CN103432079A - 含有寡核苷酸的微球体及其在制备用于治疗1型糖尿病的药物中的应用 - Google Patents

含有寡核苷酸的微球体及其在制备用于治疗1型糖尿病的药物中的应用 Download PDF

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CN103432079A
CN103432079A CN2012105929369A CN201210592936A CN103432079A CN 103432079 A CN103432079 A CN 103432079A CN 2012105929369 A CN2012105929369 A CN 2012105929369A CN 201210592936 A CN201210592936 A CN 201210592936A CN 103432079 A CN103432079 A CN 103432079A
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特伦斯·L·斯克特
黛博拉·拉弗里尼埃
尼克·吉安努卡基斯
韦雷德·比什克－利布
拉里·R·布朗
珍妮弗·梅琴
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Baxter International Inc
University of Pittsburgh
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Abstract

本发明涉及包含用于治疗1型糖尿病的寡核苷酸的微球体，基于微球体的总重量所述寡核苷酸占微球体的约30重量百分数至约100重量百分数，所述微球体平均粒度不大于约50微米。所述寡核苷酸靶向结合初级转录物CD40、CD80、CD86和它们的组合。

Description

含有寡核苷酸的微球体及其在制备用于治疗1型糖尿病的药物中的应用
本申请为国际申请日2005年5月12日、国际申请号PCT/US2005/016689于2006年11月13日进入中国国家阶段、申请号200580015308.2、发明名称“含有寡核苷酸的微球体及其在制备用于治疗1型糖尿病的药物中的应用”的分案申请。
对有关申请的交叉引用
临时专利申请系列号60/570,273，申请日为2004年5月12日，和临时专利申请系列号60/625,483，申请日为2004年11月5日。
发明背景
发明的技术领域
本发明广泛地涉及AS-寡核苷酸的微球体递送以诱导树突状细胞的耐受性，尤其是在非肥胖糖尿病的（NOD）小鼠模型中。更特别地，本发明涉及通过利用完全水溶性条件制备的微球体的药物递送技术，其中微球体中引入了反义（AS）寡核苷酸。这些微球体用作反义方法以预防NOD小鼠体内和原位的自身免疫糖尿病症状。
发明背景
微粒、微球体和微囊是固体或者半固体的粒子，它们的直径小于1毫米，更优选小于100微米，可以由各种原料形成，包括合成的聚合物、蛋白质和多糖。微球体已经在许多不同的应用中使用，主要是分离、诊断和药物递送。
可以使用多种不同的技术从合成的聚合物、天然的聚合物、蛋白质和多糖来制备这些微球体，包括相分离、溶剂蒸发，乳化和喷雾干燥。通常聚合物形成了这些微球体的支撑结构，感兴趣的药物被加入聚合物结构中。用来形成微球体的示例性聚合物包括，如Ruiz在美国专利号5,213,812、Reid等在美国专利号5,417,986、Tice等在美国专利号4,530,840、Tice等在美国专利号4,897,268、Tice等在美国专利号5,075,109、Singh等在美国专利号5,102,872、Boyes等在美国专利号5,384,133、Tice等在美国专利号5,360,610和Southern ResearchInstitute的欧洲专利申请公开号248,531中描述的乳酸和羟基乙酸的均聚物和共聚物(PLGA)；嵌段共聚物如Illum在美国专利号4,904,479中描述的tetronic908和poloxamer407；和Cohen等在美国专利号5,149,543中描述的聚偶磷氮（polyphosphazene）。使用这些聚合物制备的微球体表现低的负载效率，常常仅能在聚合物结构中掺入少量百分数的感兴趣药物。因此实际上必须常常给予大量的微球体以达到治疗效果。
多年来，生物化学家们已经可在市场上买到球形珠子或者粒子作为一种工具。例如，结合到珠子上的抗体形成特异于特定配体的相对大的粒子。大的抗体包覆颗粒通常用于交联细胞表面上的受体用于细胞的活化，它们被结合在固相上用于免疫亲和纯化，和使用结合到颗粒的组织或者肿瘤特异性的抗体将药剂靶向需要的位置，可以用于递送随着时间缓慢释放的治疗剂。
目前可得到的微粒或者珠子的一个缺点是它们生产困难和代价高。通过这些已知的方法生产的微粒有一个宽的粒度分布，常常缺乏一致性，当活性成分处于高浓度时不能显示出长期的释放动力学。而且，用于这些已知方法的聚合物需要溶于有机溶剂中以形成微粒。因此它们必须在设计成能操作有机溶剂的特殊设备中生产。这些有机溶剂可以使包含于微粒中的蛋白质或者肽变性。当给予人类或者动物药物时，残留的有机溶剂会导致毒性。
另外，可得到的微粒很少是能充分地小到适合穿过针孔大小的尺寸，这种尺寸通常用于给予治疗或者适用于经吸入给药。例如，使用聚乳酸羟基乙酸（PLGA）制备的微粒尺寸大并有积聚的倾向。用于注射时，为除去太大的粒子必需采用会导致产品损失的尺寸选择步骤。具有用于注射的适当尺寸的PLGA粒子必须被通过大规格针给药以适应大的颗粒尺寸，这常常引起病人的不适。
通常，许多现在可用的微粒在水性介质中被激活释放出它们的内容物，因此必须把它们冻干以预防预先释放。另外，粒子如使用PLGA体系制备的那些显示出基于侵蚀和扩散的释放动力学。在这类体系中，观察到初始的破裂或者迅速地释放药物。这种破裂效力可以导致对已经被给予粒子的病人有害的副作用。
使用脂质制备的装入靶药物的微粒是已知的。例如，排列在环绕多重含水态区室的双层膜中形成粒子的脂质可被用来装入水溶性的药物用于连续的递送，如Sinil Kim在美国专利号5,422,120中所描述的。这些粒子的尺寸通常大于10微米，被设计用于关节内、鞘内、皮下和硬膜外给药。做为选择，脂质体已经被用于静脉内递送小分子。脂质体呈球状颗粒，由单一或者多重的磷脂和胆固醇双分子层组成。脂质体的尺寸在30微米或者更大，可以携带各种水溶性的或者脂溶性的药物。包括脂质组分的纯度、潜在的毒性、小泡异质性和稳定性、过量的摄入和制造或贮藏期限困难等问题阻碍了脂质体技术。
医药群体的一个目标是递送核酸到动物的细胞用于糖尿病治疗。例如，核酸可以被相对有效地递送到培养基中的细胞（体外），但是当核酸被递送给动物（体内）时，核酸酶导致高速的核酸降解。
除了保护核酸免于核酸酶消化之外，核酸的运载工具必须显示低毒，必须被细胞有效地接纳和有一种明确的、容易被制备的制剂。如临床试验所示，用于递送的病毒载体可以导致体内严重有害的、甚至致命的免疫反应。另外，这些方法在体内有诱导突变效应的可能。经由不同制剂的脂质复合物(如脂质体或者阳离子的脂质复合物）中包装核酸进行递送在体内通常是无效的，并可能产生毒性效应。核酸与各种聚合物或者与肽的复合物显示出不一致的结果，这些制剂的毒性还没有被解决。核酸也已经被装入聚合物基质中用于递送，但是在这些例子中，粒子具有宽的粒度范围，用于治疗应用的有效性还没有被证明。
因此，存在解决核酸递送问题的需要，并有需要进行微球体的开发和发展用于制造微球体的新方法。与微球体有关的细节可以参见Scott等在美国专利号6,458,387、Woiszwillo等在美国专利号6,268,053、6,090,925、5,981,719和5,599,719以及Woiszwillo在美国专利号5,578,709中的描述。这些和这里确定的所有参考文献被合并入本文中作为参考。
发明概要
根据本发明，被递送给树突状细胞的DNA以微球体进行递送。据信这样的递送方式防止了核酸酶接近微球体内部的核酸。有关AS-寡核苷酸的微球体递送，特别是在NOD鼠模型中进行，以诱导树突状细胞的耐受性。微球体是利用含水条件制备的，其中掺入了反义(AS)寡核苷酸。这些微球体被用于体内和原位抑制基因表达和防止NOD小鼠的自身免疫糖尿病症状。
在本发明的一个优选方面，靶向CD40、CD80和CD86初级转录物的三种AS-寡核苷酸被合成，寡核苷酸混合物的水溶液被制备并与聚合物溶液混合。加工之后，提供包含寡核苷酸的微球体并被施用给NOD小鼠。
通过考虑以下详细的说明，本发明的这些和其它方面、目的、特征和优点包括各种组合，将是显而易见的并能清楚地被理解。
附图简要说明
在说明的过程中，将引用附图，其中：
图1是树突状细胞在1型糖尿病中在胰腺胰岛素生成β-细胞的自身免疫破坏中的作用的示意图；
图2是包含β-半乳糖苷酶基因的质粒载体的图表；
图3显示了提供用质粒DNA微球体的转染NIH3T3成纤维细胞的证据的显微照片；
图4是裸质粒DNA和两个根据本发明的质粒DNA微球体制剂在接触DNAase后的琼脂糖电泳凝胶的显微照片；
图5是在四种不同的质粒DNA应用中β-半乳糖苷酶活性的柱状图表；
图6是AS-寡核苷酸和聚L-赖氨酸聚阳离子的微球体的扫描电子显微照片；
图7是AS-寡核苷酸和聚L-鸟氨酸聚阳离子的微球体的扫描电子显微照片显微照片；和
图8是概述在用微球体和按照用于递送三个初级转录物的其它方法处理的三个NOD小鼠组中糖尿病发生率的绘图。
优选实施方案的描述
根据需要，本发明详细的技术方案在这里公开；然而，可以理解的是这里所公开的技术方案仅仅是本发明的示例，它们可以以各种形式体现。因此，在这里公开的细节不被解释成一种限制，而仅仅作为用于主张权利的基础，和作为一种用于教导本领域的技术人员以实际上任何适当的方式不同地使用本发明的代表性基础。
预防自身免疫胰岛素-依赖的糖尿病的最优方案，其是通过制备和注射这里描述的靶向CD40、CD80和CD86的初级转录物的反义（AS）-寡核苷酸微球体来实施的。这些寡核苷酸被设计成能诱导免疫耐受性以预防NOD小鼠模型中胰岛素生成β-细胞的破坏。在图1中图解了导致这些β-细胞破坏的事件。这举例说明了在NOD鼠和人类中，1型糖尿病是如何通过胰腺胰岛素生成β-细胞的自身免疫破坏表现出来的。在临床发作时，人类有10-20%剩余的β-细胞量。这剩余量的节约可以产生适于调节葡萄糖水平的剩余胰岛素水平。本发明的微粒被提供用来干扰图1中举例说明的β-细胞的自身免疫破坏。
可以理解的是树突状细胞（DC）可以被激活，成为在所有的组织中发现的有效抗原递呈细胞并在皮下呈高浓度存在。通过T细胞尤其是在***中，这些抗原呈递树突状细胞起着免疫反应触发器的作用。
图2是包含β-半乳糖苷酶基因的质粒载体图，该质粒载体可用于转染NIH3T3成纤维细胞。通过与加入的β半乳糖苷酶x-gal（5-溴-4-氯-3-吲哚基-β-半乳糖吡喃糖苷）反应产生蓝色的细胞，NIH3T3成纤维细胞与质粒DNA微球体转染的体外证据在图3中显示。
图4举例说明了微球体保护溶液中DNA的能力。这是一张显示经质粒DNA的微球体给予核酸酶保护的琼脂糖电泳凝胶，含有质粒DNA的微球体通常如这里注解生成。在质粒样品1、2和3中，裸质粒DNA暴露于DNAse，带有拖尾表明在三个水平的DNAase应用时发生了质粒DNA的降解。在颗粒1和颗粒2样品中，质粒DNA微球体制剂暴露于DNAase，没有拖尾表明微球体制剂保护质粒DNA免于降解。
图5报告了在四种不同的质粒DNA应用中的β-半乳糖苷酶活性。裸质粒DNA应用显示出极低的水平。使用lipofectamine，一种商业的作为运载工具的阳离子脂质，质粒DNA阳离子脂质复合物应用显示出稍微高一些的水平。实质上更大的活性在两种pDNA微球体中显现，即相应于图4的颗粒1的微球体1和相应于图4的颗粒2的微球体2。
在制备用于自身免疫治疗小鼠中糖尿病的微球体中，三种AS-寡核苷酸溶于水溶液中并与水溶性聚合物和聚阳离子结合。溶液一般在大约60-70℃进行温育，随后冷却至23℃，移走过量的聚合物。据信包含以下三种AS-寡核苷酸序列的微球体被生成，其中星号象征硫醇化:
Seq ID1:CD40-AS：5'C*AC*AG*C C*GA*GG*C*AA*A
GA*C*AC*C A*T*G C*AG*GG*C*A-3'
Seq ID2:CD80-AS：5'-G*GG*AA*A G*CC*AG*G A*AT*CT*A
G*AG*CC*A A*TG G*A-3'
Seq ID3:CD86-AS：5'-T*GG*GT*G C*TT*CC*G T*AA*
GT*T C*TG*GA*A C*AC*G*T*C-3'
更特别地，核酸一般包含约30和约100重量百分数之间的微球体，其具有不大于约50微米的平均粒径。它们一般如下制备。通过混合等分量的三种寡核苷酸溶液，每种溶液含有这三种类型寡核苷酸中的一种，这样制备出寡核苷酸混合物的水溶液。包含这三种类型寡核苷酸的溶液被制备。溶液优选包含大约10mg/ml寡核苷酸。这些与等分量的10mg/ml聚阳离子溶液的储备溶液混合，其中聚阳离子:寡核苷酸的体积比为约1:1到约4:1。聚乙烯基吡咯烷酮和/或聚乙二醇的聚合物溶液被制备并与其它溶液混合。多次的加热、冷却、离心和洗涤提供了一种水悬浮液，它通常可以凝固和冻干形成一种包含寡核苷酸和聚阳离子的微球体的干粉。
根据本发明的微球体是可用于质粒DNA和反义寡核苷酸和其它核酸的无病毒递送工具。它们允许体外递送β-半乳糖苷酶质粒DNA进3T3成纤维细胞。微球体保护质粒DNA阻拦核酸酶活性。在用微球体制剂转染后表达出高水平的β-半乳糖苷酶活性。
包含使人感兴趣的反义寡核苷酸的微球体下调表面细胞抗原CD40、CD80和CD86，已知这些抗原在能导致破坏胰腺的胰岛素生成β-细胞的自身免疫反应的活化中是至关重要的。这可以通过皮下注射到位于皮下的树突状细胞实施。NOD小鼠研究证明能有效的预防β细胞的自身免疫破坏。DNA和寡核苷酸微球体是有效的体外和体内的转染载体。树突状细胞看来似乎摄取寡核苷酸微球体和抑制了表面细胞抗原CD40、CD80和CD86的表达。反义寡核苷酸微球体有效地预防了NOD鼠中糖尿病的发生。
以下的实施例更进一步地解释说明了本发明的某些特征和优点。实施例不被认为是对发明的限定或者其它方面的限制。
实施例1
三种靶向CD40、CD80和CD86初级转录物的AS-寡核苷酸经匹兹堡大学（Pittsburgh，PA）的DNA合成仪合成。AS-寡核苷酸序列是:
Seq ID1:CD40-AS：5'C*AC*AG*C C*GA*GG*C*AA*A
              GA*C*AC*C A*T*G C*AG*GG*C*A-3'
Seq ID2:CD80-AS：5'-G*GG*AA*A G*CC*AG*G A*AT*CT*A
              G*AG*CC*A A*TG G*A-3'
Seq ID3:CD86-AS：5'-T*GG*GT*G C*TT*CC*G T*AA*
              GT*T C*TG*GA*A C*AC*G*T*C-3'
寡核苷酸混合物的水溶液通过混合等分量的三种寡核苷酸溶液制备，每种寡核苷酸溶液包含一种寡核苷酸，最后形成了三种寡核苷酸的10mg/ml的溶液。制备了在diH2O中的10mg/ml的聚L-赖氨酸·HBr（通过Bachem的聚L-赖氨酸·HBr直到50,000，King ofPrussia，PA）。以体积比1:1将聚L-赖氨酸·HBr加入到寡核苷酸溶液中。温和地涡旋混合物。在1M乙酸钠（Spectrum，Gardena，CA）中包含12.5%PVP（聚乙烯基吡咯烷酮,40000道尔顿,Spectrum Chemicals,Gardena,CA）和12.5%PEG（聚乙二醇，3350道尔顿，Spectrum Chemicals，Gardena，CA）的25%的聚合物溶液被制备，pH=5.5。聚合物溶液以2:1的体积比如下加入：750μl的AS-寡核苷酸，0.75ml的聚L-赖氨酸·HBr，3.0ml的PEG/PVP，总体积为4.50ml。
上述批量在70℃温育30分钟，然后冷却到23℃，冷却后，溶液变混浊和发生沉淀。然后将悬浮液进行离心，除去过量的PEG/PVP。产生的沉淀经在去离子水中重悬进行洗涤，接着离心和移去上清液。洗涤过程重复三次。水中悬浮体被冷冻干燥，形成了一种包含寡核苷酸和聚L-赖氨酸的微球体的干粉。
图6显示了1:1比率的聚L-赖氨酸:寡核苷酸物质的扫描电子显微照片(SEM）。装配的微球体的大小为0.5-4μm，平均粒度约为2.5μm。也观察到一种未知物质的沉淀。通过HPLC的补充研究证实沉淀包括残留的PEG/PVP、主要是PVP。
实施例2
靶向CD40、CD80和CD86初级转录物的AS-寡核苷酸是实施例1中的AS-寡核苷酸序列。寡核苷酸混合物的水溶液通过混合等分量的三种寡核苷酸溶液制备，每种寡核苷酸溶液包含一种寡核苷酸，最后形成了三种寡核苷酸的10mg/ml的溶液。寡核苷酸混合物的溶液被制备。制备了在diH2O中的10mg/ml的聚L-鸟氨酸·HBr（通过Sigma的聚L-鸟氨酸·HBr11,900(vis)）。聚L-鸟氨酸·HBr加入到寡核苷酸溶液中，将混合物温和地涡旋。在0.1M乙酸钠（Spectrum，Gardena，CA）中包含12.5%PVP（聚乙烯基吡咯烷酮,40000道尔顿,SpectrumChemicals,Gardena,CA）和12.5%PEG（聚乙二醇，3350道尔顿，Spectrum Chemicals，Gardena，CA）的25%的聚合物溶液被制备，pH=5.5。加入聚合物溶液。接着如实施例1所述进行温育和冲洗。制备总体积为6.0ml，其中AS-寡核苷酸1.5ml，聚L-鸟氨酸·HBr1.5ml，PEG/PVP3ml。
图7显示了1:1比率的聚L-鸟氨酸:寡核苷酸物质的SEM。装配的微球体的大小为0.2-8μm，平均粒度约为2μm。也观察到未知物质的沉淀。HPLC的补充研究能够证实沉淀包括残留的PEG/PVP、主要是PVP。
实施例3
体内试验是利用1型糖尿病的NOD小鼠模型来进行的。1型糖尿病如在图1中解释的是通过胰腺的胰岛素生成β-细胞的自身免疫破坏来表现的。在三种应用中使用AS-寡核苷酸以干扰β-细胞的自身免疫破坏。目标是通过靶向CD40、CD80和CD86的初级转录物干扰树突状细胞的功能，所述的初级转录物编码T细胞活化所需的树突状细胞表面蛋白。已知具有低水平的CD40、CD80和CD86的树突状细胞促进了体内抑制性免疫细胞网络。这些级联可以导致体内T细胞对β-细胞的反应性不足。
在第一组试验动物中，树突状细胞由NOD小鼠的骨髓祖细胞离体（）增殖而来。三种靶向CD40、CD80和CD86的初级转录物的AS-寡核苷酸的组合被添加到组织培养基中的细胞。培养后，AS-寡核苷酸转染的树突状细胞被注射入5－8周龄的有血缘关系的受体（还不是糖尿病患者）中。这是已知的离体的递送方法。
平行地，AS-寡核苷酸微球体被直接地注射进另外的同龄NOD小鼠中。在各种这样处理的小鼠上进行单次注射。另一个组的这些NOD鼠不进行治疗，作为一种对照。
图8显示了对照、未处理的NOD小鼠到23周龄时全部发生了糖尿病。离体AS-寡核苷酸转染的和重新注入树突状细胞的组（AS-ODNDC）显示出糖尿病的延缓发生，其中20%保持无糖尿病，这表明葡萄糖水平在非糖尿病范围内维持。微球体体内注射的NOD小鼠中，在43周时71%的鼠保持无糖尿病。
很清楚已经描述的本发明的技术方案是对本发明的原理应用中的一些解释性说明，本领域技术人员可以进行各种修饰而不背离本发明内涵和外延。在这里描述的各种特征可在任何组合中使用，不局限于在这里特别列出的确定组合。
Figure IDA00002694171200011

Claims (7)

1.包含寡核苷酸的通过直接给药微球体至个体用于治疗1型糖尿病的微球体，其中所述微球体含有靶向CD40的第一反义序列，靶向CD80的第二反义序列，靶向CD86的第三反义序列，其中所述第一、第二和第三寡核苷酸各自分别减少或抑制CD40、CD80和CD86的体内表达，以及其中所述寡核苷酸基于微球体总重量占所述微球体的约30重量百分数和约100重量百分数之间，所述的微球体具有的平均粒度不大于约50微米。
2.权利要求1的微球体，其中所述的寡核苷酸选自Seq ID1、SeqID2或者Seq ID3和它们的组合。
3.权利要求1的微球体在制备用于递送核酸到患有1型糖尿病的个体的药物中的应用，其中所述微球体适于静脉内、肌肉内、皮下、局部、皮内、腹膜内、口、肺、眼、鼻或者直肠途径递送。
4.权利要求1的微球体在制备用于保护非肥胖糖尿病小鼠的胰腺β-细胞免于自身免疫破坏的药物中的应用，其中所述微球体适于皮下注射。
5.权利要求1的微球体在制备用于保护个体的胰腺β-细胞免于自身免疫破坏的药物中的应用，其中所述微球体适于皮下注射。
6.权利要求1的微球体在制备用于保护人的胰腺β-细胞免于自身免疫破坏的药物中的应用，其中所述微球体适于皮下注射。
7.权利要求1的微球体在制备用于保护个体的胰腺β-细胞免于自身免疫破坏和1型糖尿病发病的药物中的应用，其中所述微球体适于皮下注射。
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