10的氨基酸序列。
11.根据权利要求7所述的多肽，所述多肽具有不超过1个氨基酸改变，条件是所述多肽不包含SEQ ID No：1或10的氨基酸序列。
12.一种多肽，所述多肽包含SEQ ID NO：11-18中任一个的具有至多5个单氨基酸改变或缺失的氨基酸序列，条件是所述多肽不包含SEQ ID No：10的氨基酸序列。
13.根据权利要求12所述的多肽，所述多肽具有至多3个单氨基酸改变，条件是所述多肽不包含SEQ ID No：10的氨基酸序列。
14.根据权利要求12所述的多肽，所述多肽具有至多3个单氨基酸改变，条件是所述多肽不包含SEQ ID No：10的氨基酸序列。
15.根据权利要求12所述的多肽，所述多肽具有至多2个单氨基酸改变，条件是所述多肽不包含SEQ ID No：10的氨基酸序列。
16.根据权利要求12所述的多肽，所述多肽具有不超过1个氨基酸改变，条件是所述多肽不包含SEQ ID No：10的氨基酸序列。
17.根据权利要求12-16中任一项所述的多肽，所述多肽前面是甲硫氨酸。
18.一种药物组合物，所述药物组合物包含根据权利要求7至17中任一项所述的多肽和药学上可接受的载体或赋形剂。
19.一种用于治疗被导致病毒性出血热的微生物感染的患者的方法，所述方法包括施用根据权利要求18所述的药物组合物。
20.根据权利要求19所述的方法，其中所述患者被来自选自由以下组成的组的科的病毒感染：丝状病毒科，布尼亚病毒科，黄病毒科和沙粒病毒科。
21.根据权利要求20所述的方法，其中所述患者被选自埃博拉病毒(EBOV)、马尔堡病毒(MARV)、裂谷热病毒(RVFV)、汉坦病毒和登革热病毒的病毒感染。
22.一种核酸分子，所述核酸分子包含编码根据权利要求1、2和7-17中任一项所述的多肽的序列。
23.根据权利要求22所述的核酸分子，所述核酸分子还包含与编码所述多肽的序列可操作地连接的表达调控序列。
24.一种重组细胞，所述重组细胞包含根据权利要求23所述的核酸分子。
25.一种产生多肽的方法，所述方法包括在适于表达所编码的多肽的条件下培养根据权利要求24所述的重组细胞，和从所述重组细胞分离所编码的多肽。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201462058639P | 2014-10-01 | 2014-10-01 | |
| US62/058,639 | 2014-10-01 | ||
| PCT/US2015/053610 WO2016054452A1 (en) | 2014-10-01 | 2015-10-01 | Ecotin variants |
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| CN107135653A true CN107135653A (zh) | 2017-09-05 |
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| CN201580054115.1A Pending CN107135653A (zh) | 2014-10-01 | 2015-10-01 | 大肠杆菌素变体 |
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| Country | Link |
|---|---|
| US (1) | US20170260251A1 (zh) |
| EP (1) | EP3201214A4 (zh) |
| CN (1) | CN107135653A (zh) |
| WO (1) | WO2016054452A1 (zh) |
Citations (5)
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|---|---|---|---|---|
| US5719041A (en) * | 1993-09-14 | 1998-02-17 | Genentech, Inc. | DNA encoding ecotin homologs |
| WO2000061634A2 (en) * | 1999-04-12 | 2000-10-19 | The Regents Of The University Of California | Ecotin-variant modulators of serine proteases |
| US20030083244A1 (en) * | 2000-04-26 | 2003-05-01 | Vernet Corine A.M. | Novel proteins and nucleic acids encoding same |
| CN102027002A (zh) * | 2008-05-16 | 2011-04-20 | 三星电子株式会社 | 用于纯化蛋白质的方法和亲和柱 |
| US20140030791A1 (en) * | 2004-04-12 | 2014-01-30 | Sandra Waugh Ruggles | Mutant MT-SP1 proteases with altered substrate specificity or activity |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994020535A1 (en) * | 1992-12-11 | 1994-09-15 | Corvas International, Inc. | ECOTIN AS A FACTOR Xa, XIa, AND XIIa INHIBITOR |
| EP2922416A4 (en) * | 2012-11-20 | 2016-07-20 | Pronutria Inc | MANIPULATED SECRETATED PROTEINS AND METHOD |
| KR20160058940A (ko) * | 2013-09-25 | 2016-05-25 | 프로뉴트리아 바이오사이언시스, 인코퍼레이티드 | 근육량, 강도 및 성능을 유지하기 위한 조성물 및 제형, 그리고 이의 생산방법 및 용도 |
-
2015
- 2015-10-01 US US15/514,044 patent/US20170260251A1/en not_active Abandoned
- 2015-10-01 CN CN201580054115.1A patent/CN107135653A/zh active Pending
- 2015-10-01 EP EP15847167.2A patent/EP3201214A4/en not_active Withdrawn
- 2015-10-01 WO PCT/US2015/053610 patent/WO2016054452A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5719041A (en) * | 1993-09-14 | 1998-02-17 | Genentech, Inc. | DNA encoding ecotin homologs |
| WO2000061634A2 (en) * | 1999-04-12 | 2000-10-19 | The Regents Of The University Of California | Ecotin-variant modulators of serine proteases |
| US20030083244A1 (en) * | 2000-04-26 | 2003-05-01 | Vernet Corine A.M. | Novel proteins and nucleic acids encoding same |
| US20140030791A1 (en) * | 2004-04-12 | 2014-01-30 | Sandra Waugh Ruggles | Mutant MT-SP1 proteases with altered substrate specificity or activity |
| CN102027002A (zh) * | 2008-05-16 | 2011-04-20 | 三星电子株式会社 | 用于纯化蛋白质的方法和亲和柱 |
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| C.GIGLIONE ET AL.: "Protein N-terminal methionine excision", 《CELL. MOL. LIFE SCI.》 * |
| LEI JIN ET AL.: "Crystal Structures of the FXIa catalytic domain in complex with Ecotin Mutants reveal substrate-like interactions", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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| WO2016054452A1 (en) | 2016-04-07 |
| US20170260251A1 (en) | 2017-09-14 |
| EP3201214A4 (en) | 2018-04-04 |
| EP3201214A1 (en) | 2017-08-09 |
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