A three-compartment non-linear model of myocardial cell conduction block during photosensitization.

This study constructed a new non-linear model of myocardial electrical conduction block during photosensitization reaction to identify the vulnerable cell population and generate an index for recurrent risk following catheter ablation for tachyarrhythmia. A three-compartment model of conductive, vulnerable, and blocked cells was proposed. To determine the non-linearity of the rate parameter for the change from vulnerable cells to conductive cells, we compared a previously reported non-linear model and our newly proposed model with non-linear rate parameters in the modeling of myocardial cell electrical conduction block during photosensitization reaction. The rate parameters were optimized via a bi-nested structure using measured synchronicity data during the photosensitization reaction of myocardial cell wires. The newly proposed model had a better fit to the measured data than the conventional model. The sum of the error until the time where the measured value was higher than 0.6, was 0.22 in the conventional model and 0.07 in our new model. The non-linear rate parameter from the vulnerable cell to the conductive cell compartment may be the preferred structure of the electrical conduction block model induced by photosensitization reaction. This simulation model provides an index to evaluate recurrent risk after tachyarrhythmia catheter ablation by photosensitization reaction. A three-compartment non-linear model of myocardial cell conduction block during photosensitization.

Clinical application of the mirror irradiation technique in photodynamic therapy for malignant glioma.

BACKGROUND: Intraoperative photodynamic therapy (PDT) using talaporfin sodium for malignant glioma is effective both in the experimental and in the clinical setting. Because the irradiation unit is fixed to the objective lens of the operating microscope, blind spots for irradiation exist. To overcome this problem, we developed a mirror reflecting system using a modified dental mirror. METHODS: The developed mirror is made of stainless steel, has a mirror-polished surface, and is rhodium coated on 1 side, which is the reflecting surface. The reflection rate was measured using He-Ne laser irradiation. The reflection intensity was measured using a laser power meter when the incident angle to the mirror was changed to 60°, 45°, and 30°, and the reflectance was calculated by the direct received light intensity from the laser. After confirming the safety of the fundamental experiment, PDT was performed with this developed mirror on 9 patients with malignant glioma (4 with recurrence and 5 newly diagnosed). RESULTS: The energy efficiency of the mirror was approximately 70 %, and apparent irregular reflection was not observed. Even during clinical use, apparent complications, such as irregular reflection, did not occur upon using the mirror in any of the patients. In all patients, recurrence did not occur in the site where mirror irradiation was performed, but in a deep site or a distant site to which sufficient laser irradiation did not reach. CONCLUSION: PDT using our newly developed mirror involves few instrumental changes compared with the conventional irradiation method, and is effective, safe, and inexpensive.

Respiration and Heat Shock Protein After Short-Term Heating/Stretch-Fixing on Smooth Muscle Cells.

PURPOSE: A treatment device without a stent is needed for peripheral stenotic artery treatment. We have proposed short-term heating balloon angioplasty, photo-thermo dynamic balloon angioplasty (PTDBA). Though smooth muscle cells (SMCs) after PTDBA are fixed in a stretched formation in a porcine model, influences of this stimulus on SMCs have not been investigated. SMC migration after vascular dilatation would be related to chronic restenosis. The aim of this study was to examine respiratory activity and recovery ability of SMCs after short-term heating/stretch-fixing in vitro for chronic phase treatment effect discussion. METHODS: SMCs on a stretch chamber were heated for 15 s with stretching and fixed in a stretched formation. SMC migration is correlated with the cell respiratory activity. The amount of ATP production was measured using a WST-8 assay for respiratory activity evaluation. The intracellular expression of heat shock protein 70 was measured by an ELISA for recovery ability evaluation. RESULTS: In the case of 60 °C heating, SMC respiratory activity after short-term heating/stretch-fixing decreased drastically in all stretching rates. In the case of 50 °C heating, SMC respiratory activity decreased and then increased. Alternatively, the recovery ability at 60 °C was greater than that at 50 °C. CONCLUSIONS: SMCs heated at 60 °C with stretching would have high recovery ability and low respiratory activity related to SMC migration. These results may be important evidence in determining the treatment condition in PTDBA.

Diffused light attenuation at 664 nm for PDT in salted cadaver brain.

BACKGROUND: We investigated light attenuation at 664 nm, which is the excitation wavelength of photodynamic therapy (PDT) using talaporfin sodium, in a salted cadaver brain. Estimation of therapeutic lesions is important to ensure the effectiveness and safety of brain tumor PDT. Previously reported optical properties of the human brain vary widely. In this study, we measured the light attenuation in brain tissue using a practical method. We employed a salted cadaver brain, in which the mechanical and optical properties can be maintained as close as possible to those under operative conditions. METHODS: A neuroendoscope was inserted into the brain until the cerebral ventricle was reached. A thin cylindrical diffuser probe was advanced 10 mm from the endoscope tip. By another path from the brain surface, an optical fiber for measurement was inserted into a puncture needle, and a pair of needles was used to puncture the tissue and reach the same cerebral ventricle in which the endoscope tip was positioned. The attenuation of light intensities in the frontal lobe and cerebellum was measured by varying the bundle tip position. The starting positions of the bundle were confirmed by the endoscopic view. The measured light intensity attenuations were fitted with an exponential curve. RESULTS: The following attenuation coefficients were obtained: 0.20 ± 0.05 mm-1 in the cerebrum and 0.27 ± 0.05 mm-1 in the cerebellum. CONCLUSION: As conventional spectroscopic measurement may overestimate attenuation in the whole tissue, in situ measurement using the withdrawal technique might be appropriate for measurement of inhomogeneous biological tissues.

Continuous Optical Monitoring of Red Blood Cells During a Photosensitization Reaction.

Background: An oxygen-enriched photosensitizer solution was created by the addition of red blood cells (RBCs) as an investigative tool for photosensitization reactions (PRs). Although the oxygen levels and reaction progress can be monitored using the optical characteristics of hemoglobin, previously this has only been done using intermittent measurements. An increase in methemoglobin concentration with irradiation time was reported. Objective: We constructed a continuous optical measurement system to study the dynamics of the PR in a photosensitizer solution containing RBCs. We also measured the relationship between hemolysis and methemoglobin production in the solution. Materials and methods: A 664 nm wavelength continuous laser beam at 60 mW/cm2 was used to drive the PR, and a broadband (475-650 nm) light beam was used to monitor the absorption spectra during the PR. The light sources were arranged perpendicularly to cross at a 1 × 10 mm cuvette. The sample in this cuvette was prepared from a low-hematocrit rabbit RBC suspension medium containing 30 µg/mL talaporfin sodium, a chlorine photosensitizer. The concentrations of oxygenated hemoglobin, deoxygenated hemoglobin, and methemoglobin were obtained using a multiple regression analysis of the measured spectra. Results: The oxygen saturation decreased continuously during the PR. The relationship between the degree of hemolysis and produced methemoglobin concentration was confirmed. Conclusions: We determined the dynamics of the oxidation and oxygen desorption of hemoglobin, as well as RBC hemolysis, during the PR. Our measurement system, which uses the properties of hemoglobin contained in RBCs, might be useful for continuous monitoring of PR dynamics.

Modified optical coefficient measurement system for bulk tissue using an optical fiber insertion with varying field of view and depth at the fiber tip.

To measure the few millimeter-scale macroscopic optical properties of biological tissue, including the scattering coefficient, while avoiding the instability that originates from sample slicing preparation processes, we performed propagated light intensity measurements through an optical fiber that punctures the bulk tissue while varying the fiber tip depth and the field of view (FOV) at the tip; the results were analyzed using the inverse Monte Carlo method. We realized FOV changes at the fiber tip in the bulk tissue using a variable aperture that was located outside the bulk tissue through a short high-numerical aperture (high-NA) multi-mode fiber with a quasi-straight shape. Using a homogeneous optical model solution, we verified the principle and operation of the constructed experimental system. A 200-µm-core-diameter silica fiber with NA of 0.5 and length of 1 m installed in a 21G needle was used. The detection fiber's shape was maintained over a radius of curvature of 30 cm. The dependences of the detected light intensity on the FOV and the depth showed better than 1.4% accuracy versus calculated dependences based on the measured optical properties of the solution. Adaptation of the method for use with complex structured biological tissue, particularly in the presence of a thick fascia, was not completely resolved. However, we believe that our specific fiber puncture-based measurement method for use in bulk tissue based on variation of the FOV with inverse Monte Carlo method-based analysis will be useful in obtaining optical coefficients while avoiding sample preparation-related instabilities.

A Three-Compartment Pharmacokinetic Model to Predict the Interstitial Concentration of Talaporfin Sodium in the Myocardium for Photodynamic Therapy: A Method Combining Measured Fluorescence and Analysis of the Compartmental Origin of the Fluorescence.

To evaluate the effectiveness of photodynamic therapy occurring in the interstitial space of the myocardium, we estimated the interstitial concentration of talaporfin sodium in the canine myocardium by constructing a three-compartment pharmacokinetic model based on measured changes in talaporfin sodium plasma concentration and myocardial fluorescence. Differential rate equations of talaporfin sodium concentration in the plasma, interstitial space, and cell compartment were developed with individual compartment volume, concentration, and rate constants. Using measured volume ratios based on histological examinations, we defined that the myocardial fluorescence consisted of the linear addition of fluorescence generated from these three compartments. The rate constants were obtained by fitting to minimize the sum of the squared errors between the measured talaporfin sodium concentrations and the calculated concentrations divided by the number of data points using the conjugate gradient method in MATLAB. We confirmed that this fitting operation may be appropriate, because a coefficient of determination between the measured talaporfin sodium changes and the calculated concentrations using our equations was 0.99. Consequently, to estimate the interstitial concentration in the canine myocardium, we propose a three-compartment pharmacokinetic model construction methodology using measured changes in talaporfin sodium plasma concentration and changes in myocardial fluorescence.

Oxygen-Enriched Photosensitizer Medium with Red Blood Cells to Study Tissue Interaction of Photosensitization Reaction.

BACKGROUND: It has been reported that the oxygen pressure of a photosensitizer medium decreases during an irradiation leading to decrease in the efficacy of the photosensitization reaction against the target cell in vitro. OBJECTIVE: The aim of this study was to obtain solutions with high dissolved oxygen levels in cultivated wells with perceiving oxygen environment and photosensitizer bleaching for photosensitization reaction studies. MATERIALS AND METHODS: We used a 10-mm-wide optical cell cuvette with a 1-mm optical path length as the well. A red blood cell (RBC) suspension with a hematocrit level of 0.625% was employed as the optical sample. The photosensitizer talaporfin sodium was added to a concentration of 30 µg/mL. The optical sample was irradiated by a 663-nm diode laser at 120 mW/cm2, for a total radiant exposure of 0-20 J/cm2, to induce a photosensitization reaction. Absorption spectra of the samples in the range of 475-700 nm were measured before and after each irradiation condition. Visible spectroscopy was selected to distinguish between the major three hemoglobin (Hb) types: oxygenated Hb, deoxygenated Hb, and met Hb. Also, this wavelength range was selected to investigate photobleaching using the Q band absorption peak. Each Hb concentration was estimated using a multiple regression analysis applied to the obtained absorption spectra. RESULTS: The relationship between oxygen saturation and the absorption peak in the Q band from the talaporfin sodium dynamics with increasing radiant exposure was revealed by our method with approximately twofold oxygen-dissolved solution. CONCLUSIONS: We could perceive the oxygen environment and the photosensitization reaction progression simultaneously with increasing dissolved oxygen by adding RBCs to the cell medium and measuring the absorption spectrum of it.

Development of a practical animal model of photodynamic therapy using a high concentration of extracellular talaporfin sodium in interstitial fluid: influence of albumin animal species on myocardial cell photocytotoxicity in vitro.

Photodynamic reaction-induced photocytotoxicity using talaporfin sodium is inhibited by serum proteins binding to talaporfin sodium. The serum albumin binding site for talaporfin sodium differs among animal species. To identify a practical animal therapeutic model, we studied the ability of human, canine, bovine, and porcine albumin to influence talaporfin sodium-induced photocytotoxicity in rat myocardial cells in vitro. Human, canine, bovine, and porcine serum albumins were used. The ratio of talaporfin sodium binding, which is strongly associated with photocytotoxicity, was measured by ultrafiltration with an albumin concentration of 0.5-20 mg/ml and 20 µg/ml talaporfin sodium to mimic interstitial fluid. Rat myocardial cell lethality was measured by the WST assay 2 h after samples were exposed to a radiant exposure of 20 J/cm2 by a red diode laser (Optical Fuel™, Sony, Tokyo, Japan) with a wavelength of 663 nm. The binding ratio dependence on albumin concentration differed among the animal species. Bovine albumin exhibited the largest difference from human albumin, with a maximum difference of 31% at 2 mg/ml albumin. The cell lethality characteristic was similar between human and canine albumin. The cell lethality dependence on albumin was not in the same order as the binding ratio. Cell lethality was lowest for human albumin with higher albumin concentrations between 5 and 20 mg/ml. There were no significant differences in cell lethality between bovine and porcine albumin and between human and canine albumin. We suggest that the canine model may be a useful animal therapeutic model for evaluating photodynamic therapy using a high concentration of the photosensitizer in the extracellular space.

Skin fluorescence following photodynamic therapy with NPe6 photosensitizer.

BACKGROUND: The second-generation photosensitizer NPe6 has strong anti-tumor effects with a much shorter photosensitive period than the first-generation photosensitizer Photofrin. Although photosensitive period has been reduced, skin photosensitivity is still a major side effect of photodynamic therapy (PDT). Therefore, we conducted a prospective study to investigate whether the NPe6 fluorescence intensity in skin after PDT could be measured effectively in human patients to improve the management of a patient's photosensitive period. METHODS: The NPe6 fluorescence measurements using a constructed fluorescence sensing system at the inside of the arm were acquired prior to and 5 and 10min after NPe6 administration as well as at the time of PDT (4-5h after administration), at discharge (2 or 3days after PDT), and at 1 or 2 weeks after PDT. Participants were interviewed as to whether they had any complications at 2 weeks after PDT. RESULTS: Nine male patients and one female patient entered this study. Nine patients were inpatients and one patient was an outpatient. All of the measurements of NPe6 fluorescence in the skin could be obtained without any complications. The spectral peak was detected at the time of discharge (2-3days after administration) in most cases and it decreased at 1 or 2 weeks after PDT. CONCLUSIONS: The fluorescence of NPe6 in the skin could be detected feasibly using the fluorescence sensing system in human patients. Measuring the relative concentration of NPe6 in the skin indirectly by measuring fluorescence intensity might be useful to predict the period of skin photosensitivity after PDT.

Effect of a photosensitization reaction performed during the first 3 min after exposure of rat myocardial cells to talaporfin sodium in vitro.

To better understand the mechanism of photodynamic cardiac ablation, we studied the effects of a photosensitization reaction (PR) performed during the first 3 min after rat myocardial cells were exposed to talaporfin sodium. A 3-mm-square microelectrode array with 64 electrodes was used to continuously measure the action potentials of the myocardial cells. A 30 µg/mL talaporfin sodium solution, a chlorine photosensitizer, was used, along with a 663-nm red diode laser (86 mW/cm2 for up to 600 s). The first trough of the measured action potential waveform corresponding to Na+ dynamics decreased exponentially with increasing PR duration. The average decay time of the exponential function from PR onset was 20.1 s. Marked morphological changes in the myocardial cells was observed after the PR. These results indicated that the behavior of the action potential waveform measured by the microelectrode array might be used as a less invasive method to evaluate the electrophysiological effects of a PR on myocardial cells.

Temperature Influence on Myocardial Cell Cytotoxicity of the Extracellular Photosensitization Reaction with Talaporfin Sodium and Serum Proteins at 17°-37°C.

BACKGROUND: We investigated the binding of talaporfin sodium with albumin and its photocytotoxicity during temperature changes by measuring absorbance spectra. The targeted tissue temperature differs according to the procedure. The photocytotoxicity efficiency should be investigated quantitatively because efficiency changes arising from temperature changes are expected. MATERIALS AND METHODS: The temperature dependence of talaporfin sodium binding with human serum albumin (0-20 mg/mL), high-density lipoprotein (0-0.04 mg/mL), and low-density lipoprotein (0-0.14 mg/mL) was investigated at 17°C, 27°C, and 37°C by measurement of absorbance spectra. Cell lethality was measured using a water-soluble tetrazolium-8 assay at 2 h after the photosensitization reaction at 17°C and 37°C. RESULTS: The binding ratios of talaporfin sodium with high-density lipoprotein decreased by 6.3% and those with low-density lipoprotein decreased by 12.8% when the temperature increased from 17°C to 37°C. Cell lethality increased significantly with a temperature rise from 17°C to 37°C at irradiation exposure of 20 and 40 J/cm2 and talaporfin sodium concentration of 20 µg/mL. CONCLUSIONS: From our in vitro data, we can predict that the change in photocytotoxicity efficiency would be negligible with a temperature decrease of <5°C from the body temperature in the case of photodynamic ablation with a short drug-light interval.

Irradiance dependence of the conduction block of an in vitro cardiomyocyte wire.

BACKGROUND: To obtain therapeutic condition precisely by in vitro experiment, we studied the irradiance dependence of the electrical conduction blockage caused by a photodynamic reaction using a high extracellular concentration of talaporfin sodium on a novel in vitro cardiomyocyte electrical conduction wire. METHODS: The cardiomyocyte wires were constructed on patterned cultivation cover glass, which had cultivation areas 60µm in width, and a maximum length of 10mm. The talaporfin sodium concentration was set to 20µg/mL. The photodynamic reaction with a high extracellular photosensitizer concentration was performed with a short time interval (approximately 15min) between photosensitizer exposure and irradiation. A 663-nm laser was applied to the cardiomyocyte wire, and the irradiance was varied between 3 and 120mW/cm2. The cardiomyocyte electrical conduction was evaluated using the cross-correlation function of intracellular Ca2+ probe fluorescence brightness from an upper and lower section outside the laser irradiation area of a wire every 10s, which lasted up to 600s. RESULTS: The onset of electrical conduction blockage was defined by an 85% decrease in the cross-correlation function, compared with its initial value. The time for the electrical conduction blockage decreased from 600 to 300s as the irradiance was increased. Also, the probability of electrical conduction blockage was found to increase with increasing irradiance. CONCLUSIONS: We found a strong dependence on the irradiance for the time and probability of electrical conduction blockage.

The Noninvasive Treatment for Sentinel Lymph Node Metastasis by Photodynamic Therapy Using Phospholipid Polymer as a Nanotransporter of Verteporfin.

Aim. The usefulness of photodynamic therapy (PDT) for treating sentinel lymph node (SLN) metastasis was evaluated. Materials and Methods. Verteporfin, a hydrophobic photosensitizer, forms a soluble aggregate with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). The concentrations of verteporfin were determined by measuring the fluorescence emitted at 700 nm. Seven days after the inoculation of A431 cells at the forearm of BALB/c nude mice, PMB-verteporfin was injected at dorsum manus and 75 J of light energy was delivered for 1 minute. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure, light exposure alone, PMB-verteporfin injection alone, and no treatment groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs, were harvested and evaluated. Results. The concentration of verteporfin in SLN was significantly higher than other organs. The combination of PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis (13%) comparing with no treatment group (52%), light exposure alone group (57%), and PMB-verteporfin injection alone group (46%). Conclusions. These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer and represent a potential alternative procedure to SLNB.

Phototoxicity of Vascular Endothelial Cells Caused by Contact with Talaporfin Sodium for 15-120 Min: In Vitro and In Vivo Studies.

OBJECTIVE: To reveal the mechanism of vascular patency in the myocardium after photosensitization immediately after talaporfin sodium (TS) injection in a canine model, we investigated acute injury to vascular endothelial cells (VECs) in vitro and in vivo. BACKGROUND DATA: There are many reports of vascular shutdown within the target region in photodynamic therapy with TS. Vascular patency within healthy canine myocardium in which a photosensitization reaction starts immediately after injection of TS has been reported. MATERIALS AND METHODS: TS fluorescence in human umbilical vein endothelial cells and cell lethality were measured with drug contact time (DCT) up to 120 min at 20 µg/mL. Dependence of radiant exposure on cell lethality with 60 min DCT was investigated using two albumin concentrations that corresponded to those in plasma and interstices. Irradiation (21 mW/cm) outside the adventitia of canine cervical veins for 167 or 667 sec was emitted through a diffuser probe 30 min after intravenous injection of TS (2.5 mg/kg). Veins were extracted ∼30 min after the reaction and stained with von Willebrand factor. RESULTS: Intracellular fluorescence increased, but not cell lethality, with increasing DCT. Cell lethality increased gradually and reached 100% over 20 J/cm2 in the albumin concentration in the interstices. Normal VECs were found at the acute phase over 20 J/cm2 with a TS concentration in plasma of ∼14 µg/mL in vivo. CONCLUSIONS: VEC injury after a photosensitization reaction to healthy tissue shortly after TS injection might be low enough for the blood vessels to be patent.

Dependence of damage within 10min to myocardial cells by a photodynamic reaction with a high concentration of talaporfin sodium outside cells in vitro on parameters of laser irradiation.

BACKGROUND: To investigate the immediate occurrence of irreparable severe damage to myocardial cells up to 10min after a photodynamic reaction with a high concentration of photosensitizer outside cells, we measured the damage response time and the parameters that govern the response time via rat myocardial Ca(2+) concentration. In our proposed method for catheter ablation of tachyarrhythmia by photodynamic reaction, there are two components to the electrical conduction block: an immediate electrical conduction block of several tens of seconds to several minutes, and a permanent electrical conduction block. METHODS: Rat myocardial intracellular Ca(2+) concentration changes before, during and after the photodynamic reaction with a high concentration of photosensitizer outside myocardial cells were continuously observed using a Fluo-4 AM Ca(2+) probe. Talaporfin sodium with 663-nm excitation was used to induce the photodynamic reaction. Talaporfin concentration was 10-30µg/ml, radiant exposure was 10-40J/cm(2), and irradiance was 30-290mW/cm(2). We evaluated the response time of irreparable severe damage to myocardial cells, according to Ca(2+) concentration. RESULTS: The response time of the defined severe damage occurrence to myocardial cells ranged from 200 to 500s. The response time decreased with increasing irradiance and photosensitizer concentration, but exhibited no significant change with total radiant exposure. CONCLUSIONS: We found that severe myocardial cell damage caused by a photodynamic reaction with a high concentration of photosensitizer outside cells occurred within a few minutes, which might be useful for catheter ablation for tachyarrhythmia that needs immediate response during the ablation procedure.

Electrical superior vena cava isolation using photodynamic therapy in a canine model.

AIMS: With the new era of multi-tip radiofrequency or balloon ablation catheters replacing the point-to-point ablation strategy, we aimed to determine the feasibility of a ring-laser catheter ablation technology to electrically isolate the superior vena cava (SVC) by exploring the advantages of the limitless catheter tip size possibly with the photodynamic therapy (PDT)-mediated ablation. METHODS AND RESULTS: We developed a first-generation prototype of a circular-laser-mapping catheter by fitting a 7 cm plastic optical fibre onto a circular variable-loop Lasso™ mapping catheter. Following SVC venography, both the laser catheter and another ring catheter for monitoring the SVC potentials were placed at the SVC. After the systemic infusion of a photosensitizer (talaporfin sodium), we initiated the irradiation with an output of 1 W in three canines and 0.3 W in four. The creation of electrical isolation as well as occurrence of phrenic nerve injury, sinus node injury, and SVC stenosis were evaluated before, immediately after, and 1 month after the procedure. A PDT-mediated SVC isolation was successfully performed in all seven canines. The isolation was completed with a laser irradiation of 70.4 ± 71.4 J/cm under 30.9 ± 5.0 µg/mL of a photosensitizer without any sinus node injury, phrenic nerve palsy, or SVC stenosis in both the acute and chronic evaluations. The minimum isolation time of 270 s was not correlated with the laser input power or the photosensitizer concentration. CONCLUSION: The electrical SVC isolation was successfully and instantly achieved using the PDT laser-ring catheter without any complications.

Comparison of myocardial cell survival 2 h and 24 h after extracellular talaporfin sodium-induced photodynamic reaction.

BACKGROUND: We have proposed an application of photodynamic reaction for less-heated myocardial ablation which employs talaporfin sodium. Intracellular photodynamic reactions with ongoing uptake have the ability to induce apoptosis over time, raising the possibility of extending the lesion depth. The objective of this study was to understand how, in myocardial cells, the late cell survival levels change by incubation time with talaporfin sodium, and what dependence talaporfin sodium uptake has on the duration of incubation with talaporfin sodium in vitro. METHODS: Rat myocardial cells were incubated with talaporfin sodium for 5-360 min and intracellular concentrations measured using a fluorescence micro-plate reader after wash. Cell survival was measured using a water-soluble tetrazolium assay at 2 and 24 h after a photodynamic reaction using a red diode laser of 660 nm, following 15-180 min of incubation with talaporfin sodium. Cells were stained with Hoechst 33342 to observe nuclear changes. RESULTS: Intracellular talaporfin sodium concentration increased with incubation time, with a marked increase between 0 and 60 min. Cell survival at 24 h decreased by 20% when the duration of incubation with talaporfin sodium was extended from 15 to 30 min. Following incubation time of 30-180 min with talaporfin sodium, cell survival was decreased by approximately 30% between measurements at 2 and 24 h. The intracellular talaporfin sodium concentration that induced higher levels of late cell death with cell nuclei fragmentation in these cells was approximately 0.2 µg/mL. CONCLUSION: We obtained the characteristics of late cell death occurrence and talaporfin sodium uptake to myocardial cell with various incubation times with talaporfin sodium.

Evaluation of human and bovine serum albumin on oxidation characteristics by a photosensitization reaction under complete binding of talaporfin sodium.

BACKGROUND: In order to investigate the therapeutic interaction of an extra-cellular photosensitization reaction, we evaluated the oxidation characteristics of human and bovine serum albumin by this reaction with talaporfin sodium under complete binding with albumin by spectroscopic analysis in a cell-free solution. METHODS: The solution was composed of 20µg/ml talaporfin sodium and 2.1mg/ml human or bovine serum albumin. A 662nm laser light was used to irradiate the solution. Visible absorbance spectra of solutions were measured to obtain the oxidized and non-oxidized relative densities of albumin and talaporfin sodium before and after the photosensitization reaction. The defined oxidation path ratio of talaporfin sodium to albumin reflected the oxidation of the solution. Absorbance wavelengths at approximately 240 and 660nm were used to calculate normalized molecular densities of oxidized albumin and talaporfin sodium, respectively. RESULTS AND CONCLUSIONS: The oxidation path ratio of talaporfin sodium to albumin when binding human serum albumin was approximately 1.8 times larger than that of bovine serum albumin during the photosensitization reaction from 1 to 50J/cm(2). We hypothesized that the oxidation path ratio results might have been caused by talaporfin sodium binding affinity or binding location difference between the two albumins, because the fluorescence lifetimes of talaporfin sodium bound to human and bovine serum albumin were 7.0 and 4.9ns, respectively. Therefore, the photodynamic therapeutic interaction might be stronger with human serum albumin than with bovine serum albumin in the case of extracellular photosensitization reaction.

Effects of albumin binding on photocytotoxicity of extracellular photosensitization reaction using talaporfin sodium to rat myocardial cells.

BACKGROUND: We previously proposed a new treatment for tachyarrhythmia using an extracellular photosensitization reaction occurring in the interstitial space of myocardia shortly after the injection of talaporfin sodium. Using myocardial cells, we studied the photocytotoxicity of this extracellular photosensitization reaction between talaporfin sodium and albumin. METHODS: The albumin concentrations tested spanned the physiological range found in the interstitial space (0-15 mg/ml) while the talaporfin sodium concentration were varied from 0 to 40 µg/ml. The reactions were conducted in 96-well plates. To obtain the binding ratio and the amount of energy deposited into the photosensitizer, we measured the change in the absorbance spectra of talaporfin sodium solutions containing different concentrations of albumin. RESULTS: Photocytotoxicity to myocardial cell due to the reaction decreased when physiological concentrations of albumin were added to the reaction mix, and decreased sharply when the molar concentration ratio of albumin to talaporfin sodium was between 0.3 and 1.2. A monotonic binding ratio was obtained, ranging from 10 to 80%, at albumin concentrations of 0.1-1.0 mg/ml. We found that the lethality of the extracellular photosensitization reaction towards myocardial cells had a threshold albumin concentration, even though the energy deposited into the talaporfin sodium solution was calculated to be almost constant (4.23 ± 0.19 J/well) in the presence of 0-15 mg/ml albumin. CONCLUSIONS: Based on the likely concentration of albumin in the interstitial space, we conclude that the photodynamic efficacy of talaporfin, under conditions used here, will markedly decrease if the albumin level exceeds 0.65 mg/ml.