Predicting human pharmacokinetics of liposomal temoporfin using a hybrid in silico model.

Over the years, the performance of the liposomal formulations of temoporfin, Foslip® and Fospeg®, was investigated in a broad array of cell-based assays and preclinical animal models. So far, little attention has been paid to the influence of drug release and liposomal stability on the plasma concentration-time profile. The drug release is a key attribute which impacts product quality and the in vivo efficacy of nanocarrier formulations. In the present approach, the in vitro drug release and the drug-protein transfer of Foslip® and Fospeg® was determined using the dispersion releaser technology. To analyze the stability of both formulations in physiological fluids, nanoparticle tracking analysis was applied. A comparable drug release behavior and a high physical stability with a vesicle size of approximately 92 ± 2 nm for Foslip® and at 111 ± 5 nm for Fospeg® were measured. The development of a novel hybrid in silico model resulted in an optimal representation of the in vivo data. Based on the information available for previous formulations, the model enabled a prediction of the performance of Foslip® in humans. To verify the simulations, plasma concentration-time profiles of a phase I clinical trial were used. An absolute average fold error of 1.4 was achieved. Moreover, a deconvolution of the pharmacokinetic profile into different fractions relevant for the in vivo efficacy and safety was achieved. While the total plasma concentration reached a cmax of 2298 ng/mL after 0.72 h, the monomolecular drug accounted for a small fraction of the photosensitizer with a cmax of 321 ng/mL only.

A new level of in vivo singlet molecular oxygen luminescence measurements.

BACKGROUND: Singlet oxygen is known to be the main mediator of the photodynamic effect. The kinetics of its generation and deactivation allows for insights in the microenvironment and efficacy of the photodynamic effect. Therefore, it is highly desirable to perform direct and time resolved measurements of singlet molecular oxygen (1O2) as well as data analysis during the therapy. METHODS: In this work, tumors grown on the CAM of chicken embryos as well as blood vessels were scanned after injection of the photosensitizer Foslip®, yielding time resolved singlet molecular oxygen luminescence. Using a custom-made trifurcated fiber, it is possible to simultaneously detect time resolved NIR luminescence as well as spectrally resolved UV/VIS fluorescence. RESULTS: After photosensitizer application the singlet oxygen luminescence kinetics for tumors grown on the CAM of chicken embryos as well as for mixed venous and arterialized blood were recorded. Data was analyzed by traditional fitting as well as a novel and robust approach, reducing the time resolved data to a a meaningful minimum. Both approaches show the differences between blood of different oxygen saturation as well as tumor tissue. CONCLUSIONS: This work shows for the first time the possibility of deducing the oxygen content during photodynamic therapy by measuring singlet oxygen kinetics in tissue. If more oxygen is consumed - due to chemical quenching during PDT - than is subsequently diffused, oxygen depletion occurs, resulting in inefficiency of the photodynamic effect. These results represent a major step towards live monitoring of therapy success and thus towards the possibility of direct control of PDT efficiency in real time.

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells.

PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Mosquito larvae control by photodynamic inactivation of their intestinal flora - a proof of principal study on Chaoborus sp.

Mosquitoes are carriers of dangerous infectious disease pathogens all over the world. Owing to travelling and global warming, tropical disease-carrying species such as Aedes, Anopheles and Culex spread beyond tropical and subtropical zones, even to Europe. The aim of this study is to investigate the potential of photodynamic agents to combat mosquito larvae. Three different photosensitizers were tested on Chaoborus sp. larvae: TMPyP and TPPS as antimicrobial photosensitizers, and mTHPC as a PDT drug against eukaryotic animal and human cells. Chaoborus sp. is a commercially available harmless species developing translucent larvae similar to the larvae of Aedes, Anopheles and Culex. The uptake of photosensitizers by the larvae was tested by fluorescence microscopy. All tested photosensitizers were observed in the intestinal tract of the living larvae, and none of the photosensitizers was found in the larval tissues. In phototoxicity tests, mTHPC and TPPS did not have any effect on the larvae, while TMPyP killed the larvae efficiently. TPPS is an antimicrobial photosensitizer, mainly phototoxic to Gram-positive bacteria. TMPyP is well known as an efficient photosensitizer against Gram-negative bacteria like most species of the intestinal flora. From this result, we conclude that the photodynamic inactivation of the intestinal flora leads to the death of mosquito larvae. The feasibility of mosquito larvae control by photodynamic inactivation of their intestinal flora instead of the direct killing of the larvae is a promising alternative to other highly toxic insecticides. Compared to insecticides and other biochemical toxins, photosensitizers are not dark toxic. No resistance against photosensitizers is known so far. Thus, the dilution of the active substances by being distributed in the environment, which promotes the development of resistance in biocides of all kinds, does not pose danger. Thus, it reduces the potential side effects on environment and human health.

Advanced in silico modeling explains pharmacokinetics and biodistribution of temoporfin nanocrystals in humans.

Foscan®, a formulation comprising temoporfin dissolved in a mixture of ethanol and propylene glycol, has been approved in Europe for palliative photodynamic therapy of squamous cell carcinoma of the head and neck. During clinical and preclinical studies it was observed that considering the administration route, the drug presents a rather atypical plasma profile as plasma concentration peaks delayed. Possible explanations, as for example the formation of a drug depot or aggregation after intravenous administration, are discussed in current literature. In the present study an advanced in silico model was developed and evaluated for the detailed description of Foscan® pharmacokinetics. Therefore, in vitro release data obtained from experiments with the dispersion releaser technology investigating dissolution pressures of various release media on the drug as well as in vivo data obtained from a clinical study were included into the in silico models. Furthermore, precipitation experiments were performed in presence of biorelevant media and precipitates were analyzed by nanoparticle tracking analysis. Size analysis and particle fraction were also incorporated in this model and a sensitivity analysis was performed. An optimal description of the in vivo situation based on in vitro release and particle characterization data was achieved, as demonstrated by an absolute average fold error of 1.21. This in vitro-in vivo correlation provides an explanation for the pharmacokinetics of Foscan® in humans.

In vitro photodynamic inactivation (PDI) of pathogenic germs inducing onychomycosis.

Onychomycosis is a fungal nail infection caused primarily by the dermatophytes Trichophyton rubrum and Trichophyton interdigitale or, less frequently, by molds like Aspergillus spp. and Scopulariopsis brevicaulis. Photodynamic treatment of onychomycosis is considered a promising future therapy to overcome the frequent failure of currently used antifungals. In this study, we tested the potential of three photosensitizers for photodynamic inactivation of the onychomycosis causing pathogens T. rubrum, T. interdigitale and S. brevicaulis. Photosensitizers used are 10,15,20-Tetrakis(1-methylpyridinium-4-yl) porphyrintetra(p-toluenesulfonate) (TMPyP), 5,10,15-tris-(1-methylpyridinium-2-yl)corrolato-(trans-dihydroxo)phosphorus(V) (PCor+) and 2',4',5',7'-tetrabromo-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one (Eosin Y). The phototoxic effects caused by the cationic photosensitizers (PCor+ and TMPyP) were tested on suspension cultures of spores as well as on fungi during growth on surfaces where both photosensitizers cause high phototoxicity. The anionic Eosin Y was tested on surface-growing fungi only and induces remarkable phototoxic effects on dermatophytes and molds. In all cases, no spore regrowth was detected after PDI. This study is considered a first step towards successful and cost efficient treatment of onychomycosis.

Electron beam functionalized photodynamic polyethersulfone membranes - photophysical characterization and antimicrobial activity.

Polymer membranes are powerful filtration tools in medicine and water treatment. Their efficiency and operational lifetime is limited by biofouling caused by microorganisms. This study describes the development of photodynamical active antimicrobial polymer membranes in a one-pot functionalization step using a well-known photosensitizer (PS). Commercially available polyethersulfone (PES) membranes for microfiltration were doped with the polycationic PS TMPyP using electron beam irradiation. These membranes were characterized in terms of binding stability and quantification of the PS and membrane morphology. Furthermore, the photodynamic ability was verified by time resolved singlet oxygen luminescence scans and successfully tested against the Gram-negative bacterium E. coli under low dose white light illumination resulting in the reduction in cell survival of 6 log10 units. Finally, in preliminarily experiments the photodynamic action against the Gram-positive bacteria M. luteus and the Gram-negative P. fluorescence and the mold C. cladosporioides was demonstrated. These promising results show the high photodynamic potential of electron beam functionalization of PES membranes with TMPyP. It preserves the photodynamic abilities of the immobilized PS resulting in efficient photodynamic inactivation of bacteria and mold on the membrane surface. The uprising worldwide spread of antibiotic resistant bacteria makes the development of new antibacterial strategies an inevitable challenge. The photodynamic inactivation of bacteria and its adaptation for antimicrobial surfaces, e.g. filtration membranes for water treatment, displays many advantages in terms of a wide application range, low mutagenic potential and environmental compatibility.

In vivo singlet molecular oxygen measurements: Sensitive to changes in oxygen saturation during PDT.

BACKGROUND: Direct singlet molecular oxygen detection is known to be a valuable tool for understanding photodynamic action. It could become useful for optimizing illumination schedules in photodynamic therapy. The method of time resolved singlet molecular oxygen luminescence detection can give insights into generation of singlet oxygen and its interaction with the environment and therefore possibly allows monitoring the treatments efficacy. Due to high requirements for sensitivity as well as time resolution it has not yet been used in situ. The latest improvements in the detection system make in vivo time resolved singlet molecular oxygen luminescence detection possible. METHODS: In this work, blood vessels in the chicken embryo CAM-model were scanned after injection of the photosensitizer Foslip®, yielding time resolved singlet molecular oxygen luminescence. A custom-made trifurcated fiber in combination with a dye laser, a photomultiplier tube and a fiber spectrometer was utilized for simultaneous excitation, singlet molecular oxygen luminescence and photosensitizer fluorescence detection. RESULTS: Singlet oxygen luminescence kinetics for mixed venous and arterialized blood in chicken embryos using the CAM-model were recorded. The data analysis resulted in two distinct and distinguishable photosensitizer triplet lifetimes corresponding to the high and low oxygen partial pressures in the oxygen-rich arterialized blood and oxygen-poor mixed venous blood. CONCLUSIONS: The sensitivity of direct singlet molecular oxygen luminescence detection to different oxygen partial pressures could be shown in vivo. Therefore, this study is a first step towards optimizing the illumination conditions of photodynamic treatment in situ by real time monitoring of the oxygen partial pressure within the target tissue.

Photodynamic inactivation of Escherichia coli - Correlation of singlet oxygen kinetics and phototoxicity.

Photodynamic inactivation (PDI) of bacteria may play a major role in facing the challenge of the ever expanding antibiotic resistances. Here we report about the direct correlation of singlet oxygen luminescence kinetics and phototoxicity in E. coli cell suspension under PDI using the widely applied cationic photosensitizer TMPyP. Through direct access to the microenvironment, the time resolved investigation of singlet oxygen luminescence plays a key role in understanding the photosensitization mechanism and inactivation pathway. Using the homemade set-up for highly sensitive time resolved singlet oxygen luminescence detection, we show that the cationic TMPyP is localized predominantly outside the bacterial cells but in their immediate vicinity prior to photodynamic inactivation. Throughout following light exposure, a clear change in singlet oxygen kinetics indicates a redistribution of photosensitizer molecules to at least one additional microenvironment. We found the signal kinetics mirrored in cell viability measurements of equally treated samples from same overnight cultures conducted in parallel: A significant drop in cell viability of the illuminated samples and stationary viability of dark controls. Thus, for the system investigated in this work - a Gram-negative model bacteria and a well-known PS for its PDI - singlet oxygen kinetics correlates with phototoxicity. This finding suggests that it is well possible to evaluate PDI efficiency directly via time resolved singlet oxygen detection.

Synthesis, Photophysics and PDT Evaluation of Mono-, Di-, Tri- and Hexa-PEG Chlorins for Pointsource Photodynamic Therapy.

Pointsource photodynamic therapy (PSPDT) is a newly developed fiber optic method aimed at the delivery of photosensitizer, light and oxygen to a diseased site. Because of a need for developing photosensitizers with desirable properties for PSPDT, we have carried out a synthetic, photophysical and phototoxicity study on a series of PEGylated sensitizers. Chlorin and pheophorbide sensitizers were readily amenable to our synthetic PEGylation strategy to reach triPEG and hexaPEG galloyl pheophorbides and mono-, di-, triPEG chlorins. On screening these PEG sensitizers, we found that increasing the number of PEG groups, except for hexaPEGylation, increases phototoxicity. We found that three PEG groups but not less or more were optimal. Of the series tested, a triPEG gallyol pheophorbide and a triPEG chlorin were the most efficient at generating singlet oxygen, and produced the highest phototoxicity and lowest dark toxicity to Jurkat cells. A detailed kinetic analysis of the PEGylated sensitizers in solution and cell culture and media is also presented. The data provide us with steps in the development of PSPDT to add to the PDT tools we have in general.

Photodynamic inactivation of biofilm building microorganisms by photoactive facade paints.

This study was performed as a proof of concept for singlet oxygen generating facade paint as an alternative to conventional biocide containing facade paint for the prevention of biofilm growth on outdoor walls. Biofilms on outdoor walls cause esthetic problems and economic damage. Therefore facade paints often contain biocides. However commercially available biocides may have a series of adverse effects on living organisms as well as harmful environmental effects. Furthermore, biocides are increasingly designed to be more effective and are environmentally persistent. Thus, an eco-friendly and non-harmful to human health alternative to conventional biocides in wall color is strongly recommended. The well-known photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) was used as an additive in a commercially available facade paint. The generation of singlet molecular oxygen was shown using time resolved 2D measurements of the singlet oxygen luminescence. The photodynamic activity of the photosensitizer in the facade paint was demonstrated by phototoxicity tests with defined mold fungi and a mixture of microorganisms harvested from native outdoor biofilms as model organisms. It was proven in general that it is possible to inhibit the growth of biofilm forming microorganisms growing on solid wall paint surfaces by the cationic photosensitizer TMPyP added to the facade paint using daylight conditions for illumination in 12h light and dark cycles.

Photodynamic inactivation of mold fungi spores by newly developed charged corroles.

The photodynamic effect, originally used in photodynamic therapy (PDT) for the treatment of different diseases, e.g. of cancer, has recently been introduced for the inactivation of bacteria. Mold fungi, which provoke health problems like allergies and diseases of the respiratory tract, are even more resistant and their biology is also very different. This study presents the development of four new photosensitizers, which, in combination with low doses of white light, inhibit the germination of mold fungi spores. Two of them even cause lethal damage to the conidia (spores) which are responsible for the spreading of mold fungi. The photoactivity of the newly synthesized corroles was obtained by their application on three different mold fungi: Aspergillus niger, Cladosporium cladosporoides, and Penicillium purpurgenum. To distinguish between inactivation of germination and permanent damage, the fungi were first incubated under illumination for examination of photosensitizer-induced growth inhibition and then left in darkness to test the survival of the conidia. None of the compounds displayed dark toxicity, but all of them attenuated or prevented germination when exposed to light, and the positively charged complexes induced a complete damage of the conidia.

Photosensitizer loaded HSA nanoparticles II: in vitro investigations.

The photosensitizing efficiency of human serum albumin (HSA) nanoparticles loaded with the photosensitizers meta-tetra(hydroxy-phenyl)-chlorin (mTHPC) and meta-tetra(hydroxy-phenyl)-porphyrin (mTHPP) was investigated in vitro. The endocytotic intracellular uptake, and the time dependent drug release caused by nanoparticle decomposition of the PS loaded HSA nanoparticles were studied on Jurkat cells in suspension. The photoxicity as well as the intracellular singlet oxygen ((1)O(2)) generation were investigated in dependence on the incubation time. The obtained results show that HSA nanoparticles are promising carriers for the clinical used mTHPC (Foscan). After release the ((1)O(2)) generation as well as the phototoxicity are more efficient compared with mTHPC applied without the HSA nanoparticles.

Photosensitizer loaded HSA nanoparticles. I: Preparation and photophysical properties.

Photodynamic therapy (PDT) is a promising option in the treatment of cancer. Efficient photosensitizers are available but many of them have insufficient physico-chemical properties for parenteral application. We have established nanoparticles consisting of human serum albumin (HSA) as a drug carrier system for 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrine (mTHPP) and 5,10,15,20-tertrakis(m-hydroxyphenyl)chlorin (mTHPC), two well-known photosensitizers. Nanoparticle loading was performed in water/ethanol mixtures in the presence of dissolved HSA acting as solubilizer for photosensitizers. The HSA concentration was optimized to exclude precipitation in the nanoparticle suspension and to increase binding to nanoparticles. Additionally, the influence of pH and incubation time on drug adsorption was investigated. A freeze drying method was established for mTHPC loaded nanoparticles and the storage stability of the freeze dried formulation was tested. PDT related photophysical parameters of drug loaded HSA nanoparticles, especially singlet oxygen generation, are presented. Both preparations were able to generate singlet oxygen with low quantum yield. In contrast, efficient singlet oxygen generation was obtained when Jurkat cells were incubated with mTHPP and mTHPC loaded HSA nanoparticles. This indicates that the photosensitizer molecules were successfully released from the nanoparticles that were taken up by the cells. Therefore, the efficiency of HSA nanoparticles as drug carriers for photosensitizers was proven under in vitro conditions.

New insights to primary photodynamic effects--Singlet oxygen kinetics in living cells.

The kinetics of chemical singlet oxygen quencher consumption inside living cells during low dose illumination was revealed via time resolved singlet oxygen luminescence detection. Deviations of the measured data from the common theoretical model for (1)O(2) kinetics forced the authors to consider a one-dimensional diffusion model for description of the kinetics of singlet oxygen generated by membrane localized photosensitizers. Our observations reconcile seemingly contradictory reports presenting different values for the efficiency of singlet oxygen interaction with cells.

Novel photosensitizer-protein nanoparticles for photodynamic therapy: photophysical characterization and in vitro investigations.

In this work two types of pheophorbide-HSA (Pheo-HSA) nanoparticles, PHSA40 and PHSA100, were prepared and their photophysical and photosensitizing properties were investigated. Due to intramolecular interactions the singlet oxygen quantum yield of PHSA40 and PHSA100 is very low (less than 0.1). Intracellular uptake and phototoxicity of pheophorbide a as well as of the Pheo-HSA nanoparticles were studied in Jurkat cells. The HSA nanoparticles do not influence the amount of dye accumulation in cells. After 24h incubation, PHSA40 and PHSA100 showed a higher phototoxicity than Pheo. The reason for this behavior is an efficient nanoparticle decomposition in the cellular lysosomes. The process of drug release during incubation of cells with Pheo-HSA nanoparticles was illustrated by fluorescence lifetime imaging (FLIM) and confocal laser scanning microscopy (CLSM). The final phototoxicity of Pheo-HSA is at the same scale as induced by free Pheo. The drug release ability of HSA nanoparticles shows the possibility to use such formulations as drug carriers in PDT treatment. Therefore, this work constructs a standard for further investigation and optimization of photosensitizer-HSA drug carrier system.