CN1269530C - 用于结合血管生成性靶组织以在手术中显影肿瘤边缘的抗体-染料结合物 - Google Patents
用于结合血管生成性靶组织以在手术中显影肿瘤边缘的抗体-染料结合物 Download PDFInfo
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- CN1269530C CN1269530C CNB00813295XA CN00813295A CN1269530C CN 1269530 C CN1269530 C CN 1269530C CN B00813295X A CNB00813295X A CN B00813295XA CN 00813295 A CN00813295 A CN 00813295A CN 1269530 C CN1269530 C CN 1269530C
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Abstract
本发明涉及适合于结合新形成的血管的抗体-染料结合物以及在手术中显影病理血管生成的应用。
Description
本发明涉及适合于结合新形成的血管结构的抗体-染料结合物以及在手术中显影病理血管生成的应用。
在成年人机体中,除非常少的个别情况(如孕龄妇女的周期)外,不发生新的血管形成。但是,在许多疾病中却可观察到新的血管形成。发生新血管形成的过程在此称为血管生成,而且作为对某些信号的应答而发生。
血管生成是一个优选在病灶的边缘区域发生的过程。由病灶的中心释放因子,扩散至病灶的边缘区域。这些因子也称为血管生成刺激剂。如果这些血管生成刺激剂到达病灶边缘区域周围的健康组织,预先没有与病灶成为一体的血管被刺激形成新的血管芽。由这些血管芽生长的血管在病灶的边缘区域中形成新的毛细血管网络。通过该过程,总是可以确保对病灶的合适营养输送。现已证明特别重要的是,肿瘤及其转移物的生长取决于诱导血管生成的能力。
在治疗局部的病灶时,手术疗法目前是标准的措施。其在治疗肿瘤时具有非常大的重要意义。但是已经证明,尽管手术技术已有很大的提高,但局部复发的数量仍是相当大的,这是因为在人体中的手术很少能够大规模地除去病灶。在许多器官(如脑)中,必须避免大规模除去病灶,以得到健康组织。损坏健康器官的风险将随着手术介入的剧烈程度而增加。
但是,在完成手术除去肿瘤后,对肿瘤边缘区域的组织学研究表明,相当数量的肿瘤不能被完全除去,而且肿瘤残余仍留在体内。其他的肿瘤生长以及肿瘤转移都有可能源自于这些肿瘤残余。在手术治疗期间精确表明疾病过程相对于健康组织的范围能够使得完全除去病灶,并最大程度地使健康组织不受影响。
用于显影病灶的染料是众所周知的(Poon,W.S.等人,J.Neurosurgery(1992)76:679-686;Haglund,M.M.等人,Neurosurgery(1996)38:308-317)。它们优选从肿瘤细胞直接吸收或者非特异性地累积于肿瘤的细胞外空间。因为即使在健康组织中的浓度也可检测该机理,所以所用物质的特异性和灵敏度较低。
可用于通过选择性地显影病灶的边缘区域而在手术中显出病灶轮廓的化合物目前还是未知的。
血管生成优选发生在病灶的边缘区域中。通过显影血管生成,也可显影健康组织的范围。用于检测病灶中血管生成的抗体是已知的,而且可用于显影组织学组织切片中的新形成血管,用于检测病灶中的各种蛋白或者作为治疗物质的载体分子。
然而,可用于通过选择性地显影病灶的边缘区域而在手术中显出病灶轮廓的与染料结合的抗体,即所谓的抗体-染料结合物仍是未知的。
因此,本发明的目的是制备用于手术中显影肿瘤边缘的抗体-染料结合物。根据本发明的抗体-染料结合物中的抗体针对血管生成过程中特异性的物质。根据本发明的抗体-染料结合物包括可通过其浓集而光学显影的染料。
因为血管生成在病灶的边缘区域最强,在此产生最大的光学信号。
因此,根据本发明的抗体-染料结合物适合于通过手术中的光学诊断显影相对于健康组织的病灶范围,即所谓的边缘区域。其结果是,可完全除去病灶,而最大程度地不影响健康组织。
针对血管原性组织中强烈表达而且在相邻组织中仅非常低水平表达的物质的抗体是已知的(WO 96/01653)。
针对血管生长因子的受体、炎症介质所结合的内皮细胞上的受体、基质分子所结合的内皮细胞上的受体、以及在新血管形成中特异性表达的基质蛋白的抗体,对于抗体-染料结合物是特别有用的(Brekken等人,Cancer Res.(1998)58:1952-9;以及Schold,S.C.Jr.等人,Invest.Radiol.(1993)28:488-96)。
抗基质蛋白EDB-纤连蛋白的抗体或者抗体片段是优选的。EDB-纤连蛋白(EDBFN)也称为胎性癌纤连蛋白,是纤连蛋白的剪接变体,可在血管生成过程中特异性地形成新形成血管。抗EDB-纤连蛋白的抗体的特别优点包括:在除去病灶时不会由于手术中的损伤在健康组织中新形成任何EDB-纤连蛋白。在此方面,在手术过程中都保持该特异性。但是,在接近病灶的健康组织中,在手术中仍然新形成抗内皮细胞上的生长因子受体或者炎症介质而且在肿瘤边缘区域中特异性表达的抗体。
在本发明的抗体-染料结合物中特别优选的是抗EDB-纤连蛋白的抗体L19和E8(Viti,F.等人,Cancer Res.(1999)59:347-352)。此等抗体染料结合物也是本发明的主题。
已知的抗体与染料结合,该染料在组织中的浓集可光学检测,而且可在手术中显影病灶边缘区域的轮廓。
根据本发明的抗体-染料结合物的优点是,其可用于选择性地荧光染色新血管原性阶段的组织。荧光染色是肿瘤特异性的,而且产生可以高信噪比检测的荧光信号。
用于荧光成像的抗体-染料结合物对于经皮非侵入性地显影肿瘤也是已知的(Neri,D.等人,Nature Biotechnology(1997)15:1271-1275)。但是,优选累积于病灶边缘区域中的抗体-染料结合物仍是未知的。
用于手术中肿瘤显影的蛋白-染料结合物也是已知的。
这些结合物的缺陷是,特别缺氧而且代谢营养不足的肿瘤细胞吸收结合物。但是,因为肿瘤边缘区域中的组织是可良好血管化的,而且在此方面细胞被供给充足的氧和营养,所以已知的蛋白-染料结合物不可能充分地累积。
然而,根据本发明的抗体-染料结合物很大程度上独立于病灶的代谢状态。
虽然可以用不同的方式对病灶的范围进行光学检测,但通常优选检测相应的刺激光所诱发的染料特异性荧光辐射。根据发射波长,在此情况下荧光可直接用肉眼检测或者用显微镜检测,并同时任选地用成像检测系统进行数字记录,在显示器上观看。
400-650nm光谱范围的荧光辐射是可肉眼检测的。特别优选的波长是450-600nm。使用可见光的特别优点是,检测荧光的技术成本低。用合适的激光或者激光二极管产生的刺激光连接至光纤光导器中,并通过后者传递到待诊断的区域。用大规模的辐射面积可对手术中的肿瘤边缘实施检测。反射的刺激光被滤光器(例如,研究人员配戴的滤光镜)阻断,而仅有染料特异性的荧光被观察到(肉眼观察)。或者,可用显微镜来检测荧光(显微镜观察)。通过可见光在组织中的小的穿透深度(几毫米),可按此方式检测位于表面上的新血管形成。
光谱范围的可见光的另一个优点是,在组织中的穿透深度以及由组织中的发射小。因此,可检测的信号不会被来自更深部分的组织的信号扭曲,而且可特异性地确定在表面上可见的组织结构。
本发明的主题还包括抗体-染料结合物,其中所述染料诱发可见光范围的光信号。
但是,使用其中染料吸收近红外范围的光(NIR,600-900nm)的抗体-染料结合物可检测更深层组织(最大达1cm)的新血管形成,这是因为NIR光的组织吸收性更弱,因此具有更大的组织穿透深度。不可能用肉眼观察荧光,但可通过设置在目标组织区域上的CCD摄像机(电耦装置摄像机)来进行。肉眼检测和显微镜检测是可能的。如果必须评估被遮盖的区域(如血液),在抗体-染料结合物中使用在NIR光谱范围中有吸收并发出荧光的染料的优点发生作用。
从光物理的角度看,在400-800nm的光谱范围具有最大吸收而且在500-900nm的范围内具有至少一个最大荧光的染料对于抗体-染料结合物是合适的。
本发明的主题还包括抗体-染料结合物,其特征在于,在仅使用可见光或者近红外光范围的确定波长时,染料诱发荧光信号。
抗体-染料结合物中所包括的具有可肉眼检测的荧光的染料例如选自于以下组中:
荧光素、荧光素-异硫氰酸酯、羧基荧光素或者钙黄绿素、四溴荧光素或者曙红、四碘荧光素或者赤鲜红、二氟荧光素,如Oregon GreenTM488、Oregon GreenTM 500、或者Oregon GreenTM 514,羧基对甲氨基酚(Rhodol GreenTM)-染料(US 5,227,487、US 5,442,045),羧基若丹明染料(如Rhodamine GreenTM染料)(US 5,366,860);
4,4-二氟-4-硼杂-3a,4a-二氮杂-indacene,如Bodipy FL、Bodipy 493/503、或Bodipy 530/550及其衍生物(US 4,774,339、US5,187,288、US 5,248,782、US5,433,896、以及US 5,451,663);
菁染料,特别是羰花菁染料和部花菁染料;
香豆素染料,如7-氨基-4-甲基香豆素,DTPA或者四氮杂大环烯(环烯、pyclene)与铽或者铕的金属络合物或者四吡咯染料,特别是卟啉。
包含近红外染料的抗体-染料结合物例如选自于以下组中:
多亚甲基染料,如二羰花菁、三羰花菁、部花菁和氧杂菁染料(WO96/17628);若丹明染料;苯并恶嗪或者苯并噻嗪染料;四吡咯染料,特别是苯并卟啉、chorine和酞菁。
抗体-染料结合物中优选的近红外染料是在700-800nm之间具有最大吸收的花菁染料,特别是茚二羰花菁和茚三羰花菁。
在抗体-染料结合物中通常优选的染料是以上组中具有一个或者多个羧基者,该羧基在化学活化后与抗体或者抗体片段的氨基偶联。那些包含马来酰亚胺基或者溴代烷基的衍生物也是优选的,使得可以与氨基酸半胱氨酸的巯基共价偶联。另外,具有异硫氰酸基的染料也是优选的,该基团也可以与氨基反应。
再者,抗体-染料结合物中的染料必须具有高度的光稳定性,而且在光的辐射(光漂白)下不会被漂白,以确保研究期间恒定的信号。
本发明的主题是抗体-染料结合物,该结合物累积于病灶的细胞组织的边缘区域,并由此使病灶的边缘区域可光学检测。
具体而言,本发明的主题是以下通式I表示的抗体-染料结合物:
B-(F)n (I)
其中:
B代表与ED-BFN具有高度结合性的抗体或者抗体片段;
F代表选自于以下组中的染料:香豆素、荧光素、羧基荧光素、二氟荧光素、四溴荧光素、四碘荧光素、若丹明、羧基若丹明、羧基对甲氨基酚、4,4-二氟-4-硼杂-3a,4a-二氮杂-indacene、多亚甲基染料、或者四吡咯染料、或者DTPA或者环烯与铽或者铕的金属络合物及其衍生物;和
n代表1-5。
本发明的抗体-染料结合物特别优选的是,染料为花菁染料、部花菁染料、氧杂菁染料、苯乙烯基染料或者squarilium染料。
本发明的抗体-染料结合物特别优选染料部分是花菁染料,特别是羰花菁、二羰花菁或者三羰花菁。
因此,本发明还特别涉及以下抗体-染料结合物,其中通式I中的染料-(F)n是以下通式II的花菁染料:
其中:
D代表以下基团III或IV,
其中星号标记的位置代表与基团B的连接位置,以及
B代表以下基团V、VI、VII、VIII或IX,
其中
R1和R2代表C1-C4磺烷基,饱和或不饱和、直链或支链的C1-C50烷基链,该烷基链任选被最多15个氧原子、和/或最多3个羰基、和/或最多5个羟基取代,
R3代表基团-COOE1、-CONE1E2、-NHCOE1、-NHCONHE1、-NE1E2、-OE1、-OSO3E1、-SO3E1、-SO2NHE1或-E1,其中E1和E2相互独立地代表氢原子、C1-C4磺烷基、饱和或不饱和、直链或支链的C1-C50烷基链,该烷基链任选被最多15个氧原子、和/或最多3个羰基、和/或最多5个羟基取代,
R4代表氢原子或者氟、氯、溴或碘原子,
b代表2或3,
X代表氧、硫、或者基团=C(CH3)2或-(CH=CH)-,以及
L代表直接键或者连接基团,该连接基团是具有最多20个碳原子的直链或支链碳链,该碳链可被一个或多个-OH、-COOH、或SO3基团取代,和/或插入一个或多个-O-、-S-、-CO-、-CS-、-CONH-、-NHCO-、-NHCSNH-、-SO2-、PO4-、或-NH-或者芳基环。
根据本发明的抗体-染料结合物可单独使用或者配制成制剂使用。
在以制剂形式使用抗体-染料结合物时,除抗体-染料结合物外,该制剂还包含药物学上可接受的适合于肠道或非胃肠道给药的无机或有机惰性载体,如水、明胶、阿拉伯胶、乳糖、淀粉、硬脂酸镁、滑石、植物油、聚亚烷基二醇等。该药物制剂可为固体剂型,如片剂、包衣片、栓剂、胶囊,或者是液体剂型,例如溶液剂、混悬剂或乳剂。该制剂还可任选地包含辅剂如防腐剂、稳定剂、湿润剂或者乳化剂、用于改变渗透压的盐或者缓冲剂。
在非胃肠道使用时,注射液或者混悬剂、特别是抗体-染料结合物的水溶液是合适的。
还可使用载体系统,例如表面活性剂,如胆酸盐或者动物或植物磷脂,以及这些物质的混合物和脂质体或其组分。
在口服使用时,与滑石和/或烃载体或粘结剂如乳糖、玉米淀粉或马铃薯淀粉组合使用的片剂、包衣片或胶囊是特别合适的。也可以液体剂型给药,如其中添加有甜味剂的糖浆剂。
抗体-染料结合物的剂量可根据以下因素变化:给药方法、患者的年龄和体重、待治疗疾病的类型和严重程度等。在检测有限的区域时,抗体-染料结合物的适用剂量为0.5-1000mg,优选50-200mg,其中该剂量可以单剂量一次性给药或者分为两个或多个日剂量。
上述制剂和剂型也是本发明的主题。
因此,本发明还涉及药物,其包括一种或多种用于手术中显影病灶的边缘区域的抗体-染料结合物,其中该药物可单独使用或者与合适的溶剂、缓冲剂和/或载体混合使用。
根据本发明的抗体-染料结合物可用于手术治疗血管生成依赖性疾病,例如恶性肿瘤及其转移瘤、良性肿瘤、癌前组织变化、子宫内膜异位、血管瘤、和异位怀孕。
本发明的主题还包括抗体-染料结合物和药物在手术中显影病灶的应用,特别是在手术中肉眼观察或者显微观察病灶的边缘区域的应用,以及在制备用于手术治疗血管生成依赖性疾病的药物中的应用,所述疾病是恶性肿瘤及其转移瘤、良性肿瘤、癌前组织变化、子宫内膜异位、血管瘤、和异位怀孕。
染料的制备
染料是根据本领域已知的方法来制备的。适合于制备抗体-染料结合物的染料是具有羧基或者异硫氰酸基的染料,以与抗体上的氨基结合。在此情况下,特别优选的是花菁染料(Mujumdar,S.R.等人(1996)7:356-362;Flanagan,J.H.等人(1997)8:751-756;以及Licha,K.等人(1996)Proc SPIE,第2927卷,192-198)。
具有羧基的染料首先根据本领域已知的方法转化为反应性酯(如N-羟基琥珀酰亚胺酯),由此进行活化。具有异硫氰酸基团的染料可直接使用。反应性衍生物然后在缓冲液或者有机溶剂的混合物(如二甲基甲酰胺(DMF)或者二甲基亚砜(DMSO))中与抗体反应。在此情况下,使用3-100倍摩尔过量的染料。在反应完全后,未反应的部分通过超滤和/或色谱法进行分离。
也可用类似的方法制备以下染料。
制备实施例1
二-1,1′-(4-磺基丁基)茚羰花菁-5-甲酸-N-羟基琥珀酰亚胺酯
按照类似于文献中已知的方法,由1-(4-磺基丁基)-2,3,3-三甲基-3H-假吲哚和1-(4-磺基丁基)-2,3,3-三甲基-5-羧基-3H-假吲哚起始制备二-1,1′-(4-磺基丁基)茚羰花菁-5-甲酸(Cytometry 10,11-19,1989;Talanta 39,505-510,1992)。
为转化成N-羟基琥珀酰亚胺酯,0.1mmol的染料(67mg在10ml的DMF中)与分别为0.5mmol的N-羟基琥珀酰亚胺和二环己基碳化二亚胺(DCC)混合,然后在室温下搅拌24小时。添加50ml的乙醚后,过滤出固体沉淀物,再用少量的DDF重新溶解二次,沉淀物用乙醚洗涤,然后真空干燥(产率89%)。
抗体-染料结合物的制备
制备二-1,1′-(4-磺基丁基)茚羰花菁与L19抗体的结合物
抗体L19(1mg在1ml乙酸钠缓冲液50mM中,pH 8.2)与N-羟基琥珀酰亚胺酯(4mg/ml在DMSO中的溶液75μmol)混合,然后在室温下搅拌2小时。在PD10柱(Pharmacia)上用凝胶过滤进行纯制,然后使用Centricon-10管(Amicon)进行浓缩,同时得到约1mg/ml的抗体溶液。
最大吸收:555nm
最大荧光:582nm
以下实施例是用于说明本发明的抗体-染料结合物的生物应用性,但其并不仅限于该应用实施例。
应用实施例1
体内对携带肿瘤的裸鼠进行荧光成像并在活体外显微检查肿瘤组织
在注射于携带肿瘤的裸鼠后,体内检查根据本发明的化合物的成像性质。为此目的,静脉给药0.1-2μmol/kg的物质,然后在0-48小时内观察肿瘤区域中的浓度。用相应波长的光照射动物,由此诱发物质的荧光,所述光是用激光单色产生的(二极管激光、固态激光)或者是用滤光器由Hg或Xe灯的多色发光中过滤出来的。在使用如制备实施例1中描述的化合物时,使用由Nd:YAG激光产生的波长为540nm的光进行激发,以刺激试验动物,然后用增强型CCD摄像机检测>580nm波长处的荧光辐射,同时得到整体荧光图象。平行地用肉眼及照相检测荧光。由肿瘤材料制备切片,并用显微镜(Zeiss Axiovert显微镜,具有Cy3滤光器)进行研究。
在携带F9-畸胎样瘤的裸鼠中注射制备实施例1中提到的抗体-染料结合物1μmol/kg,接着可在4小时后检测荧光信号的增加,以便根据整体荧光图象与正常组织进行对比。
在制备肿瘤的表皮以及最上层组织后,荧光可以与肿瘤的边缘区域结合。显微镜评估肿瘤切片产生更高的荧光,该荧光用肿瘤边缘区域的血管进行校正。
Claims (9)
1、一种抗体—染料结合物,其优先在病灶的细胞组织的边缘区域中累积,并由此使得可以光学检测病灶的边缘区域,该结合物的特征在于是以下通式I的化合物:
B-(F)n (I)
其中:
B代表抗体L19;
(F)n代表以下通式II的花菁染料:
其中:
D代表以下基团III或IV,
其中星号标记的位置代表与基团B的连接位置,以及
B代表以下基团V、VI、VII、VIII或IX,
其中
R1和R2代表C1-C4磺烷基,
R3代表基团-COOE1或-E1,其中E1相互独立地代表氢原子,
R4代表氢原子,
b代表2或3,
X和Y代表=C(CH3)2,以及
L代表直接键或者连接基团,该连接基团是具有1-20个碳原子的直链或
支链碳链,该碳链可被一个-OH取代和/或插入1或3个-O-、
-CO-、-CONH-、-NHCO-或-NH-。
2、如权利要求1所述的抗体—染料结合物,其中染料在可见光范围内诱发光信号。
3、如权利要求1或2所述的抗体—染料结合物,其中仅在使用确定波长范围的可见光或者近红外光时染料诱发光信号。
4、一种用于手术中显影病灶边缘区域的药物,其包含一种或多种如权利要求1-3之一所述的抗体—染料结合物。
5、如权利要求4所述的药物,其是与合适的溶剂、缓冲剂和/或载体的混合物。
6、如权利要求1-3之一所述的抗体—染料结合物在制备用于手术中显影病灶的药物中的应用。
7、如权利要求1-3之一所述的抗体—染料结合物在制备用于手术中显影病灶边缘区域的药物中的应用。
8、如权利要求1-3之一所述的抗体—染料结合物在制备用于手术中肉眼观察或者显微观察病灶的边缘区域的药物中的应用。
9、如权利要求1-3之一所述的抗体—染料结合物在制备用于手术治疗血管生成依赖性疾病的药物中的应用,所述疾病是恶性肿瘤及其转移瘤、良性肿瘤、癌前组织变化、子宫内膜异位、血管瘤、和异位怀孕。
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ZA200203225B (en) | 2003-07-23 |
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