CN101479288B - 囊性纤维化的改进的治疗 - Google Patents
囊性纤维化的改进的治疗 Download PDFInfo
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- CN101479288B CN101479288B CN2007800237886A CN200780023788A CN101479288B CN 101479288 B CN101479288 B CN 101479288B CN 2007800237886 A CN2007800237886 A CN 2007800237886A CN 200780023788 A CN200780023788 A CN 200780023788A CN 101479288 B CN101479288 B CN 101479288B
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Abstract
本发明公开了一种用于治疗囊性纤维化的治疗性靶点。发现抑制非溶酶体葡糖神经酰胺酶(GBA2)在来自携带常见的ΔF508-CFTR突变的CF患者的细胞中充分地恢复氯离子流。通过位于膜双层表面顶部的酶催化中心(4),在具有能够***膜双层的基团的脱氧野尻霉素衍生物中发现了特别有效的抑制剂。
Description
技术领域
本发明属于囊性纤维化领域。特别是提供了用于治疗囊性纤维化的治疗性靶点和合适的化合物。
背景技术
囊性纤维化(cystic fibrosis,CF)是一种影响身体各个部分,特别是肺部和消化***的遗传性病变。CF在白种人中是最常见的遗传疾病,大约在每2500名新生儿中有1人患病。大约每五名患有CF的婴儿中有一人在出生时即由于其肠道被异常浓稠的胎粪堵塞而被诊断。这种病变可能需要手术。略多于一半的CF患者在婴儿时期被诊断,因为他们并不以应该具有的速度生长或增加体重。这是由于胰腺不能产生足够的脂肪分解酶而导致食物中的脂肪亚最佳吸收而导致热量摄取下降和生长延迟。未治疗的CF患者持续具有油性排便、腹部疼痛、以及体重增加问题。便秘也是一种常见症状。偶尔出现肠道完全堵塞,导致极度腹痛。
CF的特征也包括影响到肺部。在健康人中,在肺内空气通道表面上存在去除碎屑和细菌的恒定粘液流。在CF中,所述粘液过度粘稠并提供了细菌生长的理想环境。患有CF的患者处于细菌性胸部感染和肺炎的危险之中。如果患者不被尽早并且妥善地治疗,那么这些会非常难以痊愈。症状包括持久咳嗽、过量产生痰液(唾液和粘液)、喘息、以及在正常活动中气短。与CF相关的其他问题也已得到鉴别。
CF是隐性遗传的,受到影响的(CFTR)基因位于7号染色体上。所述基因编码氯离子通道CFTR。在囊性纤维化(CF)中,最常见的突变ΔF508导致CF基因蛋白氯离子通道CFTR的活性和细胞表面表达降低。在英国的白种人人群中大约每22人中有1人在其一对7号染色体中的一条上携带所述ΔF508CF突变(“携带者”)。
目前对于CFTR没有有效的治疗,并且治疗局限于缓解症状。患有CF的患者需要每日进行胸部理疗,所述理疗包括用力按摩以帮助咳出浓稠粘 液。患者还需要用抗生素迅速治疗任何胸部感染。通常的儿童时代的疫苗接种例如MMR(麻疹、腮腺炎和风疹)和DTP(白喉、破伤风和百日咳)对于患有CF的人是重要的,并且他们还应当接种针对流感和肺炎的疫苗以有助于预防胸部感染。对每一顿正餐或点心,大多数患有CF的人都需要服用提供缺失的胰腺酶以提供正常消化的胶囊。其他治疗可以包括每日口服或吸入抗生素以抑制肺部感染、吸入性抗哮喘治疗、糖皮质激素药片、营养维生素补充物特别是A和D、吸入药物(链道酶α)以使得痰液降低粘稠度、减轻便秘或提高酶补充物活性的药物、用于CF相关的糖尿病的胰岛素、用于与CF相关的肝病的药物、氧气以帮助呼吸和帮助克服生殖问题。
最常见的CF突变ΔF508产生在ER中低效折叠的突变体蛋白,显示细胞表面表达降低和氯离子流容量降低。已经考虑了一些对于CF的治疗方法。一种方法是基于基因治疗(导入正确的CFTR cDNA)。或者考虑增强内源突变体CFTR的活性作为治疗途径。通常考虑适度提高ΔF508 CFTR的氯离子传导可以是治疗性的。因此已经研究了药物性提高突变体CFTR蛋白质在ER中的折叠、药物性增加突变体CFTR蛋白质在细胞表面的表达和药物性增强通过突变体CFTR蛋白质的氯离子传导。目前,这些方法中没有任何一种产生出有效的纠正CF患者的基本问题的药物。
纠正CF需要纠正根源缺陷,即纠正上皮细胞顶膜(apical membrane)中氯离子通道CFTR的降低的活性。大部分CF患者携带突变体ΔF508-CFTR蛋白质。因此需要解决的问题特别是提高携带ΔF508-CFTR的患者的上皮细胞中的氯离子流。对于有效的治疗性纠正,在这些人中氯离子传导能力的部分提高都被认为是充分的。
发明概述
已经发现CFTR蛋白质位于上皮细胞的顶膜中富含鞘糖脂的脂筏(lipidraft)中。氯离子通道CFTR的活性受其含有鞘糖脂和神经酰胺的微环境的影响。考虑到这个因素,设想选择性地局部降低神经酰胺可能会对氯离子通道(ΔF508)-CFTR的活性具有主要的有益的刺激作用,并从而形成CF的一个治疗途径。因此本发明的一个目的是鉴别控制CFTR蛋白质的鞘脂微环境的蛋白质/酶,从而提供用于开发CF的治疗的靶点。
本发明人通过发现一种也位于上皮细胞的顶膜的酶实现此目的,所述 酶是非溶酶体葡糖神经酰胺酶。这种酶催化葡糖神经酰胺至神经酰胺的转化,并因此影响脂筏的组成。对相应基因和蛋白质的鉴别显示其与GBA2(葡萄糖苷酶,β(胆酸)2)相同,并因此也发现了GBA2在调节通过CFTR进行的氯离子传导中通过改变其脂筏微环境而具有重要作用。
通过将所述非溶酶体葡糖神经酰胺酶鉴别为靶点,发现CF的一种疗法是(优选地选择性)抑制GBA2。抑制此酶应该提高氯离子流并在患有CF的患者,特别是具有ΔF508-CFTR的患者中显示治疗效果。
因此本发明涉及用于治疗囊性纤维化的方法,所述方法包括给予需要的对象治疗有效量的GBA2抑制剂的步骤。或者换句话说本发明涉及GBA2抑制剂在制备用于治疗囊性纤维化的药物中的用途。
近来报道了Miglustat(N-丁基-1-脱氧野尻霉素)在CF上的有益的作用(Norez et al.,FEBS Letters 580(2006)2081-2086)。但是在这篇文献中设想了一种不同的作用机制,即抑制内质网α-葡萄糖苷酶。与这篇文献相比,本发明使得可以产生用于CF的化合物,所述化合物是有效得多并且特异性强得多的治疗剂。
发明详述
所述非溶酶体葡糖神经酰胺酶是一种能够有效地将葡糖神经酰胺转化为神经酰胺的酶。为了获得关于此酶的功能的知识,本发明人分析了各种组织和细胞类型中所述非溶酶体葡糖神经酰胺酶的存在。使用了一种方便的检测法:在存在5mM环己烯四醇B-环氧化物的情况下测定4-甲基伞形酮-β-葡萄糖苷的水解。在这些条件下,所述底物被溶酶体葡糖脑苷脂酶(GBA1)的降解被环己烯四醇B-环氧化物的共价连接不可逆地抑制。
本发明人发现所述非溶酶体葡糖神经酰胺酶在上皮细胞中特别丰富,并且位于其顶膜上。所述非溶酶体葡糖神经酰胺酶在来自上皮细胞的分离的顶膜中富集度是大约450倍。因此所述非溶酶体葡糖神经酰胺酶与氯离子通道CFTR位于相似的膜部分。
在分析了培养的上皮细胞的顶膜部分中的蛋白质之后鉴定了所述非溶酶体葡糖神经酰胺酶的分子性质。所述分析通过二维凝胶电泳和蛋白质染色进行。鉴别了一个候选100kDa蛋白质。所述候选蛋白被胰蛋白酶部分消化,并且一段胰蛋白酶水解肽通过MS/MS分析进行确定。所得到的氨基酸序列 与蛋白质数据库的对比提示GBA2基因可能编码所述非溶酶体葡糖神经酰胺酶。
为了证实GBA2真的编码所述非溶酶体葡糖神经酰胺酶,我们在细胞中表达了GBA2 cDNA。细胞裂解物分析发现所述非溶酶体葡糖神经酰胺酶活性基于GBA2 cDNA的表达得到提高。相反,表达GBA2 siRNA导致所述非溶酶体葡糖神经酰胺酶活性降低。预测GBA2基因产物是跨膜的100kDa蛋白质,并且其推测的催化位点位于膜顶端,参见附图。附图中示出了GBA2(具有972个氨基酸(MW 104 kDa)的蛋白质)的下列特征:(1)细胞质结构域(aa 706-927)含有二-亮氨酸基序用于分拣至PM/内体/溶酶体;(2)一个跨膜结构域;(3)富含半胱氨酸的腔内结构域(lumenal domain)(aa 1-690);(4)位于膜的腔内一侧顶端的推测的催化中心。
这个结构推测与所述酶的已知特征完全相符:膜结合型的,且当***膜双层中时其分解鞘糖脂底物。
在发现GBA2是在上皮细胞的顶膜中产生神经酰胺的原因后,本发明人推测GBA2抑制剂应当具有对CF有益的作用。因此一般说来β-葡萄糖苷酶抑制剂应当是合适的。在本发明的一个实施方案中,β-葡萄糖苷酶抑制剂被用于治疗CF。在一个实施方案中,本发明的抑制剂包含亚氨基糖(iminosugar)部分。所述亚氨基糖部分定义为单糖的结构类似物,其中一个氮原子取代了通常存在于单糖中的环上的氧原子。在一个优选的实施方案中,所述亚氨基糖包含一个5元或6元环,其中存在至少一个氮原子。优选地其他4或5个环原子是碳原子。在一个实施方案中,所述抑制剂包含具有一个6元环的单糖,其中存在一个氮原子。所述亚氨基糖部分可以具有任何单糖构型。糖类化学家已知这种构型。亚氨基糖还包括比通常的单糖缺少一或多个OH基团的化合物,例如脱氧或双脱氧等等。
包括(例如偶联至β-葡萄糖苷酶抑制剂,特别是亚氨基糖)一个使得能够***膜双层中的基团(或者换句话说能够***的基团)是有利的。这种基团也可称为亲脂基团。在亚氨基糖的情况中,这种基团可以直接或通过单糖环原子上的羟基取代基的氧原子方便地偶联至单糖环上的任何原子。在一个优选的实施方案中,能够***膜双层的基团被偶联至单糖环上的氮原子。为了能够***膜双层,优选偶联至所述亚氨基糖的基团足够疏水,或者换句话说亲脂,以与双层脂环境积极地相互作用。如果一个基团含有至少6个碳原子, 它就是足够疏水的,或亲脂的。优选地所述基团含有少于40个碳原子。因此在一个实施方案中,一个亚氨基糖用含有6-40个碳原子的基团取代。这种基团可以认为是烃链,所述烃链可以是直链或支链的、可以在中间含有一或多个杂原子例如O、S和N,并且所述烃链可以含有一或多个不饱和键,例如碳-碳双键或三键。例如已知油基链(具有一个双键(优选顺式)的C18)与脂双层很好地相互作用。不计算这种烃链上的分枝或含有碳原子的取代基,相应于少于30个碳原子或甚至24个或更少碳原子的烃链的长度通常足以有益于与膜双层相互作用。
或者包含6-40个碳原子的基团也可以被认为是包含疏水部分的基团。疏水部分的一种合适的描述是能够***膜双层的含有碳原子的环状基团。适当地,所述环状基团包含两个含有碳原子的环,或优选地甚至三个含有碳原子的环或更多。有利地,在这种多环结构中,至少两个环,优选地至少三个环,或更优选地所有环,与另一个环共享两个或更多个的碳原子。这种基团的合适的实例是胆固醇、β-胆甾烷醇、菲、金刚烷等等。将这种基团通过羟基官能团偶联至烃链(所述烃链接下来偶联至亚氨基糖,优选地偶联至亚氨基糖的环氮)是有利的。所述将疏水部分偶联至亚氨基糖的烃链包含优选地至少2个,优选地至少3个碳原子。如果在抑制剂中包含一个大的疏水部分,所述将疏水部分偶联至亚氨基糖的烃链包含优选地不多于12个,优选地不多于10个,或9个或甚至不多于8个碳原子。所述烃链可包含至少一个杂原子,例如O原子,通过所述杂原子,所述疏水部分可以偶联至所述烃链。
特别地,已知非溶酶体葡糖神经酰胺酶的有效抑制剂含有脱氧野尻霉素部分。包括(例如偶联至脱氧野尻霉素部分)一个使得能够***膜双层中的基团是有利的。一种已知的GBA2的有效抑制剂是公开于Overkleeft et al.,JBiol Chem 1998,vol.273,no.41,pp 26522-26527中的N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-1-脱氧野尻霉素(N-[5’-(adamantan-1’-yl-methoxy)-pentan]-1-deoxynojirimycin,AMP-DNM)。这个结构对于GBA2的IC50是大约1nM。
已发现通过将含有ΔF508-CFTR的上皮CF细胞与AMP-DNM一起温育可以将氯离子流恢复至被认为具有治疗作用的水平。氯离子传导能力的部分恢复就足以对氯离子传导产生主要纠正,这也被携带ΔF508-CFTR的小鼠中的提高所证实。
因此需要抑制GBA2以对氯离子通道ΔF508-CFTR产生有益作用。 AMP-DNM是一种具有治疗潜力的GBA2有效抑制剂。这种类型的化合物可以通过提高选择性而得到改进。疏水脱氧野尻霉素也已知是葡糖神经酰胺合酶的抑制剂。在一个实施方案中,其在尽可能地预防葡糖神经酰胺合酶(glucosylceramide synthase,GCS)的共抑制中被认为是特别有用的。可通过改变糖构型和改变烃链和/或改变疏水部分和改变疏水基团与糖部分的连接途径或连接方式,特别是改变偶联疏水部分和亚氨基糖部分的烃链产生更有选择性的化合物。
在一个实施方案中,本发明的一种抑制剂包含亚氨基糖,所述亚氨基糖是脱氧野尻霉素或其衍生物。衍生物包括具有不同单糖构型和/或在单糖OH基团上具有不同取代基的脱氧野尻霉素。在一个实施方案中,所述脱氧野尻霉素处于吡喃葡萄糖构型,在另一个实施方案中,所述脱氧野尻霉素处于吡喃艾杜糖(idopyranose)构型。合适地,所述抑制剂是N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-1-脱氧野尻霉素(AMP-DNM)或N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-L-艾杜-1-脱氧野尻霉素(艾杜-AMP-DNM(ido-AMP-DNM))。
在一个实施方案中,所述能够***膜双层的含有6-40个碳原子的基团包含一个大体积的烃基基团,例如叔丁基或新戊基。合适地,所述新戊基通过偶联新戊醇得到。有利地,所述叔丁基或新戊基位于所述能够***膜双层的具有6-40个碳原子的基团(特别是烃链)的末端位置。本发明的一种合适的抑制剂是N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素或N-[5’-新戊基氧基-戊基]-L-艾杜-1-脱氧野尻霉素。其他合适的抑制剂是与N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素相似的化合物,但是其中所述戊烷烃链含有更少或更多的碳原子,特别是CH2基团,例如N-[5’-新戊基氧基-丁基]-、-己基]-、-庚基]-、-辛基]-、-壬基]-、-癸基]-1-脱氧野尻霉素等等,或L-艾杜化合物。
在一个优选的实施方案中,本发明的抑制剂对于GBA2的IC50小于250nM,优选小于150nM。在另一个实施方案中,优选所述抑制剂对于GCS的IC50大于1mM,优选大于100μM。
本发明还涉及N-[5’-新戊基氧基-烃基]-1-脱氧野尻霉素或N-[5’-新戊基氧基-烃基]-L-艾杜-1-脱氧野尻霉素,其中烃基是指具有直链4-12个碳原子,优选4-11、4-10、4-9、4-8、4-7个碳原子,优选具有直链4-12个CH2基团,优选4-11、4-10、4-9、4-8、4-7个CH2基团的亚烷基。本发明还涉及这些化合物作为药物组合物和含有这种化合物以及药物学可接受的载体的药物组合物的用途。
重要的是,本发明的抑制剂的正面作用不能归因于通过抑制ER α-葡萄糖苷酶或抑制鞘糖脂合成而改善的ΔF508-CFTR的ER折叠。本发明因此示出了全新的知识,即GBA2特异性抑制剂是用于治疗囊性纤维化的高度特异性治疗剂。
实施例
AMP-DNM在含有ΔF508-CFTR的上皮CF细胞中对cAMP激活的氯离子流的作用用如下检测进行研究:
对CF15细胞(得自ΔF508纯合患者的人鼻腔上皮细胞系)进行穿孔全细胞膜片钳(perforated whole-cell patch-clamp)分析。将充满胞内溶液(电阻3-4MΩ)的膜片电极(GC150-TF10,Harvard Apparatus,USA)通过Ag/AgCl片连接至RK-400放大器(Biologic,France)。外液(mM):145NaCl,4CsCl,1MgCl2,1CaCl2,5d-葡萄糖,10TES(pH 7.4,315mOsm)。吸管内液(mM):1131-天冬氨酸,113CsOH,27CsCl,1NaCl,1EGTA,1MgCl2,3Mg-ATP(ex-temporane),10 TES(pH 7.2,含有CsOH,285mOsm)和两性霉素B(100μg/ml),每2h更新一次。仅分析输入电阻为15MΩ的细胞。平均接入电阻和全细胞电容是12±0.6MΩ和35±4.3pF(n=44)。电流通过响应电压梯度(-80至+80mV,20mV递增)获得。用pClamp 6.0.3整套软件(AxonInstruments,USA)收集数据。在细胞群上通过碘(125I)外流技术检测CFTR Cl-通道活性。
发现将上皮细胞与10nM AMP-DNM一起温育可以在含有ΔF508-CFTR的上皮CF细胞中将cAMP激活的氯离子流恢复高至正常水平的30%。这种程度的纠正被认为足以产生治疗作用。
为了测试GBA2抑制剂对CF的治疗潜力,进一步分析了AMP-DNM对得自ΔF508小鼠回肠粘膜的肠外植块(explant)的作用。使用如下检测:
使用Rotterdam ΔF508/ΔF508-CFTR小鼠(Cftrtm1 Eur)、其同窝对照(FVB自交,14-17周龄,体重在20到30g之间,在无病原体环境中用固体食物喂养)和Cftr-KO小鼠(Cftrtm2 Cam)。将剔除肌肉的肠粘膜在补充了胰岛素(10μg/ml)和***(20μg/ml)的William’s E-Glutamax培养基中培养。在不同时间点,通过反复漂洗除去化合物,之后在mini-Ussing腔中进行短路 电流(Isc)测定。用单克隆小鼠抗CFTR抗体(10μg/ml,IgG1 M3A7,Chemicon,USA)进行Western印迹和免疫印迹。用密度测定蛋白质水平并以对照组的百分比表示。对于免疫组化,将组织在4%(wt/vol)多聚甲醛中固定。用抗体R3195(1∶500)对切片(5μm)进行染色。
观察到10nM AMP-DNM在得自ΔF508小鼠回肠粘膜的肠隐窝中挽救成熟的功能性ΔF508-CFTR。
注意到在化合物对GBA2的抑制能力(通过在存在过量环己烯四醇B-环氧化物的情况下分析含有GBA2的膜对4-甲基伞形酮-β-葡萄糖苷的水解进行测定)和对氯离子流的正面作用之间存在紧密联系。
为了进一步测试结构要求,检测了具有不同间隔区(spacer)和疏水基团的化合物(参见表1)。同样注意到,GBA2的抑制能力很好地预测了氯离子流的纠正。
表1:各种能够***膜双层的基团
测试的亚氨基糖的结构(针对改善氯离子流的能力)及其在特异性检测中确定的对于GBA2和GCS的IC50值。
GBA2 GCS
N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-1-脱氧野尻霉素IC5O 1nM 0.2μM
N-壬基-1-脱氧野尻霉素 IC50 6nM 8μM
N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素 IC50 30nM >100μM
N-[6’-金刚烷基-己基]-1-脱氧野尻霉素 IC50 1nM 7.5μM
N-[5’-(金刚烷-1’-基-甲氧基)-丁基]-1-脱氧野尻霉素IC50 2nM 10μM
对比实例
N-丁基-1-脱氧野尻霉素 IC50 300nM 80μM
为了提高与抑制GCS相比抑制GBA2的特异性,发现丁酰基-间隔区或己基间隔区是有利的。所述疏水基团也可以是各种各样的。一个实例是N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素。尽管其对于GBA2的IC50不如AMP-DNM,但是这是一个相对更加特异性的抑制剂,因为其几乎不抑制GCS。
IC50值的确定
通过将酶制备物暴露于各个稀释度的抑制剂并确定酶活性降低50%时的浓度确定非溶酶体葡糖神经酰胺酶和葡糖神经酰胺合酶(GCS)的IC50值。如在J.Biol.Chem,273,26522-27(1998)第26523页在酶测定(enzyme assays)标题下所描述的进行测定。
脱氧野尻霉素的合成方案
各种合适的基于脱氧野尻霉素的抑制剂的合成在其他文献中已被很好地描述,例如J.Biol.Chem,1998,vol 273,pp26522-27和US 6,177,447。下文公开了一个用于合成N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素的方案。全部步骤对于有机化学家而言都是标准步骤,可以常规确定适当的反应条件。
Claims (4)
1.GBA2抑制剂在制备用于治疗囊性纤维化的药物中的用途,其中所述抑制剂选自N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-1-脱氧野尻霉素、N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-L-艾杜-1-脱氧野尻霉素、N-[5’-新戊基氧基-戊基]-1-脱氧野尻霉素和N-[5’-新戊基氧基-戊基]-L-艾杜-1-脱氧野尻霉素。
2.权利要求1的用途,其中所述抑制剂是N-[5’-(金刚烷-1’-基-甲氧基)-戊基]-1-脱氧野尻霉素
3.N-[5’-新戊基氧基-烃基]-1-脱氧野尻霉素或N-[5’-新戊基氧基-烃基]-L-艾杜-1-脱氧野尻霉素,其中烃基是指具有4-12个碳原子的亚烷基。
4.权利要求3的化合物作为药物的用途。
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