Lächelt Ulrich, Wittmann Valentin, Müller Katharina, Edinger Daniel, Kos Petra, Höhn Miriam, Wagner Ernst
Pharmaceutical Biotechnology, Center for System-based Drug Research, Ludwig-Maximilians-University Munich , Butenandtstrasse 5-13, 81377 Munich, Germany.
Mol Pharm. 2014 Aug 4;11(8):2631-9. doi: 10.1021/mp500017u. Epub 2014 May 2.
The antifolate drug methotrexate (MTX) can serve as a dual-functional ligand in antitumoral drug delivery, inducing both a folate receptor mediated cellular uptake and an intracellular cytotoxic action. Bioactivity of MTX however changes by conjugation; the activity can be affected by the hampered intracellular conversion to more potent poly-γ-glutamyl derivatives. Therefore, in a cancer combination therapy approach for the codelivery of cytotoxic dsRNA polyinosinic-polycytidylic acid poly(I:C), a set of molecularly precise oligo(ethanamino)amides were synthesized comprising poly(ethylene glycol) conjugated MTX ligands. The conjugates differed in the number of additional glutamic acid residues to investigate the effect of different degrees of synthetic "a priori" polyglutamylation. The bioactivity of these compounds concerning dihydrofolate reductase (DHFR) inhibition, cytotoxicity, nucleic acid binding potency, cellular uptake of poly(I:C) polyplexes, and combined antifolate/poly(I:C) toxicity was investigated. Synthetic polyglutamylation had a crucial impact on several stages of efficient poly(I:C) delivery and combined MTX cytotoxicity. DHFR inhibition of the conjugates significantly increased with increasing polyglutamate chain length. The library member with highest glutamylation degree even outperformed free MTX in direct comparison. Studies in KB cells showed the corresponding enhanced cytotoxicity by polyglutamylation. Also poly(I:C) polyplexes of the glutamylated MTX variants exhibited higher cellular uptake in the folate receptor positive cell line. Finally, a synergistic combined cytotoxicity of polyglutamylated MTX ligands and complexed poly(I:C) cargo was observed in transfected KB cells. The present structure-activity relationship study of MTX-based ligands pinpoints the concept of synthetic polyglutamylation as a promising approach for optimizing bioactivity of antifolate conjugates, which might be considered as a useful tool also in context of other drug delivery systems.
抗叶酸药物甲氨蝶呤（MTX）在抗肿瘤药物递送中可作为双功能配体，既能诱导叶酸受体介导的细胞摄取，又能产生细胞内细胞毒性作用。然而，MTX的生物活性会因共轭作用而改变；其活性可能会受到细胞内转化为更有效的聚γ-谷氨酰衍生物受阻的影响。因此，在一种用于联合递送细胞毒性双链RNA聚肌苷酸-聚胞苷酸（poly(I:C)）的癌症联合治疗方法中，合成了一组分子精确的寡（乙氨基）酰胺，其中包含聚乙二醇共轭的MTX配体。这些共轭物在额外谷氨酸残基的数量上有所不同，以研究不同程度的合成“先验”聚谷氨酸化的影响。研究了这些化合物在二氢叶酸还原酶（DHFR）抑制、细胞毒性、核酸结合能力、poly(I:C)多聚体的细胞摄取以及联合抗叶酸/聚(I:C)毒性方面的生物活性。合成聚谷氨酸化对高效递送poly(I:C)和联合MTX细胞毒性的几个阶段具有关键影响。随着聚谷氨酸链长度的增加，共轭物对DHFR的抑制作用显著增强。在直接比较中，谷氨酸化程度最高的文库成员甚至优于游离MTX。在KB细胞中的研究表明，通过聚谷氨酸化相应地增强了细胞毒性。谷氨酸化的MTX变体的poly(I:C)多聚体在叶酸受体阳性细胞系中也表现出更高的细胞摄取。最后，在转染的KB细胞中观察到谷氨酸化的MTX配体与复合的poly(I:C)货物具有协同联合细胞毒性。目前基于MTX的配体的构效关系研究指出，合成聚谷氨酸化是优化抗叶酸共轭物生物活性的一种有前景的方法，在其他药物递送系统的背景下也可能被视为一种有用的工具。
