Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery.

RGD-cryptophycin and isoDGR-cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin-binding antimitotic agent across lysosomally cleavable Val-Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin αvß3, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21-L with different expression levels of integrin αvß3, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin-based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin αvß3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective αvß3 integrin-mediated drug delivery.

ß-Glucuronidase triggers extracellular MMAE release from an integrin-targeted conjugate.

A non-internalizing αvß3 integrin ligand was conjugated to the anticancer drug MMAE through a ß-glucuronidase-responsive linker. In the presence of ß-glucuronidase, only the conjugate bearing a PEG4 spacer inhibited the proliferation of integrin-expressing cancer cells at low nanomolar concentrations, indicating important structural requirements for the efficacy of these therapeutics.

Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting.

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVß3 receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of αVß3 integrin expression: human glioblastoma U87 (αVß3+), human lung carcinoma A549 (αVß3-) and breast adenocarcinoma MDA-MB-468 (αVß3-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αVß3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (αVß3+, αVß5+, αVß6-, α5ß1+) and MDA-MB-468 (αVß3-, αVß5+, αVß6+, α5ß1-) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αVß3, but also αVß5, αVß6, and α5ß1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αVß3 (e.g., αVß5).

Tumor Targeting with an isoDGR-Drug Conjugate.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin αV ß3 . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αV ß3 receptor (IC50 =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin αV ß3 expression: human glioblastoma U87 (αV ß3 +) and U87 ß3 -KO (αV ß3 -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).