In vitro and in vivo evaluation of a paclitaxel conjugate with the divalent peptide E-[c(RGDfK)2] that targets integrin αvβ3
In vitro and in vivo evaluation of a paclitaxel conjugate with the divalent peptide E-[c(RGDfK)2] that targets integrin αvβ3
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DOI：
10.1016/j.ijpharm.2008.09.055
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发表时间：
2009-02-23
影响因子：
5.8
通讯作者：
Kratz, Felix
中科院分区：
文献类型：
--
作者：
Ryppa, Claudia;Mann-Steinberg, Hagit;Kratz, Felix
α(v)β(3) 整联蛋白在增殖的内皮细胞（例如生长肿瘤中存在的内皮细胞）以及各种来源的肿瘤细胞上过度表达。通过用含有精氨酰-甘氨酰-天冬氨酸（RGD）氨基酸序列的小肽拮抗α（v）β（3）整联蛋白，可以在体内抑制肿瘤诱导的血管生成。二价环肽 E-[c(RGDfK)(2)] 是一种新型基于配体的血管靶向剂，可结合整合素 alpha(v)beta(3)，并在 OVCAR-3 异种移植肿瘤中表现出高摄取性。在本工作中，我们通过脂肪族酯将紫杉醇的2'-OH-基团与E-[c(RGDfK)(2)]或对照肽c(RADfK)的氨基偶联，从而得到衍生物E-[c(RGDfK)(2)]-紫杉醇和c(RADfK)-紫杉醇。随后，我们使用几种成熟的体外血管生成测定研究了紫杉醇衍生物的活性：使用标准的72小时内皮细胞增殖测定，我们表明E-[c(RGDfK)2]-紫杉醇和c(RADfK-紫杉醇以与游离紫杉醇类似的方式抑制人脐静脉内皮细胞(HUVEC)的增殖(IC50值类似于0.4 nM)，这一观察结果可以用在 pH 7 下与结合物中紫杉醇酯键的半衰期相似的 2 小时来解释。相比之下，细胞暴露于三种药物 30 分钟显示出游离紫杉醇、E-[c(RGDfK)(2)]-紫杉醇和 c(RADfK)-紫杉醇之间存在明显差异，IC50 值为 10 nM，这些差异很可能是由于这三种分子进入细胞的途径不同，而带电肽的衍生物 E-[c(RGDfK)(2)]-紫杉醇与过表达的 α(v)beta(3) 整合素结合，通过受体介导的内吞作用进入细胞。仅用 E-[c(RGDfK)(2)]-紫杉醇衍生物观察到细胞附着的抑制，表明其对生长的内皮细胞具有特异性。此外，E-[c(RGDfK)(2)]-紫杉醇抑制内皮细胞迁移和毛细血管样管形成。这些结果进一步证明了它们在 OVCAR-3 异种移植模型中的抗血管生成特性。 E-[c(RGDfK)(2)] 或 E-[c(RGDfK)(2)]-紫杉醇的功效与紫杉醇的中等功效相比 (C) 2008 Elsevier B.V. 保留所有权利。
The alpha(v)beta(3) integrin is overexpressed on proliferating endothelial cells such as those present in growing tumors as well as on tumor cells of various origins. Tumor-induced angiogenesis can be inhibited in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the arginyl-glycyl-aspartic acid (RGD) amino acid sequence. The divalent cyclic peptide E-[c(RGDfK)(2)] is a novel ligand-based vascular-targeting agent that binds integrin alpha(v)beta(3) and demonstrated high uptake in OVCAR-3 xenograft tumors. in this work, we coupled the 2'-OH-group of paclitaxel through an aliphatic ester to the amino group of E-[c(RGDfK)(2)] or the control peptide c(RADfK), thus obtaining the derivatives E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel. Subsequently, we investigated the activity of the paclitaxel derivatives using several well-established in vitro angiogenesis assays: using a standard 72 h endothelial cell proliferation assay, we showed that both E-[c(RGDfK)2]-paclitaxel and c(RADfK-paclitaxel inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) in a similar manner as free paclitaxel (IC50 value similar to 0.4 nM), an observation that can be explained by the half-life of the paclitaxel ester bond in the conjugates of similar to 2 h at pH 7. In contrast, a 30-min exposure of the cells to the three drugs showed a clear difference between free paclitaxel, E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel with IC50 values of 10 nM, 25 nM, and 60 nM, respectively. These differences are very likely due to the different routes of cellular entry of these three molecules. While the hydrophobic paclitaxel diffuses rapidly through the cell membrane, the charged peptide-containing derivative E-[c(RGDfK)(2)]-paclitaxel binds to the overexpressed alpha(v)beta(3) integrin in order to enter the cells via receptor-mediated endocytosis. The differences between the derivatives were further demonstrated using an endothelial cell adhesion assay. inhibition of cell attachment was observed only with the E-[c(RGDfK)(2)]-paclitaxel derivative indicating its specificity to the growing endothelial cells. Furthermore, E-[c(RGDfK)(2)]-paclitaxel inhibited both endothelial cells migration and capillary-like tube formation. These results further demonstrate their antiangiogenic properties. In vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)(2)] or E-[c(RGDfK)(2)]-paclitaxel compared to moderate efficacy for paclitaxel. (C) 2008 Elsevier B.V. All rights reserved.