CN111491631B - 活性剂 - Google Patents
活性剂 Download PDFInfo
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- CN111491631B CN111491631B CN201880070008.1A CN201880070008A CN111491631B CN 111491631 B CN111491631 B CN 111491631B CN 201880070008 A CN201880070008 A CN 201880070008A CN 111491631 B CN111491631 B CN 111491631B
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- cells
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Abstract
本发明涉及糖酵解活化剂。本发明还涉及治疗或预防疾病或医学病症、特别是免疫相关疾病或医学病症。特别的是,本发明还涉及用于治疗或预防疾病或医学病症的活性剂,该治疗或预防通过内源性调节性T细胞(Treg)的运输来介导。
Description
发明领域
本发明通常涉及医学领域。此外,多个实施方案涉及药物。特别的是,本发明涉及可用于在患有疾病或医学病症、特别是免疫相关疾病和医学病症,或处于患有疾病或医学病症、特别是免疫相关疾病和医学病症风险中的个体中运输内源性调节性T细胞(Treg)的活性剂和组合物。
背景
定义为CD4+CD25+Foxp3+ T细胞的胸腺调节性T细胞(Treg)有助于维持对自身抗原的耐受性。Treg通过定位于淋巴样和非淋巴样组织发挥其免疫调节作用。
Treg细胞向发炎组织的迁移是其免疫调节功能的关键。相对于T效应器(Teff)细胞Treg的快速积累(其中浸润淋巴细胞中超过30%为Treg)在免疫调节过程中在发炎组织中发生(Tang等人,2012)。相反,外周血中Treg的比例很小(2-5%)进一步强调了Treg的显著迁移能力。
Teff和Treg的功能、扩增和存活的生物能学已被广泛研究。Teff和Treg细胞需要不同的代谢程序来支持其生存和分化:T辅助细胞(Th)1、Th2和Th17细胞表达高表面量的葡萄糖转运蛋白Glut1并且具有高度的糖酵解作用,而Treg细胞表达少量Glut1并且具有高体外脂质氧化率(Michalek等人,2011)。雷帕霉素的哺乳动物靶标(mTOR)通过调节T细胞代谢响应在细胞命运决定中起关键作用。T细胞活化刺激mTOR增加糖酵解并且减少脂质氧化(Wieman等人,2007)。相反,糖酵解的阻断促进Treg细胞的生成,并且这通过抑制mTOR介导的转录因子低氧诱导因子(HIF1α)的诱导而发生(Dang等人,2011;Shi等人,2011)。然而,尽管Treg主要依赖于氧化代谢,但某些特定功能可能取决于选择性转换为糖酵解的过程。响应环境线索,Treg代谢显然从mTOR依赖性和非依赖性途径中摆动(Procaccini等人,2010)。已显示Toll样受体(TLR)信号通过mTORC1信号传导、糖酵解和Glut1上调来促进Treg细胞增殖。相反,TLR诱导的mTORC1信号也降低了Treg抑制能力(Gerriets等人,2016)。
尽管运动可能是最消耗能量的细胞活动(Bernstein和Bamburg,2003年),但对T细胞迁移的代谢需求仅进行了部分研究。在先我们已经证明,Teff迁移依赖于糖酵解途径(Haas等人,2015)。然而,促进Treg迁移的代谢程序仍然未知。
由整联蛋白例如LFA-1介导的分子相互作用是T细胞运输的典型调节器。另外,分别由共刺激和共抑制受体CD28和CTLA-4产生的信号积极参与T细胞运输的调节。在淋巴结中,CD28活化促进记忆T细胞外移并且迁移到靶组织(Jain等人,2013;Mirenda等人,2007),而CTLA-4拮抗CD28的促迁移信号(Mirenda等人,2007)。效应器Treg细胞的迁移也受CD28信号调节(Muller等人,2008)。重要的是,共刺激受体还调节T细胞代谢重编程以增强糖酵解(Frauwirth等人,2002;Parry等人,2005),然而,尚未启示糖酵解与Treg细胞迁移之间的相关性。
糖酵解的过程部分地由葡萄糖激酶(GCK或GLK)控制。葡萄糖的摄取速率受到葡萄糖磷酸化为葡糖-6-磷酸(G6P)的速率限制,该过程由GCK催化。
WO2008050101描述了一组作为GCK活化剂的苯甲酰基氨基杂环基化合物,其具有有利的物理和/或药代动力学特性和/或有利的毒性特性。
WO2008050117描述了作为GCK活化剂的苯甲酰基氨基杂环基化合物,其具有更高的水溶性、更高的渗透性和/或更低的血浆蛋白结合。
WO2008075073描述了作为GCK活化剂并且可用于降低胰岛素分泌的葡萄糖阈值的晶型。
在WO0058293和WO0144216中,描述了作为葡萄糖激酶活化剂的一系列苄基氨基甲酰基化合物。通过在GCK活性与NADH产生相关联的测定中测定此类化合物的直接作用来评价此类化合物活化GCK的机制,该测定进而通过光学方法进行。
在WO03095438(取代的苯基乙酰胺)、WO03055482(羧酰胺和磺酰胺衍生物)、WO2004002481(芳基羰基衍生物)和WO03080585(氨基取代的苯甲酰基氨基杂环)中描述了其它GCK活化剂。
WO03000267描述了一组苯甲酰基氨基吡啶基羧酸,其为GCK活化剂。WO03015774描述了式(A)化合物:
其中R3为苯基或取代的杂环,但除外羧酸取代的吡啶基。
WO2004076420描述了通常为WO03015774中描述的那些的亚组的化合物,其中例如R1为(取代的)烷基醚,并且R2为(取代的)苯氧基。
Grewal等人,2014列出了用于治疗2型糖尿病的众多GCK活化剂(GKA)。预先已经注意到,在1型糖尿病与葡萄糖激酶基因之间缺乏关联(Bain等人,1992)。
已经发现了在靶点上的Treg,或已证明它们在多种疾病状态下迁移到靶点。Treg在解决多种疾病状态中也起着至关重要的作用。这些疾病状态包括多发性硬化(Denning等人,2005)、心肌炎(Stephenson等人,2016)、心肌梗死(MI)(Weirather等人,2014)、I型糖尿病(Chen等人,2005)、类风湿性关节炎(RA)(Cooles等人,2013)、系统性红斑狼疮(SLE)(Humrich&Riemekasten,2016)、银屑病(Deng等人,2016)、移植物抗宿主病(GVHD)(Zorn等人,2005)、结肠炎(Ding等人,2012)和变态反应性疾病(Denning等人,2005)。
附图描述
图1.糖酵解促进了Treg的迁移。将用指定的药物或媒介物预处理4小时的离体扩增的Treg保留,以迁移通过3μm-孔的transwell,该孔与IFN-γ-处理的同种系EC单层(A-B)分层,或响应于趋化因子CCL22通过裸滤器5μm-孔transwell(C-D)。将结果表示为24小时后(A-B,n=4,N=2)或在指定的时间点(C,n=3)迁移细胞的百分比±SD。迁移的增加倍数通过在一式三份进行的相同设计的两次试验中在6小时测定的试验迁移除以自发迁移±SD计算。
图2.活化的Treg表型以及暴露于代谢修饰药物的功能和作用。如前所述(Fu等人,2014)扩增Treg。用指定的代谢靶向药物将A-B Treg处理4-6小时,并且充分洗涤。通过基于transwell的测定评价药物暴露的Treg对CXCL10(300ng/mL)响应的迁移。将结果表示为在指定时间点迁移的细胞的平均百分比±SD。图B中显示了从相同设计的三个独立试验中在6小时获得的并且通过自发迁移标准化的数据±SD。*P<0.05。
图3.将用荧光活体染料PKH26标记的药物处理的Treg或媒介物处理的Treg i.v.注射入在先用IFN-γi.p.48小时处理的同种系受体。24小时后从指定的组织中收获细胞,并且通过流式细胞术进行分析。在图A和C中显示了来自3只动物的代表性点图。在图B和D中显示了在4只动物中回收的标记细胞的平均绝对数±SD。(N=1)
图4.A:在通过流式细胞术分析指定的表面分子的表达之前,用指定的代谢靶向药物处理Treg 4-6小时。显示了指定的表面分子的代表性直方图和平均荧光强度。N=2。B-C:通过分析钙磷脂结合蛋白V和碘化丙锭(PI)染色测定用指定的代谢靶向药物处理6小时的Treg的凋亡/坏死。将未处理的Treg和进行热诱导的凋亡的Treg分别用作阴性和阳性对照。棒状图(C)显示了凋亡和坏死的Treg细胞的平均百分比±SD。(n=4,N=3)。
图5.用指定的代谢靶向药物将Treg处理4-6小时,充分洗涤并且在培养基中单独培养过夜。通过6小时的化学激活transwell试验(106/孔)评价对CCL22(300ng/mL,A-B)CXCL10(300ng/mL,C-D)响应的药物暴露的Treg的迁移。将结果表示为一式三份进行的典型试验中迁移细胞的平均百分比±SD(A,C)。图B和图D中显示了从相同设计的三个独立试验在6小时获得的并且通过自发迁移标准化的数据±SD。*P<0.05。
图6.用2-DG(A)或乙莫克舍(B)处理Treg 4-6小时,充分洗涤并且在培养基中单独培养过夜。然后将Treg在无血清、无缓冲的XF测定培养基(Seahorse biosciences)中静置1小时,然后接种(6×105/孔)入Seahorse XF24细胞板中进行通量分析。首先向孔中注入葡萄糖。在指定的时间点(箭头)进一步注射指定的物质。ECAR=细胞外酸化率,其是糖酵解的指示。
图7.A:用塑料结合的重组(r)ICAM-1或人IgG Fc片段(Fc)刺激45分钟,然后再悬浮于包含葡萄糖摄取指示剂6-NBDG(荧光标记的葡萄糖类似物,其不会被代谢,因此如果吸收则会使细胞发荧光)的培养基10分钟的Treg的代表性的直方图。图B中显示了来自3个独立试验的平均MFI±SD。(n=3)。通过胞外通量分析仪(Seahorse)测定ICAM-1-刺激的(C)或CCL22刺激的(D)Treg的ECAR。使用IgFc或培养基作为对照,在指定的时间点添加重组分子和葡萄糖(±SD n=5,N=2),*p<0.05,**p<0.005。
图8.首先,将体外扩增的Treg在无血清、无缓冲的XF测定培养基(Seahorsebiosciences)中静置1小时,然后接种(6×105/孔)入Seahorse XF24细胞板中,进行通量分析。首先向孔注射重组ICAM-1(或人Fc作为对照,图A)或CCL22(或无葡萄糖培养基作为对照,图B)。然后在指定的时间点(箭头)进一步注射指定的物质,*P<0.05;**p<0.01;***p<0.005。
图9.CD28和CTLA4差异调节Treg细胞迁移。通过在37℃连接识别CD28或CTLA-4的激动剂抗体(Schneider等人,2005;Wells等人,2001)刺激Treg 45分钟。A-C:在基于transwell的测定中监测抗体刺激的Treg通过IFN-γ-处理的同种系内皮细胞(EC)单层(A)或响应于CXCL10趋化因子(B-C)的迁移。在图A中,将结果表示为在相同设计的三个独立试验中迁移细胞的平均百分比±SD。在图B中,将结果表示为在一式三份进行的典型试验中迁移的细胞的平均百分比±SD。图C中显示了从相同设计的三个独立试验在6小时获得的并且通过自发迁移标准化的数据±SD。
图10.用CD28和/或CTLA-4抗体刺激Treg,然后过继转移入同种系受体中,该受体已在先用IFN-γi.p.处理48小时以促进局部募集(Fu等人,2014)。16小时后评价Treg向腹膜腔的迁移。局部定位于脾作为对照测定,其中进入以被动方式发生(Fu等人,2016)。假定Treg种群是过继转移的,则在数据分析中使用绝对数量的标记细胞。CD28刺激后,腹膜腔中积累的Treg数量显著更高。伴随的CTLA-4刺激抑制CD28诱导的迁移,而CTLA-4自身触发没有任何作用。上图中显示了来自3只动物的代表性的点图。下图表示在4只动物中回收的标记细胞的平均绝对数±SD。(N=3)。
图11.通过经诱导糖酵解共刺激受体调节Treg的迁移。A-B:在分析之前,与6-NBDG温育10分钟的抗体刺激的Treg的代表性直方图。非荧光葡萄糖类似物2-DG用作阴性对照。图B显示了3个独立试验的平均MFI±SD。
图12.抗体刺激的Treg的ECAR通过流量计测定。在指定的时间点注射抗体(Ab)和D-葡萄糖±SD(A)。在未刺激的WT或CTLA-4KO Treg中测定ECAR(±SD)(B)。如箭头所示注射D-葡萄糖。
图13.在WT(A)或CTLA-4KO(B)Treg中测定ECAR(±SD),所述WT(A)或CTLA-4KO(B)Treg预先已用重组CD80或Fc片段刺激30分钟。如箭头所示注射D-葡萄糖。
图14.A:rCD80刺激的或Fc刺激的CTLA-4KO和WT Treg通过同种系IFN-γ-处理的EC单层的迁移。将结果表示为24小时迁移的细胞的平均百分比±SD。n=3,N=4。B:通过IFN-γ-处理的同种系EC单层重悬浮于无葡萄糖或葡萄糖重构培养基中的抗体刺激的Treg的迁移,将其表示为24小时后迁移的细胞的百分比±SD。(n=3,N=4)。
图15.在先IFN-γi.p.给予48小时的C57BL/6小鼠中i.p.注射PKH26标记的抗体刺激的Treg(第二栏)。此后即刻i.p.注射6-NBDG(第三栏)。1小时后取出腹膜,用DAPI复染并且通过宽视野荧光显微镜检查成像以确定浸润细胞的数目和浸润细胞对6-NBDG的摄取。代表性的图像显示在图A中。图B和图C中分别显示了来自4个受体的10个10×视野中计数的平均细胞数±SD和来自3个10×视野的10-12个细胞中6-NBDG的平均总细胞荧光±SD。(n=4,N=2),*p<0.05;**p<0.01;***p<0.005。
图16.促迁移刺激诱导Treg的代谢重编程。A-B:通过蛋白质印迹刺激抗体后4小时,测定Treg中指定酶的表达。在图B中,显示了在3个独立试验中通过密度计分析测定的平均相对表达±SD。
图17.A:通过在指定的时间点经蛋白质印迹测定的CD28刺激的或LFA-1刺激的Treg对指定的酶的表达。B-C:通过RT-PCR测定通过抗体刺激的Treg对GCK(B)和GCKR(C)的相对mRNA表达水平。GCKR=葡萄糖激酶调节蛋白;其结合GCK并且阻断酶活性。
图18.A-B:通过在指定的时间点经共聚焦显微镜检查测定的CD28刺激的或LFA-1刺激的Treg对指定酶的表达。通过共聚焦显微镜检查测定通过抗体刺激的Treg的细胞蛋白质表达。在图B中,显示了使用Image J软件测定的平均MFI±SEM。N=3。比例尺20μm。
图19.使用流量计进行用抗-CD3加抗-CD28抗体刺激24小时的CD4+T细胞的ECAR和OCR的实时分析。首先向孔中注射入CLT或媒介物。在指定的时间点(虚线)进行第二次注射,从而将D-葡萄糖导入孔中。随后,在指定的时间点依次注射入糖酵解调节剂(A)或细胞呼吸调节剂(B)。(N=2),**p<0.01,***p<0.005。OCR=耗氧率:线粒体呼吸和脂肪酸氧化(FAO)的间接测量值。
图20.A:将AZD1656(GCK活化剂,1μM)处理的Treg和媒介物处理的Treg(在无胰岛素的培养基中2小时)用不同的活体荧光染料标记,并且共同注射入在先接受IFN-γi.p.48小时的同种系受体中。24小时后从腹膜或脾中回收细胞,并且通过流式细胞术分析。显示了代表性的点图。棒状图表示回收的标记细胞的平均绝对数±SD(n=4,N=2)。B-C:通过流式细胞术测定用AZD1656(B)或克霉唑(CLT,1μM,2小时,C)或单独的媒介物处理后,通过用同种异体DC刺激的Treg对PCNA的表达。NS,未刺激的对照Treg。显示了代表性的直方图。(n=3,N=3)。
图21.A:用重组ICAM-1或Fc对照活化的Treg细胞的ECAR。按指示添加CLT或媒介物以及其它影响糖酵解的药物。B:CLT或媒介物处理的Treg进行CD28或同种型匹配的抗体刺激,用不同的活体荧光染料标记并且i.v.注射入在先接受IFN-γi.p.48小时的同种系受体中。24小时后从腹膜(P)或脾(S)中回收细胞,并且通过流式细胞术分析。显示了代表性的点图。柱图表示回收的标记细胞的平均绝对数±SD(n=3,N=2)。*p<0.05;***p<0.005;****p<0.001。
图22.GCK的药理学活化显著延迟了皮肤同种异体移植排斥。C57B16小鼠接受B6Kd皮肤移植或同种系皮肤作为对照。移植后,一些接受者用GCK活化剂AZD1656(每日20mg/kg)治疗2周。N=9
图23.带有功能丧失的GCKR等位基因的Treg细胞显示出增强的运动性。A:与携带WT等位基因(P446)的个体相比,等位基因P446L携带者中的Treg(CD4+CD25highCD127low)细胞数/mL。代表性的点图显示在图B中。n=25。图显示了两个研究群中定义为(CD45RA+)、中枢记忆(CD45RO+、CD62L+、CCR7+)和效应器记忆(CD45RO+、CD62L-、CCR7-)的CD4+淋巴细胞亚群。
图24.通过transwell测定来自8个P446L-GCKR携带者或WT-GCKR个体的Treg(A)和Tconv(B)对趋化因子CCL19/21的迁移响应。
图25.在图A-B中,显示了来自P446L-GCKR携带者(n=8)和WT-GCKR(n=8)个体的表达图24中迁移试验中使用的趋化因子CCL19/21受体CCR7的T细胞百分比,*p<0.05。
发明概述
活化的调节性T-细胞(Treg)向发炎组织的迁移对其免疫调节功能至关重要。尽管对Treg分化过程中的代谢重编程进行了广泛研究,但Treg转运的生物能学仍然无法确定。本发明人已经研究了在体外和体内迁移Treg的代谢需求。
通过比较LFA-1介导的促迁移信号和CD28介导的促迁移信号作为工作模型,本发明人研究了体外和体内迁移Treg细胞的生物能学。因此,本发明人在本文中定义了在小鼠和人中在Treg迁移期间参与的代谢重编程新的特异性途径。
本发明人已经发现,糖酵解有助于Treg迁移,并且是由终止葡萄糖激酶(GCK)诱导的促迁移刺激引发。随后,GCK通过与肌动蛋白结合促进细胞骨架重排。缺乏此途径的Treg具有功能抑制性,但不能迁移至皮肤同种异体移植物并且抑制排斥。类似地,在GCK调节蛋白基因中功能缺失突变的人携带者-导致GCK活性增加-已经减少循环活化的Treg数量。这些Treg展示出增强的迁移活性,但具有相似的抑制功能,而传统的T细胞不受影响。因此,GCK依赖性糖酵解调节Treg迁移。因此,本发明涉及Treg中糖酵解的活化,以治疗或预防疾病和医学病症,特别是免疫介导的那些疾病和医学病症。
因此,在一个方面,本发明提供了用于治疗或预防疾病或医学病症的糖酵解活化剂,该治疗或预防通过运输内源性调节性T细胞(Treg)介导。
在另一个方面,本发明提供了用于个体的运输内源性调节性T细胞(Treg)的糖酵解活化剂,所述个体患有疾病或医学病症或处于患有疾病或医学病症的风险中。
在另一个方面,本发明提供了糖酵解活化剂,其用于治疗或预防疾病或医学病症,所述疾病或医学病症通过将内源性调节性T细胞(Treg)运输至组织或器官而缓解。
在另一个方面,本发明提供了糖酵解活化剂,其用于调节个体的免疫应答。
在另一个方面,本发明提供了药物组合物,其包含用于如本文所述用途的糖酵解活化剂和药学上可接受的载体、媒介物、稀释剂或赋形剂。
在另一个方面,本发明提供了治疗或预防个体的免疫介导的疾病或病症的方法,该方法包括给个体施用糖酵解活化剂。
在另一个方面,本发明提供了调节个体的免疫应答的方法,该方法包括给个体施用糖酵解活化剂。
详细描述
如本文所用,单数形式“一个”、“一种”和“该”包括单数和复数指代物,上下文另外明确指出的除外。
如本文所用,术语“包含”与“包括”同义,并且是包括性的或开放式的,并且不排除其它的、未列举的成员、要素或方法步骤。术语“包含”、“包括”也包括术语“由…组成”。
糖酵解活化剂
在本发明的上下文中,“糖酵解活化剂”是能够活化、刺激、诱导、催化或增加细胞中糖酵解的生化过程速率的任何活性剂或化合物。所述活性剂或化合物本质上可以是例如生物或化学的,例如小分子或抗体。所述活性剂或化合物可以例如直接或间接靶向糖酵解的过程,或者它可以直接作用于参与糖酵解途径的酶的一种或多种。
一种参与糖酵解过程的这样的酶为葡萄糖激酶(GCK、GLK或GK)。因此,在一个实施方案中,本发明的糖酵解活化剂为GCK活化剂(也称为葡萄糖激酶活化剂或GKA)。在一个实施方案中,GKA防止或抑制GCK与GCK调节蛋白(GCKR)之间的相互作用。
用于确定酶例如GCK的活性的方法和测定法是本领域技术人员众所周知的。例如,在WO2008050101中描述了用于测定GCK活性的特定测定法,其中通过将GCK与ATP和葡萄糖一起温育来测定重组GCK的酶活性。产物形成的速率可以通过将测定与G6P脱氢酶、NADP/NADPH系统偶联并且测定340nm处光密度随时间的线性增加来测定。
许多GCK活化剂是本领域已知的,并且在本文引用的参考文献中描述。
在本发明的一个实施方案中,GCK-活化剂为式(I)化合物或其药学上可接受的盐:
其中:
R1选自异丙基、丁-2-基、环戊基、1,1,1-三氟丙-2-基、1,3-二氟丙-2-基、丁-1-炔-3-基、1-羟基丙-2-基、2-羟基丁-3-基、1-羟基丁-2-基、四氢呋喃基、四氢吡喃基、1-甲氧基丙-2-基、1-甲氧基丁-2-基、2-羟基丙-1-基、2-甲氧基丙-1-基、2-羟基丁-1-基、2-甲氧基丁-1-基、1-氟甲氧基丙-2-基、1,1-二氟甲氧基丙-2-基和1-三氟甲氧基丙-2-基;
HET-1是5元或6元的C连接的杂芳基环,其含有在2-位的氮原子和任选1或2个独立地选自O、N和S的另外的环杂原子;该环任选在任何氮原子上(如果不由此被季铵化)被选自R7的取代基取代和/或在一个或两个可利用的碳原子上被独立地选自R6的取代基取代;
R2选自-C(O)NR4R5和-SO2NR4R5;
R3选自甲基、三氟甲基和卤素;
R4和R5与它们所连接的氮原子一起形成4-7元饱和或部分不饱和的杂环基环,其任选含有1或2个独立地选自O、N和S的另外的杂原子(除连接的N原子之外),其中-CH2-基团可以任选被-C(O)-代替,并且,其中环中的硫原子可以任选被氧化成S(O)或S(O)2基团;该环任选在可利用的碳原子上被1或2个独立地选自R8的取代基取代和/或在可利用的氮原子上被选自R9的取代基取代;或者R4和R5与它们所连接的氮原子一起形成6-10元双环饱和或部分不饱和的杂环基环,其任选含有1个另外的氮原子(除了连接的N原子之外),其中-CH2-基团可以任选被-C(O)-代替;该环任选在可利用的碳上被选自羟基和R3的1个取代基取代或在可利用的氮原子上被甲基取代;
R6独立地选自(1-4C)烷基、卤素、羟基(1-4C)烷基、(1-4C)烷氧基(1-4C)烷基、(1-4C)烷基S(O)p(1-4C)烷基、氨基(1-4C)烷基、(1-4C)烷基氨基(1-4C)烷基和二(1-4C)烷基氨基(1-4C)烷基;
R7独立地选自(1-4C)烷基、卤代(1-4C)烷基、二卤代(1-4C)烷基、三卤代(1-4C)烷基、羟基(1-4C)烷基、(1-4C)烷氧基(1-4C)烷基、(1-4C)烷基S(O)p(1-4C)烷基、氨基(1-4C)烷基、(1-4C)烷基氨基(1-4C)烷基和二(1-4C)烷基氨基(1-4C)烷基;
R8选自羟基、(1-4C)烷氧基、(1-4C)烷基、氨基羰基、(1-4C)烷基氨基羰基、二(1-4C)烷基氨基羰基、(1-4C)烷基氨基、二(1-4C)烷基氨基、(1-4C)烷氧基(1-4C)烷基、羟基(1-4C)烷基和-S(O)p(1-4C)烷基;
R9选自(1-4C)烷基、-C(O)(1-4C)烷基、氨基羰基、(1-4C)烷基氨基羰基、二(1-4C)烷基氨基羰基、(1-4C)烷氧基(1-4C)烷基、羟基(1-4C)烷基和-S(O)p(1-4C)烷基;
其中X1、X2和X3各自独立地选自CH、N、S和O;X4不存在(构成5-元环),或选自CH、N、O和S;条件是X1、X2、X3和X4的至少一个为CH,并且条件是环内不存在O-O、O-S或S-S键。
在本发明的一个实施方案中,GCK-活化剂选自:AZD1656、吡格列丁(a.k.a.R1440或RO4389620)、AZD6370、GKA 50、YH-GKA、PSN-010、LY2121260、灵芝多糖B、异泽兰黄素、glucolipsin A、glucolipsin B、sinogliatin、GKM-001、TTP-399、SY-004、TMG-123、阿比鲁肽、AM-9514、AMG-0696、AMG-1694、AMG-3969、LCZ-960、AZD-1092、AZD-5658、ARRY-403、BMS-820132、GKM-002、LY-2608204、MK-0941、R-1511、RO-281675、ZYGK-1、OP-286CR、CM-3、DS-7309、LY-2599506、PF-04937319、PF-04991532、TAK-329及其药学上可接受的盐。
吡格列丁具有如下结构式:
并且化学名称为(2R)-2-(3-氯-4-甲基磺酰基苯基)-3-[(1R)-3-氧代环戊基]-N-吡嗪-2-基丙酰胺。
AZD1656具有如下结构式:
并且化学名称为3-[5-(氮杂环丁烷-1-羰基)吡嗪-2-基]氧基-5-[(2S)-1-甲氧基丙-2-基]氧基-N-(5-甲基吡嗪-2-基)苯甲酰胺。
在本发明的一个实施方案中,GCK-活化剂为3-{[5-(氮杂环丁烷-1-基羰基)吡嗪-2-基]氧基}-5-{[(1S)-1-甲基-2-(甲氧基)乙基]氧基}-N-(5-甲基吡嗪-2-基)苯甲酰胺。
AZD6370具有如下结构式:
并且化学名称为3-(((1S)-2-羟基-1-甲基乙基)氧基)-N-(1-甲基-1H-吡唑-3-基)-5-(4-(甲基磺酰基)苯氧基)苯甲酰胺。
GKA 50具有如下结构式:
并且化学名称为6-[[3-[(1S)-2-甲氧基-1-甲基乙氧基]-5-[(1S)-1-甲基-2-苯基乙氧基]苯甲酰基]氨基-3-吡啶甲酸。
YH-GKA具有如下结构式:
PSN-010具有如下结构式:
LY2121260具有如下结构式:
并且化学名称为(1R,2S)-2-环己基-1-(4-甲基磺酰基苯基)-N-(1,3-噻唑-2-基)环丙烷-1-甲酰胺。
异泽兰黄素具有如下结构式:
并且为来源于魁蒿(Artemisia princeps)的黄酮。
Glucolipsin A具有如下结构式:
并且为来源于Steptomyces purpurogeniscleroticus的糖脂。
Glucolipsin B具有如下结构式:
并且为来源于瓦辛尼诺卡菌(Nocardia vaccinii)的糖脂。
在优选的实施方案中,GCK-活化剂为AZD1656。在优选的实施方案中,GCK-活化剂为GKA 50。
本领域技术人员还已知并且在本发明中使用的是葡萄糖激酶活化剂(GKA),其列在如下文献中:US20130252973A1、US20130165452A1、WO2013086397A1、US20130131113A1、EP2582706A1、US20130029939A1、EP2543667A1、WO2012150202A1、US20120277242A1、EP2513103A1、US20120252814A1、US20120225887A1、US20120214735A1、US20120184544A1、US20120178765A1、US20120165375A1、US20120149704A1、US20120142636A1、EP2444397A1、US20120088760A1、EP2406230A1和Grewal等人,2014,它们各自均通过引用整体并入本文。
本发明的糖酵解活化剂的细胞靶标可以选自如下的一种或多种:醛缩酶、AMP活化的蛋白激酶(AMP激酶)、CD28、CD80、CTLA-4、烯醇化酶、GCK、GCK调节蛋白(GCKR)、己糖激酶I(HKI)、己糖激酶II(HKII)、ICAM-1和LFA-1。
疾病或医学病症
本发明的糖酵解活化剂和化合物应用于治疗或预防疾病和医学病症。
在一个实施方案中,疾病或医学病症是免疫介导的疾病或医学病症。此类疾病或病症的特征在于存在不需要或异常的免疫应答。因此,在一个实施方案中,免疫介导的疾病或医学病症是自身免疫障碍。本文使用的“自身免疫障碍”是指由生物体针对其自身健康细胞或组织的免疫应答引起的任何疾病或病症。在另一个实施方案中,疾病或医学病症是移植相关障碍。本文使用的“移植相关障碍”是指与患者或个体接受器官、组织或细胞移植有关或由于其而发生的任何障碍,包括生物药物例如单克隆抗体的免疫排斥。移植相关障碍的实例包括移植排斥、同种异体移植排斥、血管化同种异体移植排斥、细胞移植排斥(即干细胞,胰β细胞)和移植物抗宿主病(GVHD)。
在一个实施方案中,免疫介导的疾病或医学病症、例如自身免疫障碍选自:移植排斥、同种异体移植排斥、移植物抗宿主病(GVHD)、系统性红斑狼疮(SLE)、多发性硬化(MS)、银屑病、I型糖尿病、桥本甲状腺炎、自身免疫性甲状腺炎(AITD)、心肌炎、心肌梗死(MI)、变态反应,病毒、细菌或寄生虫感染,癌症、炎性肠病(IBD)、类风湿性关节炎、自身免疫性胃炎、结肠炎、抗肾小球基底膜肾炎、自身免疫性肝炎、原发性胆汁性肝硬化(PBC)、斑秃、自身免疫性孕酮皮炎、自身免疫性荨麻疹、寻常型天疱疮、自身免疫性多内分泌综合征(APS;除外3型APS,又称为IPEX综合征)、自身免疫性胰腺炎、格雷夫斯病、舍格伦综合征、腹部疾病、溃疡性结肠炎、抗磷脂综合征、自身免疫性溶血性贫血、自身免疫性血小板减少性紫癜、恶性贫血、混合性结缔组织病(MCTD)、未分化结缔组织病(UCTD)、银屑病关节炎、复发性多发性软骨炎、风湿热、皮肌炎、重症肌无力、多发性肌炎、急性弥漫性脑脊髓炎(ADEM)、格-巴综合征、桥本脑病、横贯性脊髓炎、结节病、自身免疫性葡萄膜炎、自身免疫性内耳疾病(AIED)、贝赫切特病、巨细胞动脉炎、肉芽肿合并多脉管炎(EGPA)、血管炎、湿疹。
在一个实施方案中,免疫介导的疾病或医学病症为心肌炎。在一个实施方案中,免疫介导的疾病或医学病症为移植物抗宿主病(GVHD)。在一个实施方案中,免疫介导的疾病或医学病症为移植排斥。在一个实施方案中,免疫介导的疾病或病症为类风湿性关节炎。在一个实施方案中,免疫介导的疾病或医学病症为1型糖尿病。在另一个实施方案中,免疫介导的疾病或医学病症例如自身免疫障碍不为1型糖尿病。
在另一个实施方案中,治疗的疾病或医学病症选自移植后糖尿病和缺血再灌注损伤。
自身免疫性
在体内,Treg限制了自身反应性T细胞的活化,从而阻止了它们的分化和效应器功能的获得。通过限制活化的致病细胞的供应,Treg预防或减慢了自身免疫疾病的进程。然而,这种保护性机制在自身免疫个体中显然不足,这可能归因于Treg细胞的缺乏(在特定部位)和/或在长期病程中Treg抗性病原性T细胞的发育和积累。因此,可以通过将Treg归巢到疾病部位或清除病原性T细胞、同时输注Treg以增强控制进行性组织损伤的能力来恢复这些患者中的自我耐受性。器官特异性自身免疫病症、例如甲状腺炎和胰岛素依赖型糖尿病已被归因于这种耐受机制的破坏。
1型糖尿病
1型(青少年)糖尿病是器官特异性自身免疫疾病,其由破坏产生胰岛素的胰β细胞引起。在非糖尿病患者中,胰岛细胞抗原特异性T细胞在胸腺发育中缺失或转化为T调节细胞,其主动抑制效应器对胰岛细胞抗原的响应。在青少年糖尿病患者和青少年糖尿病的NOD小鼠模型中,这些耐受机制缺失。在它们不存在的情况下,胰岛细胞抗原由人白细胞抗原(HLA)I和II类分子呈递,并且被CD8(+)和CD4(+)自反应性T细胞识别。这些自反应性细胞对胰岛细胞的破坏最终导致葡萄糖耐受不良。通过归巢Treg和/或将现存的抗原特异性效应器T细胞转化为调节表型,有害的自身免疫响应被重新定向,导致诱导抗原特异性的适应性耐受。通过诱导抗原特异性耐受来调节对自身表位的自身免疫响应可以防止正在进行的β细胞破坏。
因此,本发明可用于预防或治疗1型糖尿病的方法。当治疗1型糖尿病时,本发明的糖酵解活化剂或GCK活化剂可以作为胰岛素施用方案的组成部分来施用。在另一个实施方案中,活性剂不与胰岛素联用(例如同时、依次或分别施用)。因此,活性剂可以替代胰岛素疗法。
移植
诱导Ag特异性Treg细胞治疗器官特异性自身免疫性是重要的治疗进展,从而避免了全身免疫抑制。在骨髓移植的鼠模型中,Treg促进供体骨髓移植物植入并且降低移植物抗宿主病的发生率和严重性,而不会消除有益的移植物抗肿瘤的免疫作用。与小鼠和人的Treg共有表型和功能特征的观察结果相一致的这些发现已导致人们积极研究这些细胞在减少与人造血细胞移植相关的并发症中的用途。Treg和效应器T细胞的失衡导致移植物抗宿主疾病的发展。然而,人们对免疫调节的机制特别是Treg的同种识别特性、它们对其它免疫细胞的影响和相互作用及其抑制活性位点尚不十分了解。
来自人和试验动物模型的累积证据启示,Treg牵涉移植物抗宿主病(GVHD)的发生。Treg可以将GVHD与移植物抗肿瘤(GVT)活性分开的证明启示,可以调节其免疫抑制潜能来降低GVHD,而不会对GVT作用产生不利影响。
在一个实施方案中,本发明的活性剂可以用于增加个体的移植成功率或延迟个体的移植排斥。移植物可以是皮肤移植物。因此,活性剂可用于增加个体的移植物存活率或延迟皮肤移植物排斥。在一个实施方案中,相对于安慰剂、媒介物、无活性剂对照或其它(常规)免疫抑制剂、例如目的在于抑制效应器免疫机制(细胞或体液)的作用的那些,测定了成功率的增加或排斥的延迟。
移植后糖尿病
在英国,2型糖尿病是终末期肾衰竭的最常见原因。肾移植被广泛认为是终末期肾病患者的肾脏替代疗法的最佳形式,与仍然接受透析的患者相比,接受肾移植患者的生存期更长,并且生活质量得到改善(Reese等人,2015)。
然而,移植导致患者更高的葡萄糖负担。这与急性排斥反应、脓毒症和较差的预后有关,其中患者在移植后的早期常常不得不增加抗糖尿病药物。其原因之一在于免疫抑制药物(类固醇和神经钙蛋白抑制剂)可直接导致高血糖症。另外,随着肾功能的改善,抗糖尿病药的肾清除率更高,导致这些药物的效力降低。这些作用在移植后的初期最为明显,因为这是免疫抑制负担最强和肾功能变化最明显的时期。
短期糖酵解活化剂(例如AZD1656)可能对控制肾移植后不久的免疫抑制负担和由此糖异生作用最大的糖尿病具有有益性。另外,糖酵解活化剂在肾移植后可能具有与其抗糖尿病作用无关的额外益处,所述抗糖尿病作用涉及其对免疫功能的影响。
不希望受到理论的束缚,认为糖酵解活化剂(例如AZD1656)的治疗将在术后早期引起更多的Treg转运,从而通过其免疫抑制作用减少局部缺血-再灌注损伤,随后Treg在移植器官(例如肾脏)中的存在增加-这是改善移植物存活的标志-同时有助于控制早期的移植后糖尿病控制。
因此,本发明可用于预防或治疗移植后糖尿病,特别是肾移植后糖尿病的方法。本发明可用于预防或治疗进行肾移植的患者的糖尿病的方法。在一个实施方案中,已经向患有糖尿病的患者施用了另外的抗糖尿病药物。本发明可用于预防或治疗局部缺血-再灌注损伤的方法。本发明可用于改善移植后移植物存活的方法。本发明可用于在治疗期间增加器官移植组织(例如肾移植组织)中Treg的群体的方法。
因此,可以通过检测Treg迁移的改变例如肾移植组织中Treg的浸润、例如在3个月的治疗期间使用FACS分析测得的外周Treg群体的变化或使用本领域技术人员已知的任意适合的迁移测定法测定的体外Treg迁移的变化来评价本节所述的这种预防/治疗的效能。
因此,有效疗法的二次终点可以包括:基线与治疗活检终点之间的肾活检组织中Treg的组织学染色,基线与使用HbA1c作为标记的治疗终点之间的糖尿病控制,基线与治疗终点之间的另外的抗糖尿病药的剂量,安全性终点,即低血糖发作,使用HOMA IR定量的胰岛素抵抗,基线与治疗终点之间的移植物功能,急性排斥发作,机会致病菌(细菌和病毒)感染发作,评价遗留效果的12-个月移植物功能和糖尿病控制。
免疫调节
本发明的糖酵解活化剂可用于调控或调节个体的免疫应答,例如抑制或阻止个体的免疫应答。
本发明的糖酵解活化剂可以用于抑制或调节个体的自身免疫应答。
特别地,疾病或医学病症的治疗或预防可以通过将内源性调节性T细胞(Treg)运输至例如组织或器官、例如患病的组织或器官或移植的组织或器官介导。
因此,发现本发明的糖酵解活化剂和化合物可用于将内源性调节性T细胞(Treg)运输至患有疾病或医学病症或处于患疾病或医学病症风险中的个体的(患病的)组织或器官中。
更进一步,发现本发明的糖酵解活化剂和化合物可用于治疗或预防通过将内源性调节性T细胞运输至组织或器官、例如患病的组织或器官而缓解的疾病或医学病症。
所谓“患病的组织或器官”是指处于任何异常或病理状态中的组织或器官。例如,组织或器官可能受到免疫疾病、障碍或医学病症例如自身免疫障碍或本文所述的任何疾病或病症影响。
在一个实施方案中,Treg为天然产生的胸腺内生成的Treg(天然Treg)。在另一个实施方案中,Treg为外周产生的可诱导Treg(可诱导Treg)。
在相关的实施方案中,糖酵解活化剂可以同时抑制任何效应器T细胞应答、辅助T细胞应答或B细胞应答。有利的是,糖酵解活化剂可以与抑制效应器T细胞应答、辅助T细胞应答或B细胞应答中的任何一种的活性剂联合施用。
Treg的运输
术语“Treg的运输”通常涉及Treg的运动,例如可能涉及其运动或迁移。这种运输、归巢或迁移可能以特定的方向进行,或者是面向特定的位置或部位。
本发明的效果可以主要通过将Treg运输、归巢或迁移到疾病部位,例如发炎的组织或患病的组织或器官来实现。在优选的实施方案中,Treg细胞迁移到患病的组织或器官。在一个实施方案中,Treg细胞迁移至脾或腹膜,有利地迁移至腹膜。使Treg以这种方式运输的细胞机制是本领域技术人员已知的。
Treg迁移测定是本领域技术人员已知的,并且在本文中进一步详细描述。
治疗方法
本发明还包括在个体中治疗或预防免疫介导的障碍或病症的方法,该方法包括给个体施用糖酵解活化剂。
术语“治疗或预防”旨在涵盖任何形式的治疗、预防或诊断,并且包括治愈和预防疾病的治疗。因此,对健康个体的治疗视为疗法。除对疾病的治愈性治疗外,治疗或预防还包括缓解症状。
剂量和施用
本发明提供了药物组合物,其包含用于本发明用途的糖酵解活化剂和药学上可接受的载体、媒介物、稀释剂或赋形剂。
本发明的组合物可以是适合口服应用(例如片剂、锭剂、硬或软胶囊剂、水性或油性混悬剂、乳剂、可分散粉末或颗粒剂、糖浆剂或酏剂)、局部应用(例如乳膏剂、软膏剂、凝胶剂或水性或油性溶液剂或混悬剂)、通过吸入施用(例如,细粉或液体气雾剂)、通过吹入施用(例如,细粉)或非肠道施用(例如作为用于静脉内、皮下、肌内或肌内给药的无菌水性或油性溶液剂,或作为直肠给药栓剂)的形式。适合口服应用的剂型是优选的。
本发明的组合物可以使用本领域众所周知的常规药物赋形剂通过常规方法获得。因此,旨在口服应用的组合物可以包含例如一种或多种着色剂、甜味剂、矫味剂和/或防腐剂。
用于片剂的适合的药学上可接受的赋形剂包括例如惰性稀释剂,例如乳糖、碳酸钠、磷酸钙或碳酸钙,制粒和崩解剂,例如玉米淀粉或algenic acid;粘合剂,例如淀粉;润滑剂,例如硬脂酸镁、硬脂酸或滑石粉;防腐剂,例如对羟基苯甲酸乙酯或对羟基苯甲酸丙酯,以及抗氧化剂,例如抗坏血酸。片剂可以是不包衣的或包衣的,以改变它们的崩解和随后在胃肠道内活性成分的吸收,或在任一情况下使用本领域众所周知的常规包衣剂和方法来改善其稳定性和/或外观。
口服应用的组合物可以是硬明胶胶囊剂的形式,其中将活性成分与惰性固体稀释剂例如碳酸钙、磷酸钙或高岭土混合;或软明胶胶囊剂的形式,其中将活性成分与水或油例如花生油、液体石蜡或橄榄油混合。
水性混悬剂通常含有细粉形式的活性成分以及一种或多种助悬剂,例如羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯吡咯烷酮、黄蓍胶和阿拉伯胶;分散剂或润湿剂,例如卵磷脂或环氧烷与脂肪酸的缩合产物(例如聚氧乙烯硬脂酸酯),或环氧乙烷与长链脂族醇的缩合产物,例如十七乙烯氧基鲸蜡醇;或环氧乙烷与衍生自脂肪酸和己糖醇的偏酯的缩合产物,例如聚氧乙烯山梨醇单油酸酯,或环氧乙烷与长链脂族醇的缩合产物,例如十七乙烯氧基鲸蜡醇,或环氧乙烷与衍生自脂肪酸和己糖醇的偏酯的缩合产物,例如聚氧乙烯山梨醇单油酸酯;或环氧乙烷与衍生自脂肪酸和己糖醇酸酐的偏酯的缩合产物,例如聚乙烯脱水山梨醇单油酸酯。水性混悬剂还可以包含一种或多种防腐剂(例如Q-羟基苯甲酸乙酯或Q-羟基苯甲酸丙酯;抗氧化剂(例如抗坏血酸);着色剂;矫味剂;和/或甜味剂(例如蔗糖、糖精或阿司巴坦)。
油性混悬剂可以通过将活性成分悬浮在植物油(例如花生油、橄榄油、芝麻油或椰子油)或矿物油(例如液体石蜡)中来配制。油性混悬剂还可以包含增稠剂,例如蜂蜡、硬石蜡或鲸蜡醇。可以添加诸如上述的甜味剂和矫味剂以提供适口的口服制剂。这些组合物可以通过添加抗氧化剂例如抗坏血酸来保存。
适用于通过加水制备水性混悬液的可分散粉末和颗粒剂通常包含活性成分以及分散剂或润湿剂、助悬剂和一种或多种防腐剂。适合的分散剂或润湿剂和助悬剂通过上述的那些举例说明。
也可以存在其它赋形剂,例如甜味剂、矫味剂和着色剂。
本发明的药物组合物也可以是水包油型乳剂的形式。油相可以是植物油,例如橄榄油或花生油,或矿物油,例如液体石蜡或任何这些的混合物。适合的乳化剂可以是例如天然存在的树胶,例如阿拉伯胶或黄蓍胶,天然存在的磷脂,例如大豆、卵磷脂、衍生自脂肪酸和己糖醇酸酐的酯或偏酯(例如脱水山梨醇单油酸酯)和所述偏酯与环氧乙烷的缩合产物,例如聚氧乙烯脱水山梨醇单油酸酯。乳剂还可以包含甜味剂、矫味剂和防腐剂。
可以用甜味剂例如甘油、丙二醇、山梨醇、阿司巴坦或蔗糖配制糖浆剂和酏剂,并且还可以包含缓和剂、防腐剂、矫味剂和/或着色剂。
药物组合物也可以是无菌可注射的水性或油性混悬剂的形式,其可以根据已知的方法,使用一种或多种上述的适合的分散剂或润湿剂和助悬剂配制。无菌可注射制剂也可以是在无毒的非肠道可接受的稀释剂或溶剂中的无菌可注射溶液剂或混悬剂,例如在1,3-丁二醇中的溶液剂。
通过吸入施用的组合物可以是常规的加压气雾剂的形式,其被配置以将活性成分配置为包含细分的固体或液滴的气雾剂。可以使用常规的气雾剂抛射剂,例如挥发性氟化烃或烃,并且气雾剂装置方便地配置以配制按计量的活性成分。
有关制剂的进一步信息,读者请参见Comprehensive Medicinal Chemistry(Corwin Hansch;Chairman of Editorial Board),Pergamon Press 1990的第5卷第25.2章。
与一种或多种赋形剂合并以产生单一剂型的活性成分的量必须取决于所治疗的宿主和特定的施用途径而不同。例如,预期用于对人口服施用的制剂通常含有例如0.5mg-2g的活性剂,其与适当和方便量的赋形剂混合,所述赋形剂的含量可以占总组合物重量的约5%-约98%。本发明的糖酵解活化剂的剂量单位形式通常将包含约1mg-约500mg的活性成分,例如1、10、20、50、100、200、250或500mg的活性成分。优选地,剂量单位形式包含约100mg的活性成分。有关施用途径和剂量方案的更多信息,读者请参见ComprehensiveMedicinal Chemistry(Corwin Hansch;Chairman of Editorial Board),Pergamon Press1990的第5卷第25.3章。
根据众所周知的医学原理,用于治疗或预防目的的剂量大小自然会根据病症的性质和严重程度、动物或患者的年龄和性别以及施用途径而不同。在一个实施方案中,患者年龄为18-75岁。
在将糖酵解活化剂、例如GCK活化剂、例如AZD1656用于治疗或预防目的过程中,通常以每kg体重接受0.5mg-75mg,例如每kg体重1mg-50mg/kg,例如每kg体重5mg-30mg/kg,例如每kg体重10-25mg/kg,例如每kg体重20mg的每日剂量范围,如果需要,可以以分剂量给予。通常,当采用非肠道途径时,将施用更低的剂量。因此,例如,对于静脉内施用,通常将使用例如每kg体重0.5mg-30mg的剂量范围。类似地,对于通过吸入施用,将使用例如每kg体重0.5mg-25mg的剂量范围。然而,口服施用是优选的。在优选的实施方案中,本发明的糖酵解活化剂(例如AZD1656)可以以每日每kg体重20mg的剂量或每日两次持续1-3周、例如2周的时间施用。
在优选的实施方案中,本发明的糖酵解活化剂(例如AZD1656)可以每日施用一次、两次、三次或四次。糖酵解活化剂的过程可以持续任何时间期限,但是在优选的实施方案中,可以将本发明的糖酵解活化剂(例如AZD1656)施用1周至6个月的时间期限,例如持续1至6个月,例如2至4个月,例如3个月的时间期限。
在优选的实施方案中,本发明的糖酵解活化剂(例如AZD1656)可以通过口服途径以约1mg-约500mg、例如约100mg的剂量单位形式施用,每日一次、两次、三次或四次,持续1周至6个月,例如1至6个月,例如2至4个月,例如3个月的时间期限。在特别优选的实施方案中,本发明的糖酵解活化剂(例如AZD1656)以100mg的单位剂型通过口服途径每日施用两次,持续三个月时间期限。
盐
化合物可以作为盐、特别是药学上可接受的盐或酯存在。
化合物的药学上可接受的盐包括其适合的酸加成盐或碱式盐。适合的药用盐的综述可以在Berge等人,J Pharm Sci,66,1-19(1977)中找到。例如与强无机酸形成盐,所述的强无机酸例如无机酸例如硫酸、磷酸或氢卤酸;与强有机羧酸形成盐,所述的强有机羧酸例如未取代或取代(例如被卤素)的具有1-4个碳原子的链烷羧酸,例如乙酸;与饱和或不饱和的二羧酸形成盐,所述的二羧酸例如草酸、丙二酸、琥珀酸、马来酸、富马酸、邻苯二甲酸或四邻苯二甲酸;与羟基羧酸形成盐,所述羟基羧酸例如抗坏血酸、乙醇酸、乳酸、苹果酸、酒石酸或柠檬酸;与氨基酸形成盐,所述氨基酸例如天冬氨酸或谷氨酸;与苯甲酸形成盐;或与有机磺酸形成盐,例如未取代或取代(例如被卤素取代)的(C1-C4)-烷基-磺酸或芳基-磺酸,例如甲磺酸或对甲苯磺酸。
对异构映体/互变异构体
在上述讨论的本发明的所有方面,如果适合,本发明包括本发明化合物的所有对映异构体和互变异构体。本领域技术人员将认识到具有旋光性质(一个或多个手性碳原子)或互变异构特征的化合物。可以通过本领域已知的方法分离/制备相应的对映异构体和/或互变异构体。
立体异构体和几何异构体
本发明的某些化合物可以作为立体异构体和/或几何异构体存在-例如,它们可以具有一个或多个不对称和/或几何中心,因此可以以两种或多种立体异构和/或几何形式存在。本发明预期那些抑制剂的所有单独的立体异构体和几何异构体及其混合物的用途。权利要求中使用的术语包括这些形式,条件是所述形式保留适当的功能活性(尽管不一定达到相同程度)。
本发明还包括活性剂或其药学上可接受的盐的所有适合的同位素变体。将本发明的活性剂或其药学上可接受的盐的同位素变体定义为其中至少一个原子被具有相同原子序数但原子质量不同于自然界通常发现的原子质量的原子替代。可以掺入活性剂及其药学上可接受的盐的同位素的实例包括氢、碳、氮、钾、磷、硫、氟和氯的同位素,例如分别为2H、3H、13C、14C、15N、17O、18O、31P、32P、35S、18F和36Cl。活性剂及其药学上可接受的盐的某些同位素变体,例如其中掺入了放射性同位素(例如3H或14C)的那些可用于药物和/或底物组织分布研究。由于其易于制备和可检测性,特别优选氚代(即3H)和碳-14(即14C)同位素。另外,用同位素例如氘、即2H取代可以提供由于更高的代谢稳定性而产生的某些治疗优势,例如,增加的体内半衰期或降低的剂量要求,因此在某些情况下是优选的。本发明活性剂及其药学上可接受的盐的同位素变体通常可以通过常规方法使用适合的试剂的适当同位素变体来制备。
溶剂化物
本发明还包括本发明化合物的溶剂化物形式。权利要求中使用的术语涵盖这些形式。
多晶型物
本发明还涉及多种晶型、多晶型形式和(无水)含水形式的本发明化合物。在制药工业中已经公认,可以通过略微改变纯化方法和/或从用于合成此类化合物的溶剂中分离的方法来分离任何此类形式的化学化合物。
前药
用于本发明的化合物可以以前药的形式施用。前药是生物前体或药学上可接受的化合物,其在体内可降解以产生本发明化合物(例如本发明化合物的酯或酰胺,特别是体内可水解酯)。多种形式的前药是本领域已知的。
前药的实例如下。包含羧基或羟基的本发明化合物的体内可水解酯是例如在人或动物体内水解以产生母体酸或醇的药学上可接受的酯。用于羧基的适合的药学上可接受的酯包括C1-C6烷氧基甲基酯,例如甲氧基甲基,C1-C6烷酰基氧基甲基酯,例如新戊酰基氧基甲基,酞基酯,C3-C8环烷氧基羰基氧基,C1-C6烷基酯,例如1-环己基羰基氧基乙基;1,3-间二氧杂环戊烯-2-酮基甲基酯,例如5-甲基-1,3-间二氧杂环戊烯-2-酮基甲基;和C1-6烷氧基羰基氧基乙基酯。
包含羟基的本发明化合物的体内可水解酯包括无机酯,例如磷酸酯(包括磷酰胺环状酯)和α-酰基氧基烷基醚,以及由于该酯的体内水解分解得到母体羟基而产生的相关化合物。α-酰基氧基烷基醚的实例包括乙酰氧基甲氧基和2,2-二甲基丙酰基氧基-甲氧基。
羟基的体内可水解的成酯基团的选择包括烷酰基,苯甲酰基,苯基乙酰基和取代的苯甲酰基和苯基乙酰基,烷氧基羰基(产生烷基碳酸酯),二烷基氨基甲酰基和N-(二烷基氨基乙基)-N-烷基氨基甲酰基(产生氨基甲酸酯),二烷基氨基乙酰基和羧基乙酰基。
组合治疗
本文所述的糖酵解活化活性可以作为单独的治疗或与一种或多种其它物质和/或用于所治疗适应征的治疗组合使用。这种联合治疗可以通过同时、依次或分开施用治疗的各个组分来实现。同时治疗可以以单一片剂或在分开的片剂中进行。
在一个实施方案中,本发明的糖酵解活化剂与免疫抑制剂,细胞治疗,致耐受性树突细胞治疗,抗炎剂和/或激素替代治疗例如胰岛素或甲状腺激素组合或联合施用。激素替代疗法对于自身免疫障碍的治疗特别有利,例如胰岛素对于I型糖尿病的治疗。
如本文所用,“同时”用于表示两种活性剂同时施用。因此,“依次”施用可以允许一种活性剂在例如另一种活性剂之后的5分钟、10分钟或几个小时之内施用,条件是第一种施用的活性剂的循环半衰期应使其同时存在于治疗有效量中。施用组分之间的时间延迟将取决于组分的确切性质、它们之间的相互作用以及它们各自的半衰期而不同。
与“依次”相反,“分开”在本文中用来表示施用一种活性剂与另外的活性剂之间的间隔是显著的,即当施用第二种活性剂时,第一种施用的活性剂可以不再以治疗有效量存在于血流中。在一个优选的实施方案中,第二种活性剂在第一种活性剂之后至少2小时,更优选至少4小时,甚至更优选至少8小时,甚至更优选仍至少12或24或48或72小时施用。在一个特别优选的实施方案中,第二种活性剂在第一种活性剂之后至少24小时施用。
另外的方面
尽管本文所述的用途和方法通常适用于内源性Treg细胞,但也认为通过活化糖酵解途径诱导Treg运输或迁移可用于引发自体和/或同种异体Treg细胞。
例如,认为可以将患者自身的Treg细胞分离出来,使用糖酵解活化剂活化,并且重新导入患者体内。Treg可以任选被扩增。
因此,本文还提供了运输或动员Treg细胞的离体方法,该方法包括通过使Treg细胞与糖酵解活化剂接触来活化Treg细胞中的糖酵解的步骤。该方法可以包括例如在糖酵解活化之前诱导Treg增殖的步骤。
因此,本文还提供了增加Treg细胞(例如,过继转移的Treg)定位于炎性位点的效率的离体方法,该方法包括用GCK活化剂预处理自体和/或同种异体Treg细胞的步骤。可以将这样的预处理步骤定义为将Treg细胞与含有GCK活化剂的培养基在37℃一起温育至少2小时。该方法可以包括例如在糖酵解活化之前,诱导Treg细胞增殖或扩增Treg细胞的步骤。建立了这样的扩增和增殖方法,并且是本领域技术人员已知的。
上述方法可以包括任选例如从待治疗的个体(即自体)或从另外的个体或来源(即同种异体)分离Treg细胞的步骤。
因此,本发明还提供了治疗本文所述的免疫介导的疾病或医学病症的方法,该方法包括以下步骤:
a)从待治疗的个体中分离Treg细胞和/或从另外的个体或来源中分离Treg细胞;
b)扩增来自步骤(a)中的Treg细胞以获得用于治疗的足够数量;
c)将来自步骤(b)的Treg细胞与GCK活化剂接触或用其进行预处理;
d)将来自步骤(c)中的Treg细胞施用入待治疗的个体。
可以使用任何适合的给药方案将来自步骤(c)的Treg细胞施用或输注入个体。例如,可以以每kg体重Treg的测量剂量,以及以多种给药方案(例如一次或多次输注,其中在多种间隔输注)施用Treg。
因此,本文还提供了分离的、糖酵解活化的Treg细胞。本文还提供了用于医学的糖酵解活化的Treg细胞,其中所述Treg是任选分离的。在一个实施方案中,糖酵解活化的Treg是GCK活化的Treg或AZD1656处理的Treg。这些方面的分离的Treg细胞可以是自体Treg细胞。分离的Treg细胞可以在GCKR基因中包含突变,例如在GCKR基因中缺失,例如可以缺失整个GCKR基因。这样的突变导致GCK和Treg迁移的抑制解除。
实施例
现在通过以下非限制性实施例描述本发明。
材料
小鼠.本研究的试验中使用的所有小鼠均为7-11周。C57BL/6、BALB/c和CBA/Ca小鼠购自Charles River(UK)。D Wraith教授(University of Birmingham)提供了来自4周龄CTLA-4KO小鼠(H-2u单倍型)的切下的二级淋巴器官。
微血管内皮细胞的分离.如先前所述分离鼠肺微血管内皮细胞(Marelli-Berg等人,2000)。
骨髓来源的树突细胞(BMDC)的分离.从WT BALB/c(H2-d)小鼠获得骨髓来源的DC。取出7至10周龄的雌性小鼠的股骨和胫骨,并且使用27号针头(Becton Dickinson,Cat#302200)用PBS冲洗BM细胞。用裂解缓冲液(Sigma-Aldrich,Cat#R7757)从细胞悬液中裂解红细胞。如下所述,将BM细胞(5×106)每孔接种在6孔板(Helena bioscience,Cat#92006)中的DC培养基中。
树突细胞的培养.在补充有10%FCS、2mM谷氨酰胺、50IU/mL青霉素、50μg/mL链霉素、50μM 2-ME和2%鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)的RPMI 1640培养基(Gibco,Cat#21875-034)中培养骨髓来源的树突细胞,所述鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)得自GMCSF杂交瘤上清液(来自Imperial College,London,UK的Jian-Guo Chai博士的赠品)。在5%CO2的存在下于37℃培养细胞。在第3和5天,将新鲜的培养基添加到平板中。
对于Treg-DC共培养,收集未成熟的BMDC并且在培养的第6天使用。对于功能测定,将未成熟的DC用100ng/mL脂多糖(LPS)(Invivogen,Cat#tlrl-3pelps)成熟过夜,并且在分离后7至10天使用。
H2-d异种特异性Treg的培养.使用 FlowCompTM小鼠CD4+CD25+Treg细胞试剂盒(Invitrogen Dynal,Cat#11463D)从脾和淋巴结中分离出CD4+CD25+Treg细胞。对于极高纯度的Treg细胞(>99%),通过荧光活化的细胞分选从Foxp3-GFP报告基因小鼠中获得CD4+CD25+Foxp3+细胞。为了扩增,每周用照射或丝裂霉素C(Sigma-Aldrich,Cat#M4287)-灭活的未成熟BALB/c衍生的(H2-d)DC以5:1比例(Treg:DC)刺激分离自C57BL/6(H2-b)小鼠的Treg细胞(Fu等人,2014)。将共培养物维持在补充有10U/mL IL-2的完全T细胞培养基中。每周收获细胞并且在24-孔组织培养(Helena bioscience,Cat#92024)板中以1.5×106Treg/孔的最佳密度接种。培养两周后,CD4+Foxp3+细胞的百分比大于95%。为了用于功能测定,刺激后6-8天使用Treg。
抗体介导的T细胞活化.通过在37℃在补充20U/mL重组IL-2(Roche,West Sussex,UK)的完全T细胞培养基中用板结合的抗-CD3(1μg/mL,eBiosciences,Cat#16-0032-85)和板结合的抗-CD28(5μg/mL,eBiosciences,Cat#16-0281-86)多克隆刺激LN细胞7天得到活化的T细胞。通过将抗体在200μL Tris缓冲液(pH 8.5)中于37℃温育1小时来进行组织培养板的抗体包被。
方法
淋巴细胞跨内皮迁移和趋化性测定.接种用IFN-γ处理48-72小时的原代微血管EC(3×104),并且在EC培养基中含有3-μm孔径(Costar,Cat#CLS3472-48EA)聚碳酸酯膜的2%明胶包被的Transwell插入物(直径为6.5mm)上培养16小时至单层。将重悬于迁移培养基(补充有2%胎牛血清的RPMI 1640)中的T细胞(5×105)添加到每个插入物中,并且使其迁移通过单层。将孔体积也用新鲜的迁移介质代替。通过血细胞计数器对历经24小时的不同时间点的孔介质中存在的细胞进行计数来确定迁移的T细胞数量。为了测定趋化性,将T细胞接种到具有5或3-μm孔径(Costar,Cat#CLS3421-48EA)聚碳酸酯膜的TranswellTM裸滤组织培养孔插入物(直径6.5mm)上,并且将含有趋化因子的迁移培养基放置在孔的底部。通过血细胞计数器测定迁移的细胞数。
CD28、CTLA-4和LFA-1信号传导的诱导.如先前所述(Schneider等人,2005;Wells等人,2001),通过抗体刺激诱导CD28和CTLA-4信号传导。为了通过CD28和CTLA-4共受体诱导共刺激信号以进行功能测定,将细胞与靶向共受体功能结构域的抗体一起温育。为了诱导CD28信号传导,将T细胞用仓鼠抗-小鼠CD28(5μg/5×106个细胞)(克隆:37.52,Bio-Rad,Cat#MCA1363)和山羊抗-仓鼠免疫球蛋白(Ig)(2.5μg/5×106个细胞)(Bio-Rad,Cat#STAR104)的混合物处理T细胞如每个图分别所述的不同的时间点。类似地,通过将T细胞与仓鼠抗-小鼠CTLA-4(5μg/5×106个细胞)(克隆:UC10-4F10-11,Becton Dickinson,Cat#553718)和山羊抗-仓鼠免疫球蛋白(Ig)(2.5μg/5×106个细胞)(Bio-Rad,Cat#STAR104)的混合物一起温育实现CTLA-4信号传导。使用仓鼠IgG同种型对照来观察抗体刺激的任何非特异性作用(Bio-Rad,Cat#MCA2356)。为了诱导LFA-1信号传导,将细胞与2μg/5×106个细胞重组小鼠ICAM-1-人IgG Fc嵌合体(2μg/mL,R&D Systems,Cat#796-IC-050)或人IgG-Fc片段(R&D Systems,Cat#110-HG)作为对照-塑料结合或与小鼠抗-人IgG(1μg/mL,MK1A6,Bio-Rad Cat#MCA647G连接)-一起温育如每个图分别所述不同时间点。在用于试验之前,用PBS洗涤T细胞。
活T细胞的荧光标记.为了用荧光探针标记T细胞,将T细胞用PBS洗涤,计数并且以107/mL的终浓度重悬于PBS中。如果必要,在重新悬浮之前,使用Ficoll-Paque密度梯度离心去除死细胞。使用制造商的说明书用细胞膜的细胞连接染料PKH26(Sigma-Aldrich,Cat#PKH26GL-1KT)标记T细胞。加入终浓度为5μM的PKH26,并且将细胞在室温温育5分钟。通过向细胞悬液中加入等体积的FBS使反应灭活,并且将细胞在含10%FBS的PBS中洗涤10分钟。通过在室温在含有3.3μm CFSE或1.3μm DDAO-SE终浓度的PBS中温育T细胞10-15分钟,用琥珀酰亚胺酯染料CFSE(Invitrogen,Cat#C1157)或DDAO-SE(Invitrogen,Cat#C34553)标记T细胞。通过加入等体积的FBS终止反应,然后用含有10%FBS的PBS洗涤细胞10分钟。
腹膜中的T细胞募集.为了观察T细胞的体内募集,我们使用了在先描述的T细胞募集模型(Mirenda等人,2007)。将PKH26或CFSE或DDAO-SE标记的T细胞(107)静脉内注射(i.v.)到48至72小时前通过腹膜内注射(i.p.)hd接受IFN-γ(600U)的小鼠中。16小时后,使用流式细胞术分析通过腹腔灌洗回收的标记T细胞。此外,还分析了Treg在脾中的定位,其中以被动方式进入(Fu等人,2016),以确保在所有接受者中都注射或共同注射了相似数量/比例的标记细胞(内部对照)。
酵母多糖诱导的腹膜炎.在第0天,在无菌盐水溶液中给小鼠腹膜内注射酵母多糖(1mg/小鼠,Cat#4250SIGMA)以诱发腹膜炎。注射后72小时处死小鼠,并且将获得的组织样品用于流式细胞术分析。
宽视野重叠合法荧光显微镜检查.切下组织样品,将其包埋在最佳切割温度化合物(OCT;Thermo Fisher Scientific,Cat#12678646)中,速冻并且保存直至分析。将冷冻的组织切片置于Polysine包被的显微镜载玻片(VWR International,Cat#47100)上,风干,然后用冰冷的丙酮(Sigma,Cat#534064)固定10分钟。用PBS洗涤组织切片,用血清封闭3小时,并且在4℃使用提及的一抗染色24小时。过量的抗体用PBS洗涤掉,并且将组织在室温用指定的二抗以及DAPI(4’,6-二脒基-2-苯基吲哚)(Invitrogen/LifeTechnologies,Cat#D1306)染色30分钟。洗涤载玻片,将其固定在ProLong Gold Antifade试剂(Invitrogen/Life Technologies,Cat#P36930)中,并且使用配备AxioCam MRm冷却单色数码相机和Apotome 2成像单元的Zeiss Z1荧光显微镜(Carl Zeiss,UK)进行可视化。使用PlanApochromat 20×/0.8NA物镜和Axiovision软件4.8版(Carl Zeiss,UK)获取图像。为了染色,将Treg在R10培养基中培养并且用3.7%甲醛固定。固定后,分别在37℃分别用抗GCK、抗-Na,K-ATP酶和1ng/mL的四甲基罗丹明B异硫氰酸酯缀合的鬼笔环肽(Sigma-Aldrich,Cat#P1951)染色30分钟。随后是二抗Alexa 555山羊抗-小鼠Ig(Biolegend,Cat#405324)和FITC驴抗-兔IgG(最小x-反应性)抗体(Biolegend 406403)。将盖玻片充分洗涤,风干,并且在载玻片上用DAPI(Vector Laboratories Cat#z0603)固定在Vectorshield固定介质中。
体外6-NBDG摄取测定.用PBS洗涤新鲜分离的T细胞或培养的T细胞,并且重悬于含有多种提及的信号传导抗体的无葡萄糖T细胞培养基(Gibco,Cat#11879-020)中,并且在37℃与5%CO2温育45分钟。然后将无糖T细胞培养基中终浓度为400μM的6-NBDG(LifeTechnologies,Cat#N23106)加入细胞中,并且将细胞再温育10-15分钟。最终,将细胞用温PBS洗涤两次,并且重悬于流式细胞术缓冲液中并且置于冰上。使用流式细胞术进行立即分析,以观察T细胞的荧光摄取。
体外6-NBDG摄取测定(人研究).用PBS洗涤新鲜分离的CD3+T细胞,并且在R2(补充2%胎牛血清的RPMI 1640)中以重组CCL19和CCL21(200ng/mL–Peprotech Cat#300-29B和300-35)在37℃与5%CO2下培养106/mL。将终浓度为400μM 6-NBDG(Life Technologies,Cat#N23106)添加至细胞,并且温育0、15、30和60分钟。最后,将细胞用温PBS洗涤两次,对CD4+T细胞和Treg染色并且置于冰上。使用流式细胞术进行立即分析以观察Treg的荧光摄取。
体内6-NBDG摄取测定.为了测定T细胞体内的葡萄糖摄取活性,将PKH26标记的T细胞(3×106个)i.p.注射入小鼠。此后对小鼠即刻给予二次i.p.注射6NBDG(无菌水中400μM)。1小时时间期限后,处死小鼠,并且收集肠系膜(引流)淋巴结和脾,用于流式细胞术分析。进行腹膜的宽视野显微镜检查以观察标记的T细胞流入腹膜。使用图像分析软件ImageJ进一步分析浸润膜的PKH26+T细胞的6NBDG摄取(绿色荧光)。手动计数10倍放大倍数视野图像中标记细胞的数量,以确定T细胞浸润的差异。
ECAR和OCR的测定.使用XF分析仪(Seahorse biosciences)对受抗体刺激的T细胞的胞外酸化率(ECAR)和耗氧率(OCR)进行实时生物能分析。将T细胞在无血清、无缓冲的XF测定培养基(Seahorse biosciences,Cat#102365-100)中培养1小时。然后将细胞接种(6×105个/孔)到海马XF24细胞板中用于分析。通过添加寡霉素(1μM)、FCCP(1μM)、抗霉素A(1μM)、鱼藤酮(1μM)、D-葡萄糖(10mM)、2-去氧-D-葡萄糖(2DG,50mM;均来自Seahorsebiosciences,Cat#103020-100和103015-100)来实现T细胞利用代谢途径的扰动剖析。使用以下测定条件进行用Seahorse系统的试验:2分钟混合物;2分钟等待;和4-5分钟测量。计算代谢参数。在至少一式三份的孔中进行试验。
表面染色.对于表面染色,将细胞重悬(107/mL),并且在4℃用荧光染料缀合的抗体在100μL流式细胞术缓冲液中染色30分钟,该缓冲液由含0.1%叠氮化钠(Sigma-Aldrich,Cat#S2002-25G)和1%FBS的PBS制成。CCR7抗体染色在37℃进行30分钟。基于制造商的说明计算用于染色的最佳抗体浓度。染色后,洗涤细胞并且用流式细胞术缓冲液重悬并且立即分析。可选择的是,为了进行延迟分析,将细胞在4℃的固定缓冲液(含1%甲醛的流式细胞术缓冲液(Sigma-Aldrich,Cat#15,812-7)中固定30分钟,洗涤并且在4℃在流式细胞术缓冲液中保存。
胞内染色.对于胞内Foxp3染色,使用eBioscience Anti-Mouse/Rat Foxp3Staining Set APC(克隆FJK-16S,Thermo Fisher Scientific Cat#17-5773-82)试剂盒。如上所述,将细胞重悬(107/mL)并且用表面抗原染色,然后使用固定/透化工作溶液在4℃固定/透化30分钟,所述固定/透化工作溶液通过混合1份固定/透化浓缩物(eBioscience,Cat#00-5123)与3份固定/透化稀释剂(eBioscience,Cat#00-5223)制成。然后将细胞在1×透化缓冲液(eBioscience,Cat#00-8333)中洗涤两次,并且在4℃在1×透化缓冲液中用荧光染料缀合的Foxp3抗体染色30分钟。用1×透化缓冲液进行最终洗涤,然后将细胞离心并且重悬于200uL流式细胞术缓冲液中。对于T细胞增殖研究,用未成熟的Balb/c DC刺激Treg。3小时后,将Treg固定并且用冰冷的70%乙醇透化,然后染色用于增殖细胞核抗原(PCNA,克隆PC10,BioLegend Cat#307908)。
体外AKT磷酸化(人研究).用PBS洗涤新鲜分离的浓缩CD4+CD25-T细胞(Tconv)和CD4+CD25+(Treg)T细胞,并且在R2(补充2%胎牛血清的RPMI 1640)中培养5×106/mL。将0.5×106CD4+CD25-(Tconv)和CD4+CD25+(Treg)铺在96孔-板上,并且在37℃与5%CO2下用趋化因子CCL19和CCL21(1μg/mL–Peprotech Cat#300-29B和300-35)刺激15分钟或不刺激15分钟。立即将细胞固定,以在黑暗中于RT用2%甲醛停止刺激10分钟。洗涤细胞,并且重悬于100%冰冷的甲醇中,并且在冰上温育30分钟。随后,将细胞用PBS 2%FBS洗涤两次,并且在黑暗中于4℃对pAKT-s473(eBioscience,Cat#17-9715-41)染色30分钟。洗涤细胞,重悬于PBS+2%FBS中,并且立即通过流式细胞术分析。
蛋白质印迹和共免疫沉淀.使全细胞裂解液在Nonidet P-40裂解缓冲液[50mMHepes(pH 8.0)、350mM NaCl、1%Nonidet P-40、1mM EDTA、1mM Na3VO4、1mM NaF、20mM甘油-2-磷酸、1mM PMSF、1mM DTT、10μg/mL抑肽酶、10μg/mL亮肽素和蛋白酶抑制剂混合物(RocheCat#11836145001)]中裂解。通过SDS/PAGE分离通过标准Bradford测定法(Bio-RadCat#5000001)测定的等量蛋白质,并且转移至硝化酸纤维素膜(GE Healthcare LifeSciences Cat#10600002)。将膜在室温于5%乳/TBS-Tween 20(Sigma Cat#P1379)中封闭2小时,并且在4℃与以下列出的一抗一起温育过夜。随后加入HRP缀合的二抗(1:5000;Amersham Bioscience Cat#NA934)。然后展开薄膜。使用ImageJ(NIH)量化条带的强度。对于共免疫沉淀试验,在RIPA缓冲液中制备细胞提取物。首先在4℃用蛋白质G-琼脂糖珠(Sigma Cat#P3296)预清洗250μg总蛋白质提取物1小时,在4℃与5μg抗-GCK Ab(SantaCruz Biotechnology Cat#SC7908)一起温育过夜,然后在4℃与蛋白G珠一起再温育16小时。将最终的沉淀重悬于10mM Tris HCl,pH 7.4中,补充有1mM PMSF,并且通过蛋白质印迹分析B肌动蛋白(Santa Cruz Cat#SC161)。
定量实时PCR(qRT-PCR).收获组织并且将其保存在-80℃RNA后(QiagenCat#76104)中,直到进行处理为止。根据制造商的说明,使用Trizol试剂(Life TechnologiesCat#15596)纯化RNA,并且使用吸收测量评价其质量和数量。根据制造商的说明(AppliedBiosystems Cat#4374966)进行逆转录。使用SYBR Green Supermix(Biorad Cat#1725120)在CFX连接光循环仪(Biorad Cat#1855200)中进行基因表达分析。使用ΔΔCt方法(Livak和Schmittgen,2001)计算表达,并且对看家基因(GAPDH)校准。使用在线工具(Primer3Plus)设计qPCR引物,每个引物对使用至少一个外显子结合位点。扩增的热循环曲线为95℃持续10分钟,然后进行40个95℃持续15秒和54℃持续1分钟的循环。扩增在95℃持续10分钟,然后进行40个95℃持续15秒和60℃持续1分钟的循环。为了确保扩增特异性,将熔解曲线程序设置如下:在PCR循环后立即进行95℃持续15秒,60℃持续1分钟和95℃持续15秒。试验一式三份进行。
用于基因沉默的慢病毒制备.HEK293T细胞在10cm细胞培养皿中生长至70%汇合,并且使用磷酸钙方法用上述质粒转染。转染后48和72小时收获上清液,并且在超速离心机中浓缩100倍。将等分试样储存在-80℃。对于Treg的转导,将细胞接种在6-孔板中,并且在DMEM中培养至60-70%汇合。在5μg/mL Polybrene(Sigma-Aldrich Cat#107689)存在下,将慢病毒添加到细胞中,并且将6-孔板在室温以2,300rpm离心90分钟,然后在37℃与5%CO2下温育8小时。24小时后除去病毒;用PBS洗涤T细胞两次,并且在完全DMEM(Lifetechnologies,Cat#1852730)中温育24小时。
研究人群.Progressione della Lesione Intimale Carotidea(PLIC)Study(CHECK研究的子研究)是对米兰北部地区总人口的大规模调查(n=2.606)(Baragetti等人,2015;Lorenz等人,2012;Norata等人,2009;Norata等人,2006),其后进入了Center forthe Study of Atherosclerosis,Bassini Hospital(Cinisello Balsamo,Milan,Italy)。本研究得到了Scientific Committee of the Università degli Studi di Milano(“Cholesterol and Health:Education,Control and Knowledge-Studio CHECK((SEFAP/Pr.0003)-参考编号Fa-04-Feb-01)在2001年2月4日批准。根据赫尔辛基宣言获得了个体知情同意。
如先前所述(Norata等人,2007),使用Flexigene DNA试剂盒(Qiagen,Milan,Italy)提取基因组DNA。使用TaqMan等位基因鉴别测试,可在整个人群中获得p.Leu446ProGCKR错义突变的基因分型。对16名个体亚组进行试验分析,其中8名Leu 446-GCKR和8名446-GCKR的年龄和性别相匹配。获得了有关病史和正在进行的疗法的信息;计算体重指数(BMI,Kg/m2),并且禁食过夜后,从肘前静脉采集血样,以测定脂质分布、葡萄糖水平、肝酶、白细胞计数和亚级分,如前所述(Ammirati等人,2012;Baragetti等人,2015;Norata等人,2006)。
血液表面和胞内染色(人研究).对于表面染色,在RT(室温)于黑暗中,在50μL由含有2%FBS和2μM EDTA的PBS制成的MACS缓冲液中,用荧光染料缀合的抗体对100μL全血进行染色30分钟。基于制造商的说明计算用于染色的最佳抗体浓度。染色后,将红细胞在RT用2mL 1步固定/裂解溶液(eBioscience,Cat#00-5333-54)裂解20分钟,洗涤并且用MACS缓冲液重新悬浮,并且立即进行分析。
可选择的是,对于胞内染色,分离外周血单核细胞(PBMC)(如下所述)并且用于染色(1步固定/裂解溶液与某些胞内染色不相容)。将细胞重悬(107/mL),并且在50μL具有表面抗原的MACS缓冲液中染色。对于胞内Foxp3和Ki67染色,使用eBioscience Anti-Mouse/Rat Foxp3染色试剂盒(Cat#77-5775-40)。使用通过将1份固定/透化浓缩物与3份固定/透化稀释剂混合制成的固定/透化工作溶液,在4℃固定/透化细胞。然后将细胞在1×透化缓冲液中洗涤两次,并且在1×透化缓冲液中用荧光染料缀合的Foxp3和-Ki67抗体在4℃染色30分钟。用1×透化缓冲液进行最后洗涤,然后将细胞离心并且重悬于200μL MACS缓冲液中。
PBMC分离和CD4+CD25+ Treg纯化(人研究).对于每个个体,将30mL血液(补充EDTA)分入两个15mL的falcon,并且以1000×g旋转12分钟。弃去血浆,并且小心地收集富含白细胞和血小板的血浆与红细胞之间的界面(血沉棕黄层),用冷PBS稀释,并且使其在3mLFicoll-PlaqueTM PREMIUM(GE-Healthcare,Cat#17-5442-03)上分层。在以250×g离心35分钟后,小心地收集PBMC层,并且用10mL冷PBS以180×g离心3次,12分钟以去除血小板。计数PBMC,并且根据制造商的说明,使用人CD4+CD25+调节性T细胞分离试剂盒(MiltenyiBiotec.,Cat#130-09-301)进行CD4+CD25+ Treg纯化。用血细胞计数器对纯化的CD4+CD25+Treg或CD4+CD25-Tconv进行计数,并且用于迁移和抑制测定。
Treg和Tconv迁移测定(人研究).将每个个体的300μL Treg和Tconv(1×105)重悬于迁移培养基(补充2%胎牛血清的RPMI 1640)中,并且在孔径为5-μm聚碳酸酯膜(Costar,Cat#CLS3421-48EA)的TranswellTM插入物(直径6.5mm)上培养。相对于置于孔底的迁移培养基或趋化因子CCL19和CCL21(200ng/mL–Peprotech Cat#300-29B和300-35),使细胞迁移1、2、4和12小时。用血细胞计数器测定迁移细胞的数目,并且将数据表示为相对于培养细胞的迁移百分比。
Treg抑制测定(人研究).将CD4+CD25-Tconv重悬于MACS缓冲液(107/mL)中,并且用琥珀酰亚胺基酯染料CFSE(2μM-Invitrogen,Cat#C1157)在室温黑暗中标记10分钟。用MACS缓冲液洗涤细胞3次,并且以360×g离心5分钟。在37℃,将96孔板的U-型底部用抗-人CD3纯化的抗体(5μg/mL-eBioscience,Cat#14-0039-82)包被1小时。将Tconv重悬于(106/mL)完全培养基(补充10%FBS、1mM丙酮酸钠、10mM Hepes、50μMβ-MeOH和Pen/strep/谷氨酰胺的RPMI 1640)加50U/mL IL-2(Peprotech;Cat#200-02)和2μg/mL抗-人CD28纯化抗体(eBioscience,Cat#14-0289-82),并且铺板(1×105/100μL)。将CD4+CD25+Treg洗涤并且重悬于完全培养基中,并且通过用完全培养基系列稀释Treg,按照如下比例(Tconv:Treg):1:1、1:0.5、1:0.25和1:0添加至Tconv。将96孔-板以120×g旋转1分钟以收集底部的细胞,并且使其增殖4天。然后收集细胞,对于每个个体,将在Treg存在下增殖细胞的百分比与Tconv:Treg 1:0的条件(100%增殖)进行比较。
定量和统计学分析.通过从看家基因中获取目标基因的CT值、然后将其与野生型对照样品校准,使用ΔΔCT方法分析qPCR数据。将结果传输到prism,然后进行图形呈现和统计分析。结果以每组的平均值±SD给出。使用双尾不配对的Student’s t检验和Mann-Whitney检验对数据进行分析。将p值小于0.05视为具有显著性。考虑到多重比较,使用应用Bonferroni校正的单向方差分析或应用Dunn氏检验后的Kruskal-Wallis对来自海马的试验数据组进行分析。如果适合,“n”代表生物重复样本数。通过rs1260326GCKR多态性的TT与CC基因型(按年龄和性别调整)之间的ANCOVA(协方差分析)模型分析人数据。如果是正态分布,则变量表示为平均值(标准偏差,S.D.);或如果是非正态分布,则表示为中位值(四分位间距,IQR)(Shapiro-Wilk检验)。进行T检验以比较正态分布的变量和U-Mann Whitney来比较非正态分布的变量(报告了每个变量的P值;p小于0.05为显著性的)。针对每个变量进行Grubb的离群值检测。对于人结果,将数据报告为每组的平均值±SEM。使用双尾不配对的Student’s t检验和Mann-Whitney检验分析数据。将p值小于0.05视为显著性的。
实施例1-Treg迁移需要糖酵解途径的参与
我们探索了像常规T细胞(Tconv)一样,Treg通过利用葡萄糖类似物2-脱氧葡萄糖(2-DG)抑制这种途径来利用糖酵解进行迁移的可能性。暴露于2-DG的Treg在体外(图1A,C-D和图2A-B)和体内(图3A-B)均不能有效迁移。除了在暴露于药物后进行充分洗涤外,通过裸露的transwell抑制Treg趋化作用还排除了在这些情况下药物对内皮的间接作用。相反,使用可通过AMP激酶刺激糖酵解的二甲双胍激活糖酵解增加Treg运动性(图1B,C-D和图2A-B)和运输(图3C-D)。在所使用的剂量下,没有一种药物会影响迁移相关受体的Treg表达或存活力(图4A-C)。为了证实一旦将经体外处理的T细胞注射到受体小鼠中,这些化合物各自的作用都得以保留,将Treg细胞暴露于多种药物4小时,充分洗涤并且在单独的培养基中再温育16小时。在没有化合物的情况下长时间温育后,对药物处理的Treg运动性的影响仍然很明显(图5A-D,图6A-B)。乙莫克舍是一种乙酰辅酶A羰基化酶(ACC)磷酸化抑制剂,并且用于表明Treg迁移不需要脂肪酸氧化(FAO)。
为了证实促迁移刺激对糖酵解途径的诱导,我们随后测试了粘附分子整联蛋白LFA-1(T细胞迁移的关键介体)的参与对Treg中需氧糖酵解的影响。固定化或抗体连接的重组小鼠ICAM-1(rICAM-1)(LFA-1的配体)用于此目的。
首先,我们使用不能被己糖激酶磷酸化并且以其荧光形式蓄积在胞质中的葡萄糖类似物6-[N-(7-硝基苯并(nitrobemz)-2-氧杂-1,3-二唑-4-基)氨基]-2-去氧葡萄糖(6-NBDG)评估LFA-1-诱导的葡萄糖摄取。用塑料结合的rICAM-1或人IgGFc片段(对照)刺激Treg,并且在30分钟后通过流式细胞术测定6-NBDG的摄取。如图7A-B中所示,LFA-1刺激显著增加了6-NBDG的摄取。
其次,我们测定了LFA-1参与或暴露于趋化因子CCL22(CCR4配体)对胞外酸化率(ECAR)的影响,从而量化了质子产生作为乳酸产生的替代,由此反映了总的糖酵解通量。LFA-1或CCR4(图7C-D)刺激后,葡萄糖供应时ECAR的增加得到显著增强。线粒体呼吸的量度耗氧率(OCR)不受影响(图8A-B)。
实施例2-CD28和CTLA-4通过调节糖酵解途径调节Treg迁移
首先,我们证实CD28的抗体活化增强了离体扩增的Treg TEM(图9A)以及体外趋化性(图9B-C)和体内迁移(图10),而不会影响相关受体的表达(数据未显示)。此外,我们观察到,尽管单独触发CTLA-4不会影响Treg迁移,但与CD28的共连接消除了CD28诱导的迁移(图9A-B)。
其次,我们测定了CD28和CTLA-4信号对Treg中糖酵解途径的影响。与用同型对照和二抗处理相比,CD28触发的6-NBDG摄取(图11A-B)和糖酵解通量(图12A)显著增加。相反,CTLA-4刺激本身并不会影响葡萄糖摄取或ECAR,但是当共连接时,CTLA-4信号阻止CD28诱导的葡萄糖摄取和ECAR增加。耗氧率(OCR)不受任何一种共刺激受体触发影响(数据未显示)。
我们通过分析CTLA-4缺陷Treg的代谢活性和迁移,进一步探索了共刺激信号与运动的代谢调节之间的关联性。在这些试验中,我们将CD28和CTLA-4共有的配体重组(r)CD80用作刺激物。与WT Treg相比,CTLA-4缺陷Treg自发地展示出对葡萄糖响应的ECAR延长增加(图12B)-甚至考虑到更高的酸化基线。正如预期的,由于同时存在CD28和CTLA-4参与,重组CD80活化的WT Treg细胞的ECAR保持不变(图13A),而CTLA-4缺陷Treg进一步增加了其对葡萄糖添加的糖酵解响应(图13B)。尽管CTLA-4KO Treg OCR自发高于其WT对应物,但它不受CD28或CTLA-4信号影响(数据未显示)。
同时,我们用rCD80刺激的CTLA-4KO Treg通过IFN-γ-活化的同种系EC测试了TEM。如图14A中所示,用重组CD80刺激的CTLA-4KO展示出通过EC增强的迁移,而表达CTLA-4的Treg对这种刺激无响应,这支持CTLA-4信号抑制CD28诱导的糖酵解和迁移的结论。
为了研究是否通过调节糖酵解的调节发生CD28和CTLA-4对Treg迁移的控制,我们分析了葡萄糖耗尽培养基中TEM期间CD28和/或CTLA-4刺激的作用。如图14B中所示,葡萄糖耗尽阻止了由CD28信号诱导的运动性的增加。结果不受与葡萄糖缺乏相关的细胞窘迫影响,因为在葡萄糖充足和不足的条件下基线低水平的迁移仍然保持相似。
体内通过监测CD28刺激的迁移Treg对6-NBDG的摄取来分析CD28-糖酵解-迁移轴。为此目的,我们使用了组织浸润模型,其中定量了注入腹膜腔中的Treg浸润腹膜的能力(Mirenda等人,2007)。选择该模型是因为6-NBDG荧光随时间会迅速损失,因此从技术上讲不可能长期跟踪静脉内转移的T细胞。将Treg用PKH26标记(红色荧光;图15A,第二列),然后在有或没有CTLA-4触发的情况下进行CD28连接,或者在48小时前用IFN-γ处理的同种系小鼠i.p.注射之前接受同种型匹配和二抗作为对照。细胞之后立即进行i.p.注射6-NBDG(绿色荧光;图15A,第三栏),因此可以通过标记的Treg体内平行评价组织浸润和葡萄糖摄取。如图15A-C中所示,CD28活化显著增加了渗透腹膜并且显示6-NBDG摄取的Treg数量(黄色荧光,合并;图15A,第四栏),表明Treg迁移与体内葡萄糖摄取直接相关。通过CTLA-4共连接可防止这些作用。
实施例3-迁移前刺激诱导代谢程序重排
在先我们已经证实,在Tconv细胞中,迁移过程中糖酵解的活化是通过己糖激酶(HK)I的转录和转录后调节而发生(Haas等人,2015)。因此,我们在CD28和CTLA-4刺激后4小时通过扩展的Treg分析了许多糖酵解酶的表达。CD28刺激导致Treg适度增加HKI、烯醇酶和醛缩酶表达(图16A-B)。预料不到的是,观察到HK同工酶葡萄糖激酶(HKIV或GCK)的表达是最大幅度地增加,HK同工酶是肝细胞和胰β细胞功能的限速酶,在先尚未报道其T细胞的表达。伴随的CTLA-4触发抑制CD28诱导的酶表达。
我们也将分析扩展到了HKII,并且观察到,与GCK一样,该酶的表达显著增强,不仅在CD28信号刺激后而且在重组ICAM-1触发LFA-1后5分钟出现(图17A),这启示这两种刺激也都通过转录后机制增强了酶的表达。
然后,我们测定了GCK、HKI和HKII基因的转录,实际上其通过LFA-1或CD28刺激而增加(图17B,未显示HKI和HKII的数据)。正如预期的,CTLA-4触发阻止了CD28的酶诱导。在这组试验中,GCK调节蛋白(GCKR)基因(图17C)即转录后的调节剂的转录可阻断游离的胞质GCK(Farrelly等人,1999)。CD28和LFA-1活化后GCK和GCKR的表达和共定位的共聚焦分析证实,CD28和LFA1刺激均会减少GCKR表达,伴随显著增加GCK的利用率(图18A-B)。
为了解决HKII和GCK活性对Treg运动的相对贡献,用GCK活化剂AZ D1656(Bonn等人,2012)或HKII选择性抑制剂克霉唑(CLT)处理Treg,其抑制CD3/CD28刺激的Tconv细胞中糖酵解通量和呼吸作用(图19A-B)(van der Windt等人,2013)。GCK活化显著增强了Treg向发炎组织的迁移(图20A),但不影响Treg的分裂(图20B)。反之亦然,CLT有效抑制Treg(图20C)响应于同种异体DC的PCNA上调,但不影响rICAM-1介导的诱导糖酵解(图21A)或Treg体内迁移至发炎的腹膜(图21B)。
实施例4-GCK活化对皮肤应答(grant)排斥的影响
为了确定Treg使用GCK来维持其运动性的选择性,我们比较了GCK药理活化对在C57BL/6接受者中移植的B6Kd衍生的皮肤移植物的存活的影响。移植后,每天用选择性GCK活化剂AZD1656(10mg/kg,每天两次)对一些接受者处理2周。设计该时间表的目的在于在异常响应的早期促进内源性Treg的迁移。对照组单独接受具有媒介物的盐水溶液。如图22所示,单独使用AZD1656进行短期治疗足以显著延迟皮肤移植物的排斥。
实施例5-来自功能缺失的GCKR基因纯合携带者的人Treg展示出增强的运动力
为了测试上述鉴定的途径在人系统中的生理相关性,我们分析了来自GCKR基因(C至T,P446L)功能缺失多态性纯合子携带者的循环Treg(定义为CD25high CD127low)的数量和功能行为。P446L-GCKR基因对GCK的抑制活性降低,并且与禁食血糖降低和肝中GCK活性增加相关的甘油三酯合成增加有关(Beer等人,2009)。
与WT等位基因的携带者相比,GCKR基因的罕见等位基因P446L携带者的循环Treg数量显著减少(图23A-B),而其它CD4+ T细胞群体未受影响(数据未显示)。重要的是,与WT-GCKR Treg相比,P446L-GCKR Treg展示出趋化因子诱导的运动性显著增加,而Tconv细胞迁移未受影响(图24A-B)。P446L-GCKR Treg的抑制能力和表型与WT-GCKR Treg的抑制能力和表型没有显著差异(数据未显示),包括CCR7的表达(图25A-B),CCR7为迁移测定中所用趋化因子的受体。此外,在P446L-和WT-GCKR Treg和Tconv中,趋化因子诱导的GCK活化上游的分子事件、包括葡萄糖摄取和丝氨酸473位上的Rictor-依赖性AKT磷酸化相差无几(数据未显示),这进一步支持了GCK可利用性增加在增强P446L Treg运动性中的关键作用。
1.1讨论
本发明人已经研究了维持胸腺Treg迁移的代谢途径,以及这些途径如何通过促迁移信号而参与。
数据表明,迁移刺激诱导Treg向需氧糖酵解的代谢重编程。Treg增殖过程中糖酵解调节与迁移之间的二分法反映在参与这些细胞响应的酶促机制中。常规T细胞抗原激活后的代谢重编程涉及己糖激酶活性的显著增加,这取决于HK同工酶表达从HKI到HKII的转录开关,其对葡萄糖具有更高的亲和力(Bosca等人,1988;Marjanovic等人,1990;Wang等人,2011)。此外,-并且作为mTORC2活化的可能结果-促迁移信号显著增强GCK表达。已证明GCK是肝中mTORC2-介导的信号传导下游的主要靶标之一(Hagiwara等人,2012)。
本发明人提供了该途径在人免疫系统中起作用的证据。GCKR功能缺失型变体的携带者中循环Treg的数量显著减少,而突变型Treg展示出显著增强的运动能力,这启示在组织中的定位增加。重要的是,Tconv的迁移不受GCKR抑制作用缺失和GCK活性增加的影响。
通过迁移Treg优先利用GCK是一个有意义的特征。与其它己糖激酶不同,GCK对葡萄糖的亲和力要低得多,这在生理血浆葡萄糖范围内(S0.5≈7mM),并且对糖酵解代谢物葡糖6-磷酸的抑制作用敏感性较低(Lenzen,2014)。Treg使用GCK可能解释了在先的报告,即响应CD28刺激,与Tconv细胞相比,Treg细胞的极化速率延迟(Muller等人,2008)。已显示CD28刺激的Treg经历早期和晚期迁移波,这可能反映了我们观察到的GCK表达的转录后和转录增加。
在免疫应答期间,CD28和CTLA-4充当代谢开关的能力至少部分解释了它们对T细胞分裂和功能的相反作用。例如,激活后短暂的CTLA-4表达可用于关闭糖酵解,从而支持将长寿命记忆CD8+ T细胞重编程为FAO(O’Sullivan等人,2014),由此通过有利于诱导无能促进T细胞响应的收缩期并且维持体内平衡(Zheng等人,2009)。
尽管已经很好地建立了CTLA-4在调节效应子免疫性方面的功能作用,但尚未完全了解CTLA-4对Treg功能的贡献。CTLA-4-缺陷型小鼠展示出正常数量的Treg,但显然在其体内抑制功能方面存在缺陷(Wing等人,2008),但在体外却没有(Tang等人,2004)。本文的观察结果表明,通过拮抗CD28诱导的迁移信号,CTLA-4是Treg定位体内(例如)的组织保留而不是其抑制活性所必需的。
CD28Y170F Treg响应先天性免疫活化的迁移受损也表明CD28信号指示Treg的动员和从淋巴组织-其中CD28参与可能发生在与活化的DC相互作用期间-到血流的重新分布。
总而言之,本研究描述了用于通过促迁移刺激对Treg中诱导的运动性和迁移性进行代谢调节的新途径。由于参与增殖Treg细胞的代谢重编程的信号传导介体不同于调节运动性的那些,因此这些酶的选择性靶向允许在治疗环境中调节不同的Treg功能。
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Claims (23)
1.糖酵解活化剂在制备用于治疗或预防自身免疫障碍或移植相关障碍的药物中的用途,所述治疗或预防通过运输内源性调节性T细胞(Treg)介导,其中糖酵解活化剂为葡萄糖激酶(GCK)-活化剂AZD1656:
或其药学上可接受的盐。
2.根据权利要求1的用途,其中自身免疫障碍或移植相关障碍选自:移植排斥、系统性红斑狼疮(SLE)、多发性硬化(MS)、银屑病、I型糖尿病、桥本甲状腺炎、自身免疫性甲状腺炎(AITD)、心肌炎、心肌梗死(MI)、病毒或细菌感染、类风湿性关节炎、自身免疫性胃炎、抗肾小球基底膜肾炎、自身免疫性肝炎、原发性胆汁性肝硬化(PBC)、斑秃、自身免疫性孕酮皮炎、自身免疫性荨麻疹、寻常型天疱疮、自身免疫性多内分泌综合征(APS;除外3型APS,又称为IPEX综合征)、自身免疫性胰腺炎、格雷夫斯病、舍格伦综合征、抗磷脂综合征、自身免疫性溶血性贫血、自身免疫性血小板减少性紫癜、恶性贫血、混合性结缔组织病(MCTD)、未分化结缔组织病(UCTD)、银屑病关节炎、复发性多发性软骨炎、风湿热、皮肌炎、重症肌无力、多发性肌炎、急性弥漫性脑脊髓炎(ADEM)、格-巴综合征、桥本脑病、横贯性脊髓炎、结节病、自身免疫性葡萄膜炎、自身免疫性内耳疾病(AIED)、贝赫切特病、巨细胞动脉炎、肉芽肿合并多脉管炎(EGPA)、血管炎和腹部疾病。
3.根据权利要求1的用途,其中自身免疫障碍为心肌炎。
4.根据权利要求1的用途,其中移植相关障碍为移植物抗宿主病(GVHD)或同种异体移植排斥。
5.根据权利要求1的用途,其中移植相关障碍为移植排斥。
6.根据权利要求1的用途,其中自身免疫障碍为I型糖尿病。
7.根据权利要求1的用途,其中自身免疫障碍为类风湿性关节炎。
8.根据权利要求1的用途,其中自身免疫障碍或移植相关障碍选自移植后糖尿病和局部缺血-再灌注损伤。
9.根据权利要求1-8任一项的用途,其中将所述糖酵解活化剂与一种或多种另外的治疗剂联合施用。
10.根据权利要求9的用途,其中所述另外的治疗剂选自:免疫抑制剂、细胞治疗、致耐受性树突细胞治疗、抗炎剂和激素替代治疗。
11.根据权利要求1-7和10任一项的用途,其中所述糖酵解活化剂以1mg-500mg的剂量单位形式施用。
12.根据权利要求11的用途,其中所述糖酵解活化剂以1、10、20、50、100、200、250或500mg的剂量单位形式施用。
13.根据权利要求12的用途,其中所述糖酵解活化剂以100mg的剂量单位形式施用。
14.根据权利要求1-7、10、12和13任一项的用途,其中通过口服途径施用所述糖酵解活化剂。
15.根据权利要求1-7、10、12和13任一项的用途,其中将所述糖酵解活化剂每日施用1次、2次、3次或4次。
16.根据权利要求15的用途,其中将所述糖酵解活化剂每日施用2次。
17.根据权利要求1-7、10、12、13和16任一项的用途,其中将所述糖酵解活化剂施用1周-6个月的时间期限。
18.根据权利要求17的用途,其中将所述糖酵解活化剂施用
(a)1-6个月的时间期限;或
(b)2-4个月的时间期限;或
(c)3个月的时间期限。
19.根据权利要求1-7、10、12、13、16和18任一项的用途,其中通过口服途径,以100mg的剂量单位形式,每日2次,施用所述糖酵解活化剂3个月的时间期限。
20.根据权利要求1-7、10、12、13、16和18任一项的用途,其中通过口服途径,以每kg体重0.5mg的每日剂量施用所述糖酵解活化剂。
21.糖酵解活化剂在制备用于治疗或预防疾病或医学病症的药物中的离体用途,其中治疗或预防是通过运输或动员Treg细胞介导的,包括通过使Treg细胞与糖酵解活化剂接触来活化Treg细胞中的糖酵解的步骤,其中糖酵解活化剂为GCK-活化剂AZD1656或其药学上可接受的盐。
22.GCK活化剂在制备用于治疗或预防疾病或医学病症的药物中的离体用途,其中治疗或预防是通过增加Treg细胞定位于炎性部位的效率介导的,包括用GCK活化剂预处理自体和/或同种异体Treg细胞的步骤,其中GCK活化剂为AZD1656或其药学上可接受的盐。
23.根据权利要求21或22的用途,其中制备药物包括下列步骤:
a)从待治疗的个体中分离Treg细胞和/或从另外的个体或来源中分离Treg细胞;
b)扩增来自步骤(a)的Treg细胞以获得用于治疗的足够数量;
c)将来自步骤(b)的Treg细胞与GCK-活化剂AZD1656或其药学上可接受的盐接触或用GCK-活化剂AZD1656或其药学上可接受的盐预处理来自步骤(b)的Treg细胞。
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