SummaryA photosensitizer is defined as a chemical entity, which upon absorption of light induces a chemical or physical alteration of another chemical entity. Some photosensitizers are utilized therapeutically such as in photodynamic therapy (PDT) and for diagnosis of cancer (fluorescence diagnosis, FD). PDT is approved for several cancer indications and FD has recently been approved for diagnosis of bladder cancer. The photosensitizers used are in most cases based on the porphyrin structure. These photosensitizers generally accumulate in cancer tissues to a higher extent than in the surrounding tissues and their fluorescing properties may be utilized for cancer detection. The photosensitizers may be chemically synthesized or induced endogenously by an intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) or 5-ALA esters. The therapeutic effect is based on the formation of reactive oxygen species (ROS) upon activation of the photosensitizer by light. Singlet oxygen is assumed to be the most important ROS for the therapeutic outcome. The fluorescing properties of the photosenisitizers can be used to evaluate their intracellular localization and treatment effects. Some photosensitizers localize intracellularly in endocytic vesicles and upon light exposure induce a release of the contents of these vesicles, including externally added macromolecules, into the cytosol. This is the basis for a novel method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, gene-encoding plasmids, adenovirus, peptide-nucleic acids and the chemotherapeutic drug bleomycin. The background and present status of PDT, FD and PCI are reviewed.

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 (AlPcS(2a)) as photosensitizer. AlPcS(2a), 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 (TPCS(2a)) has therefore been developed by di-imide reduction of disulfonated tetraphenyl porphine (TPPS(2a)). The synthesized TPCS(2a) 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 TPPS(2a) and absorbs light at 652 nm. As prerequisites for a PCI photosensitizer TPCS(2a) was found to localize in intracellular granules assumed to be endocytic vesicles. In cells in culture TPCS(2a)-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 TPCS(2a)-mediated PCI of bleomycin in CT26.CL25 carcinomas growing subcutaneously in athymic mice. TPCS(2a)-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. TPCS(2a)-PDT and -PCI induced indistinguishably strong edema the first 3-4 days after TPCS(2a)-administration and only weak erythema the first day after TPCS(2a) 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 TPCS(2a) 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. TPCS(2a) was found to be a clinically suitable PCI photosensitizer for photochemical activation of molecules that do not readily penetrate the cellular plasma membrane.

Photodynamic therapy using topical methyl 5-aminolevulinate (MAL) is a new treatment modality for basal cell carcinoma (BCC) and actinic keratosis (AK). MAL induces endogenous porphyrins, which act as photosensitizers. Pharmacokinetic studies of the porphyrin-inducing effect of MAL creams (Metvix) applied in different concentrations (16-160 mg/g) and application times are presented. Surface fluorescence measurements were used to monitor porphyrin accumulation in 18 superficial BCCs and 32 AKs. For both lesion types, the fluorescence increased during the first 13 of 28 hours of continuous MAL application. A 20-fold site-to-site variation was observed, and there were no significant MAL concentration dependencies. The selectivity between lesions and normal skin was 10-fold during the first hours and decreased throughout the application time. Fluorescence microscopy images of tissue sections from 32 nodular BCCs were analyzed to calculate the porphyrin content in tumor tissue as a function of depth. Significant correlation to MAL concentration was seen within the tumors treated for 3 hours. Increase to 18-hour MAL application enhanced the fluorescence levels in superficial tumor layers, but not in deep layers. In conclusion, application of the 160 mg/g cream for 3 hours gave advantageous porphyrin distributions for all types of lesions.

Treatment with ALA PDT induced pronounced necrosis in tumors only if the light was delivered at a low rate. The treatment prolonged the survival for tumor-bearing animals.