Expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL is greatly elevated in many advanced cancers, especially those resistant to standard therapies, such as radiation or chemotherapy. It has been suggested that those two proteins would be attractive targets for the development of new cancer treatments. Photodynamic therapy (PDT) with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, specifically attack the anti-apoptotic protein Bcl-2, generating a variety of oxidized, complexed, and cleaved photoproducts. The closely related protein Bcl-xL is also a target of Pc 4-PDT. In a recent study employing transient transfection of an expression vector encoding deletion mutants of Bcl-2, we identified the membrane anchorage regions of the protein that are required to form the photosensitive target. In spite of the demonstrated photodamage to Bcl-2 (and Bcl-xL), how the photodamage translates into changes in the sensitivity of cells to PDT-induced apoptosis or other modes of cell death is not clear, and it also remains unclear how elevated amounts of anti-apoptotic proteins in tumors might make them more or less responsive to PDT. In the present study, we have studied the PDT response of MCF7 human breast cancer cells overexpressing wild-type Bcl-2 or certain deletion mutants either in a transient or stable mode. We show that cells expressing modestly elevated amounts (<10-fold increase) of Bcl-2 and in which the pro-apoptotic protein Bax is not upregulated do not differ from the parental cells with respect to PDT-induced cell killing. In contrast, cells expressing higher amounts (>50-fold increase) of Bcl-2 or certain mutants are made significantly more resistant to the induction of apoptosis and the loss of clonogenicity upon exposure to Pc 4-PDT. In the presence of high levels of Bcl-2, extensive photodamage requires higher PDT doses. We conclude that Pc 4-PDT targets Bcl-2 and Bcl-xL, eliminating one mechanism that protects the tumor cells from other types of therapy. However, it is possible that cells expressing very high levels of the anti-apoptotic proteins might still be resistant to PDT. The data suggest that PDT with a non-vascular-targeting photosensitizer might be effective in a combination treatment in which Bcl-2 and Bcl-xL are first photodamaged before delivery of a second agent.

We had previously described the use of relatively hydrophobic bile acids, notably UDCA (ursodeoxycholate) for the promotion of the apoptotic response to photodynamic therapy. Further study revealed that this effect occurred only when the target for photodamage was the anti-apoptotic protein Bcl-2. The efficacy of lysosomal photodamage, leading to a cleavage of the protein Bid, was not influenced by UDCA. Moreover, the apoptotic cell death resulting from treatment of cells with the non-peptidic Bcl-2 inhibitor HA 14-1 was also promoted by UDCA. These results are consistent with the proposal that the pro-apoptotic effects of UDCA are directed against Bcl-2, promoting inactivation by HA 14-1 or photodamage.

An in vivo model has been developed for the study (PDT) and anti-angiogenic treatments. Significant damage to the vasculature of the chick chorioallantoic membrane (CAM) was observed immediately following PDT with 5-aminolevulinic acid (ALA). Multicell human glioma spheriods were placed on the CAM at day five of embryonic development, however, neovascularization was not observed.

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates over extended periods in order to increase selective tumor cell kill through apoptosis. The focus of the present work is on mPDT treatment of malignant brain tumors, in which selectivity between damage to tumor cells versus normal brain tissue is critical. Previous studies have shown that low-dose PDT using aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. In order to produce enough tumor cell kill to be an effective therapy, multiple PDT treatments, such as hyperfractionation or metronomic delivery, are likely requried, based on the levels of apoptosis achieved and model calculations of tumor growth rates. mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of interstitial devices for extended light delivery while allowing free movement. In rat models ALA administration via the drinking water has been accomplished at significant doses for up to 10 days, and ex vivo spectrofluorimetry of tumore, normal brain and other tissues post mortem demonstrates a 3-4 increase in the tumor-to-brain concentration of PpIX, without toxicity. Prototype light sources and delivery devices are also shown to be practical, either using a laser diode or light emitting diode (LED) coupled to an implanted optical fiber in the case of the rat model or a directly-implanted LED in rabbits. The combined delivery of both drug and light over an extended period, with survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.

The combination of verteporfin-based photodynamic therapy (PDT) wiht radiaiton therapy from an orthovoltage device has been examiend in the radiation induced fibrosarcoma tumor model. PDT with verteporfin using a 3 hour delay between injection and the time of optical irradiation has been shown to cause a significant rise in overlal tumor oxygenation. It was huypothesized that this mechanism arises from the reduced oxygen consumption from cells where the PDT has targeted the mitochondria and shut down cellular respiration. Tumor blood flow was measured and found to be still be patent immediately following therapy. This increasing oxygenation was thought to provide an opportunity to increase the radiation sensitivity of the tumor immediately following PDT. When this type of treatment was combined with radiation therapy, a delay in the tumor regrowth time demonstrated that the combined effect was greater than additive. Further study of this phenomenon will provide a more complete mechanistic understanding of the effect and possibly provide a viable pre-treatment for radiation therapy of tumore that increases the therapeutic ratio. This effect could be used to either increase the radiaton dose without increasing the side effects or decrease the dose needed for the same effect on the tumor.

In order to better understand light dosimetry issues for photodynamic therapy (PDT), we have used various tumor and normal tissue geometries to develop a diffusion model of light transport in tissues. We hypothesize that tumor tissues with curved surfaces will have significantly different internal fluence distributions, as compared to tissues with flat surfaces. Using a mouse subcutaneous tumor and rear limb muscle model we compared the internal fluence values within the tissue. In addition, numerical simulations for these corresponding tissue geometries and laser light incidence angles were made. Assuming that the relative photon fluence in the tissue can be accurately modeled by the diffusion equation, we used a finite element approach to approximate the distribution inside the tissue. Meshes with different geometries (flat and curved with different curvatures) were used in this study to mimic the tumor and leg geometries of the murine tumors treated in the lab. Results suggest that tissues surface geometries and incidence angle of light can significantly alter the photon fluence inside the tissue. The photon fluence difference for an 8 mm diameter, curved surface mouse tumor vs. flat muscle tissue can be as high as 20%. In general, the greater the tissues curvature, the greater the potential loss in light fluence is. In summary, our data demonstrates the importance of tissue surface geometry and the incidence angle of light in determining optimal PDT light dosimetry, and indicates that comparisons between tissue geometries must be carried out with attention to differences in the internal optical distribution.

The success of photodynamic therapy with verteporfin is partially determined by the pharmacokinetic distribution of the sensitizer at the time of treatment. In this study tumor blood flow changes in the RIF-1 murine tumor model and tumor resopnse using the regrowth assay were measured, comparing two different intervals between drug and light administration. Blood flow measurements were taken with a laser Doppler system monitoring continuously over 1 hour and periodically up to 6 hours after treatment. Treatment after the longer interval caused significantly less flow decrease, to only 50% of the initial flow in 6 h. Hoechst staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors after the longer time interval, the regrowth rate was not signifincalty different from that of the control, indicating that only the 15-min interval group caused serious damage to the vascular bed of the tumor. These studies support the hypothesis that temporal pharmacokinetic changes in the photosensitizer distribution between the tumor parenchyma and blood vessels can significantly alter the mechanism of tumor targeting during therapy.

Small circular light fields (<= 2 cm diameter) are sometimes used for photodynamic therapy of skin and recurrent breast cancers on the chest wall. These fields have lateral dimensions comparable to the effective mean free path of photons in the turbid medium, which causes reduced light fluence rate compared to that of a broad beam of uniform incident irradiance. We have compared Monte-Carlo simulation with in-vivo dosimetry for circular fields (R = 0.25, 0.35, 0.5, 0.75, 1, 2, 3, and 8 cm) in a liquid phantom composed of intralipid and ink (μ_s' = 4 20^-1/cm and μ_a = 0.1 cm^-1) for wavelengths between 532 and 730 nm. We used anisotropy g = 0.9 and the index of refraction n = 1.4 for all Monte-Carlo simulations. The measured light fluence rate agrees with Monte-Carlo simulation to within 10%, with the measured value lower than that of the Monte-Carlo simulation on tissue surface. The ratio of the peak fluence rates between a circular beam and a broad beam under tissue is 0.58 - 0.96 or 0.84 - 1.00 for R between 0.5 - 2 cm and mueff = 1.1 or 2.0 cm^-1, respectively. The ratio of peak fluence rate and incident irradiance for the broad beam is 5.9 and 6.4 for μ_eff = 1.1 and 2.0 cm^-1, respectively. The optical penetration depth delta varies from 0.34 - 0.48 cm for R between 0.5 and 2 cm, with the corresponding δ= 0.51 cm for a broad beam. The ratio of fluence rate and incident irradiance above tissue surface is 1.4 - 1.8 or 1.9 - 2.2 for R between 0.5 - 2 cm and mueff = 1.1 or 2.0 1/cm, respectively. At depth of 0.2 cm inside tissue, Off-axis ratio OAR, defined as the ratio of fluence rate at off-axis distance r to that on the central axis, varies between 0.91 - 0.54 or 0.93 - 0.52 for off-axis distances r between 0.6 and 1.0 cm and μ_eff = 1.1 or 2.0 cm^-1, respectively. In conclusion, in-vivo light dosimetry agrees with Monte-Carlo simulation for small field dosimetry provided the isotropic detector is corrected for the blind spot. The light fluence rates for small circular fields are substantially lower than that of the broad beam of the same incident irradiance.

Photodynamic therapy (PDT) employs a combination of photosensitizing chemical, light, and oxygen Knowledge of tissue optical properties, including absorption (μ_a) and reduce scattering coefficients (μ_s’), makes possible to derive blood oxygen saturation, light penetration depth, and drug concentration, which are important to ensure PDT treatment efficacy at the specific wavelengths. We have developed an absorption spectroscopy system to measure μ_a and μ_s’ in the spectral range 600-800nm using a contact linear probe with a source fiber and multiple source-detector separation distances less than 1 cm. The μ_a and μ_s’ were recovered based on diffusion approximations of the photon transport equation. We measured tissue optical properties among various organs of patients with intraperitoneal malignancies for an on-going Phase II PDT protocol. The results from 12 patients showed various effective penetration depth from site to site and from organ to organ. The percentage oxygen saturation (%StO2) are similar before and after PDT. Before PDT, meff (mean (standard deviation) (number of patients)) in cm-1 at 630nm are 2.4 (0.2) (12) in small bowel, 2.2(0.4) (9) in large bowel, 4.2(2.7) (7) in tumor, 3.3 (0.3) (10) in peritoneum, 2.7 (0.3) (11) in skin, and 10.1 (0.6) (10) in liver. %S_tO₂ is 60-80% for most organs but 30-40% for tumor.

Photodynamic therapy has been applied to Barrett's esophagus and has been shown in prospective randomized studies to eliminate dysplasia as well as decrease the occurrence of cancer. However, the therapy isnot always effective and there are issues with residual areas of Barrett's mucosa despite therapy. There has not been a good explanation for these residual areas and they seem to imply that there may exist a biological mechanisms by which these cells may be resistant to photodynamic therapy. It was our aim to determine if known abnormalities in Barrett's mucosa could be correlated with the lack of response of some of these tissues. We examined the tissue from mulitpel patients who had resonse to therapy as well as those who did not respond. We assessed the tissue for p53 mutations, inactivatino of p16, ploidy status, cell proliferation, telomerase activity, and degree of dysplasia. Interestingly, the only genetic marker than was found to be correlated with lack of reonse was p53 and telomerase activity. This suggests that cells that have lost mechanisms for cell death such as apoptosis or telomere shortengin may be more resistant to photodynamic therapy. In this study, we examined patients before and after PDT for telomerase activity.

Fluorescence-guided brain tumor resection may help the neurosurgeon to identify tumor margins that merge imperceptibly into the normal brain tissue and are difficult to identify under white light illumination even using an operating microscope. We compared the amount of residual tumor after white light resection using an operating microscope versus that after fluorescnece-guided resection of an intracranial VX2 tumor in a preclinical model using our previously developed co-axial fluorscence imaging and spectroscopy system, exciting and detecting PpIX fluorescence at 405nm and 635nm respectively. Preliminary results: No fluorescence was present in 3 non-tumor-bearing animals. Fluorescence was present in all 15 tumor-bearing animals after white light resection was completed. To date in 4 rabbits, a decrease in residual tumor was found when using additional fluorescence guided resection compared to white light resection only. Conclusions: ALA induced PpIX fluorescence detects tumor margins not seen under an operation microscope using while light. Using fluorescence imaging to guide tumor resection resulted in a 3-fold decrease in the amount of residual timor. However, these preliminary results indicate that also an additional amount of normal brain is resected, which will be further investigated.

In two randomized prospective studies of brain tumor PDT more than 180 patients have been accrued. At the Toronto site we recognized two patients who developed a lower motor neuron (LMN) facial paralysis in the week following the PDT treatment. In both cases a temporal lobectomy was undertaken and the residual tumor cavity was photo-illuminated. The surface illuminated included the temporal fossa floor, thus potentially exposing the facial nerve to the effect of PDT. The number of frontal, temporal, parietal, and occipital tumors in this cohort was 39, 24, 12 and 4, respectively. Of the 24 temporal tumors 18 were randomized to Photofrin-PDT. Of these 18 a temporal lobectomy was carried out exposing the middle fossa floor as part of the tumor resection. In two of the 10 patients where the lobectomy was carried out and the fossa floor was exposed to light there occurred a postoperative facial palsy. Both patients recovered facial nerve function in 6 and 12 weeks, respectively. 46 J/cm² were used in the former and 130 J/cm² in the latter. We did not encounter a single post-operative LMN facial plasy in the 101 phase 2 patients treated with Photofrin-PDT. Among 688 supratentorial brain tumor operations in the last decade involving all pathologies and all locations no case of early post-operative LMN facial palsy was identified in the absence of PDT. One further patient who had a with post-PDT facial palsy was identified at the Denver site. Although it is possible that these patients had incidental Bell's palsy, we now recommend shielding the temporal fossa floor during PDT.

In this study, photodynamic therapy (PDT) mediated with a novel, second generation photosensitizer Tookad (palladium-bacteriopheophorbide, WST09, STEBA Biotech, France), is investigated as an alternative modality in the treatment of prostate cancer. In vivo normal canine prostate and spontaneous advanced prostate cancer are used as the animal model. PDT was performed by irradiating the surgically exposed prostates with a diode laser (763 nm, 150 mW/cm) to activate the i.v. infused photosensitizer. The effects of drug concentration, drug-light interval, and light fluence rate on the PDT efficacy were studied. The prostates and adjacent tissues (bladder and underlying colon) were harvested and subjected to histopathological examination. During the one-week to 3-month period post PDT treatment, the dogs recovered well with little or no urethral complications. Prostatic urethra and prostate adjacent tissues (bladder and underlying colon) were well preserved. Light irradiation delivered during drug infusion or within 15 min post drug infusion induced the similar extend of damages. PDT induced prostate lesions in both normal and cancerous prostate were characterized by marked hemorrhagic necrosis and atrophy. Maximum lesion size of over 3 cm in dimension could be achieved with a single 1-cm interstitial treatment, suggesting the therapy is very effective in ablating cancerous prostatic tissue. In conclusion, the second generation photosensitizer Tookad mediated PDT may provide an effective alternative to treat prostate cancer.

PDT in prostate cancer will likely be implemented clinically with patients who have failed prior ionizing radiation therapy (RT). The current study is to develop an in vivo model to evaluate the effects of PDT on prostatic tissue after RT. To produce a physiological and anatomical environment in prostate similar to that in patients who have failed RT, canine prostates (n=4) were subjected to a definitive course of ionizing radiation therapy (2.7 Gy x 20 fractions) 5 to 6 months prior to PDT. A laparotomy was performed to expose the prostate for PDT. Second generation photosensitizer Tookad (Palladium-Bacteriopheophorbide, Steba Biotech, The Netherlands) acts primarily on tissue vasculature and is very effective in destroying normal prostatic tissue, as shown by our prior studies. Due to the extremely fast clearance of the photosensitizer, interstitial light irradiation (760 nm, 50-200 J/cm, 150 mW/cm from a 1 cm diffuser fiber) was delivered 4 minutes after the onset of Tookad infusion (i.v. 2.5 mg/ml, 2 mg/kg, total infusion time 10 min). The prostates were harvested for histopathology one week after PDT. At one week, the lesions were characterized by acute hemorrhagic necrosis with patchy sub-capsular hyperemia and edema. The maximum lesion diameter for 50, 100 and 200 J/cm PDT was approximately 15, 20 and 28 mm, respectively. The lesion size is well correlated with light fluence and comparable to that in prostates treated with identical PDT doses but without prior-RT. Under light-microscopy, the PDT induced necrosis is clearly distinguishable from the radiation induced fibrosis. No urethral lesions were observed. Dyer’s Verhoeff stain showed the loss of stromal connective tissue and the acinar collagen in the PDT treated area. There was no noticeable damage on the bladder or underlying colon section. In conclusion, Tookad-PDT can effectively destroy prostate tissue with prior-RT induced fibrosis, thus, may provide an alternative modality for those prostate-cancer patients who have failed RT.

We studied the repetition-rate dependence of PDT cytotoxicity and relation between PDT cytotoxicity and both oxygen consumption and photobleaching during PDT in vitro. Mice renal carcinoma cells were incubated wtih second-generation photosensitizier, PAD-S31, and were irradiated with 670-nm nanoseconds pulsed light from YAG-OPO system. Four repetition rates of 30, 15, 5 and 3 Hz were investigated, provided that the incident peak intensity and the total light dose adjusted to 1.2 MW/cm2 and 40 J/cm2, respectively. We found limited cytotoxicity about 40% at 30 and 15 Hz and sufficient cytotoxicity about 80% at 5 and 3 Hz. The oxygen measurement during irradiation revealed that the 5- and 30Hz irradiation caused slow oxygen consumption, while rapid oxygen consumption followed by a rapid recovery of oxygenation at 30 and 15 Hz. Interestingly, the fluorescence measurement during irradiation also demonstrated that photobleaching discontinued in the same period of oxygen recovery at 30 and 15 Hz. These discontinued oxygen consumption and photobleaching at 30 and 15 Hz might have limited effective total fluence and resulted in suppressed cytotoxicity. These results suggest that the PDT efficacy using a pulsed laser significantly depends on the pulse repetition rate probably due to different oxygen consumption process during PDT.

Changes in blood flow and oxygenation during and after PDT provide information about tumor vessel and cellular damage. The characterization of these changes may improve our understanding of PDT mechanisms and help predict treatment efficacy. We have designed a hybrid system that can non-invasively measure in vivo hemodynamic changes and provide independent information about tumor oxygenation and blood flow. Diffuse correlation spectroscopy (DCS) monitors blood flow by measuring the optical phase shifts caused by moving blood cells, while diffuse photon density wave (DPDW) spectroscopy measures tissue absorption and scattering. When mounted on a camera, our unique probe allows non-contact measurements that avoid compressing the tumor and altering blood flow. An optical filter mounted in front of the camera lens cut off light below 650nm, which allowed monitoring of blood flow during PDT. The utility of the hybrid system was demonstrated by monitoring the hemodynamic changes during and after PDT in mice bearing the experimental radiation-induced fibrosarcoma (RIF). For the first time, we non-invasively and continually monitored the in vivo flow changes during PDT. Relative oxygen consumption was calculated using flow values measured by DCS and oxygenation measured by a broadband absorption spectrometer. During PDT an initial rapid increase in blood flow was found, followed by a decrease and slow recovery. After PDT, substantial and continued reductions in blood saturation, blood flow and oxygen consumption were found after 3 hours, suggesting that permanent damage to tumor cells and blood vessels had occurred. The comparison of flow values after PDT as measured by DCS and by Power Doppler ultrasound (CWFA) demonstrated that both techniques non-invasively detected similar global changes in tumor blood flow or perfusion after PDT.

In this paper we present results from experiments to develop a real-time, optical monitor for singlet molecular oxygen produced during photodynamic therapy. Using a pulsed diode laser and a sensitive photomultiplier tube, we have obtained signals from singlet oxygen during and following pulsed laser excitation. Several photosensitizers were used, and we obtained strong signals even in the presence of protein laden environments. Values obtained for the lifetimes of the singlet oxygen state and the photosensitizer triplet state are compared to literature values.

Photodynamic Therapy (PDT) using photosensitizer Photosense (PS) in dose 0.5 mg per kg of body weight have been provided in 24 patients with breast cancer. In 22 patients with T1-T2N0M0 primary tumor was treated as the preoperative treatment, radical mastectomy has been fulfilled 7-10 days after PDT with subsequent histological examination. 2 patients had recurrencies of breast cancer with lymph node metastases after radiotherapy. Fluorescent diagnostics of tumor, accumulation of PS in tumor, adjacent tissue, skin before and during PDT was fulfilled with spectranalyzer LESA-01. We used semiconductive laser for PDT - λ = 672+2nm, P=1,5 W, interstitial irradiation 2-24 hours after PS injection has been done in light dose 150-200 J/cm3, 1-3 irradiations with interval 24-48 hours and total light dose 400-600 J/cm3 depending mostly of size and fluorescent data. Partial regression of tumor with pathomorphosis of 2-4 degrees has been found in 19 cases. Our experience shows pronounced efficacy of PDT for treating breast cancer as preoperative modality and as palliation in cases of recurrencies.

Porphycenes are currently under investigation for use in Photodynamic therapy, which is a promising treatment for cancer. These materials, which display preferential uptake in cancerous cells, also exhibit high fluorescence yields, and can be used for tumour detection. Problems with steady-state fluorescence techniques such as background autofluorescence can be eliminated by the use of time-resolved techniques. Improved contrast can be obtained with time-resolved techniques because of the differing fluorescence lifetimes between autofluorescence and longer-living exogenous photosensitisers. An imaging system was constructed using a fast (200 ps) gated CCD camera and a pulsed 635 nm laser diode. A tissue phantom composed of polymethyl methacrylate (PMMA) with thirty-six wells of varying diameter and depth (10 mm to 1 mm) was assembled to test the system. The system was used to record images of a porphycene derivative within the wells at differing concentrations in an organic solvent. A tissue imitator was placed on top of the PMMA block at varying thickness. 10-4 M zinc phthalocyanine tetrasulfonate was also placed on top of the block to mimic autofluorescence. The results indicate that the time-gated imaging system can prevent background excitation scatter and fluorescence from a shorter-lived fluorophore from distorting the fluorescence signal from a longer-lived photosensitiser.

To investigate the spectral specialities of stomach cancer serum for diagnosis, fluorescence and Raman spectra of normal, stomach cancer, esophagus cancer and atophic gastritis sera were measured in the visible region in this study. All spectra except esophagus cancer were characterized by three sharp peaks. The intensity of each peak was different in different spectrum. After sampels were radiated by laser, fluorescence weakened along wiht red shift of its band center, and spectral changes of normal and stomach cancer cases were different from other samples. It was also observed that spectral changes of atophic gastritis were very similar with stomach cancer after radiated by laser, however, there are still some distinctions that can be used to differentiate them from each other. A notable difference is that the relative intensity of peak C excited by 488.0nm is higher than excited by 514.5nm in spectrum of stomach cancer, whereas lower in other cases. We utilized it as a criterion and got an accuracy of 80.77% in stomach cancer detection.

Evaluating the light distribution in the esophagus is central to understanding how to optimize treatment of Barrett's esophagus with PDT. The light distribution of a cylindrical light diffuser in the esophagus was investigated by Monte Carlo simulations and experiments in esophageal simulating phantoms. Simulation of light transport in the esophagus requires knowledge of several biological parameters including radius, height, mucosal wall thickness and optical parameters such as reduced scattering coefficient, absorption coefficient, scattering anisotropy and refractive index. Results of Monte Carlo simulations were tested by measuring the light fluence rate in an esophagus phantom (absorption coefficient = 0.27 mm^-1, reduced scattering coefficient = 1.9 mm^-1, g = 0.9, n= 1.37) irradiated at 633nm with a 5cm long cylindrical light diffuser. We evaluated the light distribution within the esophagus for the on-axis and off-axis treatment fiber conditions both experimentally and mathematically. Results showed the reflected light from the walls of the esophagus increased the incident light dose at all points being treated by a factor of 1.6, compared to the dose given off by the fiber itself. Also, when the fiber was moved from one side of the esophagus to the other, it increased the light dose to the proximal area by a factor of 2, and decreased the light dose to the distal region by a factor of 2 in the phantom study, and in Monte Carlo simulation, factor of 18 and factor of 3 respectively.

The aim of the present work is to analyze the histological changes on hamster buccal mucosa caused by the topical use of 7,12-dimethylbenzanthracene (DMBA) and exposition to a 220 µJ/pulse nitrogen laser light (@ 337 nm) at an average power of 2,3 mW. Twenty-one hamsters divided into two experimental groups were treated six times with DMBA. One hamster was kept as control. Group I was composed by ten hamsters and was submitted only to DMBA. Group II, also with ten hamsters, received the same treatment as group I and was exposed to the laser radiation. The time duration of each irradiation section was 10 seconds. All the treatment happened in alternated days. The histological analysis took place twice, after the end of the treatment and after sixty days. Both experimental groups presented dilatation of vessels, thickening of the epithelial tissue and the presence of inflammatory infiltrates. The preliminary results indicates that in group II the number of dilated vessels and its new area are much more significant than in group I.

Tumor hypoxia, either pre-existing or as a result of oxygen bleaching during Photodynamic Therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT induced cell killing. To overcome the effect of tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin PDT. Our previous study has demonstrated that, in an in vivo model, tumor control can be improved by normobaric or hyperbaric 100% oxygen supply. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combined therapy is investigated in this study by using an in vivo/in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma (MCA) in hind legs of C3H mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air or carbogen or 100% normobaric or hyperbaric 100% oxygen), was delivered through an optical fiber with a microlens to animals who received 12.5 mg/kg Photofrin 24 hours prior to light irradiation. Tumors treated with PDT were harvested and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that, when combined with hyperoxygenation, the cell killing rate immediately after a PDT treatment is significantly improved over that treated without hyperoxygenation, suggesting an enhanced direct cell killing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation, it can be more effective in controlling hypoxic tumors. H&E stain revealed that PDT induced tumor necrosis and hemorrhage. In conclusion, by using an in vivo/in vitro assay, we have shown that PDT combined with hyper-oxygenation can enhance direct cell killing and improve tumor cure.

Tumor hypoxia, either pre-existing or as a result of oxygen bleaching during Photodynamic Therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT induced cell killing. To overcome the effect of tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin PDT. Our previous study has demonstrated that, in an in vivo model, tumor control can be improved by normobaric or hyperbaric 100% oxygen supply. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combined therapy is investigated in this study by using an in vivo/in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma (MCA) in hind legs of C3H mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air or carbogen or 100% normobaric or hyperbaric 100% oxygen), was delivered through an optical fiber with a microlens to animals who received 12.5 mg/kg Photofrin 24 hours prior to light irradiation. Tumors treated with PDT were harvested and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that, when combined with hyperoxygenation, the cell killing rate immediately after a PDT treatment is significantly improved over that treated without hyperoxygenation, suggesting an enhanced direct cell killing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation, it can be more effective in controlling hypoxic tumors. H&E stain revealed that PDT induced tumor necrosis and hemorrhage. In conclusion, by using an in vivo/in vitro assay, we have shown that PDT combined with hyper-oxygenation can enhance direct cell killing and improve tumor cure.

The technique and the device for studying of phototoxic properties of photosensitizers in vitro on cell monolayers in 96-well microplate was developed. It allows to irradiate independently each well of microtiter plate, and study simultaneously all points of dependence of phototoxic effect on the light dose for certain conditions of investigation. Also it allows to study and compare at the same conditions the dependence of phototoxic effect on the light dose for different concentrations of photosensitizer or for several photosensitizers. Developed device includes powerful source of light based arc xenon lamp with elliptical reflector, special filters and multi-fiber bundle. To determine the degree of the cell viability under the photodynamic treatment proliferative test with fluoresceinediacetate was used. Using this device and technique phototoxic action of a number of different derivatives of aluminium phthalocyanines was studied. The results of this screening have a good correlation with the results obtained in vivo on mice.

Photodynamic Therapy (PDT) is a promising modality for tumor treatment that combines a photosensitizing agent and visible light resulting in the production of cytotoxic reactive oxygen species leading to cell death. Bioluminescence detection/imaging is a noninvasive technique that uses luciferase gene transfection together with administration of luciferin to generate detectable visible light. It can provide real-time assessment of tumor growth and therapeutic response. The aim of this study is to investigate the potential fo bioluminescence following animolevulinic acid (ALA)-mediated PDT. The in vitro results show a decrease of luminescence, with an excellent correlation to the number of viable cells. In vivo, the tumor growth was monitored using a cooled CCD camera, and ALA-PDT was performed 7-10 days post tumor implantation. The results show a decrease of the bioluminescence signal from the tumor that corresponds to a decrease of viable cells within the tumor, followed by re-growth at the sub-curative PDT doses used.