Cathepsin B-Cleavable Polymeric Photosensitizer Prodrug for Selective Photodynamic Therapy: In Vitro Studies.

Cathepsin B is a lysosomal cysteine protease that plays an important role in cancer, atherosclerosis, and other inflammatory diseases. The suppression of cathepsin B can inhibit tumor growth. The overexpression of cathepsin B can be used for the imaging and photodynamic therapy (PDT) of cancer. PDT targeting of cathepsin B may have a significant potential for selective destruction of cells with high cathepsin B activity. We synthesized a cathepsin B-cleavable polymeric photosensitizer prodrug (CTSB-PPP) that releases pheophorbide a (Pha), an efficient photosensitizer upon activation with cathepsin B. We determined the concentration dependant uptake in vitro, the safety, and subsequent PDT-induced toxicity of CTSB-PPP, and ROS production. CTSB-PPP was cleaved in bone marrow cells (BMCs), which express a high cathepsin B level. We showed that the intracellular fluorescence of Pha increased with increasing doses (3-48 µM) and exerted significant dark toxicity above 12 µM, as assessed by MTT assay. However, 6 µM showed no toxicity on cell viability and ex vivo vascular function. Time-dependent studies revealed that cellular accumulation of CTSB-PPP (6 µM) peaked at 60 min of treatment. PDT (light dose: 0-100 J/cm2, fluence rate: 100 mW/cm2) was applied after CTSB-PPP treatment (6 µM for 60 min) using a special frontal light diffuser coupled to a diode laser (671 nm). PDT resulted in a light dose-dependent reduction in the viability of BMCs and was associated with an increased intracellular ROS generation. Fluorescence and ROS generation was significantly reduced when the BMCs were pre-treated with E64-d, a cysteine protease inhibitor. In conclusion, we provide evidence that CTSB-PPP showed no dark toxicity at low concentrations. This probe could be utilized as a potential imaging agent to identify cells or tissues with cathepsin B activity. CTSB-PPP-based PDT results in effective cytotoxicity and thus, holds great promise as a therapeutic agent for achieving the selective destruction of cells with high cathepsin B activity.

Chemo-manipulation of tumor blood vessels by a metal-based anticancer complex enhances antitumor therapy.

Human pleural mesothelioma is an incurable and chemoresistant cancer. Using a nude mouse xenograft model of human pleural mesothelioma, we show that RAPTA-T, a compound undergoing preclinical evaluation, enhances tumor vascular function by decreasing blood vessel tortuosity and dilation, while increasing the coverage of endothelial cells by pericytes and vessel perfusion within tumors. This in turn significantly reduces the interstitial fluid pressure and increases oxygenation in the tumor. Consequently, RAPTA-T pre-treatment followed by the application of cisplatin or liposomal cisplatin (Lipoplatin) leads to increased levels of the cytotoxin in the tumor and enhanced mesothelioma growth inhibition. We demonstrate that the vascular changes induced by RAPTA-T are related, in part, to the inhibition of poly-(ADP-ribose) polymerase 1 (PARP-1) which is associated to tumor vascular stabilization. These findings suggest novel therapeutic implications for RAPTA-T to create conditions for superior drug uptake and efficacy of approved cytotoxic anti-cancer drugs in malignant pleural mesothelioma and potentially other chemoresistant tumors.

Intra-Arterial Drug and Light Delivery for Photodynamic Therapy Using Visudyne®: Implication for Atherosclerotic Plaque Treatment.

UNLABELLED: Photodynamic therapy (PDT), which is based on the activation of photosensitizers with light, can be used to reduce plaque burden. We hypothesized that intra-arterial photosensitizer administration and photo-activation will lead to high and rapid accumulation within the plaque with reduced systemic adverse effects. Thus, this "intra-arterial" PDT would be expected to have less side effects and due to the short time involved would be compatible with percutaneous coronary interventions. AIM: We characterized the dose-dependent uptake and efficacy of intra-arterial PDT using Liposomal Verteporfin (Visudyne®), efficient for cancer-PDT but not tested before for PDT of atherosclerosis. METHODS AND RESULTS: Visudyne® (100, 200, and 500 ng/ml) was perfused for 5-30 min in atherosclerotic aorta isolated from ApoE(-/-) mice. The fluorescence Intensity (FI) after 15 min of Visudyne® perfusion increased with doses of 100 (FI-5.5 ± 1.8), 200 (FI-31.9 ± 1.9) or 500 ng/ml (FI-42.9 ± 1.2). Visudyne® (500 ng/ml) uptake also increased with the administration time from 5 min (FI-9.8 ± 2.5) to 10 min (FI-23.3 ± 3.0) and 15 min (FI-42.9 ± 3.4) before reaching saturation at 30 min (FI-39.3 ± 2.4) contact. Intra-arterial PDT (Fluence: 100 and 200 J/cm(2), irradiance-334 mW/cm(2)) was applied immediately after Visudyne® perfusion (500 ng/ml for 15 min) using a cylindrical light diffuser coupled to a diode laser (690 nm). PDT led to an increase of ROS (Dihydroethidium; FI-6.9 ± 1.8, 25.3 ± 5.5, 43.4 ± 13.9) and apoptotic cells (TUNEL; 2.5 ± 1.6, 41.3 ± 15.3, 58.9 ± 6%), mainly plaque macrophages (immunostaining; 0.3 ± 0.2, 37.6 ± 6.4, 45.3 ± 5.4%) respectively without laser irradiation, or at 100 and 200 J/cm(2). Limited apoptosis was observed in the medial wall (0.5 ± 0.2, 8.5 ± 4.7, 15.3 ± 12.7%). Finally, Visudyne®-PDT was found to be associated with reduced vessel functionality (Myogram). CONCLUSION: We demonstrated that sufficient accumulation of Visudyne® within plaque could be achieved in short-time and therefore validated the feasibility of local intravascular administration of photosensitizer. Intra-arterial Visudyne®-PDT preferentially affected plaque macrophages and may therefore alter the dynamic progression of plaque development.

Optimization of drug combinations using Feedback System Control.

We describe a protocol for the discovery of synergistic drug combinations for the treatment of disease. Synergistic drug combinations lead to the use of drugs at lower doses, which reduces side effects and can potentially lead to reduced drug resistance, while being clinically more effective than the individual drugs. To cope with the extremely large search space for these combinations, we developed an efficient combinatorial drug screening method called the Feedback System Control (FSC) technique. Starting with a broad selection of drugs, the method follows an iterative approach of experimental testing in a relevant bioassay and analysis of the results by FSC. First, the protocol uses a cell viability assay to generate broad dose-response curves to assess the efficacy of individual compounds. These curves are then used to guide the dosage input of each drug to be tested in combination. Data from applied drug combinations are input into the differential evolution (DE) algorithm, which predicts new combinations to be tested in vitro. This process identifies optimal drug-dose combinations, while saving orders of magnitude in experimental effort. The complete optimization process is estimated to take ∼4 weeks. FSC does not require insight into the disease mechanism, and it has therefore been applied to find combination therapies for many different pathologies, including cancer and infectious diseases, and it has also been used in organ transplantation.

Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease.

Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD). In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.

A streamlined search technology for identification of synergistic drug combinations.

A major key to improvement of cancer therapy is the combination of drugs. Mixing drugs that already exist on the market may offer an attractive alternative. Here we report on a new model-based streamlined feedback system control (s-FSC) method, based on a design of experiment approach, for rapidly finding optimal drug mixtures with minimal experimental effort. We tested combinations in an in vitro assay for the viability of a renal cell adenocarcinoma (RCC) cell line, 786-O. An iterative cycle of in vitro testing and s-FSC analysis was repeated a few times until an optimal low dose combination was reached. Starting with ten drugs that target parallel pathways known to play a role in the development and progression of RCC, we identified the best overall drug combination, being a mixture of four drugs (axitinib, erlotinib, dasatinib and AZD4547) at low doses, inhibiting 90% of cell viability. The removal of AZD4547 from the optimized drug combination resulted in 80% of cell viability inhibition, while still maintaining the synergistic interaction. These optimized drug combinations were significantly more potent than monotherapies of all individual drugs (p < 0.001, CI < 0.3).

Study of the influence of over-the-counter vitamin supplement intake on urine fluorescence to optimize cancer detection by fluorescence cystoscopy.

Fluorescence cystoscopy (FC) efficiently enhances the detection and improves the therapeutic management of early bladder cancer. During an FC, about 150 ml of water is needed to inflate the bladder. The water is quickly diluted by urine which can be fluorescent. If this bladder washout fluid (BWF) becomes fluorescent, the FC images are frequently degraded. Unfortunately, it is unclear which elements of the diet may contribute to this background fluorescence. We propose to start this exploration with over-the-counter (OTC) vitamin supplements. To this end, we measured excitation­emission matrices of urine samples and the kinetics of modifications of urine fluorescence obtained from nine healthy volunteers before, during, and after intake of a commercially available OTC vitamin supplement. The pharmacokinetics shows that the BWF fluorescence values reach a maximum 8 to 10 h after vitamin intake. They decrease in the half-day that follows and reach values close to baseline ~1 day afterward. Based on these results, we conclude that, in order to avoid degradations of fluorescence images, it is likely best that the intake of OTC vitamin supplements be avoided during the week preceding an FC.

Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer.

Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation (p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis (p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly (p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases.

Discovery of a low order drug-cell response surface for applications in personalized medicine.

The cell is a complex system involving numerous components, which may often interact in a non-linear dynamic manner. Diseases at the cellular level are thus likely to involve multiple cellular constituents and pathways. As some drugs, or drug combinations, may act synergistically on these multiple pathways, they might be more effective than the respective single target agents. Optimizing a drug mixture for a given disease in a particular patient is particularly challenging due to both the difficulty in the selection of the drug mixture components to start out with, and the all-important doses of these drugs to be applied. For n concentrations of m drugs, in principle, n(m) combinations will have to be tested. As this may lead to a costly and time-consuming investigation for each individual patient, we have developed a Feedback System Control (FSC) technique which can rapidly select the optimal drug-dose combination from the often millions of possible combinations. By testing this FSC technique in a number of experimental systems representing different disease states, we found that the response of cells to multiple drugs is well described by a low order, rather smooth, drug-mixture-input/drug-effect-output multidimensional surface. The main consequences of this are that optimal drug combinations can be found in a surprisingly small number of tests, and that translation from in vitro to in vivo is simplified. This points to the possibility of personalized optimal drug mixtures in the near future. This unexpectedly simple input-output relationship may also lead to a simple solution for handling the issue of human diversity in cancer therapeutics.

Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix®.

Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate thebladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degradesfluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studiedtheir fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C).A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320∕420 nm, FWHM =50∕100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455∕525 nm, FWHM = 80∕50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine's main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370­430 nm to 395­415 nm would reduce the BWF background by a factor 2.

In vivo measurement of tissue oxygenation by time-resolved luminescence spectroscopy: advantageous properties of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate.

Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO2) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO2 and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber­based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO2 dependence at a low drug dose (1 mg∕kg) and low fluence (120 mJ∕cm2 at 470 nm). The phototoxic threshold was found to be at 10 J∕cm2 with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO2 measurement. Finally, an illustrative application of this pO2 measurement approach in a hypoxic tumor environment is presented.

Low-Dose Vascular Photodynamic Therapy Decreases Tumor Interstitial Fluid Pressure, which Promotes Liposomal Doxorubicin Distribution in a Murine Sarcoma Metastasis Model.

INTRODUCTION: Solid tumors are known to have an abnormal vasculature that limits the distribution of chemotherapy. We have recently shown that tumor vessel modulation by low-dose photodynamic therapy (L-PDT) could improve the uptake of macromolecular chemotherapeutic agents such as liposomal doxorubicin (Liporubicin) administered subsequently. However, how this occurs is unknown. Convection, the main mechanism for drug transport between the intravascular and extravascular spaces, is mostly related to interstitial fluid pressure (IFP) and tumor blood flow (TBF). Here, we determined the changes of tumor and surrounding lung IFP and TBF before, during, and after vascular L-PDT. We also evaluated the effect of these changes on the distribution of Liporubicin administered intravenously (IV) in a lung sarcoma metastasis model. MATERIALS AND METHODS: A syngeneic methylcholanthrene-induced sarcoma cell line was implanted subpleurally in the lung of Fischer rats. Tumor/surrounding lung IFP and TBF changes induced by L-PDT were determined using the wick-in-needle technique and laser Doppler flowmetry, respectively. The spatial distribution of Liporubicin in tumor and lung tissues following IV drug administration was then assessed in L-PDT-pretreated animals and controls (no L-PDT) by epifluorescence microscopy. RESULTS: L-PDT significantly decreased tumor but not lung IFP compared to controls (no L-PDT) without affecting TBF. These conditions were associated with a significant improvement in Liporubicin distribution in tumor tissues compared to controls (P < .05). DISCUSSION: L-PDT specifically enhanced convection in blood vessels of tumor but not of normal lung tissue, which was associated with a significant improvement of Liporubicin distribution in tumors compared to controls.

Correlations between photoactivable porphyrins' fluorescence, erythema and the pain induced by PDT on normal skin using ALA-derivatives.

BACKGROUND: Photodynamic therapy (PDT) with precursors of photoactivable porphyrins is a well-established treatment modality for skin pathologies as well as hair removal. Pain is a major side effect thereof, and it affects the treatment compliance and acceptance. METHODS: Five male subjects underwent a PDT procedure on normal skin, either with a diode laser (635 nm) or a lamp (405 nm), 3 or 6h after application of various precursors of photoactivable porphyrins (ALA 1M; Metvix(®) 1M; ALA-DGME 1M; ALA-DGME 3.66 M). Light doses ranged from 30 to 150 J/cm(2) and irradiances were 100 or 180 mW/cm(2). Fluorescence measurements were performed just before PDT, pain was quantified during PDT, and erythema was determined 24h afterwards. RESULTS: Because precursor ALA-DGME was very selective for the pilosebaceous apparatus vs. the epidermis, we solely carried out the PDTs using this precursor. In the absence of light, no pain was reported. An increase in pain was observed when increasing the irradiance. A correlation was observed between the follicular fluorescence and the maximal pain score during PDT. A correlation was observed between follicular fluorescence and skin erythema, and between pain score and skin erythema. CONCLUSIONS: With our well-controlled PDT parameters and homogenous subjects' conditions, we showed that pain could be reduced by reducing irradiance during PDT procedures. With the various correlations observed, we conclude that both pain and PaP fluorescence are useful tools to predict the post-PDT tissue effects (side effects and outcome). We suggest that A∂ nerve fibres would be the best candidate as first generators of PDT-induced pain.

Near-infrared 808 nm light boosts complex IV-dependent respiration and rescues a Parkinson-related pink1 model.

Mitochondrial electron transport chain (ETC) defects are observed in Parkinson's disease (PD) patients and in PD fly- and mouse-models; however it remains to be tested if acute improvement of ETC function alleviates PD-relevant defects. We tested the hypothesis that 808 nm infrared light that effectively penetrates tissues rescues pink1 mutants. We show that irradiating isolated fly or mouse mitochondria with 808 nm light that is absorbed by ETC-Complex IV acutely improves Complex IV-dependent oxygen consumption and ATP production, a feature that is wavelength-specific. Irradiating Drosophila pink1 mutants using a single dose of 808 nm light results in a rescue of major systemic and mitochondrial defects. Time-course experiments indicate mitochondrial membrane potential defects are rescued prior to mitochondrial morphological defects, also in dopaminergic neurons, suggesting mitochondrial functional defects precede mitochondrial swelling. Thus, our data indicate that improvement of mitochondrial function using infrared light stimulation is a viable strategy to alleviate pink1-related defects.

Photodynamic therapy for polypoidal choroidal vasculopathy.

The first effective therapy for exudative macular degeneration (AMD) was Photodynamic Therapy (PDT). Diagnosis of the disease was to a large extent by fluorescein angiography (FA). Distinguishing between the leaky choroidal neovessels (CNV) associated with exudative AMD, and the polypoidal structures associated with Polypoidal Choroidal Vasculopathy (PCV) is not always easy using FA alone. The switch to Indocyanine Green angiography helped to pinpoint PCV, and thus to study the efficacy of photodynamic therapy of this particular form of retinal disease, which is more frequently encountered among pigmented individuals. The results appear to be quite promising, and in the year following treatment only a small fraction of the patients had to be retreated. Alternatively, treating PCV with repeated intravitreal VEGF blocking agents was not as successful as it was in the treatment of wet AMD. However, combining PDT-induced angio-occlusion of the polypoidal lesions with anti-vascular endothelial growth factor therapy was shown to be quite effective, and the combination of PDT with an anti-angiogenic agent as well as a steroid, in a triple therapy, was recently also shown to be a quite promising option. In the present article we review the data on PDT of PCV, including combination therapies and alternative treatments. We also report on similarities and differences between AMD and PCV.

Correlation between protoporphyrin IX fluorescence intensity, photobleaching, pain and clinical outcome of actinic keratosis treated by photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with Metvix® is a good therapeutic option to treat actinic keratosis, but it presents drawbacks (pain, lesion recurrences, heterogeneous outcome), emphasizing the possible need to individualize treatment. OBJECTIVE: We assessed whether PDT clinical outcome and pain during treatment were correlated with protoporphyrin IX fluorescence intensity and photobleaching. METHODS: 25 patients were treated by Metvix PDT. The outcome was evaluated after 1.3 (±0.4), 7.6 (±1.8), 13.2 (±1.2) and 33.6 (±3.0) months. After administration of Metvix, red light (632 ± 10 nm) was delivered with a light-emitting diode panel device. The outcome was assessed on a cosmetoclinical scale. RESULTS: All patients who showed a fluorescence level before PDT treatment above a certain threshold had a complete recovery at 33.6 (±3.0) months. CONCLUSION: Our approach could be used to individualize PDT treatment based on the pretreatment fluorescence level, and to predict its long-term outcome.

Synthesis and characterization of a new class of anti-angiogenic agents based on ruthenium clusters.

New triruthenium-carbonyl clusters derivatized with glucose-modified bicyclophosphite ligands have been synthesized. These compounds were found to have cytostatic and cytotoxic activity and depending on the number of bicyclophosphite ligands, and could be tuned for either anti-cancer or specific anti-angiogenic activity. While some compounds had a broad cellular toxicity profile in several cell types others showed endothelial cell specific dose-dependent anti-proliferative and anti-migratory efficacy. A profound inhibition of angiogenesis was also observed in the in vivo chicken chorioallantoic membrane (CAM) model, and consequently, these new compounds have considerable potential in drug design, e.g. for the treatment of cancer.

Selective photodetection and photodynamic therapy for prostate cancer through targeting of proteolytic activity.

Frequent side effects of radical treatment modalities and the availability of novel diagnostics have raised the interest in focal therapies for localized prostate cancer. To improve the selectivity and therapeutic efficacy of such therapies, we developed a minimally invasive procedure based on a novel polymeric photosensitizer prodrug sensitive to urokinase-type plasminogen activator (uPA). The compound is inactive in its prodrug form and accumulates passively at the tumor site by the enhanced permeability and retention effect. There, the prodrug is selectively converted to its photoactive form by uPA, which is overexpressed by prostate cancer cells. Irradiation of the activated photosensitizer exerts a tumor-selective phototoxic effect. The prodrug alone (8 µmol/L) showed no toxic effect on PC-3 cells, but upon irradiation the cell viability was reduced by 90%. In vivo, after systemic administration of the prodrug, PC-3 xenografts became selectively fluorescent. This is indicative of the prodrug accumulation in the tumor and selective local enzymatic activation. Qualitative analysis of the activated compound confirmed that the enzymatic cleavage occurred selectively in the tumor, with only trace amounts in the neighboring skin or muscle. Subsequent photodynamic therapy studies showed complete tumor eradication of animals treated with light (150 J/cm(2) at 665 nm) 16 hours after the injection of the prodrug (7.5 mg/kg). These promising results evidence the excellent selectivity of our prodrug with the potential to be used for both imaging and therapy for localized prostate cancer.

Photodynamic drug delivery enhancement in tumours does not depend on leukocyte-endothelial interaction in a human mesothelioma xenograft model.

OBJECTIVES: The pre-treatment of tumour neovessels by low-level photodynamic therapy (PDT) improves the distribution of concomitantly administered systemic chemotherapy. The mechanism by which PDT permeabilizes the tumour vessel wall is only partially known. We have recently shown that leukocyte-endothelial cell interaction is essential for photodynamic drug delivery to normal tissue. The present study investigates whether PDT enhances drug delivery in malignant mesothelioma and whether it involves comparable mechanisms of actions. METHODS: Human mesothelioma xenografts (H-meso-1) were grown in the dorsal skinfold chambers of 28 nude mice. By intravital microscopy, the rolling and recruitment of leukocytes were assessed in tumour vessels following PDT (Visudyne(®) 400 µg/kg, fluence rate 200 mW/cm(2) and fluence 60 J/cm(2)) using intravital microscopy. Likewise, the distribution of fluorescently labelled macromolecular dextran (FITC-dextran, MW 2000 kDa) was determined after PDT. Study groups included no PDT, PDT, PDT plus a functionally blocking anti-pan-selectin antibody cocktail and PDT plus isotype control antibody. RESULTS: PDT significantly enhanced the extravascular accumulation of FITC-dextran in mesothelioma xenografts, but not in normal tissue. PDT significantly increased leukocyte-endothelial cell interaction in tumour. While PDT-induced leukocyte recruitment was significantly blunted by the anti-pan-selectin antibodies in the tumour xenograft, this manipulation did not affect the PDT-induced extravasation of FITC-dextran. CONCLUSIONS: Low-level PDT pre-treatment selectively enhances the uptake of systemically circulating macromolecular drugs in malignant mesothelioma, but not in normal tissue. Leukocyte-endothelial cell interaction is not required for PDT-induced drug delivery to malignant mesothelioma.

Photodynamic induced uptake of liposomal doxorubicin to rat lung tumors parallels tumor vascular density.

BACKGROUND: Visudyne®-mediated photodynamic therapy (PDT) at low drug/light conditions has shown to selectively enhance the uptake of liposomal doxorubicin in subpleural localized sarcoma tumors grown on rodent lungs without causing morphological alterations of the lung. The present experiments explore the impact of low-dose PDT on liposomal doxorubicin (Liporubicin™) uptake to different tumor types grown on rodent lungs. MATERIAL AND METHODS: Three groups of Fischer rats underwent subpleural generation of sarcoma, mesothelioma, or adenocarcinoma tumors on the left lung. At least five animals of each group (sarcoma, n = 5; mesothelioma, n = 7; adenocarcinoma, n = 5) underwent intraoperative low-dose (10 J/cm(2) at 35 mW/cm(2) ) PDT with 0.0625 mg/kg Visudyne® of the tumor and the lower lobe. This was followed by intravenous (IV) administration of 400 µg Liporubicin™. After a circulation time of 60 min, the tumor-bearing lung was processed for HPLC analyses. At least five animals per group underwent the same procedure but without PDT (sarcoma, n = 5; mesothelioma, n = 5; adenocarcinoma, n = 6). Five untreated animals per group underwent CD31 immunostaining of their tumors with histomorphometrical assessment of the tumor vascularization. RESULTS: Low-dose PDT significantly enhanced Liporubicin™ uptake to all tumor types (sarcoma, P = 0.0007; mesothelioma, P = 0.001; adenocarcinoma, P = 0.02) but not to normal lung tissue compared to IV drug administration alone. PDT led to a significantly increased ratio of tumor to lung tissue drug uptake for all three tumor types (P < 0.05). However, the tumor drug uptake varied between tumor types and paralleled tumor vascular density. The vascular density was significantly higher in sarcoma than in adenocarcinoma (P < 0.001) and mesothelioma (P < 0.001), whereas there was no significant difference between adenocarcinoma and mesothelioma. CONCLUSION: Low-dose Visudyne®-mediated PDT selectively enhances the uptake of systemically administered liposomal doxorubicin in tumors without affecting the drug uptake to normal lung. However, drug uptake varied significantly between tumor types and paralleled tumor vascular density.