Paclitaxel trevatide

点击上方的 行舟Drug ▲ 添加关注脑靶向肽偶联药物研究进展来源《中国药学杂志》2024年作者崔娜，史亚军，白敏，王胜正，丁一陕西中医药大学药学院；空军军医大学第一附属医院药剂科；空军军医大学药学系摘要中枢神经系统（centralnervous system，CNS）疾病严重危害人类的生命健康，但由于血脑屏障（blood–brainbarrier，BBB）的存在，缺乏将药物输送到大脑的有效技术，严重影响其相关药物的开发成功率，致使这类疾病的治疗效果往往不尽如人意。因此，迫切需要一种新技术解决上述问题。脑靶向肽偶联药物由脑靶向肽、连接基团和有效载荷这三部分组成，其利用生物相关的内源性转运机制使生物活性分子透过BBB，并到达脑实质，已成为一种有前景的CNS药物输送系统。本文简要介绍了脑靶向肽、连接基团、有效载荷的种类及特征等，并列举了一些常见的脑靶向肽偶联药物，以及此类药物面临的挑战和改进的方法，以期为后续CNS药物的设计和开发提供新思路。关键词多肽偶联药物；脑靶向肽；连接基团；有效载荷；中枢神经系统；血脑屏障_正文_中枢神经系统（central nervous system，CNS）疾病是世界范围内的主要健康问题之一，尤其是脑卒中、脑癌、神经退行性疾病等。目前，这些疾病还没有行之有效的治疗方法，究其主要原因之一是大多数药物无法充分穿过血脑屏障（blood-brain barrier，BBB）。因此，克服此生物障碍仍然是开发CNS药物的主要挑战。BBB的基本组成部分是神经血管单元（neurovascular unit，NVU），主要由脑毛细血管内皮细胞和脑星形胶质细胞构成。此外，其他细胞也有助于BBB的形成和维持，包括神经元、少突细胞、小胶质细胞和肥大细胞等。然而，与存在于CNS外的大多数器官中的高渗透性脉管系统不同，BBB表现出高的跨内皮电阻（transendothelial electrical resistance，TEER）以及低的转胞率和细胞旁渗透性。研究表明，这都是由于紧密连接（tight junction，TJ）蛋白复合物构成的膜内颗粒形成的链网络相对有效地堵塞了脑微血管内皮细胞（cerebral microvascular endothelial cell，BMEC）之间的细胞旁通路。该结构虽然阻止了有毒物质或病原体进入CNS，但也显著限制了治疗和诊断药物进入大脑的可能性。此外，BBB内皮细胞中存在的ATP结合盒转运体（ABC转运体）将一些可能穿过BBB的化合物外排回血液，进一步限制了CNS中药物和成像探针的可用性。因此，只有极少数的分子被有效地输送到大脑。为了克服限制药物输送到大脑的生物屏障，研究者已经尝试开发了多种策略。其中，一种具有前景的给药策略引起了研究者们的广泛关注。该策略属于一种非侵入给药方式，以脑靶向肽作为载体，将药物以化学键的方式与脑靶向肽偶联，脑靶向肽可以穿过BBB，将药物带入脑实质，从而达到治疗的目的。其结构与作用机制均与抗体偶联药物（antibody drug conjugates，ADC）相似，具体结构见图1A。图 1 脑靶向多肽偶联药物的结构及其常见的穿过BBB 的运输过程A—脑靶向多肽偶联药物的基本结构;B—脑靶向多肽偶联药物通过血脑屏障的主要运输路线图但与ADC相比，脑靶向肽偶联药物由于多肽自身的特性而具有分子量更低，易于大规模合成，成本更低，穿透BBB能力更好且低免疫原性等优点，见表1。由此可见，脑靶向肽偶联药物在精准治疗上已表现出巨大潜力。本文将对脑靶向多肽偶联药物各结构作简单的介绍，并总结一些常见的脑靶向多肽偶联药物，以及此类药物面临的挑战和改进的方法。1脑靶向肽偶联药物的结构1.1 脑靶向肽靶向部分在脑靶向肽偶联药物中起着至关重要的作用。首先，它通过对药物的修饰，可以改变药物的理化性质，增加药物的吸收；其次，它可以将药物输送到指定部位，提高选择性，降低毒副作用，增加疗效。由此可以看出，脑靶向肽对药物的疗效、药代动力学和治疗指标都有显著影响。因此，选择一个合适的多肽是至关重要的。理想的脑靶向肽应具有较强的目标结合亲和力、高稳定性、低免疫原性、高效内化和较长的血浆半衰期等特点。通常选择的脑靶向肽为靶向大脑内特定受体的多肽。这类多肽可以通过受体介导的胞吞作用（receptor-mediated transcytosis，RMT）特异性摄取某些大分子，其具体过程如图1B所示。常见的靶向大脑内特定受体的多肽见表2。据研究，一种针对乳腺癌患者癌细胞脑转移情况的脑靶向肽偶药物ANG4043已经被合成，即Angiopep-2多肽偶联曲妥珠单抗。曲妥珠单抗可以靶向人表皮生长因子受体2（human epidermal growth factor receptor-2，HER2）的胞外结构域，使HER2阳性乳腺癌患者的生存率显著提高。但当癌细胞转移到大脑内时，由于其脑渗透能力差，它就缺乏疗效。因此，研究者选择靶向低密度脂蛋白受体相关蛋白1（low-density lipoprotein receptor-related proteins1，LRP1）的多肽Angiopep-2，与曲妥珠单抗偶联。LRP1是低密度脂蛋白（Low Density Lipoprotein，LDL）受体家族的一员，在BBB毛细血管内皮细胞上高度表达，并已被证明在脑毛细血管内皮细胞中转运多种配体，包括乳铁蛋白（lactoferrin，LF）、载脂蛋白E（apolipoprotein E，ApoE）和β-淀粉样肽等。该多肽偶联药物既保留了对HER2受体的体外结合亲和力和对HER2阳性BT-474乳腺导管癌细胞的抗增殖效力，又增加了脑内皮细胞的吸收。此外，测量颈动脉内递送后的脑暴露水平，发现该多肽偶联物以1.6×10-3mL/(g·s)的脑进入率穿透血脑屏障。最后，在脑内异种移植BT-474细胞的小鼠模型中，表明给予ANG4043治疗后可明显提高生存率。所以，这项研究表明Angiopepe-2与抗HER2单抗的结合可以增加脑内皮细胞的吸收以及BBB的通透性。ANG4043的这些特征导致BT-474脑肿瘤暴露水平较高，可以在全身治疗后延长生存期。既往研究表明狂犬病病毒糖肽（RVG）是由狂犬病病毒G蛋白中的29个氨基酸组成的多肽片段。RVG肽作为特异性配体，可识别γ-基丁酸（γ-aminobutyric acid，GABA）和烟碱乙酰胆碱受体（nAchR，Nicotinic acetylcholine receptor），促进病毒转运至CNS。因此，RVG肽常常用于修饰药物递送系统，以提高药物脑内递送效率。THR是通过噬菌体展示技术筛选出的12聚体多肽，可通过与转铁蛋白受体（TfR）结合，透过BBB，运输金纳米粒到达CNS。这些数据进一步验证了脑靶向多肽药物偶联策略可以作为神经肿瘤学和其他中枢神经神经系统疾病的治疗新方法。某些细胞穿透肽（cell-penetrating peptides，CPPs）也常作为CNS药物的递送载体。CPPs指一般不超过30个氨基酸的多肽。通常表现出有助于跨细胞膜转运的两亲性和净正电荷特征。它们仅通过与暴露的细胞膜相互作用，便可独立地进入细胞，其机制可能为吸附介导的胞吞作用（Adsorptive-mediated transcytosis，AMT），具体过程如图1B所示。这是一种非特异性胞吞作用，由某些大分子的带正电部分与含有阴离子肝素蛋白多糖的脑内皮细胞带负电的膜之间的静电相互作用触发。此外，它们由于具有高细胞渗透性和低免疫原性的特点，也表现出不依赖于受体的特征。这也是它们被认为是安全的和高效的原因。但此类多肽往往缺乏一定的选择性。常见的相关细胞穿透肽见表3。如TAT多肽是来自人类疫缺陷病毒1型（human immunodeficiency virus，HIV-1）的转录激活物TAT蛋白的转导结构域。它可以偶联异源蛋白质或纳米粒等，使其透过BBB。另外，它还可以运送量子点穿过BBB到达脑实质。Liu等在载环丙沙星的纳米胶束表面偶联TAT多肽，结果显示偶联TAT多肽，可以使该胶束穿过BBB，从而增强了人星形胶质细胞对胶束的摄取。SynB1作为一种来自于抗菌肽蛋白1的载体材料，在递送药物透过BBB方面已显示出一定优势。有实验表明SynB1在不影响BBB完整性的情况下，可以显著增强大脑中的吗啡-6-葡糖醛酸酯（M6G，Morphin-6-Glucuronid）的含量。1.2 连接基团完美的多肽偶联药物只有在到达指定部位后才会释放药物，而连接基团在这方面显得尤为重要。即一旦多肽偶联药物到达目标后，连接基团被裂解，药物以完全活性状态释放。所以，连接基团在循环时必须稳定，优先在目标部位裂解，从而确保最大剂量药物到达目标。然而，大多数连接基团在全身给药后，从进入血液那一刻开始就被裂解，当到达指定部位后，所剩不多的药物才被细胞吸收。因此，设计多肽偶联药物选择连接基团时，为避免干扰多肽与其受体的结合亲和力及药效，需要考虑到其所在的微环境，如其长度、稳定性、释放机制、官能团、亲水性/疏水性和其他特性。目前，多肽偶联药物的连接基团分为可裂解基团和不可裂解基团（丁二酰硫醚、肟和三唑等）两大类。可裂解基团又分为pH敏感型（缩醛，酮和碳酸酯）、酶敏感型（酯酶和酰胺酶、氨基甲酸酯）、氧化还原敏感型（二硫键）三大类。在多肽偶联药物全身给药后，最不稳定的位点首先被裂解，其次是其他位点，最终成为包括未改性药物、具有部分连接基团的药物或带有连接基团的氨基酸在内的混合物。虽然在靶向治疗药物的开发中，可裂解的连接基团比不可裂解的连接基团更受青睐。但不可裂解基团具有不会因外部刺激而被裂解（如化学诱导的刺激）的优点，因而在循环中具有更高的稳定性，故其可能不会像可裂解基团那样在血液中就被裂解，并在到达目标部位之前在血液中过早释放一些药物。因此，脑靶向多肽和药物的具体连接方式，应根据实际需求确定。脑胶质瘤是最常见和最致命的原发性恶性脑肿瘤。由于存在药物溶解性差、缺乏肿瘤选择性、穿过BBB的渗透性差、以及广泛的肿瘤内和肿瘤间异质性等棘手问题，大多数治疗在临床上往往以失败告终。但一种只在CNS中表达的细胞外基质（extracellular matrix，ECM）糖蛋白，称为brevican（Bcan），被发现在脑胶质瘤中表现为上调，并与肿瘤侵袭性和侵袭性增加有关。其被称为dg-Bcan的去糖基化亚型仅在人类高级别胶质瘤（包括胶质瘤）组织样本中发现，并在整个肿瘤组织中表达出肿瘤特异性和一致性。这强调了其作为一种新型胶质瘤特异性标记物因而具有开发新的靶向药物的潜力。于是，研究者筛选了一个名为BTP-7靶向肽，该肽在血清中稳定，可以穿过BBB，特异性结合dg-Bcan。于是，就将该多肽与喜树碱（camptothecin，CPT）通过二硫键偶联，得到多肽偶联药物BTP-7-CPT。实验结果表明，该多肽偶联药物在体外对患者源性脑胶质瘤干细胞表现出抑制作用，在人源性脑胶质瘤颅内异种移植（patient-derived tumor xenograft，PDX）小鼠模型中增加了对肿瘤部位的药物输送。而且，与健康脑组织相比，该多肽偶联药物还增强了肿瘤毒性，并延长了动物的生存期。但该化合物仍存在一些不足，虽然天然BTP-7在人血清中稳定超过12小时，但BTP-7-CPT多肽偶联药物在1小时内完全降解，这表明在还未到达肿瘤部位时，血液中的二硫键很可能已经被裂解。因此，需要提高该化合物在血液中的连接基团的稳定性。1.3 有效载荷脑靶向肽偶联药物中的有效载荷，不只局限于治疗药物，还可以是造影剂。将药物与脑靶向多肽偶联后，可以改变药物的理化性质，如溶解度、选择性和半衰期等，使药物具有选择性，可以提高原本药物的疗效，减少毒副作用，改善溶解度，增加吸收。以治疗脑胶质瘤为例，常用的是传统的化疗药物，如紫杉醇（paclitaxel，PTX）、CPT或阿霉素（doxorubicin，DOX）等。其主要是通过各种机制来阻止癌细胞的有丝分裂或促进细胞凋亡，从而达到治疗目的。这类药物虽然有一定的疗效，但也有一定的缺点，如毒副作用大、溶解度差。选择合适的脑靶向肽偶联就可以解决这些问题。研究者根据该策略设计了PTX偶联双功能肽SynB3和序列为PVGLIG的基质金属蛋白酶2（Matrix metalloproteinase-2，MMP-2）敏感肽，用于脑胶质瘤的治疗。实验结果表明，1）SynB3-PVGLIG-PTX与MMP-2表现出较强的亲和力，它可以通过聚合形成带正电荷的特殊结构来提高水溶性；2）MMP-2裂解后，SynB3-PVGLIG-PTX可控制释放PTX，这表明SynB3-PVGLIG-PTX对胶质瘤细胞具有特异性的细胞毒性；3）SynB3-PVGLIG-PTX在体内外均能有效抑制胶质瘤细胞的增殖、迁移和侵袭。此外，SynB3-PVGLIG-PTX的抑制率显著高于阳性药替莫唑胺(Temozolomide，TMZ)和PTX；4）联合使用MMP-2敏感的多肽和CPPs（SynB3），既增强了BBB渗透性，又提高了SynB3-PVGLG-PTX的胶质瘤靶向效应，使药物在治疗期间具有低不良反应的高抗肿瘤活性。除此之外，脑靶向肽偶联药物还可以用于向肿瘤细胞传递小干扰RNA（siRNA）。一旦被靶向，siRNA可以抑制翻译和随后的蛋白质合成。这种结合策略，还有助于对抗耐药性。但如果能够使用不同的成像方式对发病部位的结构功能成像，确定病变部位和状态，就可以更加精准地诊断和治疗疾病。例如美国食品和药物管理局（Food And Drug Administration，FDA）批准的首个含放射性核素的多肽偶联药物111In-DTPA-Octreotide（octrescan），用于治疗神经内分泌肿瘤。然而，研究表明奥曲肽（octreotide）扫描对治疗肿瘤的作用有限，因此该药物就主要用于诊断。而大脑的结构和功能更为复杂，如果能对其成像观察病理生理状态，可以达到更好的治疗效果。近年来为了同时解决药物定位、药物释放和药物疗效的相关问题，已经探索了将药物和显像剂组合在分子或纳米级平台上的多成分结构，称为治疗诊断剂。这种方法，有望显著改善治疗效果欠佳的脑部疾病，如体内分子神经成像等。Ben Woods等设计了[[99mTcO4]−CPepH3]复合物，PepH3多肽为脑靶向肽，CPepH3为客体，99mTcO4放射性标记为主体。研究表明，该复合物能够穿过BBB，其脑积累量高于其他用于脑靶向传递的肽。CPepH3结构呈笼状结构，该笼状支架可以使用不同的技术进行正交成像，如为正电子发射断层扫描成像术（Positron Emission Tomography，PET）设计的客体笼和为磁共振成像（magnetic resonance imaging，MRI）设计的主体笼等。同样，这些超分子金属基结构的坚固性和模块化组成可以引入靶向部分，如多肽或抗体等。目前，该实验室正在探索实现具有不同功能的异质笼，同时封装抗癌药物在内，如顺铂等。文章内容由凡默谷小编查阅文献选取，排版与编辑为原创。如转载，请尊重劳动成果，注明【来源：凡默谷公众号】。2常见的脑靶向肽偶联药物2.1 ANG1005(GRN1005)ANG1005是由一分子Angiopep-2多肽和三分子的PTX通过可裂解的琥珀酰酯偶联而成的，Angiopep-2以LRP-1为靶点，使其穿过血-脑脊液屏障（Blood-Cerebrospinal fluid Barrier，BCB）和BBB，进入肿瘤细胞，在肿瘤细胞中裂解出PTX以发挥其抗肿瘤活性。原位脑灌注实验显示，ANG1005进入大脑的量比PTX更大，并且可以绕过BBB上的P-糖蛋白（P-glycoprotein，P-gp）。体外实验证明ANG1005对人类癌细胞株的抗肿瘤效力与PTX相似；体内实验证明ANG1005对人肿瘤异种移植的抑制作用比PTX更有效，可显著提高脑内植入U87MG胶质母细胞瘤细胞或NCI-H460肺癌细胞的小鼠的存活率。在I期研究中，单次静脉注射该多肽偶联药物，在3-6小时后切除的复发性胶质瘤中可以检测到治疗浓度的ANG1005，这为其通过BBB转运和肿瘤渗透提供了有力证据。在复发性乳腺癌脑转（brain metastases from breast cancer，BCBM）、伴或不伴有软脑膜癌症（leptomeningeal carcinomatosis subset，LMC）的II期临床研究中，ANG1005每3周静脉注射600mg/m2，77%颅内和86%颅外患者获益，主要表现为病情稳定或更好；在软脑膜癌症中，79%的患者颅内疾病得到控制，估计中位总生存期为8.0个月（95%置信区间，5.4~9.4个月）。一项注册号为NCT03613181的III期临床试验正在进行中，目的是观察与医生选择的最佳药物相比，ANG1005是否可以延长新近诊断为LMC并曾治疗过脑转移的HER2阴性乳腺癌患者的生存期。2.2 DA-TP10帕金森病（Parkinson's disease，PD）是一种常见的进行性神经退行性疾病，但治疗效果并不完全令人满意。目前PD治疗的主要难点之一是药物BBB渗透性较差。研究者设计选用细胞穿透肽TP10作为药物载体，采用“click”反应，偶联多巴胺（Dopamine，DA）。实验结果表明，该多肽偶联药物具有比DA更好的药代动力学和药效学性能。它可穿过BBB，进入脑组织，对儿茶酚-O-甲基转移酶的O-甲基化反应的敏感性较低，甚至低于DA，对多巴胺1型受体（dopamine receptor1，D1）和多巴胺2型受体（dopamine receptor2，D2）受体的亲和力较高，如在D1受体的情况下，远高于DA。在药物诱导的PD临床前动物模型中，与左旋多巴（L-DOPA）相比，抗帕金森活性更明显。因此，治疗PD的药物与CPPs的结合可能会成为一种治疗PD的新策略。2.3 M6G-AngM6G在脑内注射后的镇痛效果比吗啡高50倍。然而，M6G的脑穿透性明显低于吗啡，从而影响了其疗效。因此，设计了M6G-Ang多肽偶联药物，即1个Angiopep-2多肽通过二硫键偶联3个M6G。实验证明，静脉注射或皮下注射An2-M6G多肽偶联药物，与M6G、吗啡单体相比，M6G-Ang增加了BBB通透性，其比同等剂量的吗啡或M6G具有更大、更持久的镇痛活性，且An2-M6G表现出减少便秘副作用的优点。这些结果表明使用脑靶向多肽载体作为一种新型的透过BBB的技术，可应用于CNS疾病的治疗。3目前脑靶向肽偶联药物面临的挑战及改进方法尽管脑靶向肽偶联药物有很多优点，但仍存在一些局限性。由于其分子量较生物大分子低，稳定性较差，肾脏可快速清除，半衰期和循环周期较短，可能导致药物疗效有限。此外，因为胃肠道有多种酶类，会使多肽在胃肠道内降解，故此类药物不适合口服给药，常用的给药方式是静脉注射，致使患者依从性差。目前，研究者也通过不同的技术来克服这些挑战。3.1 对多肽自身结构的修饰目前，研究者常采用环化肽（头尾环化、二硫键环化等）、拟肽、订书肽、bicycle策略、用D-氨基酸代替L-氨基酸等，增强多肽的稳定性，以此来延长半衰期。RAP12肽来源于受体相关蛋白(receptor associated protein，RAP)的微型化，与LRP-1具有结合亲和力。然而，当其脱离稳定蛋白环境时，RAP12表现出弱的α-螺旋片段。考虑到α-螺旋结构是该肽中介导配体-受体相互作用的常见结构基序，研究者利用肽吻合器技术合成了订书肽stapled RAP12（ST-RAP12）。经验证，与RAP12相比，优化后的ST-RAP12具有更高的α-螺旋含量、与LRP-1的结合亲和力和血清稳定性。此外，ST-RAP12表现出对bEnd.3细胞、U87胶质瘤细胞和人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）内化增强。而且，ST-RAP12在体外克服BBB和血脑肿瘤屏障（blood–brain tumorbarrier，BBTB）的能力也增强了。进一步应用ST-RAP12肽修饰高分子材料，构建ST-RAP12胶束。实验结果表明，该胶束在体内外可以有效穿透BBB/BBTB并靶向胶质瘤。此外，ST-RAP12胶束能有效地向胶质瘤传递PTX，延长胶质瘤荷瘤小鼠的生存时间，抑制肿瘤血管生成，诱导胶质瘤细胞凋亡，具有明显的抗胶质瘤作用。处于临床试验阶段的BT1718，是由双环肽通过可裂解的二硫键与美登素（mertansine，DM1）偶联得到的。双环肽可以特异性结合膜型1基质金属蛋白酶（MT1-MMP），MT1-MMP在乳腺癌、肺癌、卵巢癌、结肠癌等恶性肿瘤中过表达。与ADC相比，BT1718分子量低，分布良好，可快速穿透并“杀死”肿瘤细胞，对晚期实体瘤有治疗作用。已有大量研究表明Angiopep-2修饰的纳米载体显著增加了脑分布。Wei等构建了Angiopep-2的反向异构体，命名为DAngiopep-2，建立了脑靶向药物输送系统。虽然在体外实验中，DAngiopep-2被大脑毛细血管内皮细胞摄取的效率比LAngiopep-2低，但它表现出了增强的稳定性，并且修饰后的胶束比LAngiopep-2修饰后的胶束在正常的大脑和颅内胶质母细胞瘤细胞中具有更高的分布。3.2 与化学大分子的结合修饰多肽的电荷也与药物清除率有关。带负电荷的多肽序列比带正电荷的有更长的半衰期，这是因为肾小球内膜上阴离子电荷的存在限制了尿液中阴离子化合物的过滤。此外，还可通过增加多肽的大小和血浆蛋白结合，以防止结合物通过肾脏被过滤出来。有一种策略是将聚乙二醇（polyethylene glycol，PEG）与脑靶向肽药物偶联，可以延长半衰期。PEG的固有性质使其成为改性的理想候选材料：便宜、亲水、生物相容性、非免疫原性。它是最广泛使用的非天然聚合物之一，用于增加肽的溶解度、降低免疫反应和提高肽的生物利用度。PEG聚合物分子中的每个氧原子都能结合两到三个水分子，这样会大大增加附着在其上的化合物的质量和溶解度。FDA也批准了多种聚乙二醇化蛋白质，如PEG-牛腺苷脱氨酶和PEG-α-干扰素等。但分子量至少为30kDa聚乙二醇化的多肽会导致其在各种器官中的空泡化，如肾脏、肝脏、脾脏、骨髓等。因此，PEG天然替代品被开发出来。最突出的替代品是PASylation和XTEN。XTEN是由丙氨酸（A）、谷氨酸（E）、甘氨酸（G）、脯氨酸（P）、丝氨酸（S）和苏氨酸（T）这六种化学稳定氨基酸的非重复随机片段组成的遗传融合多肽。这些氨基酸的选择是基于避免可能影响蛋白质溶解度、活性或稳定性原则。艾塞那肽与XTEN偶联可显著改善肽的药代动力学，将其在大鼠、小鼠或猴子体内的半衰期分别延长65、71或125倍。PASylation化是指脯氨酸（P）、丙氨酸（A）和丝氨酸（S）的聚合物。由这些氨基酸组成的聚合物被认为对肽的水力动态体积与PEG有类似的影响，并且是可生物降解的，已经成功应用于超过10种的第一代生物制剂，包括人类生长激素、瘦素、促红细胞生成素、艾塞那肽、尿酸酶和凝血因子等。3.3 剂型修饰胃肠道的生理机能阻碍了大多数口服蛋白质制剂的临床转化。胃中含有刺激性的酸和酶，为了确保药物发挥作用，必须保护其和传递载体不受影响。有一些方法可以提高多肽类药物的口服生物利用度，如使用耐酸涂层、肠道酶抑制剂、粘液穿透肽和渗透增强剂等。Lamson 等研究发现<100nm的阴离子纳米颗粒可以充当物理化学渗透增强剂，促进蛋白质的口服输送。需要注意的是，这里描述的纳米颗粒不是通过作为运输载体移动穿过肠上皮，而是通过结合肠表面的受体介导紧密连接的开放，即纳米颗粒通过结合整合素和激活肌球蛋白轻链激酶（myosin-light-chain kinase，MLCK）增加肠道通透性，且这种作用是可逆的，不会导致肠组织坏死或炎症。此外，使用酸稳定涂层，是通过在表面涂上pH敏感的肠道聚合物来实现的，这种聚合物只有在达到肠道更中性的pH值时才会溶解，导致涂层破裂和包裹物释放。4小结由于BBB的存在，发现和开发新的治疗各种CNS疾病有效药物是非常具有挑战性的。脑靶向肽偶联药物作为一种非侵入性药物输送系统，可以透过BBB，将药物带入脑实质。与ADC相比，低分子质量的脑靶向肽偶联药物可能有高渗透性，高效的细胞运输，低免疫原性，以及更容易的合成和纯化。这使得它成为继ADC后，靶向给药领域又一研究热点。影响脑靶向肽偶联药物成为治疗CNS疾病有效药物的因素有：①药物运送到大脑的效率；②脑内药物的分布情况；③有效药物的释放和脑内的蓄积量；④与药物释放和体内稳定性有关的连接基团的选择；⑤受体饱和性等。因此，脑靶向肽偶联药物各部分的选择和设计尤为重要。但由于多肽的特性，也使其应用受到一定的限制。目前，研究者也发明了许多新技术，用来克服这些缺点，如对其结构修饰、与生物大分子偶联和剂型修饰等。随着相关技术的发展，如来自噬菌体、酵母展示、嗜神经病毒和干细胞的肽库的新型筛选平台以及基于特定靶点的计算机模拟设计，研究者将开发出更好的BBB选择性、更高的转运能力、更强的代谢稳定性的脑靶向肽。相信在未来脑靶向肽偶联药物输送系统可以取得实质性进展，在CNS疾病治疗中发挥其独特优势。参考文献详见《中国药学杂志》2024年文章信息源于公众号凡默谷，登载该文章目的为更广泛的传递行业信息，不代表赞同其观点或对其真实性负责。文章版权归原作者及原出处所有，文章内容仅供参考。本网拥有对此声明的最终解释权，若无意侵犯版权，请联系小编删除。学如逆水行舟，不进则退；心似平原走马，易放难收。行舟Drug每日更新 欢迎订阅+医药大数据|行业动态|政策解读

GPCRs, as key signal transduction molecules on the cell surface, regulate various physiological processes and constitute one-third of the targets for clinical drug applications. Based on structural similarities, GPCRs are divided into several families, including A, B, C, etc. Although most drugs targeting GPCRs are small molecules, many GPCRs have endogenous ligands that are peptides containing fewer than 50 amino acids. According to the pharmacology guidelines of the International Union of Basic and Clinical Pharmacology (IUPHAR), about one-third (64 types) of class A GPCRs bind to endogenous peptides; 20 types of class B GPCRs are activated by 15 endogenous peptides, and these receptors are classified according to the ligands they recognize into families such as calcitonin, corticotropin-releasing factor, glucagon, parathyroid hormone (although it has 84 amino acids, it is still typically considered a peptide), vasoactive intestinal peptide (VIP), or pituitary adenylate cyclase-activating peptide (PACAP), etc. Typically, endogenous peptides can bind to GPCRs on the cell surface, bridging paracrine and autocrine signaling and regulating the downstream signaling pathways of GPCRs. Peptides are one of the largest and oldest categories of intercellular chemical messengers. The pioneering development of using these naturally occurring peptides as therapeutic drugs dates back to the application of insulin in the 1920s. Insulin primarily acts on tyrosine kinase receptors, and the development of this therapy has made full use of the outstanding pharmacokinetic properties of peptides: high affinity, selectivity, and potency. Consistent with the observed effects of insulin, most other peptide therapies are well-tolerated with fewer side effects. However, naturally occurring peptides are generally not suitable as effective therapeutic drugs. The development of peptide drugs is limited by factors such as poor pharmacokinetic properties (short half-life, rapid degradation, high clearance), as well as insufficient oral bioavailability due to low intestinal stability and poor permeability. Therefore, to make most peptides effective drugs, strategies that address these issues need to be developed. Peptide-based therapeutic drugs are experiencing a resurgence. Emerging innovative strategies include extending the half-life, stabilizing peptide chains, and enhancing resistance to proteolytic degradation, all of which significantly improve pharmacokinetic properties and oral bioavailability. A prime example of these strategies might be the development of glucagon-like peptide-1 (GLP-1) receptor agonists, which possess enhanced resistance to proteolytic degradation and reduced renal clearance. Numerous successful market products and a large array of novel approaches in preclinical stages (such as peptide stabilization, cyclization, and glycosylation) stem from these efforts. This success has also spurred the development of multifunctional peptides that combine agonist activities against two or more GPCRs within the same peptide, based on relatively high sequence homology and similar binding sites. Complementary pharmacokinetic strategies are expanding the variety of drugs acting on specific GPCRs. Approved peptide therapeutic drugsMost of the peptide drugs targeting GPCRs that have been approved for clinical use or are under development function as agonists, used to substitute for or enhance the low levels of endogenous peptides. The following figure summarizes peptide drugs targeting GPCRs. Within class A GPCRs, representative receptor targets include the μ and κ opioid receptors (abbreviated as MOR and KOR, respectively) for pain relief; oxytocin and vasopressin receptors for inducing labor; as well as apelin and angiotensin receptors for the treatment of cardiovascular diseases; and somatostatin receptors for treating acromegaly and Cushing's disease, among others. Peptide therapies targeting Class B receptors include GLP-1 receptor agonists for the treatment of Type 2 Diabetes (T2D), GLP-2 receptor agonists for the treatment of Short Bowel Syndrome, as well as other synthetic or modified peptide hormones. Currently, a few peptide antagonists have entered clinical trials, most of which target Class A GPCRs. For example, antagonists that block the action of Gonadotropin-Releasing Hormone (such as degarelix and abarelix) are used for cancer treatment. Synthesis of Endogenous Peptide AnalogsThe marketed synthetic endogenous peptide drugs targeting Class A and Class B GPCRs include those targeting the angiotensin receptor, melanocortin receptor, thyrotropin-releasing hormone receptor, vasopressin receptor, oxytocin receptor, calcitonin receptor, glucagon receptor, parathyroid hormone receptor, among others. According to statistics, peptide drugs targeting Class A GPCRs are mainly high-affinity (pKi=8.4) and high-efficacy (pEC50=8.5) agonists. However, they typically exhibit a very short plasma half-life following intravenous administration, with a median value of about 5.3 minutes. This reflects their rapid degradation by peptidases or their higher rate of excretion, particularly through the kidneys. Theoretically, the levels of peptide drugs are expected to decrease to less than 1% of the original dose within 6 half-lives, which is approximately 32 minutes. Lastly, endogenous peptides usually have a low rate of binding to plasma proteins, ensuring that most peptides in circulation are unbound and can directly interact with and activate the corresponding receptors. However, unbound peptides are also highly susceptible to renal clearance and cleavage by serum or tissue proteases, both of which can reduce the plasma half-life. For all Class B GPCRs, the pharmacokinetic and pharmacodynamic median values of their endogenous ligands are similar to those of Class A receptor peptides: high efficacy (pEC50=9.9), accompanied by a short plasma half-life (15 minutes) and a low volume of distribution (approximately 9.9 L). It is worth mentioning that the short plasma half-lives of at least two classes of peptides have been utilized for clinical benefit. The peptide drug with the shortest plasma half-life is Angiotensin II. Synthetic analogs of this Angiotensin II are used to treat critically ill patients with distributive shock due to sepsis, where abnormal distribution of blood to the smallest vessels leads to insufficient systemic blood supply, potentially resulting in death. This drug binds to the AT1 receptors on vascular smooth muscle, and after intravenous administration, it effectively raises blood pressure within a median time of 5 minutes. Its short half-life (less than 1 minute) ensures that the possibility of hypertensive crisis due to overdose is minimal. Similarly, oxytocin, which was sequenced and synthesized in the 1950s, is used to induce labor and immediately enhance contractions upon intravenous administration, but its effects wear off within an hour, reflecting its short half-life of several minutes. Modified peptidesTaking Semaglutide as an example, modifications and redesigns have significantly extended its half-life (approximately 168 hours). This extension is achieved by using a fatty acid linker, which allows the molecule to form non-covalent reversible interactions with albumin, thereby reducing renal excretion. Simultaneously, alpha-aminobutyric acid has been introduced at position 8 of Semaglutide to reduce metabolism mediated by Dipeptidyl Peptidase-4 (DPP-4). Additionally, Albiglutide and Dulaglutide utilize different protein linking strategies. These two GLP-1 receptor agonists are covalently linked to large molecules (albumin and the Fc fragment of human IgG4, respectively). In Albiglutide, the GLP-1 sequence at position 8 is also modified by substituting glycine with alanine near the DPP-4 cleavage site to reduce metabolism. Another exemplary case is Lutathera ([177Lu]-dotatate) used for the treatment of neuroendocrine tumors, representing a recent example of peptide receptor radionuclide therapy. In this compound, the peptide dotatate, an agonist of the somatostatin receptor, is labeled with lutetium [177]. After the peptide binds to somatostatin receptors highly expressed on tumor surfaces, the radiation emitted causes tumor cell death, while the limited range of the particles minimally affects the surrounding healthy tissue due to their short penetration depth. Peptide Design Optimization StrategiesRecent approvals of peptide drugs have shown trends such as high sustained target efficacy combined with increased plasma half-lives, reduced enzymatic metabolism, and decreased renal clearance. For example, degarelix is a synthetic derivative of GnRH that blocks the binding of endogenous peptides to pituitary receptors and is used to treat prostate cancer. After subcutaneous injection, degarelix forms a drug depot at the injection site, with the drug slowly releasing into the bloodstream, producing a plasma half-life of 42-70 days. Clearance is unaffected, primarily through hepatic-biliary hydrolysis and renal excretion of unchanged drug. Additionally, lanreotide is a somatostatin agonist that functions mainly by binding to SST2 and SST5 receptors, used to inhibit growth hormone release to treat acromegaly; it also forms a drug depot at the injection site with a 22-day plasma half-life and includes non-natural amino acids. Peptides, being charged and hydrophilic molecules, typically lack oral bioavailability. Decades after the synthetic modification of peptides, most peptide drugs still possess the intrinsic drawback of low membrane permeability, which limits their oral bioavailability and tissue distribution, including distribution to the CNS. Emisphere's use of a pharmacologically inactive small molecule enhancer, N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC), co-formulated with Semaglutide, results in the oral drug semaglutide having increased transcellular permeability and about 4% bioavailability. Experimental methods to enhance brain permeability include linking the drug neurotensin to the brain barrier-penetrating peptide angiopep-2, thus increasing the blood-brain barrier crossing capability by approximately tenfold through receptor-mediated transcytosis. This approach is sufficient to reverse pain behaviors in animal models of neuropathic and bone cancer pain. Cyclization helps in resistance to metabolism, and its hydrophobicity enhances absorption by intestinal cells. Despite very low bioavailability via the oral route (0.08%-0.16%), this is sufficient to achieve clinically effective plasma concentrations. Desmopressin is one example of an orally administered peptide drug, showcasing the potential of engineering peptides to possess oral activity. The new generation of peptide drugs often incorporates unnatural amino acid residues, such as D-amino acids, N-methyl amino acids, or residues with unnatural side chains. Chemical modifications—non-amino acid monomers added to peptides during synthesis—are also becoming increasingly important. These modifications include N-terminal modifications, C-terminal capping, fatty acids, and polyethylene glycol (PEG) groups. Among 49 peptide drugs approved for targeting GPCRs studied by researchers, 22 contain only natural amino acids, 3 contain at least one unnatural amino acid, 3 have at least one chemical modification, and 21 contain both chemical modifications and unnatural amino acids. Classic Case: Development of GLP-1 Agonist Peptides Exenatide is the first clinically-approved GLP-1 receptor agonist. In 1992, scientists identified a novel peptide hormone, exendin-4, from the saliva of the Heloderma suspectum. Exendin-4 shares many pharmacological properties with GLP-1: enhancing insulin secretion and reducing plasma glucose levels. However, unlike GLP-1, exendin-4 is resistant to enzymatic cleavage and inactivation by DPP4. As exenatide, it was approved in 2005 as an adjunct therapy for Type 2 Diabetes (T2D) and as a monotherapy in 2009. Although exenatide is widely used in the treatment of T2D, its short plasma half-life requires frequent (twice daily) subcutaneous injections, which limits its efficacy, leads to poor patient compliance, and increases the risk of additional side effects such as injection site infections. Following the success of exenatide, researchers have adopted multiple strategies to increase the plasma stability and half-life of GLP-1 receptor agonists, to reduce renal clearance, and to enhance oral bioavailability. These strategies are generally divided into two approaches. One approach focuses on extending the plasma half-life of exenatide, which led to the development of once-weekly Exenatide (QW) and lixisenatide. Exenatide QW involves reformulating exenatide into microspheres composed of the biodegradable polymer poly(D,L-lactide-co-glycolide), thus extending the dosing interval to once weekly. In lixisenatide, modifications to the exenatide sequence, including the addition of a lysine tail at the C-terminus, confer resistance to DPP4 enzyme cleavage and prolong the plasma half-life. The second strategy focuses on modifying the natural GLP-1 peptide, resulting in the production of liraglutide, albiglutide, dulaglutide, and the recently approved semaglutide. Liraglutide, approved in 2009, is a human GLP-1 receptor agonist based on the natural GLP-1 peptide, with an amino acid substitution (Lys34Arg) and a C16 palmitic acid side chain attached via a glutamyl spacer at position 26. These modifications increase its binding to serum albumin, significantly reducing renal clearance and DPP4 enzyme cleavage. The modified GLP-1 is released from albumin at a slow, steady rate, resulting in slower degradation and reduced renal clearance compared to the mature endogenous form of GLP-1, GLP-17-37. Semaglutide, approved in 2017, incorporates the non-natural amino acid α-aminoisobutyric acid at position 8 to reduce DPP4 enzyme cleavage, and contains a substituted C18 dicarboxylic fatty acid at Lys26 that provides strong albumin binding. Both albiglutide and dulaglutide are peptide fusion protein variants. Each carries protection against DPP4 enzyme cleavage due to a substitution at position 8, and both include two GLP-1 molecules, either fused to human serum albumin (albiglutide) or covalently linked via a small peptide connector to the human IgG4-Fc heavy chain (dulaglutide), to increase the molecule's half-life through recycling and/or interaction with the neonatal Fc receptor (FcRn) or by increasing molecular weight. Reference: https://www.nature.com/articles/s41573-020-0062-z