Rational Design of RGD–Albumin Conjugates for Targeted Delivery of the VEGF‐R Kinase Inhibitor PTK787 to Angiogenic Endothelium

Angiogenesis, the formation of new blood vessels out of preexisting capillaries, is a prominent feature during the pathogenesis of cancer. Antiangiogenic therapies are therefore extensively investigated for combating this disease. The newest treatments consist of antibodies or kinase inhibitors that block the signaling by VEGF, one of the most prominent angiogenic modulators. Herein we propose specific targeting of the VEGF receptor (VEGF-R) kinase inhibitor PTK787 to angiogenic vasculature in tumor tissue. Our conjugates combine the action of a kinase inhibitor with specificity for disease-controlling target cells. This can result in a far greater restriction in drug action and thus an improvement in safety and efficacy. This may be relevant in view of side effects associated with VEGF-R inhibition, such as vomiting, hypertension, and embolism. We have developed three new classes of drug carriers consisting of human serum albumin (HSA), cyclic RGD peptides, and polyethylene glycol (PEG) (Scheme 1A). HSA served as a biocompatible and biodegradable carrier with low polydispersity, thus allowing characterization of the final macromolecular conjugates by mass spectrometry. HSA was equipped with cyclic RGD peptides as targeting ligands that bind with high affinity to the target receptor avb3-integrin, [6] which is overexpressed on angiogenic endothelium. This restricted expression profile and the good accessibility of endothelial cells make them an ideal target for drug delivery. 8] We applied either a short alkyl linker that enables introduction of a high number of RGD peptides in the carrier (RGD-HSA), or an extended polyethylene glycol linker that presents the RGD peptide at the distal end of the PEG chain (RGDPEG-HSA), but which leads to lower RGD incorporation. The use of such a PEG linker furthermore affects the distribution of the conjugates by the stealth effect of PEG, and increases the solubility and decreases the immunogenicity of the products. A third carrier was designed by combination of the short alkyl linker for RGD incorporation together with separately attached monofunctional PEG groups (RGD-HSA-PEG). Previous studies have shown that it is very important to introduce multiple RGD peptides in the conjugates to allow multivalent receptor interactions to facilitate binding and internalization by target cells. We therefore optimized the attachment of the targeting ligand followed by subsequent conjugation of the drug. RGD coupling was carried out with a 22-fold molar excess of N-hydroxysuccinimide ester (SIA) or a 50-fold molar excess of vinylsulfone polyethylene glycol-N-hydroxysuccinimide ester (VS-PEG-NHS, 3.5 kDa) over HSA. The RGD peptide cACHTUNGTRENNUNG(RGDf(e-S-acetylthioacetyl)K) was added in slight excess over the added linker (25-fold respective 55-fold molar excess over HSA). After purification of the products RGD-HSA and RGDPEG-HSA, a fraction of RGD-HSA was further modified with monofunctional mPEG-SMB (5 kDa) in a ratio of 20:1 to obtain RGD-HSA-PEG. MALDI-TOF MS analysis elegantly demonstrated the incorporation of RGD and PEG in the carriers (Figure 1, Table 1). Attachment of RGD to the compounds could be furthermore deduced from the binding studies with target cells, as discussed below.