Thomas Jeffery Wieman

PHOTODYNAMIC THERAPY IN THE TREATMENT OF PANCREATIC CARCINOMA: DIHEMATOPORPHYRIN ETHER UPTAKE and PHOTOBLEACHING KINETICS

Abstract Results of dihematoporphryin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40‐60 μg DHE g−1 of tissue at an injected dose of 40 mg kg−1. Previously concentrations on this order have primarily been found in organs of the reticuloendothelial system. Two intra‐pancreatic carcinoma models, one of acinar origin (rat) and one of ductal origin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared with normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630‐nm light from a dye laser (75 mW cm−2, 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration.

In Vivo and In Vitro Photodynamic Studies with Benzochlorin Iminium Salts Delivered by a Lipid Emulsion

Benzochlorin iminium salts (Bis) are hydrophobic photosensitizers based on an octaethylbenzochlorin nucleus that absorb in the near‐IR region of the visible spectrum. In these studies the photodynamic activities of the zinc, copper and metal‐free BI derivatives were compared in vivo in C3H‐HeJ mice bearing a mammary adenocarcinoma tumor line. In vitro studies were also performed with the radiation‐induced fibrosarcoma tumor line. An argon‐pumped Ti‐sapphire laser tuned to deliver light between 710 and 800 nm or an Oriel arc‐lamp filtered to deliver broadband light above 590 nm were used as light source. A lipid emulsion was used as the delivery system for sensitizers in all studies. A pronounced solvent dependence was observed for the Q band for each of all iminium salts examined. As an example, the metal‐free (BI) derivative had an absorption maximum at 798 nm in dichloromethane and at 727 nm in serum. The action spectra showed a greater PDT response at blue‐shifted wavelengths for each of the three iminium salts both in vivo and in vitro. Among the three derivatives, the zinc analog (ZnBI) produced the greatest tumor regression at the low drug/light dose of 0.7 (μ mole/kg and 200 J/cm2. These results indicate that iminium salts have characteristics that may make them promising third‐generation photosensitizers.

Photodynamic Therapy Trials with Lutetium Texaphyrin (Lu-Tex) in Patients with Locally Recurrent Breast Cancer

Photodynamic therapy (PDT) of locally recurrent breast cancer has been limited to treatment of small lesions because of non- selective necrosis of adjacent normal tissues in the treatment field. Lutetium Texaphyrin (PCI-0123, Lu-Tex) is a photosensitizer with improved tumor localization that is activated by 732 nm light, which can penetrate through larger tumors. We have evaluated Lu-Tex in a Phase I trial and in an ongoing Phase II trial in women with locally recurrent breast cancer with large tumors who have failed radiation therapy. Patients received Lu-Tex intravenously by rapid infusion 3 hours before illumination of cutaneous or subcutaneous lesions. In Phase I, Lu-Tex doses were escalated from 0.6 to 7.2 mg/kg in 7 cohorts. Sixteen patients with locally recurrent breast cancer lesions were treated. Dose limiting toxicities above 5.5 mg/kg were pain in the treatment field during therapy, and dysesthesias in light exposed areas. No necrosis of normal tissues in the treated field was noticed. Responses were observed in 60% of evaluable patients [n equals 15, 27% complete remission (CR), 33% partial remission (PR)], with 63% of lesions responding (n equals 73: 45% CR, 18% PR). In Phase II, 25 patients have been studied to date, receiving two treatments ranging from 1.0 to 3.0 mg/kg at a 21 day interval. Treatment fields up to 480 cm2 in size were treated successfully and activity has been observed. Patients have experienced pain at the treatment site but no tissue necrosis. These studies demonstrate the feasibility of Lu-Tex PDT to large chest wall areas in women who have failed radiation therapy for the treatment of locally recurrent breast cancer. Treatment conditions are currently being optimized in the ongoing Phase II trials.

Use of scanning Doppler velocimetry to monitor vascular changes during photodynamic therapy

We are investigating the use of Scanning Doppler Velocimetry to evaluate changes in tissue perfusion that occur during Photodynamic Therapy (PDT). In initial studies, this technique was used to assess changes in tissue perfusion in mice given PDT using Photofrin, Purlytin, or Lutrin. There was a rapid and complete loss of perfusion in both normal tissue and tumor in animals given PDT using Photofrin. Using Purlytin, there was immediate loss of perfusion at the tumor site. For PDT using Lutrin, there was a lot of perfusion in the tumor with relatively no change in the surrounding normal skin, confirming the high degree of selectivity of PDT with this photosensitizer. These reductions in persistent through at least 24 h after PDT.

Studies on the absence of photodynamic mechanism in the normal pancreas

Extraction procedures to quantitate porfimer sodium concentration in tissues were correlated with fluorescence measurements made in vivo, on hamster and rat normal pancreas and intra-pancreatic tumors. The uptake of photosensitizer has been shown to be high in both normal and malignant pancreatic tissues, in both animal models studied. Photobleaching of the drug, as evidenced by both techniques within the pancreatic tumor, occurs in a typical manner during PDT, with resultant tissue destruction. In contrast, when the normal pancreas is exposed to PDT, a negligible photobleaching effect, as well as a lack of tissue response, is observed. The lack of observable response is corroborated by a lack of measurable physiological response. Both serum amylase and serum glucose show acute changes up to 12 hours post treatment but quickly return to normal. HPLC analysis shows that the drug extracted from both the normal pancreas and intrapancreatic tumor is essentially the same as that extracted from other tissues and similar to that which has been injected into the animal. Fluorescence microscopy has shown that at time points between 12-120 hours the drug is associated with lymphatic channels. This would not, however, necessarily preclude normal tissue destruction. Similar results have been found with other photosensitizers. Understanding the lack of response in the pancreas may lead to a deeper understanding of the diseased state which is normally refractory to all therapy as well as understanding the fundamental concepts of the mechanisms of PDT.

An Investigation Of Photodynamic Therapy In The Treatment Of Pancreatic Carcinoma: Dihematoporphyrin Ether Uptake And Photobleaching Kinetics

Results of dihematoporphyrin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40-60 μg DHE/g of tissue at an injected dose of 40 mg/kg. Previously concentrations on this order have only been found in organs of the reticuloendothelial system. Two intrapancreatic carcinoma models, one of acinar origin (rat) and one of ductal orgin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared to normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630 nm light from a dye laser (75 mW/cm2, 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet, the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration. These results represent the emergence of an entirely new modality, with substantial potential for the treatment of cancer of the pancreas.

Differential vascular response and relationship to tumor response with photodynamic therapy using WST-09 (TOOKAD)

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

Modulation of photodynamic activity with Photofrin: effect of dose, time interval, fluence, and delivery system

A goal of our laboratory is to accurately define the parameters of light dose and drug dose that contribute to tissue destruction after Photodynamic therapy (PDT). Using Photofrin as sensitizer, we examined a range of drug doses, various intervals between injection and light treatment, and various fluence rates. The effect of Photofrin photosensitizer encapsulated in liposomal delivery vehicle was also studied. Three liposome delivery vehicles were chosen to deliver the photosensitizer in vivo: DPPC/cholesterol, DMPC/HPC and stealth liposomes. Tumor response and microvessel behaviour were examined in tumor and surrounding skin in a mouse model. Under these conditions, better selectivity of tissue damage was seen using some of the treatment. These data might be used to design better clinical protocols for patient care. In memory of Dr. Victor Fingar (Supported by R01 CA51771).

Automatic segmentation and quantification of fluorescing microspheres adhering to capillary endothelial cells in the rat lung

Adhesion molecules present in the cellular membrane of the endothelium provide sites of leukocyte adherence as a first step in the process of leukocyte migration into the interstitium. New evidence suggests the same adhesion proteins may be responsible for the spread of metastatic tumors by providing a location for tumor cell attachment. A method was sought to quantitate the degree of adhesion molecule expression in the pulmonary capillary endothelium using a recently developed animal model which allows for viewing the lung surface in vivo. Videoimages of the pulmonary vascular system were gathered using this new lung chamber technique. A fully automated digital image processing and analysis (DIPA) system was also developed to estimate the level of intercellular adhesion molecule-1 (ICAM-1) expression on the capillary endothelial cells in these videoimages. Fluorescent microspheres were immunologically bound to the ICAM-1 molecules present on the endothelial cell surface. The DIPA system then located and quantified the fluorescent spots present in the videoimages. The ability of this system to locate and measure the fluorescence was compared with human measurements of the same images.

Mechanisms of vessel damage in photodynamic therapy (Invited Paper)

Vessel constriction and platelet aggregation are observed within the first minutes of light exposure to photosensitized tissues and lead to blood flow stasis, tissue hypoxia, and nutrient depravation. The mechanism for these vessel changes remains unknown, although the release of eicosanoids is implicated. We propose the following hypothesis: Photodynamic therapy results in specific perturbations of endothelial cells which results in a combination of membrane damage, mitochondrial damage, and rearrangement of cytoskeletal proteins. This results in cellular stress which leads to interruption of tight junctions along the endothelium and cell rounding. Cell rounding exposes the basement membrane proteins causing activation of platelets and leukocytes. Activated platelets and leukocytes release thromboxane and other eicosanoids. These eicosanoids induce vasoconstriction, platelet aggregation, increases in vessel permeability, and blood flow stasis.