CN105592854A - 使吸烟的不利作用减少的新方法 - Google Patents
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Abstract
本发明涉及弹性蛋白酶抑制剂,优选发新素(fahsin)用于治疗或预防肺气肿、COPD或肺癌中的应用。优选地,所述弹性蛋白酶抑制剂通过吸入,优选通过吸入烟草烟雾来给予。本发明还包括包含所述弹性蛋白酶抑制剂的吸烟制品。
Description
发明领域
本发明涉及健康与医药领域,更具体地,涉及预防或治疗肺疾病(例如COPD、肺气肿和肺癌)的领域,或者,克服吸烟带来的问题的领域,更具体地,涉及用于减少烟雾吸入的不利作用的香烟、烟草和其它吸烟材料。
背景技术
吸烟有害健康。自从二十世纪七十年代以来,收集到越来越多的证据显示吸烟(尤其是吸香烟(cigarettes),以及其他形式的吸烟)对吸烟者与未吸烟的旁人的健康的负面影响。众所周知,并且也由多次临床实验确定了,香烟烟雾在肺结构的弹性内容物(例如,气管、支气管、细支气管和肺泡系统)的退化中起关键作用。更具体地,肺气肿是局部肺泡壁损伤或破坏。
肺泡壁中的毛细管是最重要的解剖学特征。它们形成缠结的网络,并且由富含弹性和网硬蛋白纤维的精细的纤维间质支持。巨噬细胞在肺泡空间中发生,并且形成抵抗入侵细菌的第一道防御机制。根据“弹性蛋白酶:抗弹性蛋白酶”假说,香烟烟雾会导致炎症,以及后续的蛋白水解酶超过其天然抑制剂的规模向肺中释放。在缺乏正常修复的情况下,蛋白水解导致组织破坏和气道扩大。该弹性蛋白酶:抗弹性蛋白酶假说已经主导了近四十年的COPD研究。1963年,Laurell和Eriksson(LaurellCB,ErikssonS.ScandJClinInvest1963,15:132-140)报道了如下观察结果:不生成或生成不足量的α-1-抗胰蛋白酶(α1AT)(嗜中性弹性蛋白酶(NE)的主要抑制剂)的患者发展早发性肺气肿。紧随其后,Gross等(GrossP等.ArchEnvironHealth1965,11:50-58)将木瓜蛋白酶,一种弹性蛋白酶,慢慢地输入大鼠的肺,导致肺气肿。随后,研究者们能够通过将其它弹性酶(包括人嗜中性弹性蛋白酶)慢慢地输入实验动物的气道来诱导肺气肿(SeniorRM等JClinInvest1980,66:859-862;JanoffA等.AmRevRespirDis1977,115:461-478;SniderGL等.AmRevRespirDis1984,129:155-160)。总之,这些观察结果确证NE作为蛋白酶最有可能引起肺气肿中的组织破坏。吸烟的关系也已由Shapiro等确证(ShapiroS等.AmJPathol.2003163(6):2329–2335)。
无疑,在多项研究中,已将肺癌的发生与吸烟相关联。肺癌没有单一的形式,并且其可由支气管癌、肺泡癌、支气管腺瘤和间充质瘤组成。特别是支气管癌,已将其与吸烟(香烟)相关联。观察到该癌症的解剖学变化是上皮的变化,例如,毛细胞损失、基底细胞增生、鳞状细胞化生和非典型细胞结构。尽管已经显示弹性蛋白酶抑制会受到存在于香烟烟雾中并且被证明具有致癌性的化学化合物(如多芳环烃)的致癌活性影响,人们假设弹性蛋白酶抑制会影响伴随癌发生的级联事件,例如,癌的免疫组分的作用。
尽管已表明弹性蛋白酶抑制剂可用于治疗肺气肿(例如Koraki,T.等,2002,Am.J.Resp.Crit.Caremed.166:496-500;Wright,J.等,2003,Eur.Resp.J.22:77-81),迄今为止未见这些化合物在肺气肿领域的商业应用。
仍然需要对于具体蛋白酶(例如,弹性蛋白酶、胰蛋白酶、糜蛋白酶、组织蛋白酶G等)具有特异性的新型蛋白酶抑制剂。
发明内容
本发明的发明人现已发现发新素(fahsin)可用于预防选自肺气肿、COPD和肺癌的肺疾病。例如在治疗或预防肺疾病(具体为肺气肿)中的应用可优选地通过吸入,尤其是通过吸入烟雾(更具体地,烟草(例如香烟)烟雾)来实现。
本发明还包括用于治疗或预防肺气肿的吸烟制品,例如香烟,包括电子香烟(e-cigarette)、烟斗烟草、雪茄或烟卷(joint)。优选地,所述吸烟制品包含弹性蛋白酶抑制剂,优选地,选自下组:发新素、瓜梅素(guamerin)、匹瓜梅素(piguamerin)、希如斯塔辛(hirustasin)、德拉斯塔辛(bdellastasin)和瓜梅素的突变体、在第6个半胱氨酸残基之后包含亮氨酸残基的匹瓜梅素、希如斯塔辛和德拉斯塔辛。进一步优选地,所述弹性蛋白酶抑制剂以掺混物形式或由烟草或大麻表达的蛋白质形式包含在所述烟草或大麻中。或者,所述香烟是过滤嘴香烟或电子香烟,并且,所述弹性蛋白酶抑制剂存在于过滤嘴中。又或者,所述吸烟制品是香烟或卷烟,并且所述弹性蛋白酶抑制剂存在于卷烟纸中。
在另一个实施方式中,所述弹性蛋白酶抑制剂是重组生成的发新素。
本发明还包括转基因烟草或大麻,其包含弹性蛋白酶抑制剂,优选地是发新素。因此,本发明包括含有所述转基因烟草或大麻的香烟。
本发明的另一个部分是预防或减轻肺气肿、COPD或肺癌的方法,所述方法包括抽吸本发明的吸烟制品。
本发明的另一个实施方式是预防或减轻肺气肿、COPD或肺癌的方法,所述方法包括吸入弹性蛋白酶抑制剂,优选是发新素,更优选是重组发新素。
本发明还包括一种改善吸烟者的肺功能的方法,所述方法通过给予弹性蛋白酶抑制剂,优选地通过吸入给予所述抑制剂来进行。优选地,在所述方法中,所述吸入是吸入烟雾,其中所述烟雾包含所述抑制剂。
在另一个实施方式中,本发明提供发新素、瓜梅素、匹瓜梅素、希如斯塔辛和德拉斯塔辛的新型突变体。
附图说明
图1:五种不同安逖斯塔辛(antistasin)型丝氨酸蛋白酶抑制剂的一级氨基酸序列的比对。所述蛋白质中具有相似间距的半胱氨酸残基以粗体表示。反映所述抑制剂的特异性的反应位点(P1)氨基酸残基以下划线表示。
图2.不同瓜梅素突变体对人嗜中性弹性蛋白酶的抑制。X轴指示突变体P1位的残基(met是野生型),空白是仅有底物,而最大(max)是底物+弹性蛋白酶。短线表示自孵育开始之后的时间。Y轴显示检测到的A405-A540的差。
图3.不同浓度的发新素突变体对组织蛋白酶G的抑制。突变体指示和y轴与图2中类似。
图4.瓜梅素突变体对不同蛋白酶的抑制。A:组织蛋白酶G,B:糜蛋白酶,C:弹性蛋白酶,D:胰蛋白酶,E:纤溶酶,F:凝血酶
发明详述
近期发现的弹性蛋白酶抑制剂之一是发新素,其源自尼罗河水蛭(Limnatisnilotica)(DeBruin,E.等,FEMSYeastRes.5:1069-1077,2005;WO96/13585)。在该公开文件中已证明,发新素是对人嗜中性弹性蛋白酶(hNE)具有特异性的蛋白酶,并且是其它重要的血液源性丝氨酸蛋白酶,例如纤溶酶、凝血酶、tPA、凝结因子VIIa、Xa、XIa和XIIa所无法比拟的。这使其成为本发明的理想候选物。
此外,尽管发新素是肽化合物,其在重组技术的协助下易于生成,并且已证明其具有相当的稳定性。所述氨基酸序列(GenBankDQ097891.1)和编码所述氨基酸序列的核苷酸序列(GenBankAAY85799.1)已示于图1。如DeBruin等(同上)所述,发新素类似于其它安逖斯塔辛型蛋白酶抑制剂,它们以特定的距离具有10个半胱氨酸残基的共有序列:
C(X4)CS(X4)C(X4)CXC(X4)CL(X3)C(X6)DXNGC(X3)CXC
其中X可以是任何氨基酸,并且C、L、N、G和C具有其氨基酸命名中的常规含义。
对于NE的特异性归因于第6个半胱氨酸残基之后的亮氨酸残基。认为具有上述共有序列的肽可用于本发明,并且将具有NE-抑制效应。
发新素之外,其它安逖斯塔辛型丝氨酸蛋白酶(NE)抑制剂也可用于本发明。示例有:瓜梅素、匹瓜梅素、希如斯塔辛和德拉斯塔辛。这些化合物的氨基酸序列为:
瓜梅素(日本医蛭(Hirudonipponia)):vdenaedthglcgektcspaqvclnnecactaircmifcpngfkvdengceypctca
匹瓜梅素(日本医蛭):tdcggktcseaqvckdgkcvcvigqcrkycpngfkkdengctfpctca
希如斯塔辛(欧洲医蛭(Hirudomedicinalis)):tqgntcggetcsaaqvclkgkcvcnevhcrirckyglkkdengceypcscakasq
德拉斯塔辛(欧洲医蛭):fdvnshttpcgpvtcsgaqmcevdkcvcsdlhckvkcehgfkkddngceyacicadapq
发新素的这些替代物中,特别优选的是突变的瓜梅素,其中,第6个半胱氨酸残基之后的甲硫氨酸残基被改成亮氨酸残基:vdenaedthglcgektcspaqvclnnecactairclifcpngfkvdengceypctca。已显示(结果未显示),所述突变的瓜梅素对化学和生物氧化作用均不敏感,并且,该突变的蛋白质还似乎是NE分子的强抑制剂(如野生型发新素)。据信,将该特定残基(参见图1),其在上述的其它分子中是精氨酸残基(匹瓜梅素和希如斯塔辛)或赖氨酸残基(德拉斯塔辛),改变为亮氨酸残基,也能够提供对NE具有提高的反应性的突变蛋白,并且其比野生型蛋白质更加稳定。
由此,所述在第6个半胱氨酸残基之后具有亮氨酸残基的突变蛋白也是本发明的部分。
在研究可用于本发明的突变体时制得的其它突变体包括多种发新素突变体,其中,P1位点(即,第6个半胱氨酸残基之后的残基)已被改变。制备的若干突变体中,P1=Arg,P1=Ile,P1=Met且P1是Val。这些突变体通过定点诱变来制备。发新素-Ile(即,第6个半胱氨酸残基之后的残基是Ile)似乎是对弹性蛋白酶极具特异性的抑制剂,并且不抑制糜蛋白酶、组织蛋白酶G和蛋白酶3。这表示,该突变体非常合适于其中特定的弹性蛋白酶是致病因素的疾病,例如,肺气肿和牛皮癣。并且,该突变体能够非常良好地适用于由酶,人嗜中性弹性蛋白酶(HNE)引起的与组织破坏相关的关节炎、齿龈炎、牙周炎和其它炎性病症。因此,本发明还涉及该发新素-Ile突变体作为治疗性化合物的应用,尤其是用于治疗与嗜中性弹性蛋白酶相关联的炎性疾病,具体有肺气肿、牙周炎、关节炎等。认为,对于肺气肿和牙周炎的治疗,优选地以经口方式进行给予。对于肺气肿治疗,可通过任何形式的吸入器进行给予,但有利的是通过本文所述的电子香烟给予。对于牙周炎,也可采用电子香烟递送,但该化合物也可在牙膏、香口胶或提供该化合物在口腔中的释放的其它给予形式中提供。
同样地,所述发新素-Val和发新素-Met突变体也可以与上述关于发新素-Ile相同的方式用作弹性蛋白酶-抑制剂,尽管其效果相对于发新素-Ile突变体和野生型发新素而言特异性较弱。
第二种非常有用的发新素突变体是发新素-Arg。该化合物,尽管其与野生型发新素(无法特异性抑制弹性蛋白酶)仅差一个氨基酸,但惊人的是,它是胰蛋白酶的极佳抑制剂(并且其也抑制凝结因子Xa、XIa和XIIa)。因为这些作用,认为发新素-Arg适用于抑制凝结和纤维蛋白溶解。并且,发新素-Arg可用于治疗胰腺炎。发新素-Arg也是强于其它发新素突变体的组织蛋白酶G抑制剂。这意味着它也可被用作组织蛋白酶G抑制剂,因此,其可能有利于治疗或预防炎症,特别是导致水肿的炎症,以治疗或预防光老化。并且,其增强发新素-Arg的抗血栓形成作用。
对于瓜梅素,除了上述的Leu突变体以外,还已制备在P1位具有Ile、Arg、Lys或Val的其它突变体(参见图1)。如上所述,该Leu突变体在抑制人嗜中性弹性蛋白酶方面最为有效,尽管野生型(P1位为Met)也显示一些效果。其它三种突变体效果较差。然而,Arg突变体似乎是糜蛋白酶的最佳抑制剂,Leu突变体名列第二。这两种也是组织蛋白酶G的最佳抑制剂。然而,显示Lys突变体是胰蛋白酶和纤溶酶的特异性抑制剂,而对于其它突变体,仅瓜梅素-Arg能够显示一些作用。凝血酶几乎不被任何所述突变体抑制。
此外,先前设想的弹性蛋白酶抑制剂在肺疾病(例如COPD和肺气肿)领域中的应用已集中于已经确立了肺气肿之后的应用。应清楚的是,弹性蛋白酶抑制剂在香烟中的应用是意在预防肺气肿的发作或抑制肺气肿的进一步发展。因此,本发明还提供通过发新素来预防肺气肿或预防肺气肿的发展。
所述应用尤其有利于预防或减少吸烟(尤其是吸香烟)的有害作用。此外,已证明(Kozumi,F.等,1999,Clin.Pharmacol.Ther,66:501-508)吸烟者的肺对弹性蛋白酶抑制剂的摄取可相对于非吸烟者的摄取而有所增加,这协助进行非常适合于对抗吸烟的有害作用的治疗。
本发明的NE抑制剂可用于吸入器中,以预防或治疗疾病,例如COPD、肺气肿和肺癌。此外,NE抑制剂的给予可用于改善或减轻其中肺凝结或因其它因素而导致长期功能受损的病症。这类病症包括:哮喘、由细菌或其它微生物所致的肺炎,例如肺炎球菌、葡萄球菌、流感嗜血杆菌、绿脓假单胞菌、粘膜炎莫拉菌、支原体、肺炎性披衣菌、嗜肺军团菌、呼吸道合胞病毒(RSV)、腺病毒、衣原体、曲霉菌、普通感冒、由石棉沉着病所致的肺组织破坏或损伤、空气污染等。理想地,NE抑制剂(例如发新素)的吸入可通过吸入,尤其是通过吸入器的吸入或通过电子烟(电子香烟)的吸入来递送。
对于吸烟者,本发明的NE抑制剂,尤其是发新素,具体为重组发新素,可以任何可行的方式包括在吸烟制品内。首先,所述肽能够在香烟中所含的烟草或其它植物材料(大麻)中重组表达。可利用用于获得毕赤酵母(Pichiapastoris)中的重组表达的示例中已用的相似的表达构建体的重组生成,但当然,其需要适应于植物中的表达。
有多种方式能够将重组核酸转入植物细胞,例如农杆菌(Agrobacterium)介导的转化。然而,除了农杆菌(Agrobacterium)感染以外,若想实践本发明,还有其它手段来将DNA有效递送至受体植物细胞。据信,用于将DNA递送至植物细胞的合适的方法实际上包括能够使DNA导入细胞的任何方法,例如通过DNA的直接递送,例如通过PEG介导的原生质体转化,通过干燥/抑制介导的DNA摄取(Potrykus等,Mol.Gen.Genet.,199:183-188,1985),通过电穿孔(美国专利号5,384,253),通过用碳化硅纤维刺激(Kaeppler等,1990;美国专利号5,302,523;和美国专利号5,464,765),以及通过DNA被覆的颗粒的促进(美国专利号5,550,318;美国专利号5,538,877;和美国专利号5,538,880)。通过应用例如这些技术,实际上,来自任何植物物种的细胞均可被稳定转化,并且这些细胞将发展成转基因植物。
如果采用农杆菌介导的转化,优选采用基本强毒的农杆菌宿主细胞,例如根癌农杆菌(A.tumefaciens),示例有菌株A281或及其衍生的菌株,或本领域可得的其它强毒株。这些农杆菌菌株携带源自Ti质粒pTiBo542的毒力区域的DNA区,所述Ti质粒pTiBo542包含virB、virC和virG基因。根癌农杆菌(A.tumefaciens)的毒力(vir)基因产物协助T-DNA的加工及其向植物细胞内的转化。Vir基因表达受virA和virG控制,由此,virA在感知诱导信号之后会通过磷酸化激活virG。VirG,进而诱导virB、C、D、E的表达。这些蛋白质基因编码涉及DNA的转化。认为pTiBo542的增强的毒力是由该Ti质粒上的高强毒virG基因所致(Chen等Mol.Gen.Genet230:302-309,1991)。
在将核酸转化进入植物或植物细胞之后,必须确定哪些植物或植物细胞已经具有所述核酸。例如,这可通过采用可选择的标志物或报告基因来实现。最广泛用于植物转化的选择性标志物或选择基因有,细菌新霉素磷酸转移酶基因(nptI、nptII和nptIII基因),其赋予对选择剂卡那霉素的抗性,如EP131623所述,和细菌aphIV基因,如EP186425所述,其赋予对潮霉素的抗性。EP275957公开了来自绿色产色链霉菌(Streptomycesviridochromogenes)的乙酰转移酶基因的应用,其赋予对除草剂草丁膦的抗性。赋予对除草剂草甘膦的相对抗性的植物基因描述于EP218571。该抗性基于编码5-烯醇莽草酸酯-3-磷酸合酶(EPSPS)的基因的表达,其相对耐受N-磷酸甲基甘氨酸。某些氨基酸例如赖氨酸、苏氨酸或赖氨酸衍生物氨乙基半胱氨酸(AEC)和色氨酸类似物如5-甲基色氨酸也可用作选择剂,因其以高浓度施加时具有抑制细胞生长的能力。在该选择系统中,可选择的标志物基因的表达导致转基因细胞生成过量的氨基酸,这允许转基因在选择压力下生长。报告基因的合适示例有β-葡糖苷酸酶(GUS)、β-半乳糖苷酶、荧光素酶和绿色荧光蛋白(GFP)。
或者,转化体可通过分析编码发新素的核酸或表达所述核苷酸序列的发新素蛋白质的存在来检测。
作为农杆菌转化的替代方式,和Wypijewski(2001,ActaBiochim.Polon.48-3:657-661)示范了用于完整BY-2烟草培养细胞的电穿孔法,通过表达报告子绿色荧光蛋白(GFP)的质粒的表达来示范。电穿孔法包括在包含5mMCaCl2、10mMNaCl、8.7%甘油、0.4M蔗糖和10mM管道缓冲剂的缓冲系统(pH6.8)中,在30μg质粒存在下,诱导细胞质壁分离15-20分钟。然后,使细胞经历真空处理,之后对其进行冰上孵育,随后通过对其施加2kV/cm的脉冲(持续80μs)来进行电穿孔处理。电脉冲之后,细胞再次冰上孵育10分钟,室温下放置10分钟,之后,通过添加不含蔗糖的BY培养基,以在三个步骤中将蔗糖浓度从0.4M降至0.05M,使细胞去质壁分离。然后,将去质壁分离的细胞转移至BY培养基,并在后续的时日中监测GFP表达。电穿孔之后,测定转染效率为50%,而细胞活力为70%,但在随后的时日中降低。因此,似乎在该过程中,导入DNA的处理对细胞的活力和再生能力具有负面作用。
更为巧妙的方法见述于Chen,C.-P.等(2007,FEBSLett.581:1891-1897),其采用基于多聚精氨酸的肽来在完整的绿豆和大豆的根中递送GFP蛋白质和GFP表达载体。在该示例中,生成九聚-Arg肽作为载体,并且将10μg与10μg的质粒在总体积为50μl的PBS中于37℃预先孵育30分钟。随后,使绿豆和大豆的根在该DNA-肽溶液中浸没30分钟,随后清洗。监测GFP的表达,并显示其在整个根的处理之后的24小时和48小时之间发生。
可采用表达编码本发明的弹性蛋白酶抑制剂的核酸的其它替代性方法。
当发新素在烟草或大麻中重组生成时,可通过将其与其它烟草掺混或应用其本身来将该烟草或大麻包括在香烟内。还可通过将烟草浸没在弹性蛋白酶抑制剂的溶液中,然后干燥所述烟草来将其添加至烟草或其它吸烟材料。
发新素,或任何其它本发明的弹性蛋白酶抑制剂,还可被包含在用于卷制香烟的卷烟纸中。由此,该卷烟纸可由能够表达发新素的重组植物(例如,转基因大米)的浆料产生,或可在制备卷烟纸的过程中将发新素添加至所述浆料。或者,可在卷制香烟生成之后或在卷制香烟之前使发新素被覆所述卷烟纸。
此外,可在制备吸烟材料的过程中将弹性蛋白酶抑制剂添加至所述材料。就此而言,可将其以蛋白质粉末形式与烟草掺混或包封在载体材料中。并且,可将其添加至过滤嘴香烟中的过滤材料。在实验部分,已显示将包含丙烯酸盐的珠的r发新素(rFahsin)插入香烟的滤嘴获得了测试对象的肺功能的显著改善。并且,可通过将过滤材料浸没在抑制剂溶液中,并使其干燥,然后生成香烟滤嘴,来将所述弹性蛋白酶抑制剂纳入所述过滤材料。
最后,如果(香烟)烟雾通过其中释放所述抑制剂的材料或烟斗而被吸入,则弹性蛋白酶抑制剂可伴随该烟雾被吸入。这可以是额外的过滤材料,但也可以是通过用户缓释所述弹性蛋白酶抑制剂的载体材料,例如,在开始吸烟之前添加至烟斗中。
当然,其中包含所述弹性蛋白酶抑制剂的吸烟制品可以是任何吸烟制品,例如,香烟、雪茄、小雪茄、烟斗、卷烟、水烟或任何其它吸烟材料。优选地,所述吸烟制品是香烟,因为其应用最为广泛,并且其被认为与肺疾病的病因最为相关。
吸烟材料中所用的弹性蛋白酶抑制剂优选地是发新素,更优选地是重组发新素。发新素具有的主要益处是,它热稳定性极高,从而不会被高温烟雾破坏。在一项测试中,已将发新素加热至123℃,但并没有出现熔解曲线。在该高温处理之后,该蛋白质并没有丧失活性。所述弹性蛋白酶抑制剂可以0.001~100mg/kg吸烟材料的浓度存在于所述吸烟制品中,但优选地具有0,001~50mg/kg吸烟材料的浓度。就我们的实验来看,最小抑制浓度(MIC)似乎是4μg/1百万PMN/15分钟。然而,鉴于MIC很大程度上取决于疾病的严重程度,因此可采用具有不同量的弹性蛋白酶抑制剂的吸烟材料。通过该方式,可提供吸烟材料(轻-中-强)的数个级别,其均具有能够满足烟草烟雾的治疗或预防效果的合适量的弹性蛋白酶抑制剂。
还可将所述弹性蛋白酶抑制剂在不吸入烟雾的情况下施加至肺,例如,通过应用在向支气管、细支气管或肺泡给予药物化合物时常用的标准吸入器或蒸发器。就此而言,弹性蛋白酶抑制剂,优选地是发新素或瓜梅素-Leu,可以任何可接受的药学制剂(例如干粉)或溶液形式存在于所述吸入器中。尤其优选这样的吸入器,该吸入器包含其中含有弹性蛋白酶抑制剂的溶液或悬液是能够形成气溶胶的溶液的蒸发器。产生全身性治疗效果的高效的气溶胶递送的重要参数是气溶胶云团中的粒径分布。当制剂是悬液形式时,云团的粒径受悬浮药物的粒径控制。当该制剂是溶液形式时,不存在悬浮药物颗粒的体积贡献,并且产生很大程度上由溶液中的药物浓度确定的细得多的液滴云团。将该药物通过气溶胶吸入器递送至肺从而使其被诱导进入毛细管时,该颗粒应足够小以在吸入后被递送至肺,并被吸收进入血流,即,具有约0.5μm和2.5μm范围内的有利尺寸。小于0.5μm的颗粒不具治疗益处,因为其会被再次呼出。本领域技术人员有能力采用本发明的弹性蛋白酶抑制剂生成用于吸入或蒸发器装置的药学制剂。
除了这些或多或少的医用吸入器和蒸发器之外,本发明的弹性蛋白酶抑制剂还可被纳入用于空气加湿或空气熏香的蒸发器中。在该情况中,弹性蛋白酶抑制剂应具有低浓度。家居室内空气的持续清新化将允许恒定的小量弹性蛋白酶抑制剂存在于空气中,因此允许室内居住者的恒定的吸入剂量。当然,所述应用并不限于家居室内,其还可应用于轿车、商店、办公室、公共建筑等。
吸入器的一种特定形式是电子烟或电子香烟。电子香烟或个人蒸发器(PV)是电力供能的蒸发器,其通过产生类似烟的气溶胶来模拟抽吸烟草。通常采用加热元件(称为喷雾器),其使液体溶液(称为e-液体)蒸汽化。E-液体通常包含丙二醇、植物甘油、尼古丁和香料的混合物,而其它释放不含尼古丁的风味蒸汽。对于本发明,电子香烟的应用是非常有利的,因为可将弹性蛋白酶抑制剂溶解在E-液体中,从而包含在经生成以供于吸入的气溶胶内。该溶液常在瓶中或预充在一次性弹筒中售卖,或以试剂盒形式售卖以供消费者自制其E-液体。组分是个体可得的,并且消费者可选择采用各种出售物来改变或加强其风味、尼古丁强度或浓度。
在个人蒸发器中,所述喷雾器系统可以是所谓‘烟弹雾化器’的形式,其由被液体浸透的多聚泡沫(作为e-液体固定器)围绕的喷雾器组成。烟弹雾化器可独自或与允许更大e-液体容量的罐(tank)联用。在罐中使用时,将烟弹雾化器插入塑料、玻璃或金属管并且必须在所述烟弹雾化器的侧面冲钻孔或狭缝,以允许液体到达线圈。透明雾化器或"透明雾化机(clearos)",与烟弹罐(cartotank)没什么不同,采用其中插入喷雾器的透明罐。然而,与烟弹罐不同,其内部并没有多聚泡沫材料。存在用于透明雾化器内部以确保芯的良好润湿而不溢湿线圈的多种不同的芯系统。一些依赖于重力来将e-液体带至芯和线圈组件(例如,底部线圈透明雾化器),而其它的则依赖于毛细管作用,以及某种程度上使用者在运用透明雾化器(顶部线圈透明雾化器)时搅拌e-液体。可重建的喷雾器或RBA是允许用户组装或"构建"芯和线圈本身而不是通过现成的喷雾器"头"对其进行替换的喷雾器。其还允许使用者以任何所需的电阻构建喷雾器。"重建"喷雾器所需的材料通常比配合透明雾化器的常用的预制可替换芯和线圈组件便宜很多。这些可重建的喷雾器主要分为两类:可重建的罐式喷雾器(RTA)和可重建的液滴喷雾器(RDA)。
可重建的罐式喷雾器或RTA与透明雾化器的类似之处在于,它们采用罐或容器来容纳液体并将液体带至线圈。通常,它们容纳的e-液体远多于其RDA类似物。
另一方面,可重建的液滴喷雾器或RDA缺乏容器部分,并且相较于RTA而言容纳非常少的液体,但通常也会小很多。它们通常仅由喷雾器"构建台(buildingdeck)"和"顶盖"组成,所述构建台能够接受一个或多个线圈,所述顶盖能够覆盖可连接接口管(mouthpiece)的线圈。使用者需要通过在裸芯和线圈组件上滴加液体来手动保持喷雾器湿润。
实施例1
重组发新素的生成和表征
发新素生成和纯化按照DeBruin,E.等,FEMSYeastRes.5:1069-1077,2005所述进行。简言之,合成的发新素基因通过四个长寡核苷酸的重叠延伸PCR构建,所用密码子针对宿主毕赤酵母(Pichiapastoris)进行优化:
FA-1:5’-GGGGTATCTCTCGAGAAAAGAGACGACAACTGTGGTGGTAAGGTTTGTTCTAAGGGTCAA-3’
FA-2:5’-AATCAAACATCTAATTGAGTACACTCACAGTGACCGGTCGTGACACAATTGACCCTTAGAACAAAC-3’
FA-3:5’-CCAATTAGATGTTTGATTTTCTGTCCAAACGGTTTCGCTGTTGACGAGAACGGTTGTGAG-3’
FA-4:5’-GCTGGCGGCCGCTCATTGGTGCTTACAAGAACATGGCAACTCACAACCGTTCTCGTC-3’
在采用pGEMT-简易克隆试剂盒(普洛麦格(Promega),美国威斯康星州麦迪逊)克隆PCR产物和后续的DNA测序之后,采用XhoI和NotI限制性核酸内切酶(英杰公司,美国加利福尼亚州卡尔斯巴德)将合适的基因被克隆进入毕赤酵母载体pPIC9。
毕赤酵母GS115(his4,参见Cregg,J.等,Mol.Cell.Biol.5:3376-3385,1985)通过电穿孔转化。在转化前,质粒pPIC9发新素用SalI(英杰公司)线性化。在选择板上于30℃生长3天后,选取数个集落,采用载体引物和(英杰公司)进行PCR确认。
在选择产生r发新素的毕赤酵母(P.pastoris)转化体之后,在5升BioFlo3000发酵罐(新布伦瑞克科学公司(NewBrunswickScientific),美国新泽西州艾迪逊市)中于补充有0.2%(v/v)PTM1-痕量盐(英杰公司)的最小基底盐培养基中发酵。进行甲醇分批补料式发酵(Potter,K.等,ProteinExpr.Purif.19:393-402,2000),并利用针对20mMTris-缓冲剂(pH8,0)的透析过夜来从发酵物料中纯化r发新素。采用阴离子交换色谱在SP琼脂糖FF柱上分离r发新素,并采用柠檬酸缓冲剂(20mM,pH4.0)中的1MNaCl在探测器(GE医疗集团)上洗脱。利用发色试验,测定包含r发新素的色谱分离部分对NE的活性,并且汇集活性分离部分,随后对20mMTris-HCl(pH8.0)透析以去除NaCl。在Q-琼脂糖快速流动或Q-琼脂糖高效仪上的最后的阴离子交换色谱步骤中,获得由HPLC(C8反相)测定的基本纯(>90%)。
测量r发新素的若干特点:
-r发新素与以模拟通过胃肠道期间遇到的环境的0.32%(w/v)胃蛋白酶和10mMHCl(pH2.0)的溶液孵育显示r发新素完全失活。
r发新素与仅10mMHCl(pH2.0)的孵育不影响活性。因此,失活由胃蛋白酶导致,其甚至能够以低十倍的剂量使r发新素完全失活。
-r发新素是具有极高的热稳定性。在加热至123℃1小时之后,r发新素没有失去其NE抑制活性。该特点使其极为合适于通过(香烟)烟雾递送。
-pH稳定性。以2.0、4.0、6.0、8.0或10.0的pH在60℃孵育48小时之后,似乎没有丧失特定活性。
-r发新素与等摩尔浓度的DDT的孵育减少了r发新素的特定活性。
-r发新素通过形成r发新素-NE复合物来抑制NE,正如NE的天然人拮抗剂,α1-抗胰蛋白酶(AAT)。然而,与AAT-NE复合物不同,r发新素-NE复合物不显示任何促炎活性,并且很可能不被从体内快速清除。
-r发新素与AAT不同,其抵抗化学和生物氧化作用。这在慢性炎症中是一项重要益处,所述慢性炎症如同发生COPD的那些,存在许多活化的嗜中性粒细胞其导致形成活性氧物质(ROS)。此外,r发新素不受烟雾中的氧或活性化合物的化学氧化作用影响。
-r发新素能够中和因用f-MLP(N-甲酰基-甲硫氨酰基-亮氨酰-苯丙氨酸)刺激嗜中性粒细胞而释放的人NE。r发新素还能够离体中和弹性蛋白酶活性,即,在患者齿龈液体中。并且,r发新素似乎在该齿龈液体中稳定长达72小时。
实施例2
采用纯化的发新素的体外细胞毒性测试
L-929小鼠成纤维细胞(BioWhittaker,#3C0840)以冷冻储存培养物贮存在液氮中。对于实验而言,使其在补充有热失活的小牛血清(10%v/v)、非必需氨基酸(1%v/v)、L-谷氨酰胺(2mM)和庆大霉素(50μg/ml)的达氏改良伊氏培养基(DMEM)中生长。细胞在37℃湿润孵育箱中常规培养。
通过胰蛋白酶处理收获接近融和的L-929细胞培养物,并使其重悬于培养基中。采用Bürker-Türk计数腔室来对细胞计数。
将四份重组生成的蛋白质的测试样品(两种r发新素形式,均以两种不同方式纯化)冻干,并在使用前溶解于500μl培养基,获得约1.59–3.64mg/ml的浓度,灭菌并进行连续稀释。
细胞毒性测定采用MTT试验(Mosmann,T.,J.Immunol.Meth.65:55-63,1983)进行。该试验通过评估细胞的将MTT降解成其对应的MTT-甲产物的代谢能力来测定细胞活力。简言之,使细胞与含有0.5mgMTT/ml的100μl培养基孵育1小时。在孵育之后,小心地移除MTT培养基,并采用1mlDMSO提取MTT-甲产物至少1小时。采用伯乐(Biorad)多孔板酶标仪检测540nm波长和655nm的参照波长处的吸光度。采用不含r发新素的培养基作为阴性对照,并采用0.1%SDS溶液作为阳性对照。
四份测试样品和对照的结果显示,重组发新素样品不诱导细胞毒性作用(仅显示发新素的最高浓度,表1)。并且,观察到该L-929细胞没有形貌变化。
表1.重组发新素的细胞毒性作用
实施例3
抽吸发新素对肺功能的作用
跟踪观察抽吸包含发新素的香烟的14人(44-71岁的8位男性,和44-69岁的6位女性)。就该测试而言,包含发新素的香烟通过将重组发新素溶解于水中并将该溶液包装于丙烯基珠中来制备。然后,利用迷你手术刀和钳将丙烯基珠手工插入常规香烟的过滤材料。这导致每根香烟的发新素含量为约0,04μg/香烟(十倍MIC,1200香烟/kg吸烟材料)。
实验对象的吸烟习惯从约10支~约25支香烟/天不等。采用FEV1测试(Donahue,J.F.,COPD2:111-124,2005)在实验开始时和实验开始后4、6和12周测试参与者的肺功能。该FEV1采用哮喘监测器(Asma-1,肺活量描记器,英国白金汉)检测。在该实验开始之前,所有对象具有数年的吸烟史,并且其肺功能似乎已经劣于相当年龄和性别对象的正常水平。如表2所示,当抽吸包含发新素的香烟时,FEV1值一般会随着改善,并且观察到平均16%的增加。
该实验还显示发新素在由香烟烟雾加热之后仍具有活性。
实施例4
发新素突变体
通过在用于产生重组发新素的菌株中进行定点诱变制备具有不同P1位氨基酸的发新素突变体(即,第6个半胱氨酸残基之后的Leu残基)(实施例1,毕赤酵母GS115)。对其进行数项蛋白质分析的测试,以测试对于其它(丝氨酸)蛋白酶的活性。这些突变体对于组织蛋白酶G的作用的示例示于图3。
实施例5
瓜梅素突变体
以与用于发新素的相同的方式制备表达瓜梅素和瓜梅素突变体的酵母菌株。显示瓜梅素Lys突变体强力抑制弹性蛋白酶(图2和图4C)。在图4中,给出了瓜梅素及其突变体对6种不同蛋白酶的抑制作用。
对于这些分析,使PBS/0.2%吐温20中的25μl的突变体样品或其在相同溶剂中的稀释物与25μl的蛋白酶在37℃预先孵育60分钟。然后添加合适的底物(50μl),并且使该混合物在37℃反应1小时(弹性蛋白酶,胰蛋白酶),2小时(组织蛋白酶G,糜蛋白酶,凝血酶)或4小时(纤溶酶)。
孵育之后,在405和540埃处测量吸光度,并将这些值的差绘制于图4A-F。
Claims (22)
1.用于预防肺疾病的发新素,所述肺疾病选自下组:肺气肿、COPD和肺癌。
2.用于通过吸入,尤其是通过吸入烟雾,更具体地,烟草烟雾来治疗或预防肺疾病,尤其是肺气肿的发新素。
3.吸烟制品,例如香烟,包括电子香烟、烟斗烟草、雪茄或卷烟,所述吸烟制品用于治疗或预防肺气肿。
4.如权利要求3所述的吸烟制品,其特征在于,所述制品包含弹性蛋白酶抑制剂,所述弹性蛋白酶抑制剂优选选自下组:发新素、瓜梅素、匹瓜梅素、希如斯塔辛、德拉斯塔辛和在第6个半胱氨酸残基之后包含亮氨酸残基的瓜梅素、匹瓜梅素、希如斯塔辛和德拉斯塔辛的突变体。
5.如权利要求4所述的吸烟制品,其特征在于,所述弹性蛋白酶抑制剂以掺混物形式或以由烟草或大麻表达的蛋白质形式被包含在所述烟草或大麻中。
6.如权利要求4所述的吸烟制品,其特征在于,所述香烟是过滤嘴香烟或电子香烟,并且,所述弹性蛋白酶抑制剂存在于所述过滤嘴中。
7.如权利要求4所述的吸烟制品,其特征在于,所述吸烟制品是香烟或卷烟,并且,所述弹性蛋白酶抑制剂存在于卷烟纸中。
8.如权利要求4-6中任一项所述的吸烟制品,其特征在于,所述弹性蛋白酶抑制剂是重组生成的发新素。
9.转基因烟草或大麻,其包含弹性蛋白酶抑制剂,优选是发新素。
10.香烟,其包含如权利要求9所述的转基因烟草或大麻。
11.预防或缓解肺气肿、COPD或肺癌,或改善肺的状况的方法,所述方法包括抽吸如权利要求3-8中任一项所述的吸烟制品。
12.预防或缓解肺气肿、COPD或肺癌,或改善肺的状况的方法,所述方法包括吸入弹性蛋白酶抑制剂,所述弹性蛋白酶抑制剂优选地选自下组:发新素、瓜梅素、匹瓜梅素、希如斯塔辛、德拉斯塔辛,和在第6个半胱氨酸残基之后包含亮氨酸残基的瓜梅素、匹瓜梅素、希如斯塔辛和德拉斯塔辛的突变体,更优选是发新素。
13.用于改善吸烟者的肺功能的方法,所述方法通过给予弹性蛋白酶抑制剂,优选地通过吸入给予所述抑制剂来进行。
14.如权利要求13所述的方法,其特征在于,所述吸入是吸入烟雾,其中所述烟雾包含所述抑制剂。
15.如权利要求13或14所述的方法,其特征在于,所述吸入是通过电子香烟的吸入,或者,其中所述烟雾由电子香烟产生。
16.如权利要求13~15中任一项所述的方法,其特征在于,所述弹性蛋白酶抑制剂选自下组:发新素、瓜梅素、匹瓜梅素、希如斯塔辛、德拉斯塔辛,和在第6个半胱氨酸残基之后包含亮氨酸残基的瓜梅素、匹瓜梅素、希如斯塔辛和德拉斯塔辛的突变体。
17.瓜梅素的突变体,其在第6个半胱氨酸残基之后包含亮氨酸、精氨酸、异亮氨酸、赖氨酸或缬氨酸残基。
18.匹瓜梅素的突变体,其在第6个半胱氨酸残基之后包含亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸或缬氨酸残基。
19.希如斯塔辛的突变体,其在第6个半胱氨酸残基之后包含亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸或缬氨酸残基。
20.德拉斯塔辛的突变体,其在第6个半胱氨酸残基之后包含亮氨酸、精氨酸、异亮氨酸、甲硫氨酸或缬氨酸残基。
21.发新素的突变体,其在第6个半胱氨酸残基之后包含精氨酸、异亮氨酸、甲硫氨酸、赖氨酸或缬氨酸残基,优选地,其中所述突变体包含精氨酸残基。
22.如权利要求21所述的发新素的突变体,所述发新素的突变体用于治疗,优选地用于治疗或预防胰腺炎、炎症、水肿、光老化或血栓形成。
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US20160345631A1 (en) | 2005-07-19 | 2016-12-01 | James Monsees | Portable devices for generating an inhalable vapor |
US10279934B2 (en) | 2013-03-15 | 2019-05-07 | Juul Labs, Inc. | Fillable vaporizer cartridge and method of filling |
US20160366947A1 (en) | 2013-12-23 | 2016-12-22 | James Monsees | Vaporizer apparatus |
US10159282B2 (en) | 2013-12-23 | 2018-12-25 | Juul Labs, Inc. | Cartridge for use with a vaporizer device |
US10076139B2 (en) | 2013-12-23 | 2018-09-18 | Juul Labs, Inc. | Vaporizer apparatus |
USD842536S1 (en) | 2016-07-28 | 2019-03-05 | Juul Labs, Inc. | Vaporizer cartridge |
PL3498115T3 (pl) | 2013-12-23 | 2021-12-20 | Juul Labs International Inc. | Systemy urządzeń do odparowywania |
USD825102S1 (en) | 2016-07-28 | 2018-08-07 | Juul Labs, Inc. | Vaporizer device with cartridge |
US10058129B2 (en) | 2013-12-23 | 2018-08-28 | Juul Labs, Inc. | Vaporization device systems and methods |
CN112155255A (zh) | 2014-12-05 | 2021-01-01 | 尤尔实验室有限公司 | 校正剂量控制 |
MX2018009703A (es) | 2016-02-11 | 2019-07-08 | Juul Labs Inc | Cartuchos de fijacion segura para dispositivos vaporizadores. |
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US10405582B2 (en) | 2016-03-10 | 2019-09-10 | Pax Labs, Inc. | Vaporization device with lip sensing |
FR3049866B1 (fr) | 2016-04-07 | 2019-09-06 | Nfl Biosciences | Extrait de feuilles de tabac et utilisation pour le traitement de l'addiction au tabac |
USD849996S1 (en) | 2016-06-16 | 2019-05-28 | Pax Labs, Inc. | Vaporizer cartridge |
USD836541S1 (en) | 2016-06-23 | 2018-12-25 | Pax Labs, Inc. | Charging device |
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