Synthesis and Biological Evaluation of RGD–Cryptophycin Conjugates for Targeted Drug Delivery

Abstract

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvβ3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)–cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvβ3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

Size‐Dependent Cellular Uptake of RGD Peptides

“…Cellular internalization was studied in vitro. M21 cells overexpress α v β 3 , whereas the variant M21L lacks α v and thus does not express α v integrins . As described previously, uptake of monomeric RGD peptides is fluid‐phase dependent, whereas for multimeric peptides internalization is clathrin‐dependent .…”

“…However, the pathway of uptake was only studied sparingly and reports are conflicting. For example, studies by us revealed colocalization of an RGD‐cryptophycin‐52 conjugate with lysosomes in WM‐115 cells (overexpressing α v β 3 ) and in contrast, an RGD‐cryptophycin‐55 conjugate was internalized both by M21 (overexpressing α v β 3 ) and M21L (α v knockout) cells . Likewise, cytotoxic RGD‐α‐amanitin conjugates did not provide specific uptake .…”

Synthesis and Biological Characterization of Monomeric and Tetrameric RGD‐Cryptophycin Conjugates

“…An alkyne‐functionalized PEG5‐linker was introduced to the N ‐terminus of the linker to allow the reaction with the azido‐functionalized monomeric ( 3 ) or tetrameric ( 4 ) integrin ligands (Scheme ). The conjugate 5 containing the monomer RGD ligand was synthesized as previously reported, whereas multimeric RGD compound 4 was achieved using a modular convergent strategy that involves the oxime ligation of aldehyde‐RGD and RAFT that displays 4 aminooxy groups (see the Supporting Information) …”

“…In recent years, considerable research efforts have been devoted to the development of SMDCs based on cryptophycins, afamily of microtubule targeting agents, that are characterized with outstanding potencya nd retained activity against multidrug-resistant (MDR) cancer cell lines. [24][25][26][27][28] Remarkably,t he synthetic cryptophycin-55 glycinate (1, Figure 1) displays adequate stability, exhibits cytotoxic activity in the subnanomolar range and showsh igh antitumor activity in vivo against MDR tumors. [26,29] We have previously reported that conjugates of monomeric c(RGDfK) ligandsa nd cryptophycin-55 glycinated isplayh igh potencya gainst the M21a nd M21-L human melanoma cells.…”

“…[24][25][26][27][28] Remarkably,t he synthetic cryptophycin-55 glycinate (1, Figure 1) displays adequate stability, exhibits cytotoxic activity in the subnanomolar range and showsh igh antitumor activity in vivo against MDR tumors. [26,29] We have previously reported that conjugates of monomeric c(RGDfK) ligandsa nd cryptophycin-55 glycinated isplayh igh potencya gainst the M21a nd M21-L human melanoma cells. [26] However, we aimed to improve the tumort argetingp roperties of RGD-cryptophycinc onjugatesu sing multivalent ligands.…”