Received for publication November 10, 2006.
Revised February 1, 2007.
Accepted for publication February 2, 2007.

Metabolic profile of a peptide-conjugated chlorin-type photosensitizer targeting neuropilin-1: an in vivo and in vitro study

Abstract

Since angiogenic endothelial cells of the tumor vasculature represent an interesting target to potentiate the anti-vascular 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 (VEGF) receptor specific-heptapeptide (H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH, noted ATWLPPR), and demonstrated that TPC-Ahx-ATWLPPR binds to neuropilin-1 (NRP-1). Since peptides often display low stability in biological fluids, we examined the in vivo and in vitro stability of this conjugate by HPLC and MALDI-TOF mass spectrometry. TPC-Ahx-ATWLPPR was stable in vitro in human and mouse plasma for at least 24h at 37°C but, following intravenous injection in glioma-bearing nude mice, was degraded in vivo to various rates, depending on the organ considered. TPC-Ahx-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 TPC-Ahx-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.

Key words: drug targeting, HPLC, mass spectrometry, metabolite identification, metabolite kinetics