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Photodynamic therapy is a therapy that utilizes a specialized drug, a photosensitizer, which is not toxic by itself, but upon activation with light, is activated and leads to the production of singlet oxygen species. This production of singlet oxygen species starts a cascade that leads to cell death. Photodynamic therapy is a potential treatment for cancer patients, and it might be helpful to promote human health by providing alternative treatment to cancer. In-vitro study with Visudyne (VD) in KB carcinoma cells unveiled the mechanism of cell injury to cell death upon photodynamic therapy (PDT). In this study, we investigated real time monitoring of the production of singlet oxygen (SO), reactive oxygen species (ROS), mitochondria integrity and chromatin integrity in VD treated live cells by irradiating with a mercury lamp light source and 665–675 nm and 480–560 nm emission filter. Upon PDT we also observed cytoskeleton remodeling and cytochrome –C release using immunofluorescence techniques. We investigate the cytotoxicity of PDT and performed quantitative analysis for SO, ROS and mitochondrial potential change using colocalization analysis between VD and mitochondria.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumour in adults. Since glioma stem cells (GSCs) are associated with therapeutic resistance as well as the initiation and recurrence in GBM, therapies targeting GSCs are considered to be effective for long‐term survival in GBM. Several reports suggested that oxidative phosphorylation (OXPHOS) of cancer stem cells is important for their survival; however, the requirement of OXPHOS in GSCs remains unclear. Few effective and safe agents that target GSC mitochondria are available in clinical settings. In this study, we demonstrated that GSCs had high OXPHOS activity compared with isogenic differentiated GSCs and that GSC survival depended on their OXPHOS activity. Remarkably, we showed that complexes III and IV had broad therapeutic windows and that the expression levels of mitochondrial DNA‐coded components of complexes III and IV were elevated in GSCs compared with differentiated GSCs. Moreover, our search of the Food and Drug Administration‐approved drugs for those targeting GSC mitochondria revealed that verteporfin (Visudyne®), a drug approved for macular degeneration, was a novel GSC‐specific cytotoxic compound that reduced OXPHOS activity. Importantly, the cytotoxic effect of verteporfin was specific to GSCs without any toxicity to normal cells, and the IC50 of approximately 200 nm was ten times less than its maximum blood concentration in humans. Overall, these findings indicated that high mitochondrial OXPHOS of GSCs is a potential GSC‐specific vulnerability and that clinically available drugs, such as verteporfin, might become novel GSC‐specific cytotoxic agents. [ABSTRACT FROM AUTHOR]

Background and objectivePulsed dye laser (PDL) treatment of cutaneous vascular lesions is associated with variable and unpredictable efficacy. Thus, alternative treatment modalities are needed. Previous in vitro and in vivo studies have demonstrated enhanced selective vascular destruction with benzoporphyrin derivative (BPD) monoacid ring A photodynamic therapy (PDT) followed immediately by PDL irradiation (PDT+PDL). Here, we evaluate PDT alone, PDL alone, and PDT+PDL protocols using an optimized in vivo rodent dorsal window chamber model.Study design/materials and methodsA dorsal window chamber was surgically installed on male Golden Syrian hamsters. BPD solution was administered intravenously via a jugular venous catheter. Evaluated interventions included: (1) Control (no BPD, no light); (2) Control (BPD, no light); (3) PDT alone (lambda = 576 nm; 25, 50, 75, or 96 J/cm2 radiant exposure; 15 minutes post-BPD injection); (4) PDL alone at 7 J/cm2 (585 nm, 1.5 milliseconds pulse duration, 7 mm spot); and (5) PDT (25 or 75 J/cm2)+PDL (7 J/cm2). Laser speckle imaging was used to monitor blood flow dynamics before, immediately after, and 1, 3, and 5 days post-intervention.ResultsPerfusion reduction on day 1 post-intervention was achieved with PDT>50 J/cm2, PDL alone, and PDT+PDL. However, by day 5 post-intervention, recovery of flow was observed with PDT alone at 50 J/cm2 (-15.1%) and PDL alone (+215%). PDT (75 J/cm2)+PDL resulted in the greatest prolonged reduction in vascular perfusion (-99.8%).ConclusionsOur in vivo data suggest that the PDT+PDL therapeutic protocol can result in enhanced and persistent vascular shutdown compared to PDT or PDL alone. The PDT+PDL approach has potential for considerable superficial vascular destruction and should be considered as a treatment modality for cutaneous vascular lesions. Monitoring of blood flow changes for as long as possible is crucial for accurate assessment of light-based vascular interventions.

Photodynamic therapy is a therapy that utilizes a specialized drug, a photosensitizer, which is not toxic by itself, but upon activation with light, is activated and leads to the production of singlet oxygen species. This production of singlet oxygen species starts a cascade that leads to cell death. Photodynamic therapy is a potential treatment for cancer patients, and it might be helpful to promote human health by providing alternative treatment to cancer. In-vitro study with Visudyne (VD) in KB carcinoma cells unveiled the mechanism of cell injury to cell death upon photodynamic therapy (PDT). In this study, we investigated real time monitoring of the production of singlet oxygen (SO), reactive oxygen species (ROS), mitochondria integrity and chromatin integrity in VD treated live cells by irradiating with a mercury lamp light source and 665–675 nm and 480–560 nm emission filter. Upon PDT we also observed cytoskeleton remodeling and cytochrome –C release using immunofluorescence techniques. We investigate the cytotoxicity of PDT and performed quantitative analysis for SO, ROS and mitochondrial potential change using colocalization analysis between VD and mitochondria. [ABSTRACT FROM AUTHOR]

• Photodynamic therapy with verteporfin causes cell death in glioma cells. • Verteporfin is a potential drug for targeting glioblastoma pharmacologically. • Verteporfin can be used as an optical imaging dye in fluorescence guided surgery. • Verteporfin can be used to make glioblastoma susceptible for photodynamic therapy. Stummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm. To elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP. Human glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer. Significantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1). This study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT. [ABSTRACT FROM AUTHOR]

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) induces damage to the endothelium, which can lead to increased vascular permeability and, under intensive PDT conditions, even to platelet aggregation, vasoconstriction, and blood flow stasis. Eventually, ischemia, hypoxia, and inflammation can occur, resulting in angiogenesis. We studied the sequence of the vascular events after Visudyne-PDT in the chicken chorioallantoic membrane (CAM) at day 11 of development. Using epi-fluorescence microscopy, we monitored the regrowth of capillaries in the PDT treated area. Immediately after irradiation, the treatment resulted in blood flow arrest. And 24 h post PDT, sprouting of new blood vessels was observed at the edge of the PDT zone. Neovessels looping out from the edge of the PDT zone gave rise to specialized endothelial tip structures guiding the vessels towards the center of the treated area. At 48 h almost all of the treated area was repopulated with functional but morphologically altered vasculature. These observations also showed reperfusion of some of the vessels that had been closed by the PDT treatment. CAM samples were immunohistochemically stained for Ki-67 showing proliferation of endothelial cells in the PDT area. Also, several markers of immature and angiogenic blood vessels, such as αVβ3-integrin, vimentin and galectin-1, were found to be enhanced in the PDT area, while the endothelial maturation marker intercellular adhesion molecule (ICAM)-1 was found to be suppressed. These results demonstrate that the new vascular bed is formed by both neo-angiogenesis and reperfusion of existing vessels. Both the quantitative real-time RT-PCR profile and the response to pharmacological treatment with Avastin, an inhibitor of angiogenesis, suggest that angiogenesis occurs after PDT. The observed molecular profiling results and the kinetics of gene regulation may enable optimizing combination therapies involving PDT for treatment of cancer and other diseases. [ABSTRACT FROM AUTHOR]

Verteporfin photodynamic therapy (PDT) is approved for the treatment of predominantly classic subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration (AMD), as well as for subfoveal CNV due to pathologic myopia and ocular histoplasmosis syndrome. Verteporfin PDT addresses the underlying pathology of ocular vascular disorders through its angio-occlusive mechanism of action, which reduces both visual acuity loss and the underlying leakage associated with lesions. Verteporfin PDT has also been associated with encouraging treatment outcomes in case studies involving patients with choroidal vascular disorders such as polypoidal choroidal vasculopathy, central serous chorioretinopathy, choroidal haemangioma, angioid streaks, and inflammatory CNV, i.e. conditions currently considered as non-standard indications of verteporfin PDT. In many studies, outcomes were better than expected based on the natural courses of each of these conditions. Although the anti-vascular endothelial growth factor (VEGF) therapies, ranibizumab and pegaptanib, have been approved for CNV due to AMD, their role in these other choroidal vascular disorders remains to be established. We summarize current literature that has documented the use of verteporfin PDT in these conditions. The complex pathogenesis of CNV provides a rationale for investigating combination approaches comprising verteporfin PDT and anti-VEGF therapies. Randomized controlled studies are warranted to confirm the preliminary results of verteporfin PDT as a monotherapy or in combination with anti-VEGF therapies in the treatment of a variety of choroidal vascular conditions. [ABSTRACT FROM AUTHOR]

objectives In Ontario, Canada, in a cohort of all people initially aged 50-54 years, modeling whether the Age-Related Eye Disease Study (AREDS) antioxidant supplementation for stage 3 and 4 AMD would decrease the costs of photodynamic treatment with Visudyne. perspective Third party payer, the Ontario Health Insurance Plan. methods Using reported risk reductions, prevalence data by age and sex from the Beaver Dam studies, and yearly costs: AREDS C$182.50, potential savings were calculated as the difference or incremental cost between the estimated medical costs for the untreated cohort of $17,000 for Visudyne treatment of individuals with neovascularization and the same cohort if stage 3 and 4 AMD patients were treated with antioxidants, decreasing progression to neovascularization. Different scenarios were explored for sensitivity analysis of direct cost savings. results For the Ontario cohort of approximately 788,000 aged 51-55 years in 2001, for photodynamic therapy of the untreated cohort, modeled costs were $1.7 billion. AREDS treatment costs would be $513 million. AREDS would reduce photodynamic therapy costs, a net saving of $431 million, a saving of $547 per person in the total cohort, or $6,753 per stage 3 and 4 patient treated. To explore the sensitivity of this model to AMD incidence rather than prevalence data, Framingham incidence data were incorporated in the model: net savings of $70.3 million were modeled using Framingham incidence data. conclusion Under reasonable assumptions, if the case progresses to wet AMD (1) AREDS with Visudyne is less expensive than Visudyne alone in every five-year time period for the cohort that is age 50-54 right now until they become 75-79; thus, the lifetime cost is lower; (2) AREDS with Visudyne yields more QALYs than Visudyne alone in every five-year interval; (3) under all but the most extreme assumptions, the conclusions reached are robust. Even when AREDS costs a little more, it yields more QALYs at a reasonable cost per QALY. Thus, AREDS antioxidant supplementation appears to be a dominant strategy for macular degeneration. Applied to the whole Canadian population, the potential medical cost savings for Visudyne treatment of neovascular AMD are $5.6 billion dollars in direct costs. These values would be tenfold higher for the USA, because of the currency and population size differences. [ABSTRACT FROM AUTHOR]

We previously observed that antiangiogenic photodynamic therapy (PDT), namely, laser irradiation at 15 min after administration of photosensitizer, by using stable liposomal benzoporphyrin derivative monoacid ring A (BPD-MA), in which the liposomes were composed of dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, cholesterol, and dipalmitoylphosphatidylglycerol (10:10:10:2.5 as a molar ratio), was quite effective for cancer treatment. On the other hand, Visudyne, a commercialized liposomal formulation of BPD-MA, is based on more fluid lipids, namely, dimyristoylphosphatidylcholine and egg yolk phosphatidylglycerol, and is thought to be less stable in the presence of serum. The data of spin column chromatography indicated a little faster transfer of BPD-MA from Visudyne to lipoprotein fraction when Visudyne was incubated with serum than when the stable liposomal BPD-MA was used. The phototoxicity of Visudyne against a human endothelial cell line, ECV304, was almost the same as that of stable liposomal BPD-MA after PDT treatment. Therefore, we examined the antiangiogenic scheduling of PDT with Visudyne. Tumor growth of Meth-A sarcoma-bearing mice was strongly suppressed when the antiangiogenic scheduling was performed with Visudyne, namely, irradiation at 15 min after injection of the drug, in comparison with the conventional scheduling in which laser irradiation is done at 3 h post-injection. This greater effectiveness of PDT at 15 min was suggested to be caused by hemostasis, based on observations made in a dorsal air sac angiogenesis model. Visudyne-mediated antiangiogenic PDT cured 40 or 60% of Meth-A-bearing mice completely when 0.25 or 0.5 mg/kg BPD-MA, respectively, was used. These data suggest that the antiangiogenic scheduling is effective in Visudyne-mediated cancer PDT despite the transferring of BPD-MA from the liposomal fraction to lipoproteins in the bloodstream. [Copyright &y& Elsevier]

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contribute to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer-associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment has a significant impact on the prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient-derived PCAFs that simulate heterogeneous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab (a clinically approved anti-EGFR antibody) photoimmunoconjugates (PICs) of a benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne), which is currently in clinical trials for PDT of PDAC. We demonstrate that both Visudyne-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-, 19-, and 14-fold more phototoxic in spheroids with 50, 75, and 90% PCAFs, respectively, as compared to Visudyne-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute homogeneously within spheroids with a higher stromal content and the mechanistically different modes of action of the two formulations. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.

Lipid composition and lipid degradation are critical to the stability of liposomal formulations which can impact the safety and efficacy of the drug. Herein we developed and validated an ultrahigh performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) method for determining phospholipid composition and phospholipid degradation products in a verteporfin liposomal formulation (Visudyne). The high mass accuracy (<5 ppm) of the QTOF method coupled with database searching (SimLipid) and comparison with known standards accurately identified and quantified the phospholipid compositions and lipid degradation products. The analysis of Visudyne indicated that more than 50% (w/w) of the total phospholipids are composed of phosphatidylcholine (PC) 14:0–14:0 and major phosphatidylglycerol (PG) species found are PG 16:0–18:2, PG 16:0–18:1, PG 18:0–18:2, and PG 18:0–18:1. The LC-MS method developed is capable of separating structural isomers such as PG 18:1–18:1 versus PG 18:0–18:2 and the separation of PG stereoisomers, such as PG 18:1–18:1 cis and PG 18:1–18:1 trans. The major lipid degradation products in Visudyne includes lysophosphatidylcholine and a few saturated and unsaturated lysophosphatidylglycerols, and free fatty acids (FFA). Each degradation product is less than 1% of the total phospholipids (w/w). In addition, the lipid profiles of naturally sourced egg PG from six different vendors were compared with the PG composition in Visudyne. Differences in lipid composition in egg PGs from different vendors were observed and the PG composition in Visudyne is matched with the lipid profile of the some of the egg PGs from different vendors. Drug developers can utilize this method to assess raw materials and lipid-based drug product quality and regulatory scientists can monitor the quality of the drug available in the market using this validated method. [Display omitted] • UPLC-QTOF-MS method was developed to determine the lipid composition and lipid degradation products in Visudyne. • More than 50% (w/w) of the total phospholipids are composed of PC 14:0–14:0 and major PG species. • The major lipid degradation products in Visudyne are LPC, LPG and FFA. • Each degradation product is less than 1% of the total phospholipids (w/w). [ABSTRACT FROM AUTHOR]

The L-type calcium channel blocker fendiline has been shown to interfere with Ras-dependent signaling in K-Ras mutant cancer cells. Earlier studies from our lab had shown that treatment of pancreatic cancer cells with fendiline causes significant cytotoxicity and interferes with proliferation, survival, migration, invasion and anchorage independent growth. Currently there are no effective therapies to manage PDACs. As fendiline has been approved for treatment of patients with angina, we hypothesized that, if proven effective, combinatorial therapies using this agent would be easily translatable to clinic for testing in PDAC patients. Here we tested combinations of fendiline with gemcitabine, visudyne (a YAP1 inhibitor) or tivantinib (ARQ197, a c-Met inhibitor) for their effectiveness in overcoming growth and oncogenic characteristics of PDAC cells. The Hippo pathway component YAP1 has been shown to bypass K-Ras addiction, and allow tumor growth, in a Ras-null mouse model. Similarly, c-Met expression has been associated with poor prognosis and metastasis in PDAC patients. Our results presented here show that combinations of fendiline with these inhibitors show enhanced anti-tumor activity in Panc1, MiaPaCa2 and CD18/HPAF PDAC cells, as evident from the reduced viability, migration, anchorage-independent growth and self-renewal. Biochemical analysis shows that these agents interfere with various signaling cascades such as the activation of Akt and ERK, as well as the expression of c-Myc and CD44 that are altered in PDACs. These results imply that inclusion of fendiline may improve the efficacy of various chemotherapeutic agents that could potentially benefit PDAC patients. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT), is a clinically-approved light-based anti-cancer treatment modality in which a photoactivatable photosensitizer is irradiated with an appropriate wavelength of light to generate cytotoxic molecules to kill cancer cells. In this article, we describe an in vitro PDT protocol using a 3-dimensional (3D) model of ovarian cancer that was established on beds of Matrigel. PDT was performed using a liposomal formulation of verteporfin photosensitizer (Visudyne ® ). The cancer cells were genetically-labeled with the fluorescent protein mCherry to facilitate the evaluation of the treatment response. This protocol is advantageous because the mCherry fluorescence is an indicator of cell viability, eliminating the need for external dyes, which often exhibit limited penetration and diffusion into 3D organoids. Additionally, Visudyne PDT achieves significant tumor-killing efficacy in a 3D model for ovarian cancer., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

Background: Low-dose, Visudyne®-mediated photodynamic therapy (photo-induction) was shown to selectively enhance tumor vessel transport causing increased uptake of systemically administered chemotherapy in various tumor types grown on rodent lungs. The present experiments explore the efficacy of photo-induced vessel modulation combined to intravenous (IV) liposomal cisplatin (Lipoplatin®) on rodent lung tumors and the feasibility/toxicity of this approach in porcine chest cavities., Material and Methods: Three groups of Fischer rats underwent orthotopic sarcoma (n = 14), mesothelioma (n = 14), or adenocarcinoma (n = 12) implantation on the left lung. Half of the animals of each group had photo-induction (0.0625 mg/kg Visudyne®, 10 J/cm(2) ) followed by IV administration of Lipoplatin® (5 mg/kg) and the other half received Lipoplatin® without photo-induction. Then, two groups of minipigs underwent intrapleural thoracoscopic (VATS) photo-induction (0.0625 mg/kg Visudyne®; 30 J/cm(2) hilum; 10 J/cm(2) apex/diaphragm) with in situ light dosimetry in combination with IV Lipoplatin® administration (5 mg/kg). Protocol I (n = 6) received Lipoplatin® immediately after light delivery and Protocol II (n = 9) 90 minutes before light delivery. Three additional animals received Lipoplatin® and VATS pleural biopsies but no photo-induction (controls). Lipoplatin® concentrations were analyzed in blood and tissues before and at regular intervals after photo-induction using inductively coupled plasma mass spectrometry., Results: Photo-induction selectively increased Lipoplatin® uptake in all orthotopic tumors. It significantly increased the ratio of tumor to lung Lipoplatin® concentration in sarcoma (P = 0.0008) and adenocarcinoma (P = 0.01) but not in mesothelioma, compared to IV drug application alone. In minipigs, intrapleural photo-induction combined to systemic Lipoplatin® was well tolerated with no toxicity at 7 days for both treatment protocols. The pleural Lipoplatin® concentrations were not significantly different at 10 and 30 J/cm(2) locations but they were significantly higher in protocol I compared to II (2.37 ± 0.7 vs. 1.37 ± 0.7 ng/mg, P  < 0.001)., Conclusion: Visudyne®-mediated photo-induction selectively enhances the uptake of IV administered Lipoplatin® in rodent lung tumors. Intrapleural VATS photo-induction with identical treatment conditions combined to IV Lipoplatin chemotherapy is feasible and well tolerated in a porcine model. Lasers Surg. Med. 47:807-816, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)