Metabolic Profile of a Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1: An in Vivo and in Vitro Study

Abstract

ABSTRACT:Because angiogenic endothelial cells of the tumor vasculature represent an interesting target to potentiate the antivascular effect of photodynamic therapy, we recently described the conjugation of a photosensitizer [5-(4-carboxyphenyl)-10,15,20-triphenylchlorin (TPC)], via a spacer [6-aminohexanoic acid (Ahx)], to a vascular endothelial growth factor receptor-specific heptapeptide [H-AlaThr-Trp-Leu-Pro-Pro-Arg-OH (ATWLPPR)] and showed that TPCAhx-ATWLPPR binds to neuropilin-1. Because peptides often display low stability in biological fluids, we examined the in vivo and in vitro stability of this conjugate by high-performance liquid chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry. TPC-Ahx-ATWLPPR was stable in vitro in human and mouse plasma for at least 24 h at 37°C but, following i.v. injection in glioma-bearing nude mice, was degraded in vivo to various rates, depending on the organ considered. TPCAhx-A was identified as the main metabolic product, and biodistribution studies suggested that its appearance in plasma mainly resulted from the degradation of the peptidic moiety into organs of the reticuloendothelial system. According to in vitro cell culture experiments, TPC-Ahx-ATWLPPR was also significantly degraded after incorporation in human umbilical vein endothelial cells (HUVEC), mainly into TPC-Ahx-A and to a lesser extent into TPCAhx-AT and TPC-Ahx-ATWLPP. TPC-Ahx-ATWLPPR mostly localized into lysosomes, and when HUVEC were treated with the lysosomal enzymes' inhibitor ammonium chloride, this resulted in a significant decrease of the peptide degradation. This study provides essential information for the choice of the time of activation of the photosensitizer (drug-light interval) not to be exceeded and for the future design of more stable molecules.

Response Surface Methodology: An Extensive Potential to Optimize in vivo Photodynamic Therapy Conditions

“…In practice, the four factors usually considered are the administered PS dose, the time interval between PS administration and light irradiation (drug-light interval [DLI]), the light fluence, and the light fluence rate (12). In our approach, to limit the number of experiments to be carried out, a DLI of 4 hours was selected, corresponding to relevant PS levels in plasma and tumor, low PS levels in skin, and low degradation of the peptidic moiety (13,14). Fixing this parameter, determination of the optimal PDT dosimetry can be expressed as the optimal choice of the three remaining factors (PS dose, light fluence, and fluence rate), i.e., the selection of values leading to an optimal PDT response.…”

Neuropilin-1 Targeting Photosensitization-Induced Early Stages of Thrombosis via Tissue Factor Release

“…Although increased uptake of photosensitizer by tumor tissue was solely attributable to the PEG moiety, conjugation to APRPG appeared necessary to suppress tumor growth efficiently. For the first time, we propose to characterize the vascular effect of PDT using a peptide-conjugated photosensitizer targeting NRP-1 (19). As a DLI of 4 h, corresponded to the highest incorporation of TPC-Ahx-ATWLPPR in endothelial cells lining the vessels, has been chosen (17), and since our molecular marker is mainly over expressed by angiogenic vascular endothelial cells (33), the supposed initiating event would be a morphological and functional alteration of endothelium.…”

“…TPC was purchased from Frontier Scientific (Logan, UT). TPC-Ahx-ATWLPPR was synthesized and purified as described previously (15,19). Stock solutions in dimethylsulfoxide (2 mM) were used.…”

Vascular-Targeted Photodynamic Therapy (VTP)

“…Our study demonstrated that TPC-Ahx-ATWLPPR accumulated up to 25-fold more in human umbilical vein endothelial cells (HUVEC) than free TPC over a 24 hours period. The accumulation of the conjugated photosensitizer was related to NRP-1-dependent mechanisms but also to non-specific mechanisms (Thomas et al, 2008, Tirand et al, 2007. In vivo biodistribution studies in nude mice xenografted with U87 human malignant glioma cells revealed significant tumour level to normal ratios as early as 1 hour after intravenous injection of TPC-Ahx-ATWLPPR.…”