Effect of Photofrin-mediated photocytotoxicity on a panel of human pancreatic cancer cells.

BACKGROUND AND OBJECTIVE: Pancreatic cancer is a leading cause of cancer-related deaths in men and women. Early clinical studies suggest that photodynamic therapy (PDT) might be a useful modality in the management of this deadly disease. In this study, the photocytotoxicity of Photofrin-mediated PDT on different human pancreatic cancer cells (BxPc-3, HPAF-II, Mia PaCa-2, MPanc-96, PANC-1 and PL-45) was examined. MATERIALS AND METHODS: After co-incubating cancer cells with Photofrin (0-10 µg/ml) for 4h, the cells were irradiated with 0-6J/cm(2) of 630 nm light. The effect of Photofrin PDT on the survival of cells were examined using tetrazolium-based colorimetric assay and clonogenic assay. PDT-induced apoptosis was analyzed by flow cytometry. Expressions of apoptosis-related proteins were determined by western blot analysis. RESULTS: Photofrin PDT strongly inhibited the survival of pancreatic cancer cells. A small portion of cells (<15%) underwent apoptosis 24h after PDT at LD50. Cleavage of caspase-3, caspase-8, caspase-9 and PARP after PDT were also confirmed. BxPc-3, Mia PaCa-2, MPanc-96, and PANC-1 cells were more sensitive and HPAF-II and PL-45 cells less sensitive. CONCLUSION: Photofrin PDT can induce apoptosis and inhibit survival of human pancreatic cancer cells.

Self-expandable metal stents and trans-stent light delivery: are metal stents and photodynamic therapy compatible?

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.

The effect of Tookad-mediated photodynamic ablation of the prostate gland on adjacent tissues--in vivo study in a canine model.

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).

Magnetic resonance imaging correlated with the histopathological effect of Pd-bacteriopheophorbide (Tookad) photodynamic therapy on the normal canine prostate gland.

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.

Effects of photodynamic therapy on peripheral nerve: in situ compound-action potentials study in a canine model.

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.

Studies of a vascular-acting photosensitizer, Pd-bacteriopheophorbide (Tookad), in normal canine prostate and spontaneous canine prostate cancer.

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.

Effects of Pd-bacteriopheophorbide (TOOKAD)-mediated photodynamic therapy on canine prostate pretreated with ionizing radiation.

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.

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy.

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.

Laser-assisted penetration of topical anesthetic in adults.

OBJECTIVE: To determine whether pretreatment of skin with erbium:YAG (Er:YAG) laser-assisted delivery facilitates the penetration of lidocaine cream to provide anesthesia suitable for needlesticks after just 5 minutes. DESIGN: Trial 1 was a double-blind randomized controlled trial, whereas trial 2 was a nonblinded randomized controlled trial. SETTING: The study was conducted in 2 facilities, an academic and a private clinical research unit. PARTICIPANTS: A total of 320 healthy volunteers, aged 18 to 65 years and of any Fitzpatrick skin phototype. INTERVENTIONS: Trial 1 involved an Er:YAG laser pretreatment to disrupt the stratum corneum followed by an application of 4% lidocaine cream on one arm, and a laser pretreatment plus placebo on the other arm. Trial 2 involved an application of 4% lidocaine cream alone on one arm, and a laser pretreatment followed by an application of 4% lidocaine cream on the other arm. MAIN OUTCOME MEASURE: Self-reported pain perception on a 100-mm visual analog scale after quick insertion and removal of a 25-gauge hypodermic needle on the treatment sites. RESULTS: Data from the 2 trials showed that there was a 62% pain reduction with laser pretreatment plus lidocaine compared with laser pretreatment plus placebo, and a 61% pain reduction with laser pretreatment plus lidocaine, compared with lidocaine alone. The decrease in pain in both trials was statistically significant (P<.001). Adverse events reported 48 hours after treatment were few and mild. CONCLUSION: Treatment with the Er:YAG laser followed by lidocaine cream is a safe, effective, and efficient means of inducing skin anesthesia that significantly reduces the pain of hypodermic needle insertion.

Preclinical studies in normal canine prostate of a novel palladium-bacteriopheophorbide (WST09) photosensitizer for photodynamic therapy of prostate cancers.

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.

Laser assisted delivery of topical anesthesia for intramuscular needle insertion in adults.

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.

Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy.

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.