Tumor Targeting with an isoDGR-Drug Conjugate

Abstract

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin α_V β₃ . 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 α_V β₃ 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 α_V β₃ expression: human glioblastoma U87 (α_V β₃ +) and U87 β₃ -KO (α_V β₃ -). 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).

Keywords: antitumor agents; cancer; drug delivery; integrins; peptidomimetics.

Figure 1

Structures of the integrin ligands cyclo[DKP‐isoDGR] (1) and cyclo[DKP‐RGD] (3), and of the corresponding SMDCs cyclo[DKP‐isoDGR]‐Val‐Ala‐PTX (2) and cyclo[DKP‐RGD]‐Val‐Ala‐PTX (4).

Scheme 1

Synthesis isoDGR peptidomimetic 5. Reagents and conditions: a) TFA/CH₂Cl₂ 1:2, RT, 2 h; b) Cbz‐Arg(Mtr)‐OH, HATU, HOAt, iPr₂NEt, DMF, 0 °C to RT, overnight, 94 % over 2 steps; c) [Pd(PPh₃)₄], N‐methylaniline, CH₂Cl₂, 0 °C, 1 h, 88 %; d) HATU, HOAt, iPr₂NEt, DMF, 0 °C to RT, overnight, 86 %; e) piperidine, DMF, 2 h, RT, 67 %; f) 13, HATU, HOAt, iPr₂NEt, DMF, 0 °C to RT, overnight, 95 %; g) H_2, 10 % Pd/C, THF/H₂O 1:1, overnight, RT, 95 %; h) HATU, HOAt, iPr₂NEt, DMF/CH₂Cl₂ 1:1 (1.4 mm), 0 °C to RT, overnight, 79 %; i) TFA/TMSBr/thioanisole/EDT/phenol 70:14:10:5:1, 2 h, RT, 47 %. (TFA=trifluoroacetic acid, HATU=1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate, TMSBr=bromotrimethylsilane, EDT=1,2‐ethanedithiol).

Scheme 2

Synthesis of isoDGR–drug conjugate 2. Reagents and conditions: a) DIC, NHS, DMF, 0 °C to RT, overnight; b) 5, CH₃CN/PBS 1:1; pH 7.3–7.6, 0 °C to RT, overnight; c) TFA/CH₂Cl₂ 1:2, 1 h, RT, 55 % over three steps; d) 4‐Nitrophenylchloroformate, pyridine, CH₂Cl₂, −50 °C to −20 °C; 4 h, 69 %; e) 22, iPr₂NEt, DMF, 0 °C to RT, overnight, 55 %. (PBS=phosphate‐buffered saline).

Figure 2

Flow cytometry experiments on U87 and U87 β₃‐KO cells to assess the different α_Vβ₃ integrin expression. Cells were incubated with the secondary antibody (CF488A‐goat anti‐mouse IgG, Biotium 20 011), or with the anti‐α_Vβ₃ antibody (clone LM609‐Millipore MAB 1976) followed by the secondary antibody, see the Supporting Information.