CN102241734B - 一类抗凝血多肽及其应用 - Google Patents
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Abstract
一类抗凝血多肽及其应用,属于生物医药领域。由SEQ ID NO.1所示氨基酸序列组成的多肽;将SEQ ID NO.1所示氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而形成的,且能选择性抑制凝血因子XIa的衍生多肽。该类抗凝血多肽是凝血因子XIa的选择性抑制剂,具有较强抗凝活性,能明显延长血浆活化部分凝血活酶时间,而对凝血酶原时间基本无影响。该类抗凝血多肽具较强抗血栓形成作用,可作为制备预防和治疗血栓性疾病药物应用,而且副作用小。
Description
技术领域
本发明属于生物医药领域。具体的说,本发明涉及一类抗凝血多肽与该抗凝血多肽在制备预防和治疗血栓性疾病中的应用。
技术背景
血栓性疾病尤其是心脑血管栓塞性疾病是严重危害人们身体健康的常见病,其致死率及致残率也高。虽然目前有较多药物如肝素(包括低分子量肝素LMWH如伊诺肝素)、华法林、血小板聚集抑制剂(如阿司匹林)、水蛭素等药物应用于临床,但他们同时也还存在一定的副作用,其中,出血是目前临床抗凝抗栓治疗最常见、最严重的并发症之一,因此,发明出血副作用小的抗凝抗栓药物具有非常重要的现实意义。临床数据及近年来一系列的动物实验研究表明,凝血因子XIa(fXIa)是一个出血副作用小的抗凝抗栓治疗新靶点,发明针对该靶点的药物或许是解决目前困扰临床抗凝抗栓治疗引起的出血并发症问题的有效途径之一。
在临床上,fXI升高能促进静脉血栓形成(Meijers JCM et al.N Engl J Med.2000;342:696-701),而fXI先天缺乏严重患者的深静脉血栓、缺血性脑卒中发生率少(Salomon O et al.Blood.2008,111:4113-4117.),这些现象提示fXI能促进血栓形成。另一方面,fXI先天性缺乏患者通常只引起温和出血,很少有自发性出血(Seligsohn U.J Thromb Haemost.2009,7(suppl):84-87),这也意味着当fXIa缺乏或被抑制时,对止血功能影响不明显,即出血副作用小。
在动物实验中,fXI基因敲除小鼠在进行外科手术时不会引起过量出血。fXI缺陷小鼠能显著减少用不同机械及化学方法引起的静脉及动脉血栓形成,而且止血功能正常。针对fXI抗体能有效减少大鼠、兔及狒狒的血栓形成,并且对出血时间无影响(RennéT et al.J Thromb Haemost.2009,Suppl 1:79-83)。抗fXI单抗aXIMab能减少狒狒体内人造血管血栓的形成及凝血酶的产生,而对出血时间、血小板聚集活性无影响,因此作者认为“阻断fXI比其它抗栓剂具有出血副作用小”(Tucker et al.Blood.2009,113:936-944)。在最近的试验中,针对fXI的反义核酸(antisense oligonucleotides,ASOs)能够有效抗各种静脉血栓及动脉血栓形成,与伊诺肝素(enoxaparin)及华法林(warfarin)比较,还不引起出血;联合用药时,ASOs能增加伊诺肝素及氯吡格雷(clopidogrel)的抗栓活性,也 不增加出血(Zhang H et al.Blood.2010,116(22):4684-4692),因此被认为是安全的抗凝。
利用基因敲除手段还证实,敲除凝血因子包括组织因子(TF)、fVII、fV、fX 及凝血酶原基因鼠不能存活(Mackman N.Arterioscler Thromb Vasc Biol2005;25:2273-2281),fVIII-/-及fIX-/-缺陷鼠虽然能够生存,但常会引起严重出血,类似于人类fVIII或fIX缺乏引起的血友病;与上述凝血因子缺乏或不能生存,或引起严重出血相比较的是,fXI基因敲除鼠,即fXI-/-鼠能够生存,并且止血功能正常,还能抗血栓形成(Gailani D,RennéT.J Thromb Haemost2007;5:1106-1112)。
上述结果提示fXIa(或fXI)被抑制或缺乏时出血副作用小,即意味着针对凝血因子XIa的药物具有出血副作用小的优点,而且可能优于目前针对凝血酶、fX、fVII等靶点的应用的抗凝抗栓治疗药物或正研发中新药。但目前针对fXIa靶点的候选药物分子还寥寥无几,除前述提到的抗fXI抗体、fXI反义核酸外,目前还有类肽物(peptidomimetics)、一些化学小分子及一种分离自海绵的分子对fXIa具有抑制作用(Schumacher WA et al.Arterioscler Thromb Vasc Biol.2010,30(3):388-392),尚缺少针对凝血因子XIa的选择性肽类抑制剂。
钩虫是一类寄生在哺乳动物及人体的吸血寄生虫,其寄生在肠道,以摄取宿主血液为食。钩虫能分泌抗凝成份,使咬附的宿主伤口不易凝血,造成流血不止,该特点类似于水蛭吸血后宿主伤口不易凝血。本发明人课题组发现的源自犬钩虫的抗凝肽AcaNAP10具有很强的抗凝活性,对凝血途径中组织因子与活化的凝血因子VII复合物(TF/fVIIa)及凝血因子XIa(fXIa)的均具有较好抑制作用(Li D et al.Biochem Biophys Res Commun.2010,392:155-159),是国际上首个,也是目前唯一对TF/fVIIa及fXIa均有抑制作用的抗凝物质。
为获得针对凝血因子XIa的选择性抑制剂,我们对AcaNAP10进行了结构改造,结果在AcaNAP10的基础上发明了对凝血因子XIa具选择性抑制作用的系列抗凝血多肽,这些选择性抑制凝血因子XIa的抗凝血多肽可望应用于制备预防和治疗血栓性疾病药物,而且副作用小。
发明内容
本发明的目的在于提供一类抗凝血多肽及其应用,该类抗凝血多肽是凝血因子XIa(fXIa)的选择性抑制剂,具有较强抗凝活性,能明显延长血浆活化部 分凝血活酶时间(aPTT),而对凝血酶原时间(PT)基本无影响。该抗凝血多肽可应用于制备药物以预防和治疗血栓性疾病,并且具有出血副作用小的特点。
本发明提供了一类抗凝血多肽,该类抗凝血多肽为由SEQ ID NO.1所示氨基酸序列组成的多肽,或将SEQ ID NO.1所示氨基酸序列组成的多肽经过一个或几个氨基酸残基的取代、缺失或添加而形成的能选择性抑制凝血因子XIa的衍生多肽。
本发明提供了一类抗凝血多肽,该类抗凝血多肽含有由SEQ ID NO.2,或SEQ ID NO.3,或SEQ ID NO.4,或SEQ ID NO.5,或SEQ ID NO.6,或SEQ IDNO.7,或SEQ ID NO.8,或SEQ ID NO.9,或SEQ ID NO.10所示氨基酸序列组成的多肽。
与犬钩虫抗凝肽10(AcaNAP10)对TF/fVIIa及fXIa均有抑制作用相比较,本发明提供的抗凝血多肽是凝血因子XIa(fXIa)的选择性抑制剂,对凝血因子XIa(fXIa)具有较强的抑制活性,而对组织因子与凝血因子VIIa复合物(TF/fVIIa)基本无抑制活性;本发明提供的抗凝血多肽能明显延长血浆活化部分凝血活酶时间(aPTT),而对凝血酶原时间(PT)基本无影响。
本发明的一个应用在于应用发明的抗凝血多肽制备预防和治疗血栓性疾病药物。包含本发明的多肽具有明显抗血栓形成作用,而且对止血功能、血小板聚集活性无明显影响,可用于作为或发展成为出血副作用小的抗血栓药物。
本发明的其它方面由于本文技术内容的公开,对本领域技术人员是易于理解并实施的。例如,包含本发明的抗凝血多肽与其它抗凝血、抗栓药物联合用药可减少副作用,利用基因工程方法或化学合成方法生产本发明中的抗凝血多肽,包含本发明抗凝血多肽药物组合物在临床中的应用,包含本发明的抗凝血多肽在作为抗凝制剂中的应用。
本发明抗凝血多肽的优点是:本发明提供的抗凝血多肽是凝血因子XIa的选择性多肽抑制剂,该类抗凝血多肽不但能够制备作为有效预防和治疗血栓性疾病药物应用,而且又不影响人体和动物的正常止血功能,出血副作用小,对于解决目前临床抗凝抗栓治疗出现的出血副并发症具有重大意义。
附图说明
图1NAPTin-1对凝血因子XIa、Xa及fVIIa/TF的抑制作用
具体实施方式
下面结合具体实例,进一步阐述本发明。应理解,这些实施仅用于说明本发明而不用于限制本发明的范围。凡是依照本发明公开内容所做出的等同替换,均属于本发明的保护范围。
实施例1
选择性抑制凝血因子XIa的抗凝血多肽的获得
根据钩虫抗凝肽AcaNAP10编码序列(Li D et al.Biochem Biophys Res Commun.2010,392:155-159),设计系列引物用PCR方法对AcaNAP10羧基末端序列进行逐个氨基酸残基缺失突变。结果表明,AcaNAP10羧基末端缺失相应氨基酸残基后,就获得了能选择性抑制凝血因子XIa,而对TF/fVIIa无抑制或具有弱抑制作用的抗凝血多肽。其中,NAPTin-1见SEQ ID NO.1所示氨基酸序列;NAPTin-2见SEQ ID NO.2所示氨基酸序列;NAPTin-3见SEQ ID NO.3所示氨基酸序列;NAPTin-4见SEQ ID NO.4所示氨基酸序列;NAPTin-5见SEQ ID NO.5所示氨基酸序列;NAPTin-6见SEQ ID NO.6所示氨基酸序列;NAPTin-7见SEQID NO.7所示氨基酸序列;NAPTin-8见SEQ ID NO.8所示氨基酸序列;NAPTin-9见SEQ ID NO.9所示氨基酸序列。NAPTin-10见SEQ ID NO.10所示氨基酸序列
实施例2
本发明提供的抗凝血多肽的抗凝活性
本实施例中的抗凝血多肽由本发明人实验室通过在大肠杆菌中重组表达、分离纯化制备。通过检测活化部分凝血活酶时间(aPTT)及凝血酶原时间(PT)观察本发明提供的抗凝血多肽的抗凝活性。
凝血酶原时间(PT)测定:取一定浓度的蛋白纯化产物10μl和45μl正常人血浆混匀37℃孵育15min后,加入37℃预温的45μl PT试剂(MDC Hemostasis,Germany),用酶标仪Elx808(BioTek,United States)监测OD630nm值变化情况,推算凝固时间即为PT值。每个浓度均重复3次。
活化部分凝血活酶时间(aPTT)测定:取一定浓度的蛋白纯化产物10μl和50μl正常人血浆混匀后,加入20μl aPTT试剂(MDC Hemostasis,Germany)37℃孵育15min后,加入37℃预温的0.005mol/L的CaCl2溶液20μl,用Elx808酶标仪(BioTek,United States)监测OD630nm值变化,推算aPTT值。每个浓度均重复3次,取各浓度aPTT值均数与空白对照组aPTT值比较即为该浓度延 长aPTT倍数。
各抗凝血多肽延长aPTT及PT见表1,结果表明:在100nM及500nM浓度下,NAPTin-1、NAPTin-2、NAPTin-3、NAPTin-4、NAPTin-5、NAPTin-6、NAPTin-7、NAPTin-8及NAPTin-9均能显著延长aPTT,但NAPTin-1、NAPTin-2、NAPTin-3、NAPTin-4、NAPTin-5、NAPTin-6及NAPTin-7无延长PT活性,NAPTin-8及NAPTin-9在100nM下基本不延长PT,在500nM下能部分延长不到1.6倍PT。
表1本发明提供的抗凝血多肽对aPTT及PT的影响
与生理盐水对照组比较,*p<0.01;与生理盐水对照组比较,#p<0.01
实施例3
本发明提供的抗凝血多肽对凝血因子的抑制作用
本实施例中的抗凝血多肽由本发明人实验室通过在大肠杆菌中重组表达、分离纯化制备。用发色底物法检测各抗凝血肽对凝血因子的作用。人凝血因子 IIa(即凝血酶)、Xa、XIa、XIIa、EGR-fXa购自美国Haematologic TechnologiesInc.,重组fVIIa为Novo-Nordisk(Denmark)产品,重组可溶性组织因子rsTF购自北京Protgen公司。凝血因子Xa的发色底物用S2765,凝血因子VIIa及IIa的发色底物用S2288,凝血因子Xia及XIIa的发色底物用S2366。
在96孔酶标板上,每孔100μl反应体系中进行。50μL凝血因子IIa、Xa、XIa、XIIa(终浓度为1nM)分别与10μL相应不同浓度抗凝血肽(或PBS对照组)混合【观测对fVIIa的作用时,使用50μL fVIIa+TF(1μM)+EGR-fXa(终浓度为500nM)】。在25℃孵育15分钟后,加入40μL发色底物(终浓度为400μM);。用Elx808酶标仪(BioTek,United States)监测OD405nm波长光吸收值变化情况,计算反应速度,根据加入抗凝血多肽时的反应速度与未加抗凝血多肽时的反应速度比推算各抗凝血多肽对凝血因子的抑制情况。
结果表明,抗凝血多肽在100nM浓度下,NAPTin-1、NAPTin-2、NAPTin-3、NAPTin-4、NAPTin-5、NAPTin-6、NAPTin-7、NAPTin-8及NAPTin-9均能抑制超过90%以上的凝血因子XIa活性,但对IIa、Xa、XIIa及fVIIa/TF均无抑制作用;在200nM浓度下,均能完全抑制凝血因子XIa的活性,对IIa、Xa、XIIa也无抑制活性。200nM浓度的NAPTin-1、NAPTin-2、NAPTin-3、NAPTin-4、NAPTin-5、NAPTin-6及NAPTin-7对fVIIa/TF无明显抑制作用,但NAPTin-8及NAPTin-9能抑制15%的fVIIa/TF活性。
上述结果表明,我们发明的抗凝血肽是凝血因子XIa的选择性抑制剂。
检测不同浓度的NAPTin-1对凝血因子XIa、Xa及fVIIa/TF的抑制作用,作图见附图1,图中V0为加入NAPTin-1前酶反应速度,V为加入不同浓度NAPTin-1后酶反应速度,V/V0为速率比,反映NAPTin对凝血因子的抑制活性。随NAPTin-1浓度的增加,fXIa的活性逐渐被抑制,抑制fXIa的IC50约为18.7nM,而100nM的NAPTin-1对fVIIa/TF及fXa均无明显抑制作用。
实施例4
抗凝血多肽对小鼠尾出血时间的影响
取SPF级BALb/c小鼠(由广东医学院实验动物中心提供)130只,雌雄各半,体重19-23g,随机分为生理盐水对照组,重组抗凝血多肽NAPTin-1、NAPTin-3、 NAPTin-5、NAPTin-7低、中高剂量组,静脉给药后10分钟后,在距尾尖1.5mm处剪断,待血液自行溢出开始计时,每隔30s用滤纸吸去血滴1次,直至用滤纸吸时无血为止,记录断尾尖开始至吸不出血为止的时间,即是尾出血时间,以均数±标准差表示。结果见表2.
表2NAPTin-1、NAPTin-3、NAPTin-5及NAPTin-7对小鼠尾出血时间的影响
表2中,与生理盐水对照组比较,低剂量组(100μg·kg-1)、中剂量组(1mg·kg-1)和高剂量组(5mg·kg-1)的NAPTin-1、NAPTin-3、NAPTin-5及NAPTin-7对出血时间均无明显影响,表明本发明所述抗凝血肽对小鼠的正常止血功能无明显影响,作为药物应用时,出血副作用小。
实施例5
重组抗凝血多肽NAPTin-1对大鼠动-静脉旁路血栓形成的影响
取SPF级雄性SD大鼠(由广东医学院实验动物中心提供)50只,体重300-350g,随机分为生理盐水对照组,阳性对照组(100U·kg-1肝素钠,上海第一生化药业有限公司),重组抗凝血多肽NAPTin-1低剂量组(100μg·kg-1)、中剂 量组(1mg·kg-1)和高剂量组(5mg·kg-1)。大鼠用10%水合氯醛(250mg/kg体重)腹腔注射麻醉,仰位固定,分离气管插管。手术分离出右颈总动脉及左颈外静脉.在一内径1.5mm、长22cm、充满50U/mL的肝素生理盐水的硅化聚乙烯管内,内置一根长5cm的4号手术缝合线.将聚乙烯管的一端***右颈总动脉,另一端***左颈外静脉。然后由尾静脉分别给予相应的药物,给药后5min,打开动脉夹,血液由右颈总动脉流经聚乙烯管返回左颈外静脉。开放血流15min后中断血流,迅速取出丝线称重,总重量减去丝线重量为血栓湿质量;抑制率=(生理盐水对照组血栓湿重-给药组血栓湿重)/生理盐水对照组血栓湿重×100%。结果见表3,与生理盐水对照组比较,各重组抗凝血多肽NAPTin-1均能显著抑制大鼠动-静脉旁路血栓形成(p<0.01)。
表3NAPTin-1对大鼠动-静脉旁路血栓形成的影响
与生理盐水对照组比较,*p<0.01)
实施例6
重组抗凝血多肽NAPTin-1对大鼠颈总动脉血栓形成的影响
取SPF级雄性SD大鼠(由广东医学院实验动物中心提供)50只,体重300-350g。动物随机分为5组,即假手术组(除不用FeCl3外,其余步骤同其他组)、模型组、重组抗凝血多肽NAPTin-1低剂量组(100μg·kg-1)、中剂量组(1mg·kg-1)和高剂量组(5mg·kg-1)。大鼠用10%水合氯醛(250mg/kg体重)腹腔注射麻醉后,沿颈正中线切开,钝性分离右侧颈动脉1cm长,放入0.6cm宽的封口胶条,给药5min后,用浸有20%FeCl3溶液的滤纸(1.0cm×0.5cm)环裹分离备用的颈总动脉段,并用封口胶条封住。15min后,取下滤纸条。40min后,结扎滤纸条两端血管,精确剪下滤纸条包裹的血管段,用洁净滤纸吸干血管内余血,精确称量含血栓的血管湿重,取出血栓后的血管再称重,两者相减即为该0.5cm长血管段内血栓的质量。假手术组用生理盐水代替FeCl3浸泡的滤纸条。结果见表4,
表4重组抗凝血多肽NAPTin-1对大鼠颈总动脉血栓形成的影响
与模型组比较,*p<0.01
实施例7
抗凝血多肽NAPTin-1抗大鼠静脉血栓形成实验。
SPF级雄性SD大鼠40只(广东医学院实验动物中心提供),体重300-350g。随机分成4组,即生理盐水对照组,重组抗凝血多肽NAPTin-1低剂量组(100μg·kg-1)、中剂量组(1mg·kg-1)和高剂量组(5mg·kg-1),每组10只大鼠。大鼠腹腔注射10%水合氯醛(250mg/kg体重)麻醉,剖腹分离下腔静脉,于左肾静脉下方置一丝线备结扎血管用,自静脉注射不同浓度受试药物,5min后结扎下腔静脉造成淤血,然后关闭腹腔,4h后再次打开腹腔,于结扎下方2.0cm处夹闭血管,纵行剖开检查有无血栓形成,以正常对照组为基准计算各组血栓形成比率。结果见表5,表明抗凝血多肽NAPTin-1具有显著的抗大鼠静脉血栓形成作用。
表5重组抗凝血多肽NAPTin-1对大鼠下腔静脉血栓形成的影响
*P<0.01vs生理盐水对照组
实施例8
重组抗凝血多肽NAPTin-1对大鼠凝血时间的影响
取SPF级雄性SD大鼠(由广东医学院实验动物中心提供)40只,体重300-350g,随机分成4组。静脉给药2分钟后,心脏取血,注入加有3.8%柠檬酸钠的塑料管内(血∶抗凝剂=9∶1),轻轻摇均。1500×g离心10min,分离血浆。测定aPTT、PT。结果见表6。各剂量组NAPTin-1均能显著延长aPTT,但对PT无影响,亦表明其出血副作用小。
表6重组抗凝血多肽NAPTin-1对大鼠凝血时间的影响
*P<0.01vs生理盐水对照组。
Claims (3)
1.一种抗凝血多肽,其特征在于:
(a)由SEQ ID NO.1所示氨基酸序列组成的、且能选择性抑制凝血因子XIa,而对凝血因子Xa、XIIa、IIa及fVIIa/TF均无明显抑制作用的多肽;或者
(b)由SEQ ID NO.2、或SEQ ID NO.3、或SEQ ID NO.4、或SEQ ID NO.5、或SEQID NO.6、或SEQ ID NO.7、或SEQ ID NO.8、或SEQ ID NO.9、或SEQ ID NO.10所示氨基酸序列组成的、且能选择性抑制凝血因子XIa,而对凝血因子Xa、XIIa、IIa及fVIIa/TF均无明显抑制作用的多肽。
2.如权利要求1所述的一种抗凝血多肽的应用,其特征在于在制备预防和治疗血栓性疾病药物中的应用。
3.如权利要求2所述的一类抗凝血多肽的应用,其特征在于,将所述的一种抗凝血多肽作为活性成份直接或与药学上可接受的载体制成制剂。
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CN201110097205.2A CN102241734B (zh) | 2011-04-15 | 2011-04-15 | 一类抗凝血多肽及其应用 |
US14/111,470 US9243044B2 (en) | 2011-04-15 | 2012-04-05 | Anticoagulant polypeptide and applications thereof |
AU2012100391A AU2012100391A4 (en) | 2011-04-15 | 2012-04-05 | Anticoagulant Peptides and Uses thereof |
EP12752184.7A EP2698378B1 (en) | 2011-04-15 | 2012-04-05 | Anticoagulant polypeptide and applications thereof |
PCT/CN2012/073509 WO2012116663A1 (zh) | 2011-04-15 | 2012-04-05 | 一类抗凝血多肽及其应用 |
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CN102241734B (zh) | 2011-04-15 | 2014-04-02 | 广东医学院 | 一类抗凝血多肽及其应用 |
CN113201049B (zh) * | 2015-08-05 | 2022-10-04 | 陕西麦科奥特科技有限公司 | 有抗凝血和抗血小板活性的多靶点化合物及制法和用途 |
CN105175510B (zh) * | 2015-10-22 | 2018-06-01 | 江苏大学 | 噬菌体展示技术筛选的具有抗凝血活性的多肽 |
CN107267539A (zh) * | 2016-04-06 | 2017-10-20 | 沈阳药科大学 | 一种高效获得重组蛋白的大肠埃希菌可溶性表达载体 |
CN108586582B (zh) * | 2018-07-04 | 2020-07-10 | 中国科学院昆明动物研究所 | 一种抗凝多肽fx18及其应用 |
CN110857318B (zh) * | 2018-08-22 | 2022-09-23 | 广州医科大学附属肿瘤医院 | 一种抗血栓多肽cystatin-T及其制备方法和应用 |
CN110981953B (zh) * | 2019-12-13 | 2021-10-08 | 首都医科大学 | 一种多肽和多肽的应用及包含多肽的组合物 |
CN113354713B (zh) * | 2021-06-16 | 2022-07-26 | 昆明医科大学第一附属医院 | 一种多肽及其在制备抗血小板聚集的药物中的应用 |
CN113429459B (zh) * | 2021-06-16 | 2022-07-26 | 昆明医科大学第一附属医院 | 一种抗血小板多肽及其药物组合物与其应用 |
CN113956339B (zh) * | 2021-10-28 | 2023-02-24 | 中国药科大学 | 宽体金线蛭抗凝血因子XIa多肽及其应用 |
CN118059209A (zh) * | 2024-03-07 | 2024-05-24 | 北京胜元优奇生物科技有限公司 | 一种包含生物活性肽的保健品或药品以及在治疗心脑血管疾病中的应用 |
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Title |
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Identification of an anticoagulant peptide that inhibits both fⅪa and fⅦa/tissue factor from the blood-feeding nematode Ancylostoma caninum;LI,D.等;《Biochemical and Biophysical Research Communications》;20100205;第392卷(第2期);摘要和图1 * |
LI,D.等.Identification of an anticoagulant peptide that inhibits both fⅪa and fⅦa/tissue factor from the blood-feeding nematode Ancylostoma caninum.《Biochemical and Biophysical Research Communications》.2010,第392卷(第2期), |
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EP2698378B1 (en) | 2015-09-09 |
EP2698378A1 (en) | 2014-02-19 |
EP2698378A4 (en) | 2014-02-19 |
US20140323404A1 (en) | 2014-10-30 |
CN102241734A (zh) | 2011-11-16 |
WO2012116663A1 (zh) | 2012-09-07 |
AU2012100391A4 (en) | 2012-05-10 |
US9243044B2 (en) | 2016-01-26 |
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