Kristian Berg

5-Aminolevulinic acid-based photodynamic therapy : Clinical research and future challenges

Photodynamic therapy (PDT) for cancer patients has developed into an important new clinical treatment modality in the past 25 years. PDT involves administration of a tumor‐localizing photosensitizer or photosensitizer prodrug (5‐aminolevulinic acid [ALA], a precursor in the heme biosynthetic pathway) and the subsequent activation of the photosensitizer by light. Although several photosensitizers other than ALA‐derived protoporphyrin IX (PpIX) have been used in clinical PDT, ALA‐based PDT has been the most active area of clinical PDT research during the past 5 years. Studies have shown that a higher accumulation of ALA‐derived PpIX in rapidly proliferating cells may provide a biologic rationale for clinical use of ALA‐based PDT and diagnosis. However, no review updating the clinical data has appeared so far.

THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3‐hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i. e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3–6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01–0.02 μm, which corresponds to a lifetime of 0.01–0.04 μs of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.

5-Aminolevulinic Acid-Based Photodynamic Therapy: Principles and Experimental Research

The iron(I1) complex of protoporphyrin IX (PpIX)? (heme) is bound to different proteins to form key biomolecules (hemoproteins) such as hemoglobin, myoglobin, cytochromes, catalase, peroxidase and tryptophan pyrrolase. The lives of the cells and of the body as a whole is therefore crucially dependent upon the biosynthesis and metabolism of porphyrins. Almost all types of cells of the human body, with the exception of mature red blood cells, are equipped with a machinery to synthesize heme. In the first step of the heme biosynthetic pathway 5-aminolevulinic acid (ALA) is formed from glycine and succinyl CoA. The synthesis of ALA is regulated by the amount of heme in the cell. The last step in the formation of heme is the incorporation of iron into PpIX and takes place in the mitochondria under the action of the enzyme, ferrochelatase. By adding exogenous ALA, PpIX may accumulate because of the limited capacity of ferrochelatase. Porphobilinogen deaminase (PBGD) is another enzyme that is active in the heme synthesis pathway (catalyzing the formation of uroporphyrinogen from porphobilinogen [PBG]). The activity of this enzyme is higher in some tumors (1-3), while that of ferrochelatase is lower (2-7), so that PpIX accumulates with some degree of selectivity in tumors. Because PpIX is an efficient photosensitizer, ALA has been introduced as a drug for clinical photodynamic therapy (PDT) of cancer (8,9). Photodynamic therapy involves systemic administration of a tumor-localizing photosensitizer and its subsequent activation by light of an appropriate wavelength to create a pho-

Photochemotherapy of cancer: experimental research.

Worldwide, photochemotherapy ( P a ) * is being evaluated as a new modality of cancer treatment. It is based on injection of photosensitizing and tumor localizing dyes followed by exposure of the tumor region to high fluence rates of light, usually from a laser. A variety of different types of tumors respond to PCT. However, PCT is efficient only in cases where the entire tumor can be reached by light. Thus, tumors thicker than 5-7 mm are rarely eradicated by PCT, unless the light is applied interstitially through fibers. Extracorporal PCT, with 8methoxypsoralen as sensitizer, of T-cells of patients suffering from the erythrodermic form of cutaneous T-cell lymphoma has been reported to give good clinical results. Extracorporal PCT for elimination of residual tumor cells from autologous bone marrow grafts is also being evaluated. The biological effects of surgical lasers, like the C0,-laser and the Nd:YAG-laser, are based on the heating that occurs when the laser radiation is absorbed by tissues. Unlike this, PCT is based on photochemical reactions and not on tissue heating. A combination of injection of a tumor localizing and photosensitizing dye and local exposure of the tumor region to light constitutes the basis for PCT. The fact that certain porphyrins administered to tumor-bearing animals tend to accumulate in tumor tissue has been known for more than 50 years (Policard, 1924). Since most porphyrins exhibit a characteristic red fluorescence, their tumor localizing properties can be used for tumor detection (Lipson, 1976; Pope et al . , 1991). The photosensitizing properties of porphyrins, together with their tumor-localizing properties, were first exploited for therapeutic purposes by Auler and Banzer in 1943 (Auler and Banzer, 1943). The first cancer patients were treated with PCT in 1976 (Kelly and Snell, 1976). Since that time a number of clinical trials have been performed (Dougherty, 1987). Also a large number of experimental investigations have been carried out (Moan, 1986a; Henderson and Dougherty; 1992). In the following some of the most recent knowledge gained through experimental and clinical research on PCT will be reviewed.

PHOTOSENSITIZING EFFICIENCIES, TUMOR‐ and CELLULAR UPTAKE OF DIFFERENT PHOTOSENSITIZING DRUGS RELEVANT FOR PHOTODYNAMIC THERAPY OF CANCER: Section IV – Cellular Photosensitization

Abstract Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)‐di‐hexyl‐ether, HP‐di‐ethyl‐ether, tetra (3‐hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS4) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e6) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X‐114/H20 partition coefficients at different pH‐values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption‐, fluorescence‐excitation‐ and fluorescence‐emission spectra of the cell‐bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3‐diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes.

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.

Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX

The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.

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.

Lysosomes and Microtubules as Targets for Photochemotherapy of Cancer

Most of the photosensitizers used in experimental and clinical photochemotherapy (PCT)? of cancer (i.e. photodynamic therapy [PDT]) localize extranuclearly in cells: in the plasma membrane, in mitochondria, in endoplasmic reticulum, in Golgi apparatus and in lysosomes. The intracellular localization is dependent upon the chemical properties of the sensitizer, i.e. its hydrophobicity, charge and amphiphilic character (1). The intracellular localization seems also to be to some extent cell line dependent. The identity of the intracellular targets responsible for photochemically induced cell death depends on the parameters mentioned above. The special cases of lysosomes and microtubules (MT) as targets for photochemical treatment of cells in culture will be discussed.