Hans J. Schuitmaker

Photodynamic Inactivation of the Dermatophyte Trichophyton rubrum

Abstract Dermatophytes are fungi that can cause infections (known as tinea) of the skin, hair and nails because of their ability to use keratin. Superficial mycoses are probably the most prevalent of infectious diseases worldwide. One of the most distinct limitations of the current therapeutic options is the recurrence of the infection and duration of treatment. The present study shows that Trichophyton rubrum in suspension culture is susceptible to photodynamic treatment (PDT), a completely new application in this area. T. rubrum could be effectively killed with the use of the light-activated porphyrins deuteroporphyrin monomethylester (DP mme) and 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B). The photodynamic efficacy was compared with that of some other photosensitizers that are well known in the field of PDT: the porphyrins deuteroporphyrin and hematoporphyrin, the drug Photofrin and several phthalocyanines. It was demonstrated that with the use of broadband white light, the phthalocyanines and Photofrin displayed a fungistatic effect for about 1 week, whereas all the porphyrins caused photodynamic killing of the dermatophyte. Sylsens B was the most effective sensitizer and showed no dark toxicity; therefore, in an appropriate formulation, it could be a promising candidate for the treatment of various forms of tinea. For Sylsens B and DP mme, which displayed the best results, a concentration-dependent uptake by T. rubrum was established.

Increase of the Photosensitizing Efficiency of the Bacteriochlorin a by Liposome-Incorporation

To describe the action mechanisms of Bacteriochlorin a (BCA), a second generation photosensitizer, in phosphate buffer (PB) and in dimyristoyl phosphatidylcholine (DMPC) liposomes we carried out oxygen consumption and ESR measurements. In PB, where BCA was in a monomer-dimer equilibrium, our results suggested that the oxygen consumption was related to the BCA monomers concentration in solution. Incorporation of BCA in DMPC liposomes, by promoting the monomerization of BCA, increased 9-fold the oxygen consumption in comparison to the value in PB. The use of specific singlet oxygen quenchers (Azide and 9,10-Anthracenedipropionic acid) in ESR and oxygen consumption experiments allowed us to assert that BCA was mainly a type II sensitizer when it was incorporated in DMPC. Finally, the cell survival of WiDr cells after a PDT treatment was measured for cells incubated with BCA in cell culture medium and cells incubated with BCA in DMPC. Irrespective of the dye concentration, the cell survival was lower when liposomes were used. This effect could be the result of a better BCA monomerization and/or a different BCA uptake in cells.

Photodynamic Treatment of the Dermatophyte Trichophyton rubrum and its Microconidia with Porphyrin Photosensitizers

The application of photosensitizers for the treatment of fungal infections is a new and promising development within the field of photodynamic treatment (PDT). Dermatophytes, fungi that can cause infections of the skin, hair and nails, are able to feed on keratin. Superficial mycoses are probably the most prevalent of infectious diseases in all parts of the world. One of the most important restrictions of the current therapeutic options is the return of the infection and the duration of the treatment. This is especially true in the case of infections of the nail (tinea unguium) caused by Trichophyton rubrum, an anthropophilic dermatophyte with a worldwide distribution. Recently, we demonstrated that 5,10,15‐tris(4‐methylpyridinium)‐20‐phenyl‐[21H,23H]‐porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester were excellent photosensitizers toward T. rubrum when using broadband white light. This study demonstrates the photodynamic activity of these photosensitizers with red light toward both a suspension culture of T. rubrum and its isolated microconidia. The higher penetration depth of red light is important for the PDT of nail infections. In addition, we tested the photodynamic activity of a newly synthesized porphyrin, quinolino‐[4,5,6,7‐efg]‐7‐demethyl‐8‐deethylmesoporphyrin dimethylester, displaying a distinct peak in the red part of the spectrum. However, its photodynamic activity with red light toward a suspension culture of T. rubrum appeared to be only fungistatic. Sylsens B was the best photosensitizer toward both T. rubrum and its microconidia. A complete inactivation of the fungal spores and destruction of the fungal hyphae was found. In studies into the photostability, Sylsens B appeared to be photostable under the conditions used for fungal PDT. A promising result of this study is the demonstration of the complete degradation of the fungal hyphae in the time after the PDT and the inactivation of fungal spores, both with red light. These results offer the ingredients for a future treatment of fungal infections, including those of the nail.

Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen During the Photohemolysis of Human Erythrocytes with Bacteriochlorin A

Abstract— Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate‐buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K‐leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K‐leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K‐leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin‐trapping techniques that BCA is able to generate 02∼* and OH* radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline‐iV‐oxide (DMPO) gave an ESR spectrum characteristic of the DMPO‐hydroxyl radical spin adduct DMPO‐OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH * and CO2‐1 radical adducts, indicating the presence of free OH*. Production of DMPO‐OH was partly inhibited by superoxide dismutase (SOD), catalase and desferoxamine, suggesting that the iron‐catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH *. The complementary inhibition of DMPO‐OH production by azide and 9,10‐anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO‐OH and free OH*. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye‐induced hemolysis of erythrocytes was well caused by a mixed type 1/type 2 mechanism.

Positively charged porphyrins: a new series of photosensitizers for sterilization of RBCs

BACKGROUND:u2002 Photodynamic treatment could be a way to inactivate pathogens in RBCs. The objective of this study was to characterize the virucidal activity and RBC‐damaging activity of a series of cationic porphyrins. Using the most efficacious photosensitizer, various in‐vitro human RBC quality variables and in‐vivo RBC survival in Rhesus monkeys were evaluated.

Decreased expression of HLA class I on ocular melanoma cells following in vitro photodynamic therapy

Our objective was to investigate whether photodynamic therapy (PDT) influences the expression of HLA Class I and beta 2-microglobulin molecules on cultured uveal melanoma cells. Uveal melanoma cells were incubated with hematoporphyrin esters (HPE) and illuminated using red light. HLA expression on cells was determined by flowcytometry. PDT treatment induced an immediate reduction in expression of HLA Class I and beta 2-microglobulin, followed by a transient increase in expression after 2 h. Normalization occurred after 6 h. Treatment of ocular melanoma cells with PDT temporally alters the expression of HLA Class I and beta 2-microglobulin, which may affect anti-tumor-immune responses.

Non-specific systemic immune suppression induced by photodynamic treatment of lymph node cells with bacteriochlorin a

Although photodynamic therapy is being used increasingly for the treatment of cancer, its effect on immune responses has received little attention. This aspect was studied in a rat model. Rats were given (intravenously) lymph node cells which were simultaneously exposed to a photosensitizer and light. As a result of this treatment the animals showed a decreased contact hypersensitivity response. Most important is that the generated immunosuppression proved to be non-specific. The consequence of this finding may be that photodynamic therapy of tumours can inhibit the immune response against malignant cells.

Fluorescence, Absorption and Electron Spin Resonance Study of Bacteriochlorin a Incorporation into Membrane Models

Analysis of the bacteriochlorin a absorption spectra suggests the existence of a monomer-dimer equilibrium, particularly intense in phosphate buffer and favored by a decrease of the pH. The dye in methanolic solution is predominantly in monomeric form. Fluorescence and electron spin resonance nitroxide spin labeling measurements indicate that incorporation into the lipid phase of dimyristoyl-L-alpha-phosphatidylcholine liposomes induces dye monomerization. Moreover, the molecules are bound in the external surface of the vesicles and a complete incorporation is ensured by a lipid-to-dye ratio greater than 125.

Development of a Test System for Mutagenicity of Photosensitizers Using Drosophila melanogaster

In the past few years, there has been an increase in the application of photosensitizers for medical purposes. A good standardized test system for the evaluation of the mutagenic potentials of photosensitizers is therefore an indispensable device. In the standard Ames test, white light itself was proven to be mutagenic and the result influenced by the light source. Lack of a reliable positive control is another problem in many genotoxicity test systems used for the evaluation of mutagenicity of photosensitizers. Based on the validated somatic mutation and recombination test, known as SMART, and using Drosophila melanogaster, we developed the Photo‐SMART and demonstrated that methylene blue, known to induce photomutagenicity, can act as a positive control in the presented test system. The SMART scores for the loss of heterozygosity caused predominantly by homologous mitotic recombination. The Photo‐SMART can be used to detect photogenotoxicity caused by short‐lived photoproducts or by stable photoproducts or both. We demonstrated the Photo‐SMART to be a good standardized test system for the evaluation of mutagenic potentials of the photosensitizer 5,10,15‐tris(4‐methylpyridinium)‐20‐phenyl‐[21H,23H]‐porphine trichloride (TPP). We demonstrated that TPP was mutagenic using the Photo‐SMART. For hematoporphyrin, the results of the Photo‐SMART indicate the absence of mutagenicity.

Corneal neovascularization in rats as a model for photothrombotic therapy using bacteriochlorin a and an argon laser

Abstract• Background: The presence of vessels has a negative influence on corneal transplant survival. Closure of such vessels prior to transplantation may improve the transplant results, and this might be achieved by irradiating the vessels with argon laser light after intravenous administration of a photosensitizer, e.g. bacteriochlorin a (BCA). A suture-induced corneal neovascularization model in rats was set up to test this hypothesis. • Methods: Suture-induced vessels in the cornea of male Wistar rats were irradiated with argon laser light after intravenous administration of BCA. We applied irradiation of varying energy levels and duration and assessed the changes in the vessels by slit-lamp examination, fluorescein angiography and histology. • Results: Suture-induced corneal vessels in the rat could be used effectively to study photothrombosis therapy. Intravenous administration of BCA prior to irradiation (λ=514.5 nm) of the corneal vessels led to vessel closure at lower energy levels and of longer duration than occurred with laser treatment alone. • Conclusion: Suture-induced corneal neovascularization in the rat can be used as a model to study the efficacy of photothrombosis therapy. BCA can be used to enhance the rate and duration of vessel closure.