Search

Background and Objectives: Obstructive non-small cell lung cancer and obstructive esophageal cancer are US FDA approved indications of photodynamic therapy (PDT). The usefulness of PDT for the treatment of cholangiocarcinoma is currently under clinical investigation. Endoscopic stenting for lumen restoration is a common palliative intervention for those indications. It is important to assess whether self-expandable metal stents are compatible with trans-stent PDT light delivery., Study Design/materials and Methods: Direct effects of various components of metal biliary (n = 2), esophageal (n = 2), and bronchial (n = 1) stents on PDT light transmittance and distribution were examined using a point or linear light source (630 or 652 nm diode laser). Resected pig biliary duct and esophageal wall tissues were used to examine the feasibility of PDT light delivery through the fully expanded metal stents., Results: While using a point light source, the metal components (thread and joint) of the stent could cause a significant shadow effect. The liner material (polytetrafluoroethylene or polyurethane) could cause various degrees of light absorption. When the stent was covered with a thin layer of biliary duct and esophageal tissues containing all wall layers, the shadow effect could be mitigated due to tissue scattering., Conclusions: This study clearly demonstrates that it is feasible to combine stenting and PDT for the treatment of luminal lesions. PDT light dose should be adjusted to counteract the reduction of light transmittance caused by the metal and liner materials of stent., ((c) 2008 Wiley-Liss, Inc.)

Photodynamic therapy (PDT) mediated with vascular acting photosensitizer Tookad (Pd-bacteriopheophorbide) was investigated as an alternative modality for treating prostate cancer. Photodynamic effects on the prostate gland and its adjacent tissues were evaluated in a canine model. Interstitial prostate PDT was performed by irradiating individual lobes with a cylindrical diffuser fiber at various drug/light doses. The sensitivity of the adjacent tissues to Tookad PDT was determined by directly irradiating the surface of the bladder, colon, abdominal muscle and pelvic plexus with a microlens fiber at various drug/light doses. The prostate and adjacent tissues were harvested one-week after the treatment and subjected to histopathological examination. PDT-induced prostate lesions were characterized by marked hemorrhagic necrosis. The bladder, colon, abdominal muscle and pelvic plexus appeared to be sensitive to PDT although the Tookad PDT-induced responses in these tissues were minimal compared to that of the prostate gland at the same dose levels. Nevertheless, the protection of the adjacent tissues should be taken into consideration during the total prostate ablation process due to their sensitivity to PDT. The sensitivity of the prostatic urethra is worth further investigation. Direct intraurethral irradiation might provide an ideal means to determine the sensitivity of the prostatic urethra and might lead to transurethral PDT protocols for the management of benign prostatic hyperplasia (BHP).

Background and Objective: To determine the optimal magnetic resonance imaging (MRI) methodology to assess photodynamic therapy (PDT)-induced histopathological responses in the prostate., Study Design/materials and Methods: Laparotomy was performed in five healthy dogs. Cylindrical diffuser was placed in the prostates to deliver light of 50-300 J/cm at 150 mW/cm and 763 nm to activate IV-injected Tookad (1 mg/kg b.w.). Fast spin echo (FSE) T2-weighted, post-contrast-enhanced T1-(CE-T1) and diffusion weighted images (DWI) were obtained pre- and 2 days, 7 days, and 1 month post-PDT. Radiological-histopathological correlation was performed at 7 days (n = 4) and 1 month (n = 1) after PDT. A qualitative assessment of signal changes and apparent diffusion coefficient (ADC) mapping was performed., Results: At 2 or 7 days post-PDT, there was good spatial correlation between PDT-induced hemorrhagic necrosis and unenhanced regions on CE-T1 images. There was a rapidly and persistently enhancing rim corresponding to edema and inflammation. FSE T2 and DWI showed altered signal but did not clearly define necrosis in all cases. At 1 month, it was hard to correlate MR images to histopathologic changes as they represented a mixture of necrosis and developing fibrosis, which led to a mixed signal intensity and less demarcated contrast enhancement., Conclusions: At 7 days after PDT, gadolinium DTPA contrast-enhanced MRI is superior to DWI and T2 imaging in assessing the boundary of Tookad PDT-induced tissue necrosis in the normal canine prostate., (Copyright 2006 Wiley-Liss, Inc.)

Background and Objectives: Photodynamic therapy (PDT) mediated with Tookad (Pd-bacteriopheophorbide, WST09) was investigated pre-clinically as part of a program to develop an alternative modality for treating prostate cancer., Study Design/materials and Methods: Spontaneous canine prostate cancer and normal canine prostate were used as the animal models. Interstitial PDT was performed by IV infusion of the photosensitizer and irradiating the prostates with a diode laser (763 nm). The prostates were harvested 1-week post-PDT and subjected to histopathologic examinations. The effects of the drug doses and light doses were studied for one- and two-session PDT. Pharmacokinetics were studied using HPLC assay. The feasibility of using perfusing CT scans for assessing PDT lesions was also evaluated., Results: Tookad is a vascular-acting drug and clears rapidly from the circulation. Tookad-PDT-induced lesions, in both normal and cancerous prostates, were characterized by marked hemorrhagic necrosis., Conclusions: Tookad-PDT is very effective in ablating prostatic tissue through its vascular effects., (Copyright 2005 Wiley-Liss, Inc.)

Objective: Our aim is to investigate the effects of photodynamic therapy (PDT) on peripheral nerve conductivity., Background Data: Interstitial PDT has been demonstrated as a promising treatment modality for prostate cancer. However, the sensitivity of nerves, in the immediate vicinity of the prostate gland, to PDT procedures has not been studied. This study attempts to establish an in situ canine model to evaluate direct PDT effect on peripheral nerves., Methods: PDT was performed by irradiating the cutaneous branches of the saphenous nerve at 763 nm with light doses of 50-200 J/cm2 after i.v. infusion of the photosensitizer Tookad (0-2 mg/kg). Evoked compound-action potentials (CAP) were recorded directly from the surface of the saphenous nerve. The latencies to onset and conduction velocities were determined during PDT and 1-week post-PDT., Results: Nerve and surrounding tissue damage corresponded well with drug/light doses. With Tookad doses of 2 mg/kg, treatment with 50 J/cm2 induced little change in saphenous nerve conduction properties. However, treatment with 100 J/cm2 resulted in localized nerve injury and decreases in nerve conduction velocities, and treatment with 200 J/cm2 severely damaged the nerve., Conclusions: This canine model adequately demonstrates effects of Tookad PDT on peripheral nerves. Direct irradiation of 100-200 J/cm2 can alter nerve conduction and induce nerve damage. Therefore, possible side effects of interstitial PDT on the pelvic plexus need to be investigated in future studies.

The aim of this study was to evaluate the effects of photodynamic therapy (PDT) using a novel palladium bacteriopherophorbide photosensitizer TOOKAD (WST09) on canine prostate that had been pretreated with ionizing radiation. To produce a physiological and anatomical environment in canine prostate similar to that in patients for whom radiotherapy has failed, canine prostates (n = 4) were exposed to ionizing radiation (54 Gy) 5 to 6 months prior to interstitial TOOKAD-mediated PDT. Light irradiation (763 nm, 50-200 J/cm at 150 mW/cm from a 1-cm cylindrical diffusing fiber) was delivered during intravenous infusion of TOOKAD at 2 mg/kg over 10 min. Interstitial measurements of tissue oxygen profile (pO(2)) and of local light fluence rate were also measured. The prostates were harvested for histological examination 1 week after PDT. The baseline pO(2) of preirradiated prostate was in the range 10-44 mmHg. The changes in relative light fluence rate during PDT ranged from 12 to 43%. The acute lesions were characterized by hemorrhagic necrosis, clearly distinguishable from the radiotherapy-induced pre-existing fibrosis. The lesion size was correlated with light fluence and comparable to that in unirradiated prostate treated with a similar TOOKAD-PDT protocol. There was no noticeable damage to the urethra, bladder or adjacent colon. The preliminary results obtained from a small number of animals indicate that TOOKAD-PDT can effectively ablate prostate pretreated with ionizing radiation, and so it may provide an alternative modality for those prostate cancer patients for whom radiotherapy has failed.

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. 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.

Photodynamic therapy (PDT) uses light to activate a photosensitizer to achieve localized tumor control. In this study, PDT mediated by a second-generation photosensitizer, palladium-bacteriopheophorbide WST09 (Tookad) was investigated as an alternative therapy for prostate cancer. Normal canine prostate was used as the animal model. PDT was performed by irradiating the surgically exposed prostate superficially or interstitially at 763 nm to different total fluences (100 or 200 J/cm2; 50, 100 or 200 J/cm) at 5 or 15 min after intravenous administration of the drug (2 mg/kg). Areas on the bladder and colon were also irradiated. The local light fluence rate and temperature were monitored by interstitial probes in the prostate. All animals recovered well, without urethral complications. During the 1 week to 3 month post-treatment period, the prostates were harvested for histopathological examination. The PDT-induced lesions showed uniform hemorrhagic necrosis and atrophy, were well delineated from the adjacent normal tissue and increased linearly in diameter with the logarithm of the delivered light fluence. A maximum PDT-induced lesion size of over 3 cm diameter could be achieved with a single interstitial treatment. There was no damage to the bladder or rectum caused by scattered light from the prostate. The bladder and rectum were also directly irradiated with PDT. At 80 J/cm2, a full-depth necrosis was observed but resulted in no perforation. At 40 J/cm2, PDT produced minimal damage to the bladder or rectum. On the basis of optical dosimetry, we have estimated that 20 J/cm2 is the fluence required to produce prostatic necrosis. Thus, the normal structure adjacent to the prostate can be safely preserved with careful dosimetry. At therapeutic PDT levels, there was no structural or functional urethral damage even when the urethra was within the treated region. Hence, Tookad-PDT appears to be a promising candidate for prostate ablation in patients with recurrent, or possibly even primary, prostate cancer.

Photodynamic therapy (PDT) requires molecular oxygen during light irradiation to generate reactive oxygen species. Tumor hypoxia, either preexisting or induced by PDT, can severely hamper the effectiveness of PDT. Lowering the light irradiation dose rate or fractionating a light dose may improve cell kill of PDT-induced hypoxic cells but will have no effect on preexisting hypoxic cells. In this study hyperoxygenation technique was used during PDT to overcome hypoxia. C3H mice with transplanted mammary carcinoma tumors were injected with 12.5 mg/kg Photofrin and irradiated with 630 nm laser light 24 h later. Tumor oxygenation was manipulated by subjecting the animals to 3 atp (atmospheric pressure) hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyperoxygenation. With hyperoxygenation up to 80% of treated tumors showed no regrowth after 60 days. In comparison, when animals breathed room air, only 20% of treated tumors did not regrow. To explore the effect of hyperoxygenation on tumor oxygenation, tumor partial oxygen pressure was measured with microelectrodes positioned in preexisting hypoxic regions before and during the PDT. The results show that hyperoxygenation may oxygenate preexisting hypoxic cells and compensate for oxygen depletion induced by PDT light irradiation. In conclusion, hyperoxygenation may provide effective ways to improve PDT efficiency by oxygenating both preexisting and treatment-induced cell hypoxia.

Background and Objectives: Currently there is no safe, effective, and rapid means to eliminate the pain associated with a needle insertion through the skin. It is hypothesized that ablation of the stratum corneum layer using a low energy Erbium(Er):YAG laser would allow rapid local anesthesia from a lidocaine product., Study Design/materials and Methods: Eighty volunteers participated in a placebo-controlled, double blind, cross-over study employing the Norwood-Abbey (Chelsea Heights, Victoria, Australia) laser anesthesia device (LAD) and two lidocaine preparations. Upper-arm skin ablation was followed by a 5-minute application of study treatment. Pain scores were registered immediately following a needle insertion., Results: Comparing the combined lidocaine preparations to placebo, there was a statistically significant reduction in pain when the LAD was employed (P < 0.001). The median pain reduction for lidocaine was 51.3% (95% CI = [40.9, 76.1])., Conclusions: Use of the low energy Er:YAG LAD device in combination with a 5-minute application of lidocaine significantly reduced the pain associated with a needle insertion., (Copyright 2002 Wiley-Liss, Inc.)

Background: Previous reports suggest that ischemic conditions rapidly reduce the capacity of human albumin to bind exogenous cobalt. A new assay based on human albumin-cobalt binding (ACB) may help detect early myocardial ischemia. We investigated altered ACB during the first 24 hours after transient ischemia induced during elective percutaneous transluminal coronary angioplasty (PTCA). We then compared ACB assay results with creatine kinase isoenzyme (CK-MB), myoglobin, and cardiac troponin I (cTn-I) values after PTCA., Methods and Results: In 41 patients undergoing elective PTCA, plasma samples were tested for the ACB assay, CK-MB, myoglobin, and cTn-I before, immediately after, and 6 and 24 hours after PTCA. Thirteen additional patients served as a control group with albumin-cobalt assays performed before and after diagnostic coronary catheterization without angioplasty. ACB assay results demonstrated a significant mean percent difference (10.1%) immediately after PTCA compared with baseline (P < .000001) and returned to baseline by 6 hours after PTCA. ACB assay differences immediately after PTCA were significantly greater than in the control group (10.1% vs -0.9%, P < .001). Mean CK-MB, myoglobin, and cTn-I values were not elevated above baseline immediately after PTCA but were significantly elevated above baseline 6 and 24 hours after PTCA., Conclusions: These preliminary results suggest that human albumin undergoes a significant reduction in its capacity to bind exogenous cobalt soon after transient coronary occlusion during human PTCA and before significant elevations of CK-MB, myoglobin, or cTn-I. Further confirmatory investigations are warranted to determine if the ACB assay is a useful diagnostic test for early myocardial ischemia.

Objective: To review the currently available data of photodynamic therapy (PDT) optical dosimetry for possible prostatic applications., Summary Background Data: PDT is a new cancer treatment modality often used as an alternative tumor treatment method. Recently, PDT has been suggested as an alternative therapy for prostatic carcinoma and BPH., Methods: PDT: utilizes light and a preadministered photosensitizer drug to achieve localized tumor control. This article reviews currently available data on optical dosimetry of PDT in both human and canine prostates., Results: At 630 nm, a common wavelength used for Photofrin PDT, results indicate that light penetration is similar in cancerous and normal prostatic tissue. Because of limited light penetration, multiple fiber irradiation is necessary if eradicating the entire prostate glad is the ultimate goal. The available data also show that dynamic changes occur in light fluence rate distribution during PDT irradiation., Conclusions: PDT can be used to destroy prostatic tissue. Real-time optical dosimetry is necessary if accurate lesion volume control is desired.

Objective: To determine the depth of tissue destruction and the minimum light dose required for necrosis in interstitial photodynamic therapy (PDT), as a prerequisite for the investigational therapy of patients., Materials and Methods: Seven adult beagle dogs were given 2 mg/kg of the photosensitizer Photofrin intravenously and two controls received none. After 24 h, 450 J/cm of 630 nm wavelength laser light was delivered interstitially to the prostate via a 2 cm long diffuser fibre. Seven single-fibre treatments were performed in five sensitized dogs and two single-fibre treatments in the controls. The two remaining sensitized dogs had two fibres placed 10 mm apart within the prostate to determine the coalescence of PDT lesions. The penetration depth of light was measured in all prostates, and after PDT the extent of necrosis was assessed histologically., Results: The mean (standard deviation, SD) radius of PDT destruction around each diffuser was 5.3 (1.4) mm and PDT lesions overlapped in prostates treated with two fibres placed 10 mm apart. There was no observable tissue damage in the controls. The mean (SD) minimum light dose required for PDT necrosis was 84 (64) J/cm2. Differences among animals in the light penetration depth were small, with a mean of 2.14 (0.2) mm, and did not correlate with the depth of necrosis (P = 0.07). Bleeding around the optical diffuser fibre impeded light penetration., Conclusion: Interstitial PDT in the canine prostate using Photofrin produced modest volumes of tissue necrosis. The minimum light dose required to induce necrosis was variable because bleeding was unpredictable in relation to the optical fibre.

Background: This study was designed to investigate the relationships among primary tumor size, lymphatic vessel diameters, the incidence of sentinel lymph node (SLN) metastasis and lymphatic clearance from murine footpad melanomas., Methods: Lymphatic clearance (LC) of [99mTc]HSA from the middle of the footpad of syngeneic C57BL/6 mice, with or without primary melanomas (sizes varying from 1 to 5 mm in anteroposterior diameter), was quantitated using a gamma scintillation detection system. Lymphatic vessel diameters (LD) were measured after injection of aniline blue dye into footpad tumors. The incidence of SLN, femoral lymph node (FLN), and lung metastases was recorded., Results: Metastasis to SLNs increased as tumor growth progressed (r = 0.976, p = 0.001), and there was a correlation between tumor size and both FLN (p = 0.041) and lung (p = 0.055) metastases. There was also a correlation between lymph node metastasis and LC (r = 0.83, p = 0.04) and LD (r = 0.84, p = 0.04)., Conclusions: These studies support the hypothesis that lymph flow and LD is increased in experimental murine melanomas and this relates to both primary tumor size and to lymphatic and hematogenous metastasis.

Objectives: To determine the clearance of a radionuclide from various sizes of footpad melanomas via lymphatics and to measure the diameters of these vessels., Design: Nonrandomized animal study., Setting: A hospital research laboratory., Subjects: C57BL/6 mice., Interventions: Female mice were injected in the right rear footpad with B16 F10 cells that were allowed to grow to either 1, 2, 3, 4, or 5 mm in anteroposterior diameter. Clearance from feet with or without tumors of injected technetium Tc 99m human serum albumin (99mTcHSA) was assessed for 200 minutes. Calf lymphatic diameters were measured using aniline blue dye., Results: The clearance of the injected 99mTcHSA from mouse footpads without tumors was 1.26 +/- 0.18 x 10(-4) mL/min x cm3 of tissue. Clearance increased 2.24-fold to 2.82 +/- 0.12 x 10(-4) mL/min x cm3 of tissue from 1-mm tumors and to 6.20 +/- 0.08, 6.11 +/- 0.13, 6.91 +/- 0.58, and 7.23 +/- 0.48 x 10(-4) mL/min x cm3 of tissue from 2-, 3-, 4-, and 5-mm tumors, respectively (P < .05). Calf lymphatic diameters increased from 75.41 +/- 9.72 microns in naive nontumor-bearing mice to 93.51 +/- 7.12, 111.61 +/- 27.07, 126.69 +/- 25.20, 124.43 +/- 24.75, and 127.44 +/- 25.35 microns in mice bearing 1-, 2-, 3-, 4-, and 5-mm tumors, respectively (P < .01)., Conclusions: There was a size-dependent, direct correlation between increasing tumor size in the footpad and increasing diameter of lymphatics draining the footpad. Clearance of injected 99mTcHSA from these tumors also exhibited a similar positive correlation with tumor size.

The optical absorption and transport scattering coefficients of normal prostate tissue have been measured in vivo in dogs. The measurements were made at 630 nm before and during treatment by Photofin photodynamic therapy using interstitial optical fiber fluence-rate detectors. Corresponding measurements were made ex vivo, at 1 week after treatment, in the contralateral lobe. The optical properties were derived by applying a diffusion theory model to the fluence rates measured at two different source-detector fiber distances. While the in vivo pretreatment and in vivo contralateral post-treatment absorption and scattering values are self-consistent and in agreement with published data, significant changes were observed in the light fluence rates, and hence in the derived optical properties, during light irradiation. The possible causes of such changes are considered, and the implications for light dosimetry in photodynamic therapy are discussed.

Photodynamic therapy (PDT) and hyperthermia are two alternative tumor treatment modalities currently being investigated in clinical trials. It has been suggested that, due to the differences in cell-killing mechanisms, synergetic tumor responses may be achieved if the two modalities are combined in appropriate sequences. This hypothesis is tested in the current study by delivering graded PDT doses during a transient tumor reoxygenation period after a hyperthermia treatment, or delivering graded hyperthermia doses when the tumor becomes acidic and hypoxic after a PDT treatment. The results indicate that the latter combination sequence has a profound effect on tumor response. While treating the tumors with PDT followed by hyperthermia evokes a synergetic tumor response, reversing the sequence results only in an additive effect. Possible mechanisms associated with tissue oxygenation are discussed.

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10(16) photons/cm3. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.

Tumor oxygenation after a photodynamic therapy (PDT) treatment is a critical factor for understanding the posttreatment metabolic pathway of the tumor. It also provides important information for designing combination therapy of PDT and other oxygen-dependent anticancer modalities. In this study, mammary carcinoma in flank and hind leg of C3H mice were subjected to PDT at either subcurative or curative level (12.5 mg/kg Photofrin; 200 or 600 J/cm2, respectively). The before and post-PDT tumor oxygenation was measured with an oxygen-sensitive microelectrode. The data revealed that tumor oxygenation at the time of PDT has a profound effect on posttreatment tumor oxygenation, which may largely be due to an interplay between direct PDT cytotoxicity and PDT damage to the tumor microvasculature. Transient reoxygenation occurred after PDT, which may provide a window for improved combination therapy for other oxygen-dependent modalities.

Purpose: To determine if prior tumor irradiation influences tumor pO2 changes in mice breathing oxygen (100%) at normal and elevated pressure., Methods and Materials: Single-point pO2 measurements were performed in nonirradiated and previously irradiated (72 h) isotransplanted MCaIV tumors in C3H/Sed mice. Continuous recordings were performed at the same tumor locus under air breathing, followed by 100% oxygen and oxygen at three atmospheres pressure. Following decompression and induction of pentobarbital anesthesia, the procedure was repeated at the same locus. Six nonirradiated and five irradiated tumors were evaluated under the three gas breathing conditions +/- anesthesia., Results: The mean, median, and range of pO2 values did not differ under air-breathing conditions in the nonirradiated vs. previously irradiated tumors. However, prior irradiation substantially enhanced the tumor pO2 increase when the inspired gas phase was switched from air to 100% oxygen at 1 or 3 atmospheres pressure. In four of six nonirradiated tumors, 100% oxygen breathing resulted in a pO2 increase of < 4 mmHg; in the irradiated tumors, the minimum increase was 16 mmHg. Pentobarbital anesthesia did not significantly influence the results obtained., Conclusion: These data indicate that the efficacy of oxygen breathing increases during tumor treatment, and suggests that oxygen breathing is a simple nontoxic method for reducing or eliminating radiobiologic hypoxia during therapy.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.

Photodynamic therapy has been used in the management of patients with malignant brain tumors even though the effects of this form of treatment on the adjacent normal brain are incompletely characterized. The authors examined, in sequential experiments, morphologic alterations affecting the cerebral cortex in rats injected with Photophrin II and exposed to light. Initially, minimal cell alterations, including cisternal swelling of both endoplasmic reticulum and Golgi apparatus, involved only neurons located in the superficial layers of the cerebral cortex exposed to light. These changes spread, over a period of several hours, from the surface to the bottom of the cortex and eventually involved the entire cortical segment exposed to light. The earliest structural signs of lethal injury to neurons developed over a period of 18 hours after porphyrins had been photoactivated and astrocytes had been severely damaged. Signs of lethal injury to neurons included an increase in the number of mitochondrial cristae and appearance of amorphous electron-dense deposits within swollen mitochondria. The appearance of these alterations was followed by segregation of intracytoplasmic organelles and fragmentation of nuclear and cytoplasmic membranes. The tissue changes, including those involving neurons, eventually progressed to coagulation necrosis at 48 hours. These observations suggest that prophyrins injected to rats (48 hours before photoactivation) cause swelling and necrosis of astrocytes. This is followed by neuronal necrosis, which appears at two time intervals; the initial neuronal necrosis occurs after the astrocytic disintegration. A second type of neuronal alteration appears after microvessels become thrombosed and ischemia is likely to develop.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.

The light fluence distributions of 632.8 nm light incident on the exposed surface of normal rat brain in vivo have been measured using an interstitial, stereotactically-mounted optical fiber detector with isotropic response. The dependence of the relative fluence rate on depth and the spatial distribution of fluence were compared for incident beam diameters of 3 and 5 mm. The fluence rate at depth of 1-6 mm along the optical axis within the brain tissue was approximately 70% greater for a 5 mm diameter beam than for a 3 mm beam, at the same incident fluence rate, although the plots of the relative fluence rate vs depth were parallel over the depth range 1-6 mm. The depths of necrosis resulting from photodynamic treatment of brain tissue using the photosensitizer Photofrin and irradiation by 632 nm light with 3 and 5 mm incident beams were also measured. The observed difference in necrosis depths was consistent with the measured difference in fluence. The importance of beam size in photodynamic treatment with small diameter incident light fields is discussed.

It has been clearly established that changes in intratumor pO2 and pH occur following hyperthermia, and it has been hypothesized that these changes may, in some way, be related to the ultimate response (i.e., cure) of the lesion. The purpose of this study was twofold: first, to examine the changes in intratumor pH during the course of a hyperthermia treatment at biologically related end point "doses"; second, to examine the response of pO2 after treatment in a different lesion transplant site. During hyperthermia treatment of the tumor transplanted in the leg, intratumor pH was found to drop from a control value of 6.74 +/- 0.17 to 6.47 +/- 0.13 within 15 min following the start of treatment. The values then remained relatively constant throughout the remainder of the treatment (either 1 or 2 h at 43.5 degrees C). Following the subcurative (10% tumor cures at 30 days; 60 min at 43.5 degrees C) treatment the pH began to rise immediately, while after the higher dose (60% tumor cures at 30 days; 120 min at 43.5 degrees C) a slight rise in pH was followed by a continuous drop in pH for up to 4 h, as we have reported previously. Oxygen response in the two transplant sites (leg and flank) was found to be remarkably different even though the tumor cure rate was identical for a given hyperthermia "dose" in terms of time and temperature. In the leg, only very low levels of oxygen can be measured in the tumor 24 h after treatment with either "dose" studied (all measured pO2 values less than or equal to 5 mm Hg). In the flank, the tumor response is dependent on hyperthermia "dose." Only 28% of measured oxygen values are less than or equal to 5 mm Hg 24 h following a subcurative "dose," while 4 h following the higher "dose" there is a nonsignificant trend toward hypoxia (approximately 65% of values less than or equal to 5 mm Hg) with a subsequent shift toward reoxygenation. These latter observations are contrary to results reported previously and tend to contradict some current theories regarding the physiological mechanisms associated with hyperthermia treatment.

Light activation of circulating hematoporphyrin derivatives has been used in the treatment of selected brain tumors. The effects of this photodynamic therapy on the non-neoplastic, adjacent brain tissue are incompletely characterized. We studied in adult Fisher rats the time-dependent (1 hour to 7 days) effects of photoactivated Photofrin II. Our protocol was comparable to that used in the treatment of human brain tumors. Structural and functional changes spread from the treatment surface and from the center to the periphery to involve the entire cerebral cortex exposed under a 5 mm craniectomy. The sequential changes spreading from the surface to the deepest cortical layer involve first astrocytes (1 hour), then endothelial cells and, ultimately, neurons. Thrombi were first noted in the microvasculature after 18 hours and coagulation necrosis of the entire area at risk occurred only after 48 hours. The results suggest that the photosensitizing agent crosses the intact blood-brain barrier and enters the astrocytic compartment where it becomes cytotoxic upon light activation. A comparison between the focal brain lesions of photodynamic therapy and those induced by middle cerebral artery occlusion suggests that cell damage evolves along different paths in these two forms of brain injury.

Although the sequence and time interval effects of combined photodynamic therapy (PDT) and hyperthermia tumor treatments have been studied using survival curves, tumor regrowth, and cloning assays, the metabolic response to combined treatment measured by nuclear magnetic resonance (NMR) spectroscopy has not yet been clarified. In this study, mammary carcinoma in the flank of C3H mice was subjected to PDT (12.5 mg/kg Photofrin II, 632 +/- 1 nm at 200 J/cm2) and water bath hyperthermia (43.5 degrees C, 30 min) with no delay or 4 h delay between treatments. In vivo 31P-NMR spectroscopy was employed to measure energy metabolism and pH of the tumors before and serially after treatment for up to 1 week. The data revealed significant differences in the time course of high energy phosphate levels between treatment combinations, which may reflect the biological effectiveness of the combined treatments. Our observations indicate that 31P-NMR spectroscopy can be used to evaluate the metabolic response of tumors to treatment with combined PDT and hyperthermia.

1H Magnetic resonance imaging (MRI) was used to study the effects of photodynamic therapy (PDT) on normal rat brain (n = 5) using T1-, T2-, diffusion-, and proton density (rho)-weighted images. Rats received intraperitoneal injections of 12.5 mg/kg of Photofrin II, and 48 hours later the dural area over the frontal cortex was treated with 35 J/cm2 of light (632 +/- 1 nm). The T1-, T2-, and diffusion-weighted images revealed an evolving high contrast region of brain that corresponded to the PDT-treated area. Lesioned brain exhibited significant increases in T1 and T2 relaxation times at 1 day (P less than 0.01) and 3 days (T1, P = 0.018; T2, P less than 0.01) after treatment, compared with the contralateral equivalent volume of nonlesioned brain. Water proton diffusion coefficient (DW) in the lesioned area decreased at 1 day (P = 0.026) and increased at 3 days (P = 0.012) compared with nonlesioned brain. An increase in the proton density ratio (rho D/rho O) from PDT (rho D) versus nonlesioned side (rho O) was found 3 days after PDT treatment (P = 0.03). The data indicate that the biophysical parameters obtained from magnetic resonance imaging scans, T1, T2, DW, and proton density, can be used to monitor changes in an evolving photochemically induced lesion.

In vivo 31P NMR spectroscopy and pH microelectrodes were employed to measure the energy metabolism and pH of a mammary carcinoma in the flank of the C3H mouse before and serially up to a week after various hyperthermia treatments. Water bath hyperthermia was used to treat the tumor at 43.5 degrees C for 30 min (TCD0/30, NMR measurement only), 1 h (TCD10/30), and 2 h (TCD60/30), respectively. The data indicate that, except at 4 h after TCD60/30 treatment, all pH values measured by NMR (pHn) were significantly higher (P less than or equal to 0.001) compared to pH values measured by microelectrodes (pHe) at all treatment levels and times. The magnitude of the difference between pHn and pHe (delta pH) was significantly decreased from the pretreatment level only at 4 h after hyperthermia treatment (0.51 pH units for TCD60/30 and 0.21 pH units for TCD10/30). The ratio of beta-nucleoside triphosphate to inorganic phosphate (beta-NTP/Pi) and pHn were more sensitive to hyperthermia treatment than pHe. The beta-NTP/Pi ratio failed to recover to the pretreatment ratio after 1 or 2 h hyperthermia treatment, while a total recovery was observed within 72 h for 30 min hyperthermia treatment. Our data suggest that the temporal profile of beta-NTP/Pi, pHn, and delta pH may be indicative of the biological outcome of hyperthermia treatment.

The response of normal brain to photodynamic therapy (PDT) was investigated in 62 Fisher rats. The animals were injected i.p. with Photofrin II (12.5 mg/kg). Forty-eight hours following injection, an area of dura 5 mm in diameter over the frontal cortex was photoactivated with red light (632 +/- 2 nm) at 100 mW cm-2, with no contributing thermal increases, at optical energy doses ranging from 1-140 J cm-2 from an argon-pumped dye laser. Appropriate controls were also prepared. Brain tissue samples for histological analysis were taken 24 h following PDT treatment. Maximum lesion depth perpendicular to the pial brain surface, was measured using an eyepiece micrometer. Lesions of increasing depth were generated as the incident optical energy dose was increased. Fitting the depth of necrosis to a natural log dependence of incident optical dose yielded a slope of 0.83 mm/ln J cm-2 (r2 = 0.99). The intercept of 1.47 J cm-2 indicated the energy dose below which no normal tissue damage would occur at the incident laser intensity of 100 mW cm-2. The smallest lesions consisted almost exclusively of isolated neuronal injury and neuropil vacuolation, suggestive of an early ischemic lesion. Damage at the upper energy levels (35-140 J cm-2) consisted of complete coagulative necrosis identical to that induced by an arterial occlusion. The existence of viable tissue alongside neurons in various stages of necrosis at low energy levels (less than 35 J cm-2) is suggestive of reversible injury and possibly clinically relevant treatment levels.

C57BL/6 mice with syngeneic B16-F10 melanomas were treated 7 days after tumor inoculation into the footpad with local hyperthermia (HT) of 43.5 degrees C for 90 min. A combination of local 30 Gy X-irradiation (XRT) given 2, 4 or 12 h after HT cured the primary tumor in 34/35 mice, with irreversible damage to normal foot tissues in most of the animals. When 7.5, 10 or 15 Gy XRT were delivered 4, 18 or 24 h after HT, there were only a small number of cures and also a much smaller incidence of irreversible normal tissue damage. HT alone resulted in a significant (P less than 0.001) increase in metastases to regional lymph nodes (RLN) and the lungs. The 'curative' doses of combined XRT and HT resulted in a significant (P less than 0.001) decrease in metastasis to RLN and to the lungs. Conversely, subcurative doses of combined therapy resulted in an increase in RLN and lung metastasis (P less than 0.001). Abdominal lymph node metastasis, not usually seen in control mice, is markedly increased after HT alone or in combination with subcurative XRT (P less than 0.001). The overall survival of mice treated with HT alone is decreased (P less than 0.0028). The survival of mice treated with HT followed 4, 18 or 24 h later with 10 Gy XRT is further decreased (P less than 0.0025). These data show that subcurative HT, or XRT plus HT, increases the incidence of spontaneous metastasis in this syngeneic mouse melanoma model. Curative doses prevent this effect on metastasis, but there is an unacceptable incidence of irreversible damage to the tumor-bearing foot. The cause(s) of this phenomenon are not known.

The metabolic response of normal rat brain to photodynamic therapy (PDT) was studied over a 1 week interval using in vivo 31P-NMR spectroscopy. Rats injected with 12.5 mg/kg Photofrin II were submitted to brain photoactivation 48 h after drug administration with either 140 or 70 J/cm2 light (630 +/- 1 nm) from an Argon dye laser. Control studies, animals not given drug or light, animals submitted only to brain illumination without drug, and animals given drug but no light, were also performed. The data revealed a transient metabolic degradation; a decrease in the ratio of beta-nucleotriphosphate to inorganic phosphate (P less than 0.001) at 24 h after PDT treatment was followed by a return to pretreatment spectral values. Brain tissue alkalosis was also noted, with significant (P less than 0.05) differences in brain tissue pH detected at 72 h post treatment between 70 J/cm2 PDT vs control studies and at 1 week post treatment between 140 J/cm2 vs 70 J/cm2, 140 J/cm2 vs no light-no drug and 140 J/cm2 vs drug only. The data suggest that there is no difinitive metabolic marker from 31P-NMR spectroscopy that can identify necrotic brain tissue caused by PDT. Phosphorus-31 NMR data are also presented which suggest that PDT damage to brain is not solely the result of microvascular occlusion causing ischemic necrosis.

Considerable attention has been paid to the modification of intratumor pH in response to hyperthermia. It has been hypothesized that observed reductions in intralesional pH are involved in the ultimate response of tissue to hyperthermia treatment. Further, it has been shown that significant differences exist in hyperthermia-induced changes in blood flow between tumor and normal tissue in many systems. Changes in blood flow are hypothesized to be related to observed changes in pH. Since reduced blood flow is not observed in normal tissue under normal treatment conditions, changes in pH in normal tissue have not been considered significant in their response to hyperthermia treatment. However, this conclusion has not been verified or documented experimentally. The purpose of this study was to examine the distribution of pH in normal tissue (muscle) as a function of time following hyperthermia treatments which in the same animal system resulted in subcurative (TCD 10/30) or curative (TCD 90/30) tumor (mammary adenocarcinoma) responses. The observed distribution of pH in normal tissue was compared with that obtained in tumors under identical conditions. The results indicate that some post-treatment changes in muscle pH do occur following hyperthermia, but that these changes are small compared to those observed in tumors. More importantly, unlike the response observed in tumors, no hyperthermia dose dependency is observed in the muscle response. From these studies it can be concluded that changes in normal muscle pH are probably not associated with normal tissue response to hyperthermia.

Photodynamic therapy utilizes the tumor localizing drug dihematoporphyrin ether and red laser light to produce both direct tumor cell destruction via damage to mitochondrial membranes, and also indirect cell kill via destruction of the tumor vasculature. As a first step towards examining the mechanistic relationship between metabolic and vascular effects of photodynamic therapy, murine RIF-1 tumors were treated with a subcurative treatment (500 J/cm2). Tumor metabolic status was monitored using in vivo 31P NMR before, during and after the treatment. The tumor blood flow immediately before and after treatment was measured by direct intratumor injection of D2O saline and observation of the tracer signal clearance from the tumor via 2H NMR. During the photodynamic therapy treatment, significant decreases were observed for the nucleoside triphosphate concentrations, tumor pH and tumor blood flow, while inorganic phosphate concentrations increased. Animals treated with laser light alone and those not given any treatment, demonstrated no significant changes in tumor metabolic status, tumor pH or tumor blood flow. Further studies are required to determine whether tumor blood flow or metabolic status is affected first.

The metabolic response of mammary carcinoma in the C3H mouse to photodynamic therapy (PDT) was measured using in vivo 31P nuclear magnetic resonance (31P-NMR) spectroscopy and pH microelectrodes. Twenty-four hours after administration of Photofrin II (12.5 mg/kg), the tumor was subjected to photoactivation using an argon dye laser. Optical treatment doses were 200, 400, and 600 J/cm2 and corresponded to the following tumor control doses: TCD10/30, TCD50/30, and TCD90/30, respectively. In vivo 31P-NMR spectra and pH micro-electrode measurements were obtained prior to treatment and at 4, 24, 48, and 72 h and 1 week post-treatment. The data revealed a significant (P less than 0.0002) alkalosis as indicated by the pH measured by NMR compared to pH measured by microelectrodes at all treatment levels and time points. Spectral differences between treatment groups were apparent as early as 4 h after treatment. The ratio of beta-nucleoside triphosphate to inorganic phosphate at 4 h after treatment was significantly (P less than 0.01) smaller for 600 J/cm2 treatment than for 200 J/cm2 treatment. At curative (600 J/cm2) levels, from 48 h on, no phosphate resonances were detected in the spectra. The pH measured by NMR transiently decreased from pretreatment levels after 200 and 400 J/cm2 treatment (P less than 0.002, P less than 0.009, respectively), while no change in pH from pretreatment values was found after 600 J/cm2 treatment. The data suggest that the early metabolic response of mammary carcinoma to PDT, as indicated by 31P-NMR spectroscopy, is dose dependent, and may be a sensitive indicator of biological outcome to treatment.

Metastasis to distant sites is mediated by various receptors on the surface of tumor cells. B16-F1 melanomas surviving 43.5 degrees C heat in vitro for 15 minutes and cultured for 10 days bind significantly increased amounts of the basement membrane protein laminin. Motility of heat-resistant B16-F1 cells in vitro toward the chemoattractant laminin is significantly increased. The increased expression of putative laminin receptors may be associated with increased metastasis of melanomas after subcurative hyperthermia.

Hematoporphyrin derivative photodynamic therapy has very important clinical applicability in the diagnosis and treatment of transitional cell carcinoma of the bladder, but many aspects of the photodynamic process are yet to be elucidated. This paper investigates the role of dihematoporphyrin ether (DHE) concentration, the duration of light exposure, and the initial size of the tumors in the treatment of a transplantable murine transitional cell tumor system. The best results were noted in tumors less than six mm. in diameter when treated with 15 mg./kg. DHE and exposed to 100 to 180 minutes of visible light. Animals with small initial tumor size combined with higher DHE concentration and longer light exposure time were most likely to show tumor response.

Following hyperthermia a dramatic drop in intra-lesional pH has occurred in a variety of experimental tumor systems. To date, no direct observations have been made that document the time course of such changes or the recovery from such changes over prolonged periods of time. These experiments were designed to measure intralesional pH as a function of time following "doses" of hyperthermia related to specific biological end points. All studies were conducted in the C3H-mammary carcinoma tumor model system. Intralesional temperature was continuously monitored throughout treatment and post treatment pH was measured with microelectrodes at several specific time points ranging from 4 hrs to 7 days. The pretreatment control value of mean pH was 6.73. At the TCD90 "dose" level the mean value of pH dropped to a level of 6.22 +/- .095 while following the TCD10 "dose" the value obtained was 6.55 +/- .148. Recovery of the pH to higher values followed similar time courses returning to maximum values approximately 3 days post treatment. The recovery, plateau levels of pH were however, separated by approximately .3 pH units with the higher "dose" curve always at lower values than that achieved following the lower "dose".

Sterile L-2-oxothiazolidine-4-carboxylate, given as a neutral 100 mmol solution at a dose of 8 mmol/kg subcutaneously, caused an increase in total glutathione concentration in the brain of treated rats. This finding could be important in understanding the role of glutathione in the central nervous system.

We have characterized the oxygen distribution in V-79 spheroids which were grown by the spinner flask method. Using ultramicroelectrodes with tip diameters of 1-5 microns and a perfusion system whereby the spheroids' milieu could be maintained and controlled, we found plateau pO2 values of less than 10 mm Hg in spheroids of greater than 500 microns in diameter. Data from experiments using respiration inhibitory drugs indicate that the characteristics of the outer layers of cells is the major determinant of the oxygen profile in the interior. Parallel control radiobiological experiments confirmed the control pO2 measurements, and formed the basis for the experiments using the potential indirect radiation sensitizers: Chlorambucil and Mustargen.

Photoradiation therapy is emerging as a promising technique for combating cancer. Fundamentally, this approach consists of two steps: hematoporphyrin derivative (HPD) is used to selectively sensitize cancer cells to visible light; after an appropriate time interval, light is introduced into the tumor via a laser-fiber optic system to trigger the cytotoxic action of HPD. The present investigation was initiated to determine the therapeutic potential of HPD in combination with a chemiluminescent activator in treating mice which had been transplanted with tumors.

We heated Wistar rats (n = 10) to 41.5 +/- 0.2 degrees C for 15 minutes, 24 hours before the induction of forebrain cerebral ischemia. We subjected 23 rats to forebrain ischemia without prior heating. Ischemic cell damage in the medial, lateral, and overall CA 1/2 hippocampus, inferior frontal cortex, and dorsal-lateral striatum was significantly (p less than 0.05) less severe in heated animals than in nonheated animals.

In the past several years numerous investigators have employed various mouse tumor systems and a variety of heating methods to examine the effects of hyperthermia in vivo. These studies have led, in some cases, to different results in tumor response in what appear to be similar experiments. Our results indicate that these discrepancies may in part be attributable to variations in intratumor temperature and depend strongly upon the method and conditions of heating employed. In all cases, we have employed the C3H mammary carcinoma tumor system with the tumor implanted in the right hind limb of the mouse. All tumors were 7-9 mm in diameter at the time of the experiments. Water bath heating was employed with the animals under one of four possible conditions: the presence or absence of anesthesia, and the use or lack of core cooling. Thermometry was performed with an array of multiple microthermocouples, each less than 150 micron in diameter, implanted in the tumor in a grid-like pattern. Significant variations (1.0 degrees C for no anesthesia, p less than .01; 1.3 degrees C for Thorazine/Ketamine, p less than .001) in intratumor temperature were found across the tumor in the unperturbed state. These variations were essentially eliminated under all hyperthermic conditions. Control of core (rectal) temperature (with or without anesthesia) reduced the intratumor temperatures even though the water bath temperature was held constant resulting in variations between water bath and tumor temperatures of from 0.1-0.8 degrees C.

We evaluated the effects of local X-irradiation on microscopic or small macroscopic primary melanomas in the feet of C57BL/6 mice and the subsequent development of spontaneous femoral lymph node (LN) metastases. Doses of 30, 40, 55, 62.5, or 72.5 Gy often cured the foot tumor and metastases to regional femoral lymph nodes were relatively uncommon. Doses of 3.75, 7.5, 10, 15, and 20 Gy were associated with a dose-dependent regrowth delay of the foot tumor treated at microscopic size. Foot melanomas that were not cured spread to regional femoral LNs more frequently (P less than 0.001). The relative risk of developing femoral LN metastasis increased 2.55 times for each 1-mm increase in the anteroposterior diameter of the primary foot tumor in mice with 20 days of primary tumor exposure and increased 4.87 times for each 1-mm increase in mice with 100 days of primary tumor exposure. Although tumors treated with subcurative doses of irradiation had a longer period of time to metastasize to regional LNs for each 1-mm increase in primary tumor size, this variable alone did not account for the increased incidence of metastasis seen with irradiation.