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528 Background: Novel therapies are required for BCG-unresponsive, high risk non-muscle invasive bladder cancer. We report the interim results of a Phase II Clinical Study of Intravesical Photo Dynamic Therapy (PDT) in patients with BCG-Unresponsive Carcinoma In-Situ (CIS) with or without papillary disease. Methods: Out of a planned 125 patients, 42 patients have been enrolled and treated with two Study Treatments (Day 0 and Day 180) consisting of an intravesical instillation of the photosensitizer TLD-1433 (0.70 mg/cm2) followed by activation with a 520 nm intravesical laser under general anesthesia (Study Device TLC-3200) to a total of 90 J/cm2 of laser light. The primary outcome assessed was efficacy, evaluated by Complete Response (CR), at any point in time. The secondary outcome was duration of CR at 12 months, post initial CR. A tertiary objective is safety, evaluated by the incidence and severity of Adverse Events, Grade 4 or higher that do not resolve within 450 days post treatment. Patients with a negative cystoscopy and positive cytology have been defined as indeterminate response (IR), as these patients remain under investigation for lower and upper tract urothelial carcinoma. Results: Interim analyses included the first 42 patients, along with 3 patients treated in a preceding Phase Ib NMIBC clinical study assessing the safety of TLD-1433 PDT who weretreated at the same parameters, for a total of 45 patients. Data for the primary and secondary outcomes are listed in the table.The interim clinical data demonstrates a 90 day CR of 50% and a duration of response at 360 and 450 days of 35% and 21%, respectively.There have been eight Serious Adverse Events (SAE) identified (2 Grade II (tachycardia, hematuria), 3 Grade III (acute kidney injury, cellulitis), 2 Grade IV (urosepsis, depression/anxiety) and 1 Grade V). None of the SAEs were deemed to be directly related to the PDT. Conclusions: The interim data support that treatment with Photo Dynamic Therapy provides a viable treatment option for patients with BCG unresponsive CIS (+/- papillary disease) with an acceptable ongoing safety profile. Clinical trial information: NCT03945162 . [Table: see text]

International audience; Photodynamic therapy (PDT) is an approved medical technique to treat certain forms of cancer. It has been used to complement traditional anticancer modalities such as surgery, chemotherapy or radiotherapy, and in certain cases, to replace these treatments. One critical parameter of PDT is the photosensitizer (PS); historically, a purely organic macrocyclic tetrapyrrole-based structure. This short review surveys two recent clinical examples of metal complexes, namely TOOKAD®-Soluble and TLD-1433, which have ideal photophysical properties to act as PDT PSs. We highlight the important role played by the metal ions in the PS for PDT activity.

Significance: Photodynamic therapy (PDT) could become a treatment option for nonmuscle invasive bladder cancer when the current high morbidity rate associated with red light PDT and variable PDT dose can be overcome through a combination of intravesical instillation of the photosensitizer and the use of green light creating a steep PDT dose gradient. Aim: To determine how a high PDT selectivity can be maintained throughout the bladder wall considering other efficacy determining parameters, in particular, the average optical properties of the mucosal layer governing the fluence rate multiplication factor, as well as the bladder shape and the position of the emitter in relationship to the bladder wall. Approach: We present three irradiance monitoring systems and evaluate their ability to enable selective bladder PDT considering previously determined photodynamic threshold values for the bladder cancer, mucosa and urothelium in a preclinical model, and the photosensitizer’s specific uptake ratio. Monte Carlo-based light propagation simulations performed for six human bladders at the time of therapy for a range of tissue optical properties. The performance of one irradiance sensing device in a clinical phase 1B trial is presented to underline the impact of irradiance monitoring, and it is compared to the Monte Carlo-derived dose surface histogram. Results: Monte Carlo simulations showed that irradiance monitoring systems need to comprise at least three sensors. Light scattering inside the bladder void needs to be minimized to prevent increased heterogeneity of the irradiance. The dose surface histograms vary significantly depending on the bladder shape and bladder volume but are less dependent on tissue optical properties. Conclusions: We demonstrate the need for adequate irradiance monitoring independent of a photosensitizer’s specific uptake ratio.

Even though a patient has a good immune system, tumors are shielded from it, because tumors grow by suppressing the host’s immune-response by various mechanisms. They are keeping their local microenvironment immune suppressed by producing immune suppressive cytokines like IL-10 and TGF-b, express immune checkpoint ligands like Programmed Death Ligand 1 (PDL1), and harbor immune suppressive cells like Tregs and MDSCs. To overcome these barriers a stronger anti-tumour immune-response is essential. We evaluated a whole cell vaccine with extracorporeal Rutherrin®-PDT treated cancer cells (RuVaCareTM) to break the suppressive barrier in the RG2-glioblastoma model. Rutherrin®-PDT induced strong immunogenic cell death (ICD) in glioblastoma cells in-vitro. RuVaCareTM supernatants showed significantly higher level of extracellular ATP, which is known to induce recruitment of antigen presenting cells (APCs) and their activation by eliciting an effective anti-tumour immune-response. Extracellular calreticulin (CRT) is one of the hallmarks of ICD; its expression went up in more than 85% cells undergoing Rutherrin®-PDT mediated cell death. There was a close to 10 times increase in expression of HSP 70 in RuVaCareTM. Immunostimulatory cytokines IFNa, IL-1b and GMCSF expression is high in the RuVaCareTM. In-vivo efficacy of the RuVaCare™ was evaluated in orthotopic RG2 rat glioblastoma model. There was a significant increase (~43% with 2-time vaccine and 87% in the 6-time vaccine) in survival in the RuVaCare™ vaccinated groups compared to unvaccinated controls. Increased intratumoral CD8+T-cell numbers are shown to be correlated with increased survival in glioblastoma rats, with RuVaCare™ there was a significant increase in the number of CD8+T-cells.

Glioblastoma is a highly aggressive and common brain cancer in adults with a grave prognosis, and aggressive radio and chemotherapy provide only a 15 months median survival. We evaluated the tolerability, and efficacy of the Ruthenium-based photosensitizer TLD-1433 in the formulation with apo-Transferrin (Rutheriin®) in the RG2, rat glioblastoma model. The specific tumour uptake ratio, PDT threshold, of the RG2 rat glioblastoma models and normal brain in vivo were determined as well as the survival post-PDT and the extent of CD8+T cell infiltration post-PDT. Results were compared with those obtained by 5-ALA-induced PpIX mediated PDT in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill is compared during in-vitro and in vivo studies. A significantly lower absorbed energy was enough to achieve LD50 with Rutherrin® versus -PpIX mediated PDT. Rutherrin® provides higher selective uptake ratio (SUR>20) in RG2 tumours compared to normal brain, whereas the SUR for ALA-induced PpIX was 10.6 in the same tumour model. To evaluate the short-term tissue response in vivo enhanced T2-weighted MR images provided the spatial extent of edema, which is twice post PpIX-PDT versus Rutherrin®-PDT suggesting reduced non-specific damage typically associated with a secondary wave of neuronal damage. A significant survival increase was observed in Rutherrin® treated rats bearing RG2 versus PpIX-PDT for the selected treatment conditions, associated with an increased CD8+T cell infiltration in the tumours. Rutherrin®-PDT was well tolerated providing safe and effective treatment of RG2 -induced glioblastoma.

For patients failing standard Bacillus Calmette-Guerin based immunotherapy for Non-Muscle Invasive Bladder Cancer (NMIBC), PDT may delay or prevent cystectomy. A Phase IB clinical trial evaluated the feasibility and safety of TLD1433, a novel Ruthenium coordination-complex as photosensitizer (PS) for PDT. The clinical trial combined PS instillation for one hour and the use of strongly attenuated green (525nm) light to reduce PDT caused damage to the bladder wall. The low and high PS doses were defined as 0.35mg and 0.7mg TLD1433 per cm^2 bladder surface (N=3 each) and 90+/-9J/cm^2 as target radiant exposure on the bladder wall. The PS concentration in the urine and blood at 24hrs post instillation was below 1ng/ml indicating rapid drug clearing. In all patients, the average target radiant exposure was attained as verified by irradiance sensors in the bladder. The average measured irradiance was ~ 15mW/cm^2, never exceeding 35mW/cm2 at the sensor positions. At 30 days post-treatment, all patients receiving the low PS dose tolerated the procedure well with no grade 3, 4 or 5 AEs. Three patients were then treated at the Therapeutic Dose, again with no grade 3, 4 or 5 AEs, and an identical pharmacokinetic profile to the half dose. At half dose, all patients had recurrent, but no progressive NMIBC noted at the 180-day cystoscopy. At therapeutic dose, 2 of 3 patients were tumour-free at the 180-day cystoscopy. Moderate bladder irritability was reported at full dose which primarily resolved within 90 days.

Recurrent Non-Muscle Invasive Bladder Cancer (NMIBC) is a diffuse disease, and patients have failed standard BCG therapy face prophylactic cystectomy. PDT fell out of favour due to its variable outcome, and high morbidity. To overcome PDT associate toxicity to the bladder’s muscle layer, the use of shorter wavelength and instillation of the photosensitizer were suggested. While either approach was shown to improve the outcome in animal models they have not previously combined in human studies. Additionally, the effects of highly variable tissue optical properties of the bladder and its shape have not been studied. Here, we present surface dose histograms derived from light propagation simulation in 6 human bladders using CT images for anatomical detail and the FullMonte software package. The ability of a single light sensor versus 3 or 12 light sensors to measure the average irradiance on the bladder surface was evaluated as a function of the bladder wall’s tissue optical properties. Results show that the irradiance in non-spherical bladders can vary over an order of magnitude, but the irradiance histograms are affected little by displacement of the emitter inside the bladder void. As the surface area monitored by a single sensor depends strongly on the bladder shape, the responsivity of a single sensor to the average bladder irradiance can vary equally. Twelve light sensors monitor the entire bladder surface almost complete and hence their average responsivity is constant to the average irradiance on the bladder largely independent of shape. The dependency of the sensor’s response on the tissue optical properties is also lower.

Background Glioblastoma is an aggressive brain cancer in adults with a grave prognosis, aggressive radio and chemotherapy provide only a 15 months median survival. Methods We evaluated the tolerability and efficacy of the Ruthenium-based photosensitizer TLD-1433 with apo-Transferrin (Rutherrin) in the rat glioma 2 (RG-2) model. The specific tumor uptake ratio and photodynamic therapy (PDT) threshold of the rat glioblastoma and normal brain were determined, survival and CD8+T-cell infiltration post-therapy were analyzed. Results were compared with those obtained for 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX)-mediated photodynamic therapy in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill was determined for in vitro and in vivo studies. Results A significantly lower absorbed energy was sufficient to achieve LD50 with Rutherrin versus PpIX-mediated PDT. Rutherrin provides a higher specific uptake ratio (SUR) >20 in tumors versus normal brain, whereas the SUR for ALA-induced PpIX was 10.6. To evaluate the short-term tissue response in vivo, enhanced T2-weighted magnetic resonance imaging (MRI) provided the spatial extent of edema, post PpIX-PDT at twice the cross-section versus Rutherrin-PDT suggesting reduced nonspecific damage, typically associated with a secondary wave of neuronal damage. Following a single therapy, a significant survival increase was observed in rats bearing glioma for PDT mediated by Rutherrin versus PpIX for the selected treatment conditions. Rutherrin-PDT also demonstrated an increased CD8+T-cell infiltration in the tumors. Conclusion Rutherrin-PDT was well tolerated providing a safe and effective treatment of RG-2 glioma.

Median survival for Glioblastoma patients remains at around 15 months after surgery, radiation, and chemotherapy but varies widely between surgeons and centres. Interstitial Photodynamic Therapy (iPDT) mediated by ALA induced PpIX or other photosensitizers is investigated at various clinics. To enable iPDT treatment planning, photosensitizer transport characteristics needs to be known in order to optimize photoactivation. A method using contrast enhanced, functional magnetic resonance imaging, compartment modelling, spatial frequency domain imaging (SFDI) and inductively coupled plasma mass spectrometry (ICP-MS) is proposed to predict and validate the localization characteristics of two photosensitizers in murine Glioblastoma models.

INTRODUCTION AND OBJECTIVES:To achieve a pre-defined photosensitizer dose and photon irradiance for PDT of NMIBC, independent of a patient’s bladder volume, shape and bladder wall optical propertie...

While Photofrin mediated PDT for bladder cancer was the first approved indication for this technique, it failed to attract the confidence of urologists as a treatment option, primarily due to the high incidence of incontinence linked to PDT damage to the bladder muscle. To mitigate this hazard a phase I clinical trial using instillation of the Ru(II) coordination complex TLD1433 and 530 nm activation light was initiated. To achieve the intended drug doses of 0.35 and 0.7 mg/cm2 and a radiant exposure of 90 J/cm2 the concentration of the instillation was adjusted to each patients' bladder volume and the irradiance was measured at up to 12 positions in the bladder. Irradiance monitoring proved helpful in adjusting the irradiation time to the bladder wall albedo and also for increased light scattering and absorption due to turbidity built up in the bladder void. The initial multiplication factors of the bladders (n=6) ranged from 1.1 to 2.8. Monte Carlo simulations based on CT-scans from all 6 participants approximate the range of irradiances observed during these studies. Nevertheless, a fraction of the surface can see a multiple of the average irradiance whereas other regions (typically less than 5% of the surface area) see significantly less than the average irradiance. These variations are due to the actual bladder shape and are somewhat independent of the position of the spherical emitter. Fitting of the measured surface irradiance to the simulated dose surface histograms enables extraction of the bladder wall and bladder void’s optical properties.

Disruption of cell respiration and metabolic changes accompanies many tissue disorders including neurodegenerative diseases and cancers. Fluorescence lifetime imaging microscopy (FLIM), a non-invasive and information-rich technique, can be invoked to reveal these changes. The method allows for monitoring of fluorescent intrinsic metabolic coenzymes, first of all, NADH (nicotinamide adenine dinucleotide), on the level of single cells and can be applied to living tissue. The ratio between protein-bound and free NADH gives information about the balance between oxidative phosphorylation and glycolysis in cells. There is a correlation between cellular metabolic activity, redox ratio and fluorescence lifetime of NADH. In combination with laser scanning microscopy, the time-correlated single photon counting (TCSPC) technology enables FLIM NADH mapping of the biomedical samples. On the other hand, TCSPC provides quantification of the phosphorescence lifetime of oxygen-sensing molecules. Accordingly, the oxygendependent quenching of phosphorescence of compounds such as transition metal complexes can be employed for evaluation of oxygen partial pressure (pO2) by PLIM (phosphorescence lifetime imaging microscopy). We demonstrate correlated FLIM/PLIM imaging, which provides simultaneous mapping of NADH and oxygen in living cells. Continuous FLIM/PLIM imaging enables to monitor changes in oxygen levels and cell metabolic status dynamically during PDT and provides new opportunities in theranostics.

Significance: Photodynamic therapy (PDT) could become a treatment option for nonmuscle invasive bladder cancer when the current high morbidity rate associated with red light PDT and variable PDT dose can be overcome through a combination of intravesical instillation of the photosensitizer and the use of green light creating a steep PDT dose gradient. Aim: To determine how a high PDT selectivity can be maintained throughout the bladder wall considering other efficacy determining parameters, in particular, the average optical properties of the mucosal layer governing the fluence rate multiplication factor, as well as the bladder shape and the position of the emitter in relationship to the bladder wall. Approach: We present three irradiance monitoring systems and evaluate their ability to enable selective bladder PDT considering previously determined photodynamic threshold values for the bladder cancer, mucosa and urothelium in a preclinical model, and the photosensitizer’s specific uptake ratio. Monte Carlo-based light propagation simulations performed for six human bladders at the time of therapy for a range of tissue optical properties. The performance of one irradiance sensing device in a clinical phase 1B trial is presented to underline the impact of irradiance monitoring, and it is compared to the Monte Carlo-derived dose surface histogram. Results: Monte Carlo simulations showed that irradiance monitoring systems need to comprise at least three sensors. Light scattering inside the bladder void needs to be minimized to prevent increased heterogeneity of the irradiance. The dose surface histograms vary significantly depending on the bladder shape and bladder volume but are less dependent on tissue optical properties. Conclusions: We demonstrate the need for adequate irradiance monitoring independent of a photosensitizer’s specific uptake ratio.

During photodynamic therapy (PDT), disruption of cell respiration and metabolic changes could be one of the first events. Photophysical characteristics of the photosensitizer (PS) and its specific redox potential define consumption of molecular oxygen followed by generation of reactive oxygen species. The potential PS TLD1433 is based on transition metal Ru(II) and possess an oxygen-dependent luminescence. This enables the study of oxygen consumption by PS-phosphorescence lifetime imaging (PLIM) and simultaneously changes the cellular metabolic state by nicotinamide adenine dinucleotide (NAD(P)H)-fluorescence lifetime imaging (FLIM). Within this study, localization and cellular function of TLD1433 is investigated in bladder carcinoma cells using time-resolved and confocal laser scanning microscopy. Simultaneous FLIM/PLIM of NAD(P)H and TLD1433 during PDT correlated oxygen consumption, redox state and cellular energy metabolism. Our investigations aimed to provide a personalized protocol in theranostic PDT procedures and demonstrate the potential use of TLD1433 PDT also under hypoxic conditions, which are otherwise difficult to treat.

Background: The majority of cancers upregulate their transferrin receptor (Tf-R) to satisfy their higher Fe3+ requirements for proliferation. TLD-1433 can bind to transferrin to form Rutherrin, which is a promising photosensitizer with stable chemical structure and higher tissue selectivity. Methods: To investigate the effect of Rutherrin®-mediated photodynamic treatment (PDT), we used non-small lung cancer cell lines H2170, A549, and H460. Subcutaneous tumors were treated with Rutherrin-mediated PDT, 4hrs post intravenous administration. The treatment parameters10 mg/kg Rutherrin and 600 Jcm-2 808 nm radiation. In an orthotopic A549 tumor model, the presence of tumor after inoculation in lungs was confirmed by microCT. Tissue samples were collected for Inductively Coupled Mass Spectrometry to quantify the Rutherrin concentrations via a Ru isotope in tumor and normal lung tissue. Results: Evaluation of TfR expression by flow cytometric and western blotting showed that almost all cancer cells express TfR. In in-vitro cytotoxicity assay, all cancer cell lines showed high cell kill by PDT at 100nM Rutherrin concentrations. In the subcutaneous tumor model, PDT after Rutherrin injection significantly inhibited the tumor growth and histopathology showed extensive necrosis at 24 hrs, which was confirmed with lowered Ki67 staining. In an orthotopic model, the lung lobe with tumor retained more Rutherrin than the contralateral lung, showing specific tumor uptake. Conclusion: These results support the hypothesis that safe and efficient Rutherrin-mediated PDT is feasible due to improved photosensitizer localization to lung tumors tissue. Selective irradiation of the cancer lesions by strategic placement of the light source remains a requirement.

A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations. The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

The photo-physical and photo-biological properties of two small (2 kDa), novel Ru(ii) photosensitizers (PSs) referred to as TLD1411 and TLD1433 are presented. Both PSs are highly water-soluble, provide only very limited luminescence emission at 580-680 nm following excitation at 530 nm, and demonstrate high photostability with less than 50% photobleaching at radiant exposures H = 275 J cm(-2) (530 nm irradiation). It was previously shown that these two photosensitizers exhibit a large singlet oxygen ((1)O2) quantum yield (Φ (Δ) ∼0.99 in acetonitrile). Their photon-mediated efficacy to cause cell death (λ = 530 nm, H = 45 J cm(-2)) was tested in vitro in colon and glioma cancer cell lines (CT26.WT, CT26.CL25, F98, and U87) and demonstrated a strong photodynamic effect with complete cell death at concentrations as low as 4 and 1 μM for TLD1411 and TLD1433, respectively. Notably, dark toxicity was negligible at concentrations less than 25 and 10 μM for TLD1411 and TLD1433, respectively. The ability of the PSs to initiate Type I photoreactions was tested by exposing PS-treated U87 cells to light under hypoxic conditions (pO20.5%), which resulted in a complete loss of the PDT effect. In vivo, the maximum tolerated doses 50 (MTD50) were determined to be 36 mg kg(-1) (TLD1411) and 103 mg kg(-1) (TLD1433) using the BALB/c murine model. In vivo growth delay studies in the subcutaneous colon adenocarcinoma CT26.WT murine model were conducted at a photosensitizer dose equal to 0.5 and 0.2 MTD50 for TLD1411 and TLD1433, respectively. 4 hours post PS injection, tumours were irradiated with continuous wave or pulsed light sources (λ = 525-530 nm, H = 192 J cm(-2)). Overall, treatment with continuous wave light demonstrated a higher tumour destruction efficacy when compared to pulsed light. TLD1433 mediated PDT resulted in statistically significant longer animal survival compared to TLD1411. Two-thirds of TLD1433-treated mice survived more than 100 days (p0.01) whereas TLD1411-treated mice did not survive longer than 20 days. Here we present evidence that two novel PSs have very potent photo-biological properties and are able to cause PDT-mediated cell death in both in vitro cell culture models and in vivo tumour regression.

Ru(II) dyads derived from organic units that impart low-lying 3IL excited states combine the most attractive features of organic photosensitizers with those of coordination complexes. The result is a bichromophoric system with excited-state lifetimes that are significantly longer than those associated with traditional 3MLCT states. Incorporation of α-oligothiophenes as the organic chromophore leads to systems that act as dual Type I/II photosensitizers, opening up the possibility of treating hypoxic tumors with photodynamic therapy (PDT) and overcoming problems with in vivo dosimetry. These photosensitizers, particularly those that consist of three thiophene units and higher, are remarkable DNA binders and photocleavers when exposed to light, exhibiting no interference with DNA structural integrity in the absence of a light-trigger. Such light-responsive agents localize in the nuclei of cells without the need for a carrier and produce a potent PDT response with minimal dark toxicity. This phototherapeutic effect translates directly to animals and is superior to the clinical agent Photofrin® in this model. These Ru(II) dyads can be activated with light in the PDT window, despite very low molar extinction coefficients in this region, and this phenomenon can be attributed to the efficiency with which these agents operate. The ability to activate these prodrugs with ultraviolet to near-infrared light marks an unprecedented versatility that can be exploited to match treatment depth to tumor invasion depth without compromising potency, giving rise to photosensitizers for multiwavelength PDT.

Cancer remains a major global malaise requiring the advent of new, efficient and low-cost treatments. Photodynamic therapy, which combines a photosensitizer and photons to produce cytotoxic reactive oxygen species, has been established as an effective cancer treatment but has yet to become mainstream. One of the main limitations has been the paucity of photosensitizers that are effective over a wide range of wavelengths, can exert their cytotoxic effects in hypoxia, are easily synthesized and produce few if any side effects. To address these shortfalls, three new osmium-based photosensitizers (TLD1822, TLD1824 and TLD1829) were synthesized and their photophysical and photobiological attributes determined. These photosensitizers are panchromatic (i.e. black absorbers), activatable from 200 to 900 nm and have strong resistance to photobleaching. In vitro studies show photodynamic therapy efficacy with both red and near-infrared light in normoxic and hypoxic conditions, which translated to good in vivo efficacy of TLD1829 in a subcutaneous murine colon cancer model.

nude mice. Once tumors had developed (average volume 70 mm), they were treated with 8 intravesical injections of miR-145. Synthetic miR-145 (100 nM) complexed with 100 ml of LipofectamineTM RNAiMAX reagent was added to 50 ml of PBS and delivered 8 times intravesically every other day. Induction of sub-cellular pathways was assessed by Western blot analyses in in Vitro and in Vivo. RESULTS: The expression level of miR-145 in 253J B-V cells was markedly down-regulated compared with that in normal human urothelial cells. Transfection of human 253J B-V cells with miR-145 resulted in a pronounced growth suppression that occurred in a dosedependent fashion. Western blotting analysis showed that the expression levels of all FSCN1, E-cadherin, b-catenin, and catenin d-1 were significantly down-regulated in the miR-145-treated 253J B-V cells. Also, cleaved form of PARP and down-regulation of c-Myc and socs7 were observed. Hoechst33342 nuclear staining indicated a typical apoptotic appearance such as condensed chromatin and nuclear fragmentation in the miR-145-treated 253J B-V cells. The ectopic expression of miR-145 significantly decreased cell migration of 253J BV cells approximately 30%. preparing Kaplan-Maier plots, with the survival duration analyzed for significance by performing log-rank survival analysis. The intravesical treatment with miR-145 significantly prolonged animal survival in mice bearing orthotopic 253J B-V xenografts when compared with that for the controls (Log rank p 1⁄4 0.018). CONCLUSIONS: The present findings for the first time showed that miR-145 was an effective tumor suppressor miRNA against bladder cancer and that its intravesical delivery effectively inhibited the tumorigenic phenotypes without any adverse effects. Thus we propose that the use of miR-145 by the intravesical approach can become a successful therapeutic strategy for bladder cancer.

Metal-based photosensitizers are of interest as their absorption and chemical binding properties can be modified via the use of different ligands. Ru(2+) based photosensitizers are known to be effective photodynamic therapy (PDT) agents against bacteria, whereas use for oncological indications in vivo has not been demonstrated with the same level of evidence. We present data showing that premixing the Ru(2+)-complex TLD1433 with transferrin increases the molar extinction coefficient, including longer activation wavelengths, reduces photobleaching rates, and reduces the toxicity of the complex improving overall PDT efficacy. As the transferrin receptor is upregulated in most malignancies, premixing the Ru(2+) complex with transferrin converts the active pharmaceutical ingredient TLD1433 into a drug of potentially considerable clinical utility.

Background Vapor-based fumigant systems for disinfection of health care surfaces and spaces is an evolving technology. A new system (AsepticSure) uses an ozone-based process to create a highly reactive oxidative vapor with broad and high-level antimicrobial properties. Methods Ozone gas at 50-500 ppm was combined with 3% hydrogen peroxide vapor in a test chamber and upscaled in rooms measuring 82 m 3 and 90 m 3 in area. Test organisms included methicillin-resistant Staphylococcus aureus , vancomycin-resistant enterococcus, Escherichia coli , Pseudomonas aeruginosa , Clostridium difficile , and Bacillus subtilis spores dried onto steel discs or cotton gauze pads. Results The combination of 80-ppm ozone with 1% hydrogen peroxide vapor achieved a very high level of disinfection, with a ≥6 log 10 reduction in the bacteria and spores tested on steel discs and MRSA tested on cotton gauze during a 30- to 90-minute exposure. The entire system was scalable such that it achieved the same high level of disinfection in both the 81-m 3 and 90-m 3 rooms in 60-90 minutes. Conclusion The ozone hydrogen peroxide vapor system provides a very high level of disinfection of steel and gauze surfaces against health care-associated bacterial pathogens. The system is an advanced oxidative process providing a rapid and effective means of disinfecting health care surfaces and spaces.

Administration of VP025 (Vasogen Inc.), a novel drug formulation based on phospholipid nanoparticles incorporating phosphatidylglycerol, has previously been shown to have a neuroprotective effect in the brain. We examined the effect of VP025 in a rat model of Parkinson's disease, the 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle. VP025 or phosphate-buffered saline (PBS) was administered to rats 14 days, 13 days and 1 day before the unilateral 6-OHDA lesion. Functional integrity of nigrostriatal dopaminergic neurons was assessed 7 and 21 days later by amphetamine-induced rotational testing and we observed that rotational counts were significantly less in rats that were pretreated with VP025 compared with PBS-pretreated 6-OHDA-lesioned rats. Neurochemical analysis at 10 and 28 days after lesion revealed that VP025 protected against a 6-OHDA-induced decrease in concentrations of striatal dopamine and its metabolites. Immunocytochemical studies of the ipsilateral substantia nigra showed that VP025 significantly inhibited 6-OHDA-induced loss of dopaminergic neurons. We also observed that increases in immunostaining for activated microglia and for activated p38 in dopaminergic neurons of 6-OHDA-lesioned rats were prevented by VP025. This study shows that VP025 has significant protective effects on the 6-OHDA-lesioned nigrostriatal pathway and may therefore have potential for the treatment of Parkinson's disease.

Background: Contact hypersensitivity (CHS) is a Th1-mediated immune response that can be down-regulated by immunosuppressive agents such as cyclosporine and environmental stimuli such as ultraviolet light. Recently, an immunomodulation therapy, VAS972, has been developed which is believed to down-regulate the Th1 arm of the immune response. This VAS972 involves modifying autologous blood by controlled exposure to the oxidizing agent ozone and UVC light, at an elevated temperature ex vivo. The processed blood is then administered by intramuscular injection. Objective: To further evaluate the immune modulating effect of VAS972. Methods: We examined the effect of VAS972 treatment on CHS. Contact hypersensitivity was induced with dinitro-fluorobenzene (DNFB) in animals receiving VAS972-processed blood, control blood, or saline. A preliminary study was also conducted to evaluate the effect of plasma and cellular fractions of processed blood. Results: Mice injected with VAS972-processed blood demonstrated a significantly lower (46%) CHS response than controls. Histologic examination of challenged ear skin from control mice displayed edema with a significant lymphocytic infiltration, whereas animals administered processed blood demonstrated a reduction in lymphocytic infiltration. Mice injected with either plasma or the cellular fraction of the VAS972-treated blood also demonstrated a significant suppression (49% and 41%, respectively). Conclusion: The results of this study demonstrated that VAS972 suppresses CHS and cellular infiltration. Furthermore, the plasma and cellular components of the VAS972 treatment were also able to induce immunosuppression. This further supports the hypothesis that VAS972 down-regulates the Th1 arm of the immune response.

Summary Background The introduction of new disinfection and sterilization methods, such as antimicrobial photodynamic therapy, is urgently needed for the healthcare industry, in particular to address the pervasive problem of antibiotic resistance. This study evaluated the efficacy and the mechanisms of photodynamic antimicrobial chemotherapy (PACT), also known as photodynamic inactivation (PDI) of microorganisms, induced by novel Ru(II)-based photosensitizers against Staphylococcus aureus and methicillin-resistant S. aureus strains. Methods The photodynamic antibacterial effects of a new class of Ru(II)-based photosensitizers (TLD1411 and TLD1433) were evaluated against a strain of S. aureus (ATCC 25923) and a methicillin-resistant strain of S. aureus (MRSA, ATCC 33592). Bacterial samples were dosed with a range of photosensitizer concentrations (0.3–12 μM) and exposed to 530 nm light (90 J cm −2 ) in normoxic conditions (ambient atmosphere) and in hypoxic conditions (0.5% O 2 ). Results Both photosensitizers exerted photodynamic inactivation (PDI) of the microorganisms in normoxia, and this activity was observed in the nanomolar regime. TLD1411 and TLD1433 maintained this PDI potency under hypoxic conditions, with TLD1433 becoming even more active in the low-oxygen environment. Conclusion The observation of activity in hypoxia suggests that there exists an oxygen-independent, Type I photoprocess for this new class of compounds in addition to the typical Type II pathway mediated by singlet oxygen. The intrinsic positive charge of the Ru(II) metal combined with the oxygen independent activity demonstrated by this class of photosensitizers presents a new strategy for eradicating both gram-positive and gram-negative bacteria regardless of oxygenation level.

Up regulation of iNOS gene expression is playing a role in the initiation of the anti-inflammatory and tissue protective mechanisms related to nitric oxide (NO) for continuous wave red and infrared as well as 905nm superpulsed laser therapy (SPLT). The iNOS expression before and after laser therapy was evaluated in a zymosan-induced acute arthritis model, in knee joints of young ( 15 weeks and 35 weeks) FVB/N-Tg (iNOS-luc) mice by bioluminescence imaging.

Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

Objective: A randomized placebo-controlled clinical trial to evaluate an adjunctive treatment modality for pain associated with knee disorders was conducted utilizing a therapeutic laser system (low energy, non-surgical). Subjects and methods: The therapeutic laser system utilized a dual wavelength, multiple diode laser cluster probe with five super-pulsed 905 nm near-infrared (NIR) laser diodes, each emitting at 40 mW average power and four continuous wave 660 nm visible (VIS) red laser diodes, each emitting at 25 mW. It was used as an adjunctive modality providing 12 treatments, three times a week to a homogeneous patient population (n = 126), in combination with standardized chiropractic techniques, to evaluate effectiveness on subjects presenting with osteoarthritis and knee pain. The primary endpoint was measured by the visual analog scale (VAS) to assess pain levels on a scale of 0 – 10. The success criteria for an individual patient in this study were identified as an improvement of 30 % or more in the VAS from baseline to 12th treatment and/or an improvement of 20 % or more in the VAS from baseline to 30-day follow-up evaluation. Results: The data obtained in the study demonstrated that the present therapeutic laser system provided significant pain relief and osteoarthritic improvements in all primary evaluation criteria, with a statistical and clinical significance of p < 0.01 in VAS from baseline to the 30-day follow-up.

Background: A common change that occurs with age in the central nervous system is an increase in microglial-associated inflammation. This is usually coupled with an increase in the concentration of the inflammatory cytokine interleukin-1β (IL-1β) in the hippocampus and an inhibition in long-term potentiation. Objectives: To assess the effects of a novel preparation of phospholipid nanoparticles incorporating phosphatidylglycerol, VP025, on inflammatory changes in hippocampus of aged and lipopolysaccharide (LPS)-treated rats. Methods/Results: We report that a possible initial target cell of the putative anti-inflammatory actions of VP025 may be macrophages, as VP025 is engulfed by, and has the capacity to alter the activity of, these cells. VP025 reversed the increase in IFN-γ concentration in supernatant taken from peritoneal macrophages harvested from LPS-treated rats. In addition, markers of microglial activity, major histocompatibility complex class II (MHC II) mRNA expression, CD40 expression and IL-1β concentration were increased, and CD200 expression was reduced, in the hippocampus of these rats. VP025 reversed changes in CD40, IL-1β and CD200 in aged rats, and also restored long-term potentiation in aged and LPS-treated rats. Conclusions: We conclude that VP025 has the ability to modulate the activity of macrophage, microglia and neurons in response to stressors such as ageing and LPS treatment.

Failure of the ubiquitin-proteasome system to degrade abnormal proteins may underlie the accumulation of alpha-synuclein and dopaminergic neuronal degeneration that occurs in Parkinson's disease. Consequently, a reduction of functional proteasome activity has been implicated in Parkinson's disease. VP025 (Vasogen Inc.) is a preparation of phospholipid nanoparticles incorporating phosphatidylglycerol that has been shown to have neuroprotective effects. We show that VP025 prevents the deficits in motor coordination and dopamine observed in a proteasome inhibitor rat model of PD. Thus, VP025 may have a therapeutic effect on the impairment of dopaminergic-mediated motor activity induced by proteasome inhibition.