Anders Høgset

Porphyrin‐related photosensitizers for cancer imaging and therapeutic applications

A 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 internalization provides time- and space-controlled endolysosomal escape of therapeutic molecules.

A successful cure of cancer by biopharmaceuticals with intracellular targets is dependent on both specific and sufficient delivery of the drug to the cytosol or nuclei of malignant cells. However, cytosolic delivery and efficacy of membrane-impermeable cancer therapeutics are often hampered by the sequestration and degradation of the drugs in the endolysosomal compartments. Hence, we developed photochemical internalization (PCI) as a site-specific drug delivery technology, which bursts the membrane of endocytic vesicles inducing release of entrapped drugs to the cytosol of light exposed cells. The principle of PCI has been demonstrated in >80 different cell lines and 10 different xenograft models of various cancers in different laboratories demonstrating its broad application potential. PCI-induced endosomal escape of protein- or nucleic acid-based therapeutics and some chemotherapeutics will be presented in this review. With a joint effort by life scientists the PCI technology is currently in a Phase I/II clinical trial with very promising initial results in the treatment of solid tumors.

5‐Aminolevulinic Acid, but not 5‐Aminolevulinic Acid Esters, is Transported into Adenocarcinoma Cells by System BETA Transporters

Abstract 5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis–Menten kinetics (Km = 8–10 mM and Vmax = 18–20 nmol·(mg protein × h)−1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32°C. The activation energy was 112 kJ·mol−1 in the temperature range 15°–32°C and 26 kJ·mol−1 above 32°C. Transport of 5-ALA was Na+ and partly Cl−-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65–75%, by the β-amino acids, β-alanine and taurine and by γ-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-β-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM β-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 μM) was not significantly influenced by β-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by β-amino acid and GABA carriers in this cell line.

Site-Specific Drug Delivery by Photochemical Internalization Enhances the Antitumor Effect of Bleomycin

Purpose: Photochemical internalization is under development for improving macromolecular therapy by inducing photochemical damage to endocytic vesicles. This damage leads to the release of therapeutic macromolecules entrapped in endocytic vesicles into the cytosol. The macromolecules may in this way be able to interact with therapeutic targets instead of being degraded by lysosomal hydrolases. Bleomycin is used in several standard cancer chemotherapy regimens. Its hydrophilic and relatively large chemical structure limits its ability to penetrate membrane structures, which causes the accumulation of bleomycin in endocytic vesicles. The purpose of this study was to evaluate the therapeutic potential of aluminum phthalocyanine disulfonate (AlPcS2a)–based photochemical delivery of bleomycin. Experimental Design: Three tumors of different origin were grown s.c. in BALB/c (nu/nu) mice. The photosensitizer AlPcS2a and bleomycin were systemically administered and the tumor area was exposed to red light when the tumor volume had reached 100 mm3. The tumor volume was measured frequently after treatment and the time for the tumor volume to reach 800 to 1,000 mm3 was selected as the end point. Results: The photochemical delivery of bleomycin induced a delayed tumor regrowth, and in two out of three tumor models, lead to 60% complete response, whereas no complete responses were seen after treatment with bleomycin alone. A statistical model to assess synergism was established. Combination of the photochemical treatment and bleomycin was found to induce a synergistic delay in tumor growth. Conclusion: AlPcS2a-based photochemical internalization of bleomycin induces a synergistic inhibition of tumor growth in three different tumor models. This treatment combination should be further considered for clinical utilization.

Photochemical transfection : A new technology for light-induced, site-directed gene delivery

The development of methods for specific delivery of therapeutic genes into target tissues is an important issue for the further progress of in vivo gene therapy. In this article we report on a novel technology, named photochemical transfection, to use light to direct a precise delivery of therapeutic genes to a desired location. The technology makes use of photosensitizing compounds that localize mainly in the membranes of endosomes and lysosomes. On illumination these membrane structures will be destroyed, releasing endocytosed DNA into the cell cytosol. Using a green fluorescent protein gene as a model we show that illumination of photosensitizer-treated cells induces a substantial increase in the efficiency of transfection by DNA-poly-L-lysine complexes. Thus, in a human melanoma cell line the light treatment can increase the transfection efficiency more than 20-fold, reaching transfection levels of about 50% of the surviving cells. In this article various parameters of importance for the use of this technology are examined, and the potential use of the technology in gene therapy is discussed.

Photochemical Internalization: A New Tool for Drug Delivery

The utilisation of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action. Photochemical internalisation (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby, endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment.

Photochemical disruption of endocytic vesicles before delivery of drugs: a new strategy for cancer therapy.

The development of methods for specific delivery of drugs is an important issue for many cancer therapy approaches. Most of macromolecular drugs are taken into the cell through endocytosis and, being unable to escape from endocytic vesicles, eventually are degraded there, which hinders their therapeutic usefulness. We have developed a method, called photochemical internalization, based on light-induced photochemical reactions, disrupting endocytic vesicles specifically within illuminated sites e.g. tumours. Here we present a new drug delivery concept based on photochemical internalization-principle – photochemical disruption of endocytic vesicles before delivery of macromolecules, leading to an instant endosomal release instead of detrimental stay of the molecules in endocytic vesicles. Previously we have shown that illumination applied after the treatment with macromolecules substantially improved their biological effect both in vitro and in vivo. Here we demonstrate that exposure to light before delivery of protein toxin gelonin improves gelonin effect in vitro much more than light after. However, in vitro transfection with reporter genes delivered by non-viral and adenoviral vectors is increased more than 10- and six-fold, respectively, by both photochemical internalization strategies. The possible cellular mechanisms involved, and the potential of this new method for practical application of photochemical internalization concept in cancer therapy are discussed.

Evaluation of Different Photosensitizers for Use in Photochemical Gene Transfection

Abstract Many potentially therapeutic macromolecules, e.g. transgenes used in gene therapy, are taken into the cells by endocytosis, and have to be liberated from endocytic vesicles in order to express a therapeutic function. To achieve this we have developed a new technology, named photochemical internalization (PCI), based on photochemical reactions inducing rupture of endocytic vesicles. The aim of this study was to clarify which properties of photosensitizers are important for obtaining the PCI effect improving gene transfection. The photochemical effect on transfection of human melanoma THX cells has been studied employing photosensitizers with different physicochemical properties and using two gene delivery vectors: the cationic polypeptide polylysine and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Photochemical treatment by photosensitizers that do not localize in endocytic vesicles (tetra[3-hydroxyphenyl]porphyrin and 5-aminolevulinic acid–induced protoporphyrin IX) do not stimulate transfection, irrespective of the gene delivery vector. In contrast, photosensitizers localized in endocytic vesicles stimulate polylysine-mediated transfection, and amphiphilic photosensitizers (disulfonated aluminium phthalocyanine [AlPcS2a] and meso-tetraphenylporphynes) show the strongest positive effect, inducing approximately 10-fold increase in transfection efficiency. In contrast, DOTAP-mediated transfection is inhibited by all photochemical treatments irrespective of the photosensitizer used. Neither AlPcS2a nor Photofrin affects the uptake of the transfecting DNA over the plasma membrane, therefore photochemical permeabilization of endocytic vesicles seems to be the most likely mechanism responsible for the positive PCI effect on gene transfection.

Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy

A main issue for further clinical progress of cancer gene therapy is to develop technologies for efficient and specific delivery of therapeutic genes to tumor cells. In this work, we describe a photochemical treatment that substantially improves gene delivery by adenovirus, one of the most efficient gene delivery vectors known. Transduction of two different cell lines was studied by microscopy, flow cytometry, and an enzymatic assay, employing a β-galactosidase–encoding adenovirus. The photochemical treatment induced a >20-fold increase in gene transduction, compared with conventional adenovirus infection, both when measured as the percentage of cells transduced, and when measured as the total β-galactosidase activity in the cell population. The effect was most pronounced at lower virus doses, where in some cases the same transduction efficiency could be achieved with a 20 times lower virus dose than with conventional infection. Photochemical treatments are already in clinical use for cancer therapy, and generally are very specific and have few side effects. The photochemical internalization technology described thus has a clear potential for improving both the efficiency and the specificity of gene delivery in cancer gene therapy, making it possible to achieve efficient site-specific in vivo gene delivery by adenoviral vectors.

Disulfonated tetraphenyl chlorin (TPCS2a), a novel photosensitizer developed for clinical utilization of photochemical internalization

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.