The phototoxicity of PDT in cell culture can be promoted by the relatively hydrophilic bile acid UDCA (ursodeoxycholic acid). This was attributed to a conformational change in the anti-apoptotic protein Bcl-2, leading to an enhanced sensitivity to photodamage by sensitizers that target sites of Bcl-2 localization. UDCA also promoted the binding and inactivation of Bcl-2 by the non-peptidic antagonist HA14- 1, suggesting that UDCA may also be useful for promoting chemotherapy designed to target Bcl-2. In tumor-bearing animals, addition of UDCA to a PDT protocol involving the tin etiopurpurin SnET2 resulted in enhanced cancer control, but there was no effect on the extent of PDT-induced vascular shut-down. These results are consistent with the propo proposal that UDCA only promotes direct tumor cell kill. In this report, we have sal summarized recent research relating to mode of action of UDCA as it effects the on the efficacy of photodynamic therapy where Bcl-2 is among the PDT targets, and discuss the implications of the results.

Cardiolipin is a unique phospholipid containing two phosphatidyl glycerol moieties and four fatty acids per molecule. It is found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. The acridine derivative, nonyl-acridine orange (NAO), is a highly specific probe of cardiolipin, with a binding affinity approximately two orders of magnitude greater than that for binding to other anionic phospholipids. We recently reported that when NAO is bound in the mitochondria of human prostate cancer PC-3 cells and activated at 488 nm, NAO could transfer fluorescence resonance energy to the phthalocyanine photosensitizer Pc 4. This observation indicates that one site of Pc 4 binding is very near to NAO and therefore very near to cardiolipin. The average distance between the two fluorophores was calculated to be 7 nm. In the present study, we have extended the observation to the endogenously synthesized photosensitizer, protoporphyrin IX, an intermediate in heme biosynthesis that is used for photodynamic therapy of several types of malignant and non-malignant conditions. Protoporphyrin IX is generated in the mitochondria but is known to bind to other cellular sites as well, especially the endoplasmic reticulum. The ability of this molecule to accept resonance energy from NAO in cells is consistent with a localization of at least some of the molecules in the mitochondria either on the inner membrane, the site of cardiolipin, or within about 10 nm of it. Since protoporphyrin IX binds with high affinity to the peripheral benzodiazepine receptor, a component of the permeability transition pore complex that forms at contact sites between the inner and outer membranes, our observations provide evidence for the close association of several critical molecules for mitochondrial functions and suggest that cardiolipin may be an early oxidative target during PDT with at least two photosensitizers.

Bacteriopheophorbide molecules are second-generation photosensitizers with promise for PHotodynamic Therapy applications due largely to their absorption peaks in the near-Infrared region. Palladium bcteriopheophorbide, also called TOOKAD, has been successfully evaluated in several pre-clinical animal models. In this study the effect on tumor and normal vasculature was evaluated using an intravital vascular model on mouse cremaster muscle implanted with the RIF tumor. For tumor response studies, the same RIF tumor was implanted intradermally on the right flank and regression was evaluated for 42 days or until the tumor reached a 12 mm diameter. A light dose 300 J/cm² were delivered at 763 nm with power density of 100 mW/cm². Photosensitizer dose was 4 mg/kg body weight. Mice were treated immediately, 10 minutes, 30 minutes, or 24 hours after injection. Only the higher light dose (300 J/cm²) delivered 10 minutes after injection produced a reproducible and complete vascular and tumor response after PDT in these animals. In the cremaster-tumor model, arterioles and venules partially shutdown as early as 40 minutes after the beginning of treatment, while tumor neovasculature was irreversibly closed within 20 minutes of treatment. Tumor response studies demonstrated that the magnitude of vascular stasis correlates with tumor regression studies. Further studies using this photosensitizer are warranted, given its short clearance time and its near-Infrared activation wavelength.

Photodynamic therapy (PDT) mediated with vascular acting photosensitizer pd-bacteriopheophorbide (Tookad), is investigated as an alternative modality for the total ablation of prostate cancer. In vivo normal canine prostate is used as the animal model. Interstitial PDT was performed by irradiating the surgically exposed prostates with a diode laser (763 nm, 150 mW/cm) to activate the IV infused photosensitizer drug. The prostate and its adjacent tissues were harvested and subjected to histopathological examination. At one-week post PDT, the animals recovered well with little or no urethral complications. Prostatic urethra and prostate adjacent tissues (bladder and underlying colon) were well preserved. PDT induced prostate lesions were characterized by marked hemorrhagic necrosis. Prostate lesions could be detected by MRI scan as early as 48 h post PDT. Maximum lesion size of 1.5 cm³ and 2.9 cm³ could be achieved at 50 J/cm and 100 J/cm, respectively, with interstitial treatment using a single 1-cm diffuser fiber, suggesting the Tookad-PDT is very effective in ablating prostatic tissue. Pharmacokinetic studies show that the photosensitizer is cleared rapidly from the circulation. In conclusion, the novel photosensitizer Tookad mediated PDT may provide an effective alternative to treat localized prostate cancer.

The dark toxicity and singlet oxygen quantum yields of the zinc(II) complexes of the nido- and closo-carboranylporphyrins 4 and 5 are reported. Both carboranylporphyrins show very low toxicity towards V79 hamster lung fibroblast cells (IC₅₀ > 500 μM), display similar absorption and fluorescence emission spectra, and are efficient producers of singlet oxygen (quantum uilds of 0.72 in CD₃OD and 0.45 in CHCl₃ were determined for 4 and 5, respectively). Our results indicate that the presence of nido- or closo-carborane cages bound via methylene linkages to the meso-phenyl groups, do not substantially alter the photosensitizing properties of porphyrin macrocycles. It is suggested that carboanylporphyrins could have dual application in the boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) treatment of malignant brain tumors.

Photodynamic therapy (PDT) is now a reasonably well-known therapeutic option and is approved as a first line treatment of age-related macular degeneration (AMD), a non-oncologic condition. For most cancer applications PDT is approved mainly as a palliative or adjunctive treatment often when all other options have failed. As the modality evolves toward becoming a first-line or curative option, long-term effects of processes involved will need to be studied. Cellular and tissue responses to PDT are more complex than responses to the more conventional therapies, perhaps because PDT is inherently a binary (or ternary) therapy. In addition to the nature and localization of the photosensitizer (PS), the timing of illumination after administration, the mode of administration and the PS and light doses, the efficacy and selectivity of responses are also determined by the physiology and geometry of tumors, the inherent survivability of tumor cells (in circulation and other anatomic sites) and cellular and molecular responses to PDT. The overall outcome of photodynamic treatment in the long term is determined by a combination, in varying degrees, of all of the above factors. In order to enhance and broaden the application of PDT to complex anatomical sites, an understanding of these factors would be useful. In the laboratory, the outcome is also dependent on the specific animal models being studied. This manuscript discusses preliminary studies along these lines using a variety of tools and implications, if any, of the results obtained.

Nasopharyngeal carcinoma (NPC), endemic in Southern China, is ranked as the fourth leading cause of cancer deaths in Hong Kong. In an effort to develop new PDT agents for cancer treatment, with a particular emphasis on the NPC, we have investigated the benzochlorin-type photosensitizers. The chromophore is functionalized with side chains containing polar and/or cationic groups. Synthetic methods have been developed for such purposes; e.g. 5-chlorosulfonyloctaethylbenzochlorin and 5-bromooctaethylbenzochlorin are used as versatile precursors for the preparation of ammonium and amidinium salts. Preliminary in vitro study of the photodynamic activity of the synthetic compounds indicated that the cationic benzochlorin derivatives display significant photocytotoxicity towards NPC cells.

Pulsed photodynamic effect on Ps.aeruginosa has been investigated in vitro with the use of methylene blue (MB) as a photosensitizer. Bacterial suspensions were illuminated with 665-nm, nanosecond light pulses under the various drug and light dose conditions. Bacterial survival fraction decreased with increasing the drug and light doses, while no significant bacterial cell death was observed when the bacteria were washed after incubation with the drug. These results suggest a two-step photo-bactericidal mechanism; the photodynamic effect first induces membrane damage through which MB molecules were translocated into the bacterial cytoplasm, and then caused bacterial cell death.

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. Although many workers have used photodynamic therapy (PDT) to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections by Gram-negative bacteria in living mice. However these infected wound models used a short time after infection (30 min) before PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. We used Staphylococcus aureus stably transformed with a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in Gram-positive bacteria. These engineered bacteria emitted bioluminescence allowing the progress of the infection to be monitored in both space and time with a lowlight imaging charged couple device (CCD) camera. One million cells were injected into one or both thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four hours later the bacteria had multiplied more than one hundred-fold, and poly-L-lysine chlorin(e6) conjugate or free chlorin(e6) was injected into one area of infected muscle and imaged with the CCD camera. Thirty-minutes later red light from a diode laser was delivered as a surface spot or by interstitial fiber into the infection. There was a lightdose dependent loss of bioluminescence (to < 5% of that seen in control infections) not seen in untreated or light alone treated infections, but in some cases the infection recurred. Conjugate alone led to a lesser reduction in bioluminescence. Infections treated with free chlorin(e6) responded less and the infection subsequently increased over the succeeding days, probably due to PDT-mediated tissue damage. PDT-treated infected legs healed better than legs with untreated infections. This data shows that PDT may have applications in drug-resistant soft-tissue infections.

Therapeutic options for patients with locally recurrent prostate cancer after treatment with radiation therapy are limited. An ongoing Phase I trial of interstitial photodynamic therapy (PDT) with the photosensitizer motexafin lutetium (MLu) was initiated in year 2000 for men with locally recurrent prostate cancer. The primary objective of this trial is to determine the maximally tolerated dose of motexafin lutetium-mediated PDT. Twelve men with biopsy-proven recurrent prostate cancer and no evidence of distant metastatic disease have been enrolled. Pre-treatment evaluation included an MRI of the prostate, bone scan, laboratory studies, cystoscopy, and transrectal ultrasound. Treatment plans were generated based upon the ultrasound findings. PDT dose was escalated by increasing the motexafin lutetium dose, increasing the 732 nm light dose, and decreasing the drug-light interval. Motexafin lutetium doses ranged from 0.5 to 2 mg/kg administered IV 3, 6, or 24 hours prior to 732 nm light delivery. The light dose measured in real time with in situ spherical detectors was 25-100 J/cm2 for all patients. Light was delivered through optical fibers inserted through a transperineal brachytherapy template in the operating room and optical property measurements were made before and after light therapy. Prostate biopsies were obtained before and after light delivery for spectrofluorometric measurements of photosensitizer uptake. Twelve patients have completed protocol treatment on eight dose levels without dose-limiting toxicity. Grade I PDT-related genitourinary symptoms were observed. One patient had Grade II urinary urgency that was urinary catheter-related. No rectal or other GI PDT-related toxicities were observed. Measurements of motexafin lutetium in prostate tissue demonstrated the presence of photosensitizer at all dose levels. Conclusions: Motexafin lutetium-mediated PDT designed to treat comprehensively the entired prostate gland has been well-tolerated at the doses studied to date.

Background and Objective: Photodynamic therapy (PDT) may be used for ablation of high grade dysplasia and/or early cancer (HGD/T1) in Barrett's esophagus. A complication of PDT is esophageal stricture. The objective of this study was to find the lowest light dose to potentially reduce the incidence of strictures while effectively ablating HGD/T1. Materials and Methods: Patients (n=113) with HGD/T1 received an intravenous injection of porfimer sodium (2 mg/kg). Three days later, laser light (630 nm) was delivered using a cylindrical diffuser inserted in a 20 mm.diameter PDT balloon. Patients were treated at light doses of 115 J/cm, 105 J/cm, 95 J/cm and 85 J/cm. The efficacy was determined by four quadrant biopsies of the treated area three months after PDT. The formation of stricture was determined by the incidence of dysphagia and the need for esophageal dilation. Strictures were considered mild if they required less than 6 dilations, and severe if 6 or more dilations were required. Efficacy and incidence of strictures were tabulated as a function of light dose. Results: Using 115 J/cm, there were 17% of patients with residual HGD/T1 after one treatment. However, when the light doses of 105 J/cm, 95 J/cm and 85 J/cm were used, the residual HGD/T1 after one PDT session was increased to 33%, 30%, and 32% respectively. The overall incidence of strictures (mild and severe) was not correlated to the light dose. However, the incidence of severe strictures was directly proportional to the light dose. Using the light dose of 115 J/cm, 15.3% of patients developed severe strictures compared to about 5% in the groups of patients who received the lower light doses. Conclusions: Decreasing the light dose below 115 J/cm doubled the rate of residual HGD/T1 after one treatment while reducing the incidence of severe strictures to one-third of cases from 115 J/cm. The results may be used to evaluate the risks and benefits of different light doses.

For most Photodynamic Therapy (PDT) applications a diffuse, broad and uniform source of irradiation is needed to obtain the most effective and consistent treatment. Since many treatments are within the patient's body, an effective compact fiber optic delivery system is needed for the activation of the photsensitizer drub at the site of the tissue to be treated. High Numerical Aperture (NA) optical fibers have benefits for PDT treatments but possibly even more so for PDT diagnostic applications These are summarized and new optical fibers with high and ultra high NAs are described. Properties of these fibers are presented as well as advantages they have over other fibers for delivering light in various PDT applications. Silica fibers with enhanced effective NAs approaching 0.6 are described.

Oblique incident light fields are sometimes unavoidable for photodynamic therapy of skin cancers, e.g., for large fields on uneven surface. We have performed Monte-Carlo simulation for circular fields (R = 0.25, 0.35, 0.5, 1, 2, 3, and 8 cm) for reduced scattering coefficient μ_s' = 10 cm^-1 and attenuation coefficient μ_a = 0.1-1.0 cm^-1. We used anisotropy g = 0.9 and the index of refraction n = 1.4 for all Monte-Carlo simulations. Compared to a broad beam of normal incidence, the peak fluence rate along the central-axis for a slanted beam is increased for otherwise the same geometrical conditions and optical properties. The effective attenuation coefficient is slightly decreased for a slanted beam compared to a normal incident beam. The beam profile for a slanted beam at a fixed depth is no longer symmetrical but is higher towards the lateral side of beam incidence. Since the broad beam with finite radius R can be considered as a convolution of a pencil beam, solution for a slanted pencil beam can be used to determine the light fluence distribution for circular beams with oblique beam incidence. An analytical solution can be obtained for the pencil beam obliquely incident on a semiinfinite medium. The solution can be approximated using the diffusion or P3 theory with one point source or two point sources located at appropriate depths with appropriate weights along the beam pathlength inside the phantom, with corresponding image sources to fulfill the extended boundary condition. The analytical solution agrees well with Monte-Carlo Simulation at depths z > 2cosθ_t/µ’_t, θ_t is the incident angle after refraction at the interface. Measurements using an isotropic detector were made in a liquid phantom composed of intralipid and ink to verify the Monte-Carlo simulation results.

Photodynamic therapy (PDT) utilizes photon energy to activate a pre-administered photosensitizer drug in tissue to achieve a localized tumor control. PDT cell killing mechanism is directly related to the reactive oxygen species (ROS) produced during the photochemical reactions. Conventional PDT dosimetry evaluates distributions of the photosensitizer drug, photon propagation and absorption, and availability of molecular oxygen in the target tissue. Yet, the ultimate bullet for the damaging effect is ROS. An evaluation of ROS production during PDT should provide a more direct marker for PDT. Fluoresceinyl Cypridina Luciferin Analog (FCLA) is a chemiluminescence probe that specifically interacts with ROS (singlet oxygen and/or superoxide). The work is a preliminary investigation of the feasibility using FCLA as a means to evaluate ROS production in PDT.

A continuing challenge in photodynamic therapy is the accurate in vivo determination of the optical properties of the tissue being treated. We have developed a method for characterizing the absorption and scattering spectra of prostate tissue undergoing PDT treatment. Our current prostate treatment protocol involves interstitial illumination of the organ via cylindrical diffusing optical fibers (CDFs) inserted into the prostate through clear catheters. We employ one of these catheters to insert an isotropic white light point source into the prostate. An isotropic detection fiber connected to a spectrograph is inserted into a second catheter a known distance away. The detector is moved along the catheter by a computer-controlled step motor, acquiring diffuse light spectra at 2 mm intervals along its path. We model the fluence rate as a function of wavelength and distance along the detector’s path using an infinite medium diffusion theory model whose free parameters are the absorption coefficient μ_a at each wavelength and two variables A and b which characterize the reduced scattering spectrum of the form μ’_s = Aλ^-b. We analyze our spectroscopic data using a nonlinear fitting algorithm to determine A, b, and μ_a at each wavelength independently; no prior knowledge of the absorption spectrum or of the sample’s constituent absorbers is required. We have tested this method in tissue simulating phantoms composed of intralipid and the photosensitizer motexafin lutetium (MLu). The MLu absorption spectrum recovered from the phantoms agrees with that measured in clear solution, and μ_a at the MLu absorption peak varies linearly with concentration. The µ’_s spectrum reported by the fit is in agreement with the known scattering coefficient of intralipid. We have applied this algorithm to spectroscopic data from human patients sensitized with MLu (2 mg kg^-1) acquired before and after PDT. Before PDT, the absorption spectra we measure include the characteristic MLu absorption peak. Using our phantom data as a calibration, we have determined the pre-treatment MLu concentration to be approximately 2 to 8 mg kg^-1. After PDT, the concentration is reduced to 1 to 2.5 mg kg^-1, an indication of photobleaching induced by irradiation. In addition, absorption features corresponding to the oxygenated and deoxygenated forms of hemoglobin indicate a reduction in tissue oxygenation during treatment.

New techniques research mechanisms of photdynamic reactions at somatic frog nerve was approved. Dosimetry PDT with minimum time resolution ~1ms determined by changing the amplitude of compound action potential of somatic frog nerve. Light-induced inactivation of dynamic response of somatic frog nerve on electrical pulsed excitation was study ex vivo. The light-sensitive dyes: methylene blue (Mb), Indocianin green and eryhtrocin-B has been used on photodynamic induced inactivation of the processes generation nerve pulses. Inactivation of consequence action potential of somatic frog nerve using excitation of electical pulsed was achieved by irradiation with He-Ne laser light in a red spectral region (λ=633 nm, power level 2-20 mW), diode laser (λ=805 nm, P<0.1-1 W/cm²) in the case of Indocianin green and YAG:Nd laser (λ=532 nm, P~1mW) for eryhtrocin-B. It was discovered that methylene blue, Indocainine green and erytrocin-B decrease of the amplitude compound action potential of the ensemble neurons. The possible cell death mechanism was connected with damage of the sodium potassium adenosine triphosphatase (K-Na ATP) active transport which decrease of amplitude of compound action potential and decrease lifetime ionic channel of membrane nerve.

Photodynamic therapy (PDT) and fluorescent diagnostics (FD) with Radaclorine (RadaPharma, Russia) (RC) have been provided in 32 patients with T1-4 stage basal cell carcinoma (BCC) and in 81 patients with Photsense. Pharmacocynetic studies with detecting the borders of tumor growth and intensity of accumulation of photosensizers in tumor, normal tissues and visualization have been done by Spectral-fluorescent Complex and spectranalyser LESA-01 (He-Ne-laser, λ=633nm). We've got fluorescence of all tumors and additional fluorescence zones were found, cytological verification of BCC was got in most of cases. The fluorescent signs of RC in normal skin were found till 5 days after injection. As a source of light for PDT we used simeconductive lasers: Milon - λ = 660+2nm, light dose was 200-300 J/cm² and Biospec (λ+672+2nm), multiple laser surface and interstitial irradiation was performed 24 hours after PS injection with total light dose till 400-600 J/cm². 2 months after PDT with RC complete response (CR) in 65.6% of cases, partial response-in 34.4% of cases. The efficacy of PDT with PS was higher (CR-84.0%, PR-14.8%). Our experience show pronounced efficacy of PDT with RC for BCC without side effects and very short skin toxicity.

Photodynamic Therapy (PDT) and fluorescent diagnostics (FD) using Photosense have been provided in 26 patients with breast cancer (BC) and in 108 patients with skin metastases of BC. In 22 patients with T1-T2N0M0 primary tumor PDT was preoperative treatment, with radical mastectomy 7-10 days after PDT. 4 patients had residual tumor after radiotherapy. FD was fulfilled with spectranalyser. We used semiconductive laser for PDT-λ=672+2nm, P=1,5 W, interstitial irradiation 2-24 hours after PS injection in light dose 150-200 J/cm³ in patients with primary tumor and multiple surface irradiations (1-4) with interval 24-48 hours and total light dose 400-600 J/cm² for metastases. Partial regression of tumor with pathomorphosis of 2-4 degree has been found in 23 cases in first group. Treating metastases we had overall response rate of 86,9% with complete response (CR) in 51,5% and partial response in 35,4%. In a year after PDT in 52 patients with CR we had CR in 36,6%, local recurrences in 23,1%, progression (distant [lung or bone] metastasis) in 40,4% of cases. Our experience show pronounced efficacy of FD for detecting tumor borders and PDT for treating BC as preoperative modality and as palliation in cases of recurrencies.

This study represents the first reported use of photodynamic therapy in bone and specifically, as a treatment for spinal metastases. A metastatic model in rat confirmed the efficacy of benzoporphyrin derivative-monoacid-mediated PDT for treating lesions within the spine and appendicular bone. Fluorimetry confirmed the selective accumulation of drug into the tumor(s) at 3 hours post-injection. 48 hrs post light delivery into the vertebral body of the rat spine loss of bioluminescent signal and histological analyses of sectioned spine confirmed MT-1 tumor cell kill in vivo as previously confirmed in vitro using an established cell viability assay. Porcine vertebrae provided a model comparable to that of human for light propagation and PDT response. Light measurements were recorded at 2.5 mm increments as the detector probe was retracted out of the vertebral body away from a diffusing fiber at 70-90° planar angle to it. At 30 minutes or 1hr post BPD-MA administration (6 mg/m²), light (648 J, 150 mW/cm, 690 nm) was delivered to vertebrae L1 and/or L2. Vertebrae were harvested and sectioned for histology 48 hrs following PDT. Light propagation was plotted as distance (μm) from the emitting source. Results support the application of PDT to the treatment of primary or metastatic lesions within bone.