Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers that locate in endocytic vesicles, and that upon activation by light induce a release of macromolecules from the endocytic vesicles. PCI has been shown to stimulate delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane. The preclinical evaluation of PCI has been performed with aluminum phthalocyanine disulfonate (AlPcS2a) as photosensitizer. AlPcS2a, due to its large number of isomers potentially with batch-to-batch ratio variations, is not an optimal photosenstizer for clinical use. Disulfonated tetraphenyl chlorin (TPCS2a) has therefore been developed by di-imide reduction of disulfonated tetraphenyl porphine (TPPS2a). The synthesized TPCS2a contains 3 isomers as shown by HPLC with low (<4%) inter-batch variation with respect to isomer formation, less than 0.5% (w/w) of the starting material TPPS2a and absorbs light at 652 nm. As prerequisites for a PCI photosensitizer TPCS2a was found to localize in intracellular granules assumed to be endocytic vesicles. In cells in culture TPCS2a-PCI induced activation of gelonin as seen by enhanced cytotoxicity, increased transfection efficacy by an enhanced green fluorescence protein (EGFP)-encoding plasmid, induced gene silencing by siRNA towards EGFP and induced in a synergistic manner tumor growth delay by TPCS2a-mediated PCI of bleomycin in CT26.CL25 carcinomas growing subcutaneously in athymic mice. TPCS2a-PCI of bleomycin was found superior to meso-tetraphenyl chlorin-based photodynamic therapy (mTHPC-PDT) with respect to inhibition of tumor growth. The tumor growth delay by PCI of bleomycin was independent of the time of bleomycin administration between 3 h prior to light to immediately after light, while bleomycin administered 24 h prior to or 24 h after the light exposure induced suboptimal or only additive effects on tumor growth delay respectively. TPCS2a-PDT and -PCI induced indistinguishably strong edema the first 3–4 days after TPCS2a-administration and only weak erythema the first day after TPCS2a administration. In contrast, mTHPC-PDT induced moderate edema the first 7 days after mTHPC administration, but strong erythema resulting in open wounds and escar formation the first 2–3 days after mTHPC administration. The pharmacokinetic properties of TPCS2a were evaluated in athymic mice. The plasma pharmacokinetics was best fit to a 2-compartment model with half-lives of 0.78 and 36 hrs. TPCS2a was found to be a clinically suitable PCI photosensitizer for photochemical activation of molecules that do not readily penetrate the cellular plasma membrane.
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